Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses.

PubMed

Clark, Lars E; Mahmutovic, Selma; Raymond, Donald D; Dilanyan, Taleen; Koma, Takaaki; Manning, John T; Shankar, Sundaresh; Levis, Silvana C; Briggiler, Ana M; Enria, Delia A; Wucherpfennig, Kai W; Paessler, Slobodan; Abraham, Jonathan

2018-05-14

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.

Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses

PubMed Central

Brouillette, Rachel B.; Phillips, Elisabeth K.; Ayithan, Natarajan

2017-01-01

ABSTRACT The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies. PMID:28100617

Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

PubMed

Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

2017-04-01

The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies. Copyright © 2017 American Society for Microbiology.

Self-Association of Lymphocytic Choriomeningitis Virus Nucleoprotein Is Mediated by Its N-Terminal Region and Is Not Required for Its Anti-Interferon Function

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin

2012-01-01

Arenaviruses have a bisegmented, negative-strand RNA genome. Both the large (L) and small (S) genome segments use an ambisense coding strategy to direct the synthesis of two viral proteins. The L segment encodes the virus polymerase (L protein) and the matrix Z protein, whereas the S segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC). NPs are the most abundant viral protein in infected cells and virions and encapsidate genomic RNA species to form an NP-RNA complex that, together with the virus L polymerase, forms the virus ribonucleoprotein (RNP) core capable of directing both replication and transcription of the viral genome. RNP formation predicts a self-association property of NPs. Here we document self-association (homotypic interaction) of the NP of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), as well as those of the hemorrhagic fever (HF) arenaviruses Lassa virus (LASV) and Machupo virus (MACV). We also show heterotypic interaction between NPs from both closely (LCMV and LASV) and distantly (LCMV and MACV) genetically related arenaviruses. LCMV NP self-association was dependent on the presence of single-stranded RNA and mediated by an N-terminal region of the NP that did not overlap with the previously described C-terminal NP domain involved in either counteracting the host type I interferon response or interacting with LCMV Z. PMID:22258244

Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses

PubMed Central

Trombley, Adrienne R.; Wachter, Leslie; Garrison, Jeffrey; Buckley-Beason, Valerie A.; Jahrling, Jordan; Hensley, Lisa E.; Schoepp, Randal J.; Norwood, David A.; Goba, Augustine; Fair, Joseph N.; Kulesh, David A.

2010-01-01

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan™-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies. PMID:20439981

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory

PubMed Central

Alimonti, Judie; Leung, Anders; Jones, Shane; Gren, Jason; Qiu, Xiangguo; Fernando, Lisa; Balcewich, Brittany; Wong, Gary; Ströher, Ute; Grolla, Allen; Strong, James; Kobinger, Gary

2014-01-01

Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route. PMID:25059478

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory.

PubMed

Alimonti, Judie; Leung, Anders; Jones, Shane; Gren, Jason; Qiu, Xiangguo; Fernando, Lisa; Balcewich, Brittany; Wong, Gary; Ströher, Ute; Grolla, Allen; Strong, James; Kobinger, Gary

2014-07-25

Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route.

Virions at the gates: receptors and the host-virus arms race.

PubMed

Coffin, John M

2013-01-01

All viruses need to bind to specific receptor molecules on the surface of target cells to initiate infection. Virus-receptor binding is highly specific, and this specificity determines both the species and the cell type that can be infected by a given virus. In some well-studied cases, the virus-binding region on the receptor has been found to be unrelated to the receptor's normal cellular function. Resistance to virus infection can thus evolve by selection of mutations that alter amino acids in the binding region with minimal effect on normal function. This sort of positive selection can be used to infer the history of the host-virus "arms race" during their coevolution. In a new study, Demogines et al. use a combination of phylogenetic, structural, and virological analysis to infer the history and significance of positive selection on the transferrin receptor TfR1, a housekeeping protein required for iron uptake and the cell surface receptor for at least three different types of virus. The authors show that only two parts of the rodent TfR1 molecule have been subject to positive selection and that these correspond to the binding sites for two of these viruses-the mouse mammary tumor virus (a retrovirus) and Machupo virus (an arenavirus). They confirmed this result by introducing the inferred binding site mutations into the wild-type protein and testing for receptor function. Related arenaviruses are beginning to spread in human populations in South America as the cause of often fatal hemorrhagic fevers, and, although Demogines et al. could find no evidence of TfR1 mutations in this region that might have been selected as a consequence of human infection, the authors identified one such mutation in Asian populations that affects infection with these viruses.

The reversibility of virus attachment to mineral surfaces

USGS Publications Warehouse

Loveland, J.P.; Ryan, J.N.; Amy, G.L.; Harvey, R.W.

1996-01-01

Virus transport through groundwater is limited by attachment to mineral surfaces and inactivation. Current virus transport models do not consider the implications of the reversibility of virus attachment to minerals. To explore the reversibility of virus attachment to mineral surfaces, we attached PRD1, a bacteriophage considered to be a good model of enteric viruses, to quartz and ferric oxyhydroxide-coated quartz surfaces over a range of pH values in equilibrium 'static columns'. Following attachment, we detached the viruses by replacing the pore solution with solutions of equal and higher pH. The extent of virus attachment followed an attachment 'edge' that occurred at a pH value about 2.5-3.5 pH units above the pH(IEP) of the mineral surfaces. Viruses attached below this edge were irreversibly attached until the pH of the detachment solution exceeded the pH value of the attachment edge. Viruses attached above this edge were reversibly attached. Derjaguin-Landau-Verwey-Overbeek (DEVO) potential energy calculations showed that the attachment edge occurred at the pH at which the potential energy of the primary minimum was near zero, implying that the position of the primary minimum (attractive or repulsive) controlled the equilibrium distribution of the viruses. The results suggest that the reversibility of virus attachment must be considered in virus transport models for accurate predictions of virus travel time.

Seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses.

PubMed

van Riel, Debby; den Bakker, Michael A; Leijten, Lonneke M E; Chutinimitkul, Salin; Munster, Vincent J; de Wit, Emmie; Rimmelzwaan, Guus F; Fouchier, Ron A M; Osterhaus, Albert D M E; Kuiken, Thijs

2010-04-01

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses-two seasonal influenza viruses and pandemic H1N1 virus-attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.

Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

PubMed

Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

2012-10-12

The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

Detection of viruses in used ventilation filters from two large public buildings.

PubMed

Goyal, Sagar M; Anantharaman, Senthilvelan; Ramakrishnan, M A; Sajja, Suchitra; Kim, Seung Won; Stanley, Nicholas J; Farnsworth, James E; Kuehn, Thomas H; Raynor, Peter C

2011-09-01

Viral and bacterial pathogens may be present in the air after being released from infected individuals and animals. Filters are installed in the heating, ventilation, and air-conditioning (HVAC) systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. These filters process enormous volumes of air. This study was undertaken to determine the utility of sampling used ventilation filters to assess the types and concentrations of virus aerosols present in buildings. The HVAC filters from 2 large public buildings in Minneapolis and Seattle were sampled to determine the presence of human respiratory viruses and viruses with bioterrorism potential. Four air-handling units were selected from each building, and a total of 64 prefilters and final filters were tested for the presence of influenza A, influenza B, respiratory syncytial, corona, parainfluenza 1-3, adeno, orthopox, entero, Ebola, Marburg, Lassa fever, Machupo, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis viruses. Representative pieces of each filter were cut and eluted with a buffer solution. Attempts were made to detect viruses by inoculation of these eluates in cell cultures (Vero, MDCK, and RK-13) and specific pathogen-free embryonated chicken eggs. Two passages of eluates in cell cultures or these eggs did not reveal the presence of any live virus. The eluates were also examined by polymerase chain reaction or reverse-transcription polymerase chain reaction to detect the presence of viral DNA or RNA, respectively. Nine of the 64 filters tested were positive for influenza A virus, 2 filters were positive for influenza B virus, and 1 filter was positive for parainfluenza virus 1. These findings indicate that existing building HVAC filters may be used as a method of detection for airborne viruses. As integrated long-term bioaerosol sampling devices, they may yield valuable information on the epidemiology and aerobiology of viruses in air that can inform the development of methods to prevent airborne transmission of viruses and possible deterrents against the spread of bioterrorism agents. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Vaccine Platforms to Control Arenaviral Hemorrhagic Fevers.

PubMed

Carrion, Ricardo; Bredenbeek, Peter; Jiang, Xiaohong; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S

2012-11-20

Arenaviruses are rodent-borne emerging human pathogens. Diseases caused by these viruses, e.g., Lassa fever (LF) in West Africa and South American hemorrhagic fevers (HFs), are serious public health problems in endemic areas. We have employed replication-competent and replication-deficient strategies to design vaccine candidates potentially targeting different groups "at risk". Our leader LF vaccine candidate, the live reassortant vaccine ML29, is safe and efficacious in all tested animal models including non-human primates. In this study we showed that treatment of fatally infected animals with ML29 two days after Lassa virus (LASV) challenge protected 80% of the treated animals. In endemic areas, where most of the target population is poor and many live far from health care facilities, a single-dose vaccination with ML29 would be ideal solution. Once there is an outbreak, a fast-acting vaccine or post-exposure prophylaxis would be best. The 2(nd) vaccine technology is based on Yellow Fever (YF) 17D vaccine. We designed YF17D-based recombinant viruses expressing LASV glycoproteins (GP) and showed protective efficacy of these recombinants. In the current study we developed a novel technology to clone LASV nucleocapsid within YF17D C gene. Low immunogenicity and stability of foreign inserts must be addressed to design successful LASV/YFV bivalent vaccines to control LF and YF in overlapping endemic areas of West Africa. The 3(rd) platform is based on the new generation of alphavirus replicon virus-like-particle vectors (VLPV). Using this technology we designed VLPV expressing LASV GP with enhanced immunogenicity and bivalent VLPV expressing cross-reactive GP of Junin virus (JUNV) and Machupo virus (MACV), causative agents of Argentinian and Bolivian HF, respectively. A prime-boost regimen required for VLPV immunization might be practical for medical providers, military, lab personnel, and visitors in endemic areas.

Attachment anxiety is related to Epstein–Barr virus latency

PubMed Central

Fagundes, Christopher P.; Jaremka, Lisa M.; Glaser, Ronald; Alfano, Catherine M.; Povoski, Stephen P.; Lipari, Adele M.; Agnese, Doreen M.; Yee, Lisa D.; Carson, William E.; Farrar, William B.; Malarkey, William B.; Chen, Min; Kiecolt-Glaser, Janice K.

2015-01-01

Attachment theory provides a framework for understanding individual differences in chronic interpersonal stress. Attachment anxiety, a type of relationship insecurity characterized by worry about rejection and abandonment, is a chronic interpersonal stressor. Stress impacts cellular immunity, including herpes-virus reactivation. We investigated whether attachment anxiety was related to the expression of a latent herpesvirus, Epstein–Barr virus (EBV), when individuals were being tested for breast or colon cancer and approximately 1 year later. Participants (N = 183) completed a standard attachment questionnaire and provided blood to assess EBV viral capsid antigen (VCA) IgG antibody titers. Individuals with more attachment anxiety had higher EBV VCA IgG antibody titers than those with less attachment anxiety. The strength of the association between attachment anxiety and antibody titers was the same at both assessments. This study is the first to show an association between latent herpesvirus reactivation and attachment anxiety. Because elevated herpesvirus antibody titers reflect poorer cellular immune system control over the latent virus, these data suggest that high attachment anxiety is associated with cellular immune dysregulation. PMID:24945717

A Small Molecule Inhibits Virion Attachment to Heparan Sulfate- or Sialic Acid-Containing Glycans

PubMed Central

Colpitts, Che C.

2014-01-01

ABSTRACT Primary attachment to cellular glycans is a critical entry step for most human viruses. Some viruses, such as herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV), bind to heparan sulfate, whereas others, such as influenza A virus (IAV), bind to sialic acid. Receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. However, no molecule that inhibits the attachment of viruses in both groups has yet been identified. Epigallocatechin gallate (EGCG), a green tea catechin, is active against many unrelated viruses, including several that bind to heparan sulfate or to sialic acid. We sought to identify the basis for the broad-spectrum activity of EGCG. Here, we show that EGCG inhibits the infectivity of a diverse group of enveloped and nonenveloped human viruses. EGCG acts directly on the virions, without affecting the fluidity or integrity of the virion envelopes. Instead, EGCG interacts with virion surface proteins to inhibit the attachment of HSV-1, HCV, IAV, vaccinia virus, adenovirus, reovirus, and vesicular stomatitis virus (VSV) virions. We further show that EGCG competes with heparan sulfate for binding of HSV-1 and HCV virions and with sialic acid for binding of IAV virions. Therefore, EGCG inhibits unrelated viruses by a common mechanism. Most importantly, we have identified EGCG as the first broad-spectrum attachment inhibitor. Our results open the possibility for the development of small molecule broad-spectrum antivirals targeting virion attachment. IMPORTANCE This study shows that it is possible to develop a small molecule antiviral or microbicide active against the two largest groups of human viruses: those that bind to glycosaminoglycans and those that bind to sialoglycans. This group includes the vast majority of human viruses, including herpes simplex viruses, cytomegalovirus, influenza virus, poxvirus, hepatitis C virus, HIV, and many others. PMID:24789779

Cotransport of clay colloids and viruses through water-saturated vertically oriented columns packed with glass beads: Gravity effects.

PubMed

Syngouna, Vasiliki I; Chrysikopoulos, Constantinos V

2016-03-01

The cotransport of clay colloids and viruses in vertically oriented laboratory columns packed with glass beads was investigated. Bacteriophages MS2 and ΦX174 were used as model viruses, and kaolinite (ΚGa-1b) and montmorillonite (STx-1b) as model clay colloids. A steady flow rate of Q=1.5 mL/min was applied in both vertical up (VU) and vertical down (VD) flow directions. In the presence of KGa-1b, estimated mass recovery values for both viruses were higher for VD than VU flow direction, while in the presence of STx-1b the opposite was observed. However, for all cases examined, the produced mass of viruses attached onto suspended clay particles were higher for VD than VU flow direction, suggesting that the flow direction significantly influences virus attachment onto clays, as well as packed column retention of viruses attached onto suspended clays. KGa-1b hindered the transport of ΦX174 under VD flow, while STx-1b facilitated the transport of ΦX174 under both VU and VD flow directions. Moreover, KGa-1b and STx-1b facilitated the transport of MS2 in most of the cases examined except of the case where KGa-1b was present under VD flow. Also, the experimental data were used for the estimation of virus surface-coverages and virus surface concentrations generated by virus diffusion-limited attachment, as well as virus attachment due to sedimentation. Both sedimentation and diffusion limited virus attachment were higher for VD than VU flow, except the case of MS2 and STx-1b cotransport. The diffusion-limited attachment was higher for MS2 than ΦΧ174 for all cases examined. Copyright © 2015 Elsevier B.V. All rights reserved.

Short report: duration of tick attachment required for transmission of powassan virus by deer ticks.

PubMed

Ebel, Gregory D; Kramer, Laura D

2004-09-01

Infected deer ticks (Ixodes scapularis) were allowed to attach to naive mice for variable lengths of time to determine the duration of tick attachment required for Powassan (POW) virus transmission to occur. Viral load in engorged larvae detaching from viremic mice and in resulting nymphs was also monitored. Ninety percent of larval ticks acquired POW virus from mice that had been intraperitoneally inoculated with 10(5) plaque-forming units (PFU). Engorged larvae contained approximately 10 PFU. Transstadial transmission efficiency was 22%, resulting in approximately 20% infection in nymphs that had fed as larvae on viremic mice. Titer increased approximately 100-fold during molting. Nymphal deer ticks efficiently transmitted POW virus to naive mice after as few as 15 minutes of attachment, suggesting that unlike Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, no grace period exists between tick attachment and POW virus transmission.

Fluorescence molecular painting of enveloped viruses.

PubMed

Metzner, Christoph; Kochan, Feliks; Dangerfield, John A

2013-01-01

In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment. Results from the FMP-flow cytometry approach were verified by immunoblotting and enzyme-linked immunosorbent assay. Since the modification strategy is applicable to a broad range of proteins and viruses, variations of this method may be useful in a range of research and applied applications from bio-distribution studies to vaccine development and targeted infection for gene delivery.

MS-2 and poliovirus transport in porous media: Hydrophobic effects and chemical perturbations

NASA Astrophysics Data System (ADS)

Bales, Roger C.; Li, Shimin; Maguire, Kimberly M.; Yahya, Moyasar T.; Gerba, Charles P.

1993-04-01

In a series of pH 7 continuous-flow column experiments, removal of the bacteriophage MS-2 by attachment to silica beads had a strong, systematic dependence on the amount of hydrophobic surface present on the beads. With no hydrophobic surface, removal of phage at pH 5 was much greater than at pH 7. Release of attached phage at both pH values did occur, but was slow; breakthrough curves exhibited tailing. Poliovirus attached to silica beads at pH 5.5 much more than at pH 7.0, and attachment was also slowly reversible. Time scales for phage and poliovinis attachment were of the order of hours. The sticking efficiency factor (α), reflecting microscaie physicochemical influences on virus attachment, was in the range of 0.0007-0.02. Phage release was small but measurable under steady state conditions. Release was enhanced by lowering ionic strength and by introducing beef extract, a high-ionic-strength protein solution. Results show that viruses experience reversible attachment/detachment (sometimes termed sorption), that large chemical perturbations are needed to induce rapid virus detachment, and that viruses should be quite mobile in sandy porous media. Even small amounts of hydrophobic organic material in the porous media (≥0.001%) can retard virus transport.

Transport and fate of viruses in sediment and stormwater from a Managed Aquifer Recharge site

NASA Astrophysics Data System (ADS)

Sasidharan, Salini; Bradford, Scott A.; Šimůnek, Jiří; Torkzaban, Saeed; Vanderzalm, Joanne

2017-12-01

Enteric viruses are one of the major concerns in water reclamation and reuse at Managed Aquifer Recharge (MAR) sites. In this study, the transport and fate of bacteriophages MS2, PRD1, and ΦX174 were studied in sediment and stormwater (SW) collected from a MAR site in Parafield, Australia. Column experiments were conducted using SW, stormwater in equilibrium with the aquifer sediment (EQ-SW), and two pore-water velocities (1 and 5 m day-1) to encompass expected behavior at the MAR site. The aquifer sediment removed >92.3% of these viruses under all of the considered MAR conditions. However, much greater virus removal (4.6 logs) occurred at the lower pore-water velocity and in EQ-SW that had a higher ionic strength and Ca2+ concentration. Virus removal was greatest for MS2, followed by PRD1, and then ΦX174 for a given physicochemical condition. The vast majority of the attached viruses were irreversibly attached or inactivated on the solid phase, and injection of Milli-Q water or beef extract at pH = 10 only mobilized a small fraction of attached viruses (<0.64%). Virus breakthrough curves (BTCs) were successfully simulated using an advective-dispersive model that accounted for rates of attachment (katt), detachment (kdet), irreversible attachment or solid phase inactivation (μs), and blocking. Existing MAR guidelines only consider the removal of viruses via liquid phase inactivation (μl). However, our results indicated that katt > μs > kdet > μl, and katt was several orders of magnitude greater than μl. Therefore, current microbial risk assessment methods in the MAR guideline may be overly conservative in some instances. Interestingly, virus BTCs exhibited blocking behavior and the calculated solid surface area that contributed to the attachment was very small. Additional research is therefore warranted to study the potential influence of blocking on virus transport and potential implications for MAR guidelines.

Spatial Distribution of Viruses Associated with Planktonic and Attached Microbial Communities in Hydrothermal Environments

PubMed Central

Nunoura, Takuro; Kazama, Hiromi; Noguchi, Takuroh; Inoue, Kazuhiro; Akashi, Hironori; Yamanaka, Toshiro; Toki, Tomohiro; Yamamoto, Masahiro; Furushima, Yasuo; Ueno, Yuichiro; Yamamoto, Hiroyuki; Takai, Ken

2012-01-01

Viruses play important roles in marine surface ecosystems, but little is known about viral ecology and virus-mediated processes in deep-sea hydrothermal microbial communities. In this study, we examined virus-like particle (VLP) abundances in planktonic and attached microbial communities, which occur in physical and chemical gradients in both deep and shallow submarine hydrothermal environments (mixing waters between hydrothermal fluids and ambient seawater and dense microbial communities attached to chimney surface areas or macrofaunal bodies and colonies). We found that viruses were widely distributed in a variety of hydrothermal microbial habitats, with the exception of the interior parts of hydrothermal chimney structures. The VLP abundance and VLP-to-prokaryote ratio (VPR) in the planktonic habitats increased as the ratio of hydrothermal fluid to mixing water increased. On the other hand, the VLP abundance in attached microbial communities was significantly and positively correlated with the whole prokaryotic abundance; however, the VPRs were always much lower than those for the surrounding hydrothermal waters. This is the first report to show VLP abundance in the attached microbial communities of submarine hydrothermal environments, which presented VPR values significantly lower than those in planktonic microbial communities reported before. These results suggested that viral lifestyles (e.g., lysogenic prevalence) and virus interactions with prokaryotes are significantly different among the planktonic and attached microbial communities that are developing in the submarine hydrothermal environments. PMID:22210205

Dynamic analysis of fixed-free single-walled carbon nanotube-based bio-sensors because of various viruses.

PubMed

Gupta, A; Joshi, A Y; Sharma, S C; Harsha, S P

2012-09-01

In the present study, the vibrations of the fixed-free single-walled carbon nanotube (SWCNT) with attached bacterium/virus on the tip have been investigated. To explore the suitability of the SWCNT as a bacterium/virus detector device, first the various types of virus have been taken for the study and then the resonant frequencies of fixed-free SWCNT with attachment of those viruses have been simulated. These resonant frequencies are compared with the published analytical data, and it is shown that the finite element method (FEM) simulation results are in good agreement with the analytical data. The results showed the sensitivity and suitability of the SWCNT having different length and different masses (attached at the tip SWCNT) to identify the bacterium or virus.

Temperature dependency of virus and nanoparticle transport and retention in saturated porous media

USDA-ARS?s Scientific Manuscript database

The influence of temperature (4 and 20 °C) on virus and nanoparticle attachment in columns packed with quartz sand was studied under various physiochemical conditions. Fitted values of the attachment rate coefficient (katt) and the solid fraction that contributed to attachment (Sf) were found to be...

Polymer-attached zanamivir inhibits synergistically both early and late stages of influenza virus infection

PubMed Central

Lee, Chia Min; Weight, Alisha K.; Haldar, Jayanta; Wang, Ling; Klibanov, Alexander M.; Chen, Jianzhu

2012-01-01

Covalently conjugating multiple copies of the drug zanamivir (ZA; the active ingredient in Relenza) via a flexible linker to poly-l-glutamine (PGN) enhances the anti-influenza virus activity by orders of magnitude. In this study, we investigated the mechanisms of this phenomenon. Like ZA itself, the PGN-attached drug (PGN-ZA) binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. Unlike monomeric ZA, however, PGN-ZA also synergistically inhibits early stages of influenza virus infection, thus contributing to the markedly increased antiviral potency. This inhibition is not caused by a direct virucidal effect, aggregation of viruses, or inhibition of viral attachment to target cells and the subsequent endocytosis; rather, it is a result of interference with intracellular trafficking of the endocytosed viruses and the subsequent virus-endosome fusion. These findings both rationalize the great anti-influenza potency of PGN-ZA and reveal that attaching ZA to a polymeric chain confers a unique mechanism of antiviral action potentially useful for minimizing drug resistance. PMID:23185023

TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection.

PubMed

Wang, Jing; Qiao, Luhua; Hou, Zhouhua; Luo, Guangxiang

2017-01-15

Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope. TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand, PS, may serve as novel targets for antiviral intervention. Copyright © 2017 American Society for Microbiology.

The Z Proteins of Pathogenic but Not Nonpathogenic Arenaviruses Inhibit RIG-i-Like Receptor-Dependent Interferon Production

PubMed Central

Xing, Junji; Ly, Hinh

2014-01-01

ABSTRACT Arenavirus pathogens cause a wide spectrum of diseases in humans ranging from central nervous system disease to lethal hemorrhagic fevers with few treatment options. The reason why some arenaviruses can cause severe human diseases while others cannot is unknown. We find that the Z proteins of all known pathogenic arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Lassa, Junin, Machupo, Sabia, Guanarito, Chapare, Dandenong, and Lujo viruses, can inhibit retinoic acid-inducible gene 1 (RIG-i) and Melanoma Differentiation-Associated protein 5 (MDA5), in sharp contrast to those of 14 other nonpathogenic arenaviruses. Inhibition of the RIG-i-like receptors (RLRs) by pathogenic Z proteins is mediated by the protein-protein interactions of Z and RLRs, which lead to the disruption of the interactions between RLRs and mitochondrial antiviral signaling (MAVS). The Z-RLR interactive interfaces are located within the N-terminal domain (NTD) of the Z protein and the N-terminal CARD domains of RLRs. Swapping of the LCMV Z NTD into the nonpathogenic Pichinde virus (PICV) genome does not affect virus growth in Vero cells but significantly inhibits the type I interferon (IFN) responses and increases viral replication in human primary macrophages. In summary, our results show for the first time an innate immune-system-suppressive mechanism shared by the diverse pathogenic arenaviruses and thus shed important light on the pathogenic mechanism of human arenavirus pathogens. IMPORTANCE We show that all known human-pathogenic arenaviruses share an innate immune suppression mechanism that is based on viral Z protein-mediated RLR inhibition. Our report offers important insights into the potential mechanism of arenavirus pathogenesis, provides a convenient way to evaluate the pathogenic potential of known and/or emerging arenaviruses, and reveals a novel target for the development of broad-spectrum therapies to treat this group of diverse pathogens. More broadly, our report provides a better understanding of the mechanisms of viral immune suppression and host-pathogen interactions. PMID:25552708

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells.

PubMed

Gitman, A G; Kahane, I; Loyter, A

1985-05-21

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.

The Laminin Receptor Is a Cellular Attachment Receptor for Classical Swine Fever Virus

PubMed Central

Chen, Jianing; He, Wen-Rui; Shen, Liang; Dong, Hong; Yu, Jiahui; Wang, Xiao; Yu, Shaoxiong; Li, Yongfeng; Li, Su; Luo, Yuzi; Sun, Yuan

2015-01-01

ABSTRACT Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The Erns and E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV Erns protein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane protein genes, we identified the laminin receptor (LamR) to be another attachment receptor. We demonstrate the involvement of LamR together with HS in virus attachment, and we elucidate the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle of flaviviruses and the development of antiviral strategies for flaviviruses. PMID:25694590

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens

PubMed Central

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping

2017-01-01

ABSTRACT The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received recombinant rabies viruses carrying only the CDV attachment protein according to the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies virus-based vaccine against CDV induces protective immune responses against both pathogens. PMID:28148801

Transport and retention of bacteria and viruses in biochar-amended sand.

PubMed

Sasidharan, Salini; Torkzaban, Saeed; Bradford, Scott A; Kookana, Rai; Page, Declan; Cook, Peter G

2016-04-01

The transport and retention of Escherichia coli and bacteriophages (PRD1, MS2 and ФX174), as surrogates for human pathogenic bacteria and viruses, respectively, were studied in the sand that was amended with several types of biochar produced from various feedstocks. Batch and column studies were conducted to distinguish between the role of attachment and straining in microbe retention during transport. Batch experiments conducted at various solution chemistries showed negligible attachment of viruses and bacteria to biochar before or after chemical activation. At any given solution ionic strength, the attachment of viruses to sand was significantly higher than that of biochar, whereas bacteria showed no attachment to either sand or biochar. Consistent with batch results, biochar addition (10% w/w) to sand reduced virus retention in the column experiments, suggesting a potential negative impact of biochar application to soil on virus removal. In contrast, the retention of bacteria was enhanced in biochar-amended sand columns. However, elimination of the fine fraction (<60μm) of biochar particles in biochar-amended sand columns significantly reduced bacteria retention. Results from batch and column experiments suggest that land application of biochar may only play a role in microbe retention via straining, by alteration of pore size distribution, and not via attachment. Consequently, the particle size distribution of biochar and sediments is a more important factor than type of biochar in determining whether land application of biochar enhances or diminishes microbial retention. Copyright © 2016 Elsevier B.V. All rights reserved.

A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

PubMed

Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

2014-04-01

Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

The Globoside Receptor Triggers Structural Changes in the B19 Virus Capsid That Facilitate Virus Internalization▿

PubMed Central

Bönsch, Claudia; Zuercher, Christoph; Lieby, Patricia; Kempf, Christoph; Ros, Carlos

2010-01-01

Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections. PMID:20826697

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

PubMed

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping; Schnell, Matthias J; von Messling, Veronika

2017-04-15

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received recombinant rabies viruses carrying only the CDV attachment protein according to the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies virus-based vaccine against CDV induces protective immune responses against both pathogens. Copyright © 2017 American Society for Microbiology.

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

PubMed Central

Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

2014-01-01

Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

Viral entry mechanisms: the increasing diversity of paramyxovirus entry

PubMed Central

Smith, Everett Clinton; Popa, Andreea; Chang, Andres; Masante, Cyril; Dutch, Rebecca Ellis

2009-01-01

The paramyxovirus family contains established human pathogens such as measles virus and human respiratory syncytial virus, and emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus-cell membrane fusion required for entry. While common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family related to receptor binding and the potential mechanisms of fusion triggering. PMID:19878307

Sulfated Glycans and Related Digestive Enzymes in the Zika Virus Infectivity: Potential Mechanisms of Virus-Host Interaction and Perspectives in Drug Discovery.

PubMed

Pomin, Vitor H

2017-01-01

As broadly reported, there is an ongoing Zika virus (ZIKV) outbreak in countries of Latin America. Recent findings have demonstrated that ZIKV causes severe defects on the neural development in fetuses in utero and newborns. Very little is known about the molecular mechanisms involved in the ZIKV infectivity. Potential therapeutic agents are also under investigation. In this report, the possible mechanisms of action played by glycosaminoglycans (GAGs) displayed at the surface proteoglycans of host cells, and likely in charge of interactions with surface proteins of the ZIKV, are highlighted. As is common for the most viruses, these sulfated glycans serve as receptors for virus attachment onto the host cells and consequential entry during infection. The applications of (1) exogenous sulfated glycans of different origins and chemical structures capable of competing with the virus attachment receptors (supposedly GAGs) and (2) GAG-degrading enzymes able to digest the virus attachment receptors on the cells may be therapeutically beneficial as anti-ZIKV. This communication attempts, therefore, to offer some guidance for the future research programs aimed to unveil the molecular mechanisms underlying the ZIKV infectivity and to develop therapeutics capable of decreasing the devastating consequences caused by ZIKV outbreak in the Americas.

Potential for Aedes albopictus and Ochlerotatus j. japonicus to Change the Field Ecology of Arboviruses of Human Health Importance in the Mid-Atlantic Region of the United States

DTIC Science & Technology

2001-10-16

attachment, the viral attachment proteins on the surface of the virion bind to receptors on the microvillar membrane of the mosquito s midgut ...Penetration refers to the process through which the virions enter the midgut cells; arboviruses enter cell through receptor -mediated endocytosis. During...inactivation of the virus by digestive enzymes in the lumen of the midgut , and the absence or reduced number of cellular receptor sites for virus attachment

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

PubMed Central

Felt, Sébastien A.; Droby, Gaith N.

2017-01-01

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy. PMID:28566376

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed

Gitman, A G; Graessmann, A; Loyter, A

1985-11-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed Central

Gitman, A G; Graessmann, A; Loyter, A

1985-01-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA. PMID:2997783

Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization ofmore » the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.« less

Activation of Paramyxovirus Membrane Fusion and Virus Entry

PubMed Central

Jardetzky, Theodore S.; Lamb, Robert A.

2014-01-01

The paramyxoviruses represent a diverse virus family responsible for a wide range of human and animal diseases. In contrast to other viruses, such as HIV and influenza virus, which use a single glycoprotein to mediate host receptor binding and virus entry, the paramyxoviruses require two distinct proteins. One of these is an attachment glycoprotein that binds receptor, while the second is a fusion glycoprotein, which undergoes conformational changes that drive virus-cell membrane fusion and virus entry. The details of how receptor binding by one protein activates the second to undergo conformational changes have been poorly understood until recently. Over the past couple of years, structural and functional data have accumulated on representative members of this family, including parainfluenza virus 5, Newcastle disease virus, measles virus, Nipah virus and others, which suggest a mechanistic convergence of activation models. Here we review the data indicating that paramyxovirus attachment glycoproteins shield activating residues within their N-terminal stalk domains, which are then exposed upon receptor binding, leading to the activation of the fusion protein by a ‘provocateur’ mechanism. PMID:24530984

Nano-mechanical Resonantor Sensors for Virus Detection

NASA Astrophysics Data System (ADS)

Bashir, Rashid

2005-03-01

Micro and nanoscale cantilever beams can be used as highly sensitive mass detectors. Scaling down the area of the cantilever allows a decrease in minimum detectable mass limit while scaling down the thickness allows the resonant frequencies to be within measurable range. We have fabricated arrays of silicon cantilever beams as nanomechanical resonant sensors to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams were in the range of 4-5 μm in length, 1-2 μm in width and 20-30 nm in thickness. The virus particles we used in the study were vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of the cantilever beams, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the cantilever beam surface forms the basis of the detection scheme. We have demonstrated the detection of a single vaccinia virus particle with an average mass of 9.5 fg. Specific capture of the antigens requires attachment of antibodies, which can be in the same range of thickness as these cantilever sensors, and can alter their mechanical properties. We have attached protein layers on both sides of 30nm thick cantilever beams and we show that the resonant frequencies can increase or decrease upon the attachment of protein layers to the cantilevers. In certain cases, the increase in spring constant out-weighs the increase in mass and the resonant frequencies can increase upon the attachment of the protein layers. These devices can be very useful as components of biosensors for the detection of air-borne virus particles.

Capsid functions of inactivated human picornaviruses and feline calicivirus.

PubMed

Nuanualsuwan, Suphachai; Cliver, Dean O

2003-01-01

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72 degrees C), and physiological temperature (37 degrees C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37 degrees C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72 degrees C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37 degrees C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72 degrees C inactivation is the capsid and that the target of thermal inactivation (37 degrees C versus 72 degrees C) is temperature dependent.

Modes of Paramyxovirus Fusion: a Henipavirus perspective

PubMed Central

Lee, Benhur; Akyol-Ataman, Zeynep

2011-01-01

Henipavirus is a new genus of paramyxovirus that uses protein-based receptors (EphrinB2 and EphrinB3) for virus entry. Paramyxovirus entry requires the coordinated action of the fusion (F) and attachment viral envelope glycoproteins. Receptor binding to the attachment protein triggers F to undergo a conformational cascade that results in membrane fusion. The accumulation of structural and functional studies on many paramyxoviral fusion and attachment proteins, including recent structures of Nipah and Hendra virus G bound and unbound to cognate ephrinB receptors, indicate that henipavirus entry and fusion differs mechanistically from paramyxoviruses that use glycan-based receptors. PMID:21511478

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

PubMed

Golden, Joseph W; Maes, Piet; Kwilas, Steven A; Ballantyne, John; Hooper, Jay W

2016-01-20

Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT(50)), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever

PubMed Central

Golden, Joseph W.; Maes, Piet; Kwilas, Steven A.; Ballantyne, John

2016-01-01

ABSTRACT Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT50), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products. PMID:26792737

Crispen's Five Antivirus Rules.

ERIC Educational Resources Information Center

Crispen, Patrick Douglas

2000-01-01

Provides rules for protecting computers from viruses, Trojan horses, or worms. Topics include purchasing commercial antivirus programs and keeping them updated; updating virus definitions weekly; precautions before opening attached files; macro virus protection in Microsoft Word; and precautions with executable files. (LRW)

New World Bats Harbor Diverse Influenza A Viruses

PubMed Central

Tong, Suxiang; Zhu, Xueyong; Li, Yan; Shi, Mang; Zhang, Jing; Bourgeois, Melissa; Yang, Hua; Chen, Xianfeng; Recuenco, Sergio; Gomez, Jorge; Chen, Li-Mei; Johnson, Adam; Tao, Ying; Dreyfus, Cyrille; Yu, Wenli; McBride, Ryan; Carney, Paul J.; Gilbert, Amy T.; Chang, Jessie; Guo, Zhu; Davis, Charles T.; Paulson, James C.; Stevens, James; Rupprecht, Charles E.; Holmes, Edward C.; Wilson, Ian A.; Donis, Ruben O.

2013-01-01

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses. PMID:24130481

Computer Virus Protection

ERIC Educational Resources Information Center

Rajala, Judith B.

2004-01-01

A computer virus is a program--a piece of executable code--that has the unique ability to replicate. Like biological viruses, computer viruses can spread quickly and are often difficult to eradicate. They can attach themselves to just about any type of file, and are spread by replicating and being sent from one individual to another. Simply having…

Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production.

PubMed

El Najjar, Farah; Schmitt, Anthony P; Dutch, Rebecca Ellis

2014-08-07

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.

Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

PubMed Central

El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

2014-01-01

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

The Mechanism of Henipavirus Fusion: Examining the Relationships between the Attachment and Fusion Glycoproteins

DTIC Science & Technology

2009-04-01

USA.) Abstract: The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated...of the fusion activity of F. Key words: Hendra virus; Nipah virus; Henipavirus; Paramyxovirus; Viral entry Hendra virus (HeV) and Nipah virus...distribution unlimited 13. SUPPLEMENTARY NOTES Virologica Sinica, April 2009, 24(2) 110-120 14. ABSTRACT The henipaviruses, represented by Nipah

Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region.

PubMed Central

Pontisso, P; Petit, M A; Bankowski, M J; Peeples, M E

1989-01-01

Hepatitis B virus particles contain three related viral envelope proteins, the small, middle, and large S (surface) proteins. All three proteins contain the small S amino acid sequence at their carboxyl terminus. It is not clear which of these S proteins functions as the viral attachment protein, binding to a target cell receptor and initiating infection. In this report, recombinant hepatitis B surface antigen (rHBsAg) particles, which contain only virus envelope proteins, were radioactively labeled, and their attachment to human liver membranes was examined. Only the rHBsAg particles containing the large S protein were capable of directly attaching to liver plasma membranes. The attachment was saturable and could be prevented by competition with unlabeled particles or by a monoclonal antibody specific for the large S protein. In the presence of polymerized human serum albumin, both large and middle S protein-containing rHBsAg particles were capable of attaching to the liver plasma membranes. Small S protein-containing rHBsAg particles were not able to attach even in the presence of polymerized human serum albumin. These results indicate that the large S protein may be the viral attachment protein for hepatocytes, binding directly to liver plasma membranes by its unique amino-terminal (pre-S1) sequence. These results also indicate that polymerized human serum albumin or a similar molecule could act as an intermediate receptor, attaching to liver plasma membranes and to the amino acid sequence (pre-S2) shared by the middle and large S proteins but not contained in the small S protein. Images PMID:2649690

Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

PubMed Central

Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

2015-01-01

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz san

USGS Publications Warehouse

Ryan, Joseph N.; Harvey, Ronald W.; Metge, David W.; Elimelech, Menachem; Navigato, Theresa; Pieper, Ann P.

2002-01-01

Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer, followed by the slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is ∼3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

PubMed Central

Garner, Omai B.; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C.; Park, Arnold; Bowden, Thomas A.; Freiberg, Alexander N.

2014-01-01

ABSTRACT Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. PMID:25505064

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsvitov, Marianna; Frampton, Arthur R.; Shah, Waris A.

2007-04-10

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry andmore » gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.« less

Multilamellar Structures and Filament Bundles Are Found on the Cell Surface during Bunyavirus Egress

PubMed Central

Sanz-Sánchez, Laura; Risco, Cristina

2013-01-01

Inside cells, viruses build specialized compartments for replication and morphogenesis. We observed that virus release associates with specific structures found on the surface of mammalian cells. Cultured adherent cells were infected with a bunyavirus and processed for oriented sectioning and transmission electron microscopy. Imaging of cell basal regions showed sophisticated multilamellar structures (MLS) and extracellular filament bundles with attached viruses. Correlative light and electron microscopy confirmed that both MLS and filaments proliferated during the maximum egress of new viruses. MLS dimensions and structure were reminiscent of those reported for the nanostructures on gecko fingertips, which are responsible for the extraordinary attachment capacity of these lizards. As infected cells with MLS were more resistant to detachment than control cells, we propose an adhesive function for these structures, which would compensate for the loss of adherence during release of new virus progeny. PMID:23799021

Pathogen transmission in relation to duration of attachment by Ixodes scapularis ticks.

PubMed

Eisen, Lars

2018-03-01

The blacklegged tick, Ixodes scapularis, is the primary vector to humans in the eastern United States of the deer tick virus lineage of Powassan virus (Powassan virus disease); the protozoan parasite Babesia microti (babesiosis); and multiple bacterial disease agents including Anaplasma phagocytophilum (anaplasmosis), Borrelia burgdorferi and Borrelia mayonii (Lyme disease), Borrelia miyamotoi (relapsing fever-like illness, named Borrelia miyamotoi disease), and Ehrlichia muris eauclairensis (a minor causative agent of ehrlichiosis). With the notable exception of Powassan virus, which can be transmitted within minutes after attachment by an infected tick, there is no doubt that the risk of transmission of other I. scapularis-borne pathogens, including Lyme disease spirochetes, increases with the length of time (number of days) infected ticks are allowed to remain attached. This review summarizes data from experimental transmission studies to reinforce the important disease-prevention message that regular (at least daily) tick checks and prompt tick removal has strong potential to reduce the risk of transmission of I. scapularis-borne bacterial and parasitic pathogens from infected attached ticks. The most likely scenario for human exposure to an I. scapularis-borne pathogen is the bite by a single infected tick. However, recent reviews have failed to make a clear distinction between data based on transmission studies where experimental hosts were fed upon by a single versus multiple infected ticks. A summary of data from experimental studies on transmission of Lyme disease spirochetes (Bo. burgdorferi and Bo. mayonii) by I. scapularis nymphs indicates that the probability of transmission resulting in host infection, at time points from 24 to 72 h after nymphal attachment, is higher when multiple infected ticks feed together as compared to feeding by a single infected tick. In the specific context of risk for human infection, the most relevant experimental studies therefore are those where the probability of pathogen transmission at a given point in time after attachment was determined using a single infected tick. The minimum duration of attachment by single infected I. scapularis nymphs required for transmission to result in host infection is poorly defined for most pathogens, but experimental studies have shown that Powassan virus can be transmitted within 15 min of tick attachment and both A. phagocytophilum and Bo. miyamotoi within the first 24 h of attachment. There is no experimental evidence for transmission of Lyme disease spirochetes by single infected I. scapularis nymphs to result in host infection when ticks are attached for only 24 h (despite exposure of nearly 90 experimental rodent hosts across multiple studies) but the probability of transmission resulting in host infection appears to increase to approximately 10% by 48 h and reach 70% by 72 h for Bo. burgdorferi. Caveats to the results from experimental transmission studies, including specific circumstances (such as re-attachment of previously partially fed infected ticks) that may lead to more rapid transmission are discussed. Published by Elsevier GmbH.

Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins

NASA Astrophysics Data System (ADS)

Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard

1991-06-01

Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.

Assembly of Oriented Virus Arrays by Chemo-Selective Ligation Methods and Nanolithography Techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camarero, J A; Cheung, C L; Lin, T

2002-12-02

The present work describes our ongoing efforts towards the creation of nano-scaled ordered arrays of protein/virus covalently attached to site-specific chemical linkers patterned by different nanolithograpy techniques. We will present a new and efficient solid-phase approach for the synthesis of chemically modified long alkyl-thiols. These compounds can be used to introduce chemoselective reacting groups onto gold and silicon-based surfaces. Furthermore, these modified thiols have been used to create nanometric patterns by using different nanolithography techniques. We will show that these patterns can react chemoselectively with proteins and/or virus which have been chemically or recombinantly modified to contain complementary chemical groupsmore » at specific positions thus resulting in the oriented attachment of the protein or virus to the surface.« less

Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine

USDA-ARS?s Scientific Manuscript database

E2, the major envelope glycoprotein of Classical Swine Fever Virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity dis...

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry.

PubMed

Plemper, Richard K; Brindley, Melinda A; Iorio, Ronald M

2011-06-01

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.

The Importance of Quality of Care: Effects of Perinatal HIV Infection and Early Institutional Rearing on Preschoolers' Attachment and Indiscriminate Friendliness

ERIC Educational Resources Information Center

Dobrova-Krol, Natasha A.; Bakermans-Kranenburg, Marian J.; Van IJzendoorn, Marinus H.; Juffer, Femmie

2010-01-01

Background: The rearing environment of human immunodeficiency virus (HIV)-infected children is often compromised, putting these children at additional risks. Positive caregiving may ameliorate the impact of adverse circumstances and promote attachment security. The goal of the present study was to examine the attachment relationships of…

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

PubMed

Garner, Omai B; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C; Park, Arnold; Bowden, Thomas A; Freiberg, Alexander N; Lee, Benhur; Baum, Linda G

2015-03-01

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Determination of protection zones for Dutch groundwater wells against virus contamination--uncertainty and sensitivity analysis.

PubMed

Schijven, J F; Mülschlegel, J H C; Hassanizadeh, S M; Teunis, P F M; de Roda Husman, A M

2006-09-01

Protection zones of shallow unconfined aquifers in The Netherlands were calculated that allow protection against virus contamination to the level that the infection risk of 10(-4) per person per year is not exceeded with a 95% certainty. An uncertainty and a sensitivity analysis of the calculated protection zones were included. It was concluded that protection zones of 1 to 2 years travel time (206-418 m) are needed (6 to 12 times the currently applied travel time of 60 days). This will lead to enlargement of protection zones, encompassing 110 unconfined groundwater well systems that produce 3 x 10(8) m3 y(-1) of drinking water (38% of total Dutch production from groundwater). A smaller protection zone is possible if it can be shown that an aquifer has properties that lead to greater reduction of virus contamination, like more attachment. Deeper aquifers beneath aquitards of at least 2 years of vertical travel time are adequately protected because vertical flow in the aquitards is only 0.7 m per year. The most sensitive parameters are virus attachment and inactivation. The next most sensitive parameters are grain size of the sand, abstraction rate of groundwater, virus concentrations in raw sewage and consumption of unboiled drinking water. Research is recommended on additional protection by attachment and under unsaturated conditions.

Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry

PubMed Central

2013-01-01

Background We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry. Results Extensive analysis of the tannins’ mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses. Conclusions CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified. PMID:23924316

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. PMID:26863313

78 FR 12759 - Draft Guidance for Industry on Attachment to Guidance on Antiviral Product Development-Conducting...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-25

...--Conducting and Submitting Virology Studies to the Agency: Guidance for Submitting Hepatitis C Virus... sponsors in submitting hepatitis C virus (HCV) clinical virology data, which are important for supporting...

Interactions of Cryptosporidium parvum, Giardia lamblia, Vaccinal Poliovirus Type 1, and Bacteriophages φX174 and MS2 with a Drinking Water Biofilm and a Wastewater Biofilm▿

PubMed Central

Helmi, Karim; Skraber, Sylvain; Gantzer, Christophe; Willame, Raphaël; Hoffmann, Lucien; Cauchie, Henry-Michel

2008-01-01

Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, φX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination. PMID:18281435

Interactions of Cryptosporidium parvum, Giardia lamblia, vaccinal poliovirus type 1, and bacteriophages phiX174 and MS2 with a drinking water biofilm and a wastewater biofilm.

PubMed

Helmi, Karim; Skraber, Sylvain; Gantzer, Christophe; Willame, Raphaël; Hoffmann, Lucien; Cauchie, Henry-Michel

2008-04-01

Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, phiX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination.

Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corey, Elizabeth A.; Iorio, Ronald M.; Program in Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655

2009-01-05

Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation ofmore » fusion by these two paramyxoviruses.« less

Risk assessment of virus infections in the Oder estuary (southern Baltic) on the basis of spatial transport and virus decay simulations.

PubMed

Schernewski, G; Jülich, W D

2001-05-01

The large Oder (Szczecin) Lagoon (687 km2) at the German-Polish border, close to the Baltic Sea, suffers from severe eutrophication and water quality problems due to high discharge of water, nutrients and pollutants by the river Oder. Sewage treatment around the lagoon has been very much improved during the last years, but large amounts of sewage still enter the Oder river. Human pathogenic viruses generally can be expected in all surface waters that are affected by municipal sewage. There is an increasing awareness that predisposed persons can be infected by a few infective units or even a single active virus. Another new aspect is, that at least polioviruses attached to suspended particles can be infective for weeks and therefore be transported over long distances. Therefore, the highest risk of virus inputs arise from the large amounts of untreated sewage of the city of Szczecin (Poland), which are released into the river Oder and transported to the lagoon and the Baltic Sea. Summer tourism is the most important economical factor in this coastal region and further growth is expected. Human pathogenic viruses might be a serious problem for bathing water quality and sustainable summer tourism. The potential hazard of virus infections along beaches and shores of the Oder lagoon and adjacent parts of the Baltic Sea is evaluated on the basis of model simulations and laboratory results. We used two scenarios for the Older Lagoon considering free viruses and viruses attached to suspended particle matter. The spatial impact of the average virus release in the city of Szczecin during summer (bathing period) was simulated with a hydrodynamic and particle tracking model. Simulations suggest that due to fast inactivation, free viruses in the water represent a risk only in the river and near the river mouth. On the other hand, viruses attached to suspended matter can affect large areas of the eastern, Polish part of the lagoon (Grosses Haff). At the same time the accumulation of viruses on suspended particulate matter increases the likelihood of an infection after incorporation of such a particle. There is no evidence, that there is a risk of virus infections in the western part of the lagoon (Kleines Haff) or along the outer Baltic Sea coast.

[Birth and death process of computer viruses].

PubMed

Segawa, Katsunori; Nakano, Tatsuya; Nakata, Kotoko; Hayashi, Yuzuru

2006-01-01

The daily variations in the number of computer viruses found attaching to e-mails and the number of accesses to the home page of a national institute in Japan are examined. The power spectral densities (PSD) of the variation in the computer viruses show a time-correlation characteristic of Markov process, but the daily access number does not (identified as white noise). Like biological viruses, the variation in the computer viruses can be described by the birth-and-death model known as a Markov process.

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses

PubMed Central

Hendricks, Gabriel L.; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C.; Viswanathan, Karthik; Albers, Leila; Comolli, James C.; Shriver, Zachary; Knipe, David M.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Trevejo, Jose M.

2016-01-01

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as ‘molecular sinks’ and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. PMID:25637710

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

PubMed

Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

2018-03-01

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

DIESEL EXHAUST PARTICLES ENHANCE INFLUENZA VIRUS INFECTIVITY BY INCREASING VIRUS ATTACHMENT

EPA Science Inventory

Despite vaccination and antiviral therapies, influenza infections continue to cause large scale morbidity and mortality every year. Several factors, such as age and nutritional status can affect the incidence and severity of influenza infections. Moreover, exposure to air polluta...

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

PubMed Central

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

1997-01-01

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells.

PubMed

Bose, Sayantan; Song, Albert S; Jardetzky, Theodore S; Lamb, Robert A

2014-04-01

Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

PubMed Central

Bose, Sayantan; Song, Albert S.; Jardetzky, Theodore S.

2014-01-01

ABSTRACT Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the “triggering” interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active “headless” mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed “stalk exposure model” to other paramyxoviruses and propose an “induced fit” hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. IMPORTANCE Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed “stalk exposure model” first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an “induced fit” hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. PMID:24453369

Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells.

PubMed Central

Lonberg-Holm, K; Whiteley, N M

1976-01-01

Attachment, ""tight binding'' and eclipse of radioactive poliovirus 2 (P2) and human rhinovirus 2 (HRV 2) were investigated. The activation energy for attachment of both HRV2 and P2 was about 13 kcal/mol. HRV2 differed from P2 in two respects: the Arrhenius plot for attachment of HRV2 showed a break at 15 to 19 degrees C when the cells were first treated several hours at 0 degrees C, and attachment of HRV2 was inhibited by treatment of cells with metabolic poisons able to reduce cellular ATP by more than 90%. Tight binding was determined by isolation of a specific P2-membrane complex or by loss of EDTA dissociability of HRV2. Tight binding of both viruses was slowed by 0.01 M iodoacetamide but not by 0.02 M F-; F- plus 0.002 M CN- slowed tight binding of HRV2 but not of P2. Eclipse, the irreversible alteration of parental virions, was detected by isolation of cell-associated subviral particles or by loss of cell-associated infectious virus. Eclipse of both viruses is slowed by iodoacetamide or F-. It seems likely that the early steps of infection with picornaviruses may be sensitive to alterations in the cell membrane produced by metabolic inhibitors or by treatment at low temperature. PMID:184301

Crispen's Five Antivirus Rules.

ERIC Educational Resources Information Center

Crispen, Patrick Douglas

2000-01-01

Explains five rules to protect computers from viruses. Highlights include commercial antivirus software programs and the need to upgrade them periodically (every year to 18 months); updating virus definitions at least weekly; scanning attached files from email with antivirus software before opening them; Microsoft Word macro protection; and the…

New Insights into the Hendra Virus Attachment and Entry Process from Structures of the Virus G Glycoprotein and Its Complex with Ephrin-B2

DTIC Science & Technology

2012-11-05

organization of measles virus fusion complexes. J Virol 83: 10480–10493. 24. Bishop KA, Hickey AC, Khetawat D, Patch JR, Bossart KN, et al. (2008...its receptor, sialyllactose. Structure 13: 803–815. 38. Colf LA, Juo ZS, Garcia KC (2007) Structure of the measles virus hemagglutinin. Nat Struct Mol...Biol 14: 1227–1228. 39. Hashiguchi T, Kajikawa M, Maita N, Takeda M, Kuroki K, et al. (2007) Crystal structure of measles virus hemagglutinin provides

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

USDA-ARS?s Scientific Manuscript database

E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, media...

Full-Genome Sequence of Infectious Laryngotracheitis Virus (Gallid Alphaherpesvirus 1) Strain VFAR-043, Isolated in Peru

PubMed Central

Bendezu Eguis, Jorge; Montesinos, Ricardo; Fernández-Díaz, Manolo

2018-01-01

ABSTRACT We report here the first genome sequence of infectious laryngotracheitis virus isolated in Peru from tracheal tissues of layer chickens. The genome showed 99.98% identity to the J2 strain genome sequence. Single nucleotide polymorphisms were detected in five gene-coding sequences related to vaccine development, virus attachment, and viral immune evasion. PMID:29519822

Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.

PubMed

Reiter, Dirk M; Frierson, Johnna M; Halvorson, Elizabeth E; Kobayashi, Takeshi; Dermody, Terence S; Stehle, Thilo

2011-08-01

Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.

Host cell recognition by the henipaviruses: Crystal structures of the Nipah G attachment glycoprotein and its complex with ephrin-B3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Kai; Rajashankar, Kanagalaghatta R.; Chan, Yee-Peng

2008-07-28

Nipah virus (NiV) and Hendra virus are the type species of the highly pathogenic paramyxovirus genus Henipavirus, which can cause severe respiratory disease and fatal encephalitis infections in humans, with case fatality rates approaching 75%. NiV contains two envelope glycoproteins, the receptor-binding G glycoprotein (NiV-G) that facilitates attachment to host cells and the fusion (F) glycoprotein that mediates membrane merger. The henipavirus G glycoproteins lack both hemagglutinating and neuraminidase activities and, instead, engage the highly conserved ephrin-B2 and ephrin-B3 cell surface proteins as their entry receptors. Here, we report the crystal structures of the NiV-G both in its receptor-unbound statemore » and in complex with ephrin-B3, providing, to our knowledge, the first view of a paramyxovirus attachment complex in which a cellular protein is used as the virus receptor. Complex formation generates an extensive protein-protein interface around a protruding ephrin loop, which is inserted in the central cavity of the NiV-G {beta}-propeller. Analysis of the structural data reveals the molecular basis for the highly specific interactions of the henipavirus G glycoproteins with only two members (ephrin-B2 and ephrin-B3) of the very large ephrin family and suggests how they mediate in a unique fashion both cell attachment and the initiation of membrane fusion during the virus infection processes. The structures further suggest that the NiV-G/ephrin interactions can be effectively targeted to disrupt viral entry and provide the foundation for structure-based antiviral drug design.« less

Effect of goethite coating and humic acid on the transport of bacteriophage PRD1 in columns of saturated sand

NASA Astrophysics Data System (ADS)

Foppen, J. W. A.; Okletey, S.; Schijven, J. F.

2006-05-01

The transport of bacteriophage PRD1, a model virus, was studied in columns containing sediment mixtures of quartz sand with goethite-coated sand and using various solutions consisting of monovalent and divalent salts and humic acid (HA). Without HA and in the absence of sand, the inactivation rate of PRD1 was found to be as low as 0.014 day - 1 (at 5 ± 3 °C), but in the presence of HA it was much lower (0.0009 day - 1 ), indicating that HA helps PRD1 to survive. When the fraction of goethite in the sediment was increased, the removal of PRD1 also increased. However, in the presence of HA, C/ C0 values of PRD1 increased by as much as 5 log units, thereby almost completely eliminating the effect of addition of goethite. The sticking efficiency was not linearly dependent on the amount of goethite added to the quartz sand; this is apparently due to surface charge heterogeneity of PRD1. Our results imply that, in the presence of dissolved organic matter (DOM), viruses can be transported for long distances thanks to two effects: attachment is poor because DOM has occupied favourable sites for attachment and inactivation of virus may have decreased. This conclusion justifies making conservative assumptions about the attachment of viruses when calculating protection zones for groundwater wells.

Effect of pH on bacteriophage transport through sandy soils

USGS Publications Warehouse

Kinoshita, Takashi; Bales, Roger C.; Maguire, Kimberley M.; Gerba, Charles P.

1993-01-01

Effects of pH and hydrophobicity on attachment and detachment of PRD-1 and MS-2 in three different sandy soils were investigated in a series of laboratory-column experiments. Concentrations of the lipid-containing phage PRD-1 decreased 3–4 orders of magnitude during passage through the 10–15-cm-long columns. Attachment of the lipid-containing phage PRD-1 was insensitive to pH and was apparently controlled by hydrophobic interactions in soil media. The less-hydrophobic phage MS-2 acted conservatively; it was not removed in the columns at pH's 5.7–8.0. The sticking efficiency (α) in a colloid-filtration model was between 0.1 and 1 for PRD-1, indicating a relatively high removal efficiency. Phage attachment was reversible, but detachment under steady-state conditions was slow. An increase in pH had a moderate effect on enhancing detachment. Still, these soils should continue to release phage to virus-free water for days to weeks following exposure to virus-containing water. In sandy soils with a mass-fraction organic carbon as low as a few hundredths of a percent, pH changes in the range 5.7–8.0 should have little effect on retention of more-hydrophobic virus (e.g., PRD-1), in that retardation will be dominated by hydrophobic effects. Sharp increases in pH should enhance detachment and transport of virus previously deposited on soil grains. A more hydrophilic virus (e.g., MS-2) will transport as a conservative tracer in low-carbon sandy soil.

Ebola images emerge from the cave.

PubMed

Diamond, Michael S; Fremont, Daved H

2008-08-14

Ebola virus causes a lethal hemorrhagic disease for which no therapy or vaccine is currently approved. Recently, the crystal structure of the Ebola virus glycoprotein in complex with a human neutralizing antibody was illuminated, providing a path from the shadows toward understanding cellular attachment, viral fusion, and immune evasion.

Detection of Ebola Virus RNA through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates

DTIC Science & Technology

2016-08-02

301- 619-4768(f). 1 2 3 4 5 6 7 8 Title: Detection of Ebola Virus RNA through Aerosol Sampling of Animal Biosafety Level 9 4...5 6 To whom it may concern, 7 8 My colleagues and I are submitting the attached manuscript, Detection of Ebola Virus 9 RNA through Aerosol...embedded in the texts. This is the first report demonstrating detection of Ebola virus 17 RNA from animal rooms housing infected nonhuman primates and

Bacteriophage PRD1 batch experiments to study attachment, detachment and inactivation processes

NASA Astrophysics Data System (ADS)

Sadeghi, Gholamreza; Schijven, Jack F.; Behrends, Thilo; Hassanizadeh, S. Majid; van Genuchten, Martinus Th.

2013-09-01

Knowledge of virus removal in subsurface environments is pivotal for assessing the risk of viral contamination of water resources and developing appropriate protection measures. Columns packed with sand are frequently used to quantify attachment, detachment and inactivation rates of viruses. Since column transport experiments are very laborious, a common alternative is to perform batch experiments where usually one or two measurements are done assuming equilibrium is reached. It is also possible to perform kinetic batch experiments. In that case, however, it is necessary to monitor changes in the concentration with time. This means that kinetic batch experiments will be almost as laborious as column experiments. Moreover, attachment and detachment rate coefficients derived from batch experiments may differ from those determined using column experiments. The aim of this study was to determine the utility of kinetic batch experiments and investigate the effects of different designs of the batch experiments on estimated attachment, detachment and inactivation rate coefficients. The experiments involved various combinations of container size, sand-water ratio, and mixing method (i.e., rolling or tumbling by pivoting the tubes around their horizontal or vertical axes, respectively). Batch experiments were conducted with clean quartz sand, water at pH 7 and ionic strength of 20 mM, and using the bacteriophage PRD1 as a model virus. Values of attachment, detachment and inactivation rate coefficients were found by fitting an analytical solution of the kinetic model equations to the data. Attachment rate coefficients were found to be systematically higher under tumbling than under rolling conditions because of better mixing and more efficient contact of phages with the surfaces of the sand grains. In both mixing methods, more sand in the container yielded higher attachment rate coefficients. A linear increase in the detachment rate coefficient was observed with increased solid-water ratio using tumbling method. Given the differences in the attachment rate coefficients, and assuming the same sticking efficiencies since chemical conditions of the batch and column experiments were the same, our results show that collision efficiencies of batch experiments are not the same as those of column experiments. Upscaling of the attachment rate from batch to column experiments hence requires proper understanding of the mixing conditions. Because batch experiments, in which the kinetics are monitored, are as laborious as column experiments, there seems to be no major advantage in performing batch instead of column experiments.

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

PubMed Central

Ng, Wy Ching; Liong, Stella; Tate, Michelle D.; Irimura, Tatsuro; Denda-Nagai, Kaori; Brooks, Andrew G.; Londrigan, Sarah L.

2014-01-01

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca2+-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca2+-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV. PMID:24257596

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

PubMed

Wong, Joyce J W; Young, Tracy A; Zhang, Jiayan; Liu, Shiheng; Leser, George P; Komives, Elizabeth A; Lamb, Robert A; Zhou, Z Hong; Salafsky, Joshua; Jardetzky, Theodore S

2017-10-03

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses.

PubMed

Hendricks, Gabriel L; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C; Viswanathan, Karthik; Albers, Leila; Comolli, James C; Shriver, Zachary; Knipe, David M; Kurt-Jones, Evelyn A; Fygenson, Deborah K; Trevejo, Jose M; Wang, Jennifer P; Finberg, Robert W

2015-04-01

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as 'molecular sinks' and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. Copyright © 2015 Elsevier B.V. All rights reserved.

Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis.

PubMed

Nauwynck, H J; Duan, X; Favoreel, H W; Van Oostveldt, P; Pensaert, M B

1999-02-01

Porcine alveolar macrophages (AMphi) are the dominant cell type that supports the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AMphi in a dose-dependent manner. Binding of PRRSV to AMphi increased gradually and reached a maximum within 60 min at 4 degrees C. By confocal microscopy, it was shown that different degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating was prevented. These results indicate that PRRSV entry into AMphi involves attachment to a specific virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus replication.

Mxra8 is a receptor for multiple arthritogenic alphaviruses.

PubMed

Zhang, Rong; Kim, Arthur S; Fox, Julie M; Nair, Sharmila; Basore, Katherine; Klimstra, William B; Rimkunas, Rebecca; Fong, Rachel H; Lin, Hueylie; Poddar, Subhajit; Crowe, James E; Doranz, Benjamin J; Fremont, Daved H; Diamond, Michael S

2018-05-01

Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.

Transport and recovery of bacteriophage PRD1 in a sand and gravel aquifer: Effect of sewage-derived organic matter

USGS Publications Warehouse

Pieper, A.P.; Ryan, J.N.; Harvey, R.W.; Amy, G.L.; Illangasekare, T.H.; Metge, D.W.

1997-01-01

To test the effects of sewage-derived organic matter on virus attachment, 32P-labeled bacteriophage PRD1, linear alkylbenzene sulfonates (LAS), and tracers were injected into sewage-contaminated (suboxic, elevated organic matter) and uncontaminated (oxic, low organic matter) zones of an iron oxide-coated quartz sand and gravel aquifer on Cape Cod, MA. In the uncontaminated zone, 83% of the PRD1 were attenuated over the first meter of transport by attachment to aquifer grains. In the contaminated zone, 42% of the PRD1 were attenuated over the first meter of transport. Sewage-derived organic matter contributed to the difference in PRD1 attenuation by blocking attachment sites in the contaminated zone. At greater distances down gradient (to a total transport distance of 3.6 m), a near-constant amount of PRD1 continued to break through, suggesting that aquifer grain heterogeneities allowed a small amount of reversible attachment. Injection of an LAS mixture (25 mg L-1), a common sewage constituent, remobilized 87% of the attached PRD1 in the contaminated zone, but only 2.2% in the uncontaminated zone. LAS adsorption promoted virus recovery in the contaminated zone by altering the PRD1-surface interactions; however, the amount of LAS adsorbed was not sufficient to promote release of the attached PRD1 in the uncontaminated zone.

Role of sulfatide in vaccinia virus infection.

PubMed

Perino, Julien; Foo, Chwan Hong; Spehner, Daniele; Cohen, Gary H; Eisenberg, Roselyn J; Crance, Jean-Marc; Favier, Anne-Laure

2011-07-01

Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. We demonstrate that the VACV-WR (VACV Western-Reserve strain) displays no binding to Cer (ceramide) or to Gal-Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3' sulfogalactosylceramide. The interaction between Sulf and VACV-WR resulted in a time-dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV-WR. Together the results suggest that Sulf could play a role as an alternate receptor for VACV-WR and probably other Orthopoxviruses.

Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki

PubMed Central

Lee, Jin-Ching; Chang, Fang-Rong; Chen, Shu-Rong; Wu, Yu-Hsuan; Hu, Hao-Chun; Wu, Yang-Chang; Backlund, Anders; Cheng, Yuan-Bin

2016-01-01

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2–9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 μg/mL), which is better than that of the positive control, 2′CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS–NP computational analysis. The ChemGPS–NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space. PMID:27517937

Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode.

PubMed

Sayhi, Maher; Ouerghi, Oussama; Belgacem, Kamel; Arbi, Marwa; Tepeli, Yudum; Ghram, Abdeljalil; Anik, Ülkü; Österlund, Lars; Laouini, Dhafer; Diouani, Mohamed Fethi

2018-06-01

Influenza is a viral infectious disease considered as a source of many health problems and enormous socioeconomic disruptions. Conventional methods are inadequate for in-field detection of the virus and generally suffer from being laborious and time-consuming. Thus, studies aiming to develop effective alternatives to conventional methods are urgently needed. In this work, we developed an approach for the isolation and detection of influenza A virus subtype H9N2. For this aim, two specific influenza receptors were used. The first, anti-matrix protein 2 (M2) antibody, was attached to iron magnetic nanoparticles (MNPs) and used for the isolation of the virus from allantoic fluid. The second biomolecule, Fetuin A, was attached to an electrochemical detectable label, gold nanoparticles (AuNPs), and used to detect the virus tacking advantage from fetuin-hemagglutinin interaction. The MNP-Influenza virus-AuNP formed complex was isolated and treated by an acid solution then the collected gold nanoparticles were deposited onto a screen printed carbon electrode. AuNPs catalyzes the hydrogen ions reduction in acidic medium while applying an appropriate potential, and the generated current signal was proportional to the virus titer. This approach allows the rapid detection of influenza virus A/H9N2 at a less than 16 HAU titer. Copyright © 2018 Elsevier B.V. All rights reserved.

Paramyxovirus fusion and entry: multiple paths to a common end.

PubMed

Chang, Andres; Dutch, Rebecca E

2012-04-01

The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

PubMed

Okamatsu, Masatoshi; Feng, Fei; Ohyanagi, Tatsuya; Nagahori, Noriko; Someya, Kazuhiko; Sakoda, Yoshihiro; Miura, Nobuaki; Nishimura, Shin-Ichiro; Kida, Hiroshi

2013-02-01

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Predicting “Airborne” Influenza Viruses: (Trans-) mission Impossible?

PubMed Central

Sorrell, E.M.; Schrauwen, E.J.A.; Linster, M.; De Graaf, M.; Herfst, S.; Fouchier, R.A.M.

2011-01-01

Repeated transmission of animal influenza viruses to humans has prompted investigation of the viral, host, and environmental factors responsible for transmission via aerosols or respiratory droplets. How do we determine – out of thousands of influenza virus isolates collected in animal surveillance studies each year – which viruses have the potential to become “airborne”, and hence pose a pandemic threat? Here, using knowledge from pandemic, zoonotic and epidemic viruses, we postulate that the minimal requirements for efficient transmission of an animal influenza virus between humans are: efficient virus attachment to (upper) respiratory tissues, replication to high titers in these tissues, and release and aerosolization of single virus particles. Investigating “airborne” transmission of influenza viruses is key to understand – and predict – influenza pandemics. PMID:22440921

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction*

PubMed Central

Altgärde, Noomi; Eriksson, Charlotta; Peerboom, Nadia; Phan-Xuan, Tuan; Moeller, Stephanie; Schnabelrauch, Matthias; Svedhem, Sofia; Trybala, Edward; Bergström, Tomas; Bally, Marta

2015-01-01

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells. PMID:26160171

Rapid bacteriophage MS2 transport in an oxic sandy aquifer in cold climate: Field experiments and modeling

NASA Astrophysics Data System (ADS)

Kvitsand, Hanne M. L.; Ilyas, Aamir; Østerhus, Stein W.

2015-12-01

Virus removal during rapid transport in an unconfined, low-temperature (6°C) sand and gravel aquifer was investigated at a riverbank field site, 25 km south of Trondheim in central Norway. The data from bacteriophage MS2 inactivation and transport experiments were applied in a two-site kinetic transport model using HYDRUS-1D, to evaluate the mechanisms of virus removal and whether these mechanisms were sufficient to protect the groundwater supplies. The results demonstrated that inactivation was negligible to the overall removal and that irreversible MS2 attachment to aquifer grains, coated with iron precipitates, played a dominant role in the removal of MS2; 4.1 log units of MS2 were removed by attachment during 38 m travel distance and less than 2 days residence time. Although the total removal was high, pathways capable of allowing virus migration at rapid velocities were present in the aquifer. The risk of rapid transport of viable viruses should be recognized, particularly for water supplies without permanent disinfection.

A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin-Glycan Interactions.

PubMed

Li, Wenkai; Feng, Xinru; Yan, Xing; Liu, Keyi; Deng, Le

2016-06-01

Most therapeutical nucleic acid aptamers tend to inhibit protein-protein interactions and thereby function as antagonists. Attachment of the influenza virus surface glycoprotein hemagglutinin (HA) to sialic acid-containing host cell receptors (glycan) facilitates the initial stage of viral infection. Inhibition of the attachment may result in an antiviral effect on the proliferation of the influenza virus. To develop therapeutically interesting agents, we selected two single-stranded DNA (ssDNA) aptamers specific to the HA protein of H1N1 influenza virus (A/Puerto Rico/8/1934) through a procedure of systematic evolution of ligands by exponential enrichment. As it showed a higher binding affinity for HA protein (Kd = 78 ± 1 nM), aptamer 1 was tested for its ability to interfere with HA-glycan interactions using chicken red blood cell hemagglutination and microneutralization assays, which demonstrated that it significantly suppressed the viral infection in host cells. These results indicate that the isolated ssDNA aptamer may be developed as an antiviral agent against influenza through appropriate therapeutic formulation.

Impact of virus surface characteristics on removal mechanisms within membrane bioreactors.

PubMed

Chaudhry, Rabia M; Holloway, Ryan W; Cath, Tzahi Y; Nelson, Kara L

2015-11-01

In this study we investigated the removal of viruses with similar size and shape but with different external surface capsid proteins by a bench-scale membrane bioreactor (MBR). The goal was to determine which virus removal mechanisms (retention by clean backwashed membrane, retention by cake layer, attachment to biomass, and inactivation) were most impacted by differences in the virus surface properties. Seven bench-scale MBR experiments were performed using mixed liquor wastewater sludge that was seeded with three lab-cultured bacteriophages with icosahedral capsids of ∼30 nm diameter (MS2, phiX174, and fr). The operating conditions were designed to simulate those at a reference, full-scale MBR facility. The virus removal mechanism most affected by virus type was attachment to biomass (removals of 0.2 log for MS2, 1.2 log for phiX174, and 3 log for fr). These differences in removal could not be explained by electrostatic interactions, as the three viruses had similar net negative charge when suspended in MBR permeate. Removals by the clean backwashed membrane (less than 1 log) and cake layer (∼0.6 log) were similar for the three viruses. A comparison between the clean membrane removals seen at the bench-scale using a virgin membrane (∼1 log), and the full-scale using 10-year old membranes (∼2-3 logs) suggests that irreversible fouling, accumulated on the membrane over years of operation that cannot be removed by cleaning, also contributes towards virus removal. This study enhances the current mechanistic understanding of virus removal in MBRs and will contribute to more reliable treatment for water reuse applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus.

PubMed

Bradel-Tretheway, Birgit G; Liu, Qian; Stone, Jacquelyn A; McInally, Samantha; Aguilar, Hector C

2015-07-01

Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses. Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Bacteriophage PRD1 batch experiments to study attachment, detachment and inactivation processes.

PubMed

Sadeghi, Gholamreza; Schijven, Jack F; Behrends, Thilo; Hassanizadeh, S Majid; van Genuchten, Martinus Th

2013-09-01

Knowledge of virus removal in subsurface environments is pivotal for assessing the risk of viral contamination of water resources and developing appropriate protection measures. Columns packed with sand are frequently used to quantify attachment, detachment and inactivation rates of viruses. Since column transport experiments are very laborious, a common alternative is to perform batch experiments where usually one or two measurements are done assuming equilibrium is reached. It is also possible to perform kinetic batch experiments. In that case, however, it is necessary to monitor changes in the concentration with time. This means that kinetic batch experiments will be almost as laborious as column experiments. Moreover, attachment and detachment rate coefficients derived from batch experiments may differ from those determined using column experiments. The aim of this study was to determine the utility of kinetic batch experiments and investigate the effects of different designs of the batch experiments on estimated attachment, detachment and inactivation rate coefficients. The experiments involved various combinations of container size, sand-water ratio, and mixing method (i.e., rolling or tumbling by pivoting the tubes around their horizontal or vertical axes, respectively). Batch experiments were conducted with clean quartz sand, water at pH 7 and ionic strength of 20 mM, and using the bacteriophage PRD1 as a model virus. Values of attachment, detachment and inactivation rate coefficients were found by fitting an analytical solution of the kinetic model equations to the data. Attachment rate coefficients were found to be systematically higher under tumbling than under rolling conditions because of better mixing and more efficient contact of phages with the surfaces of the sand grains. In both mixing methods, more sand in the container yielded higher attachment rate coefficients. A linear increase in the detachment rate coefficient was observed with increased solid-water ratio using tumbling method. Given the differences in the attachment rate coefficients, and assuming the same sticking efficiencies since chemical conditions of the batch and column experiments were the same, our results show that collision efficiencies of batch experiments are not the same as those of column experiments. Upscaling of the attachment rate from batch to column experiments hence requires proper understanding of the mixing conditions. Because batch experiments, in which the kinetics are monitored, are as laborious as column experiments, there seems to be no major advantage in performing batch instead of column experiments. Copyright © 2013 Elsevier B.V. All rights reserved.

[Research progress on ebola virus glycoprotein].

PubMed

Ding, Guo-Yong; Wang, Zhi-Yu; Gao, Lu; Jiang, Bao-Fa

2013-03-01

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

An antivirally active sulfated polysaccharide from Sargassum horneri (TURNER) C. AGARDH.

PubMed

Hoshino, T; Hayashi, T; Hayashi, K; Hamada, J; Lee, J B; Sankawa, U

1998-07-01

A sulfated polysaccharide was isolated from the hot water extract of a brown alga, Sargassum horneri (TURNER) C. AGARDH. Fucose was detected as the main component sugar of this polysaccharide. This compound showed potent antiviral activity against herpes simplex virus type 1, human cytomegalovirus and human immunodeficiency virus type 1. Time-of-addition experiments suggested that it inhibited not only the initial stages of viral infection, such as attachment to and penetration into host cells, but also later replication stages after virus penetration.

Lactoferrin for prevention of common viral infections.

PubMed

Wakabayashi, Hiroyuki; Oda, Hirotsugu; Yamauchi, Koji; Abe, Fumiaki

2014-11-01

Although lactoferrin has many biological functions, the host-protective effects against pathogenic microorganisms including bacteria, fungi, and viruses are regarded as one of the most important. Here, we review research on the protective role of lactoferrin administration against common viral infections. Many studies have shown the in vitro antiviral activity of lactoferrin against viral pathogens that cause common infections such as the common cold, influenza, gastroenteritis, summer cold, and herpes, where lactoferrin inhibits mainly viral attachment to the target cells. Recently, studies indicating the in vivo protective effects of lactoferrin by oral administration against common viral infections have been increasing. For instance, norovirus is an extremely important emerging human pathogen that causes a majority of gastroenteritis outbreaks worldwide that may be a target candidate for lactoferrin. Lactoferrin consumption reduced the incidence of noroviral gastroenteritis in children and a similar effect was observed in a wide range of ages in a preliminary survey. A recent in vitro study reported that lactoferrin inhibits both cellular attachment of the murine norovirus, a virus closely-related to the human norovirus, and viral replication in the cells by inducing antiviral cytokines interferon (IFN)-α/β. Lactoferrin administration also enhances NK cell activity and Th1 cytokine responses, which lead to protection against viral infections. In conclusion, lactoferrin consumption may protect the host from viral infections through inhibiting the attachment of a virus to the cells, replication of the virus in the cells, and enhancement of systemic immune functions. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

PubMed Central

2012-01-01

Background It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical and initial imaging analysis indicated that productive fusion events occur predominantly within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Quantitative monitoring of the fraction of individual viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques. PMID:22935135

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

2012-02-05

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S.

2012-05-24

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.

2013-10-01

Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using pointmore » mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.« less

Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamb, Robert A.; Paterson, Reay G.; Jardetzky, Theodore S.

2006-01-05

Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process-an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxovirusesmore » (parainfluenza viruses 1-5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described.« less

Paramyxovirus Fusion and Entry: Multiple Paths to a Common End

PubMed Central

Chang, Andres; Dutch, Rebecca E.

2012-01-01

The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens. PMID:22590688

Role of Jumonji c-domain containing protein 6 (JMJD6) in infectivity of foot-and-mouth disease virus

USDA-ARS?s Scientific Manuscript database

Foot-and-mouth disease virus (FMDV) can utilize as many as three distinct groups of receptor molecules to attach and enter a susceptible host cell. Four integrin heterodimers (alphavBeta1, alphavBeta3, alphavBeta6, and alphavBeta8) can function as the primary receptor for FMDV field strains. FMDV ...

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

PubMed

Xu, Kai; Chan, Yee-Peng; Bradel-Tretheway, Birgit; Akyol-Ataman, Zeynep; Zhu, Yongqun; Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z Hong; Broder, Christopher C; Aguilar, Hector C; Nikolov, Dimitar B

2015-12-01

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model.

PubMed

Gabbard, Jon D; Dlugolenski, Daniel; Van Riel, Debby; Marshall, Nicolle; Galloway, Summer E; Howerth, Elizabeth W; Campbell, Patricia J; Jones, Cheryl; Johnson, Scott; Byrd-Leotis, Lauren; Steinhauer, David A; Kuiken, Thijs; Tompkins, S Mark; Tripp, Ralph; Lowen, Anice C; Steel, John

2014-02-01

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

Novel H7N9 Influenza Virus Shows Low Infectious Dose, High Growth Rate, and Efficient Contact Transmission in the Guinea Pig Model

PubMed Central

Gabbard, Jon D.; Dlugolenski, Daniel; Van Riel, Debby; Marshall, Nicolle; Galloway, Summer E.; Howerth, Elizabeth W.; Campbell, Patricia J.; Jones, Cheryl; Johnson, Scott; Byrd-Leotis, Lauren; Steinhauer, David A.; Kuiken, Thijs; Tompkins, S. Mark; Tripp, Ralph; Lowen, Anice C.

2014-01-01

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation. PMID:24227867

Natural History of Human Respiratory Syncytial Virus Inferred from Phylogenetic Analysis of the Attachment (G) Glycoprotein with a 60-Nucleotide Duplication

PubMed Central

Trento, Alfonsina; Viegas, Mariana; Galiano, Mónica; Videla, Cristina; Carballal, Guadalupe; Mistchenko, Alicia S.; Melero, José A.

2006-01-01

A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen. PMID:16378999

VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane

PubMed Central

Xu, Xiaopeng; Yan, Muting; Wang, Rui; Lin, Ting; Tang, Junliang; Li, Chaozheng; Weng, Shaoping

2014-01-01

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble into a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. On the basis of these facts, we redrew the putative schematic illustration of the VMBM structure. Our study suggests that the virus adopts a strategy to remodel the cellular matrix and may provide an important reference to elucidate BM functions and the mechanisms of lymphangiogenesis. PMID:24599992

Genetic Characterization of the Tick-Borne Orbiviruses

PubMed Central

Belaganahalli, Manjunatha N.; Maan, Sushila; Maan, Narender S.; Brownlie, Joe; Tesh, Robert; Attoui, Houssam; Mertens, Peter P. C.

2015-01-01

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in ‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome. PMID:25928203

Genetic characterization of the tick-borne orbiviruses.

PubMed

Belaganahalli, Manjunatha N; Maan, Sushila; Maan, Narender S; Brownlie, Joe; Tesh, Robert; Attoui, Houssam; Mertens, Peter P C

2015-04-28

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in 'conserved' Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

PubMed

Saso, Wakana; Tsukuda, Senko; Ohashi, Hirofumi; Fukano, Kento; Morishita, Ryo; Matsunaga, Satoko; Ohki, Mio; Ryo, Akihide; Park, Sam-Yong; Suzuki, Ryosuke; Aizaki, Hideki; Muramatsu, Masamichi; Sureau, Camille; Wakita, Takaji; Matano, Tetsuro; Watashi, Koichi

2018-06-22

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Tick-Borne Viruses and Biological Processes at the Tick-Host-Virus Interface

PubMed Central

Kazimírová, Mária; Thangamani, Saravanan; Bartíková, Pavlína; Hermance, Meghan; Holíková, Viera; Štibrániová, Iveta; Nuttall, Patricia A.

2017-01-01

Ticks are efficient vectors of arboviruses, although less than 10% of tick species are known to be virus vectors. Most tick-borne viruses (TBV) are RNA viruses some of which cause serious diseases in humans and animals world-wide. Several TBV impacting human or domesticated animal health have been found to emerge or re-emerge recently. In order to survive in nature, TBV must infect and replicate in both vertebrate and tick cells, representing very different physiological environments. Information on molecular mechanisms that allow TBV to switch between infecting and replicating in tick and vertebrate cells is scarce. In general, ticks succeed in completing their blood meal thanks to a plethora of biologically active molecules in their saliva that counteract and modulate different arms of the host defense responses (haemostasis, inflammation, innate and acquired immunity, and wound healing). The transmission of TBV occurs primarily during tick feeding and is a complex process, known to be promoted by tick saliva constituents. However, the underlying molecular mechanisms of TBV transmission are poorly understood. Immunomodulatory properties of tick saliva helping overcome the first line of defense to injury and early interactions at the tick-host skin interface appear to be essential in successful TBV transmission and infection of susceptible vertebrate hosts. The local host skin site of tick attachment, modulated by tick saliva, is an important focus of virus replication. Immunomodulation of the tick attachment site also promotes co-feeding transmission of viruses from infected to non-infected ticks in the absence of host viraemia (non-viraemic transmission). Future research should be aimed at identification of the key tick salivary molecules promoting virus transmission, and a molecular description of tick-host-virus interactions and of tick-mediated skin immunomodulation. Such insights will enable the rationale design of anti-tick vaccines that protect against disease caused by tick-borne viruses. PMID:28798904

Tick-Borne Viruses and Biological Processes at the Tick-Host-Virus Interface.

PubMed

Kazimírová, Mária; Thangamani, Saravanan; Bartíková, Pavlína; Hermance, Meghan; Holíková, Viera; Štibrániová, Iveta; Nuttall, Patricia A

2017-01-01

Ticks are efficient vectors of arboviruses, although less than 10% of tick species are known to be virus vectors. Most tick-borne viruses (TBV) are RNA viruses some of which cause serious diseases in humans and animals world-wide. Several TBV impacting human or domesticated animal health have been found to emerge or re-emerge recently. In order to survive in nature, TBV must infect and replicate in both vertebrate and tick cells, representing very different physiological environments. Information on molecular mechanisms that allow TBV to switch between infecting and replicating in tick and vertebrate cells is scarce. In general, ticks succeed in completing their blood meal thanks to a plethora of biologically active molecules in their saliva that counteract and modulate different arms of the host defense responses (haemostasis, inflammation, innate and acquired immunity, and wound healing). The transmission of TBV occurs primarily during tick feeding and is a complex process, known to be promoted by tick saliva constituents. However, the underlying molecular mechanisms of TBV transmission are poorly understood. Immunomodulatory properties of tick saliva helping overcome the first line of defense to injury and early interactions at the tick-host skin interface appear to be essential in successful TBV transmission and infection of susceptible vertebrate hosts. The local host skin site of tick attachment, modulated by tick saliva, is an important focus of virus replication. Immunomodulation of the tick attachment site also promotes co-feeding transmission of viruses from infected to non-infected ticks in the absence of host viraemia (non-viraemic transmission). Future research should be aimed at identification of the key tick salivary molecules promoting virus transmission, and a molecular description of tick-host-virus interactions and of tick-mediated skin immunomodulation. Such insights will enable the rationale design of anti-tick vaccines that protect against disease caused by tick-borne viruses.

DARPin-targeting of Measles Virus: Unique Bispecificity, Effective Oncolysis, and Enhanced Safety

PubMed Central

Friedrich, Katrin; Hanauer, Jan RH; Prüfer, Steffen; Münch, Robert C; Völker, Iris; Filippis, Christodoulos; Jost, Christian; Hanschmann, Kay-Martin; Cattaneo, Roberto; Peng, Kah-Whye; Plückthun, Andreas; Buchholz, Christian J; Cichutek, Klaus; Mühlebach, Michael D

2013-01-01

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics. PMID:23380817

Visualizing interactions between Sindbis virus and cells by single particle tracking

NASA Astrophysics Data System (ADS)

Williard, Mary

2005-03-01

Sindbis virus infects both mammalian and insect cells. Though not pathogenic in humans, Sindbis is a model for many mosquito- borne viruses that cause human disease, such as West Nile virus. We have used real-time single particle fluorescence microscopy to observe individual Sindbis virus particles as they infect living cells. Fluorescent labels were incorporated into both the viral coat proteins and the lipid envelope of the virus. Kinetics characteristic of free diffusion in solution, slower diffusion inside cells, attachment to spots on the cell surface, and motor protein transport inside cells have been observed. Dequenching of the membrane label is used to report membrane fusion events during the infection process. Tracking individual viral particles allows multiple pathways to be determined without the requirement of synchronicity.

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection.

PubMed

Davis, Carl W; Nguyen, Hai-Yen; Hanna, Sheri L; Sánchez, Melissa D; Doms, Robert W; Pierson, Theodore C

2006-02-01

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.

A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection.

PubMed

Riblett, Amber M; Blomen, Vincent A; Jae, Lucas T; Altamura, Louis A; Doms, Robert W; Brummelkamp, Thijn R; Wojcechowskyj, Jason A

2016-02-01

Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents with activity against RVFV, and details of its life cycle and interaction with host cells are not well characterized. We used the power of genetic screening in human cells and found that RVFV utilizes glycosaminoglycans to attach to host cells. This furthers our understanding of the virus and informs the development of antiviral therapeutics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine.

PubMed

Wang, Scarlet Xiaoyan; Michiels, Johan; Ariën, Kevin K; New, Roger; Vanham, Guido; Roitt, Ivan

2016-12-01

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine

NASA Astrophysics Data System (ADS)

Wang, Scarlet Xiaoyan; Michiels, Johan; Ariën, Kevin K.; New, Roger; Vanham, Guido; Roitt, Ivan

2016-07-01

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

PubMed

Szakács, Zoltán; Mészáros, Tamás; de Jonge, Marien I; Gyurcsányi, Róbert E

2018-05-30

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

[Role of Powassan virus in the etiological structure of tick-borne encephalitis in the Primorsky Kray].

PubMed

Leonova, G N; Isachkova, L M; Baranov, N I; Krugliak, S P

1980-01-01

Composite studies conducted annually in the Primorsky kray showed the tick-borne encephalitis virus to play the main etiological role in the group of encephalites with the spring-summer incidence. In 1976--1978, virological studies of 69 cases of the disease yielded 11 strains of tick-borne encephalitis virus. In 1978, from the blood of clinically normal woman after a tick bite strain 555 was first isolated which was identified as Powassan virus, and antigenemia was observed for 53 days using the fluorescent antibody technique. In the same period, serological examinations of the blood sera from 117 patients demonstrated antibody to tick-borne encephalitis virus in 69.2%, to Powassan virus in 4,3% and to both viruses simultaneously in 4.3%. Besides, antibody to tick-borne encephalitis virus, Powassan virus and both viruses simultaneously was found in patients with progredient forms of tick-borne encephalitis and in subjects with the history of tick attachment.

Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters.

PubMed

Lo, Michael K; Bird, Brian H; Chattopadhyay, Anasuya; Drew, Clifton P; Martin, Brock E; Coleman, Joann D; Rose, John K; Nichol, Stuart T; Spiropoulou, Christina F

2014-01-01

Nipah virus (NiV) continues to cause outbreaks of fatal human encephalitis due to spillover from its bat reservoir. We determined that a single dose of replication-defective vesicular stomatitis virus (VSV)-based vaccine vectors expressing either the NiV fusion (F) or attachment (G) glycoproteins protected hamsters from over 1000 times LD50 NiV challenge. This highly effective single-dose protection coupled with an enhanced safety profile makes these candidates ideal for potential use in livestock and humans. Published by Elsevier B.V.

Differentiation among isolates of prunus necrotic ringspot virus by transcript conformation polymorphism.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1998-09-01

A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.

Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis.

PubMed

Mader, Julia; Gallo, Antonio; Schommartz, Tim; Handke, Wiebke; Nagel, Claus-Henning; Günther, Patrick; Brune, Wolfram; Reich, Kristian

2016-01-01

Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Idiosyncratic Mòjiāng virus attachment glycoprotein directs a host-cell entry pathway distinct from genetically related henipaviruses.

PubMed

Rissanen, Ilona; Ahmed, Asim A; Azarm, Kristopher; Beaty, Shannon; Hong, Patrick; Nambulli, Sham; Duprex, W Paul; Lee, Benhur; Bowden, Thomas A

2017-07-12

In 2012, cases of lethal pneumonia among Chinese miners prompted the isolation of a rat-borne henipavirus (HNV), Mòjiāng virus (MojV). Although MojV is genetically related to highly pathogenic bat-borne henipaviruses, the absence of a conserved ephrin receptor-binding motif in the MojV attachment glycoprotein (MojV-G) indicates a differing host-cell recognition mechanism. Here we find that MojV-G displays a six-bladed β-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, in particular at host receptor-binding surfaces. We confirm the inability of MojV-G to interact with known paramyxoviral receptors in vitro, indicating an independence from well-characterized ephrinB2/B3, sialic acid and CD150-mediated entry pathways. Furthermore, we find that MojV-G is antigenically distinct, indicating that MojV would less likely be detected in existing large-scale serological screening studies focused on well-established HNVs. Altogether, these data indicate a unique host-cell entry pathway for this emerging and potentially pathogenic HNV.

Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins.

PubMed

Stone, Jacquelyn A; Vemulapati, Bhadra M; Bradel-Tretheway, Birgit; Aguilar, Hector C

2016-12-01

The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

PubMed Central

Tal-Singer, R; Peng, C; Ponce De Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

1995-01-01

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7769707

Sodium Lauryl Sulfate Abrogates Human Immunodeficiency Virus Infectivity by Affecting Viral Attachment

PubMed Central

Bestman-Smith, Julie; Piret, Jocelyne; Désormeaux, André; Tremblay, Michel J.; Omar, Rabeea F.; Bergeron, Michel G.

2001-01-01

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1NL4-3 with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 μM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 μM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases. PMID:11451679

Epitope selection from an uncensored peptide library displayed on avian leukosis virus.

PubMed

Khare, Pranay D; Rosales, Ana G; Bailey, Kent R; Russell, Stephen J; Federspiel, Mark J

2003-10-25

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.

Virus fate and transport during artificial recharge with recycled water

USGS Publications Warehouse

Anders, Robert; Chrysikopoulos, C.V.

2005-01-01

A field‐scale experiment was conducted at a research site using bacterial viruses (bacteriophage) MS2 and PRD1 as surrogates for human viruses, bromide as a conservative tracer, and tertiary‐treated municipal wastewater (recycled water) to investigate the fate and transport of viruses during artificial recharge. Observed virus concentrations were fitted using a mathematical model that simulates virus transport in one‐dimensional, homogeneous, water‐saturated porous media accounting for virus sorption (or filtration), virus inactivation, and time‐dependent source concentration. The fitted time‐dependent clogging rate constants were used to estimate the collision efficiencies for bacteriophage MS2 and PRD1 during vertical fully saturated flow. Furthermore, the corresponding time‐dependent collision efficiencies for both bacteriophage asymptotically reached similar values at the various sampling locations. These results can be used to develop an optimal management scenario to maximize the amount of recycled water that can be applied to the spreading grounds while still maintaining favorable attachment conditions for virus removal.

Viruses Occur Incorporated in Biogenic High-Mg Calcite from Hypersaline Microbial Mats

PubMed Central

De Wit, Rutger; Gautret, Pascale; Bettarel, Yvan; Roques, Cécile; Marlière, Christian; Ramonda, Michel; Nguyen Thanh, Thuy; Tran Quang, Huy; Bouvier, Thierry

2015-01-01

Using three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake “La Salada de Chiprana”, Spain, contain viruses with a diameter of 50–80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>1010 viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 109 viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as “nanobacteria” and that viruses may play a role in initiating calcification. PMID:26115121

Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus

PubMed Central

Mor, Sunil K.; Higgins, LeeAnn; Armien, Anibal; Youssef, Mohammed M.; Bruggeman, Peter J.; Goyal, Sagar M.

2018-01-01

Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O2 admixture were studied. We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid. PMID:29566061

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

PubMed

Veazey, Ronald S; Klasse, Per Johan; Schader, Susan M; Hu, Qinxue; Ketas, Thomas J; Lu, Min; Marx, Preston A; Dufour, Jason; Colonno, Richard J; Shattock, Robin J; Springer, Martin S; Moore, John P

2005-11-03

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

Zanamivir conjugated to poly-L-glutamine is much more active against influenza viruses in mice and ferrets than the drug itself

PubMed Central

Weight, Alisha K.; Belser, Jessica A.; Tumpey, Terrence M.; Chen, Jianzhu; Klibanov, Alexander M.

2014-01-01

Purpose Previously, polymer-attached zanamivir had been found to inhibit influenza A viruses in vitro far better than small-molecule zanamivir itself. The aim of this study was to identify in vitro – using the plaque reduction assay - a highly potent zanamivir-polymer conjugate, and subsequently test its antiviral efficacy in vivo. Methods By examining the structure-activity relationship of zanamivir-polymer conjugates in the plaque assay, we have determined that the most potent inhibitor against several representative influenza virus strains has a neutral high-molecular-weight backbone and a short alkyl linker. We have examined this optimal polymeric inhibitor for efficacy and immunogenicity in the mouse and ferret models of infection. Results Zanamivir attached to poly-L-glutamine is an effective therapeutic for established influenza infection in ferrets, reducing viral titers up to 30-fold for 6 days. There is also up to a 190-fold reduction in viral load when the drug is used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the drug conjugate stimulates an immune response in mice upon repeat administration. Conclusions Zanamivir attached to a neutral high-molecular-weight backbone through a short alkyl linker drastically reduced both in vitro and in vivo titers compared to those observed with zanamivir itself. Thus further development of this polymeric zanamivir for the mitigation of influenza infection seems warranted. PMID:24065587

A stabilized headless measles virus attachment protein stalk efficiently triggers membrane fusion.

PubMed

Brindley, Melinda A; Suter, Rolf; Schestak, Isabel; Kiss, Gabriella; Wright, Elizabeth R; Plemper, Richard K

2013-11-01

Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved.

Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality.

PubMed

Park, Arnold; Yun, Tatyana; Hill, Terence E; Ikegami, Tetsuro; Juelich, Terry L; Smith, Jennifer K; Zhang, Lihong; Freiberg, Alexander N; Lee, Benhur

2016-04-01

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus

PubMed Central

Ng, Wy Ching; Londrigan, Sarah L.; Nasr, Najla; Cunningham, Anthony L.; Turville, Stuart; Brooks, Andrew G.

2015-01-01

ABSTRACT It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. IMPORTANCE On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. PMID:26468543

Identification of novel target sites and an inhibitor of the dengue virus E protein.

PubMed

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P; Young, Paul R; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC(50) in the micromolar range against dengue virus type 2.

Identification of novel target sites and an inhibitor of the dengue virus E protein

NASA Astrophysics Data System (ADS)

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P.; Young, Paul R.; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC50 in the micromolar range against dengue virus type 2.

Poliovirus Cell Entry: Common Structural Themes in Viral Cell Entry Pathways

PubMed Central

Hogle, James M.

2006-01-01

Structural studies of polio- and closely related viruses have provided a series of snapshots along their cell entry pathways. Based on the structures and related kinetic, biochemical, and genetic studies, we have proposed a model for the cell entry pathway for polio- and closely related viruses. In this model a maturation cleavage of a capsid protein precursor locks the virus in a metastable state, and the receptor acts like a transition-state catalyst to overcome an energy barrier and release the mature virion from the metastable state. This initiates a series of conformational changes that allow the virus to attach to membranes, form a pore, and finally release its RNA genome into the cytoplasm. This model has striking parallels with emerging models for the maturation and cell entry of more complex enveloped viruses such as influenza virus and HIV. PMID:12142481

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roehrig, John T., E-mail: jtr1@cdc.gov; Butrapet, Siritorn; Liss, Nathan M.

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cellsmore » and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.« less

The agmatine-containing poly(amidoamine) polymer AGMA1 binds cell surface heparan sulfates and prevents attachment of mucosal human papillomaviruses.

PubMed

Cagno, Valeria; Donalisio, Manuela; Bugatti, Antonella; Civra, Andrea; Cavalli, Roberta; Ranucci, Elisabetta; Ferruti, Paolo; Rusnati, Marco; Lembo, David

2015-09-01

The agmatine-containing poly(amidoamine) polymer AGMA1 was recently shown to inhibit the infectivity of several viruses, including human papillomavirus 16 (HPV-16), that exploit cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The aim of this work was to assess the antiviral activity of AGMA1 and its spectrum of activity against a panel of low-risk and high-risk HPVs and to elucidate its mechanism of action. AGMA1 was found to be a potent inhibitor of mucosal HPV types (i.e., types 16, 31, 45, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 0.34 μg/ml and 0.73 μg/ml, and no evidence of cytotoxicity was observed. AGMA1 interacted with immobilized heparin and with cellular heparan sulfates, exerting its antiviral action by preventing virus attachment to the cell surface. The findings from this study indicate that AGMA1 is a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Inorganic Nanoparticle as a Carrier for Hepatitis B Viral Capsids

NASA Astrophysics Data System (ADS)

Dekhtyar, Yu.; Romanova, M.; Kachanovska, A.; Skrastiņa, D.; Reinhofa, R.; Pumpens, P.; Patmalnieks, A.

Virus like particles (VLP) are used to transport immune response-modulating agents to target cells to treat them. In order to deliver a high concentration of VLP to the cell, a number of VLP can be attached to a nanoparticle to be used as a nanolorry. In this study, SiO2 nanoparticles were attached to Hepatitis B VLP. Spectrophotometry measurements, electron, and fluorescent microscopy evidence showed that the SiO2 - Hepatitis B VLP complexes were formed.

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, S.-F.; Huang, Jason C.; AIDS Prevention and Research Center, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan

2008-09-05

DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing {alpha}-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capablemore » of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.« less

The great escape: viral strategies to counter BST-2/tetherin.

PubMed

Douglas, Janet L; Gustin, Jean K; Viswanathan, Kasinath; Mansouri, Mandana; Moses, Ashlee V; Früh, Klaus

2010-05-13

The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma-associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as "tetherin". However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses.

Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview.

PubMed

Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

2017-07-05

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV-host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus-host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding.

Temperature dependency of virus and nanoparticle transport and retention in saturated porous media

NASA Astrophysics Data System (ADS)

Sasidharan, Salini; Torkzaban, Saeed; Bradford, Scott A.; Cook, Peter G.; Gupta, Vadakattu V. S. R.

2017-01-01

The influence of temperature on virus (PRD1 and ΦX174) and carboxyl-modified latex nanoparticle (50 and 100 nm) attachment was examined in sand-packed columns under various physiochemical conditions. When the solution ionic strength (IS) equaled 10 and 30 mM, the attachment rate coefficient (katt) increased up to 109% (p < 0.0002) and the percentage of the sand surface area that contributed to attachment (Sf) increased up to 160% (p < 0.002) when the temperature was increased from 4 to 20 °C. Temperature effects at IS = 10 and 30 mM were also dependent on the system hydrodynamics; i.e., enhanced retention at a lower pore water velocity (0.1 m/day). Conversely, this same temperature increase had a negligible influence on katt and Sf values when IS was 1 mM or > 50 mM. An explanation for these observations was obtained from extended interaction energy calculations that considered nanoscale roughness and chemical heterogeneity on the sand surface. Interaction energy calculations demonstrated that the energy barrier to attachment in the primary minimum (ΔΦa) decreased with increasing IS, chemical heterogeneity, and temperature, especially in the presence of small amounts of nanoscale roughness (e.g., roughness fraction of 0.05 and height of 20 nm in the zone of influence). Temperature had a negligible effect on katt and Sf when the IS = 1 mM because of the large energy barrier, and at IS = 50 mM because of the absence of an energy barrier. Conversely, temperature had a large influence on katt and Sf when the IS was 10 and 30 mM because of the presence of a small ΔΦa on sand with nanoscale roughness and a chemical (positive zeta potential) heterogeneity. This has large implications for setting parameters for the accurate modeling and transport prediction of virus and nanoparticle contaminants in ground water systems.

Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus, Junín virus

PubMed Central

Belouzard, Sandrine; Cordo, Sandra M.; Candurra, Nélida A.; Whittaker, Gary R.

2014-01-01

The target cell tropism of enveloped viruses is regulated by interactions between viral proteins and cellular receptors determining susceptibility at a host cell, tissue or species level. However, a number of additional cell-surface moieties can also bind viral envelope glycoproteins and could act as capture receptors, serving as attachment factors to concentrate virus particles on the cell surface, or to disseminate the virus infection to target organs or susceptible cells within the host. Here, we used Junín virus (JUNV) or JUNV glycoprotein complex (GPC)-pseudotyped particles to study their ability to be internalized by the human C-type lectins hDC- or hL-SIGN. Our results provide evidence that hDC- and hL-SIGN can mediate the entry of Junín virus into cells, and may play an important role in virus infection and dissemination in the host. PMID:24183720

Structural and Functional Studies on the Fusion and Attachment Envelope Glycoproteins of Nipah Virus and Hendra Virus

DTIC Science & Technology

2003-01-01

epidemics, caused by Vibrio cholerae have been linked to specific seasons and biogeographical zones. In addition, the population dynamics of V. cholerae in...Climactic warming has directly affected the prevalence of RVFV by prolonging survival rates of the vector involved in disease transmission. 3 Cholera ...climate variability. The study of V. cholerae represents a model system of how climate change affects pathogens (2). Personal human behavior has

Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

PubMed

Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

2011-12-01

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

JC Polyomavirus Attachment, Entry, and Trafficking: Unlocking the Keys to a Fatal Infection

PubMed Central

Maginnis, Melissa S.; Nelson, Christian D.S.; Atwood, Walter J.

2014-01-01

The human JC polyomavirus (JCPyV) causes a lifelong persistent infection in the reno-urinary tract in the majority of the adult population worldwide. In healthy individuals infection is asymptomatic, while in immunocompromised individuals the virus can spread to the central nervous system and cause a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). There are currently very few treatment options for this rapidly progressing and devastating disease. Understanding the basic biology of JCPyV-host cell interactions is critical for the development of therapeutic strategies to prevent or treat PML. Research in our laboratory has focused on gaining a detailed mechanistic understanding of the initial steps in the JCPyV life cycle in order to define how JCPyV selectively targets cells in the kidney and brain. JCPyV requires sialic acids to attach to host cells and initiate infection, and JCPyV demonstrates specificity for the oligosaccharide lactoseries tetrasaccharide c (LSTc) with an α2,6-linked sialic acid. Following viral attachment, JCPyV entry is facilitated by the 5-hydroxytryptamine (5-HT)2 family of serotonin receptors via clathrin-dependent endocytosis. JCPyV then undergoes retrograde transport to the endoplasmic reticulum (ER) where viral disassembly begins. A novel retrograde transport inhibitor termed Retro-2cycl prevents trafficking of JCPyV to the ER and inhibits both initial virus infection and infectious spread in cell culture. Understanding the molecular mechanisms by which JCPyV establishes infection will open up new avenues for the prevention or treatment of virus-induced disease. PMID:25078361

Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

PubMed Central

Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.

2013-01-01

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger. PMID:24089572

Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

2009-03-15

Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensivemore » than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.« less

Evidence of a potential receptor-binding site on the Nipah virus G protein (NiV-G): identification of globular head residues with a role in fusion promotion and their localization on an NiV-G structural model.

PubMed

Guillaume, Vanessa; Aslan, Hamide; Ainouze, Michelle; Guerbois, Mathilde; Wild, T Fabian; Buckland, Robin; Langedijk, Johannes P M

2006-08-01

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such "escape mutants" identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of beta-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin.

Evidence of a Potential Receptor-Binding Site on the Nipah Virus G Protein (NiV-G): Identification of Globular Head Residues with a Role in Fusion Promotion and Their Localization on an NiV-G Structural Model

PubMed Central

Guillaume, Vanessa; Aslan, Hamide; Ainouze, Michelle; Guerbois, Mathilde; Fabian Wild, T.; Buckland, Robin; Langedijk, Johannes P. M.

2006-01-01

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such “escape mutants” identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of β-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin. PMID:16840334

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

PubMed

Sattler, Ursula; Khosravi, Mojtaba; Avila, Mislay; Pilo, Paola; Langedijk, Johannes P; Ader-Ebert, Nadine; Alves, Lisa A; Plattet, Philippe; Origgi, Francesco C

2014-07-01

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Chimeric bovine respiratory syncytial virus with attachment and fusion glycoproteins replaced by bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins.

PubMed

Stope, M B; Karger, A; Schmidt, U; Buchholz, U J

2001-10-01

Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background.

Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanai,R.; Kar, K.; Anthony, K.

2006-01-01

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specificmore » antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.« less

Functional Expression. Ephrin Receptor Tropism, and Heterotypic Functionality of the Attachment and Fusion Glycoproteins of Cedar Virus, a Newly Discovered Henipavirus

DTIC Science & Technology

2012-08-20

in West Africa. Despite this broad range of seropositivity in bats, HeV and NiV outbreaks have only been described in Australia, Malaysia , India...to Hendra infection (http://www.daff.qld.gov.au/4790_2900.htm). Nipah virus was described during an outbreak of encephalitis in Malaysia and...measures which brought the outbreak to an end [7], even as it caused a devastating amount of damage to the economy of Malaysia . Following the

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

PubMed

Cherpillod, P; Tipold, A; Griot-Wenk, M; Cardozo, C; Schmid, I; Fatzer, R; Schobesberger, M; Zurbriggen, R; Bruckner, L; Roch, F; Vandevelde, M; Wittek, R; Zurbriggen, A

2000-07-01

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Transport of viruses through saturated and unsaturated columns packed with sand

USGS Publications Warehouse

Anders, R.; Chrysikopoulos, C.V.

2009-01-01

Laboratory-scale virus transport experiments were conducted in columns packed with sand under saturated and unsaturated conditions. The viruses employed were the male-specific RNA coliphage, MS2, and the Salmonella typhimurium phage, PRD1. The mathematical model developed by Sim and Chrysikopoulos (Water Resour Res 36:173-179, 2000) that accounts for processes responsible for removal of viruses during vertical transport in one-dimensional, unsaturated porous media was used to fit the data collected from the laboratory experiments. The liquid to liquid-solid and liquid to air-liquid interface mass transfer rate coefficients were shown to increase for both bacteriophage as saturation levels were reduced. The experimental results indicate that even for unfavorable attachment conditions within a sand column (e.g., phosphate-buffered saline solution; pH = 7.5; ionic strength = 2 mM), saturation levels can affect virus transport through porous media. ?? Springer Science+Business Media B.V. 2008.

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

PubMed Central

Rice, W G; Schaeffer, C A; Graham, L; Bu, M; McDougal, J S; Orloff, S L; Villinger, F; Young, M; Oroszlan, S; Fesen, M R

1993-01-01

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle. Images Fig. 3 Fig. 4 PMID:7692451

78 FR 78693 - List of Approved Spent Fuel Storage Casks: Transnuclear, Inc. Standardized NUHOMS® Cask System

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-27

... dangerous to living organisms, including insects, microbes, bacteria or virus that attach to dust that.... According to his comment, ``[T]he Deer Tick has carried a spirochete bacteria for millions of years, but...

Use of PRD1 bacteriophage in groundwater viral transport, inactivation, and attachment studies

USGS Publications Warehouse

Harvey, Ronald W.; Ryan, Joseph N.

2004-01-01

PRD1, an icosahedra-shaped, 62 nm (diameter), double-stranded DNA bacteriophage with an internal membrane, has emerged as an important model virus for studying the manner in which microorganisms are transported through a variety of groundwater environments. The popularity of this phage for use in transport studies involving geologic media is due, in part, to its relative stability over a range of temperatures and low degree of attachment in aquifer sediments. Laboratory and field investigations employing PRD1 are leading to a better understanding of viral attachment and transport behaviors in saturated geologic media and to improved methods for describing mathematically subsurface microbial transport at environmentally significant field scales. Radioisotopic labeling of PRD1 is facilitating additional information about the nature of viral interactions with solid surfaces in geologic media, the importance of iron oxide surfaces, and allowing differentiation between inactivation and attachment in field-scale tracer tests.

In vitro and in vivo antiviral activity of scopadulcic acid B from Scoparia dulcis, Scrophulariaceae, against herpes simplex virus type 1.

PubMed

Hayashi, K; Niwayama, S; Hayashi, T; Nago, R; Ochiai, H; Morita, N

1988-09-01

The antiviral activity of five diterpenoids isolated from Scoparia dulcis L., Scrophulariaceae, was examined in vitro against herpes simplex virus type 1. Among these compounds, only scopadulcic acid B was found to inhibit the viral replication with the in vitro therapeutic index of 16.7. The action of scopadulcic acid B was not due to a direct virucidal effect or inhibition of virus attachment to host cells. Single-cycle replication experiments indicated that the compound interfered with considerably early events of virus growth. The influence of scopadulcic acid B on the course of the primary corneal herpes simplex virus infection was investigated by means of a hamster test model. When the treatment was initiated immediately after virus inoculation, scopadulcic acid B, when applied orally or intraperitoneally, effectively prolonged both the appearance of herpetic lesions and the survival time at the dose of 100 and 200 mg/kg per day.

Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Gaie; Jeffree, Chris E.; McDonald, Terence

2004-10-01

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of themore » virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.« less

Structural Studies of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Tetramer in Complex with Its Receptor, Sialyllactose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Ping; Thompson, Thomas B.; Wurzburg, Beth A.

2010-03-08

The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to bemore » the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.« less

La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soldan, Samantha S., E-mail: sssoldan@mail.med.upenn.ed; Hollidge, Bradley S.; Department of Neuroscience Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283

La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative tomore » LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.« less

Structural basis of efficient contagion: measles variations on a theme by parainfluenza viruses.

PubMed

Mateo, Mathieu; Navaratnarajah, Chanakha K; Cattaneo, Roberto

2014-04-01

A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious. Copyright © 2014 Elsevier B.V. All rights reserved.

Potential for Aedes albopictus and Ochlerotatus j. japonicus to Change the Field Ecology of Arboviruses of Human Health Importance in the Mid-Atlantic Region of the United States

DTIC Science & Technology

2001-10-16

in the midgut epithelial cells. Step 3 is release 17 of virus from the midgut epithelial cell into the hemolymph. Step 4 is infection of the...proteins, maturation, and budding. During attachment, the viral attachment proteins on the surface of the virion bind to receptors on the microvillar...membrane of the mosquito s midgut . Penetration refers to the process through which the virions enter the midgut cells; arboviruses enter cell

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus.

PubMed

Ng, Wy Ching; Londrigan, Sarah L; Nasr, Najla; Cunningham, Anthony L; Turville, Stuart; Brooks, Andrew G; Reading, Patrick C

2016-01-01

It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5(+)) but not late (Rab7(+)) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors.

PubMed

Anantpadma, Manu; Kouznetsova, Jennifer; Wang, Hang; Huang, Ruili; Kolokoltsov, Andrey; Guha, Rajarshi; Lindstrom, Aaron R; Shtanko, Olena; Simeonov, Anton; Maloney, David J; Maury, Wendy; LaCount, Douglas J; Jadhav, Ajit; Davey, Robert A

2016-08-01

Filoviruses are highly infectious, and no FDA-approved drug therapy for filovirus infection is available. Most work to find a treatment has involved only a few strains of Ebola virus and testing of relatively small drug libraries or compounds that have shown efficacy against other virus types. Here we report the findings of a high-throughput screening of 319,855 small molecules from the Molecular Libraries Small Molecule Repository library for their activities against Marburg virus and Ebola virus. Nine of the most potent, novel compounds that blocked infection by both viruses were analyzed in detail for their mechanisms of action. The compounds inhibited known key steps in the Ebola virus infection mechanism by blocking either cell surface attachment, macropinocytosis-mediated uptake, or endosomal trafficking. To date, very few specific inhibitors of macropinocytosis have been reported. The 2 novel macropinocytosis inhibitors are more potent inhibitors of Ebola virus infection and less toxic than ethylisopropylamiloride, one commonly accepted macropinocytosis inhibitor. Each compound blocked infection of primary human macrophages, indicating their potential to be developed as new antifiloviral therapies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology.

PubMed

Steinmetz, N F; Lin, T; Lomonossoff, G P; Johnson, J E

2009-01-01

A quintessential tenet of nanotechnology is the self-assembly of nanometer-sized components into devices. Biological macromolecular systems such as viral particles were found to be suitable building blocks for nanotechnology for several reasons: viral capsids are extremely robust and can be produced in large quantities with ease, the particles self-assemble into monodisperse particles with a high degree of symmetry and polyvalency, they have the propensity to form arrays, and they offer programmability through genetic and chemical engineering. Here, we review the recent advances in engineering the icosahedral plant virus Cowpea mosaic virus (CPMV) for applications in nano-medicine and -technology. In the first part, we will discuss how the combined knowledge of the structure of CPMV at atomic resolution and the use of chimeric virus technology led to the generation of CPMV particles with short antigenic peptides for potential use as vaccine candidates. The second part focuses on the chemical addressability of CPMV. Strategies to chemically attach functional molecules at designed positions on the exterior surface of the viral particle are described. Biochemical conjugation methods led to the fabrication of electronically conducting CPMV particles and networks. In addition, functional proteins for targeted delivery to mammalian cells were successfully attached to CPMV. In the third part, we focus on the utilization of CPMV as a building block for the generation of 2D and 3D arrays. Overall, the potential applications of viral nanobuilding blocks are manifold and range from nanoelectronics to biomedical applications.

Japanese encephalitis virus invasion of cell: allies and alleys.

PubMed

Nain, Minu; Abdin, Malik Z; Kalia, Manjula; Vrati, Sudhanshu

2016-03-01

The mosquito-borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus-induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of the cell binding domain in Nipah virus G glycoprotein using a phage display system.

PubMed

Lam, Chui-Wan; AbuBakar, Sazaly; Chang, Li-Yen

2017-05-01

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with unusual broad host tropism and is designated as a Category C pathogen by the U.S. National Institute of Allergy and Infectious Diseases. NiV infection is initiated after binding of the viral G glycoprotein to the host cell receptor. The aim of this study was to map the NiV G glycoprotein cell binding domain using a phage display system. The NiV G extracellular domain was truncated and displayed as attachment proteins on M13 phage g3p minor coat protein. The binding efficiency of recombinant phages displaying different regions of NiV G to mammalian cells was evaluated. Results showed that regions of NiV G consisting of amino acids 396-602 (recombinant phage G4) and 498-602 (recombinant phage G5) demonstrated the highest binding to both Vero (5.5×10 3 cfu/ml and 5.6×10 3 cfu/ml) and THP-1 cells (3.5×10 3 cfu/ml and 2.9×10 3 cfu/ml). However, the binding of both of these recombinant phages to THP-1 cells was significantly lower than to Vero cells, and this could be due to the lack of primary host cell receptor expression on THP-1 cells. Furthermore, the binding between these two recombinant phages was competitive suggesting that there was a common host cell attachment site. This study employed an approach that is suitable for use in a biosafety level 2 containment laboratory without the need to use live virus to show that NiV G amino acids 498-602 play an important role for attachment to host cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation of quaternary ammonium silane-coated sand filter for the removal of bacteria and viruses from drinking water.

PubMed

Torkelson, A A; da Silva, A K; Love, D C; Kim, J Y; Alper, J P; Coox, B; Dahm, J; Kozodoy, P; Maboudian, R; Nelson, K L

2012-11-01

To develop an anti-microbial filter media using an attached quaternary ammonium compound (QAC) and evaluate its performance under conditions relevant to household drinking water treatment in developing countries. Silica sand was coated with dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride via covalent silane chemistry. Filter columns packed with coated media were challenged with micro-organisms under different water quality conditions. The anti-bacterial properties were investigated by visualizing Escherichia coli (E. coli) attachment to coated media under fluorescence microscopy combined with a live/dead stain. A 9-cm columns with a filtration velocity of 18 m h(-1) achieved log(10) removals of 1·7 for E. coli, 1·8 for MS2 coliphage, 1·9 for Poliovirus type 3 and 0·36 for Adenovirus type 2, compared to 0·1-0·3 log(10) removals of E. coli and MS2 by uncoated sand. Removal scaled linearly with column length and decreased with increasing ionic strength, flow velocity, filtration time and humic acid presence. Escherichia coli attached to QAC-coated sand were observed to be membrane-permeable, providing evidence of inactivation. Filtration with QAC-coated sand provided higher removal of bacteria and viruses than filtration with uncoated sand. However, major limitations included rapid fouling by micro-organisms and natural organic matter and low removal of viruses PRD1 and Adenovirus 2. QAC-coated media may be promising for household water treatment. However, more research is needed on long-term performance, options to reduce fouling and inactivation mechanisms. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and resistance to neutralizing antibody during cynomolgus monkey infection.

PubMed

Fujii, Ken; Sudaka, Yui; Takashino, Ayako; Kobayashi, Kyousuke; Kataoka, Chikako; Suzuki, Tadaki; Iwata-Yoshikawa, Naoko; Kotani, Osamu; Ami, Yasushi; Shimizu, Hiroyuki; Nagata, Noriyo; Mizuta, Katsumi; Matsuzaki, Yoko; Koike, Satoshi

2018-05-30

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G to E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence. IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some reports, VP1-145G/Q viruses were more frequently isolated from severely affected than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper, we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a non-human primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity. Copyright © 2018 American Society for Microbiology.

Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence.

PubMed

Lopes, Ana M; Breiman, Adrien; Lora, Mónica; Le Moullac-Vaidye, Béatrice; Galanina, Oxana; Nyström, Kristina; Marchandeau, Stephane; Le Gall-Reculé, Ghislaine; Strive, Tanja; Neimanis, Aleksija; Bovin, Nicolai V; Ruvoën-Clouet, Nathalie; Esteves, Pedro J; Abrantes, Joana; Le Pendu, Jacques

2017-11-29

The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus , respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits ( Oryctolagus cuniculus ), that have recently been classified as GI strains. Yet, we could not explain the emergence of virulence since similar glycan specificities were found between several pathogenic and non-pathogenic strains. By contrast, EBHSV, recently classified as GII.1, bound to terminal β-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species ( Oryctolagus cuniculus, Lepus europaeus and Sylvilagus floridanus ) showed species-specific patterns regarding the susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagoviruses host specificity and range. IMPORTANCE Lagoviruses constitute a genus of the Caliciviridae family, comprising highly pathogenic viruses, RHDV and EBHSV, which infect rabbits and hares, respectively. Recently, non-pathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs are observed. The mechanisms responsible for the emergence of pathogenicity and host-species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely doors of virus entry. Here we studied the glycan-binding properties of novel pathogenic and non-pathogenic strains looking for a link between glycan-binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host range of the virus strains, suggesting that glycan diversity contributes to lagoviruses' host range. Copyright © 2017 American Society for Microbiology.

Glycoprotein interactions in paramyxovirus fusion

PubMed Central

Iorio, Ronald M; Melanson, Vanessa R; Mahon, Paul J

2009-01-01

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus. PMID:20161127

Computer Security-Risks, Threats, and Safeguards.

ERIC Educational Resources Information Center

Ekhaml, Leticia

2001-01-01

Describes a variety of Internet threats to computers and networks used in schools. Discusses electronic trashing; clearing hard drives; cyber spying on Web sites visited; protection against cyber spying, including disposable email accounts; password sniffers; privacy policies; email snooping; email attachments that carry viruses; and hoaxes. (LRW)

Structure of the Ebola Virus Glycoprotein Bound to An Antibody From a Human Survivor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.E.; Fusco, M.L.; Hessell, A.J.

2009-05-20

Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explainmore » why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unraveling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.« less

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action.

PubMed

Calland, Noémie; Sahuc, Marie-Emmanuelle; Belouzard, Sandrine; Pène, Véronique; Bonnafous, Pierre; Mesalam, Ahmed Atef; Deloison, Gaspard; Descamps, Véronique; Sahpaz, Sevser; Wychowski, Czeslaw; Lambert, Olivier; Brodin, Priscille; Duverlie, Gilles; Meuleman, Philip; Rosenberg, Arielle R; Dubuisson, Jean; Rouillé, Yves; Séron, Karin

2015-10-01

Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Activity of the HIV-1 Attachment Inhibitor BMS-626529, the Active Component of the Prodrug BMS-663068, against CD4-Independent Viruses and HIV-1 Envelopes Resistant to Other Entry Inhibitors

PubMed Central

Li, Zhufang; Zhou, Nannan; Sun, Yongnian; Ray, Neelanjana; Lataillade, Max; Hanna, George J.

2013-01-01

BMS-626529 is a novel small-molecule HIV-1 attachment inhibitor active against both CCR5- and CXCR4-tropic viruses. BMS-626529 functions by preventing gp120 from binding to CD4. A prodrug of this compound, BMS-663068, is currently in clinical development. As a theoretical resistance pathway to BMS-663068 could be the development of a CD4-independent phenotype, we examined the activity of BMS-626529 against CD4-independent viruses and investigated whether resistance to BMS-626529 could be associated with a CD4-independent phenotype. Finally, we evaluated whether cross-resistance exists between BMS-626529 and other HIV-1 entry inhibitors. Two laboratory-derived envelopes with a CD4-independent phenotype (one CXCR4 tropic and one CCR5 tropic), five envelopes from clinical isolates with preexisting BMS-626529 resistance, and several site-specific mutant BMS-626529-resistant envelopes were examined for their dependence on CD4 for infectivity or susceptibility to BMS-626529. Viruses resistant to other entry inhibitors (enfuvirtide, maraviroc, and ibalizumab) were also examined for susceptibility to BMS-626529. Both CD4-independent laboratory isolates retained sensitivity to BMS-626529 in CD4− cells, while HIV-1 envelopes from viruses resistant to BMS-626529 exhibited no evidence of a CD4-independent phenotype. BMS-626529 also exhibited inhibitory activity against ibalizumab- and enfuvirtide-resistant envelopes. While there appeared to be some association between maraviroc resistance and reduced susceptibility to BMS-626529, an absolute correlation cannot be presumed, since some CCR5-tropic maraviroc-resistant envelopes remained sensitive to BMS-626529. Clinical use of the prodrug BMS-663068 is unlikely to promote resistance via generation of CD4-independent virus. No cross-resistance between BMS-626529 and other HIV entry inhibitors was observed, which could allow for sequential or concurrent use with different classes of entry inhibitors. PMID:23774428

Comparative genome and evolutionary analysis of naturally occurring Beilong virus in brown and black rats.

PubMed

Woo, Patrick C Y; Wong, Annette Y P; Wong, Beatrice H L; Lam, Carol S F; Fan, Rachel Y Y; Lau, Susanna K P; Yuen, Kwok-Yung

2016-11-01

Recently, we reported the presence of Beilong virus in spleen and kidney samples of brown rats and black rats, suggesting that these rodents could be natural reservoirs of Beilong virus. In this study, four genomes of Beilong virus from brown rats and black rats were sequenced. Similar to the Beilong virus genome sequenced from kidney mesangial cell line culture, those of J-virus from house mouse and Tailam virus from Sikkim rats, these four genomes from naturally occurring Beilong virus also contain the eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'). In these four genomes, the attachment glycoprotein encoded by the G gene consists of 1046 amino acids; but for the original Beilong virus genome sequenced from kidney mesangial cell line, the G CDS was predicted to be prematurely terminated at position 2205 (TGG→TAG), resulting in a 734-amino-acid truncated G protein. This phenomenon of a lack of nonsense mutation in naturally occurring Beilong viruses was confirmed by sequencing this region of 15 additional rodent samples. Phylogenetic analyses showed that the cell line and naturally occurring Beilong viruses were closely clustered, without separation into subgroups. In addition, these viruses were further clustered with J-virus and Tailam virus, with high bootstrap supports of >90%, forming a distinct group in Paramyxoviridae. Brown rats and black rats are natural reservoirs of Beilong virus. Our results also supports that the recently proposed genus, Jeilongvirus, should encompass Beilong virus, J-virus and Tailam virus as members. Copyright © 2016 Elsevier B.V. All rights reserved.

[Metapneumovirus expands the understanding of Paramyxovirus cell fusion--a review].

PubMed

Liu, Xiaoyu; Zhang, Xiaodong; Wei, Yongwei

2014-04-04

For most viruses in Paramyxoviridae, cell fusion requires both attachment protein and fusion protein. The attachment protein is responsible for the binding to its cognate receptors, while the interaction between fusion protein and attachment protein triggers the fusion protein which is responsible for the fusion. However, the Metapneumovirus fusion in Pneumovirinae subfamily displayed different mechanism where the attachment protein is not required. The cell fusion is accomplished by fusion protein alone without the help of the attachment protein. Recent studies indicate that low pH is required for cell fusion promoted by some hMPV strains. The fusion protein of aMPV type A is highly fusogenic, whereas that of type B is low. The original fusion models for Paramyxovirus cannot explain the phenomenon above. The mechanism to regulate the cell fusion of Metapneumovirus is poorly understood. It is becoming a hot spot for the study of cell fusion triggered by Paramyxovirus where it enlarged the traditional scope of Paramyxovirus fusion. In this review, we discuss the new achievements and advances in the understanding of cell fusion triggered by Metapneumovirus.

Pharmacological inhibition of feline immunodeficiency virus (FIV).

PubMed

Mohammadi, Hakimeh; Bienzle, Dorothee

2012-05-01

Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses and causes an acquired immunodeficiency syndrome (AIDS) in domestic and non-domestic cats worldwide. Genome organization of FIV and clinical characteristics of the disease caused by the virus are similar to those of human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes and macrophages, and their replication cycle in infected cells is analogous. Due to marked similarity in genomic organization, virus structure, virus replication and disease pathogenesis of FIV and HIV, infection of cats with FIV is a useful tool to study and develop novel drugs and vaccines for HIV. Anti-retroviral drugs studied extensively in HIV infection have targeted different steps of the virus replication cycle: (1) inhibition of virus entry into susceptible cells at the level of attachment to host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus membrane with the cell membrane; (3) blockade of reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and viral DNA integration into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite much success of anti-retroviral therapy slowing disease progression in people, similar therapy has not been thoroughly investigated in cats. In this article we review current pharmacological approaches and novel targets for anti-lentiviral therapy, and critically assess potentially suitable applications against FIV infection in cats.

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

Molecular Docking Studies to Explore Potential Binding Pockets and Inhibitors for Chikungunya Virus Envelope Glycoproteins.

PubMed

Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

2017-03-11

The chikungunya virus (CHIKV) envelope glycoproteins are considered important potential targets for anti-CHIKV drug discovery due to their crucial roles in virus attachment and virus entry. In this study, using two available crystal structures of the immature and mature forms of envelope glycoproteins, virtual screenings based on blind dockings and focused dockings were carried out to identify potential binding pockets and hit compounds for the virus. The chemical library database of compounds, NCI Diversity Set II, was used in these docking studies. In addition to reproducing previously reported examples, new binding pockets were identified, e.g., Pocket 2 in the 3N40, and Pocket 2 and Pocket 3 in the 3N42. Convergences in conformational sampling in docking using AutoDock Vina were evaluated. An analysis of docking results was carried out to understand interactions of the envelope glycoproteins complexes. Some key residues for interactions, for example Gly91 and His230, are identified as possessing important roles in the fusion process.

Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus.

PubMed

Rivas-Aravena, Andrea; Muñoz, Patricio; Jorquera, Patricia; Diaz, Alvaro; Reinoso, Claudia; González-Catrilelbún, Sebastián; Sandino, Ana María

2017-08-15

The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly. Copyright © 2017. Published by Elsevier B.V.

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2015-01-01

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2015-12-03

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

Inactivation of pathogenic viruses by plant-derived tannins: strong effects of extracts from persimmon (Diospyros kaki) on a broad range of viruses.

PubMed

Ueda, Kyoko; Kawabata, Ryoko; Irie, Takashi; Nakai, Yoshiaki; Tohya, Yukinobu; Sakaguchi, Takemasa

2013-01-01

Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses.

Inactivation of Pathogenic Viruses by Plant-Derived Tannins: Strong Effects of Extracts from Persimmon (Diospyros kaki) on a Broad Range of Viruses

PubMed Central

Ueda, Kyoko; Kawabata, Ryoko; Irie, Takashi; Nakai, Yoshiaki; Tohya, Yukinobu; Sakaguchi, Takemasa

2013-01-01

Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses. PMID:23372851

Novel Insights into Cell Entry of Emerging Human Pathogenic Arenaviruses.

PubMed

Fedeli, Chiara; Moreno, Héctor; Kunz, Stefan

2018-06-22

Viral hemorrhagic fevers caused by emerging RNA viruses of the Arenavirus family are among the most devastating human diseases. Climate change, global trade, and increasing urbanization promote the emergence and re-emergence of these human pathogenic viruses. Emerging pathogenic arenaviruses are of zoonotic origin and reservoir-to-human transmission is crucial for spillover into human populations. Host cell attachment and entry are the first and most fundamental steps of every virus infection and represent major barriers for zoonotic transmission. During host cell invasion, viruses critically depend on cellular factors, including receptors, co-receptors, and regulatory proteins of endocytosis. An in-depth understanding of the complex interaction of a virus with cellular factors implicated in host cell entry is therefore crucial to predict the risk of zoonotic transmission, define the tissue tropism, and assess disease potential. Over the past years, investigation of the molecular and cellular mechanisms underlying host cell invasion of human pathogenic arenaviruses uncovered remarkable viral strategies and provided novel insights into viral adaptation and virus-host co-evolution that will be covered in the present review. Copyright © 2018. Published by Elsevier Ltd.

In search of Flavivirus inhibitors part 2: tritylated, diphenylmethylated and other alkylated nucleoside analogues.

PubMed

Saudi, Milind; Zmurko, Joanna; Kaptein, Suzanne; Rozenski, Jef; Neyts, Johan; Van Aerschot, Arthur

2014-04-09

Several flaviviruses, such as the yellow fever virus and the dengue virus cause severe and potentially lethal infection in man. Following up on our initial hit 3',5'-bistritylated uridine 1, a series of alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against dengue fever virus and yellow fever virus. Hereto, alkyl and aryl groups were attached at various positions of the sugar ring combined with subtle variation of the heterocyclic base. Among the new series of derivatives, 3',5'-di-O-trityl-5-fluoro-2'-deoxyuridine (39) was the most efficient in this series and inhibited both yellow fever virus and dengue virus replication with a 50% effective concentration (EC₅₀) of ∼1 μg/mL without considerable cytotoxicity. The other fluorinated derivatives proved more toxic. Almost all diphenylmethylated pyrimidine nucleosides with 3',5'-di-O-benzhydryl-2'-deoxyuridine (50) as the example were endowed with strong cytotoxic effects down to 1 μg/mL. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Structure of Ljungan virus provides insight into genome packaging of this picornavirus

NASA Astrophysics Data System (ADS)

Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J.; Kotecha, Abhay; Siebert, C. Alistair; Lindberg, A. Michael; Fry, Elizabeth E.; Rao, Zihe; Tuthill, Tobias J.; Stuart, David I.

2015-10-01

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Structure of Ljungan virus provides insight into genome packaging of this picornavirus.

PubMed

Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J; Kotecha, Abhay; Siebert, C Alistair; Lindberg, A Michael; Fry, Elizabeth E; Rao, Zihe; Tuthill, Tobias J; Stuart, David I

2015-10-08

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Gold nanorod vaccine for respiratory syncytial virus

NASA Astrophysics Data System (ADS)

Stone, John W.; Thornburg, Natalie J.; Blum, David L.; Kuhn, Sam J.; Wright, David W.; Crowe, James E., Jr.

2013-07-01

Respiratory syncytial virus (RSV) is a major cause of pneumonia and wheezing in infants and the elderly, but to date there is no licensed vaccine. We developed a gold nanorod construct that displayed the major protective antigen of the virus, the fusion protein (F). Nanorods conjugated to RSV F were formulated as a candidate vaccine preparation by covalent attachment of viral protein using a layer-by-layer approach. In vitro studies using ELISA, electron microscopy and circular dichroism revealed that conformation-dependent epitopes were maintained during conjugation, and transmission electron microscopy studies showed that a dispersed population of particles could be achieved. Human dendritic cells treated with the vaccine induced immune responses in primary human T cells. These results suggest that this vaccine approach may be a potent method for immunizing against viruses such as RSV with surface glycoproteins that are targets for the human immune response.

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM.

PubMed

Hashiguchi, Takao; Ose, Toyoyuki; Kubota, Marie; Maita, Nobuo; Kamishikiryo, Jun; Maenaka, Katsumi; Yanagi, Yusuke

2011-02-01

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses.

PubMed

Koch, Christine; Reichling, Jürgen; Kehm, Roland; Sharaf, Mona M; Zentgraf, Hanswalter; Schneele, Jürgen; Schnitzler, Paul

2008-11-01

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Use of PRD1 bacteriophage in groundwater viral transport, inactivation, and attachment studies

USGS Publications Warehouse

Harvey, R.W.; Ryan, J.N.

2004-01-01

PRD1, an icosahedra-shaped, 62 nm (diameter), double-stranded DNA bacteriophage with an internal membrane, has emerged as an important model virus for studying the manner in which microorganisms are transported through a variety of groundwater environments. The popularity of this phage for use in transport studies involving geologic media is due, in part, to its relative stability over a range of temperatures and low degree of attachment in aquifer sediments. Laboratory and field investigations employing PRD1 are leading to a better understanding of viral attachment and transport behaviors in saturated geologic media and to improved methods for describing mathematically subsurface microbial transport at environmentally significant field scales. Radioisotopic labeling of PRD1 is facilitating additional information about the nature of viral interactions with solid surfaces in geologic media, the importance of iron oxide surfaces, and allowing differentiation between inactivation and attachment in field-scale tracer tests. ?? 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1.

PubMed Central

Tyler, K L; Squier, M K; Rodgers, S E; Schneider, B E; Oberhaus, S M; Grdina, T A; Cohen, J J; Dermody, T S

1995-01-01

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing. PMID:7474116

Adding the Third Dimension to Virus Life Cycles: Three-Dimensional Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs

PubMed Central

Baker, T. S.; Olson, N. H.; Fuller, S. D.

1999-01-01

Viruses are cellular parasites. The linkage between viral and host functions makes the study of a viral life cycle an important key to cellular functions. A deeper understanding of many aspects of viral life cycles has emerged from coordinated molecular and structural studies carried out with a wide range of viral pathogens. Structural studies of viruses by means of cryo-electron microscopy and three-dimensional image reconstruction methods have grown explosively in the last decade. Here we review the use of cryo-electron microscopy for the determination of the structures of a number of icosahedral viruses. These studies span more than 20 virus families. Representative examples illustrate the use of moderate- to low-resolution (7- to 35-Å) structural analyses to illuminate functional aspects of viral life cycles including host recognition, viral attachment, entry, genome release, viral transcription, translation, proassembly, maturation, release, and transmission, as well as mechanisms of host defense. The success of cryo-electron microscopy in combination with three-dimensional image reconstruction for icosahedral viruses provides a firm foundation for future explorations of more-complex viral pathogens, including the vast number that are nonspherical or nonsymmetrical. PMID:10585969

Trans-infection but not infection from within endosomal compartments after cell-to-cell HIV-1 transfer to CD4+ T cells.

PubMed

Permanyer, Marc; Ballana, Ester; Badia, Roger; Pauls, Eduardo; Clotet, Bonaventura; Esté, José A

2012-09-14

Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4(+) T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delpeut, Sebastien; Noyce, Ryan S.; IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4more » (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.« less

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

PubMed Central

Talekar, Aparna; DeVito, Ilaria; Salah, Zuhair; Palmer, Samantha G.; Chattopadhyay, Anasuya; Rose, John K.; Xu, Rui; Wilson, Ian A.; Moscona, Anne

2013-01-01

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal. PMID:23903846

Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing

2005-09-01

Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in themore » mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion.« less

Structure of human Aichi virus and implications for receptor binding.

PubMed

Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Yuan, Shuai; Yamashita, Teruo; Tuthill, Tobias J; Fry, Elizabeth E; Rao, Zihe; Stuart, David I

2016-09-05

Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries 1,2 . The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life 3,4 . There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

Recent advances in the identification of the host factors involved in dengue virus replication.

PubMed

Wang, Yi; Zhang, Ping

2017-02-01

Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae and it is primarily transmitted via Aedes aegypti and Aedes albopictus mosquitoes. The life cycle of DENV includes attachment, endocytosis, protein translation, RNA synthesis, assembly, egress, and maturation. Recent researches have indicated that a variety of host factors, including cellular proteins and microRNAs, positively or negatively regulate the DENV replication process. This review summarizes the latest findings (from 2014 to 2016) in the identification of the host factors involved in the DENV life cycle and Dengue infection.

A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

PubMed

Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-04-25

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

PubMed Central

Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-01-01

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

Pharmacological Inhibition of Feline Immunodeficiency Virus (FIV)

PubMed Central

Mohammadi, Hakimeh; Bienzle, Dorothee

2012-01-01

Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses and causes an acquired immunodeficiency syndrome (AIDS) in domestic and non-domestic cats worldwide. Genome organization of FIV and clinical characteristics of the disease caused by the virus are similar to those of human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes and macrophages, and their replication cycle in infected cells is analogous. Due to marked similarity in genomic organization, virus structure, virus replication and disease pathogenesis of FIV and HIV, infection of cats with FIV is a useful tool to study and develop novel drugs and vaccines for HIV. Anti-retroviral drugs studied extensively in HIV infection have targeted different steps of the virus replication cycle: (1) inhibition of virus entry into susceptible cells at the level of attachment to host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus membrane with the cell membrane; (3) blockade of reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and viral DNA integration into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite much success of anti-retroviral therapy slowing disease progression in people, similar therapy has not been thoroughly investigated in cats. In this article we review current pharmacological approaches and novel targets for anti-lentiviral therapy, and critically assess potentially suitable applications against FIV infection in cats. PMID:22754645

Selective Destruction Of Cells Infected With The Human Immunodeficiency Virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2006-03-28

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a varient of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

Selective destruction of cells infected with human immunodeficiency virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2003-09-30

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a variant of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

PubMed Central

Sherer, Nathan M.; Jin, Jing; Mothes, Walther

2010-01-01

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

Eukaryotic-Like Virus Budding in Archaea

PubMed Central

Quemin, Emmanuelle R. J.; Chlanda, Petr; Sachse, Martin; Forterre, Patrick

2016-01-01

ABSTRACT Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission. PMID:27624130

Single-Particle Detection of Transcription following Rotavirus Entry

PubMed Central

Salgado, Eric N.; Upadhyayula, Srigokul

2017-01-01

ABSTRACT Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell. IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection. PMID:28701394

A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

PubMed

Bennett, R S; Gresko, A K; Nelson, J T; Murphy, B R; Whitehead, S S

2012-01-01

La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD₅₀) values of 0.1 and 0.5 log₁₀ PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.

ABSTRACT Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bindmore » to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease. IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.« less

Herpes Simplex Virus Type 2 Glycoprotein G Is Targeted by the Sulfated Oligo- and Polysaccharide Inhibitors of Virus Attachment to Cells▿

PubMed Central

Adamiak, Beata; Ekblad, Maria; Bergström, Tomas; Ferro, Vito; Trybala, Edward

2007-01-01

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans. PMID:17928351

GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

PubMed Central

Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

2012-01-01

Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein

PubMed Central

Zhu, Qiyun; Biering, Scott B.; Mirza, Anne M.; Grasseschi, Brittany A.; Mahon, Paul J.; Lee, Benhur; Aguilar, Hector C.

2013-01-01

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F. PMID:23283956

Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

PubMed

Bale, Shridhar; Liu, Tong; Li, Sheng; Wang, Yuhao; Abelson, Dafna; Fusco, Marnie; Woods, Virgil L; Saphire, Erica Ollmann

2011-11-01

Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2)) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2) is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2) trimeric, viral surface spike. After entry into host endosomes, GP(1,2) is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown. We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2) is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2). Further, ELISA binding data of primed GP(1,2) to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion. The results reveal that the ebolavirus GP(1,2) ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2) and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

MS2 inactivation by TiO2 nanoparticles in the presence of quartz sand

NASA Astrophysics Data System (ADS)

Syngouna, Vasiliki I.; Chrysikopoulos, Constantinos V.

2017-04-01

Virus inactivation by nanoparticles (NPs) is hypothesized to affect virus fate and transport in the subsurface. This study examines the interactions of viruses with titanium dioxide (TiO2) anatase NPs, which is a good disinfectant with unique physiochemical properties, using three different virus concentrations. The bacteriophage MS2 was used as a model virus. A series of batch experiments of MS2 inactivation by TiO2 NPs were conducted at room temperature (25 °C), in the presence of quartz sand, with and without ambient light. The virus inactivation experimental data were satisfactorily fitted with a pseudo-first order expression with a time dependent rate coefficient. Quartz sand was shown to affect MS2 inactivation by TiO2 NPs both in the presence and absence of ambient light, because, under the experimental conditions of this study, the quartz sand offers a protection to the attached MS2 against inactivation. Moreover, in most cases similar inactivation rates were observed in reactor and control tubes (absence of TiO2 NPs) suggesting that low TiO2 concentration (10 mg/L) affects only slightly MS2 inactivation with and without ambient light.

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism

NASA Astrophysics Data System (ADS)

Cagno, Valeria; Andreozzi, Patrizia; D'Alicarnasso, Marco; Jacob Silva, Paulo; Mueller, Marie; Galloux, Marie; Le Goffic, Ronan; Jones, Samuel T.; Vallino, Marta; Hodek, Jan; Weber, Jan; Sen, Soumyo; Janeček, Emma-Rose; Bekdemir, Ahmet; Sanavio, Barbara; Martinelli, Chiara; Donalisio, Manuela; Rameix Welti, Marie-Anne; Eleouet, Jean-Francois; Han, Yanxiao; Kaiser, Laurent; Vukovic, Lela; Tapparel, Caroline; Král, Petr; Krol, Silke; Lembo, David; Stellacci, Francesco

2018-02-01

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (~190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

Enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactiva ted virus.

PubMed Central

Prince, G A; Jenson, A B; Hemming, V G; Murphy, B R; Walsh, E E; Horswood, R L; Chanock, R M

1986-01-01

Cotton rats previously inoculated with Formalin-inactivated respiratory syncytial virus (RSV) were challenged intranasally with live RSV to induce an enhancement of RSV disease similar to that observed after the administration of Formalin-inactivated RSV vaccine to human infants 20 years ago. Within 24 h after infection with RSV, cotton rats developed pulmonary lesions that reached a maximum by day 4. Histologically, the lesions resembled an experimental pulmonary Arthus reaction. An action of Formalin on RSV appears to be responsible for this effect, because live virus or virus heated in the absence of Formalin did not induce enhanced immunopathology. Selected epitopes on the fusion (F) or attachment (G) or both RSV surface glycoproteins that are involved in inducing neutralizing antibodies were modified to reduce or ablate their antigenicity. However, other epitopes on the F or G or both glycoproteins were not ablated by Formalin, because cotton rats inoculated parenterally with a Formalin-inactivated virus developed a high level of F and G antibodies measurable by an enzyme-linked immunosorbent assay. At this time, the effect of Formalin on RSV cannot be localized to either the F or G glycoprotein of RSV. Images PMID:2419587

Emergence of zoonotic arboviruses by animal trade and migration

PubMed Central

2010-01-01

Arboviruses are transmitted in nature exclusively or to a major extend by arthropods. They belong to the most important viruses invading new areas in the world and their occurrence is strongly influenced by climatic changes due to the life cycle of the transmitting vectors. Several arboviruses have emerged in new regions of the world during the last years, like West Nile virus (WNV) in the Americas, Usutu virus (USUV) in Central Europe, or Rift Valley fever virus (RVFV) in the Arabian Peninsula. In most instances the ways of introduction of arboviruses into new regions are not known. Infections acquired during stays in the tropics and subtropics are diagnosed with increasing frequency in travellers returning from tropical countries, but interestingly no attention is paid on accompanying pet animals or the hematophagous ectoparasites that may still be attached to them. Here we outline the known ecology of the mosquito-borne equine encephalitis viruses (WEEV, EEEV, and VEEV), WNV, USUV, RVFV, and Japanese Encephalitis virus, as well as Tick-Borne Encephalitis virus and its North American counterpart Powassan virus, and will discuss the most likely mode that these viruses could expand their respective geographical range. All these viruses have a different epidemiology as different vector species, reservoir hosts and virus types have adapted to promiscuous and robust or rather very fine-balanced transmission cycles. Consequently, these viruses will behave differently with regard to the requirements needed to establish new endemic foci outside their original geographical ranges. Hence, emphasis is given on animal trade and suitable ecologic conditions, including competent vectors and vertebrate hosts. PMID:20377873

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus*

PubMed Central

Hendricks, Gabriel L.; Weirich, Kim L.; Viswanathan, Karthik; Li, Jing; Shriver, Zachary H.; Ashour, Joseph; Ploegh, Hidde L.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Finberg, Robert W.; Comolli, James C.; Wang, Jennifer P.

2013-01-01

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains. PMID:23362274

Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

PubMed Central

Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

2012-01-01

Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

Spread of Hepatitis B Viruses In Vitro Requires Extracellular Progeny and May Be Codetermined by Polarized Egress

PubMed Central

Funk, A.; Hohenberg, H.; Mhamdi, M.; Will, H.; Sirma, H.

2004-01-01

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells. PMID:15047813

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes.

PubMed

Gitman, A G; Loyter, A

1984-08-10

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.

Classification of viral zoonosis through receptor pattern analysis.

PubMed

Bae, Se-Eun; Son, Hyeon Seok

2011-04-13

Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell. We analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species. This study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains.

Structural Basis for Marburg Virus Neutralization by a Cross-Reactive Human Antibody

DOE PAGES

Hashiguchi, Takao; Fusco, Marnie L.; Bornholdt, Zachary A.; ...

2015-02-26

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of theirmore » NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. We find that these structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.« less

Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

PubMed

Hashiguchi, Takao; Fusco, Marnie L; Bornholdt, Zachary A; Lee, Jeffrey E; Flyak, Andrew I; Matsuoka, Rei; Kohda, Daisuke; Yanagi, Yusuke; Hammel, Michal; Crowe, James E; Saphire, Erica Ollmann

2015-02-26

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry. Copyright © 2015 Elsevier Inc. All rights reserved.

Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics.

PubMed

Jung, K M; Bae, E H; Jung, Y T; Kim, J W

2014-01-01

Avian reovirus (ARV) is an important agent of several diseases causing considerable losses in poultry farming. An outer capsid protein (σC) of ARV, is known as a virus-cell attachment protein essential for virus infectivity. In this study, the σC gene of ARV was cloned and expressed in Escherichia coli. The expressed recombinant protein was used as immunogen for raising a specific IgY antibody in laying hens. At 14 weeks post immunization, the antibody titers in serum and egg yolk reached 302,000 and 355,000, respectively. The IgY antibody was capable to neutralize ARV in BHK-21 cells and it strongly reacted in ELISA with ARV but not with heterologous viruses. The IgY antibody detected ARV in field samples of infected animal tissues in dot blot assay. These results suggest that an efficient, economic and rapid diagnostics of ARV can be performed routinely using the IgY antibody against a recombinant ARV σC protein.

Endocytic function is critical for influenza A virus infection via DC-SIGN and L-SIGN

PubMed Central

Gillespie, Leah; Roosendahl, Paula; Ng, Wy Ching; Brooks, Andrew G.; Reading, Patrick C.; Londrigan, Sarah L.

2016-01-01

The ubiquitous presence of cell-surface sialic acid (SIA) has complicated efforts to identify specific transmembrane glycoproteins that function as bone fide entry receptors for influenza A virus (IAV) infection. The C-type lectin receptors (CLRs) DC-SIGN (CD209) and L-SIGN (CD209L) enhance IAV infection however it is not known if they act as attachment factors, passing virions to other unknown receptors for virus entry, or as authentic entry receptors for CLR-mediated virus uptake and infection. Sialic acid-deficient Lec2 Chinese Hamster Ovary (CHO) cell lines were resistant to IAV infection whereas expression of DC-SIGN/L-SIGN restored susceptibility of Lec2 cells to pH- and dynamin-dependent infection. Moreover, Lec2 cells expressing endocytosis-defective DC-SIGN/L-SIGN retained capacity to bind IAV but showed reduced susceptibility to infection. These studies confirm that DC-SIGN and L-SIGN are authentic endocytic receptors for IAV entry and infection. PMID:26763587

AN ELECTRON MICROSCOPE STUDY OF NERVES INFECTED WITH HUMAN POLIOMYELITIS VIRUS

PubMed Central

De Robertis, E.; Schmitt, F. O

1949-01-01

Sciatic nerves of rhesus monkeys infected with CAM and Wis. '45 strains of human poliomyelitis virus were fixed in formalin, sectioned, fragmented, and examined in the electron microscope. Most of the neurotubules of nerves infected with the CAM strain have normal appearance but a very small number show the presence of dense particles irregularly aligned within the edges of the neurotubules. The diameters of the particles range between 160 and 500 Å, the mean being 330 Å. The particles were found in regions along the nerve which varied with the time after infection, indicating a central movement of the morphological alteration of the order of 2 mm. per hour. Relatively abundant dense particulate material was found in nerves infected with Wis. '45 strain virus and the particles were chiefly attached to the edges of the neurotubules and in the adjacent areas of the field. The dense particles appear to be associated with the virus infection but no further characterization is possible at this time. PMID:18140661

Identification of Ellagic Acid from Plant Rhodiola rosea L. as an Anti-Ebola Virus Entry Inhibitor.

PubMed

Cui, Qinghua; Du, Ruikun; Anantpadma, Manu; Schafer, Adam; Hou, Lin; Tian, Jingzhen; Davey, Robert A; Cheng, Han; Rong, Lijun

2018-03-27

The recent 2014-2016 West African Ebola virus epidemic underscores the need for the development of novel anti-Ebola therapeutics, due to the high mortality rates of Ebola virus infections and the lack of FDA-approved vaccine or therapy that is available for the prevention and treatment. Traditional Chinese medicines (TCMs) represent a huge reservoir of bioactive chemicals and many TCMs have been shown to have antiviral activities. 373 extracts from 128 TCMs were evaluated using a high throughput assay to screen for inhibitors of Ebola virus cell entry. Extract of Rhodiola rosea displayed specific and potent inhibition against cell entry of both Ebola virus and Marburg virus. In addition, twenty commercial compounds that were isolated from Rhodiola rosea were evaluated using the pseudotyped Ebola virus entry assay, and it was found that ellagic acid and gallic acid, which are two structurally related compounds, are the most effective ones. The activity of the extract and the two pure compounds were validated using infectious Ebola virus. The time-of-addition experiments suggest that, mechanistically, the Rhodiola rosea extract and the effective compounds act at an early step in the infection cycle following initial cell attachment, but prior to viral/cell membrane fusion. Our findings provide evidence that Rhodiola rosea has potent anti-filovirus properties that may be developed as a novel anti-Ebola treatment.

Identification of Ellagic Acid from Plant Rhodiola rosea L. as an Anti-Ebola Virus Entry Inhibitor

PubMed Central

Cui, Qinghua; Du, Ruikun; Anantpadma, Manu; Schafer, Adam; Hou, Lin; Tian, Jingzhen; Cheng, Han; Rong, Lijun

2018-01-01

The recent 2014–2016 West African Ebola virus epidemic underscores the need for the development of novel anti-Ebola therapeutics, due to the high mortality rates of Ebola virus infections and the lack of FDA-approved vaccine or therapy that is available for the prevention and treatment. Traditional Chinese medicines (TCMs) represent a huge reservoir of bioactive chemicals and many TCMs have been shown to have antiviral activities. 373 extracts from 128 TCMs were evaluated using a high throughput assay to screen for inhibitors of Ebola virus cell entry. Extract of Rhodiola rosea displayed specific and potent inhibition against cell entry of both Ebola virus and Marburg virus. In addition, twenty commercial compounds that were isolated from Rhodiola rosea were evaluated using the pseudotyped Ebola virus entry assay, and it was found that ellagic acid and gallic acid, which are two structurally related compounds, are the most effective ones. The activity of the extract and the two pure compounds were validated using infectious Ebola virus. The time-of-addition experiments suggest that, mechanistically, the Rhodiola rosea extract and the effective compounds act at an early step in the infection cycle following initial cell attachment, but prior to viral/cell membrane fusion. Our findings provide evidence that Rhodiola rosea has potent anti-filovirus properties that may be developed as a novel anti-Ebola treatment. PMID:29584652

A Study of Parent-Child Attachments in HIV+/AIDS Minority Families.

ERIC Educational Resources Information Center

Harris, Yvette; And Others

This study examined medical services and support services available to and utilized by minority families where a child and/or parent was identified as having Human Immunodeficiency Virus (HIV) or Acquired Immune Deficiency Syndrome (AIDS). Sixteen caregivers of children (ages 2-4) diagnosed as HIV positive or children who had been exposed to the…

Ground Based Program for the Physical Analysis of Macromolecular Crystal Growth

NASA Technical Reports Server (NTRS)

Malkin, Alexander J.

1999-01-01

In a reported period in situ atomic force microscopy was utilized in our laboratory to study mechanisms of growth and kinetics of crystallization of ten protein and virus crystals. These included canavalin, thaumatin, apoferritin, lipase, catalase, t-RNA, lysozyme, xylanase, turnip yellow mosaic virus (TYMV) and satellite tobacco mosaic virus (STMV). We have also designed and constructed in our laboratory both in situ conventional two-beam Michelson and phase shift Mach-Zenhder interferometers. Computer software for the processing of the interferometric images was developed as well. Interferometric techniques were applied for studies of growth kinetics and transport phenomena in crystallization of several macromolecular crystals. As a result of this work we have published 21 papers and have given many presentations at international and national meetings. A list of these publications and conference presentations is attached.

Complex Virus–Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview

PubMed Central

Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

2017-01-01

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV–host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus–host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding. PMID:28678154

Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies.

PubMed

Welch, Brett D; Paduch, Marcin; Leser, George P; Bergman, Zachary; Kors, Christopher A; Paterson, Reay G; Jardetzky, Theodore S; Kossiakoff, Anthony A; Lamb, Robert A

2014-10-01

Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone

PubMed Central

Wei, Qingshan; Qi, Hangfei; Luo, Wei; Tseng, Derek; Ki, So Jung; Wan, Zhe; Göröcs, Zoltán; Bentolila, Laurent A.; Wu, Ting-Ting; Sun, Ren; Ozcan, Aydogan

2014-01-01

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments. PMID:24016065

On revealing the gene targets of Ebola virus microRNAs involved in the human skin microbiome.

PubMed

Hsu, Pei-Chun; Chiou, Bin-Hao; Huang, Chun-Ming

2018-01-01

Ebola virus, a negative-sense single-stranded RNA virus, causes severe viral hemorrhagic fever and has a high mortality rate. Histopathological and immunopathological analyses of Ebola virus have revealed that histopathological changes in skin tissue are associated with various degrees of endothelial cell swelling and necrosis. The interactions of microbes within or on a host are a crucial for the skin immune shield. The discovery of microRNAs (miRNAs) in Ebola virus implies that immune escape, endothelial cell rupture, and tissue dissolution during Ebola virus infection are a result of the effects of Ebola virus miRNAs. Keratinocytes obtained from normal skin can attach and spread through expression of the thrombospondin family of proteins, playing a role in initiation of cell-mediated immune responses in the skin. Several miRNAs have been shown to bind the 3' untranslated region of thrombospondin mRNA, thereby controlling its stability and translational activity. In this study, we discovered short RNA sequences that may act as miRNAs from Propionibacterium acnes using a practical workflow of bioinformatics methods. Subsequently, we deciphered the common target gene. These RNA sequences tended to bind to the same thrombospondin protein, THSD4, emphasizing the potential importance of the synergistic binding of miRNAs from Ebola virus, Propionibacterium acnes , and humans to the target. These results provide important insights into the molecular mechanisms of thrombospondin proteins and miRNAs in Ebola virus infection.

Pet ownership among persons with AIDS in three Florida counties.

PubMed

Conti, L; Lieb, S; Liberti, T; Wiley-Bayless, M; Hepburn, K; Diaz, T

1995-11-01

Interviews were conducted among 408 adults with acquired immunodeficiency syndrome at three local health departments to determine the proportion who owned pets, their perceived attachment to their pets, and the proportion who were informed about zoonoses. Nearly half (187, or 46%) were living with pets, most commonly dogs (64%), followed by cats (38%), fish (15%), birds (8%), reptiles (3%), and rodents (2%). Most pet owners (81%) reported an attachment to their pet. Only 10% were informed of zoonoses, albeit some incorrectly. Health care providers should recognize the high pet ownership rate among persons infected with human immunodeficiency virus and correctly inform their patients of strategies to sustain a low zoonotic disease incidence.

Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.

Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainlymore » plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.« less

Coronavirus Attachment and Replication

DTIC Science & Technology

1988-03-28

is also inhibited by protease treatment (Richter, 1976). Rhabdovirus binding to host cells was inhibited by phosholipase and neuraminadase, but not...protease treatment of host cells. Therefore the rhabdovirus receptor was postulated to be a glyco- or phospholipid (Wunner et &,. 1984). More...Rabies virus and VSV competed for binding on cultured neural and non-neural cells, suggesting a common receptor for rhabdoviruses (Wunner et g1, 1984

Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli.

PubMed

Ji, Yang; Lu, Yuan; Yan, Yishu; Liu, Xinxin; Su, Nan; Zhang, Chong; Bi, Shengli; Xing, Xin-Hui

2018-03-03

The Ebola hemorrhagic fever caused by Ebola virus is an extremely dangerous disease, and effective therapeutic agents are still lacking. Platforms for the efficient production of vaccines are crucial to ensure quick response against an Ebola virus outbreak. Ebola virus glycoprotein (EbolaGP) on the virion surface is responsible for membrane binding and virus entry, thus becoming the key target for vaccine development. However, heterologous expression of this protein still faces engineering challenges such as low production levels and insoluble aggregation. Here, the authors design and compare various fusion strategies, attaching great importance to the solubility-enhancing effect, and tag removal process. It is found that a C-terminal intein-based tag greatly enhances the solubility of EbolaGP and allows one-step chromatographic purification of the untagged EbolaGP through thiol-catalyzed self-cleavage. The purified untagged EbolaGP alone or with Freund's adjuvant are highly immunogenic, as confirmed in a mouse model. Consequently, the present study puts forward a new strategy for the efficient and soluble expression of untagged immunogenic EbolaGP. The intein-based protein fusion approach may be of importance for the large-scale production of Ebola virus subunit vaccine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Receptors and routes of dengue virus entry into the host cells.

PubMed

Cruz-Oliveira, Christine; Freire, João Miguel; Conceição, Thaís M; Higa, Luiza M; Castanho, Miguel A R B; Da Poian, Andrea T

2015-03-01

Dengue is the most prevalent arthropod-borne viral disease, caused by dengue virus, a member of the Flaviviridae family. Its worldwide incidence is now a major health problem, with 2.5 billion people living in risk areas. In this review, we integrate the structural rearrangements of each viral protein and their functions in all the steps of virus entry into the host cells. We describe in detail the putative receptors and attachment factors in mammalian and mosquito cells, and the recognition of viral immunocomplexes via Fcγ receptor in immune cells. We also discuss that virus internalization might occur through distinct entry pathways, including clathrin-mediated or non-classical clathrin-independent endocytosis, depending on the host cell and virus serotype or strain. The implications of viral maturation in virus entry are also explored. Finally, we discuss the mechanisms of viral genome access to the cytoplasm. This includes the role of low pH-induced conformational changes in the envelope protein that mediate membrane fusion, and original insights raised by our recent work that supports the hypothesis that capsid protein would also be an active player in this process, acting on viral genome translocation into the cytoplasm. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses.

PubMed

Richard, Audrey Stéphanie; Zhang, Adam; Park, Sun-Jin; Farzan, Michael; Zong, Min; Choe, Hyeryun

2015-11-24

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

Comparative analysis of virulence of a novel, avian-origin H3N2 canine influenza virus in various host species.

PubMed

Lyoo, Kwang-Soo; Kim, Jeong-Ki; Kang, Bokyu; Moon, Hyoungjoon; Kim, Jongman; Song, Manki; Park, Bongkyun; Kim, Sang-Hyun; Webster, Robert G; Song, Daesub

2015-01-02

A novel avian-origin H3N2 canine influenza A virus (CIV) that showed high sequence similarities in hemagglutinin and neuraminidase genes with those of non-pathogenic avian influenza viruses was isolated in our routine surveillance program in South Korea. We previously reported that the pathogenicity of this strain could be reproduced in dogs and cats. In the present study, the host tropism of H3N2 CIV was examined by experimental inoculation into several host species, including chickens, pigs, mice, guinea pigs, and ferrets. The CIV infection resulted in no overt symptoms of disease in these host species. However, sero-conversion, virus shedding, and gross and histopathologic lung lesions were observed in guinea pig and ferrets but not in pigs, or mice. Based on the genetic similarity of our H3N2 CIV with currently circulating avian influenza viruses and the presence of α-2,3-linked rather than α-2,6-linked sialic acid receptors in the respiratory tract of dogs, we believed that this strain of CIV would have avian virus-like receptor specificity, but that seems to be contrary to our findings in the present study. Further studies are needed to determine the co-receptors of hemagglutinin or post-attachment factors related to virus internalization or pathogenesis in other animals. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Determinants of Influenza Virus Pathogenesis in Mice

PubMed Central

Katz, Jaqueline M.; York, Ian A.

2015-01-01

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account. PMID:25038937

Significance of bacteria and viruses in the carbon flow of tropical freshwater impoundments

NASA Astrophysics Data System (ADS)

Peduzzi, P.; Schiemer, F.

2003-04-01

In two types of tropical freshwater impoundments, free and particle-attached bacterial abundance and production as well as virus abundance, frequency of viral infection and virus production were investigated together with a set of environmental factors during two characteristic seasons. Organic nitrogen, phosphorus species, dissolved organic carbon and suspended solids were elevated in the wind-mixed water body of a shallow reservoir during the dry season, whereas a deeper reservoir type exhibited no obvious seasonality in these parameters. In SYBR GREEN-stained samples, bacterial abundance showed no seasonal pattern in either reservoir type. A large proportion of the overall bacterial production was associated with particulate material. Highest densities of virus particles and elevated frequency of bacteria containing mature phages were observed in the shallow reservoir during the dry season. The specific bacterial production was related to the abundance of particulate organic matter, phosphorus species and organic nitrogen. Most virus parameters were positively linked to bacterial density, production and to organic nitrogen. We calculated that between 13.2 and 46.1% of the bacterial standing stocks would be subjected to virus-mediated mortality. Carbon budgets for the microbial and organic matter compartments of these tropical freshwater reservoirs indicate prevailing autotrophy and a substantial pathway through the viral shunt. During the dry season the shallow, wind-mixed reservoir provided favorable conditions for bacterial growth and virus propagation.

Stability of and Attachment to Lettuce by a Culturable Porcine Sapovirus Surrogate for Human Caliciviruses

PubMed Central

Zhang, Zhenwen

2012-01-01

Human noroviruses (HuNoVs) are the leading cause of food-borne illness, accounting for 58% of U.S. cases. Because HuNoVs are unculturable, surrogates are needed to investigate transmission routes and evaluate disinfection methods. However, the current surrogates, feline calicivirus (FCV) and murine NoV (MNV), are less tolerant than HuNoVs to acid and chlorine, respectively. Porcine sapovirus (SaV) is the only culturable enteropathogenic calicivirus. In this study, the resistance of SaV to physicochemical treatments was compared to that of HuNoVs (by reverse transcription-PCR), FCV, and MNV (by infectivity assays). Sapovirus and HuNoV (viral RNA) showed similar resistances to heat (56°C) and to different concentrations of chlorine. However, SaV was more resistant than HuNoVs to ethanol treatment (60% and 70%). Like HuNoVs, SaV was stable at pH 3.0 to 8.0, with a <1.0 log10 50% tissue culture infective dose (TCID50) reduction at pH 3.0 compared to the value for pH 4.0 to 8.0. SaV and MNV showed similar resistances, and both were more resistant than FCV to heat inactivation (56°C). FCV was more resistant than MNV and SaV to ethanol, and all three viruses showed similar resistances to treatment with low concentrations of chlorine for 1 min. Those results indicate that SaV is a promising surrogate for HuNoVs. Next, we used SaV as a surrogate to examine virus attachment to lettuce at different pHs. Sapovirus attached to lettuce leaves significantly at its capsid isoelectric point (pH 5.0), and the attached viral particles remained infectious on lettuce after 1 week of storage at 4°C. The culturable SaV is a good surrogate for studying HuNoV contamination and transmission in leafy greens and potential disinfectants. PMID:22447610

A 32 kDa viral attachment protein of lymphocystis disease virus (LCDV) specifically interacts with a 27.8 kDa cellular receptor from flounder (Paralichthys olivaceus).

PubMed

Zhong, Ying; Fei, Chenjie; Tang, Xiaoqian; Zhan, Wenbin; Sheng, Xiuzhen

2017-06-01

The 27.8 kDa protein in flounder gill (FG) cells was previously proved to be a receptor specific for lymphocystis disease virus (LCDV) entry and infection. In this paper, a 32 kDa viral attachment protein (VAP) of LCDV specifically binding to the 27.8 kDa receptor (27.8R) was found by far-Western blotting coupled with monoclonal antibodies (MAbs) against 27.8R. The 32 kDa protein was confirmed to be encoded by the open reading frame (ORF) 038 gene in LCDV-C, and predicted to contain a putative transmembrane region, multiple N-myristoylation and glycosylation sites and phosphorylation motifs. The expression plasmid of pET-32a-ORF038 was constructed and the recombinant VAP (rVAP) was obtained. Rabbit polyclonal antibodies against the rVAP were prepared and could recognize the rVAP and 32 kDa protein in LCDV. Immunogold electron microscopy showed that the 32 kDa protein was located on the surface of LCDV particles. Immunofluorescence assay demonstrated that the rVAP could bind to the 27.8R on the cell membrane of the FG monolayer and the anti-27.8R MAbs could block the rVAP binding. Pre-incubation of the rVAP with FG cells before LCDV infection, or pre-incubation of LCDV with the antibodies against the rVAP, could significantly decrease the LCDV copy numbers (P<0.05) and delay the emergence of cytopathic effects in FG cells in a dose-dependent manner. These results indicated for the first time that the 32 kDa protein functioned as an attachment protein for the initial attachment and entry of LCDV, and the interaction of the 32 kDa VAP with the 27.8R-initiated LCDV infection.

A role for Heparan Sulfate in Viral Surfing

PubMed Central

Oh, Myung-Jin; Akhtar, Jihan; Desai, Prashant; Shukla, Deepak

2009-01-01

Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here we demonstrate that cells respond to HSV-1 infection by promoting filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum-dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane. PMID:19909728

Ultrastructure of Bacterial Cells Infected with Bacteriophage PM2, a Lipid-containing Bacterial Virus

PubMed Central

Cota-Robles, Eugene; Espejo, Romilio Torres; Haywood, Patricia Williams

1968-01-01

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal and infected cells frequently contain a myelin figure, which is found in the nucleoid region or at the periphery of the cell. The most striking finding in this investigation was that completed virions are found in the cell adjacent to or in association with the cytoplasmic membrane. This localization is precise; virions are not found elsewhere in infected cells. The completed virions occasionally appear to be attached to the cytoplasmic membrane. The virus contains a darkly staining core surrounded by a tripartite envelope of a thickness of approximately 70 A, which is identical to the thickness of the cytoplasmic membrane. Lysing cells appear to undergo extensive damage of the cytoplasmic membrane prior to rupture of the L layer of the cell wall. Images PMID:5742028

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

Virus templated plasmonic nanoclusters with icosahedral symmetry via directed assembly

NASA Astrophysics Data System (ADS)

Ratna, Banahalli; Fontana, Jake; Dressick, Walter; Phelps, Jamie; Johnson, John; Sampson, Travian; Rendell, Ronald; Soto, Carissa

2015-03-01

Controlling the spatial and orientational order of plasmonic nanoparticles may lead to structures with novel electromagnetic properties and applications such as sub-wavelength imaging and ultra-sensitive chemical sensors. Here we report the directed assembly of three-dimensional, icosahedral plasmonic nanoclusters with resonances at visible wavelengths. We show using transmission electron microcopy and in situ dynamic light scattering the nanoclusters consist of twelve gold nanospheres attached to thiol groups at predefined locations on the surface of a genetically engineered cowpea mosaic virus with icosahedral symmetry. We measured the bulk absorbance from aqueous suspensions of nanoclusters and reproduced the major features of the spectrum using finite-element simulations. Furthermore, because the viruses are easily produced in gram quantities the directed assembly approach is capable of high-throughput, providing a strategy to realize large quantities for applications. NRL summer intern under the HBCU/MI Summer Research Program.

Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site.

PubMed

Wang, Xiangxi; Zhu, Ling; Dang, Minghao; Hu, Zhongyu; Gao, Qiang; Yuan, Shuai; Sun, Yao; Zhang, Bo; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Wang, Junzhi; Fry, Elizabeth E; Stuart, David I; Rao, Zihe

2017-01-24

Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.

Semen inhibits Zika virus infection of cells and tissues from the anogenital region.

PubMed

Müller, Janis A; Harms, Mirja; Krüger, Franziska; Groß, Rüdiger; Joas, Simone; Hayn, Manuel; Dietz, Andrea N; Lippold, Sina; von Einem, Jens; Schubert, Axel; Michel, Manuela; Mayer, Benjamin; Cortese, Mirko; Jang, Karen S; Sandi-Monroy, Nathallie; Deniz, Miriam; Ebner, Florian; Vapalahti, Olli; Otto, Markus; Bartenschlager, Ralf; Herbeuval, Jean-Philippe; Schmidt-Chanasit, Jonas; Roan, Nadia R; Münch, Jan

2018-06-07

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.

Light-activated nanotube-porphyrin conjugates as effective antiviral agents

NASA Astrophysics Data System (ADS)

Banerjee, Indrani; Douaisi, Marc P.; Mondal, Dhananjoy; Kane, Ravi S.

2012-03-01

Porphyrins have been used for photodynamic therapy (PDT) against a wide range of targets like bacteria, viruses and tumor cells. In this work, we report porphyrin-conjugated multi-walled carbon nanotubes (NT-P) as potent antiviral agents. Specifically, we used Protoporphyrin IX (PPIX), which we attached to acid-functionalized multi-walled carbon nanotubes (MWNTs). We decided to use carbon nanotubes as scaffolds because of their ease of recovery from a solution through filtration. In the presence of visible light, NT-P was found to significantly reduce the ability of Influenza A virus to infect mammalian cells. NT-P may be used effectively against influenza viruses with little or no chance of them developing resistance to the treatment. Furthermore, NT-P can be easily recovered through filtration which offers a facile strategy to reuse the active porphyrin moiety to its fullest extent. Thus NT-P conjugates represent a new approach for preparing ex vivo reusable antiviral agents.

Viral entry pathways: the example of common cold viruses.

PubMed

Blaas, Dieter

2016-05-01

For infection, viruses deliver their genomes into the host cell. These nucleic acids are usually tightly packed within the viral capsid, which, in turn, is often further enveloped within a lipid membrane. Both protect them against the hostile environment. Proteins and/or lipids on the viral particle promote attachment to the cell surface and internalization. They are likewise often involved in release of the genome inside the cell for its use as a blueprint for production of new viruses. In the following, I shall cursorily discuss the early more general steps of viral infection that include receptor recognition, uptake into the cell, and uncoating of the viral genome. The later sections will concentrate on human rhinoviruses, the main cause of the common cold, with respect to the above processes. Much of what is known on the underlying mechanisms has been worked out by Renate Fuchs at the Medical University of Vienna.

Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

PubMed

Witting, S R; Vallanda, P; Gamble, A L

2013-10-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

The role of fusion activity of influenza A viruses in their biological properties.

PubMed

Jakubcová, L; Hollý, J; Varečková, E

2016-06-01

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

Zika virus - emergence, evolution, pathology, diagnosis, and control: current global scenario and future perspectives - a comprehensive review.

PubMed

Singh, Raj Kumar; Dhama, Kuldeep; Malik, Yashpal Singh; Ramakrishnan, Muthannan Andavar; Karthik, Kumaragurubaran; Tiwari, Ruchi; Saurabh, Sharad; Sachan, Swati; Joshi, Sunil Kumar

2016-09-01

This review converses the Zika virus which has attained global concern due to its rapid pandemic potential and impact on humans. Though Zika virus was first isolated in 1947, till the recent large-scale outbreak which occurred in Micronesia, in 2007, the virus was placed into the innocuous pathogen category. The World Health Organization on 1 February 2016 declared it as a 'Public Health Emergency of International Concern.' Of the note, American as well as Pacific Island strains/isolates is relatively closer to Asian lineage strains. The African and American strains share more than 87.5% and 95% homologies with Asian strains/isolates, respectively. Asian strains form independent clusters, except those isolated from China, suggesting relatively more diversity than African strains. Prevention and control are mainly aimed at the vector population (mosquitoes) with Aedes aegypti being the main species. Surveys in Africa and Asia indicated seropositivity in various animal species. However, so far its natural reservoir is unknown. There is an urgent need to understand why Zika virus has shifted from being a virus that caused mild illness to unforeseen birth defects as well as autoimmune-neurological problems. Unfortunately, an effective vaccine is not available yet. Availability of cryo-electron microscopy based on 3.8 Å resolution revealing mature Zika virus structure and the probable virus attachment site to host cell would provide critical insights into the development of antiviral treatments and vaccines.

In vitro antiherpetic and antirotaviral activities of a sulfate prepared from Mimosa scabrella galactomannan.

PubMed

Chrestani, Francielli; Sierakowski, Maria Rita; de Andrade Uchoa, Daniel Esdras; Nozawa, Carlos; Sassaki, Guilherme Lanzi; Gorin, Philip Albert James; Ono, Lucy

2009-12-01

A chemically sulfated galactomannan (BRS) from seeds of Mimosa scabrella had in vitro antiviral activity against Herpes simplex virus 1 (HSV-1), but not against Simian rotavirus A/SA11 (SiRV-A/SA11). It was examined by (13)C NMR spectroscopy, which showed the sulfate groups to be mainly at C-6 of galactose residues. BRS had a selective inhibition against HSV-1 during its attachment step, having an IC(50) lower than 2.5microg/ml, determined by plaque reduction, and a selectivity index of greater than 181, suggesting that the antiviral effect is likely due to interactions between the virus and BRS, being influenced its overall surface charge.

Infection of cells by Sindbis virus at low temperature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Gongbo; Hernandez, Raquel; Weninger, Keith

2007-06-05

Sindbis virus, which belongs to the family Togaviridae genus Alphavirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for Alphaviruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This virus-host membrane fusion model, well established by influenza virus, has been applied to other unrelated membrane-containing viruses including Alphaviruses. The other mechanism proposes direct penetration of the cell plasma membrane by the virus glycoproteins in the absence of membrane fusion. This alternate modelmore » is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311-3320] studies. We have examined the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis virus infects cells in a temperature- and time-dependent fashion.« less

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

PubMed

Iskarpatyoti, Jason A; Morse, E Ashley; McClung, R Paul; Ikizler, Miné; Wetzel, J Denise; Contractor, Nikhat; Dermody, Terence S

2012-11-25

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk. Copyright © 2012 Elsevier Inc. All rights reserved.

Envelope Protein Dynamics in Paramyxovirus Entry

PubMed Central

Plattet, Philippe; Plemper, Richard K.

2013-01-01

ABSTRACT Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics. PMID:23820396

Envelope protein dynamics in paramyxovirus entry.

PubMed

Plattet, Philippe; Plemper, Richard K

2013-07-02

Paramyxoviruses include major pathogens with significant global health and economic impact. This large family of enveloped RNA viruses infects cells by employing two surface glycoproteins that tightly cooperate to fuse their lipid envelopes with the target cell plasma membrane, an attachment and a fusion (F) protein. Membrane fusion is believed to depend on receptor-induced conformational changes within the attachment protein that lead to the activation and subsequent refolding of F. While structural and mechanistic studies have considerably advanced our insight into paramyxovirus cell adhesion and the structural basis of F refolding, how precisely the attachment protein links receptor engagement to F triggering remained poorly understood. Recent reports based on work with several paramyxovirus family members have transformed our understanding of the triggering mechanism of the membrane fusion machinery. Here, we review these recent findings, which (i) offer a broader mechanistic understanding of the paramyxovirus cell entry system, (ii) illuminate key similarities and differences between entry strategies of different paramyxovirus family members, and (iii) suggest new strategies for the development of novel therapeutics.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV

PubMed Central

Luytjes, Willem; Leenhouts, Kees; Rottier, Peter J. M.; van Kuppeveld, Frank J. M.; Haijema, Bert Jan

2016-01-01

ABSTRACT Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations. PMID:27099320

Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

NASA Astrophysics Data System (ADS)

Szuchmacher Blum, Amy; Soto, Carissa M.; Wilson, Charmaine D.; Whitley, Jessica L.; Moore, Martin H.; Sapsford, Kim E.; Lin, Tianwei; Chatterji, Anju; Johnson, John E.; Ratna, Banahalli R.

2006-10-01

Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30 nm viral particle. These quantum dot protein assemblies were studied in detail. The IgG QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV QD complexes have a local concentration of quantum dots greater than 3000 nmol ml-1, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

Topical application of polyethylenimine as a candidate for novel prophylactic therapeutics against genital herpes caused by herpes simplex virus.

PubMed

Hayashi, Kyoko; Onoue, Hiroki; Sasaki, Kohei; Lee, Jung-Bum; Kumar, Penmetcha K R; Gopinath, Subash C B; Maitani, Yoshie; Kai, Takashi; Hayashi, Toshimitsu

2014-03-01

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012(®), a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

PubMed

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

Measles Virus Fusion Protein: Structure, Function and Inhibition

PubMed Central

Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C.

2016-01-01

Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

Mechanism of chlorine inactivation of DNA-containing parvovirus H-1.

PubMed Central

Churn, C C; Bates, R C; Boardman, G D

1983-01-01

An investigation was undertaken to determine the effect of chlorine on a small DNA-containing enteric virus. Parvovirus H-1 was exposed to sodium hypochlorite in a phosphate-buffered saline solution at pH 7. Then, the whole virion, the protein capsid, or the nucleic acid was subjected to analysis. The sedimentation rate of the chlorine-treated whole virus decreased from 110S to 43S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virus demonstrated the formation of higher-molecular-weight aggregates resulting from covalent cross-linking of the capsid proteins. Electron microscopic examination revealed that the DNA was extruded as a taillike structure which remained attached to the virus particle. Furthermore, the DNA was intact and still capable of in vitro replication. The adsorption of the chlorine-treated virions to host cells was inhibited, presumably due to the effect of chlorine on the particular spatial arrangement of the capsid proteins required for adsorption. Specific sites on these proteins had become highly reactive, indicating that the initial action of chlorine on parvovirus H-1 was on the viral capsid. Images PMID:6660876

Measles Virus Fusion Protein: Structure, Function and Inhibition.

PubMed

Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C

2016-04-21

Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options.

Study of the Ability of Bifidobacteria of Human Origin to Prevent and Treat Rotavirus Infection Using Colonic Cell and Mouse Models

PubMed Central

Darveau, André; Fliss, Ismaïl

2016-01-01

Rotavirus is the leading cause of severe acute gastroenteritis among children worldwide. Despite effective vaccines, inexpensive alternatives such as probiotics are needed. The aim of this study was to assess the ability of probiotic candidate Bifidobacterium thermophilum RBL67 to inhibit rotavirus infection. Bacterial adhesion to intestinal cells and interference with viral attachment were evaluated in vitro. B. thermophilum RBL67 displayed adhesion indexes of 625 ± 84 and 1958 ± 318 on Caco-2 and HT-29 cells respectively and was comparable or superior to four other bifidobacteria, including B. longum ATCC 15707 and B. pseudolongum ATCC 25526 strains. Incubation of B. thermophilum RBL67 for 30 min before (exclusion) and simultaneously (competition) with human rotavirus strain Wa decreased virus attachment by 2.0 ± 0.1 and 1.5 ± 0.1 log10 (by 99.0% and 96.8% respectively). Displacement of virus already present was negligible. In CD-1 suckling mice fed B. thermophilum RBL67 challenged with simian rotavirus SA-11, pre-infection feeding with RBL 67 was more effective than post-infection feeding, reducing the duration of diarrhea, limiting epithelial lesions, reducing viral replication in the intestine, accelerating recovery, and stimulating the humoral specific IgG and IgM response, without inducing any adverse effect. B. thermophilum RBL67 had little effect on intestinal IgA titer. These results suggest that humoral immunoglobulin might provide protection against the virus and that B. thermophilum RBL67 has potential as a probiotic able to inhibit rotavirus infection and ultimately reduce its spread. PMID:27727323

Expanding the role of 3-O sulfated heparan sulfate in herpes simplex virus type-1 entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Donnell, Christopher D., E-mail: codonn3@uic.ed; Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612; Kovacs, Maria, E-mail: marcsika101@yahoo.co

2010-02-20

Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed,more » isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.« less

Characteristics of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion in experimentally infected cattle.

PubMed

Fowler, Veronica; Bashiruddin, John B; Belsham, Graham J; Stenfeldt, Carolina; Bøtner, Anette; Knowles, Nick J; Bankowski, Bartlomiej; Parida, Satya; Barnett, Paul

2014-02-21

Previous work in cattle illustrated the protective efficacy and negative marker potential of a A serotype foot-and-mouth disease virus (FMDV) vaccine prepared from a virus lacking a significant portion of the VP1 G-H loop (termed A(-)). Since this deletion also includes the arginine-glycine-aspartate (RGD) motif required for virus attachment to the host cell in vivo, it was hypothesised that this virus would be attentuated in naturally susceptible animals. The A(-) virus was passaged three times in cattle via needle inoculation of virus suspension delivered into the intradermal space of the tongue (intradermolingual: IDL). Included in the study were three direct contact cattle, two of which were used for the third cattle passage (by inoculation) after direct contact exposure for three days. Cattle were monitored for clinical signs and samples were collected for sequencing as well as antibody and viral genome detection by ELISA and qRT-PCR. Following needle inoculation with the A(-) virus, naïve cattle developed typical clinical signs of FMDV infection, diagnostic assays also provided positive serological and virological results. However, the contact cattle did not develop clinical signs or generate serological or virological markers indicative of FMDV infection even when the cattle were subsequently needle inoculated with 10(5) TCID50 A(-) FMDV delivered IDL following three days of direct contact exposure. The results suggest that the A(-) virus is not attentuated in cattle when inoculated IDL. This virus could be useful as a tool to understand further the natural pathogenesis, receptor usage and internalisation pathways of FMDV. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production

PubMed Central

Essaidi-Laziosi, Manel; Shevtsova, Anastasia; Gerlier, Denis; Roux, Laurent

2013-01-01

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface. PMID:24339863

In Vitro inhibitory activity of Alpinia katsumadai extracts against influenza virus infection and hemagglutination

PubMed Central

2010-01-01

Background Alpinia katsumadai (AK) extracts and fractions were tested for in vitro antiviral activities against influenza virus type A, specially human A/PR/8/34 (H1N1) and avian A/Chicken/Korea/MS96/96 (H9N2), by means of time-of-addition experiments; pre-treatment, simultaneous treatment, and post treatment. Results In pre-treatment assay, the AK extracts and AK fractions did not show significant antiviral activity. During the simultaneous treatment assay, one AK extract and five AK fractions designated as AK-1 to AK-3, AK-5, AK-10, and AK-11 showed complete inhibition of virus infectivity against A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). The 50% effective inhibitory concentrations (EC50) of these one AK extracts and five AK fractions with exception of the AK-9 were from 0.8 ± 1.4 to 16.4 ± 4.5 μg/mL against A/PR/8/34 (H1N1). The two AK extracts and three AK fractions had EC50 values ranging from <0.39 ± 0.4 to 2.3 ± 3.6 μg/mL against A/Chicken/Korea/MS96/96 (H9N2). By the hemagglutination inhibition (HI) assay, the two AK extracts and five AK fractions completely inhibited viral adsorption onto chicken RBCs at less than 100 μg/mL against both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). Interestingly, only AK-3 was found with inhibition for both viral attachment and viral replication after showing extended antiviral activity during the post treatment assay and quantitative real-time PCR. Conclusions These results suggest that AK extracts and fractions had strong anti-influenza virus activity that can inhibit viral attachment and/or viral replication, and may be used as viral prophylaxis. PMID:21062499

Genogroup IV and VI Canine Noroviruses Interact with Histo-Blood Group Antigens

PubMed Central

Breiman, Adrien; le Pendu, Jacques

2014-01-01

ABSTRACT Human noroviruses (HuNV) are a significant cause of viral gastroenteritis in humans worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism among human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I (GI), II, and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI to GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV and GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV and to compare it to the binding specificities of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs) and identified α1,2-fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry confirmed that α1,2-fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs, and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into the evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans. IMPORTANCE Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect nonhuman species and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alpha-galactosidase (α-Gal) carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood group antigens. The binding specificity of canine noroviruses is therefore very similar to that of the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses and suggests it may be possible for canine norovirus to infect humans. PMID:25008923

Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis.

PubMed

Mandl, Christian W

2005-08-01

Tick-borne encephalitis virus (TBEV) is an important human pathogen that causes severe neurological illness in large areas of Europe and Asia. The neuropathogenesis of this disease agent is determined by its capacity to enter the central nervous system (CNS) after peripheral inoculation ("neuroinvasiveness") and its ability to replicate and cause damage within the CNS ("neurovirulence"). TBEV is a small, enveloped flavivirus with an unsegmented, positive-stranded RNA genome. Mutations affecting various steps of its natural replication cycle were shown to influence its neuropathogenic properties. This review describes experimental approaches and summarizes results on molecular determinants of neurovirulence and neuroinvasiveness that have been identified for this virus. It focuses on molecular mechanisms of three particular steps of the viral life cycle that have been studied in some detail for TBEV and two closely related tick-borne flaviviruses (Louping ill virus (LIV) and Langat virus (LGTV)), namely (i) the envelope protein E and its role in viral attachment to the cell surface, (ii) the 3'-noncoding region of the genome and its importance for viral RNA replication, and (iii) the capsid protein C and its role in the assembly process of infectious virus particles. Mutations affecting each of these three molecular targets significantly influence neuropathogenesis of TBEV, particularly its neuroinvasiveness. The understanding of molecular determinants of TBEV neuropathogenesis is relevant for vaccine development, also against other flaviviruses.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragmentmore » recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.« less

Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

DOE PAGES

Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie; ...

2015-04-07

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragmentmore » recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.« less

Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus

PubMed Central

Kim, Young-Il; Pascua, Philippe Noriel Q; Kwon, Hyeok-Il; Lim, Gyo-Jin; Kim, Eun-Ha; Yoon, Sun-Woo; Park, Su-Jin; Kim, Se Mi; Choi, Eun-Ji; Si, Young-Jae; Lee, Ok-Jun; Shim, Woo-Sub; Kim, Si-Wook; Mo, In-Pil; Bae, Yeonji; Lim, Yong Taik; Sung, Moon Hee; Kim, Chul-Joong; Webby, Richard J; Webster, Robert G; Choi, Young Ki

2014-01-01

The endemicity of highly pathogenic avian influenza (HPAI) A(H5N1) viruses in Asia has led to the generation of reassortant H5 strains with novel gene constellations. A newly emerged HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in 2014. Because newly emerging high-pathogenicity H5 viruses continue to pose public health risks, it is imperative that their pathobiological properties be examined. Here, we characterized A/mallard duck/Korea/W452/2014 (MDk/W452(H5N8)), a representative virus, and evaluated its pathogenic and pandemic potential in various animal models. We found that MDk/W452(H5N8), which originated from the reassortment of wild bird viruses harbored by migratory waterfowl in eastern China, replicated systemically and was lethal in chickens, but appeared to be attenuated, albeit efficiently transmitted, in ducks. Despite predominant attachment to avian-like virus receptors, MDk/W452(H5N8) also exhibited detectable human virus-like receptor binding and replicated in human respiratory tract tissues. In mice, MDk/W452(H5N8) was moderately pathogenic and had limited tissue tropism relative to previous HPAI A(H5N1) viruses. It also induced moderate nasal wash titers in inoculated ferrets; additionally, it was recovered in extrapulmonary tissues and one of three direct-contact ferrets seroconverted without shedding. Moreover, domesticated cats appeared to be more susceptible than dogs to virus infection. With their potential to become established in ducks, continued circulation of A(H5N8) viruses could alter the genetic evolution of pre-existing avian poultry strains. Overall, detailed virological investigation remains a necessity given the capacity of H5 viruses to evolve to cause human illness with few changes in the viral genome. PMID:26038499

Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus.

PubMed

Kim, Young-Il; Pascua, Philippe Noriel Q; Kwon, Hyeok-Il; Lim, Gyo-Jin; Kim, Eun-Ha; Yoon, Sun-Woo; Park, Su-Jin; Kim, Se Mi; Choi, Eun-Ji; Si, Young-Jae; Lee, Ok-Jun; Shim, Woo-Sub; Kim, Si-Wook; Mo, In-Pil; Bae, Yeonji; Lim, Yong Taik; Sung, Moon Hee; Kim, Chul-Joong; Webby, Richard J; Webster, Robert G; Choi, Young Ki

2014-10-01

The endemicity of highly pathogenic avian influenza (HPAI) A(H5N1) viruses in Asia has led to the generation of reassortant H5 strains with novel gene constellations. A newly emerged HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in 2014. Because newly emerging high-pathogenicity H5 viruses continue to pose public health risks, it is imperative that their pathobiological properties be examined. Here, we characterized A/mallard duck/Korea/W452/2014 (MDk/W452(H5N8)), a representative virus, and evaluated its pathogenic and pandemic potential in various animal models. We found that MDk/W452(H5N8), which originated from the reassortment of wild bird viruses harbored by migratory waterfowl in eastern China, replicated systemically and was lethal in chickens, but appeared to be attenuated, albeit efficiently transmitted, in ducks. Despite predominant attachment to avian-like virus receptors, MDk/W452(H5N8) also exhibited detectable human virus-like receptor binding and replicated in human respiratory tract tissues. In mice, MDk/W452(H5N8) was moderately pathogenic and had limited tissue tropism relative to previous HPAI A(H5N1) viruses. It also induced moderate nasal wash titers in inoculated ferrets; additionally, it was recovered in extrapulmonary tissues and one of three direct-contact ferrets seroconverted without shedding. Moreover, domesticated cats appeared to be more susceptible than dogs to virus infection. With their potential to become established in ducks, continued circulation of A(H5N8) viruses could alter the genetic evolution of pre-existing avian poultry strains. Overall, detailed virological investigation remains a necessity given the capacity of H5 viruses to evolve to cause human illness with few changes in the viral genome.

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody.

PubMed

Willett, Brian J; Kraase, Martin; Logan, Nicola; McMonagle, Elizabeth L; Samman, Ayman; Hosie, Margaret J

2010-04-26

In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

Inhibitory effect of Spirogyra spp. algal extracts against herpes simplex virus type 1 and 2 infection.

PubMed

Deethae, A; Peerapornpisal, Y; Pekkoh, J; Sangthong, P; Tragoolpua, Y

2018-06-01

To determine the antiviral activities of Spirogyra spp. algal extracts against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). Spirogyra spp. was extracted using water, ethanol and methanol. Aqueous extract of Spirogyra spp. had the lowest toxicity on Vero cells with the 50% cytotoxicity concentration (CC 50 ) of 4363·30 μg ml -1 . As for potent inhibitory effect, the ethanolic extract presented the highest inhibition of viral infection on HSV-1 in the treatment during viral attachment on Vero cells with 50% inhibitory concentration (IC 50 ) and selective index (SI) values of 164·20 and 2·17 μg ml -1 . However, the methanolic extract showed the highest inhibition of HSV-2 when treated during viral attachment with IC 50 and SI values of 75·03 and 3·34 μg ml -1 . The methanolic extract of Spirogyra spp. also demonstrated significant virucidal effects on viral particles. Therefore, anti-HSV activity at various stages of the viral multiplication cycle was shown. The main active compounds in the active fractions of Spirogyra spp. ethanolic extract against HSV were found to be alkaloids, essential oils and terpenoids. The highest anti-HSV activity was obtained from the ethanolic extract of Spirogyra spp. The extract inhibited the HSV viral particles and the inhibition was during the viral attachment and the viral multiplication. Anti-HSV activity of extract of freshwater green macroalga Spirogyra spp. in Thailand was demonstrated. Therefore, anti-HSV product containing the Spirogyra spp. extract should be developed for treatment of HSV infection. © 2018 The Society for Applied Microbiology.

Molecular epidemiology of respiratory syncytial virus in The Gambia.

PubMed Central

Cane, P. A.; Weber, M.; Sanneh, M.; Dackour, R.; Pringle, C. R.; Whittle, H.

1999-01-01

Respiratory syncytial virus (RSV) infection in The Gambia occurs seasonally in association with the rainy season. This study examined the genetic variability of RSV isolates from four consecutive epidemics from 1993-6. Each epidemic was made up of a number of variants which were replaced in subsequent epidemics. Analysis of attachment (G) protein gene sequences showed that isolates were closely related to those observed in the rest of the world. However, many isolates from 1993 and 1994 were unlike other isolates observed in the developed world during this period and were more similar to isolates from 1984 in Europe. In addition, the most commonly observed genotype in the UK in the 1990s was not detected in The Gambia during this period. PMID:10098799

SJ-3366 Sam Jin Pharmaceutical.

PubMed

Baba, Masanori

2002-08-01

Sam Jin is investigating SJ-3366, a non-nucleoside reverse transcriptase inhibitor (NNRTI), for the potential treatment of HIV infection [302450]. As well as acting as an NNRTI, SJ-3366 also interferes with HIV-1 entry via an intermediate target formed after virus-cell attachment [341146], [363900]. As of June 1998, Sam Jin had been awarded a patent for SJ-3366 in South Africa, with applications pending in 22 other countries [302450].

Mechanisms of Virus-Induced Neural Cell Death

DTIC Science & Technology

2005-03-01

Y, et al. Brief CMV, with isolates being resistant to acy- report: severe infectious mononucleosis -like clovir, famciclovir, and valacyclovir, and...protease depen- by 24 h post-infection. The completion of the one-step dent process. Conversion to infectious subvirion particles growth curve precedes...apoptosis increases progres- cell attachment protein o-1 (56). sively at 24-48 h post-infection (92). ISVPs are infectious and can be formed either intra

ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5more » (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).« less

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

PubMed Central

Phan, Shannon I.; Chen, Zhenhai; Xu, Pei; Li, Zhuo; Gao, Xiudan; Foster, Stephanie L.; Teng, Michael N.; Tripp, Ralph A.; Sakamoto, Kaori; He, Biao

2014-01-01

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses - one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection. PMID:24717150

Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies

PubMed Central

Szymańska, Emilia; Orłowski, Piotr; Tomaszewska, Emilia; Bąska, Piotr; Grobelny, Jarosław; Basa, Anna; Krzyżowska, Małgorzata

2018-01-01

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection. PMID:29382085

Antiviral properties of prodelphinidin B-2 3'-O-gallate from green tea leaf.

PubMed

Cheng, Hua-Yew; Lin, Chun-Ching; Lin, Ta-Chen

2002-07-01

Prodelphinidin B-2 3-O-gallate, a proanthocyanidin gallate isolated from green tea leaf, was investigated for its anti-herpes simplex virus type 2 properties in vitro. Prodelphinidin B-2 3'-O-gallate exhibited antiviral activity with IC50 of 5.0 +/-1.0 microM and 1.6 +/-0.3 pM for XTT and plaque reduction (PRA) assays, respectively. Cytotoxicity assay had shown that prodelphinidin B-2 3'-O-gallate possessed cytotoxic effect toward Vero cell at concentration higher than its IC50. The 50% cytotoxic concentration for cell growth (CC50) was 33.3 +/- 3.7 microM. Thus, the selectivity index (SI) (ratio of IC50 to CC50) for XTT assay and PRA was 6.7 and 20.8, respectively. Prodelphinidin B-2 3'-O-gallate significantly reduced viral infectivity at concentrations 10 microM or more. Result of time-of-addition studies suggested that prodelphinidin B-2 3'-O-gallate affected the late stage of HSV-2 infection. In addition, it was also shown to inhibit the virus from attaching and penetrating into the cell. Thus, prodelphinidin B-2 3'-O-gallate was concluded to possess antiviral activity with mechanism of inhibiting viral attachment and penetration, and disturbing the late stage of viral infection.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.

PubMed

Casais, Rosa; Molleda, Lorenzo González; Machín, Angeles; del Barrio, Gloria; Manso, Alberto García; Dalton, Kevin P; Coto, Ana; Alonso, José Manuel Martín; Prieto, Miguel; Parra, Francisco

2008-10-01

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

Synthesis of 3-O-sulfonated heparan sulfate octasaccharides that inhibit the herpes simplex virus type 1 host-cell interaction

NASA Astrophysics Data System (ADS)

Hu, Yu-Peng; Lin, Shu-Yi; Huang, Cheng-Yen; Zulueta, Medel Manuel L.; Liu, Jing-Yuan; Chang, Wen; Hung, Shang-Cheng

2011-07-01

Cell surface carbohydrates play significant roles in a number of biologically important processes. Heparan sulfate, for instance, is a ubiquitously distributed polysulfated polysaccharide that is involved, among other things, in the initial step of herpes simplex virus type 1 (HSV-1) infection. The virus interacts with cell-surface heparan sulfate to facilitate host-cell attachment and entry. 3-O-Sulfonated heparan sulfate has been found to function as an HSV-1 entry receptor. Achieving a complete understanding of these interactions requires the chemical synthesis of such oligosaccharides, but this remains challenging. Here, we present a convenient approach for the synthesis of two irregular 3-O-sulfonated heparan sulfate octasaccharides, making use of a key disaccharide intermediate to acquire different building blocks for the oligosaccharide chain assembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugars blocked viral infection in a dosage-dependent manner with remarkable similarity to one another.

A role for heparan sulfate in viral surfing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Myung-Jin; Akhtar, Jihan; Desai, Prashant

2010-01-01

Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here, we demonstrate that cells respond to HSV-1 infection by enhancing filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacksmore » gB fails to surf and quantum dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.« less

Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae.

PubMed

Zhang, Minze; Veit, Michael

2018-04-01

We reported previously that carbohydrate attachment to an overlapping glycosylation site adjacent to the signal peptide of GP3 from equine arteritis virus (EAV) prevents cleavage. Here we investigated whether this unusual processing scheme is a feature of GP3s of other Arteriviridae, which all contain a glycosylation site at a similar position. Expression of GP3 from type-1 and type-2 porcine reproductive and respiratory syndrome virus (PRRSV) and from lactate dehydrogenase-elevating virus (LDV) revealed that the first glycosylation site is used, but has no effect on signal peptide cleavage. Comparison of the SDS-PAGE mobility of deglycosylated GP3 from PRRSV and LDV with mutants having or not having a signal peptide showed that GP3´s signal peptide is cleaved. Swapping the signal peptides between GP3 of EAV and PRRSV revealed that the information for co-translational processing is not encoded in the signal peptide, but in the remaining part of GP3. Copyright © 2017 Elsevier Inc. All rights reserved.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

PubMed Central

Borges, Andrew Rosa; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

2010-01-01

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3’-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC50s ranging from 0.1 – 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. PMID:20880566

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shang Liang; Hunter, Eric, E-mail: eric.hunter2@emory.ed

2010-09-01

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusionmore » mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.« less

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

PubMed

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

Electron cryo-tomographic structure of cystovirus phi 12.

PubMed

Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul

2008-03-01

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

Virus-like attachment sites as structural landmarks of plants retrotransposons.

PubMed

Ochoa Cruz, Edgar Andres; Cruz, Guilherme Marcello Queiroga; Vieira, Andréia Prata; Van Sluys, Marie-Anne

2016-01-01

The genomic data available nowadays has enabled the study of repetitive sequences and their relationship to viruses. Among them, long terminal repeat retrotransposons (LTR-RTs) are the largest component of most plant genomes, the Gypsy and Copia superfamilies being the most common. Recently it has been found that Del lineage, an LTR-RT of Gypsy superfamily, has putative virus-like attachment (vl-att) sites. This signature, originally described for retroviruses, is recognized by retroviral integrase conferring specificity to the integration process. Here we retrieved 26,092 putative complete LTR-RTs from 10 lineages found in 10 fully sequenced angiosperm genomes and found putative vl-att sites that are a conserved structural landmark across these genomes. Furthermore, we reveal that each plant genome has a distinguishable LTR-RT lineage amplification pattern that could be related to the vl-att sites diversity. We used these patterns to generate a specific quick-response (QR) code for each genome that could be used as a barcode of identification of plants in the future. The universal distribution of vl-att sites represents a new structural feature common to plant LTR-RTs and retroviruses. This is an important finding that expands the information about the structural similarity between LTR-RT and retroviruses. We speculate that the sequence diversity of vl-att sites could be important for the life cycle of retrotransposons, as it was shown for retroviruses. All the structural vl-att site signatures are strong candidates for further functional studies. Moreover, this is the first identification of specific LTR-RT content and their amplification patterns in a large dataset of LTR-RT lineages and angiosperm genomes. These distribution patterns could be used in the future with biotechnological identification purposes.

Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

PubMed

Zhao, Yuguang; Ren, Jingshan; Harlos, Karl; Jones, Daniel M; Zeltina, Antra; Bowden, Thomas A; Padilla-Parra, Sergi; Fry, Elizabeth E; Stuart, David I

2016-07-07

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

PubMed

Organtini, Lindsey J; Shingler, Kristin L; Ashley, Robert E; Capaldi, Elizabeth A; Durrani, Kulsoom; Dryden, Kelly A; Makhov, Alexander M; Conway, James F; Pizzorno, Marie C; Hafenstein, Susan

2017-01-15

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV. Copyright © 2017 American Society for Microbiology.

Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets

PubMed Central

Sun, Honglei; Pu, Juan; Wei, Yandi; Sun, Yipeng; Hu, Jiao; Liu, Litao; Xu, Guanlong; Gao, Weihua; Li, Chong; Zhang, Xuxiao; Huang, Yinhua; Chang, Kin-Chow; Liu, Xiufan

2016-01-01

ABSTRACT Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored. PMID:27122581

Feasibility studies of oncornavirus production in microcarrier cultures.

PubMed

Manousos, M; Ahmed, M; Torchio, C; Wolff, J; Shibley, G; Stephens, R; Mayyasi, S

1980-06-01

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction

PubMed Central

Witting, Scott R.; Vallanda, Priya; Gamble, Aisha L.

2013-01-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells. PMID:23698741

Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

PubMed

Xu, Jinfang; Qian, Ping; Wu, Qunfeng; Liu, Shasha; Fan, Wenchun; Zhang, Keshan; Wang, Rong; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

2014-09-01

The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

PubMed Central

Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

2010-01-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosa Borges, Andrew; Wieczorek, Lindsay; Johnson, Benitra

2010-12-05

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC{sub 50}s ranging from 0.1 to 7.4 {mu}g/ml. Inhibition of Env-mediated membrane fusion by MVC wasmore » also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. -- Research Highlights: {yields}Multivalent carbohydrates (MVCs) inhibited infection of PBMCs by HIV-1. {yields}MVCs inhibited infection by T cell line-adapted viruses. {yields}MVCs inhibited infection by primary isolates of HIV-1. {yields}MVCs inhibited Env-mediated membrane fusion.« less

An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato.

PubMed

Albert, Markus; Belastegui-Macadam, Xana; Kaldenhoff, Ralf

2006-11-01

Dodder or Cuscutaceae are holoparasitic plants subsisting on other dicotyledonous plants. The infection process is initiated by adherence of Cuscuta prehaustoria to the host surface, followed by penetration attempts by hyphae. In the case of a successful infection, these organs connect the parasite's vascular tissue to that of the host. Here we show that contact of Cuscuta reflexa prehaustoria to tomato induces the expression of a new arabinogalactan protein (AGP), attAGP, in the tomato precisely at the site of dodder attack. We show that attAGP is a plasma membrane-bound cell wall-localized protein. Using the RNAi technique and attAGP-targeted virus-induced gene silencing, we observed a correlation between attAGP expression level and force of attachment of the parasite to host tomatoes. If the expression level of attAGP was reduced, the C. reflexa attachment capability was significantly reduced, too. We conclude that C. reflexa infection induced a signal in the host leading to expression of tomato attAGP, which promotes the parasite's adherence.

Suppressing Brownian motion of individual biomolecules in solution

PubMed Central

Cohen, Adam E.; Moerner, W. E.

2006-01-01

Single biomolecules in free solution have long been of interest for detailed study by optical methods, but Brownian motion prevents the observation of one single molecule for extended periods. We have used an anti-Brownian electrokinetic (ABEL) trap to trap individual protein molecules in free solution, under ambient conditions, without requiring any attachment to beads or surfaces. We also demonstrate trapping and manipulation of single virus particles, lipid vesicles, and fluorescent semiconductor nanocrystals. PMID:16537418

The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9).

PubMed

Chiu, Mei-Wui; Shih, Hsiu-Ming; Yang, Tsung-Han; Yang, Yun-Liang

2007-05-01

Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based "Functional Yeast Array". Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.

Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development

PubMed Central

Uldahl, Kristine B.; Jensen, Signe B.; Bhoobalan-Chitty, Yuvaraj; Martínez-Álvarez, Laura; Papathanasiou, Pavlos

2016-01-01

ABSTRACT We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. IMPORTANCE Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea. This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field. PMID:27053548

Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development.

PubMed

Uldahl, Kristine B; Jensen, Signe B; Bhoobalan-Chitty, Yuvaraj; Martínez-Álvarez, Laura; Papathanasiou, Pavlos; Peng, Xu

2016-06-15

We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A 2,5-Dihydroxybenzoic Acid–Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses

PubMed Central

Lisov, Alexander; Vrublevskaya, Veronika; Lisova, Zoy; Leontievsky, Alexey; Morenkov, Oleg

2015-01-01

Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections. PMID:26501311

A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierson, Theodore C.; Sanchez, Melissa D.; Puffer, Bridget A.

2006-03-01

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) aremore » produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.« less

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.

E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adoptedmore » a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.« less

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles.

PubMed

Martinez, Osvaldo; Tantral, Lee; Mulherkar, Nirupama; Chandran, Kartik; Basler, Christopher F

2011-11-01

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

PubMed Central

Reh, Lucia; Magnus, Carsten; Schanz, Merle; Weber, Jacqueline; Uhr, Therese; Rusert, Peter; Trkola, Alexandra

2015-01-01

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application. PMID:26158270

Targeting RSV with Vaccines and Small Molecule Drugs

PubMed Central

Costello, Heather M.; Ray, William C.; Chaiwatpongsakorn, Supranee; Peeples, Mark E.

2012-01-01

Respiratory syncytial virus (RSV) is the most significant cause of pediatric respiratory infections. Palivizumab (Synagis®), a humanized monoclonal antibody, has been used successfully for a number of years to prevent severe RSV disease in at-risk infants. However, despite intense efforts, there is no approved vaccine or small molecule drug for RSV. As an enveloped virus, RSV must fuse its envelope with the host cell membrane, which is accomplished through the actions of the fusion (F) glycoprotein, with attachment help from the G glycoprotein. Because of their integral role in initiation of infection and their accessibility outside the lipid bilayer, these proteins have been popular targets in the discovery and development of antiviral compounds and vaccines against RSV. This review examines advances in the development of antiviral compounds and vaccine candidates. PMID:22335496

Microgravity

NASA Image and Video Library

2004-04-15

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

The Role of the Hendra Virus and Nipah Virus Attachment Glycoproteins in Receptor Binding and Antibody Neutralization

DTIC Science & Technology

2014-01-31

portions of the NiV-sG sequence on the 5’ ends (c: 5’- CGG AAG CTG ATG AAG CAG ATC GAG GAC-3’ , d: 5’-CTG GTG TAC TT CTT GAT CCT GGC CAG-3’). The leader...template-pcDNA-GCN(tet)-HeV-sG, forward primer: 5’- GTG GAG ATC TAC AAC ACC GGC GAC TC-3’ and reverse primer: 5’- GAG TCG CCG GTG TTG TAG ATC TCC AC-3...CCC CGC TCC GTG GCA ATA TTA CTA CTA C YA YAAHPS GAG CAG TAC GCC GCC CAT CCG TCC C F/L/W FAPHLW G TTC GCC CCC CAT CTG TGG CAA TAT TAC TAC TAC

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

PubMed Central

Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

2015-01-01

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID:26136573

Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt

PubMed Central

Watanabe, Yohei; Ibrahim, Madiha S.; Ellakany, Hany F.; Kawashita, Norihito; Mizuike, Rika; Hiramatsu, Hiroaki; Sriwilaijaroen, Nogluk; Takagi, Tatsuya; Suzuki, Yasuo; Ikuta, Kazuyoshi

2011-01-01

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential. PMID:21637809

Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses*

PubMed Central

Komarova, Anastassia V.; Combredet, Chantal; Meyniel-Schicklin, Laurène; Chapelle, Manuel; Caignard, Grégory; Camadro, Jean-Michel; Lotteau, Vincent; Vidalain, Pierre-Olivier; Tangy, Frédéric

2011-01-01

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells. PMID:21911578

Adenovirus receptors and their implications in gene delivery

PubMed Central

Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

2010-01-01

Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release

PubMed Central

Spiegel, Martin; Plegge, Teresa; Pöhlmann, Stefan

2016-01-01

Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle. PMID:27455305

Characterizing Bacteriophage PR772 as a Potential Surrogate for Adenovirus in Water Disinfection: A Comparative Analysis of Inactivation Kinetics and Replication Cycle Inhibition by Free Chlorine.

PubMed

Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

2016-03-01

Elucidating mechanisms by which pathogenic waterborne viruses become inactivated by drinking water disinfectants would facilitate the development of sensors to detect infectious viruses and novel disinfection strategies to provide safe water. Using bacteriophages as surrogates for human pathogenic viruses could assist in elucidating these mechanisms; however, an appropriate viral surrogate for human adenovirus (HAdV), a medium-sized virus with a double-stranded DNA genome, needs to be identified. Here, we characterized the inactivation kinetics of bacteriophage PR772, a member of the Tectiviridae family with many similarities in structure and replication to HAdV. The inactivation of PR772 and HAdV by free chlorine had similar kinetics that could be represented with a model previously developed for HAdV type 2 (HAdV-2). We developed and tested a quantitative assay to analyze several steps in the PR772 replication cycle to determine if both viruses being inactivated at similar rates resulted from similar replication cycle events being inhibited. Like HAdV-2, we observed that PR772 inactivated by free chlorine still attached to host cells, and viral DNA synthesis and early and late gene transcription were inhibited. Consequently, free chlorine exposure inhibited a replication cycle event that was post-binding but took place prior to early gene synthesis for both PR772 and HAdV-2.

VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

PubMed

Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

1997-07-01

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

C-type lectins do not act as functional receptors for filovirus entry into cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu

2010-12-03

Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps ofmore » virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.« less

Measles virus fusion machinery activated by sialic acid binding globular domain.

PubMed

Talekar, Aparna; Moscona, Anne; Porotto, Matteo

2013-12-01

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.

PubMed

Stone, Jacquelyn A; Nicola, Anthony V; Baum, Linda G; Aguilar, Hector C

2016-02-01

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.

Molecular analysis of Crimean-Congo hemorrhagic fever virus and Rickettsia in Hyalomma marginatum ticks removed from patients (Spain) and birds (Spain and Morocco), 2009-2015.

PubMed

Palomar, Ana M; Portillo, Aránzazu; Mazuelas, David; Roncero, Lidia; Arizaga, Juan; Crespo, Ariñe; Gutiérrez, Óscar; Márquez, Francisco J; Cuadrado, Juan F; Eiros, José M; Oteo, José A

2016-07-01

Crimean-Congo hemorrhagic fever virus (CCHFV) was detected in Spain in 2010. The presence of CCHFV in Hyalomma marginatum ticks from migratory birds passing through Morocco during the spring migration strengthened the hypothesis of the arrival of infected ticks transported by birds to the Iberian Peninsula. Furthermore, Hyalomma species are vectors of bacterial infections such as spotted fever rickettsioses. CCHFV and Rickettsia were screened in Hyalomma ticks from Spain attached to patients (n=12) and birds (n=149). In addition, Rickettsia was investigated in 52 Hyalomma ticks from Morocco (previously reported as CCHFV-infected). No sample collected in Spain showed an infection with CCHFV. Two ticks removed from patients (16.7%), as well as 47 (31.5%) and 4 (7.7%) from birds, collected in Spain and Morocco respectively, were infected with Rickettsia aeschlimannii. Rickettsia sibirica subsp. mongolitimonae was also found in 2 ticks from birds collected in Spain (1.3%). The risk of CCHFV-infected ticks attached to migratory birds to reach the North of Spain is low. This study corroborates the presence of R. aeschlimannii in Spain and Morocco, and supports that H. marginatum can be a potential vector of R. sibirica subsp. mongolitimonae in the Iberian Peninsula. Copyright © 2016 Elsevier GmbH. All rights reserved.

Transmission-blocking antibodies against mosquito C-type lectins for dengue prevention.

PubMed

Liu, Yang; Zhang, Fuchun; Liu, Jianying; Xiao, Xiaoping; Zhang, Siyin; Qin, Chengfeng; Xiang, Ye; Wang, Penghua; Cheng, Gong

2014-02-01

C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

Differential MS2 Interaction with Food Contact Surfaces Determined by Atomic Force Microscopy and Virus Recovery.

PubMed

Shim, J; Stewart, D S; Nikolov, A D; Wasan, D T; Wang, R; Yan, R; Shieh, Y C

2017-12-15

Enteric viruses are recognized as major etiologies of U.S. foodborne infections. These viruses are easily transmitted via food contact surfaces. Understanding virus interactions with surfaces may facilitate the development of improved means for their removal, thus reducing transmission. Using MS2 coliphage as a virus surrogate, the strength of virus adhesion to common food processing and preparation surfaces of polyvinyl chloride (PVC) and glass was assessed by atomic force microscopy (AFM) and virus recovery assays. The interaction forces of MS2 with various surfaces were measured from adhesion peaks in force-distance curves registered using a spherical bead probe preconjugated with MS2 particles. MS2 in phosphate-buffered saline (PBS) demonstrated approximately 5 times less adhesion force to glass (0.54 nN) than to PVC (2.87 nN) ( P < 0.0001). This was consistent with the virus recovery data, which showed 1.4-fold fewer virus PFU recovered from PVC than from glass after identical inoculations and 24 h of cold storage. The difference in adhesion was ascribed to both intrinsic chemical characteristics and the substrate surface porosity (smooth glass versus porous PVC). Incorporating a surfactant micellar solution of sodium dodecyl sulfate (SDS) into the PBS reduced the adhesion force for PVC (∼0 nN) and consistently increased virus recovery by 19%. With direct and indirect evidence of virus adhesion, this study illustrated a two-way assessment of virus adhesion for the initial evaluation of potential means to mitigate virus adhesion to food contact surfaces. IMPORTANCE The spread of foodborne viruses is likely associated with their adhesive nature. Virus attachment on food contact surfaces has been evaluated by quantitating virus recoveries from inoculated surfaces. This study aimed to evaluate the microenvironment in which nanometer-sized viruses interact with food contact surfaces and to compare the virus adhesion differences using AFM. The virus surrogate MS2 demonstrated less adhesion force to glass than to PVC via AFM, with the force-contributing factors including the intrinsic nature and the topography of the contact surfaces. This adhesion finding is consistent with the virus recoveries, which were determined indirectly. Greater numbers of viruses were recovered from glass than from PVC, after application at the same levels. The stronger MS2 adhesion onto PVC could be interrupted by incorporating a surfactant during the interaction between the virus and the contact surface. This study increases our understanding of the virus adhesion microenvironment and indicates ways to mitigate virus adhesion onto contact surfaces. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Multiple roles of genome-attached bacteriophage terminal proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redrejo-Rodríguez, Modesto; Salas, Margarita, E-mail: msalas@cbm.csic.es

2014-11-15

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid.more » Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.« less

Genomic characterization and phylogenetic position of two new species in Rhabdoviridae infecting the parasitic copepod, salmon louse (Lepeophtheirus salmonis).

PubMed

Økland, Arnfinn Lodden; Nylund, Are; Øvergård, Aina-Cathrine; Blindheim, Steffen; Watanabe, Kuninori; Grotmol, Sindre; Arnesen, Carl-Erik; Plarre, Heidrun

2014-01-01

Several new viruses have emerged during farming of salmonids in the North Atlantic causing large losses to the industry. Still the blood feeding copepod parasite, Lepeophtheirus salmonis, remains the major challenge for the industry. Histological examinations of this parasite have revealed the presence of several virus-like particles including some with morphologies similar to rhabdoviruses. This study is the first description of the genome and target tissues of two new species of rhabdoviruses associated with pathology in the salmon louse. Salmon lice were collected at different Atlantic salmon (Salmo salar) farming sites on the west coast of Norway and prepared for histology, transmission electron microscopy and Illumina sequencing of the complete RNA extracted from these lice. The nearly complete genomes, around 11,600 nucleotides encoding the five typical rhabdovirus genes N, P, M, G and L, of two new species were obtained. The genome sequences, the putative protein sequences, and predicted transcription strategies for the two viruses are presented. Phylogenetic analyses of the putative N and L proteins indicated closest similarity to the Sigmavirus/Dimarhabdoviruses cluster, however, the genomes of both new viruses are significantly diverged with no close affinity to any of the existing rhabdovirus genera. In situ hybridization, targeting the N protein genes, showed that the viruses were present in the same glandular tissues as the observed rhabdovirus-like particles. Both viruses were present in all developmental stages of the salmon louse, and associated with necrosis of glandular tissues in adult lice. As the two viruses were present in eggs and free-living planktonic stages of the salmon louse vertical, transmission of the viruses are suggested. The tissues of the lice host, Atlantic salmon, with the exception of skin at the attachment site for the salmon louse chalimi stages, were negative for these two viruses.

Genomic Characterization and Phylogenetic Position of Two New Species in Rhabdoviridae Infecting the Parasitic Copepod, Salmon Louse (Lepeophtheirus salmonis)

PubMed Central

Økland, Arnfinn Lodden; Nylund, Are; Øvergård, Aina-Cathrine; Blindheim, Steffen; Watanabe, Kuninori; Grotmol, Sindre; Arnesen, Carl-Erik; Plarre, Heidrun

2014-01-01

Several new viruses have emerged during farming of salmonids in the North Atlantic causing large losses to the industry. Still the blood feeding copepod parasite, Lepeophtheirus salmonis, remains the major challenge for the industry. Histological examinations of this parasite have revealed the presence of several virus-like particles including some with morphologies similar to rhabdoviruses. This study is the first description of the genome and target tissues of two new species of rhabdoviruses associated with pathology in the salmon louse. Salmon lice were collected at different Atlantic salmon (Salmo salar) farming sites on the west coast of Norway and prepared for histology, transmission electron microscopy and Illumina sequencing of the complete RNA extracted from these lice. The nearly complete genomes, around 11 600 nucleotides encoding the five typical rhabdovirus genes N, P, M, G and L, of two new species were obtained. The genome sequences, the putative protein sequences, and predicted transcription strategies for the two viruses are presented. Phylogenetic analyses of the putative N and L proteins indicated closest similarity to the Sigmavirus/Dimarhabdoviruses cluster, however, the genomes of both new viruses are significantly diverged with no close affinity to any of the existing rhabdovirus genera. In situ hybridization, targeting the N protein genes, showed that the viruses were present in the same glandular tissues as the observed rhabdovirus-like particles. Both viruses were present in all developmental stages of the salmon louse, and associated with necrosis of glandular tissues in adult lice. As the two viruses were present in eggs and free-living planktonic stages of the salmon louse vertical, transmission of the viruses are suggested. The tissues of the lice host, Atlantic salmon, with the exception of skin at the attachment site for the salmon louse chalimi stages, were negative for these two viruses. PMID:25402203

Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets.

PubMed

Sun, Honglei; Pu, Juan; Wei, Yandi; Sun, Yipeng; Hu, Jiao; Liu, Litao; Xu, Guanlong; Gao, Weihua; Li, Chong; Zhang, Xuxiao; Huang, Yinhua; Chang, Kin-Chow; Liu, Xiufan; Liu, Jinhua

2016-07-15

Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

PubMed Central

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina; Lien, Kathy; Tugizov, Sharof M.

2018-01-01

Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30–40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission. PMID:29277006

Structural characterization of viral epitopes recognized by broadly cross-reactive antibodies.

PubMed

Lee, Peter S; Wilson, Ian A

2015-01-01

Influenza hemagglutinin (HA) is the major surface glycoprotein on influenza viruses and mediates viral attachment and subsequent fusion with host cells. The HA is the major target of the immune response, but due to its high level of variability, as evidenced by substantial antigenic diversity, it had been historically considered to elicit only a narrow, strain-specific antibody response. However, a recent explosion in the discovery of broadly neutralizing antibodies (bnAbs) to influenza virus has identified two major supersites of vulnerability on the HA through structural characterization of HA-antibody complexes. These commonly targeted epitopes are involved with receptor binding as well as the fusion machinery and, hence, are functionally conserved and less prone to mutation. These bnAbs can neutralize viruses by blocking infection or the spread of infection by preventing progeny release. Structural analyses of these bnAbs show they exhibit striking similarities and trends in recognition of the HA and use recurring recognition motifs, despite substantial differences in their germline genes. This information can be utilized in design of novel therapeutics as well as in immunogens for improved vaccines with greater breadth and efficacy.

Characteristics of human infection with avian influenza viruses and development of new antiviral agents

PubMed Central

Liu, Qiang; Liu, Dong-ying; Yang, Zhan-qiu

2013-01-01

Since 1997, several epizootic avian influenza viruses (AIVs) have been transmitted to humans, causing diseases and even deaths. The recent emergence of severe human infections with AIV (H7N9) in China has raised concerns about efficient interpersonal viral transmission, polygenic traits in viral pathogenicity and the management of newly emerging strains. The symptoms associated with viral infection are different in various AI strains: H5N1 and newly emerged H7N9 induce severe pneumonia and related complications in patients, while some H7 and H9 subtypes cause only conjunctivitis or mild respiratory symptoms. The virulence and tissue tropism of viruses as well as the host responses contribute to the pathogenesis of human AIV infection. Several preventive and therapeutic approaches have been proposed to combat AIV infection, including antiviral drugs such as M2 inhibitors, neuraminidase inhibitors, RNA polymerase inhibitors, attachment inhibitors and signal-transduction inhibitors etc. In this article, we summarize the recent progress in researches on the epidemiology, clinical features, pathogenicity determinants, and available or potential antivirals of AIV. PMID:24096642

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization.

PubMed

Zeltina, Antra; Krumm, Stefanie A; Sahin, Mehmet; Struwe, Weston B; Harlos, Karl; Nunberg, Jack H; Crispin, Max; Pinschewer, Daniel D; Doores, Katie J; Bowden, Thomas A

2017-07-03

Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

PubMed

Agrawal, Anant; Majdi, Joseph; Clouse, Kathleen A; Stantchev, Tzanko

2018-05-23

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Antibody Against Integrin Lymphocyte Function-Associated Antigen 1 Inhibits HIV Type 1 Infection in Primary Cells Through Caspase-8-Mediated Apoptosis

PubMed Central

Walker, Tiffany N.; Cimakasky, Lisa M.; Coleman, Ebony M.; Madison, M. Nia

2013-01-01

Abstract HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus–cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs. PMID:22697794

Sub-micron filter

DOEpatents

Tepper, Frederick [Sanford, FL; Kaledin, Leonid [Port Orange, FL

2009-10-13

Aluminum hydroxide fibers approximately 2 nanometers in diameter and with surface areas ranging from 200 to 650 m.sup.2/g have been found to be highly electropositive. When dispersed in water they are able to attach to and retain electronegative particles. When combined into a composite filter with other fibers or particles they can filter bacteria and nano size particulates such as viruses and colloidal particles at high flux through the filter. Such filters can be used for purification and sterilization of water, biological, medical and pharmaceutical fluids, and as a collector/concentrator for detection and assay of microbes and viruses. The alumina fibers are also capable of filtering sub-micron inorganic and metallic particles to produce ultra pure water. The fibers are suitable as a substrate for growth of cells. Macromolecules such as proteins may be separated from each other based on their electronegative charges.

Nanosize electropositive fibrous adsorbent

DOEpatents

Tepper, Frederick; Kaledin, Leonid

2005-01-04

Aluminum hydroxide fibers approximately 2 nanometers in diameter and with surface areas ranging from 200 to 650 m.sup.2 /g have been fount to be highly electropositive. When dispersed in water they are able to attach to and retain electronegative particles. When combined into a composite filter with other fibers or particles they can filter bacteria and nano size particulates such as viruses and colloidal particles at high flux through the filter. Such filters can be used for purification and sterilization of water, biological, medical and pharmaceutical fluids, and as a collector/concentrator for detection and assay of mirobes and viruses. The alumina fibers are also capable of filtering sub-micron inorganic and metallic particles to produce ultra pure water. The fibers are suitable as a substrate for growth of cells. Macromolicules such as proteins may be separated from each other based on their electronegative charges.

3-Dimensional Protein Structure of Influenza

NASA Technical Reports Server (NTRS)

2004-01-01

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

Encapsidated Atom-Transfer Radical Polymerization in Qβ Virus-like Nanoparticles

PubMed Central

2015-01-01

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qβ VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA. PMID:25073013

Crystal Structures of Beta- and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry

PubMed Central

Aydin, Halil; Cook, Jonathan D.

2014-01-01

Membrane fusion is a key step in the life cycle of all envelope viruses, but this process is energetically unfavorable; the transmembrane fusion subunit (TM) of the virion-attached glycoprotein actively catalyzes the membrane merger process. Retroviral glycoproteins are the prototypical system to study pH-independent viral entry. In this study, we determined crystal structures of extramembrane regions of the TMs from Mason-Pfizer monkey virus (MPMV) and xenotropic murine leukemia virus-related virus (XMRV) at 1.7-Å and 2.2-Å resolution, respectively. The structures are comprised of a trimer of hairpins that is characteristic of class I viral fusion proteins and now completes a structural library of retroviral fusion proteins. Our results allowed us to identify a series of intra- and interchain electrostatic interactions in the heptad repeat and chain reversal regions. Mutagenesis reveals that charge-neutralizing salt bridge mutations significantly destabilize the postfusion six-helix bundle and abrogate retroviral infection, demonstrating that electrostatic stapling of the fusion subunit is essential for viral entry. Our data indicate that salt bridges are a major stabilizing force on the MPMV and XMRV retroviral TMs and likely provide the key energetics for viral and host membrane fusion. PMID:24131724

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus.

PubMed

Madoff, D H; Lenard, J

1982-04-01

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.

Hepatitis C Virus Infection Induces Autophagy as a Prosurvival Mechanism to Alleviate Hepatic ER-Stress Response

PubMed Central

Dash, Srikanta; Chava, Srinivas; Aydin, Yucel; Chandra, Partha K.; Ferraris, Pauline; Chen, Weina; Balart, Luis A.; Wu, Tong; Garry, Robert F.

2016-01-01

Hepatitis C virus (HCV) infection frequently leads to chronic liver disease, liver cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms by which HCV infection leads to chronic liver disease and HCC are not well understood. The infection cycle of HCV is initiated by the attachment and entry of virus particles into a hepatocyte. Replication of the HCV genome inside hepatocytes leads to accumulation of large amounts of viral proteins and RNA replication intermediates in the endoplasmic reticulum (ER), resulting in production of thousands of new virus particles. HCV-infected hepatocytes mount a substantial stress response. How the infected hepatocyte integrates the viral-induced stress response with chronic infection is unknown. The unfolded protein response (UPR), an ER-associated cellular transcriptional response, is activated in HCV infected hepatocytes. Over the past several years, research performed by a number of laboratories, including ours, has shown that HCV induced UPR robustly activates autophagy to sustain viral replication in the infected hepatocyte. Induction of the cellular autophagy response is required to improve survival of infected cells by inhibition of cellular apoptosis. The autophagy response also inhibits the cellular innate antiviral program that usually inhibits HCV replication. In this review, we discuss the physiological implications of the HCV-induced chronic ER-stress response in the liver disease progression. PMID:27223299

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer

The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in themore » cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.« less

Infection by Zika viruses requires the transmembrane protein AXL, endocytosis and low pH.

PubMed

Persaud, Mirjana; Martinez-Lopez, Alicia; Buffone, Cindy; Porcelli, Steven A; Diaz-Griffero, Felipe

2018-05-01

The recent Zika virus (ZIKV) outbreak in Brazil has suggested associations of this virus infection with neurological disorders, including microcephaly in newborn infants and Guillian-Barré syndrome in adults. Previous reports have shown that AXL, a transmembrane receptor tyrosine kinase protein, is essential for ZIKV infection of mammalian cells, but this remains controversial. Here, we have assessed the involvement of AXL in the ability of ZIKV to infect mammalian cells, and also the requirement for endocytosis and acidic pH. We demonstrated that AXL is essential for ZIKV infection of human fibroblast cell line HT1080 as the targeted deletion of the gene for AXL in HT1080 cells made them no longer susceptible to ZIKV infection. Our results also showed that infection was prevented by lysosomotropic agents such as ammonium chloride, chloroquine and bafilomycin A1, which neutralize the normally acidic pH of endosomal compartments. Infection by ZIKV was also blocked by chlorpromazine, indicating a requirement for clathrin-mediated endocytosis. Taken together, our findings suggest that AXL most likely serves as an attachment factor for ZIKV on the cell surface, and that productive infection requires endocytosis and delivery of the virus to acidified intracellular compartments. Copyright © 2018 Elsevier Inc. All rights reserved.

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

PubMed

Marcocci, M E; Amatore, D; Villa, S; Casciaro, B; Aimola, P; Franci, G; Grieco, P; Galdiero, M; Palamara, A T; Mangoni, M L; Nencioni, L

2018-05-01

The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 μg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections. Copyright © 2018 Marcocci et al.

Mechanisms of Entry and Endosomal Pathway of African Swine Fever Virus

PubMed Central

G. Sánchez, Elena; Pérez-Núñez, Daniel; Revilla, Yolanda

2017-01-01

African Swine Fever Virus (ASFV) causes a serious swine disease that is endemic in Africa and Sardinia and presently spreading in Russia and neighboring countries, including Poland and recently, the Czech Republic. This uncontrolled dissemination is a world-wide threat, as no specific protection or vaccine is available. ASFV is a very complex icosahedral, enveloped virus about 200 nm in diameter, which infects several members of pigs. The virus enters host cells by receptor-mediated endocytosis that depends on energy, vacuolar pH and temperature. The specific receptor(s) and attachment factor(s) involved in viral entry are still unknown, although macropinocytosis and clathrin-dependent mechanisms have been proposed. After internalization, ASFV traffics through the endolysosomal system. The capsid and inner envelope are found in early endosomes or macropinosomes early after infection, colocalizing with EEA1 and Rab5, while at later times they co-localize with markers of late endosomes and lysosomes, such as Rab7 or Lamp 1. A direct relationship has been established between the maturity of the endosomal pathway and the progression of infection in the cell. Finally, ASFV uncoating first involves the loss of the outer capsid layers, and later fusion of the inner membrane with endosomes, releasing the nude core into the cytosol. PMID:29117102

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

PubMed

Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

2011-02-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. Copyright © 2010 Elsevier Ltd. All rights reserved.

Influenza sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2003-09-30

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Influenza Sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2006-03-28

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Influenza Sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2005-05-17

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands

NASA Astrophysics Data System (ADS)

Schijven, Jack F.; Hoogenboezem, Wim; Hassanizadeh, S. Majid; Peters, Jos H.

1999-04-01

Removal of model viruses by dune recharge was studied at a field site in the dune area of Castricum, Netherlands. Recharge water was dosed with bacteriophages MS2 and PRD1 for 11 days at a constant concentration in a 10- by 15-m compartment that was isolated in a recharge basin. Breakthrough was monitored for 120 days at six wells with their screens along a flow line. Concentrations of both phages were reduced about 3 log10 within the first 2.4 m and another 5 log10 in a linear fashion within the following 27 m. A model accounting for one-site kinetic attachment as well as first-order inactivation was employed to simulate the bacteriophage breakthrough curves. The major removal process was found to be attachment of the bacteriophages. Detachment was very slow. After passage of the pulse of dosed bacteriophages, there was a long tail whose slope corresponds to the inactivation rate coefficient of 0.07-0.09 day-1 for attached bacteriophages. The end of the rising and the start of the declining limbs of the breakthrough curves could not be simulated completely, probably because of an as yet unknown process.

Activation of duck RIG-I by TRIM25 is independent of anchored ubiquitin.

PubMed

Miranzo-Navarro, Domingo; Magor, Katharine E

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I.

Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

PubMed Central

2017-01-01

Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. PMID:29095961

Electron Tomography Imaging of Surface Glycoproteins on Human Parainfluenza Virus 3: Association of Receptor Binding and Fusion Proteins before Receptor Engagement

PubMed Central

Gui, Long; Jurgens, Eric M.; Ebner, Jamie L.

2015-01-01

ABSTRACT In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a “heads-down” configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended “heads-up” form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN’s globular head does HN transmit its activating signal to F. PMID:25691596

Mimicking Retention and Transport of Rotavirus and Adenovirus in Sand Media Using DNA-labeled, Protein-coated Silica Nanoparticles

NASA Astrophysics Data System (ADS)

Pang, Liping; Farkas, Kata; Bennett, Grant; Varsani, Arvind; Easingwood, Richard; Tilley, Richard; Nowostawska, Urszula; Lin, Susan

2014-05-01

Rotavirus (RoV) and adenovirus (AdV) are important viral pathogens for the risk analysis of drinking water. Despite this, little is known about their retention and transport behaviors in porous media (e.g. sand filtered used for water treatment and groundwater aquifers due to a lack of representative surrogates. In this study, we developed RoV and AdV surrogates by covalently coating 70-nm sized silica nanoparticles with specific proteins and a DNA marker for sensitive detection. Filtration experiments using beach sand columns demonstrated the similarity of the surrogates' concentrations, attachment, and filtration efficiencies to the target viruses. The surrogates showed the same magnitude of concentration reduction as the viruses. Conversely, MS2 phage (a traditional virus model) over predicted concentrations of AdV and RoV by 1- and 2-orders of magnitude, respectively. The surrogates remained stable in size, surface charge and DNA concentration for at least one year. They can be easily and rapidly detected at concentrations down to one particle per PCR reaction and are readily detectable in natural waters and even in effluent. With up-scaling validation in pilot trials, the surrogates can be a useful cost-effective new tool for studying virus retention and transport in porous media, e.g. for assessing filter efficiency in water and wastewater treatment, tracking virus migration in groundwater after effluent land disposal, and establishing safe setback distances for groundwater protection.

Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

PubMed Central

Montanuy, Imma; Alejo, Ali; Alcami, Antonio

2011-01-01

Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.

PubMed

Carette, Jan E; Raaben, Matthijs; Wong, Anthony C; Herbert, Andrew S; Obernosterer, Gregor; Mulherkar, Nirupama; Kuehne, Ana I; Kranzusch, Philip J; Griffin, April M; Ruthel, Gordon; Dal Cin, Paola; Dye, John M; Whelan, Sean P; Chandran, Kartik; Brummelkamp, Thijn R

2011-08-24

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Antiviral Activity of Crude Hydroethanolic Extract from Schinus terebinthifolia against Herpes simplex Virus Type 1.

PubMed

Nocchi, Samara Requena; Companhoni, Mychelle Vianna Pereira; de Mello, João Carlos Palazzo; Dias Filho, Benedito Prado; Nakamura, Celso Vataru; Carollo, Carlos Alexandre; Silva, Denise Brentan; Ueda-Nakamura, Tânia

2017-04-01

Herpes simplex virus infections persist throughout the lifetime of the host and affect more than 80 % of the humans worldwide. The intensive use of available therapeutic drugs has led to undesirable effects, such as drug-resistant strains, prompting the search for new antiherpetic agents. Although diverse bioactivities have been identified in Schinus terebinthifolia , its antiviral activity has not attracted much attention. The present study evaluated the antiherpetic effects of a crude hydroethanolic extract from the stem bark of S. terebinthifolia against Herpes simplex virus type 1 in vitro and in vivo as well as its genotoxicity in bone marrow in mammals and established the chemical composition of the crude hydroethanolic extract based on liquid chromatography-diode array detector-mass spectrometry and MS/MS. The crude hydroethanolic extract inhibited all of the tested Herpes simplex virus type 1 strains in vitro and was effective in the attachment and penetration stages, and showed virucidal activity, which was confirmed by transmission electron microscopy. The micronucleus test showed that the crude hydroethanolic extract had no genotoxic effect at the concentrations tested. The crude hydroethanolic extract afforded protection against lesions that were caused by Herpes simplex virus type 1 in vivo . Liquid chromatography-diode array detector-mass spectrometry and MS/MS identified 25 substances, which are condensed tannins mainly produced by a B-type linkage and prodelphinidin and procyanidin units. Georg Thieme Verlag KG Stuttgart · New York.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

PubMed

Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

2017-04-01

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

The Bovine Herpesvirus 4 Bo10 Gene Encodes a Nonessential Viral Envelope Protein That Regulates Viral Tropism through both Positive and Negative Effects▿

PubMed Central

Machiels, Bénédicte; Lété, Céline; de Fays, Katalin; Mast, Jan; Dewals, Benjamin; Stevenson, Philip G.; Vanderplasschen, Alain; Gillet, Laurent

2011-01-01

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG+) cells and more infectious for GAG-negative (GAG−) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21+ cell infection and inhibits CD21− cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor. PMID:21068242

Novel molecular determinants of viral susceptibility and resistance in the lipidome of Emiliania huxleyi.

PubMed

Fulton, James M; Fredricks, Helen F; Bidle, Kay D; Vardi, Assaf; Kendrick, B Jacob; DiTullio, Giacomo R; Van Mooy, Benjamin A S

2014-04-01

Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E. huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E. huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E. huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E. huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E. huxleyi-EhV system. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Full-Genome Characterisation of Orungo, Lebombo and Changuinola Viruses Provides Evidence for Co-Evolution of Orbiviruses with Their Arthropod Vectors

PubMed Central

Mohd Jaafar, Fauziah; Belhouchet, Mourad; Belaganahalli, Manjunatha; Tesh, Robert B.; Mertens, Peter P. C.; Attoui, Houssam

2014-01-01

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein ‘outer-capsid protein 1′ (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 ‘subcore-shell protein’ as the third largest protein ‘VP3(T2)’ in the first of these groups, but the second largest protein ‘VP3(T2)’ in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors. PMID:24475112

Infection with Colorado tick fever virus among humans and ticks in a national park and forest, Wyoming, 2010.

PubMed

Geissler, Aimee L; Thorp, Emily; Van Houten, Clayton; Lanciotti, Robert S; Panella, Nicolas; Cadwell, Betsy L; Murphy, Tracy; Staples, J Erin

2014-09-01

Colorado tick fever (CTF) is an underreported tick-borne viral disease occurring in the western United States. CTF illness includes fever, headache, and severe myalgia lasting for weeks. Wyoming has one of the highest CTF incidence rates with approximately 30% of infected persons reporting tick exposure in a Wyoming National Park or Forest before symptom onset. We assessed CTF virus infections among humans and Dermacentor andersoni ticks in Grand Teton National Park (GRTE) and Bridger-Teton National Forest (BTNF). In June of 2010, 526 eligible employees were approached to participate in a baseline and 3-month follow-up serosurvey and risk behavior survey. Seropositivity was defined as antibody titers against CTF virus ≥10, as measured by the plaque reduction neutralization test. Ticks were collected at 27 sites within GRTE/BTNF and tested by RT-PCR for the CTF virus. A total of 126 (24%) employees participated in the baseline and follow-up study visits. Three (2%) employees were seropositive for CTF virus infection at baseline. During the study, 47 (37%) participants found unattached ticks on themselves, and 12 (10%) found attached ticks; however, no participants seroconverted against CTF virus. Walking through sagebrush (p=0.04) and spending time at ≥7000 feet elevation (p<0.01) were significantly associated with tick exposure. Ninety-nine percent (174/176) of ticks were D. andersoni, and all were found at ≥7000 feet elevation in sagebrush areas; 37 (21%) ticks tested positive for CTF virus and were found at 10 (38%) of 26 sites sampled. Although no GRTE or BTNF employees were infected with CTF virus during the study period, high rates of infected ticks were identified in areas with sagebrush at ≥7000 feet. CTF education and personal protection measures against tick exposure should be targeted to visitors and employees traveling to the high-risk environs identified in this study.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Visualization of Content Release from Cell Surface-Attached Single HIV-1 Particles Carrying an Extra-Viral Fluorescent pH-Sensor.

PubMed

Sood, Chetan; Marin, Mariana; Mason, Caleb S; Melikyan, Gregory B

2016-01-01

HIV-1 fusion leading to productive entry has long been thought to occur at the plasma membrane. However, our previous single virus imaging data imply that, after Env engagement of CD4 and coreceptors at the cell surface, the virus enters into and fuses with intracellular compartments. We were unable to reliably detect viral fusion at the plasma membrane. Here, we implement a novel virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment-at the plasma membrane or, possibly, in early pH-neutral vesicles. Virus particles are co-labeled with an intra-viral content marker, which is released upon fusion, and an extra-viral pH sensor consisting of ecliptic pHluorin fused to the transmembrane domain of ICAM-1. This sensor fully quenches upon virus trafficking to a mildly acidic compartment, thus precluding subsequent detection of viral content release. As an interesting secondary observation, the incorporation of the pH-sensor revealed that HIV-1 particles occasionally shuttle between neutral and acidic compartments in target cells expressing CD4, suggesting a small fraction of viral particles is recycled to the plasma membrane and re-internalized. By imaging viruses bound to living cells, we found that HIV-1 content release in neutral-pH environment was a rare event (~0.4% particles). Surprisingly, viral content release was not significantly reduced by fusion inhibitors, implying that content release was due to spontaneous formation of viral membrane defects occurring at the cell surface. We did not measure a significant occurrence of HIV-1 fusion at neutral pH above this defect-mediated background loss of content, suggesting that the pH sensor may destabilize the membrane of the HIV-1 pseudovirus and, thus, preclude reliable detection of single virus fusion events at neutral pH.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

PubMed

Lukhovitskaya, Nina I; Cowan, Graham H; Vetukuri, Ramesh R; Tilsner, Jens; Torrance, Lesley; Savenkov, Eugene I

2015-03-01

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement. © 2015 American Society of Plant Biologists. All Rights Reserved.

Hypothesis on the source, transmission and characteristics of infection of avian influenza A (H7N9) virus--based on analysis of field epidemiological investigation and gene sequence analysis.

PubMed

Ling, F; Chen, E; Liu, Q; Miao, Z; Gong, Z

2015-02-01

On 31 March 2013, the National Health and Family Planning Commission announced that human infections with influenza A (H7N9) virus had occurred in Shanghai and Anhui provinces, China. H7N9 cases were later detected in Jiangsu and Zhejiang provinces. It was estimated that the virus first spread northward along the route taken by migratory birds and then spread to neighbouring provinces with the sale of poultry. Epidemiological studies were carried out on samples from the external environment of infected cases, transmission routes, farmers markets and live poultry markets. Phylogenetic study of viral sequences from human and avian infections in Zhejiang showed that those from Shanghai and Jiangsu provinces along Taihu Lake were highly homologous with those from the external environment. This suggests that avian viruses carried by waterfowl combined with the virus carried by migratory birds, giving rise to avian influenza virus H7N9, which is highly pathogenic to humans. It is possible that the virus was transmitted by local wildfowl to domestic poultry and then to humans, or spread further by means of trading in wholesale poultry markets. As the weather has turned warm, and with measures adopted to terminate poultry trade and facilitate health communication, the epidemic in the first half of the year has been kept under control. However, the infection source in the triangular area around Taihu Lake still remains. The H7N9 epidemic will probably hit the area later in the year and next spring when the migratory birds return and may even spread to other areas. Great importance should therefore be attached to the wildfowl in Taihu Lake as the repository and disseminator of the virus: investigation and study of this population is essential. © 2014 Blackwell Verlag GmbH.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

PubMed

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Shedding of Ebola Virus Surface Glycoprotein Is a Mechanism of Self-regulation of Cellular Cytotoxicity and Has a Direct Effect on Virus Infectivity.

PubMed

Dolnik, Olga; Volchkova, Valentina A; Escudero-Perez, Beatriz; Lawrence, Philip; Klenk, Hans-Dieter; Volchkov, Viktor E

2015-10-01

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

PubMed Central

Liu, Yan; Blaum, Bärbel S.; Reiter, Dirk M.; Feizi, Ten; Dermody, Terence S.; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus. PMID:23236285

The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus.

PubMed

Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan; Blaum, Bärbel S; Reiter, Dirk M; Feizi, Ten; Dermody, Terence S; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

PubMed Central

Drannik, Anna G.; Nag, Kakon; Sallenave, Jean-Michel

2013-01-01

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa. PMID:23637403

Binding of Human GII.4 Norovirus Virus-Like Particles to Carbohydrates of Romaine Lettuce Leaf Cell Wall Materials

PubMed Central

Esseili, Malak A.

2012-01-01

Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P < 0.05) higher (1.5- to 2-fold) than that to CWM of younger leaves. Disrupting the carbohydrates of CWM or porcine gastric mucin (PGM) (a carbohydrate control) using 100 mM sodium periodate (NaIO4) significantly decreased the binding an average of 17% in younger leaves, 43% in older leaves, and 92% for PGM. In addition, lectins recognizing GalNAc, GlcNAc, and sialic acid at 100 μg/ml significantly decreased the binding an average of 41%, 33%, and 20% on CWM of older leaves but had no effect on younger leaves. Lectins recognizing α-d-Gal, α-d-Man/α-d-Glc, and α-l-Fuc showed significant inhibition on CWM of older leaves as well as that of younger leaves. All lectins, except for the lectin recognizing α-d-Gal, significantly inhibited NoV VLP binding to PGM. Collectively, our results indicate that NoV VLPs bind to lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination. PMID:22138991

Polysulfonate suramin inhibits Zika virus infection.

PubMed

Tan, Chee Wah; Sam, I-Ching; Chong, Wei Lim; Lee, Vannajan Sanghiran; Chan, Yoke Fun

2017-07-01

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log 10  PFU viral reduction with IC 50 value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor. Copyright © 2017 Elsevier B.V. All rights reserved.

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

PubMed Central

Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

Examining Merkel Cell Polyomavirus Minor Capsid Proteins | Center for Cancer Research

Cancer.gov

Merkel cell polyomavirus (MCV or MCPyV) is a recently discovered member of the viral family Polyomaviridae. It is a skin-dwelling polyomavirus species that appears to cause a rare but highly lethal form of skin cancer called Merkel cell carcinoma (MCC). Despite MCC being uncommon, chronic MCV infection of human skin is widespread, and most infected people have no known symptoms. The surface of polyomavirus virions is made up of pentameric knobs of the major capsid protein VP1. VP1 enables attachment of the virus to the cell surface, permitting infectious entry and delivery of the viral genome to host cells. The VP1 protein of previously studied polyomaviruses, such as simian virus 40 and murine polyomavirus, associates with two minor capsid proteins, VP2 and VP3, which are considered to play important roles during the infectious entry process.

Phylogenetic analysis of canine distemper virus in domestic dogs in Nanjing, China.

PubMed

Bi, Zhenwei; Wang, Yongshan; Wang, Xiaoli; Xia, Xingxia

2015-02-01

Canine distemper virus (CDV) infects a broad range of carnivores, including wild and domestic Canidae. The hemagglutinin gene, which encodes the attachment protein that determines viral tropism, has been widely used to determine the relationship between CDV strains of different lineages circulating worldwide. We determined the full-length H gene sequences of seven CDV field strains detected in domestic dogs in Nanjing, China. A phylogenetic analysis of the H gene sequences of CDV strains from different geographic regions and vaccine strains was performed. Four of the seven CDV strains were grouped in the same cluster of the Asia-1 lineage to which the vast majority of Chinese CDV strains belong, whereas the other three were clustered within the Asia-4 lineage, which has never been detected in China. This represents the first record of detection of strains of the Asia-4 lineage in China since this lineage was reported in Thailand in 2013.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fontana, Juan; Lopez-Iglesias, Carmen; Tzeng, Wen-Ping

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER andmore » CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.« less

Structure of a pentameric virion-associated fiber with a potential role in Orsay virus entry to host cells

PubMed Central

Yuan, Wang; Zhou, Ying; Wang, Tao; Demeler, Borries; Zhong, Weiwei; Tao, Yizhi J.

2017-01-01

Despite the wide use of Caenorhabditis elegans as a model organism, the first virus naturally infecting this organism was not discovered until six years ago. The Orsay virus and its related nematode viruses have a positive-sense RNA genome, encoding three proteins: CP, RdRP, and a novel δ protein that shares no homology with any other proteins. δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but the structure and function of both δ and CP-δ remain unknown. Using a combination of electron microscopy, X-ray crystallography, computational and biophysical analyses, here we show that the Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal α-helical bundle, a β-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the δ fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-δ shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-δ fibers were generated by organism-based reverse genetics. These viruses were found to be either non-viable or with poor infectivity according to phenotypic and qRT-PCR analyses. Furthermore, addition of purified δ proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner. Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ functions as a cell attachment protein to mediate Orsay entry into worm intestine cells. PMID:28241071

The Tegument Protein UL71 of Human Cytomegalovirus Is Involved in Late Envelopment and Affects Multivesicular Bodies ▿

PubMed Central

Schauflinger, Martin; Fischer, Daniela; Schreiber, Andreas; Chevillotte, Meike; Walther, Paul; Mertens, Thomas; von Einem, Jens

2011-01-01

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV. PMID:21289123

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

PubMed

Chamberlain, Kyle; Fowler, Veronica L; Barnett, Paul V; Gold, Sarah; Wadsworth, Jemma; Knowles, Nick J; Jackson, Terry

2015-09-01

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A -  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus

PubMed Central

Chamberlain, Kyle; Fowler, Veronica L.; Barnett, Paul V.; Gold, Sarah; Wadsworth, Jemma; Knowles, Nick J.

2015-01-01

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1–VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A − ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A −  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult. PMID:26296881

Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro.

PubMed

Zhao, Fu-Rong; Xie, Yin-Li; Liu, Ze-Zhong; Shao, Jun-Jun; Li, Shi-Fang; Zhang, Yong-Guang; Chang, Hui-Yun

2017-11-01

Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. FMD vaccine is the traditional way to protect against the disease, which can greatly reduce its occurrence. However, the use of FMD vaccines to protect early infection is limited. Therefore, the alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. As previously reported, LiCl has obviously inhibition effects on a variety of viruses such as transmissible gastroenteritis virus (TGEV), infectious bronchitis coronavirus (IBV), and pseudorabies herpesvirus and EV-A71 virus. In this study, our findings were the first to demonstrate that LiCl inhibition of the FMDV replication. In this study, BHK-21 cell was dose-dependent with LiCl at various stages of FMDV. Virus titration assay was calculated by the 50% tissue culture infected dose (TCID 50 ) with the Reed and Muench method. The cytotoxicity assay of LiCl was performed by the CCK8 kit. The expression level of viral mRNA was measured by RT-qPCR. The results revealed LiCl can inhibit FMDV replication, but it cannot affect FMDV attachment stage and entry stage in the course of FMDV life cycle. Further studies confirmed that the LiCl affect the replication stage of FMDV, especially the early stages of FMDV replication. So LiCl has potential as an effective anti-FMDV drug. Therefore, LiCl may be an effective drug for the control of FMDV. Based on that, the mechanism of the antiviral effect of LiCl on FMDV infection is need to in-depth research in vivo. © 2017 Wiley Periodicals, Inc.

In vitro evaluation of the antiviral properties of Shilajit and investigation of its mechanisms of action.

PubMed

Cagno, Valeria; Donalisio, Manuela; Civra, Andrea; Cagliero, Cecilia; Rubiolo, Patrizia; Lembo, David

2015-05-26

Shilajit, a herbomineral substance exuded from rocks in steep mountainous regions, has been used for thousands of years by the Indian Ayurvedic and Siddha systems of traditional medicine to relieve ailments and enhance quality of life. Although a large number of therapeutic properties have been ascribed to Shilajit, its therapeutic potential is still largely unexplored by modern research and many of its claimed bioactivities lack scientific validation. The present study was undertaken to investigate the antiviral activity of Shilajit against a panel of viruses including herpes simplex type 1 and 2 (HSV-1, HSV-2), human cytomegalovirus (HCMV), human respiratory syncytial virus (RSV), human rotavirus (HRV), and vesicular stomatitis virus (VSV). The antiviral activity of Shilajit was assayed in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virucidal and time-of-addition assays. Shilajit exhibited a dose-dependent inhibitory activity against HSV1, HSV2, HCMV, and RSV infectivity in vitro (EC50 values: 31.08μg/ml, 12.85μg/ml, 34.54μg/ml, and 30.35μg/ml, respectively), but was inactive against HRV and VSV. Humic acid, a constituent of Shilajit, displayed the same spectrum of activity. Partial virus inactivation and interference with virus attachment were both found to contribute to the antiviral activity of Shilajit. The results of the present study demonstrate that Shilajit is endowed with broad, yet specific, antiviral activity in vitro and constitutes a natural source of antiviral substances. Further work remains to be done to assess its efficacy in vivo. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

NASA Astrophysics Data System (ADS)

Zhang, Xing; Ding, Ke; Yu, Xuekui; Chang, Winston; Sun, Jingchen; Hong Zhou, Z.

2015-11-01

Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Nycholat, Corwin M.

2012-02-13

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only formore » {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.« less

Analysis of the First Genome of a Hyperthermophilic Marine Virus-Like Particle, PAV1, Isolated from Pyrococcus abyssi▿ †

PubMed Central

Geslin, C.; Gaillard, M.; Flament, D.; Rouault, K.; Le Romancer, M.; Prieur, D.; Erauso, G.

2007-01-01

Only one virus-like particle (VLP) has been reported from hyperthermophilic Euryarchaeotes. This VLP, named PAV1, is shaped like a lemon and was isolated from a strain of “Pyrococcus abyssi,” a deep-sea isolate. Its genome consists of a double-stranded circular DNA of 18 kb which is also present at a high copy number (60 per chromosome) free within the host cytoplasm but is not integrated into the host chromosome. Here, we report the results of complete analysis of the PAV1 genome. All the 25 predicted genes, except 3, are located on one DNA strand. A transcription map has been made by using a reverse transcription-PCR assay. All the identified open reading frames (ORFs) are transcribed. The most significant similarities relate to four ORFs. ORF 180a shows 31% identity with ORF 181 of the pRT1 plasmid isolated from Pyrococcus sp. strain JT1. ORFs 676 and 678 present similarities with a concanavalin A-like lectin/glucanase domain, which could be involved in the process of host-virus recognition, and ORF 59 presents similarities with the transcriptional regulator CopG. The genome of PAV1 displays unique features at the nucleic and proteinic level, indicating that PAV1 should be attached at least to a novel genus or virus family. PMID:17449623

Characterization of Bombyx mori nucleopolyhedrovirus orf68 gene that encodes a novel structural protein of budded virus.

PubMed

Iwanaga, Masashi; Kurihara, Masaaki; Kobayashi, Masahiko; Kang, WonKyung

2002-05-25

All lepidopteran baculovirus genomes sequenced to date encode a homolog of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf68 gene, suggesting that it performs an important role in the virus life cycle. In this article we describe the characterization of BmNPV orf68 gene. Northern and Western analyses demonstrated that orf68 gene was expressed as a late gene and encoded a structural protein of budded virus (BV). Immunohistochemical analysis by confocal microscopy showed that ORF68 protein was localized mainly in the nucleus of infected cells. To examine the function of orf68 gene, we constructed orf68 deletion mutant (BmD68) and characterized it in BmN cells and larvae of B. mori. BV production was delayed in BmD68-infected cells. The larval bioassays also demonstrated that deletion of orf68 did not reduce the infectivity, but mutant virus took 70 h longer to kill the host than wild-type BmNPV. In addition, dot-blot analysis showed viral DNA accumulated more slowly in mutant infected cells. Further examination suggested that BmD68 was less efficient in entry and budding from cells, although it seemed to possess normal attachment ability. These results suggest that ORF68 is a BV-associated protein involved in secondary infection from cell-to-cell. (c) 2002 Elsevier Science (USA).

Measles Fusion Machinery Is Dysregulated in Neuropathogenic Variants

PubMed Central

Jurgens, Eric M.; Mathieu, Cyrille; Palermo, Laura M.; Hardie, Diana; Horvat, Branka

2015-01-01

ABSTRACT Paramyxoviruses, including the human pathogen measles virus (MV), enter host cells by fusing their viral envelope with the target cell membrane. This fusion process is driven by the concerted actions of the two viral envelope glycoproteins, the receptor binding protein (hemagglutinin [H]) and the fusion (F) protein. H attaches to specific proteinaceous receptors on host cells; once the receptor engages, H activates F to directly mediate lipid bilayer fusion during entry. In a recent MV outbreak in South Africa, several HIV-positive people died of MV central nervous system (CNS) infection. We analyzed the virus sequences from these patients and found that specific intrahost evolution of the F protein had occurred and resulted in viruses that are “CNS adapted.” A mutation in F of the CNS-adapted virus (a leucine-to-tryptophan change present at position 454) allows it to promote fusion with less dependence on engagement of H by the two known wild-type (wt) MV cellular receptors. This F protein is activated independently of H or the receptor and has reduced thermal stability and increased fusion activity compared to those of the corresponding wt F. These functional effects are the result of the single L454W mutation in F. We hypothesize that in the absence of effective cellular immunity, such as HIV infection, MV variants bearing altered fusion machinery that enabled efficient spread in the CNS underwent positive selection. PMID:25670774

25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation.

PubMed

Shrivastava-Ranjan, Punya; Bergeron, Éric; Chakrabarti, Ayan K; Albariño, César G; Flint, Mike; Nichol, Stuart T; Spiropoulou, Christina F

2016-12-20

Lassa virus (LASV) infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H) encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC). 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA) enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy. Lassa fever is an acute viral hemorrhagic fever in humans caused by Lassa virus (LASV). No vaccine for LASV is currently available. Treatment is limited to the administration of ribavirin, which is only effective when given early in the course of illness. Cholesterol 25-hydroxylase (CH25H) is a recently identified interferon-stimulated gene (ISG); it encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC), which inhibits several viruses. Here, we identify a novel antiviral mechanism of 25HC that is dependent on inhibiting the glycosylation of Lassa virus (LASV) glycoprotein and reducing the infectivity of LASV as a means of suppressing viral replication. Since N-linked glycosylation is a critical feature of other enveloped-virus glycoproteins, 25HC may be a broad inhibitor of virus infectivity. Copyright © 2016 Shrivastava-Ranjan et al.

Comparison of Rotavirus and Norovirus transport in standardised and natural soil-water systems

NASA Astrophysics Data System (ADS)

Gamazo, P. A.; Schijven, J. F.; Victoria, M.; Alvareda, E.; Lopez, F.; Ramos, J.; Lizasoain, A.; Sapriza-Azuri, G.; Castells, M.; Colina, R.

2016-12-01

Rotavirus and Norovirus are waterborne viruses that are major causes of diarrhea and others symptoms of acute gastroenteritis. An important pathway of these viruses is groundwater. In Uruguay, as in many developed and developing countries, there are areas where the only source of water for human consumption is groundwater. In the rural area of the Salto district, groundwater is commonly used without any treatment, as it is traditionally considered as a safe source. However, virus contamination have been detected in several wells in the area. The most probable source of contamination are nearby septic systems, since the sewer coverage is scarce. This work aims to evaluate and compare the virus transport processes for a standardised soil-water systems and for the Salto aquifer system. For this, the transport of Rotavirus and Norovirus from clinic samples was studied in two sets of column experiments: 6.7 cm columns with quartz sand under saturated conditions (ionic strength 1mM, pH 7.0) and with sand from the Salto aquifer (Uruguay) (9,2% coarse sand, 47,8% medium sand, 40,5% fine sand, magnesium/calcium bicarbonate water, Ionic strength 15.1 mM, pH 7.2). Both viruses were seeded for 2 pore volumes on the columns. Samples were collected at the column outlet and viruses were enumerated by Q-PRCR. Breakthrough curves were constructed and fitted to a two-site kinetic attachment/detachment model, including blocking using Hydrus-1D. In the quartz sand column, both Rotavirus and Norovirus were removed two orders in magnitude. In the Salto sand column, Rotavirus was removed 2 log10 as well, but Norovirus was removed 4 log10. The fitting of the breakthrough curves indicated that blocking played a role for Rotavirus in the Salto sand column. These results are consistent with field observation where only Rotavirus was detected in the Salto aquifer, while similar concentrations in Salto sewer effluent was measured for these two viruses. This work, besides reporting actual parameters values for human virus transport modelling, shows the significant differences in transport that human viruses can have in standardised and natural soil-water systems.

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

PubMed Central

Cline, Troy; Baranovich, Tatiana; Govorkova, Elena A.; Schultz-Cherry, Stacey

2014-01-01

ABSTRACT Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK, all of them exhibited a reduction in viral replication in response to inhibition of FAK signaling. These findings highlight novel biphasic roles of FAK activation during IAV infection and indicate that FAK serves as a central link between receptor-mediated PI3K activation and actin reorganization during IAV infection. IMPORTANCE We found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics. PMID:24696469

Anti-pandemic influenza A (H1N1) virus potential of catechin and gallic acid.

PubMed

You, Huey-Ling; Huang, Chao-Chun; Chen, Chung-Jen; Chang, Cheng-Chin; Liao, Pei-Lin; Huang, Sheng-Teng

2018-05-01

The pandemic influenza A (H1N1) virus has spread worldwide and infected a large proportion of the human population. Discovery of new and effective drugs for the treatment of influenza is a crucial issue for the global medical community. According to our previous study, TSL-1, a fraction of the aqueous extract from the tender leaf of Toonasinensis, has demonstrated antiviral activities against pandemic influenza A (H1N1) through the down-regulation of adhesion molecules and chemokine to prevent viral attachment. The aim of the present study was to identify the active compounds in TSL-1 which exert anti-influenza A (H1N1) virus effects. XTT assay was used to detect the cell viability. Meanwhile, the inhibitory effect on the pandemic influenza A (H1N1) virus was analyzed by observing plaque formation, qRT-PCR, neuraminidase activity, and immunofluorescence staining of influenza A-specific glycoprotein. Both catechin and gallic acid were found to be potent inhibitors in terms of influenza virus mRNA replication and MDCK plaque formation. Additionally, both compounds inhibited neuraminidase activities and viral glycoprotein. The 50% effective inhibition concentration (EC 50 ) of catechin and gallic acid for the influenza A (H1N1) virus were 18.4 μg/mL and 2.6 μg/mL, respectively; whereas the 50% cytotoxic concentrations (CC 50 ) of catechin and gallic acid were >100 μg/mL and 22.1 μg/mL, respectively. Thus, the selectivity indexes (SI) of catechin and gallic acid were >5.6 and 22.1, respectively. The present study demonstrates that catechin might be a safe reagent for long-term use to prevent influenza A (H1N1) virus infection; whereas gallic acid might be a sensitive reagent to inhibit influenza virus infection. We conclude that these two phyto-chemicals in TSL-1 are responsible for exerting anti-pandemic influenza A (H1N1) virus effects. Copyright © 2017. Published by Elsevier Taiwan LLC.

Plasmonic Techniques for Viral Membrane Characterization

NASA Astrophysics Data System (ADS)

Feizpour, Amin

The lipid bilayer membrane of enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), plays an important role in key steps of the infection, including cell binding and uptake. Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. In this dissertation, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using small sample sizes is introduced. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. The performed studies reveal significant differences in the membrane of HIV-1 and Ebola VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. In addition, this technique was used in another application to improve the understanding of the relationship between the membrane PS lipid and the infectivity of HIV-2 and murine leukemia virus (MLV). The composition of the membrane, in particular the cholesterol (chol) content, determines its fluidity. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of probing the membrane fluidity on the single-virus level are required. In this dissertation, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed a plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated, for the first time, the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature on the single-VLP level. Chol extraction studies with different methyl-beta-cyclodextrin (MbetaCD) concentrations yielded a gradual decrease in polarization fluctuations as function of time. The PFTM revealed chol content and fluidity heterogeneities of an HIV-1 VLP population.

Cryo-electron tomography of bacterial viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guerrero-Ferreira, Ricardo C.; Wright, Elizabeth R., E-mail: erwrigh@emory.edu

2013-01-05

Bacteriophage particles contain both simple and complex macromolecular assemblages and machines that enable them to regulate the infection process under diverse environmental conditions with a broad range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible to observe the interactions of bacteriophages with their host cells under native-state conditions at unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of investigation and future directions in the field are also discussed.

Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

DTIC Science & Technology

2013-02-18

genome replication and production of new virions [18]. Several cellular proteins required for the function and maturation of late endosomes (LE) and...studded with a trimeric glycoprotein (GP) whose first function is to attach viral particles to the cell surface. The virions are then internalized into the...with NPC1, but at a site distinct from the C loop of NPC1 (which binds GP19 kDa). Binding to NPC1 inhibits a second function of NPC1 (i.e. in addition

Aerosol-phase Activity of Iodine Captured from a Triiodide Resin Filter on Fine Particles Containing an Infectious Virus

DTIC Science & Technology

2015-01-01

conductive tubing direc ted the aerosol into a heat sealed plastic bag (Food Saver; Jarden Consumer Solutions, Rye, NY), measuring 589 cm 9 254...a self sealing injection port attached to each bag with an adhesive. Approximately 100 mg of iodine crystals were stored at 35°C in an Erlenmeyer...was collected in a bag, 075 ml was drawn from the headspace into a 1 ml syringe and needle and immediately injected through the port into the sample

Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Li; Fokine, Andrei; O'Donnell, Erin

2010-07-22

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a 'glue' between neighboring hexameric capsomers, forming a 'cage' that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 {angstrom} resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc weremore » fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.« less

Characterisation of a type 1 Avian Paramyxovirus belonging to a divergent group.

PubMed

Briand, François-Xavier; Massin, Pascale; Jestin, Véronique

2014-01-10

Newcastle disease, induced by a type 1 Avian Paramyxovirus (APMV-1), is one of the most serious poultry diseases. APMV-1 are divided into two classes based on genetic analysis: class II strains have been recovered from wild or domestic birds and include virulent and avirulent isolates whereas class I strains have been mainly isolated from wild birds and are avirulent. Within class I, a new proposed genotype has recently been reported. The only full genome strain of this group is presently characterised from the point of view of codon usage with reference to class I and class II specificities. Class-specific residues were identified on HN and F proteins that are the two major proteins involved in cell attachment and pathogenicity. Comparison of protein patterns and codon usage for this newly identified APMV-1 strain indicates it is similar to class I viruses but contains a few characteristics close to the viruses of class II. Transmission of viruses from this recently identified divergent group from wild birds to domestic birds could have a major impact on the domestic poultry industry. Copyright © 2013 Elsevier B.V. All rights reserved.

Anti HSV-1 Activity of Halistanol Sulfate and Halistanol Sulfate C Isolated from Brazilian Marine Sponge Petromica citrina (Demospongiae)

PubMed Central

da Rosa Guimarães, Tatiana; Quiroz, Carlos Guillermo; Rigotto, Caroline; de Oliveira, Simone Quintana; Rojo de Almeida, Maria Tereza; Bianco, Éverson Miguel; Moritz, Maria Izabel Goulart; Carraro, João Luís; Palermo, Jorge Alejandro; Cabrera, Gabriela; Schenkel, Eloir Paulo; Reginatto, Flávio Henrique; Oliveira Simões, Cláudia Maria

2013-01-01

The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2. PMID:24172213

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses

PubMed Central

Macchi, Francesca; Rojas, José Manuel; Verna, Andrea Elizabeth; Sevilla, Noemí; Franceschi, Valentina; Tebaldi, Giulia; Cavirani, Sandro; Martín, Verónica; Donofrio, Gaetano

2018-01-01

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs. PMID:29556236

A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

PubMed

Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

2016-12-01

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

PubMed Central

Chou, Yi-ying; Cuevas, Christian; Carocci, Margot; Stubbs, Sarah H.; Ma, Minghe; Cureton, David K.; Evesson, Frances; He, Kangmin; Yang, Priscilla L.; Whelan, Sean P.

2016-01-01

ABSTRACT Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential. PMID:26912630

Inhibition of EV71 by curcumin in intestinal epithelial cells.

PubMed

Huang, Hsing-I; Chio, Chi-Chong; Lin, Jhao-Yin

2018-01-01

EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

PubMed

Guzman, Efrain; Taylor, Geraldine; Hope, Jayne; Herbert, Rebecca; Cubillos-Zapata, Carolina; Charleston, Bryan

2016-10-01

Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

A paramyxovirus-vectored intranasal vaccine against Ebola virus is immunogenic in vector-immune animals.

PubMed

Yang, Lijuan; Sanchez, Anthony; Ward, Jerrold M; Murphy, Brian R; Collins, Peter L; Bukreyev, Alexander

2008-08-01

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans. The virus can be transmitted by direct contact as well as by aerosol and is considered a potential bioweapon. Because direct immunization of the respiratory tract should be particularly effective against infection of mucosal surfaces, we previously developed an intranasal vaccine based on replication-competent human parainfluenza virus type 3 (HPIV3) expressing EBOV glycoprotein GP (HPIV3/EboGP) and showed that it is immunogenic and protective against a high dose parenteral EBOV challenge. However, because the adult human population has considerable immunity to HPIV3, which is a common human pathogen, replication and immunogenicity of the vaccine in this population might be greatly restricted. Indeed, in the present study, replication of the vaccine in the respiratory tract of HPIV3-immune guinea pigs was found to be restricted to undetectable levels. This restriction appeared to be based on both neutralizing antibodies and cellular or other components of the immunity to HPIV3. Surprisingly, even though replication of HPIV3/EboGP was highly restricted in HPIV3-immune animals, it induced a high level of EBOV-specific antibodies that nearly equaled that obtained in HPIV3-naive animals. We also show that the previously demonstrated presence of functional GP in the vector particle was not associated with increased replication in the respiratory tract nor with spread beyond the respiratory tract of HPIV3-naive guinea pigs, indicating that expression and functional incorporation of the attachment/penetration glycoprotein of this systemic virus did not mediate a change in tissue tropism.

Inhibition of EV71 by curcumin in intestinal epithelial cells

PubMed Central

Chio, Chi-Chong; Lin, Jhao-Yin

2018-01-01

EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections. PMID:29370243

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

In silico assessment of phosphorylation and O-β-GlcNAcylation sites in human NPC1 protein critical for Ebola virus entry.

PubMed

Basharat, Zarrin; Yasmin, Azra

2015-08-01

Ebola is a highly pathogenic enveloped virus responsible for deadly outbreaks of severe hemorrhagic fever. It enters human cells by binding a multifunctional cholesterol transporter Niemann-Pick C1 (NPC1) protein. Post translational modification (PTM) information for NPC1 is crucial to understand Ebola virus (EBOV) entry and action due to changes in phosphorylation or glycosylation at the binding site. It is difficult and costly to experimentally assess this type of interaction, so in silico strategy was employed. Identification of phosphorylation sites, including conserved residues that could be possible targets for 21 predicted kinases was followed by interplay study between phosphorylation and O-β-GlcNAc modification of NPC1. Results revealed that only 4 out of 48 predicted phosphosites exhibited O-β-GlcNAc activity. Predicted outcomes were integrated with residue conservation and 3D structural information. Three Yin Yang sites were located in the α-helix regions and were conserved in studied vertebrate and mammalian species. Only one modification site S425 was found in β-turn region located near the N-terminus of NPC1 and was found to differ in pig, mouse, cobra and humans. The predictions suggest that Yin Yang sites may not be important for virus attachment to NPC1, whereas phosphosite 473 may be important for binding and hence entry of Ebola virus. This information could be useful in addressing further experimental studies and therapeutic strategies targeting PTM events in EBOV entry. Copyright © 2015 Elsevier B.V. All rights reserved.

In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

PubMed Central

2011-01-01

Background Foot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. Results Amino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia. Conclusions FMDV quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. The RDD-containing FMD viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. The viruses expressing non-RGD receptor binding sites can replicate stably in vitro and produce typical FMD clinical disease in susceptible animals. PMID:21711567

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin.

PubMed

Kumaki, Yohichi; Wandersee, Miles K; Smith, Aaron J; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M; Bailey, Kevin W; Vest, Zachary G; Li, Joseph K-K; Chan, Paul Kay-Sheung; Smee, Donald F; Barnard, Dale L

2011-04-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

PubMed Central

Kumaki, Yohichi; Wandersee, Miles K.; Smith, Aaron J.; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M.; Bailey, Kevin W.; Vest, Zachary G.; Li, Joseph K.-K.; Chan, Paul Kay-Sheung; Smee, Donald F.; Barnard, Dale L.

2011-01-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 hours before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p<0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. PMID:21338626

The traditional use of Vachellia nilotica for sexually transmitted diseases is substantiated by the antiviral activity of its bark extract against sexually transmitted viruses.

PubMed

Donalisio, Manuela; Cagno, Valeria; Civra, Andrea; Gibellini, Davide; Musumeci, Giuseppina; Rittà, Massimo; Ghosh, Manik; Lembo, David

2018-03-01

Vachellia (Acacia) nilotica and other plants of this genus have been used in traditional medicine of Asian and African countries to treat many disorders, including sexually transmitted diseases, but few studies were performed to validate their anti-microbial and anti-viral activity against sexually transmitted infections. The present study was undertaken to explore whether the ethnomedical use of V.nilotica to treat genital lesions is substantiated by its antiviral activity against the human immunodeficiency virus (HIV), the herpes simplex virus (HSV) and the human papillomavirus (HPV). The antiviral activity of V.nilotica was tested in vitro by virus-specific inhibition assays using HSV-2 strains, sensible or resistant to acyclovir, HIV-1IIIb strain and HPV-16 pseudovirion (PsV). The potential mode of action of extract against HSV-2 and HPV-16 was further investigated by virus inactivation and time-of-addition assays on cell cultures. V.nilotica chloroform, methanolic and water bark extracts exerted antiviral activity against HSV-2 and HPV-16 PsV infections; among these, methanolic extract showed the best EC50s with values of 4.71 and 1.80µg/ml against HSV-2 and HPV-16, respectively, and it was also active against an acyclovir-resistant HSV-2 strain with an EC50 of 6.71µg/ml. By contrast, no suppression of HIV infection was observed. Investigation of the mechanism of action revealed that the methanolic extract directly inactivated the infectivity of the HPV-16 particles, whereas a partial virus inactivation and interference with virus attachment (EC50 of 2.74µg/ml) were both found to contribute to the anti-HSV-2 activity. These results support the traditional use of V.nilotica applied externally for the treatment of genital lesions. Further work remains to be done in order to identify the bioactive components. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Briggs, D.J.

1989-01-01

The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a threemore » hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.« less

A Recombinant Hendra virus G Glycoprotein-Based Subunit Vaccine Protects Ferrets from Lethal Hendra virus Challenge

PubMed Central

Pallister, Jackie; Middleton, Deborah; Wang, Lin-Fa; Klein, Reuben; Haining, Jessica; Robinson, Rachel; Yamada, Manabu; White, John; Payne, Jean; Feng, Yan-Ru; Chan, Yee-Peng; Broder, Christopher C.

2011-01-01

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are two deadly zoonotic viruses for which no vaccines or therapeutics have yet been approved for human or livestock use. In 14 outbreaks since 1994 HeV has been responsible for multiple fatalities in horses and humans, with all known human infections resulting from close contact with infected horses. A vaccine that prevents virus shedding in infected horses could interrupt the chain of transmission to humans and therefore prevent HeV disease in both. Here we characterise HeV infection in a ferret model and show that it closely mirrors the disease seen in humans and horses with induction of systemic vasculitis, including involvement of the pulmonary and central nervous systems. This model of HeV infection in the ferret was used to assess the immunogenicity and protective efficacy of a subunit vaccine based on a recombinant soluble version of the HeV attachment glycoprotein G (HeVsG), adjuvanted with CpG. We report that ferrets vaccinated with a 100 μg, 20 μg or 4 μg dose of HeVsG remained free of clinical signs of HeV infection following a challenge with 5,000 TCID50 of HeV. In addition, and of considerable importance, no evidence of virus or viral genome was detected in any tissues or body fluids in any ferret in the 100 and 20 μg groups, while genome was detected in the nasal washes only of one animal in the 4 μg group. Together, our findings indicate that 100 μg or 20 μg doses of HeVsG vaccine can completely prevent a productive HeV infection in the ferret, suggesting that vaccination to prevent the infection and shedding of HeV is possible. PMID:21689706

A recombinant Hendra virus G glycoprotein-based subunit vaccine protects ferrets from lethal Hendra virus challenge.

PubMed

Pallister, Jackie; Middleton, Deborah; Wang, Lin-Fa; Klein, Reuben; Haining, Jessica; Robinson, Rachel; Yamada, Manabu; White, John; Payne, Jean; Feng, Yan-Ru; Chan, Yee-Peng; Broder, Christopher C

2011-08-05

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are two deadly zoonotic viruses for which no vaccines or therapeutics have yet been approved for human or livestock use. In 14 outbreaks since 1994 HeV has been responsible for multiple fatalities in horses and humans, with all known human infections resulting from close contact with infected horses. A vaccine that prevents virus shedding in infected horses could interrupt the chain of transmission to humans and therefore prevent HeV disease in both. Here we characterise HeV infection in a ferret model and show that it closely mirrors the disease seen in humans and horses with induction of systemic vasculitis, including involvement of the pulmonary and central nervous systems. This model of HeV infection in the ferret was used to assess the immunogenicity and protective efficacy of a subunit vaccine based on a recombinant soluble version of the HeV attachment glycoprotein G (HeVsG), adjuvanted with CpG. We report that ferrets vaccinated with a 100 μg, 20 μg or 4 μg dose of HeVsG remained free of clinical signs of HeV infection following a challenge with 5000 TCID₅₀ of HeV. In addition, and of considerable importance, no evidence of virus or viral genome was detected in any tissues or body fluids in any ferret in the 100 and 20 μg groups, while genome was detected in the nasal washes only of one animal in the 4 μg group. Together, our findings indicate that 100 μg or 20 μg doses of HeVsG vaccine can completely prevent a productive HeV infection in the ferret, suggesting that vaccination to prevent the infection and shedding of HeV is possible. Copyright © 2011 Elsevier Ltd. All rights reserved.

Virus fate and transport during recharge using recycled water at a research field site in the Montebello Forebay, Los Angeles County, California, 1997-2000

USGS Publications Warehouse

Anders, Robert; Yanko, William A.; Schroeder, Roy A.; Jackson, James L.

2004-01-01

Total and fecal coliform bacteria distributions in subsurface water samples collected at a research field site in Los Angeles County were found to increase from nondetectable levels immediately before artificial recharge using tertiary-treated municipal wastewater (recycled water). This rapid increase indicates that bacteria can move through the soil with the percolating recycled water over intervals of a few days and vertical and horizontal distances of about 3 meters. This conclusion formed the basis for three field-scale experiments using bacterial viruses (bacteriophage) MS2 and PRD1 as surrogates for human enteric viruses and bromide as a conservative tracer to determine the fate and transport of viruses in recycled water during subsurface transport under actual recharge conditions. The research field site consists of a test basin constructed adjacent to a large recharge facility (spreading grounds) located in the Montebello Forebay of Los Angeles County, California. The soil beneath the test basin is predominantly medium to coarse, moderately sorted, grayish-brown sand. The three tracer experiments were conducted during August 1997, August-September 1998, and August 2000. For each experiment, prepared solutions of bacteriophage and bromide were sprayed on the surface of the water in the test basin and injected, using peristaltic pumps, directly into the feed pipe delivering the recycled water to the test basin. Extensive data were obtained for water samples collected from the test basin itself and from depths of 0.3, 0.6, 1.0, 1.5, 3.0, and 7.6 meters below the bottom of the test basin. The rate of bacteriophage inactivation in the recycled water, independent of any processes occurring in the subsurface, was determined from measurements on water samples from the test basin. Regression analysis of the ratios of bacteriophage to bromide was used to determine the attenuation rates for MS2 and PRD1, defined as the logarithmic reduction in the ratio during each experiment. Although the inactivation rates increased during the third tracer experiment, they were nearly two orders of magnitude less than the attenuation rates. Therefore, adsorption, not inactivation, is the predominant removal mechanism for viruses during artificial recharge. Using the colloid-filtration model, the collision efficiency was determined for both bacteriophage during the second and third field-scale tracer experiments. The collision efficiency confirms that more favorable attachment conditions existed for PRD1, especially during the third tracer experiment. The different collision efficiencies between the second and third tracer experiments possibly were due to changing hydraulic conditions at the research field site during each experiment. The field data suggest that an optimal management scenario might exist to maximize the amount of recycled water that can be applied to the spreading grounds while still maintaining favorable attachment conditions for virus removal and thereby ensuring protection of the ground-water supply.

Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Ye; Leiman, Petr G.; Li, Long

2010-02-03

The tailed bacteriophage phi29 has 12 'appendages' (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an 'autochaperone' that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function tomore » attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.« less

A novel way to go whole-cell in patch-clamp experiments.

PubMed

Inayat, Samsoon; Zhao, Yan; Cantrell, Donal R; Dikin, Dmitryi; Pinto, Lawrence H; Troy, John B

2010-11-01

With a conventional patch-clamp electrode, an Ag/AgCl wire sits stationary inside the pipette. To move from the gigaseal cell-attached configuration to whole-cell recording, suction is applied inside the pipette. We have designed and developed a novel Pushpen patch-clamp electrode, in which a W wire insulated and wound with Ag/AgCl wire can move linearly inside the pipette. The W wire has a conical tip, which can protrude from the pipette tip like a push pen, a procedure we call the Pushpen Operation. We use the Pushpen operation to impale the cell membrane in cell-attached configuration to go whole-cell without disruption of the gigaseal. We successfully recorded whole-cell currents from chinese hamster ovarian cells expressing influenza A virus protein A/M2, after obtaining whole-cell configuration with the Pushpen operation. This novel method of achieving whole-cell configuration may have a higher success rate than is the case with the conventional patch clamp technique.

Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

PubMed Central

Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

2011-01-01

Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools.

PubMed

Landowski, Matthew; Dabundo, Jeffrey; Liu, Qian; Nicola, Anthony V; Aguilar, Hector C

2014-12-01

Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses. Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. For example, drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

NASA Astrophysics Data System (ADS)

Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

2016-07-01

The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

PubMed Central

De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

2013-01-01

The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

Environmental contaminant mixtures modulate in vitro influenza infection.

PubMed

Desforges, Jean-Pierre; Bandoro, Christopher; Shehata, Laila; Sonne, Christian; Dietz, Rune; Puryear, Wendy B; Runstadler, Jonathan A

2018-09-01

Environmental chemicals, particularly organochlorinated contaminants (OCs), are associated with a ranged of adverse health effects, including impairment of the immune system and antiviral immunity. Influenza A virus (IAV) is an infectious disease of major global public health concern and exposure to OCs can increase the susceptibility, morbidity, and mortality to disease. It is however unclear how pollutants are interacting and affecting the outcome of viral infections at the cellular level. In this study, we investigated the effects of a mixture of environmentally relevant OCs on IAV infectivity upon in vitro exposure in Madin Darby Canine Kidney (MDCK) cells and human lung epithelial cells (A549). Exposure to OCs reduced IAV infectivity in MDCK and A549 cells during both short (18-24h) and long-term (72h) infections at 0.05 and 0.5ppm, and effects were more pronounced in cells co-treated with OCs and IAV than pre-treated with OCs prior to IAV (p<0.001). Pre-treatment of host cells with OCs did not affect IAV cell surface attachment or entry. Visualization of IAV by transmission electron microscopy revealed increased envelope deformations and fewer intact virions during OC exposure. Taken together, our results suggest that disruption of IAV infection upon in vitro exposure to OCs was not due to host-cell effects influencing viral attachment and entry, but perhaps mediated by direct effects on viral particles or cellular processes involved in host-virus interactions. In vitro infectivity studies such as ours can shed light on the complex processes underlying host-pathogen-pollutant interactions. Copyright © 2018 Elsevier B.V. All rights reserved.

Virus and bacteria transport in a sandy aquifer, Cape Cod, MA

USGS Publications Warehouse

Bales, Roger C.; Li, Shimin; Maguire, Kimberly M.; Yahya, Moyasar T.; Gerba, Charles P.; Harvey, Ronald W.

1995-01-01

Transport of the bacteriophage PRD-1, bacteria, and latex microspheres was studied in a sandy aquifer under natural-gradient conditions. The field injection was carried out at the U.S. Geological Survey's Toxic Substances Hydrology research site on Cape Cod. The three colloids and a salt tracer (Br−) moved along the same path. There was significant attenuation of the phage, with PRD-1 peak concentrations less than 0.001 percent of Br− peaks 6 m from the source; but the low detection limit (one per ml) enabled tracking movement of the PRD-1 plume for 12 m downgradient over the 25-day experiment. Attenuation of phage was apparently due to retention on soil particles (adsorption). Attenuation of bacteria and microspheres was less, with peak concentrations 6 m from the source on the order of 10 and 0.4 percent of Br−, respectively. Injection of a high-pH pulse of water 20 days into the experiment resulted in significant remobilization of retained phage, demonstrating that attached phage remained viable, and that PRD-1 attachment to and detachment from the sandy soil particles was highly pH dependent. Phage behavior in this experiment, i.e. attenuation at pH 5.7 and rapid resuspension at pH 6–8, was consistent with that observed previously in laboratory column studies. Results illustrate that biocolloids travel in a fairly narrow plume in sandy (relatively homogeneous) media, with virus concentrations dropping below detection limit several meters away from the source; bacteria concentrations above detection limits can persist over longer distances.

Identification of Litopenaeus vannamei BiP as a novel cellular attachment protein for white spot syndrome virus by using a biotinylation based affinity chromatography method.

PubMed

Yuan, Zengzhi; Chen, Meng; Wang, Jingting; Li, Zhuoyu; Geng, Xuyun; Sun, Jinsheng

2018-05-05

White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target. Copyright © 2018. Published by Elsevier Ltd.

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

PubMed Central

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

2016-01-01

ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. PMID:27558420

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

PubMed

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

2016-11-01

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interface of physics and biology: engineering virus-based nanoparticles for biophotonics.

PubMed

Wen, Amy M; Infusino, Melissa; De Luca, Antonio; Kernan, Daniel L; Czapar, Anna E; Strangi, Giuseppe; Steinmetz, Nicole F

2015-01-21

Virus-based nanoparticles (VNPs) have been used for a wide range of applications, spanning basic materials science and translational medicine. Their propensity to self-assemble into precise structures that offer a three-dimensional scaffold for functionalization has led to their use as optical contrast agents and related biophotonics applications. A number of fluorescently labeled platforms have been developed and their utility in optical imaging demonstrated, yet their optical properties have not been investigated in detail. In this study, two VNPs of varying architectures were compared side-by-side to determine the impact of dye density, dye localization, conjugation chemistry, and microenvironment on the optical properties of the probes. Dyes were attached to icosahedral cowpea mosaic virus (CPMV) and rod-shaped tobacco mosaic virus (TMV) through a range of chemistries to target particular side chains displayed at specific locations around the virus. The fluorescence intensity and lifetime of the particles were determined, first using photochemical experiments on the benchtop, and second in imaging experiments using tissue culture experiments. The virus-based optical probes were found to be extraordinarily robust under ultrashort, pulsed laser light conditions with a significant amount of excitation energy, maintaining structural and chemical stability. The most effective fluorescence output was achieved through dye placement at optimized densities coupled to the exterior surface avoiding conjugated ring systems. Lifetime measurements indicate that fluorescence output depends not only on spacing the fluorophores, but also on dimer stacking and configurational changes leading to radiationless relaxation-and these processes are related to the conjugation chemistry and nanoparticle shape. For biological applications, the particles were also examined in tissue culture, from which it was found that the optical properties differed from those found on the benchtop due to effects from cellular processes and uptake kinetics. Data indicate that fluorescent cargos are released in the endolysosomal compartment of the cell targeted by the virus-based optical probes. These studies provide insight into the optical properties and fates of fluorescent proteinaceous imaging probes. The cellular release of cargo has implications not only for virus-based optical probes, but also for drug delivery and release systems.

Abundance and Distribution of Enteric Bacteria and Viruses in Coastal and Estuarine Sediments—a Review

PubMed Central

Hassard, Francis; Gwyther, Ceri L.; Farkas, Kata; Andrews, Anthony; Jones, Vera; Cox, Brian; Brett, Howard; Jones, Davey L.; McDonald, James E.; Malham, Shelagh K.

2016-01-01

The long term survival of fecal indicator organisms (FIOs) and human pathogenic microorganisms in sediments is important from a water quality, human health and ecological perspective. Typically, both bacteria and viruses strongly associate with particulate matter present in freshwater, estuarine and marine environments. This association tends to be stronger in finer textured sediments and is strongly influenced by the type and quantity of clay minerals and organic matter present. Binding to particle surfaces promotes the persistence of bacteria in the environment by offering physical and chemical protection from biotic and abiotic stresses. How bacterial and viral viability and pathogenicity is influenced by surface attachment requires further study. Typically, long-term association with surfaces including sediments induces bacteria to enter a viable-but-non-culturable (VBNC) state. Inherent methodological challenges of quantifying VBNC bacteria may lead to the frequent under-reporting of their abundance in sediments. The implications of this in a quantitative risk assessment context remain unclear. Similarly, sediments can harbor significant amounts of enteric viruses, however, the factors regulating their persistence remains poorly understood. Quantification of viruses in sediment remains problematic due to our poor ability to recover intact viral particles from sediment surfaces (typically <10%), our inability to distinguish between infective and damaged (non-infective) viral particles, aggregation of viral particles, and inhibition during qPCR. This suggests that the true viral titre in sediments may be being vastly underestimated. In turn, this is limiting our ability to understand the fate and transport of viruses in sediments. Model systems (e.g., human cell culture) are also lacking for some key viruses, preventing our ability to evaluate the infectivity of viruses recovered from sediments (e.g., norovirus). The release of particle-bound bacteria and viruses into the water column during sediment resuspension also represents a risk to water quality. In conclusion, our poor process level understanding of viral/bacterial-sediment interactions combined with methodological challenges is limiting the accurate source apportionment and quantitative microbial risk assessment for pathogenic organisms associated with sediments in aquatic environments. PMID:27847499

Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

PubMed Central

Miranzo-Navarro, Domingo; Magor, Katharine E.

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I. PMID:24466302

Nosocomial transmission of respiratory syncytial virus in an outpatient cancer center.

PubMed

Chu, Helen Y; Englund, Janet A; Podczervinski, Sara; Kuypers, Jane; Campbell, Angela P; Boeckh, Michael; Pergam, Steven A; Casper, Corey

2014-06-01

Respiratory syncytial virus (RSV) outbreaks in inpatient settings are associated with poor outcomes in cancer patients. The use of molecular epidemiology to document RSV transmission in the outpatient setting has not been well described. We performed a retrospective cohort study of 2 nosocomial outbreaks of RSV at the Seattle Cancer Care Alliance. Subjects included patients seen at the Seattle Cancer Care Alliance with RSV detected in 2 outbreaks in 2007-2008 and 2012 and all employees with respiratory viruses detected in the 2007-2008 outbreak. A subset of samples was sequenced using semi-nested PCR targeting the RSV attachment glycoprotein coding region. Fifty-one cases of RSV were identified in 2007-2008. Clustering of identical viral strains was detected in 10 of 15 patients (67%) with RSV sequenced from 2007 to 2008. As part of a multimodal infection control strategy implemented as a response to the outbreak, symptomatic employees had nasal washes collected. Of 254 employee samples, 91 (34%) tested positive for a respiratory virus, including 14 with RSV. In another RSV outbreak in 2012, 24 cases of RSV were identified; 9 of 10 patients (90%) had the same viral strain, and 1 (10%) had another viral strain. We document spread of clonal strains within an outpatient cancer care setting. Infection control interventions should be implemented in outpatient, as well as inpatient, settings to reduce person-to-person transmission and limit progression of RSV outbreaks. Copyright © 2014 American Society for Blood and Marrow Transplantation. All rights reserved.

Nosocomial Transmission of Respiratory Syncytial Virus in an Outpatient Cancer Center

PubMed Central

Chu, Helen Y.; Englund, Janet A.; Podczervinski, Sara; Kuypers, Jane; Campbell, Angela P.; Boeckh, Michael; Pergam, Steven A.; Casper, Crey

2014-01-01

Background Respiratory syncytial virus (RSV) outbreaks in inpatient settings are associated with poor outcomes in cancer patients. The use of molecular epidemiology to document RSV transmission in the outpatient setting has not been well described. Methods We performed a retrospective cohort study of two nosocomial outbreaks of RSV at the Seattle Cancer Care Alliance (SCCA). Subjects included patients seen at the SCCA with RSV detected in two outbreaks in 2007-2008 and 2012, and all employees with respiratory viruses detected in the 2007-2008 outbreak. A subset of samples was sequenced using semi-nested polymerase chain reaction targeting the RSV attachment glycoprotein coding region. Results Fifty-one cases of RSV were identified in 2007-2008. Clustering of identical viral strains was detected in 10 (67%) of 15 patients with RSV sequenced from 2007-2008. As part of a multimodal infection control strategy implemented as a response to the outbreak, symptomatic employees had nasal washes collected. Of 254 employee samples, 91 (34%) tested positive for a respiratory virus, including 14 with RSV. In another RSV outbreak in 2012, 24 cases of RSV were identified; nine (90%) of 10 patients had the same viral strain, and 1 (10%) had another viral strain. Conclusions We document spread of clonal strains within an outpatient cancer care setting. Infection control interventions should be implemented in outpatient, as well as inpatient, settings to reduce person-to-person transmission and limit progression of RSV outbreaks. PMID:24607551

Stormwater runoff drives viral community composition changes in inland freshwaters.

PubMed

Williamson, Kurt E; Harris, Jamie V; Green, Jasmin C; Rahman, Faraz; Chambers, Randolph M

2014-01-01

Storm events impact freshwater microbial communities by transporting terrestrial viruses and other microbes to freshwater systems, and by potentially resuspending microbes from bottom sediments. The magnitude of these impacts on freshwater ecosystems is unknown and largely unexplored. Field studies carried out at two discrete sites in coastal Virginia (USA) were used to characterize the viral load carried by runoff and to test the hypothesis that terrestrial viruses introduced through stormwater runoff change the composition of freshwater microbial communities. Field data gathered from an agricultural watershed indicated that primary runoff can contain viral densities approximating those of receiving waters. Furthermore, viruses attached to suspended colloids made up a large fraction of the total load, particularly in early stages of the storm. At a second field site (stormwater retention pond), RAPD-PCR profiling showed that the viral community of the pond changed dramatically over the course of two intense storms while relatively little change was observed over similar time scales in the absence of disturbance. Comparisons of planktonic and particle-associated viral communities revealed two completely distinct communities, suggesting that particle-associated viruses represent a potentially large and overlooked portion of aquatic viral abundance and diversity. Our findings show that stormwater runoff can quickly change the composition of freshwater microbial communities. Based on these findings, increased storms in the coastal mid-Atlantic region predicted by most climate change models will likely have important impacts on the structure and function of local freshwater microbial communities.

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

PubMed Central

Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Kato, Shin-ichiro; Sato, Miho; Ujihara, Takako; Matsui, Hidehito; Hanaki, Hideaki; Daibata, Masanori; Matsuzaki, Shigenobu

2014-01-01

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13′ (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13′, open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses. PMID:24591378

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja Saponaria Molina

PubMed Central

Tam, Ka Ian; Roner, Michael R.

2011-01-01

Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive. We have demonstrated that Quillaja extracts have strong antiviral activities in vitro against six different viruses. In this study, we evaluated the in vivo antiviral activity of these extracts against rhesus rotavirus (RRV) using a mouse model. We established that at a dosage of 0.015 mg/mouse of saponin extract, RRV induced diarrhea can be significantly reduced from 79% to 11% when mice are exposed to 500 plaque-forming-units (PFU) for each of five consecutive days. Additionally, while a reduction of RRV induced diarrhea depended both on the concentration of virus introduced and on the amount of Quillaja extract given to each mouse, the severity and interval of diarrhea under a variety of conditions tested, in all the treated mice were greatly reduced when compared to those that did not receive the Quillaja extracts. Mechanistically, there is strong evidence that the Quillaja extracts are able to “block” rotavirus infection by inhibiting virus-host attachment through disruption of cellular membrane proteins and/or virus receptors. We believe that Quillaja extracts have promise as antivirals to reduce rotavirus infection and the severity of the disease in humans. PMID:21549151