Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

PubMed Central

Song, Haifeng; Janosko, Krisztina; Johnson, Reed F.; Qin, Jing; Josleyn, Nicole; Jett, Catherine; Byrum, Russell; Claire, Marisa St.; Dyall, Julie; Blaney, Joseph E.; Jennings, Gerald; Jahrling, Peter B.

2013-01-01

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×107 PFU) or CPXV (>102 PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection. PMID:23577120

Localized cowpox infection in a 5-month-old Rottweiler.

PubMed

von Bomhard, Wolf; Mauldin, Elizabeth A; Breuer, Wolfram; Pfleghaar, Stephan; Nitsche, Andreas

2011-02-01

Cowpox virus (CPXV) infections are a sporadic cause of localized or disseminated skin disease in domestic animals and humans in Europe. Rodents are considered the primary reservoir host for CPXV. Cats can become infected by close contact with rodents and are the most important source of human infections. Recently, public awareness has also been drawn to CPXV infections by an outbreak of rat to human infections in central Europe. In dogs, CPXV infections are rare. Here we report a case of a 5-month-old Rottweiler with a focal nodule on the muzzle. The lesion was fully excised, and recovery was uneventful. The preliminary diagnosis of a CPXV infection was established by the characteristic inclusion bodies on histopathological examination. The diagnosis was confirmed by electron microscopy and polymerase chain reaction (PCR). Sequencing of the PCR product led to a 231 bp sequence of the orthopoxvirus HA gene that was identical to a CPXV strain previously isolated from a cat. This is the third documented case of a canine CPXV infection. © 2010 The Authors. Journal compilation © 2010 ESVD and ACVD.

Variola Virus-Specific Diagnostic Assays: Characterization, Sensitivity, and Specificity

PubMed Central

Kondas, Ashley V.; Olson, Victoria A.; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard

2015-01-01

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified. PMID:25673790

Virus and Infections 2010 - BIT's first world congress.

PubMed

Garkavenko, Olga

2010-10-01

The World Congress of Virus and Infections, held in Busan, South Korea, included topics reviewing the field of zoonoses. This conference report highlights selected presentations on surveillance, epidemiology and measures for the control and prevention of zoonotic diseases. Topics discussed include human factors influencing zoonoses, the molecular epidemiology of Crimean-Congo hemorrhagic fever, the emerging Nipah virus, and the re-emergence of cowpox virus.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

PubMed

Paran, Nir; Lustig, Shlomo; Zvi, Anat; Erez, Noam; Israely, Tomer; Melamed, Sharon; Politi, Boaz; Ben-Nathan, David; Schneider, Paula; Lachmi, Batel; Israeli, Ofir; Stein, Dana; Levin, Reuven; Olshevsky, Udy

2013-07-10

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response

PubMed Central

2013-01-01

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104–120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope’s critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox. PMID:23842430

Pre-symptomatic Prediction of Illness in Mice Inoculated with Cowpox

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

Kercher, J R; Colston, Jr., B W; Langlois, R G

2007-04-19

We describe here research directed towards early (presyndromic) diagnosis of infection. By using a mouse model and a multi-component blood protein diagnostic tool we detected cowpox infection several days in advance of overt symptoms with high accuracy. We provide details of the experimental design and measurement technique and elaborate on the long-range implication of these results.

Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

PubMed

Kondas, Ashley V; Olson, Victoria A; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard; Damon, Inger K

2015-04-01

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Rodents and Risk in the Mekong Delta of Vietnam: Seroprevalence of Selected Zoonotic Viruses in Rodents and Humans

PubMed Central

Van Cuong, Nguyen; Carrique-Mas, Juan; Vo Be, Hien; An, Nguyen Ngoc; Tue, Ngo Tri; Anh, Nguyet Lam; Anh, Pham Hong; Phuc, Nguyen The; Baker, Stephen; Voutilainen, Liina; Jääskeläinen, Anne; Huhtamo, Eili; Utriainen, Mira; Sironen, Tarja; Vaheri, Antti; Henttonen, Heikki; Vapalahti, Olli; Chaval, Yannick

2015-01-01

Abstract In the Mekong Delta in southern Vietnam, rats are commonly traded in wet markets and sold live for food consumption. We investigated seroprevalence to selected groups of rodent-borne viruses among human populations with high levels of animal exposure and among co-located rodent populations. The indirect fluorescence antibody test (IFAT) was used to determine seropositivity to representative reference strains of hantaviruses (Dobrava virus [DOBV], Seoul virus [SEOV]), cowpox virus, arenaviruses (lymphocytic choriomeningitis virus [LCMV]), flaviviruses (tick-borne encephalitis virus [TBEV]), and rodent parechoviruses (Ljungan virus), using sera from 245 humans living in Dong Thap Province and 275 rodents representing the five common rodent species sold in wet markets and present in peridomestic and farm settings. Combined seropositivity to DOBV and SEOV among the rodents and humans was 6.9% (19/275) and 3.7% (9/245), respectively; 1.1% (3/275) and 4.5% (11/245) to cowpox virus; 5.4% (15/275) and 47.3% (116/245) for TBEV; and exposure to Ljungan virus was 18.8% (46/245) in humans, but 0% in rodents. Very little seroreactivity was observed to LCMV in either rodents (1/275, 0.4%) or humans (2/245, 0.8%). Molecular screening of rodent liver tissues using consensus primers for flaviviruses did not yield any amplicons, whereas molecular screening of rodent lung tissues for hantavirus yielded one hantavirus sequence (SEOV). In summary, these results indicate low to moderate levels of endemic hantavirus circulation, possible circulation of a flavivirus in rodent reservoirs, and the first available data on human exposures to parechoviruses in Vietnam. Although the current evidence suggests only limited exposure of humans to known rodent-borne diseases, further research is warranted to assess public health implications of the rodent trade. PMID:25629782

Smallpox virus resequencing GeneChips can also rapidly ascertain species status for some zoonotic non-variola orthopoxviruses.

PubMed

Sulaiman, Irshad M; Sammons, Scott A; Wohlhueter, Robert M

2008-04-01

We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.

Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease

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

Johnson, Reed F.; Hammoud, Dima A.; Lackemeyer, Matthew G.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log{submore » 10} PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases. - Highlights: • Small particle aerosol exposure of rhesus results in a severe respiratory disease. • CT findings correlated with peripheral oxygen saturation and monocyte increases. • Virus dissemination was limited and mainly confined to the respiratory tract. • CT provides insight into pathogenesis to aid development of animal models of disease.« less

Revisiting Jenner's mysteries, the role of the Beaugency lymph in the evolutionary path of ancient smallpox vaccines.

PubMed

Damaso, Clarissa R

2018-02-01

In 1796, Edward Jenner developed the smallpox vaccine consisting of pustular material obtained from lesions on cows affected by so-called cow-pox. The disease, caused by cowpox virus, confers crossprotection against smallpox. However, historical evidence suggests that Jenner might have used vaccinia virus or even horsepox virus instead of cowpox virus. Mysteries surrounding the origin and nature of the smallpox vaccine persisted during the 19th century, a period of intense exchange of vaccine strains, including the Beaugency lymph. This lymph was obtained from spontaneous cases of cow-pox in France in 1866 and then distributed worldwide. A detailed Historical Review of the distribution of the Beaugency lymph supports recent genetic analyses of extant vaccine strains, suggesting the lymph was probably a vaccinia strain or a horsepox-like virus. This Review is a historical investigation that revisits the mysteries of the smallpox vaccine and reveals an intricate evolutionary relationship of extant vaccinia strains. Copyright © 2018 Elsevier Ltd. All rights reserved.

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

PubMed

Smithson, Chad; Imbery, Jacob; Upton, Chris

2017-09-08

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses.

PubMed

Molero-Abraham, Magdalena; Glutting, John-Paul; Flower, Darren R; Lafuente, Esther M; Reche, Pedro A

2015-01-01

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses

PubMed Central

Molero-Abraham, Magdalena; Glutting, John-Paul; Flower, Darren R.; Lafuente, Esther M.; Reche, Pedro A.

2015-01-01

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes. PMID:26605344

Bayesian reconstruction of the evolutionary history and cross-species transition of variola virus and orthopoxviruses.

PubMed

Zehender, Gianguglielmo; Lai, Alessia; Veo, Carla; Bergna, Annalisa; Ciccozzi, Massimo; Galli, Massimo

2018-06-01

Variola virus (VARV), the causative agent of smallpox, is an exclusively human virus belonging to the genus Orthopoxvirus, which includes many other viral species covering a wide range of mammal hosts, such as vaccinia, cowpox, camelpox, taterapox, ectromelia, and monkeypox virus. The tempo and mode of evolution of Orthopoxviruses were reconstructed using a Bayesian phylodynamic framework by analysing 80 hemagglutinin sequences retrieved from public databases. Bayesian phylogeography was used to estimate their putative ancestral hosts. In order to estimate the substitution rate, the tree including all of the available Orthopoxviruses was calibrated using historical references dating the South American variola minor clade (alastrim) to between the XVI and XIX century. The mean substitution rate determined by the analysis was 6.5 × 10 -6 substitutions/site/year. Based on this evolutionary estimate, the time of the most recent common ancestor of the genus Orthopoxvirus was placed at about 10 000 years before the present. Cowpox virus was the species closest to the root of the phylogenetic tree. The root of VARV circulating in the XX century was estimated to be about 700 years ago, corresponding to about 1300 AD. The divergence between West African and South American VARV went back about 500 years ago (falling approximately in the XVI century). A rodent species is the most probable ancestral host from which the ancestors of all the known Orthopoxviruses were transmitted to the other mammal host species, and each of these species represented a dead-end for each new poxvirus species, without any further inter-specific spread. © 2018 Wiley Periodicals, Inc.

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

Americo, Jeffrey L.; Sood, Cindy L.; Cotter, Catherine A.

Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chestmore » and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies. - Highlights: • Wild-derived inbred CAST/Ei mice are susceptible to vaccinia virus and cowpox virus. • Morbidity and mortality from orthopoxviruses are greater in CAST/Ei than BALB/c mice. • Morbidity and mortality from herpes simplex virus type 1 are similar in both mice. • Imaging shows virus spread from nose to lungs, abdominal organs and brain. • Vaccinia virus spreads more rapidly than cowpox virus.« less

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus.

PubMed

Spik, Kristin; Shurtleff, Amy; McElroy, Anita K; Guttieri, Mary C; Hooper, Jay W; SchmalJohn, Connie

2006-05-22

DNA vaccines for Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines.

In vitro inhibition of monkeypox virus production and spread by Interferon-β

PubMed Central

2012-01-01

Background The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. Results The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-β (IFN-β) for use against monkeypox virus. We found that treatment with human IFN-β results in a significant decrease in monkeypox virus production and spread in vitro. IFN-β substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-β induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. Conclusions Our results demonstrate the successful inhibition of monkeypox virus using human IFN-β and suggest that IFN-β could potentially serve as a novel safe therapeutic for human monkeypox disease. PMID:22225589

Genome of Horsepox Virus

PubMed Central

Tulman, E. R.; Delhon, G.; Afonso, C. L.; Lu, Z.; Zsak, L.; Sandybaev, N. T.; Kerembekova, U. Z.; Zaitsev, V. L.; Kutish, G. F.; Rock, D. L.

2006-01-01

Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses. PMID:16940536

Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex.

PubMed

van Buuren, Nick; Couturier, Brianne; Xiong, Yue; Barry, Michele

2008-10-01

Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex.

Genome Sequences of Akhmeta Virus, an Early Divergent Old World Orthopoxvirus.

PubMed

Gao, Jinxin; Gigante, Crystal; Khmaladze, Ekaterine; Liu, Pengbo; Tang, Shiyuyun; Wilkins, Kimberly; Zhao, Kun; Davidson, Whitni; Nakazawa, Yoshinori; Maghlakelidze, Giorgi; Geleishvili, Marika; Kokhreidze, Maka; Carroll, Darin S; Emerson, Ginny; Li, Yu

2018-05-12

Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure to that of the cowpox virus (CPXV), but a lower sequence identity was found between AKMV and Old World OPXVs than between other known species of Old World OPXVs. Phylogenetic analysis showed that AKMV diverged prior to other Old World OPXV. AKMV isolates formed a monophyletic clade in the OPXV phylogeny, yet the sequence variability between AKMV isolates was higher than between the monkeypox virus strains in the Congo basin and West Africa. An AKMV isolate from Vani contained approximately six kb sequence in the left terminal region that shared a higher similarity with CPXV than with other AKMV isolates, whereas the rest of the genome was most similar to AKMV, suggesting recombination between AKMV and CPXV in a region containing several host range and virulence genes.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

PubMed

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-06-01

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

Development of an Epidemiological Database Management, Extraction, and Analysis System (EPISYS)

DTIC Science & Technology

1992-07-01

Hepatitis 62 1 Rabies 63 1 Mumps 64 4 Psittacosis 65 1 Coxsackie Virus Diseases 66 1 Infectious Mononucleosis 67 5 Trachoma 68 1 Other Viral Disease...Rabies Cowpox Mumps Other Viral Exanthemata Coxsackie Virus Yellow Fever Infectious Mononucleosis Dengue Other Viral Diseases. Conjunctiva Mosquito

Chromatographic removal combined with heat, acid and chaotropic inactivation of four model viruses.

PubMed

Valdés, R; Ibarra, Neysi; Ruibal, I; Beldarraín, A; Noa, E; Herrera, N; Alemán, R; Padilla, S; Garcia, J; Pérez, M; Morales, R; Chong, E; Reyes, B; Quiñones, Y; Agraz, A; Herrera, L

2002-07-03

The virus removal of protein A affinity chromatography, inactivation capacity, acid pH and a combination of high temperature with a chaotropic agent was determined in this work. The model viruses studied were sendaivirus, human immunodeficency virus (HIV-IIIb), human poliovirus type-II, human herpesvirus I and canine parvovirus. The protein A affinity chromatography showed a maximum reduction factor of 8 logs in the case of viruses larger than 120 nm size, while for small viruses (18-30 nm) the maximum reduction factor was about 5 logs. Non viral inactivation was observed during the monoclonal antibody elution step. Low pH treatment showed a maximum inactivation factor of 7.1 logs for enveloped viruses. However, a weak inactivation factor (3.4 logs) was obtained for DNA nonenveloped viruses. The combination of high temperature with 3 M KSCN showed a high inactivation factor for all of the viruses studied. The total clearance factor was 23.1, 15.1, 13.6, 20.0 and 16.0 logs for sendaivirus, HIV-IIIb, human poliovirus type-II, human herpesvirus I and canine parvovirus, respectively.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

Co-administration of the broad-spectrum antiviral, brincidofovir (CMX001), with smallpox vaccine does not compromise vaccine protection in mice challenged with ectromelia virus.

PubMed

Parker, Scott; Crump, Ryan; Foster, Scott; Hartzler, Hollyce; Hembrador, Ed; Lanier, E Randall; Painter, George; Schriewer, Jill; Trost, Lawrence C; Buller, R Mark

2014-11-01

Natural orthopoxvirus outbreaks such as vaccinia, cowpox, cattlepox and buffalopox continue to cause morbidity in the human population. Monkeypox virus remains a significant agent of morbidity and mortality in Africa. Furthermore, monkeypox virus's broad host-range and expanding environs make it of particular concern as an emerging human pathogen. Monkeypox virus and variola virus (the etiological agent of smallpox) are both potential agents of bioterrorism. The first line response to orthopoxvirus disease is through vaccination with first-generation and second-generation vaccines, such as Dryvax and ACAM2000. Although these vaccines provide excellent protection, their widespread use is impeded by the high level of adverse events associated with vaccination using live, attenuated virus. It is possible that vaccines could be used in combination with antiviral drugs to reduce the incidence and severity of vaccine-associated adverse events, or as a preventive in individuals with uncertain exposure status or contraindication to vaccination. We have used the intranasal mousepox (ectromelia) model to evaluate the efficacy of vaccination with Dryvax or ACAM2000 in conjunction with treatment using the broad spectrum antiviral, brincidofovir (BCV, CMX001). We found that co-treatment with BCV reduced the severity of vaccination-associated lesion development. Although the immune response to vaccination was quantifiably attenuated, vaccination combined with BCV treatment did not alter the development of full protective immunity, even when administered two days following ectromelia challenge. Studies with a non-replicating vaccine, ACAM3000 (MVA), confirmed that BCV's mechanism of attenuating the immune response following vaccination with live virus was, as expected, by limiting viral replication and not through inhibition of the immune system. These studies suggest that, in the setting of post-exposure prophylaxis, co-administration of BCV with vaccination should be considered

Companion Animals as a Source of Viruses for Human Beings and Food Production Animals.

PubMed

Reperant, L A; Brown, I H; Haenen, O L; de Jong, M D; Osterhaus, A D M E; Papa, A; Rimstad, E; Valarcher, J-F; Kuiken, T

2016-07-01

Companion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

PubMed

Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

2013-04-01

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

The origin of the variola virus.

PubMed

Babkin, Igor V; Babkina, Irina N

2015-03-10

The question of the origin of smallpox, one of the major menaces to humankind, is a constant concern for the scientific community. Smallpox is caused by the agent referred to as the variola virus (VARV), which belongs to the genus Orthopoxvirus. In the last century, smallpox was declared eradicated from the human community; however, the mechanisms responsible for the emergence of new dangerous pathogens have yet to be unraveled. Evolutionary analyses of the molecular biological genomic data of various orthopoxviruses, involving a wide range of epidemiological and historical information about smallpox, have made it possible to date the emergence of VARV. Comparisons of the VARV genome to the genomes of the most closely related orthopoxviruses and the examination of the distribution their natural hosts' ranges suggest that VARV emerged 3000 to 4000 years ago in the east of the African continent. The VARV evolution rate has been estimated to be approximately 2 × 10-6 substitutions/site/year for the central conserved genomic region and 4 × 10-6 substitutions/site/year for the synonymous substitutions in the genome. Presumably, the introduction of camels to Africa and the concurrent changes to the climate were the particular factors that triggered the divergent evolution of a cowpox-like ancestral virus and thereby led to the emergence of VARV.

Respiratory Syncytial Virus Isolation by Combined Continuous Flow-Isopycnic Banding Centrifugation

PubMed Central

Cline, G. B.; Coates, Helen; Anderson, N. G.; Chanock, R. M.; Harris, W. W.

1967-01-01

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition. PMID:5621468

Real-Time PCR Assay To Detect Smallpox Virus

PubMed Central

Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

2003-01-01

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

Characterization of an Artificial Swine-Origin Influenza Virus with the Same Gene Combination as H1N1/2009 Virus: A Genesis Clue of Pandemic Strain

PubMed Central

Pu, Juan; Fan, Lihong; Shi, Weimin; Hu, Yanxin; Yang, Jun; Xu, Qi; Wang, Jingjing; Hou, Dongjun; Ma, Guangpeng; Liu, Jinhua

2011-01-01

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus. PMID:21799774

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain.

PubMed

Zhao, Xueli; Sun, Yipeng; Pu, Juan; Fan, Lihong; Shi, Weimin; Hu, Yanxin; Yang, Jun; Xu, Qi; Wang, Jingjing; Hou, Dongjun; Ma, Guangpeng; Liu, Jinhua

2011-01-01

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.

Poxvirus-induced alteration of arachidonate metabolism.

PubMed Central

Palumbo, G J; Glasgow, W C; Buller, R M

1993-01-01

Recent evidence suggests that orthopoxviruses have an obligate requirement for arachidonic acid metabolites during replication in vivo and in vitro. Our report indicates that a virus family (Poxviridae) possesses multiple genes that function to regulate arachidonate metabolism. Analyses of BS-C-1 cells infected with cowpox virus or vaccinia virus detected enhanced arachidonate product formation from both the cyclooxygenase (specifically prostaglandins E2 and F2 alpha) and lipoxygenase (specifically 15-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid) pathways. In contrast, human parainfluenza type 3 or herpes simplex virus type 1 infections did not increase arachidonate metabolism. Results were consistent with a virus early-gene product either directly mediating or inducing a host factor that mediated the up-regulation of arachidonate metabolism, although vaccinia growth factor was not responsible. In addition, the cowpox virus 38-kDa protein-encoding gene, which is associated with inhibition of an inflammatory response, correlated with inhibition of formation of a product biochemically characteristic of (14R,15S)-dihydroxyeicosatetraenoic acid. We propose that orthopoxvirus-induced up-regulation of arachidonic acid metabolism during infection renders the infected cells susceptible to generation of inflammatory mediators from both the cyclooxygenase and the lipoxygenase pathways, and poxviruses, therefore, possess at least one gene (38K) that can alter the lipoxygenase-metabolite spectrum. PMID:8383332

A novel highly reproducible and lethal nonhuman primate model for orthopox virus infection.

PubMed

Kramski, Marit; Mätz-Rensing, Kerstin; Stahl-Hennig, Christiane; Kaup, Franz-Josef; Nitsche, Andreas; Pauli, Georg; Ellerbrok, Heinz

2010-04-29

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1-3 days after onset of symptoms, even when very low infectious viral doses of 5x10(2) pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID(50) (minimal monkey infectious dose 50%) of 8.3x10(2) pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

PubMed

Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

2017-04-01

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

PubMed Central

Das, Sanchita; Rundell, Mark S.; Mirza, Aashiq H.; Pingle, Maneesh R.; Shigyo, Kristi; Garrison, Aura R.; Paragas, Jason; Smith, Scott K.; Olson, Victoria A.; Larone, Davise H.; Spitzer, Eric D.; Barany, Francis; Golightly, Linnie M.

2015-01-01

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus). PMID:26381398

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

PubMed

Das, Sanchita; Rundell, Mark S; Mirza, Aashiq H; Pingle, Maneesh R; Shigyo, Kristi; Garrison, Aura R; Paragas, Jason; Smith, Scott K; Olson, Victoria A; Larone, Davise H; Spitzer, Eric D; Barany, Francis; Golightly, Linnie M

2015-01-01

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control.

PubMed

Simpson, Guy R; Han, Ziqun; Liu, Binlei; Wang, Yibing; Campbell, Gregor; Coffin, Robert S

2006-05-01

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5- to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous.

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice.

PubMed

Sakala, Isaac G; Chaudhri, Geeta; Scalzo, Anthony A; Eldi, Preethi; Newsome, Timothy P; Buller, Robert M; Karupiah, Gunasegaran

2015-12-01

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.

A Novel Highly Reproducible and Lethal Nonhuman Primate Model for Orthopox Virus Infection

PubMed Central

Kramski, Marit; Mätz-Rensing, Kerstin; Stahl-Hennig, Christiane; Kaup, Franz-Josef; Nitsche, Andreas; Pauli, Georg; Ellerbrok, Heinz

2010-01-01

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus). A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1–3 days after onset of symptoms, even when very low infectious viral doses of 5×102 pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed. We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID50 (minimal monkey infectious dose 50%) of 8.3×102 pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis. PMID

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

PubMed

Gangadhara, Sailaja; Kwon, Young-Man; Jeeva, Subbiah; Quan, Fu-Shi; Wang, Baozhong; Moss, Bernard; Compans, Richard W; Amara, Rama Rao; Jabbar, M Abdul; Kang, Sang-Moo

2017-12-19

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

PubMed Central

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-01-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses. PMID:27752100

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

NASA Astrophysics Data System (ADS)

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-10-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses.

Orthopoxvirus Genome Evolution: The Role of Gene Loss

PubMed Central

Hendrickson, Robert Curtis; Wang, Chunlin; Hatcher, Eneida L.; Lefkowitz, Elliot J.

2010-01-01

Poxviruses are highly successful pathogens, known to infect a variety of hosts. The family Poxviridae includes Variola virus, the causative agent of smallpox, which has been eradicated as a public health threat but could potentially reemerge as a bioterrorist threat. The risk scenario includes other animal poxviruses and genetically engineered manipulations of poxviruses. Studies of orthologous gene sets have established the evolutionary relationships of members within the Poxviridae family. It is not clear, however, how variations between family members arose in the past, an important issue in understanding how these viruses may vary and possibly produce future threats. Using a newly developed poxvirus-specific tool, we predicted accurate gene sets for viruses with completely sequenced genomes in the genus Orthopoxvirus. Employing sensitive sequence comparison techniques together with comparison of syntenic gene maps, we established the relationships between all viral gene sets. These techniques allowed us to unambiguously identify the gene loss/gain events that have occurred over the course of orthopoxvirus evolution. It is clear that for all existing Orthopoxvirus species, no individual species has acquired protein-coding genes unique to that species. All existing species contain genes that are all present in members of the species Cowpox virus and that cowpox virus strains contain every gene present in any other orthopoxvirus strain. These results support a theory of reductive evolution in which the reduction in size of the core gene set of a putative ancestral virus played a critical role in speciation and confining any newly emerging virus species to a particular environmental (host or tissue) niche. PMID:21994715

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication.

PubMed

Bell, Todd M; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Carey, Brian D; Lin, Shih-Chao; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A; Bailey, Charles L; Kehn-Hall, Kylene

2018-04-13

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication

PubMed Central

Bell, Todd M.; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A.; Bailey, Charles L.

2018-01-01

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection. PMID:29652799

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

A Combination in-ovo Vaccine for Avian Influenza Virus and Newcastle Disease Virus

PubMed Central

Steel, John; Burmakina, Svetlana V.; Thomas, Colleen; Spackman, Erica; García-Sastre, Adolfo; Swayne, David E.; Palese, Peter

2008-01-01

The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing; (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin – neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park, M.S., et al., 2006. Proc Natl Acad Sci U S A, 103 (21), 8203–8208). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine. PMID:18093698

A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus.

PubMed

Steel, John; Burmakina, Svetlana V; Thomas, Colleen; Spackman, Erica; García-Sastre, Adolfo; Swayne, David E; Palese, Peter

2008-01-24

The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing, (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park MS, et al. Proc Natl Acad Sci USA 2006;103(21):8203-8). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus, respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine.

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

PubMed

Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R

2001-04-01

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Therapeutic and prophylactic drugs to treat orthopoxvirus infections.

PubMed

Parker, Scott; Handley, Lauren; Buller, R Mark

2008-11-01

With the global eradication of smallpox in 1979, the causative agent, variola, no longer circulates in human populations. Other human poxvirus infections, such as those caused by vaccinia, cowpox virus and molluscum, are usually relatively benign in immunocompetent individuals. Conversely, monkeypox virus infections cause high levels of mortality and morbidity in Africa and the virus appears to be increasing its host range, virulence and demographic environs. Furthermore, there are concerns that clandestine stocks of variola virus exist. The re-introduction of aerosolized variola (or perhaps monkeypox virus) into human populations would result in high levels of morbidity and mortality. The attractiveness of variola as a bioweapon and, to a certain extent, monkeypox virus is its inherent ability to spread from person-to-person. The threat posed by the intentional release of variola or monkeypox virus, or a monkeypox virus epizoonosis, will require the capacity to rapidly diagnose the disease and to intervene with antivirals, as intervention is likely to take place during the initial diagnosis, approximately 10-15 days postinfection. Preimmunization of 'at-risk populations' with vaccines will likely not be practical, and the therapeutic use of vaccines has been shown to be ineffective after 4 days of infection with variola. However, a combination of vaccine and antivirals for those infected may be an option. Here we describe historical, current and future therapies to treat orthopoxvirus diseases.

Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

PubMed

Gao, Yan; Su, Qiudong; Yi, Yao; Jia, Zhiyuan; Wang, Hao; Lu, Xuexin; Qiu, Feng; Bi, Shengli

2015-01-01

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2) sequence (aa 368-607) of HEV (HE-ORF2) and partial virus protein 1 (VP1) sequence (aa 1-198) of HAV (HA-VP1), which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG) showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG). Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection.

Pigs with severe combined immunodeficiency (SCID) are impaired in controlling influenza A virus infection

USDA-ARS?s Scientific Manuscript database

Influenza A viruses (IAV) infect many host species, including humans and pigs. Severe Combined Immunodeficiency (SCID) is a condition characterized by a lack of T, B, and/or natural killer (NK) cells. Animal models of SCID have great value for biomedical research. Here, we evaluated the pathogenesis...

Combining mutualistic yeast and pathogenic virus - a novel method for control for codling moth control

USDA-ARS?s Scientific Manuscript database

Studies evaluated the lethal effectiveness of combining yeasts isolated from larvae of codling moth, Cydia pomonella (L.) with the codling moth granulosis virus (CpGV). Apples were treated with CpGV and three yeast species, including Metschnikowia pulcherrima Pitt and Miller, Cryptococcus tephrensis...

Safety, immunogenicity and protective efficacy in mice of a new cell-cultured Lister smallpox vaccine candidate.

PubMed

Ferrier-Rembert, Audrey; Drillien, Robert; Meignier, Bernard; Garin, Daniel; Crance, Jean-Marc

2007-11-28

It is now difficult to manufacture the first-generation smallpox vaccine, as the process could not comply with current safety and manufacturing regulations. In this study, a candidate non-clonal second-generation smallpox vaccine developed by Sanofi-Pasteur from the Lister strain has been assessed using a cowpox virus challenge in mice. We have observed similar safety, immunogenicity and protection (from disease and death) after a short or long interval following vaccination, as well as similar virus clearance post-challenge, with the second-generation smallpox vaccine candidate as compared to the traditional vaccine used as a benchmark.

The experimental production of combination forms of virus. II. A study of serial passage in the allantoic sac of agents that combine the antigens of two distinct influenza A strains.

PubMed

HIRST, G K; GOTLIEB, T

1953-07-01

Double infection of the allantoic sac with Melbourne and WSN viruses induced the formation of a combination virus, which had some of the antigenic properties of both parents and which maintained itself through serial passage in the chorioallantoic sac. In the course of prolonged passage in the egg, three varieties of combination virus were found. The first (X(1)) occurred in ten passages and was characterized as follows: X(1) was produced in and could be successfully passed from those chick embryos that had received very large inocula of virus. The X(1) hemagglutinin was efficiently inhibited by both M and W antisera. Each passage fluid, containing X(1) as the predominant strain, also contained large amounts of the parent forms M and W. It is very likely that X(1) reverted to parent types of virus at a high rate. The second variety (X(2)) arose from the passage series of X(1) and was carried for approximately 20 passages without definite alteration in its character. It differed from X(1) in that it had the ability to appear as the predominant form occasionally in embryos that had received a limiting infective dilution of virus. Because of this, it seems probable that X(2) reverted to parent types at a slower rate than X(1). Like X(1), this virus was never obtained in pure form and suspensions in which it predominated contained large amounts of M and W, especially the former. Good evidence was obtained that X(2) reverted to M and W virus while multiplying in the chorioallantoic sac. X(3) was derived from passage of X(2) virus after 32 transfers and can be characterized in several ways: (a) X(3) yielded the only X(3) fluids on passage in eggs. This was in striking contrast to X(2) which could not be passed without giving rise to some fluids in which M and W predominated, (b) The X(3) hemagglutinin was weakly inhibited by all but the highest concentrations of M serum, but was strongly inhibited by W serum. X(2) was readily inhibited by relatively small amounts of both

Fatal combined infection with canine distemper virus and orthopoxvirus in a group of Asian marmots (Marmota caudata).

PubMed

Origgi, F C; Sattler, U; Pilo, P; Waldvogel, A S

2013-09-01

A fatal combined infection with canine distemper virus (CDV) and orthopoxvirus (OPXV) in Asian marmots (Marmota caudata) is reported in this article. A total of 7 Asian marmots from a small zoological garden in Switzerland were found dead in hibernation during a routine check in the winter of 2011. The marmots died in February 2011. No clinical signs of disease were observed at any time. The viruses were detected in all individuals for which the tissues were available (n = 3). Detection of the viruses was performed by reverse transcription polymerase chain reaction. The most consistent gross lesion was a neck and thorax edema. A necrotizing pharyngitis and a multifocal necrotizing pneumonia were observed histologically. Numerous large intracytoplasmic eosinophilic inclusions were seen in the epithelial cells of the pharynx, of the airways, and in the skin keratinocytes. Brain lesions were limited to mild multifocal gliosis. Phylogenetic analysis revealed that the marmot CDV strain was closely related to the clusters of CDVs detected in Switzerland in wild carnivores during a local outbreak in 2002 and the 2009-2010 nationwide epidemic, suggesting a spillover of this virus from wildlife. The OPXV was most closely related to a strain of cowpoxvirus, a poxvirus species considered endemic in Europe. This is the first reported instance of CDV infection in a rodent species and of a combined CDV and OPXV infection.

Treatment strategies for combining immunostimulatory oncolytic virus therapeutics with dendritic cell injections.

PubMed

Wares, Joanna R; Crivelli, Joseph J; Yun, Chae-Ok; Choi, Il-Kyu; Gevertz, Jana L; Kim, Peter S

2015-12-01

Oncolytic viruses (OVs) are used to treat cancer, as they selectively replicate inside of and lyse tumor cells. The efficacy of this process is limited and new OVs are being designed to mediate tumor cell release of cytokines and co-stimulatory molecules, which attract cytotoxic T cells to target tumor cells, thus increasing the tumor-killing effects of OVs. To further promote treatment efficacy, OVs can be combined with other treatments, such as was done by Huang et al., who showed that combining OV injections with dendritic cell (DC) injections was a more effective treatment than either treatment alone. To further investigate this combination, we built a mathematical model consisting of a system of ordinary differential equations and fit the model to the hierarchical data provided from Huang et al. We used the model to determine the effect of varying doses of OV and DC injections and to test alternative treatment strategies. We found that the DC dose given in Huang et al. was near a bifurcation point and that a slightly larger dose could cause complete eradication of the tumor. Further, the model results suggest that it is more effective to treat a tumor with immunostimulatory oncolytic viruses first and then follow-up with a sequence of DCs than to alternate OV and DC injections. This protocol, which was not considered in the experiments of Huang et al., allows the infection to initially thrive before the immune response is enhanced. Taken together, our work shows how the ordering, temporal spacing, and dosage of OV and DC can be chosen to maximize efficacy and to potentially eliminate tumors altogether.

Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

NASA Technical Reports Server (NTRS)

Gridley, D. S.; Andres, M. L.; Li, J.; Timiryasova, T.; Chen, B.; Fodor, I.; Nelson, G. A. (Principal Investigator)

1998-01-01

The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.

PubMed

Nolasco, G; de Blas, C; Torres, V; Ponz, F

1993-12-15

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Modeling and Control of a Delayed Hepatitis B Virus Model with Incubation Period and Combination Treatment.

PubMed

Sun, Deshun; Liu, Fei

2018-06-01

In this paper, a hepatitis B virus (HBV) model with an incubation period and delayed state and control variables is firstly proposed. Furthermore, the combination treatment is adopted to have a longer-lasting effect than mono-therapy. The equilibrium points and basic reproduction number are calculated, and then the local stability is analyzed on this model. We then present optimal control strategies based on the Pontryagin's minimum principle with an objective function not only to reduce the levels of exposed cells, infected cells and free viruses nearly to zero at the end of therapy, but also to minimize the drug side-effect and the cost of treatment. What's more, we develop a numerical simulation algorithm for solving our HBV model based on the combination of forward and backward difference approximations. The state dynamics of uninfected cells, exposed cells, infected cells, free viruses, CTL and ALT are simulated with or without optimal control, which show that HBV is reduced nearly to zero based on the time-varying optimal control strategies whereas the disease would break out without control. At last, by the simulations, we prove that strategy A is the best among the three kinds of strategies we adopt and further comparisons have been done between model (1) and model (2).

The Appropriate Combination of Hemagglutinin and Neuraminidase Prompts the Predominant H5N6 Highly Pathogenic Avian Influenza Virus in Birds.

PubMed

Wang, Xiuhui; Zeng, Zhaoyong; Zhang, Zaoyue; Zheng, Yi; Li, Bo; Su, Guanming; Li, Huanan; Huang, Lihong; Qi, Wenbao; Liao, Ming

2018-01-01

Haemagglutinin (HA) and neuraminidase (NA) are two vital surface glycoproteins of influenza virus. The HA of H5N6 highly pathogenic avian influenza virus is divided into Major/H5 and Minor/H5, and its NA consists of short stalk NA and full-length stalk NA. The strain combined with Major/H5 and short stalk NA account for 76.8% of all strains, and the proportion was 23.0% matched by Minor/H5 and full-length stalk NA. Our objective was to investigate the influence of HA-NA matching on the biological characteristics and the effects of the epidemic trend of H5N6 on mice and chickens. Four different strains combined with two HAs and two NAs of the represented H5N6 viruses with the fixed six internal segments were rescued and analyzed. Plaque formation, NA activity of infectious particles, and virus growth curve assays, as well as a saliva acid receptor experiment, with mice and chickens were performed. We found that all the strains can replicate well on Madin-Darby canine kidney (MDCK) cells and chicken embryo fibroblasts (CEF) cells, simultaneously, mice and infection group chickens were complete lethal. However, the strain combined with Major/H5 and short stalk N6 formed smaller plaque on MDCK, showed a moderate replication ability in both MDCK and CEF, and exhibited a higher survival rate among the contact group of chickens. Conversely, strains with opposite biological characters which combined with Minor/H5 and short stalk N6 seldom exist in nature. Hence, we drew the conclusion that the appropriate combination of Major/H5 and short stalk N6 occur widely in nature with appropriate biological characteristics for the proliferation and transmission, whereas other combinations of HA and NA had a low proportion and even have not yet been detected.

A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.

PubMed Central

Rochford, R; Campbell, B A; Villarreal, L P

1987-01-01

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered. Images PMID:3025873

Evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges.

PubMed

Abdelmagid, Omar Y; Larson, Laurie; Payne, Laurie; Tubbs, Anna; Wasmoen, Terri; Schultz, Ronald

2004-01-01

The results of this study confirmed that dogs vaccinated subcutaneously with a commercially available multivalent vaccine containing modified-live canine distemper virus, canine adenovirus type 2, canine parvovirus type 2b, and canine parainfluenza virus antigens were protected against sequential experimental challenge 55 to 57 months after initial vaccination given at 7 to 8 weeks of age. All 10 vaccinates were protected against clinical diseases and mortality following parvovirus and infectious canine hepatitis experimental infections. All vaccinates were protected against mortality and 90% against clinical disease following distemper challenge. These data support at least a 4-year duration of immunity for these three "core" fractions in the combination vaccine.

Postnatal Passive Immunization of Neonatal Macaques with a Triple Combination of Human Monoclonal Antibodies against Oral Simian-Human Immunodeficiency Virus Challenge

PubMed Central

Hofmann-Lehmann, Regina; Vlasak, Josef; Rasmussen, Robert A.; Smith, Beverly A.; Baba, Timothy W.; Liska, Vladimir; Ferrantelli, Flavia; Montefiori, David C.; McClure, Harold M.; Anderson, Daniel C.; Bernacky, Bruce J.; Rizvi, Tahir A.; Schmidt, Russell; Hill, Lori R.; Keeling, Michale E.; Katinger, Hermann; Stiegler, Gabriela; Cavacini, Lisa A.; Posner, Marshall R.; Chou, Ting-Chao; Andersen, Janet; Ruprecht, Ruth M.

2001-01-01

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351–357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu+ challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200–206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu+ challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4+ T-cell decline. In contrast, all control animals had dramatic drops in their CD4+ T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1. PMID:11462019

An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein.

PubMed

Butler-Cole, Christine; Wagner, Mary J; Da Silva, Melissa; Brown, Gordon D; Burke, Robert D; Upton, Chris

2007-07-24

Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins. Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

PubMed Central

Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

2015-01-01

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox. PMID:26426117

Rational design and adaptive management of combination therapies for Hepatitis C virus infection

DOE PAGES

Ke, Ruian; Loverdo, Claude; Qi, Hangfei; ...

2015-06-30

Recent discoveries of direct acting antivirals against Hepatitis C virus (HCV) have raised hopes of effective treatment via combination therapies. Yet rapid evolution and high diversity of HCV populations, combined with the reality of suboptimal treatment adherence, make drug resistance a clinical and public health concern. We develop a general model incorporating viral dynamics and pharmacokinetics/ pharmacodynamics to assess how suboptimal adherence affects resistance development and clinical outcomes. We derive design principles and adaptive treatment strategies, identifying a high-risk period when missing doses is particularly risky for de novo resistance, and quantifying the number of additional doses needed to compensatemore » when doses are missed. Using data from large-scale resistance assays, we demonstrate that the risk of resistance can be reduced substantially by applying these principles to a combination therapy of daclatasvir and asunaprevir. By providing a mechanistic framework to link patient characteristics to the risk of resistance, these findings show the potential of rational treatment design.« less

Theaflavin-3,3′-Digallate and Lactic Acid Combinations Reduce Herpes Simplex Virus Infectivity

PubMed Central

Xu, Weimin

2013-01-01

The present study examined the efficacy of using multiple mechanisms as part of a topical microbicide to inactivate herpes simplex virus (HSV) by combining theaflavin-3,3′-digallate (TF-3) and lactic acid (LA) over the pH range of 4.0 to 5.7 to mimic conditions in the female reproductive tract. Six clinical isolates of HSV-2 and two clinical isolates of HSV-1 were almost completely inactivated when TF-3 (100 μM) was present with LA over the pH range of 4.5 to 5.7, whereas four additional HSV-1 clinical isolates required TF-3 concentrations of 250 to 500 μM for comparable virus titer reduction. LA (1%) alone at pH 4.0 reduced the titers of laboratory and clinical isolates of HSV-1 and HSV-2 by ≥5 log10, but most LA-dependent antiviral activity was lost at a pH of ≥4.5. When HSV-1 and HSV-2 were incubated at pH 4.0 without LA virus titers were not reduced. At pH 4.0, HSV-1 and HSV-2 titers were reduced 5 log10 in 20 min by LA alone. TF-3 reduced HSV-2 titers by 5 log10 in 20 to 30 min at pH 4.5, whereas HSV-1 required 60 min for comparable inactivation. Mixtures of TF-3 and LA stored at 37°C for 1 month at pH 4.0 to 5.7 maintained antiviral activity. Semen, but not cervical vaginal fluid, decreased LA-dependent antiviral activity at pH 4.0, but adding TF-3 to the mixture reduced HSV titers by 4 to 5 log10. These results indicate that a combination microbicide containing TF-3 and LA could reduce HSV transmission. PMID:23716050

Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

PubMed Central

Le Boeuf, Fabrice; Lemay, Chantal; De Silva, Naomi; Diallo, Jean-Simon; Cox, Julie; Becker, Michelle; Choi, Youngmin; Ananth, Abhirami; Sellers, Clara; Breton, Sophie; Roy, Dominic; Falls, Theresa; Brun, Jan; Hemminki, Akseli; Hinkkanen, Ari; Bell, John C.

2013-01-01

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. PMID:23221568

Yellow fever vector live-virus vaccines: West Nile virus vaccine development.

PubMed

Arroyo, J; Miller, C A; Catalan, J; Monath, T P

2001-08-01

By combining molecular-biological techniques with our increased understanding of the effect of gene sequence modification on viral function, yellow fever 17D, a positive-strand RNA virus vaccine, has been manipulated to induce a protective immune response against viruses of the same family (e.g. Japanese encephalitis and dengue viruses). Triggered by the emergence of West Nile virus infections in the New World afflicting humans, horses and birds, the success of this recombinant technology has prompted the rapid development of a live-virus attenuated candidate vaccine against West Nile virus.

Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches

PubMed Central

Fu, Xu; Wang, Zhihua; Li, Lixin; Dong, Shishang; Li, Zhucui; Jiang, Zhenzuo; Wang, Yuefei; Shui, Wenqing

2016-01-01

The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development. PMID:27403722

Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection.

PubMed

Koizumi, Yoshiki; Ohashi, Hirofumi; Nakajima, Syo; Tanaka, Yasuhito; Wakita, Takaji; Perelson, Alan S; Iwami, Shingo; Watashi, Koichi

2017-02-21

With the introduction of direct-acting antivirals (DAAs), treatment against hepatitis C virus (HCV) has significantly improved. To manage and control this worldwide infectious disease better, the "best" multidrug treatment is demanded based on scientific evidence. However, there is no method available that systematically quantifies and compares the antiviral efficacy and drug-resistance profiles of drug combinations. Based on experimental anti-HCV profiles in a cell culture system, we quantified the instantaneous inhibitory potential (IIP), which is the logarithm of the reduction in viral replication events, for both single drugs and multiple-drug combinations. From the calculated IIP of 15 anti-HCV drugs from different classes [telaprevir, danoprevir, asunaprevir, simeprevir, sofosbuvir (SOF), VX-222, dasabuvir, nesbuvir, tegobuvir, daclatasvir, ledipasvir, IFN-α, IFN-λ1, cyclosporin A, and SCY-635], we found that the nucleoside polymerase inhibitor SOF had one of the largest potentials to inhibit viral replication events. We also compared intrinsic antiviral activities of a panel of drug combinations. Our quantification analysis clearly indicated an advantage of triple-DAA treatments over double-DAA treatments, with triple-DAA treatments showing enhanced antiviral activity and a significantly lower probability for drug resistance to emerge at clinically relevant drug concentrations. Our framework provides quantitative information to consider in designing multidrug strategies before costly clinical trials.

Effects of rainfall events on the occurrence and detection efficiency of viruses in river water impacted by combined sewer overflows.

PubMed

Hata, Akihiko; Katayama, Hiroyuki; Kojima, Keisuke; Sano, Shoichi; Kasuga, Ikuro; Kitajima, Masaaki; Furumai, Hiroaki

2014-01-15

Rainfall events can introduce large amount of microbial contaminants including human enteric viruses into surface water by intermittent discharges from combined sewer overflows (CSOs). The present study aimed to investigate the effect of rainfall events on viral loads in surface waters impacted by CSO and the reliability of molecular methods for detection of enteric viruses. The reliability of virus detection in the samples was assessed by using process controls for virus concentration, nucleic acid extraction and reverse transcription (RT)-quantitative PCR (qPCR) steps, which allowed accurate estimation of virus detection efficiencies. Recovery efficiencies of poliovirus in river water samples collected during rainfall events (<10%) were lower than those during dry weather conditions (>10%). The log10-transformed virus concentration efficiency was negatively correlated with suspended solid concentration (r(2)=0.86) that increased significantly during rainfall events. Efficiencies of DNA extraction and qPCR steps determined with adenovirus type 5 and a primer sharing control, respectively, were lower in dry weather. However, no clear relationship was observed between organic water quality parameters and efficiencies of these two steps. Observed concentrations of indigenous enteric adenoviruses, GII-noroviruses, enteroviruses, and Aichi viruses increased during rainfall events even though the virus concentration efficiency was presumed to be lower than in dry weather. The present study highlights the importance of using appropriate process controls to evaluate accurately the concentration of water borne enteric viruses in natural waters impacted by wastewater discharge, stormwater, and CSOs. © 2013.

Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination.

PubMed

Manning, Nicola Mary; Bachanek-Bankowska, Katarzyna; Mertens, Peter Paul Clement; Castillo-Olivares, Javier

2017-10-20

African horse sickness is a lethal viral disease of equids transmitted by biting midges of the Genus Culicoides. The disease is endemic to sub-Saharan Africa but outbreaks of high mortality and economic impact have occurred in the past in non-endemic regions of Africa, Asia and Southern Europe. Vaccination is critical for the control of this disease but only live attenuated vaccines are currently available. However, there are bio-safety concerns over the use of this type of vaccines, especially in non-endemic countries, and live attenuated vaccines do not have DIVA (Differentiation of Infected from Vaccinated Animals) capacity. In addition, large scale manufacturing of live attenuated vaccines of AHSV represents a significant environmental and health risk and level 3 bio-safety containment facilities are required for their production. A variety of different technologies have been investigated over the years to develop alternative AHSV vaccines, including the use of viral vaccine vectors such Modified Vaccinia Ankara virus (MVA). In previous studies we demonstrated that recombinant MVA expressing outer capsid protein AHSV-VP2 induced virus neutralising antibodies and protection against virulent challenge both in a mouse model and in the horse. However, AHSV-VP2 is antigenically variable and determines the existence of 9 different AHSV serotypes. Immunity against AHSV is serotype-specific and there is limited cross-reactivity between certain AHSV serotypes: 1 and 2, 3 and 7, 5 and 8, 6 and 9. In Africa, multiple serotypes circulate simultaneously and a polyvalent attenuated vaccine comprising different AHSV serotypes is used. We investigated the potential of a polyvalent AHSV vaccination strategy based on combinations of MVA-VP2 viruses each expressing a single VP2 antigen from a specific serotype. We showed that administration of 2 different recombinant MVA viruses, each expressing a single VP2 protein from AHSV serotype 4 or 9, denoted respectively as MVA-VP2

Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas.

PubMed

Malhotra, Sandeep; Kim, Teresa; Zager, Jonathan; Bennett, Joseph; Ebright, Michael; D'Angelica, Michael; Fong, Yuman

2007-04-01

Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multimutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these 2 anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. Expression GM-CSF in vitro did not alter the infectivity, cytotoxicity, or replication of NV1034 compared to the noncytokine-secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l-6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice, respectively. In these immune-competent models, NV1034 and NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, there was no difference in the antitumor efficacy of these viruses in mice depleted of CD4+ and CD8+ T lymphocytes. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.

Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.

PubMed

Hou, Peili; Zhao, Guimin; Wang, Hongmei; He, Chengqiang; Huan, Yanjun; He, Hongbin

2018-04-01

Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of high-yield influenza B virus vaccine viruses

PubMed Central

Ping, Jihui; Lopes, Tiago J. S.; Neumann, Gabriele; Kawaoka, Yoshihiro

2016-01-01

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six “internal” influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production. PMID:27930325

Development of high-yield influenza B virus vaccine viruses.

PubMed

Ping, Jihui; Lopes, Tiago J S; Neumann, Gabriele; Kawaoka, Yoshihiro

2016-12-20

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six "internal" influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production.

Combination of Vaccine-Strain Measles and Mumps Viruses Enhances Oncolytic Activity against Human Solid Malignancies.

PubMed

Son, Ho Anh; Zhang, LiFeng; Cuong, Bui Khac; Van Tong, Hoang; Cuong, Le Duy; Hang, Ngo Thu; Nhung, Hoang Thi My; Yamamoto, Naoki; Toan, Nguyen Linh

2018-02-07

Oncolytic measles and mumps viruses (MeV, MuV) have a potential for anti-cancer treatment. We examined the anti-tumor activity of MeV, MuV, and MeV-MuV combination (MM) against human solid malignancies (HSM). MeV, MuV, and MM targeted and significantly killed various cancer cell lines of HSM but not normal cells. MM demonstrated a greater anti-tumor effect and prolonged survival in a human prostate cancer xenograft tumor model compared to MeV and MuV. MeV, MuV, and MM significantly induced the expression of immunogenic cell death markers and enhanced spleen-infiltrating immune cells. In conclusion, MM combination significantly improves the treatment of human solid malignancies.

Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice.

PubMed

Kim, Ha-Hyun; Yang, Dong-Kun; Nah, Jin-Ju; Song, Jae-Young; Cho, In-Soo

2017-06-24

Rabies is an important viral zoonosis that causes acute encephalitis and death in mammals. To date, several recombinant vaccines have been developed based on G protein, which is considered to be the main antigen, and these vaccines are used for rabies control in many countries. Most recombinant viruses expressing RABV G protein retain the G gene from attenuated RABV. Not enough is currently known about the protective effect against RABV of a combination of recombinant adenoviruses expressing the G and N proteins of pathogenic street RABV. We constructed a recombinant adenovirus (Ad-0910Gsped) expressing the signal peptide and ectodomain (sped) of G protein of the Korean street strain, and evaluated the immunological protection conferred by a single and combination of three kinds of recombinant adenoviruses (Ad-0910Gsped and Ad-0910G with or without Ad-0910 N) in mice. A combination of Ad-0910G and Ad-0910 N conferred improved immunity against intracranial challenge compared to single administration of Ad-0910G. The Ad-0910G virus, expressing the complete G protein, was more immunogenic than Ad-0910Gsped, which expressed a truncated G protein with the transmembrane and cytoplasmic domains removed. Additionally, oral vaccination using a combination of viruses led to complete protection. Our results suggest that this combination of viruses is a viable new intramuscular and oral vaccine candidate.

Sofosbuvir/ledipasvir fixed-dose combination for treatment of chronic hepatitis C virus infection in children.

PubMed

Rizza, S A; Nehra, V; Temesgen, Z

2017-08-01

The United States Food and Drug Administration recently approved sofosbuvir and the fixed-dose combination of ledipasvir/sofosbuvir for the treatment of hepatitis C virus (HCV) infection in children ages 12 to 17. These are the first direct-acting antiviral treatments approved for children and adolescents with HCV. Pharmacokinetic data confirm equivalent drug exposure in this population as that found in adults during clinical trials. The efficacy and safety of these drugs has been shown in clinical trials. Copyright 2017 Clarivate Analytics.

Patterns of orthopox virus wild rodent hosts in South Germany.

PubMed

Essbauer, Sandra; Hartnack, Sonja; Misztela, Krystian; Kiessling-Tsalos, Judith; Bäumler, Walter; Pfeffer, Martin

2009-06-01

Although cowpox virus (CPXV) infections in a variety of dead-end hosts have been investigated in Germany for more than 50 years, data on species and geographical distribution of CPXV in reservoir hosts are sparse. Here we present the first comprehensive study of 825 rodents that have been collected in Bavaria, Southern Germany. In summary, six different rodent species (Apodemus flavicollis, Myodes glareolus, Microtus arvalis, Apodemus sylvaticus, Microtus agrestis, and Arvicola amphibius) were trapped at three main trapping sites and investigated using a serum neutralization test (SNT). Prevalence of orthopox virus (OPV)-neutralizing antibodies was (with exception of one trapping site) highest in bank voles, ranging from 24.5% to 42.4%; often with SNT titers > or =96. Two up to 25% of yellow-necked mice were OPV sero-positive, but wood mice only at one site with 5.5%. Up to 7.7% of common voles were found to be OPV seroreactive, while M. agrestis and A. amphibius only sporadically showed seroreactivity. Further analyses of a subset of 450 bank voles and yellow-necked mice trapped at one site over a 18-month period revealed that male yellow-necked mice and female gravid yellow-necked mice had significantly more OPV-neutralizing antibodies. Mean body weight and OPV-seroreactivity were significantly negatively associated in male A. flavicollis. This was not due to shorter body length or smaller body mass index, but previously OPV-infected male A. flavicollis had dramatically reduced mean kidney weights. Seroreactivity in female bank voles was positively related to lung weights. We also found that both male yellow-necked mice and male bank voles with positive SNT titers had higher infestation rates with ectoparasites. We here show for the first time that A. flavicollis beside M. glareolus is a hypothetic host for CPXV, and that there are big geographical and spatial variations concerning the seroprevalence in rodent populations in South Germany.

Toward Elimination of Hepatitis B Virus Using Novel Drugs, Approaches, and Combined Modalities.

PubMed

Boucle, Sebastien; Bassit, Leda; Ehteshami, Maryam; Schinazi, Raymond F

2016-11-01

Hepatitis B virus (HBV) causes significant morbidity and mortality worldwide. The majority of chronically infected individuals do not achieve a functional and complete cure. Treated persons who achieve a long-term sustained virologic response (undetectable HBV DNA), are still at high risk of developing morbidity and mortality from liver complications. This review focuses on novel, mechanistically diverse anti-HBV therapeutic strategies currently in development or in clinical evaluation, and highlights new combination strategies that may contribute to full elimination of HBV DNA and covalently closed circular DNA from the infected liver, leading to a complete cure of chronic hepatitis B. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease

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

Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye

A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, andmore » infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. - Highlights: • Combined RSV FFG VLP vaccine is effective in inducing F specific responses. • FFG VLP vaccine confers RSV neutralizing activity and viral control in cotton rats. • Cotton rats with RSV FFG VLP vaccination do not show vaccine-enhanced disease. • Cotton rats with FFG VLP vaccine induce F specific antibody secreting cell responses. • Cotton rats with FFG VLP do not induce lung cellular infiltrates and Th2 cytokine.« less

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques.

PubMed

Simmons, Monika; Porter, Kevin R; Hayes, Curtis G; Vaughn, David W; Putnak, Robert

2006-10-01

We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.

A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments.

PubMed

Munson-McGee, Jacob H; Peng, Shengyun; Dewerff, Samantha; Stepanauskas, Ramunas; Whitaker, Rachel J; Weitz, Joshua S; Young, Mark J

2018-06-01

The application of viral and cellular metagenomics to natural environments has expanded our understanding of the structure, functioning, and diversity of microbial and viral communities. The high diversity of many communities, e.g., soils, surface ocean waters, and animal-associated microbiomes, make it difficult to establish virus-host associations at the single cell (rather than population) level, assign cellular hosts, or determine the extent of viral host range from metagenomics studies alone. Here, we combine single-cell sequencing with environmental metagenomics to characterize the structure of virus-host associations in a Yellowstone National Park (YNP) hot spring microbial community. Leveraging the relatively low diversity of the YNP environment, we are able to overlay evidence at the single-cell level with contextualized viral and cellular community structure. Combining evidence from hexanucelotide analysis, single cell read mapping, network-based analytics, and CRISPR-based inference, we conservatively estimate that >60% of cells contain at least one virus type and a majority of these cells contain two or more virus types. Of the detected virus types, nearly 50% were found in more than 2 cellular clades, indicative of a broad host range. The new lens provided by the combination of metaviromics and single-cell genomics reveals a network of virus-host interactions in extreme environments, provides evidence that extensive virus-host associations are common, and further expands the unseen impact of viruses on cellular life.

The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

PubMed

Azevedo, Adriana S; Gonçalves, Antônio J S; Archer, Marcia; Freire, Marcos S; Galler, Ricardo; Alves, Ada M B

2013-01-01

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

Three-dimensional analysis of combination effect of ellagitannins and acyclovir on herpes simplex virus types 1 and 2.

PubMed

Vilhelmova, N; Jacquet, R; Quideau, S; Stoyanova, A; Galabov, A S

2011-02-01

The effects of combinations of three nonahydroxyterphenoyl-bearing C-glucosidic ellagitannins (castalagin, vescalagin and grandinin) with acyclovir (ACV) on the replication of type-1 and type-2 herpes simplex viruses in MDBK cells were tested by the focus-forming units reduction test. Ellagitannins included in these combinations possess a high individual antiviral activity: selectivity index of castalagin and vescalagin versus HSV-1 was similar to that of ACV, and relatively lower against HSV-2. The three-dimensional analytical approach of Prichard and Shipman was used to evaluate the impact of drug-drug interactions. The combination effects of ellagitannins with acyclovir were markedly synergistic. Copyright © 2010 Elsevier B.V. All rights reserved.

Combinations of Adefovir with Nucleoside Analogs Produce Additive Antiviral Effects against Hepatitis B Virus In Vitro

PubMed Central

Delaney, William E.; Yang, Huiling; Miller, Michael D.; Gibbs, Craig S.; Xiong, Shelly

2004-01-01

Combination therapies may be required for long-term management of some patients chronically infected with hepatitis B virus (HBV). Adefovir is a nucleotide analog that has similar activity against wild-type and lamivudine-resistant HBV. In contrast to lamivudine, clinical resistance to the prodrug adefovir dipivoxil emerges infrequently. Based on its clinical efficacy and low frequency of resistance, adefovir dipivoxil may form an important component of combination regimens. We therefore investigated the in vitro antiviral efficacy of combinations of adefovir with other nucleoside analogs (lamivudine, entecavir, emtricitabine [FTC],and telbivudine [L-dT]) and the nucleotide analog tenofovir. Using a novel stable cell line that expresses high levels of wild-type HBV, we assayed the antiviral activity of each drug alone and in combination with adefovir. All two-drug combinations resulted in greater antiviral effects than treatments with single agents and could be characterized as additive by the Bliss independence model. Analysis using the Loewe additivity model indicated that adefovir exerted additive antiviral effects when combined with lamivudine, FTC, or L-dT and moderately synergistic effects when combined with entecavir or tenofovir. There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses. PMID:15388423

A novel non-toxic combined CTA1-DD and ISCOMS adjuvant vector for effective mucosal immunization against influenza virus.

PubMed

Eliasson, Dubravka Grdic; Helgeby, Anja; Schön, Karin; Nygren, Caroline; El-Bakkouri, Karim; Fiers, Walter; Saelens, Xavier; Lövgren, Karin Bengtsson; Nyström, Ida; Lycke, Nils Y

2011-05-23

Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

Pembrolizumab combined with stereotactic body radiotherapy in a patient with human immunodeficiency virus and advanced non-small cell lung cancer: a case report.

PubMed

Li, Dongqi; He, Chuanchun; Xia, Yaoxiong; Du, Yaxi; Zhang, Jing

2018-04-23

Pembrolizumab has significantly improved outcomes in patients with advanced non-small cell lung cancer. Combining programmed death-1 inhibitor with stereotactic body radiotherapy showed a slight toxicity and good benefits in recent clinical trials. However, patients infected with human immunodeficiency virus were excluded from most trials because it was assumed that their anti-tumor immunity was compromised compared with immunocompetent patients. In June 2016, a 52-year-old Chinese man presented with human immunodeficiency virus and lung adenocarcinoma (T1bN3M1b). From November 2016 to December 2016, systemic chemotherapy and palliative radiotherapy for bone metastasis of femoral neck were carried out, but the tumor progressed. In January 2017, after immunochemistry detection of programmed death-1 and programmed death-ligand 1 expression (both > 50%), pembrolizumab was started. Three weeks after pembrolizumab, we combined stereotactic body radiotherapy for the primary lung tumor. He received no comfort and his CD4 lymphocyte count was stable. Human immunodeficiency virus-ribonucleic acid remained below the limits of detection. In March 2017, after three cycles of pembrolizumab and 5 weeks of stereotactic body radiotherapy therapy, he suddenly presented with palpitations. Emergency computed tomography scanning showed massive pericardial effusion and interstitial pneumonia. So we interrupted the pembrolizumab use and initiated treatment with prednisolone 1 mg/kg; however, the tumor progressed. Then, his CD4 lymphocyte count declined. Finally he died in June 2017 due to dyscrasia. Pembrolizumab combined with SBRT therapy for patients with human immunodeficiency virus infection and non-small cell lung cancer may lead to serious immune-related adverse events and more clinical trials are needed.

Influenza Virus and Glycemic Variability in Diabetes: A Killer Combination?

PubMed Central

Hulme, Katina D.; Gallo, Linda A.; Short, Kirsty R.

2017-01-01

Following the 2009 H1N1 influenza virus pandemic, numerous studies identified the striking link between diabetes mellitus and influenza disease severity. Typically, influenza virus is a self-limiting infection but in individuals who have a pre-existing chronic illness, such as diabetes mellitus, severe influenza can develop. Here, we discuss the latest clinical and experimental evidence for the role of diabetes in predisposing the host to severe influenza. We explore the possible mechanisms that underlie this synergy and highlight the, as yet, unexplored role that blood glucose oscillations may play in disease development. Diabetes is one of the world’s fastest growing chronic diseases and influenza virus represents a constant and pervasive threat to human health. It is therefore imperative that we understand how diabetes increases influenza severity in order to mitigate the burden of future influenza epidemics and pandemics. PMID:28588558

Antiviral Evaluation of Octadecyloxyethyl Esters of (S)-3-Hydroxy-2-(phosphonomethoxy)propyl Nucleosides Against Herpesviruses and Orthopoxviruses≠

PubMed Central

Valiaeva, Nadejda; Prichard, Mark N.; Buller, R. Mark; Beadle, James R.; Hartline, Caroll B.; Keith, Kathy A.; Schriewer, Jill; Trahan, Julissa; Hostetler, Karl Y.

2009-01-01

Our previous studies showed that esterification of (S)-3-hydroxy-2-(phosphono-methoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP- and ODE- esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphono-methoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine. (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC50 values were observed with ODE-HPMPA and ODE-HPMPC while ODE-HPMPG had intermediate antiviral activity with an EC50 of 40 nanomolar. In HFF cells infected with HCMV, the lowest EC50 values were observed with ODE-HPMPC, 0.9 nanomolar. ODE -HPMPA was highly active with an EC50 of 3 nanomolar, while ODE-HPMPG and ODE-HPMPDAP were also highly active with EC50s of 22 and 77 nanomolar, respectively. Against vaccinia and cowpox viruses, ODE-HPMPG and ODE-HPMPDAP were the most active and selective compounds with EC50 values of 20 to 60 nanomolar and selectivity index values of 600 to 3,500. ODE-HPMPG was also active against ectromelia virus with an EC50 value of 410 nanomolar and a selectivity index value of 166. ODE-HPMPG and ODE-HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases. PMID:19800369

Experience with live rubella virus vaccine combined with live vaccines against measles and mumps*

PubMed Central

Smorodintsev, A. A.; Nasibov, M. N.; Jakovleva, N. V.

1970-01-01

Vaccination of pre-school children in the 1-7-years age-group for the specific prophylaxis of mumps and rubella is often difficult to arrange because of the already large number of inoculations given to these children. Combined vaccines to protect against measles, mumps and rubella should therefore be a valuable development. The existence of effective live vaccines for each of these 3 diseases makes possible the production of a single preparation suitable for subcutaneous inoculation. Tests on vaccine strains of measles (Leningrad-16), mumps (Leningrad-3) and rubella (Leningrad-8) viruses in various combinations have established that divalent or trivalent vaccines remain clinically harmless, highly immunogenic and epidemiologically effective. Single subcutaneous administrations of live measles vaccine combined with mumps or rubella vaccines or both, when given to children aged 1-8 years, brough about a high percentage of serological conversions and an increase in antibodies to a level comparable with that achieved with the corresponding monovalent vaccines. Morbidity from the 3 diseases was reduced among those vaccinated with the trivalent vaccine by 10 or more times, i.e., by about the same factor as when monovalent or divalent vaccines were used. PMID:5310140

Formation of Orthopoxvirus Cytoplasmic A-Type Inclusion Bodies and Embedding of Virions Are Dynamic Processes Requiring Microtubules

PubMed Central

Howard, Amanda R.

2012-01-01

In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix. PMID:22438543

Prolonged protection against Intranasal challenge with influenza virus following systemic immunization or combinations of mucosal and systemic immunizations with a heat-labile toxin mutant.

PubMed

Zhou, Fengmin; Goodsell, Amanda; Uematsu, Yasushi; Vajdy, Michael

2009-04-01

Seasonal influenza virus infections cause considerable morbidity and mortality in the world, and there is a serious threat of a pandemic influenza with the potential to cause millions of deaths. Therefore, practical influenza vaccines and vaccination strategies that can confer protection against intranasal infection with influenza viruses are needed. In this study, we demonstrate that using LTK63, a nontoxic mutant of the heat-labile toxin from Escherichia coli, as an adjuvant for both mucosal and systemic immunizations, systemic (intramuscular) immunization or combinations of mucosal (intranasal) and intramuscular immunizations protected mice against intranasal challenge with a lethal dose of live influenza virus at 3.5 months after the second immunization.

Combination of RNA interference and virus receptor trap exerts additive antiviral activity in coxsackievirus B3-induced myocarditis in mice.

PubMed

Stein, Elisabeth A; Pinkert, Sandra; Becher, Peter Moritz; Geisler, Anja; Zeichhardt, Heinz; Klopfleisch, Robert; Poller, Wolfgang; Tschöpe, Carsten; Lassner, Dirk; Fechner, Henry; Kurreck, Jens

2015-02-15

Coxsackievirus B3 (CVB3) is a major heart pathogen against which no therapy exists to date. The potential of a combination treatment consisting of a proteinaceous virus receptor trap and an RNA interference-based component to prevent CVB3-induced myocarditis was investigated. A soluble variant of the extracellular domain of the coxsackievirus-adenovirus receptor (sCAR-Fc) was expressed from an adenoviral vector and 2 short hairpin RNAs (shRdRp2.4) directed against CVB3 were delivered by an adeno-associated virus (AAV) vector. Cell culture experiments revealed additive antiviral activity of the combined application. In a CVB3-induced mouse myocarditis model, both components applied individually significantly reduced inflammation and viral load in the heart. The combination exerted an additive antiviral effect and reduced heart pathology. Hemodynamic measurement revealed that infection with CVB3 resulted in impaired heart function, as illustrated by a drastically reduced cardiac output and impaired contractility and relaxation. Treatment with either sCAR-Fc or shRdRp2.4 significantly improved these parameters. Importantly, the combination of both components led to a further significant improvement of heart function. Combination of sCAR-Fc and shRdRp2.4 exerted additive effects and was significantly more effective than either of the single treatments in inhibiting CVB3-induced myocarditis and preventing cardiac dysfunction. © The Author 2014. 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.

Genomic sequencing of deer tick virus and phylogeny of powassan-related viruses of North America.

PubMed

Kuno, G; Artsob, H; Karabatsos, N; Tsuchiya, K R; Chang, G J

2001-11-01

Powassan (POW) virus is responsible for central nervous system infection in humans in North America and the eastern parts of Russia. Recently, a new flavivirus, deer tick (DT) virus, related to POW virus was isolated in the United States, but neither its pathogenic potential in human nor the taxonomic relationship with POW virus has been elucidated. In this study, we obtained the near-full-length genomic sequence of the DT virus and complete sequences of 3 genomic regions of 15 strains of POW-related virus strains. The phylogeny revealed 2 lineages, one of which had the prototype POW virus and the other DT virus. Both lineages can cause central nervous system infection in humans. By use of the combination of molecular definition of virus species within the genus Flavivirus and serological distinction in a 2-way cross-neutralization test, the lineage of DT virus is classified as a distinct genotype of POW virus.

Combination drug treatment prolongs survival of experimentally infected mice with silver-haired bat rabies virus.

PubMed

Martina, Byron E E; Smreczak, Marcin; Orlowska, Anna; Marzec, Anna; Trebas, Pawel; Roose, Jouke M; Zmudzinski, Jan; Gerhauser, Ingo; Wohlsein, Peter; Baumgärtner, Wolfgang; Osterhaus, Albert D M E; Koraka, Penelope

2018-05-26

Rabies is a lethal disease in humans and animals, killing approximately 60,000 people every year. Currently, there is no treatment available, except post-exposure prophylaxis (PEP) that can be administered whenever exposure to a rabid animal took place. Here we describe the beneficial effects of a combination treatment initiated at day 4 post infection, containing anti-viral drugs and immune modulators in infected mice. Combination therapy resulted in significant increase in survival time (P < 0.05) and significantly lowers viral RNA in the brain and spinal cord (P < 0.05). Furthermore, treatment influenced markers of pyroptosis and apoptosis and early inflammatory response as measured by the levels of TNF-α. Morphological lesions were absent in rabies virus infected mice with few signs of inflammation. However, these were not significant between the different groups. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins.

PubMed

Pardo, M C; Bauman, J E; Mackowiak, M

1997-08-01

To evaluate the safety and efficacy of a live canarypox virus recombinant-canine distemper virus (CDV) combination vaccine against virulent CDV challenge exposure, and to document lack of interference among the other modified-live virus (MLV) components. 33 specific-pathogen-free (SPF) Beagle pups (7 to 10 weeks old). A canarypox virus recombinant-CDV combination vaccine was tested for safety and efficacy along with MLV components (canine adenovirus type 2, canine coronavirus, canine parainfluenza virus, and canine parvovirus) in 26 SPF Beagle pups. The combination vaccine was rehydrated with either Leptospira canicola-L icterohaemorrhagiae combination bacterin (vaccine 1) or sterile diluent (vaccine 2). An additional group of 7 seronegative SPF pups received the control MLV components devoid of the combination vaccine (vaccine 3). Two vaccinations were administered 21 days apart, either IM or SC. The dose of the combination vaccine used to inoculate these pups was 40 times lower than the recommended commercial dose. At 21 days after the booster vaccination, all pups were challenge exposed with a virulent CDV strain, then were observed for 21 days to record morbidity and mortality. Adverse local or generalized reactions were not induced by vaccinations. All vaccinates seroconverted to CDV. Serum antibody titers to MLV components were not different, with or without inclusion of the combination vaccine. After challenge exposure, morbidity and mortality in vaccinates were 0% (0/26); in control dogs, values were 100% morbidity and 86% mortality (6/7). Brain impression smear slides made from all dogs that did not survive challenge exposure were CDV positive by use of a direct fluorescein isothiocyanate method. The canarypox virus-CDV combination vaccine, administered SC or IM, is a safe product that elicits CDV seroconversion, does not interfere with other vaccine components, and protects vaccinated pups against virulent CDV challenge exposure.

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

PubMed

Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

2015-12-01

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

Enhanced Efficacy of Cidofovir Combined with Vaccinia Immune Globulin in Treating Progressive Cutaneous Vaccinia Virus Infections in Immunosuppressed Hairless Mice

PubMed Central

Dagley, Ashley; Downs, Brittney; Hagloch, Joseph; Tarbet, E. Bart

2014-01-01

The treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (CDV), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (VIG). VIG is costly, and its supply is limited, so sparing the use of VIG during treatment is an important objective. VIG sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of CDV combined with VIG to determine the effective treatments that delayed the time to death, reduced cutaneous lesion severity, and/or decreased tissue viral titers. SKH-1 hairless mice immunosuppressed with cyclophosphamide and hairless SCID mice (SHO strain) were infected cutaneously with vaccinia virus. Monotherapy, dual combinations (CDV plus VIG), or triple therapy (topical CDV, parenteral CDV, and VIG) were initiated 2 days postinfection and were given every 3 to 4 days through day 11. The efficacy assessment included survival rate, cutaneous lesion severity, and viral titers. Delays in the time to death and the reduction in lesion severity occurred in the following order of efficacy: triple therapy had greater efficacy than double combinations (CDV plus VIG or topical plus parenteral CDV), which had greater efficacy than VIG alone. Parenteral administration of CDV or VIG was necessary to suppress virus titers in internal organs (liver, lung, and spleen). The skin viral titers were significantly reduced by triple therapy only. The greatest efficacy was achieved by triple therapy. In humans, this regimen should translate to a faster cure rate, thus sparing the amount of VIG used for treatment. PMID:25385098

Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

PubMed

Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

2010-05-14

The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. Copyright 2010 Elsevier Ltd. All rights reserved.

Effect of combination antiretroviral therapy on Chinese rhesus macaques of simian immunodeficiency virus infection.

PubMed

Ling, Binhua; Rogers, Linda; Johnson, Ann-Marie; Piatak, Michael; Lifson, Jeffrey; Veazey, Ronald S

2013-11-01

Definitive treatment of HIV infection remains a critical but elusive goal, with persistence of residual virus even in the face of prolonged administration of suppressive combination antiretroviral treatment (cART) providing a source for recrudescent infection if treatment is stopped. Characterization of the residual virus and devising strategies to target it for eradication are key goals in HIV treatment research. Indian rhesus macaques (In-RM) infected with SIVmac have been widely used in such research. However, it has proven challenging to achieve and sustain clinically relevant levels of suppression (<30 vRNA copies/ml plasma) with cART in such models. As ease of viral suppression by cART is related to pretreatment levels of viral replication, and levels of replication of SIVmac239/251 are lower in Chinese rhesus macaques (Ch-RM) than in In-RM, we evaluated cART administration to SIVmac-infected Ch-RM as a potential model for studies of residual virus and eradication strategies. Four SIVmac239-infected Ch-RM received cART including reverse transcriptase inhibitors PMPA/FTC and integrase inhibitor L-870812 daily for 8 weeks. Plasma viral loads were promptly reduced to <30 copies/ml upon initiation of cART. Cell-associated SIV DNA levels in lymphocytes from the gut were also significantly reduced. Jejunal and colonic CCR5(+)CD4(+) mucosal memory T cells increased significantly; restoration of these cells was associated with reductions in immune activation. In conclusion, cART effectively suppressed viral replication to <30 vRNA copies/ml in SIVmac239-infected Ch-RM, reducing immune activation and restoring mucosal immune cell populations. SIVmac239-infected Ch-RM may be a useful model for studying responses to cART and persistent tissue reservoirs and evaluating candidate eradication strategies to cure HIV infection.

T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

PubMed Central

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A.; Edwards, David M.; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K.; Pickup, David J.; Lewinsohn, David M.; Gold, Marielle C.; Wong, Scott W.; Sacha, Jonah B.; Slifka, Mark K.; Früh, Klaus

2014-01-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

T cell inactivation by poxviral B22 family proteins increases viral virulence.

PubMed

Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A; Edwards, David M; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K; Pickup, David J; Lewinsohn, David M; Gold, Marielle C; Wong, Scott W; Sacha, Jonah B; Slifka, Mark K; Früh, Klaus

2014-05-01

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus.

PubMed

Raue, R; Hess, M

1998-08-01

Three different polymerase chain reactions (PCRs), two of them combined with restriction enzyme analysis (REA), were developed for detection and differentiation of all 12 fowl adenovirus (FAV) serotypes and the egg drop syndrome (EDS) virus. For primer construction FAV1, FAV10 and EDS virus hexon proteins were aligned and conserved and variable regions were determined. Two primer sets (H1/H2 and H3/H4) for single use were constructed which hybridize in three conserved regions of hexon genes. Each primer pair amplifies approximately half of the hexon gene including two loop regions. An amplification product was detected with both primer sets using purified DNA from all FAV1-12 reference strains. Viral EDS DNA was negative using the H1/H2 or H3/H4 primer pair. HaeII digestion of the H1/H2 amplification products differentiates between all viruses except FAV4 and FAV5. In comparison, much more clustering among genomic closely related FAV serotypes was seen after HpaII digestion of the H3/H4 PCR products. Oligonucleotides H5/H6 located in the variable regions of EDS virus hexon gene do not detect any of the FAV serotypes. The PCRs and REA described are suitable to detect all avian adenoviruses infecting chickens, to distinguish all 12 FAV reference strains and to differentiate FAVs from the EDS virus.

A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

PubMed Central

Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge

2017-01-01

Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans. PMID:28216667

Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines.

PubMed

Tscharke, David C; Karupiah, Gunasegaran; Zhou, Jie; Palmore, Tara; Irvine, Kari R; Haeryfar, S M Mansour; Williams, Shanicka; Sidney, John; Sette, Alessandro; Bennink, Jack R; Yewdell, Jonathan W

2005-01-03

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.

Combined exposure to cyanobacterial biomass, lead and the Newcastle virus enhances avian toxicity.

PubMed

Pikula, Jiri; Bandouchova, Hana; Hilscherova, Klara; Paskova, Veronika; Sedlackova, Jana; Adamovsky, Ondrej; Knotkova, Zora; Lany, Petr; Machat, Jiri; Marsalek, Blahoslav; Novotny, Ladislav; Pohanka, Miroslav; Vitula, Frantisek

2010-10-01

Under environmental conditions, wild birds can be exposed to multiple stressors including natural toxins, anthropogenic pollutants and infectious agents at the same time. This experimental study was successful in testing the hypothesis that adverse effects of cyanotoxins, heavy metals and a non-pathogenic immunological challenge combine to enhance avian toxicity. Mortality occurred in combined exposures to naturally occurring cyanobacterial biomass and lead shots, lead shots and Newcastle vaccination as well as in single lead shot exposure. Mostly acute effects around day 10 were observed. On day 30 of exposure, there were no differences in the liver accumulation of lead in single and combined exposure groups. Interestingly, liver microcystin levels were elevated in birds co-exposed to cyanobacterial biomass together with lead or lead and the Newcastle virus. Significant differences in body weights between all Pb-exposed and Pb-non-exposed birds were found on days 10 and 20. Single exposure to cyanobacterial biomass resulted in hepatic vacuolar dystrophy, whereas co-exposure with lead led to more severe granular dystrophy. Haematological changes were associated with lead exposure, in particular. Biochemical analysis revealed a decrease in glucose and an increase in lactate dehydrogenase in single and combined cyanobacterial and lead exposures, which also showed a decreased antibody response to vaccination. The combined exposure of experimental birds to sub-lethal doses of individual stressors is ecologically realistic. It brings together new pieces of knowledge on avian health. In light of this study, investigators of wild bird die-offs should be circumspect when evaluating findings of low concentrations of contaminants that would not result in mortality on a separate basis. As such it has implications for wildlife biologists, veterinarians and conservationists of avian biodiversity. Copyright 2010 Elsevier B.V. All rights reserved.

Virus reactivation: a panoramic view in human infections

PubMed Central

Traylen, Christopher M; Patel, Hersh R; Fondaw, Wylder; Mahatme, Sheran; Williams, John F; Walker, Lia R; Dyson, Ossie F; Arce, Sergio; Akula, Shaw M

2011-01-01

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is ‘quiescent’ (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus. PMID:21799704

Cowpox virus infection in two brothers with possible human to human transmission.

PubMed

Tamulyte, G; Lauraitis, J; Ganceviciene, R; Grigaitiene, J; Zouboulis, C C

2018-05-31

Two brothers from countryside were admitted to our centre with history of necrotic ulcers. Both of them were treated with oral and intravenous antibiotics, but treatment was ineffective. A 12-year-old boy had lesions on his head lasting for 4 weeks. Skin lesions started 1 week after he played with rats and neighbour's domestic calves, sheep and goats. Before the eruption occurred, the patient had subfebrile temperature and flu-like symptoms. Less severe eruption and symptoms were reported by his mother and 3-year-old sister. The 16-year-old boy had necrotic ulcers on the face. He had febrile temperature one week after his brother developed the eruption. The patient had close contact with his brother and sister but denied any contact with domestic or wild animals. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Hepatitis E virus coinfection with hepatotropic viruses in Egyptian children.

PubMed

Zaki, Maysaa El Sayed; Salama, Osama Saad; Mansour, Fathy Awaad; Hossein, Shaimaa

2008-06-01

Major hepatotropic viruses continue to be important causes of acute viral hepatitis in developing countries. This work was carried out to detect the seroprevalence of hepatitis E virus (HEV) markers in children with acute viral hepatitis due to hepatotropic viruses (A, B and C) and non-A, non-B, non-C acute hepatitis, and to ascertain the influence of HEV superinfection in individuals infected with hepatitis viruses (A, B and C). We studied prospectively 162 children with sporadic acute hepatitis who reported to our hospital. Thirteen healthy controls were also included in the study. Laboratory investigations were performed, including complete liver function tests. Complete serological profiles for hepatitis viruses A, B, C and E were evaluated. HEV immunoglobulin G was detected with highest percentage among patients with hepatitis B (56.7%), followed by patients with hepatitis C virus (52.0%), hepatitis A virus (34.1%) and combined hepatitis B and C viruses (30.0%). The detection rate among patients with non-A, non-B, non-C hepatitis was 7.1%. HEV immunoglobulin M was found in 4.5% of hepatitis A virus patients and in 3.3% of hepatitis B patients. The prevalence of HEV immunoglobulin G and immunoglobulin M correlated with the levels of hepatic aspartate aminotransferase and alanine aminotransferase in patients with dual markers of infection with hepatitis E and other viruses compared to patients with acute hepatitis due to A and C viruses. HEV serological markers are common among children with acute viral hepatitis, especially from hepatitis C and B viruses. There may be increased sensitivity to HEV coinfection in association with hepatitis B and C infections. Dual infection with HEV and other hepatotropic viruses was associated with greater elevation of aspartate and alanine aminotransferases.

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus.

PubMed

Sun, Zhihao; Qin, Tao; Meng, Feifei; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2017-10-18

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID 50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

Evolutionary ecology of virus emergence.

PubMed

Dennehy, John J

2017-02-01

The cross-species transmission of viruses into new host populations, termed virus emergence, is a significant issue in public health, agriculture, wildlife management, and related fields. Virus emergence requires overlap between host populations, alterations in virus genetics to permit infection of new hosts, and adaptation to novel hosts such that between-host transmission is sustainable, all of which are the purview of the fields of ecology and evolution. A firm understanding of the ecology of viruses and how they evolve is required for understanding how and why viruses emerge. In this paper, I address the evolutionary mechanisms of virus emergence and how they relate to virus ecology. I argue that, while virus acquisition of the ability to infect new hosts is not difficult, limited evolutionary trajectories to sustained virus between-host transmission and the combined effects of mutational meltdown, bottlenecking, demographic stochasticity, density dependence, and genetic erosion in ecological sinks limit most emergence events to dead-end spillover infections. Despite the relative rarity of pandemic emerging viruses, the potential of viruses to search evolutionary space and find means to spread epidemically and the consequences of pandemic viruses that do emerge necessitate sustained attention to virus research, surveillance, prophylaxis, and treatment. © 2016 New York Academy of Sciences.

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

The Evolution of Poxvirus Vaccines

PubMed Central

Sánchez-Sampedro, Lucas; Perdiguero, Beatriz; Mejías-Pérez, Ernesto; García-Arriaza, Juan; Di Pilato, Mauro; Esteban, Mariano

2015-01-01

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases. PMID:25853483

The evolution of poxvirus vaccines.

PubMed

Sánchez-Sampedro, Lucas; Perdiguero, Beatriz; Mejías-Pérez, Ernesto; García-Arriaza, Juan; Di Pilato, Mauro; Esteban, Mariano

2015-04-07

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

Superior Efficacy of a Human Immunodeficiency Virus Vaccine Combined with Antiretroviral Prevention in Simian-Human Immunodeficiency Virus-Challenged Nonhuman Primates.

PubMed

Le Grand, Roger; Dereuddre-Bosquet, Nathalie; Dispinseri, Stefania; Gosse, Leslie; Desjardins, Delphine; Shen, Xiaoying; Tolazzi, Monica; Ochsenbauer, Christina; Saidi, Hela; Tomaras, Georgia; Prague, Mélanie; Barnett, Susan W; Thiebaut, Rodolphe; Cope, Alethea; Scarlatti, Gabriella; Shattock, Robin J

2016-06-01

Although vaccines and antiretroviral (ARV) prevention have demonstrated partial success against human immunodeficiency virus (HIV) infection in clinical trials, their combined introduction could provide more potent protection. Furthermore, combination approaches could ameliorate the potential increased risk of infection following vaccination in the absence of protective immunity. We used a nonhuman primate model to determine potential interactions of combining a partially effective ARV microbicide with an envelope-based vaccine. The vaccine alone provided no protection from infection following 12 consecutive low-dose intravaginal challenges with simian-HIV strain SF162P3, with more animals infected compared to naive controls. The microbicide alone provided a 68% reduction in the risk of infection relative to that of the vaccine group and a 45% reduction relative to that of naive controls. The vaccine-microbicide combination provided an 88% reduction in the per-exposure risk of infection relative to the vaccine alone and a 79% reduction relative to that of the controls. Protected animals in the vaccine-microbicide group were challenged a further 12 times in the absence of microbicide and demonstrated a 98% reduction in the risk of infection. A total risk reduction of 91% was observed in this group over 24 exposures (P = 0.004). These important findings suggest that combined implementation of new biomedical prevention strategies may provide significant gains in HIV prevention. There is a pressing need to maximize the impact of new biomedical prevention tools in the face of the 2 million HIV infections that occur each year. Combined implementation of complementary biomedical approaches could create additive or synergistic effects that drive improved reduction of HIV incidence. Therefore, we assessed a combination of an untested vaccine with an ARV-based microbicide in a nonhuman primate vaginal challenge model. The vaccine alone provided no protection (and may have

Superior Efficacy of a Human Immunodeficiency Virus Vaccine Combined with Antiretroviral Prevention in Simian-Human Immunodeficiency Virus-Challenged Nonhuman Primates

PubMed Central

Le Grand, Roger; Dereuddre-Bosquet, Nathalie; Dispinseri, Stefania; Gosse, Leslie; Desjardins, Delphine; Shen, Xiaoying; Tolazzi, Monica; Ochsenbauer, Christina; Saidi, Hela; Tomaras, Georgia; Prague, Mélanie; Barnett, Susan W.; Thiebaut, Rodolphe; Scarlatti, Gabriella

2016-01-01

ABSTRACT Although vaccines and antiretroviral (ARV) prevention have demonstrated partial success against human immunodeficiency virus (HIV) infection in clinical trials, their combined introduction could provide more potent protection. Furthermore, combination approaches could ameliorate the potential increased risk of infection following vaccination in the absence of protective immunity. We used a nonhuman primate model to determine potential interactions of combining a partially effective ARV microbicide with an envelope-based vaccine. The vaccine alone provided no protection from infection following 12 consecutive low-dose intravaginal challenges with simian-HIV strain SF162P3, with more animals infected compared to naive controls. The microbicide alone provided a 68% reduction in the risk of infection relative to that of the vaccine group and a 45% reduction relative to that of naive controls. The vaccine-microbicide combination provided an 88% reduction in the per-exposure risk of infection relative to the vaccine alone and a 79% reduction relative to that of the controls. Protected animals in the vaccine-microbicide group were challenged a further 12 times in the absence of microbicide and demonstrated a 98% reduction in the risk of infection. A total risk reduction of 91% was observed in this group over 24 exposures (P = 0.004). These important findings suggest that combined implementation of new biomedical prevention strategies may provide significant gains in HIV prevention. IMPORTANCE There is a pressing need to maximize the impact of new biomedical prevention tools in the face of the 2 million HIV infections that occur each year. Combined implementation of complementary biomedical approaches could create additive or synergistic effects that drive improved reduction of HIV incidence. Therefore, we assessed a combination of an untested vaccine with an ARV-based microbicide in a nonhuman primate vaginal challenge model. The vaccine alone provided no

Structure of faustovirus, a large dsDNA virus

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

Klose, Thomas; Reteno, Dorine G.; Benamar, Samia

Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unitmore » of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.« less

Structure of faustovirus, a large dsDNA virus

DOE PAGES

Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; ...

2016-05-16

Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unitmore » of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.« less

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

PubMed

Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

2016-11-01

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

Photodynamic Effect of some Phthalocyanines on Enveloped and Naked Viruses.

PubMed

Nikolaeva-Glomb, L; Mukova, L; Nikolova, N; Kussovski, V; Doumanova, L; Mantareva, V; Angelov, I; Wöhrle, D; Galabov, A S

Activity of three photosensitizing phthalocyanine derivatives was tested for photodynamic inactivation towards two coated and two non-enveloped viruses - bovine viral diarrhea virus (BVDV), influenza virus A(H3N2), poliovirus type 1 (PV-1) and human adenovirus type 5 (HAdV5). In the case of coated viruses, a combination of virucidal and irradiation effects was registered by octa-methylpyridyloxy-substituted Ga phthalocyanine (Ga8) toward BVDV, whereas tetra-methylpyridyloxy-substituted Ga phthalocyanine (Ga4) revealed a marked photoinactivation only. No such effect was observed towards influenza A virus. In contrast, the photoinactivating potential of Ga4 and Ga8 marked very high values on naked viruses, especially on HAdV5, at which both the virucidal as well as the irradiation effects became combined.

Combined oncolytic virotherapy with herpes simplex virus for oral squamous cell carcinoma.

PubMed

Ogawa, Fumi; Takaoka, Hiroo; Iwai, Soichi; Aota, Keiko; Yura, Yoshiaki

2008-01-01

The effect of dual infection with herpes simplex virus type 1 (HSV-1) mutants on human oral squamous cell carcinoma (SCC) cells was examined. Human oral SCC cells were infected with gamma1(34.5) gene-deficient HSV-1 R849 and HSV-1 HF that has multiple mutations and induces cell fusion. Cell viability was measured by LDH release assay. Athymic mice were injected with oral SCC cells into the buccal region to induce subcutaneous tumors. Oral SCC cells were infected with R849, followed by infection with R849 or HF. Virus production was elevated by both strains of HSV-1. Although the release of LDH from R849-infected cells was increased by secondary infection with R849 or HF, the effect of HF was more remarkable. When nude mouse tumors were treated with R849, HF, R849+R849, or R849+HF, treatment with R849+HF was the most effective. These results suggest that fusion-inducing virus HF enhances the oncolytic ability of gamma1(34.5) gene-deficient HSV-1 and provides a rationale for using fusogenic viruses as enhancing agents

Treating brain tumor–initiating cells using a combination of myxoma virus and rapamycin

PubMed Central

Zemp, Franz J.; Lun, Xueqing; McKenzie, Brienne A.; Zhou, Hongyuan; Maxwell, Lori; Sun, Beichen; Kelly, John J.P.; Stechishin, Owen; Luchman, Artee; Weiss, Samuel; Cairncross, J. Gregory; Hamilton, Mark G.; Rabinovich, Brian A.; Rahman, Masmudur M.; Mohamed, Mohamed R.; Smallwood, Sherin; Senger, Donna L.; Bell, John; McFadden, Grant; Forsyth, Peter A.

2013-01-01

Background Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor–initiating cells (BTICs). Methods We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts. Results The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing “advanced” BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo. Conclusions Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients. PMID:23585629

Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip.

PubMed

Cao, Zengguo; Wang, Hualei; Wang, Lina; Li, Ling; Jin, Hongli; Xu, Changping; Feng, Na; Wang, Jianzhong; Li, Qian; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

2016-01-01

West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 10(2) copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 10(1.5) TCID50/ml and 10(1.33) TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

PubMed Central

Wernike, Kerstin; Hoffmann, Bernd

2013-01-01

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance. PMID:23303496

Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates.

PubMed

Stanković, I; Bulajić, A; Vučurović, A; Ristić, D; Milojević, K; Berenji, J; Krstić, B

2011-01-01

In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most frequently found virus at the localities of Central Serbia, while PVY and CMV were the most frequent viruses in the Vojvodina Province. Single infections were prevalent in years 2005-2007 and the most frequent were those of PVY. A triple combination of those viruses was most frequent mixed infection type in 2008. The presence of all five detected viruses was confirmed in selected ELISA-positive samples by RT-PCR and sequencing. The comparisons of obtained virus isolate sequences with those available in NCBI, confirmed the authenticity of serologically detected viruses. Phylogenetic analysis based on partial nucleocapsid gene sequences revealed a joint clustering of Serbian, Bulgarian and Montenegrin TSWV isolates into one geographic subpopulation, which was distinct from the other subpopulation of TSWV isolates from the rest of the European countries. The high incidence of viruses in Serbian tobacco crops highlights the importance of enhancing farmers knowledge towards better implementation of control strategies for preventing serious losses.

Co-infection with Influenza Viruses and Influenza-Like Virus During the 2015/2016 Epidemic Season.

PubMed

Szymański, K; Cieślak, K; Kowalczyk, D; Brydak, L B

2017-01-01

Concerning viral infection of the respiratory system, a single virus can cause a variety of clinical symptoms and the same set of symptoms can be caused by different viruses. Moreover, infection is often caused by a combination of viruses acting at the same time. The present study demonstrates, using multiplex RT-PCR and real-time qRT-PCR, that in the 2015/2016 influenza season, co-infections were confirmed in patients aged 1 month to 90 years. We found 73 co-infections involving influenza viruses, 17 involving influenza viruses and influenza-like viruses, and six involving influenza-like viruses. The first type of co-infections above mentioned was the most common, amounting to 51 cases, with type A and B viruses occurring simultaneously. There also were four cases of co-infections with influenza virus A/H1N1/pdm09 and A/H1N1/ subtypes and two cases with A/H1N1/pdm09 and A/H3N2/ subtypes. The 2015/2016 epidemic season was characterized by a higher number of confirmed co-infections compared with the previous seasons. Infections by more than one respiratory virus were most often found in children and in individuals aged over 65.

Tunable protease-activatable virus nanonodes.

PubMed

Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

2014-05-27

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

PubMed

Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

2016-05-01

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Adaptive virus detection using filament-coupled antibodies.

PubMed

Stone, Gregory P; Lin, Kelvin S; Haselton, Frederick R

2006-01-01

We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10(10) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.

Following Acute Encephalitis, Semliki Forest Virus is Undetectable in the Brain by Infectivity Assays but Functional Virus RNA Capable of Generating Infectious Virus Persists for Life.

PubMed

Fragkoudis, Rennos; Dixon-Ballany, Catherine M; Zagrajek, Adrian K; Kedzierski, Lukasz; Fazakerley, John K

2018-05-18

Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the brain, by standard infectivity assays, for around ten days. As we have shown previously, in severe combined immunodeficient (SCID) mice, infectious virus is detectable for months in the brain. Here we show that in MHC-II -/- mice, with no functional CD4 T-cells, infectious virus is also detectable in the brain for long periods. In contrast, in the brains of CD8 -/- mice, virus RNA persists but infectious virus is not detectable. In SCID mice infected with SFV, repeated intraperitoneal administration of anti-SFV immune serum rapidly reduced the titer of infectious virus in the brain to undetectable, however virus RNA persisted. Repeated intraperitoneal passive transfer of immune serum resulted in maintenance of brain virus RNA, with no detectable infectious virus, for several weeks. When passive antibody transfer was stopped, antibody levels declined and infectious virus was again detectable in the brain. In aged immunocompetent mice, previously infected with SFV, immunosuppression of antibody responses many months after initial infection also resulted in renewed ability to detect infectious virus in the brain. In summary, antiviral antibodies control and determine whether infectious virus is detectable in the brain but immune responses cannot clear this infection from the brain. Functional virus RNA capable of generating infectious virus persists and if antibody levels decline, infectious virus is again detectable.

Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells.

PubMed

Danzy, Shamika; Studdard, Lydia R; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John; Lowen, Anice C

2014-11-01

Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that

Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells

PubMed Central

Danzy, Shamika; Studdard, Lydia R.; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John

2014-01-01

ABSTRACT Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. IMPORTANCE Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

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

Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike

2009-01-20

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/{delta}F-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/{delta}F-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface,more » the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/{delta}F-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.« less

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

PubMed Central

2012-01-01

Background Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects. PMID:22236378

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

PubMed

Aitichou, Mohamed; Saleh, Sharron; Kyusung, Park; Huggins, John; O'Guinn, Monica; Jahrling, Peter; Ibrahim, Sofi

2008-11-01

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Control of cucurbit viruses.

PubMed

Lecoq, Hervé; Katis, Nikolaos

2014-01-01

More than 70 well-characterized virus species transmitted by a diversity of vectors may infect cucurbit crops worldwide. Twenty of those cause severe epidemics in major production areas, occasionally leading to complete crop failures. Cucurbit viruses' control is based on three major axes: (i) planting healthy seeds or seedlings in a clean environment, (ii) interfering with vectors activity, and (iii) using resistant cultivars. Seed disinfection and seed or seedling quality controls guarantee growers on the sanitary status of their planting material. Removal of virus or vector sources in the crop environment can significantly delay the onset of viral epidemics. Insecticide or oil application may reduce virus spread in some situations. Diverse cultural practices interfere with or prevent vector reaching the crop. Resistance can be obtained by grafting for soil-borne viruses, by cross-protection, or generally by conventional breeding or genetic engineering. The diversity of the actions that may be taken to limit virus spread in cucurbit crops and their limits will be discussed. The ultimate goal is to provide farmers with technical packages that combine these methods within an integrated disease management program and are adapted to different countries and cropping systems.

Correlation of virus load in plasma and lymph node tissue in human immunodeficiency virus infection. INCAS Study Group. Italy, Netherlands, Canada, Australia, and (United) States.

PubMed

Harris, M; Patenaude, P; Cooperberg, P; Filipenko, D; Thorne, A; Raboud, J; Rae, S; Dailey, P; Chernoff, D; Todd, J; Conway, B; Montaner, J S

1997-11-01

The impact of long-term changes in plasma viremia, produced by effective combination antiretroviral therapy, on human immunodeficiency virus (HIV) burden within tissue reservoirs is unknown. Fifteen patients who had received at least 1 year of therapy with two or three drug combinations of zidovudine, didanosine, and nevirapine had suitable samples of lymph node tissue obtained by ultrasound-guided core needle biopsy. HIV RNA was extracted from homogenized tissue samples and quantitated using a modified branched DNA assay. Results were correlated with antiretroviral treatment effect on the basis of plasma virus load measurements over the preceding 12-18 months. A statistically significant negative correlation was observed between magnitude of treatment effect on plasma viremia and lymph node virus load. These data suggest that combinations of antiretroviral drugs that produce sustained suppression of plasma HIV RNA may also be able to reduce the virus burden in lymphoid tissues.

Chikungunya Virus: In Vitro Response to Combination Therapy With Ribavirin and Interferon Alfa 2a.

PubMed

Gallegos, Karen M; Drusano, George L; D Argenio, David Z; Brown, Ashley N

2016-10-15

We evaluated the antiviral activities of ribavirin (RBV) and interferon (IFN) alfa as monotherapy and combination therapy against chikungunya virus (CHIKV). Vero cells were infected with CHIKV in the presence of RBV and/or IFN alfa, and viral production was quantified by plaque assay. A mathematical model was fit to the data to identify drug interactions for effect. We ran simulations using the best-fit model parameters to predict the antiviral activity associated with clinically relevant regimens of RBV and IFN alfa as combination therapy. The model predictions were validated using the hollow fiber infection model (HFIM) system. RBV and IFN alfa were effective against CHIKV as monotherapy at supraphysiological concentrations. However, RBV and IFN alfa were highly synergistic for antiviral effect when administered as combination therapy. Simulations with our mathematical model predicted that a standard clinical regimen of RBV plus IFN alfa would inhibit CHIKV burden by 2.5 log10 following 24 hours of treatment. In the HFIM system, RBV plus IFN alfa at clinical exposures resulted in a 2.1-log10 decrease in the CHIKV burden following 24 hours of therapy. These findings validate the prediction made by the mathematical model. These studies illustrate the promise of RBV plus IFN alfa as a potential therapeutic strategy for the treatment of CHIKV infections. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements

PubMed Central

Dolja, Valerian V.

2014-01-01

SUMMARY Viruses were defined as one of the two principal types of organisms in the biosphere, namely, as capsid-encoding organisms in contrast to ribosome-encoding organisms, i.e., all cellular life forms. Structurally similar, apparently homologous capsids are present in a huge variety of icosahedral viruses that infect bacteria, archaea, and eukaryotes. These findings prompted the concept of the capsid as the virus “self” that defines the identity of deep, ancient viral lineages. However, several other widespread viral “hallmark genes” encode key components of the viral replication apparatus (such as polymerases and helicases) and combine with different capsid proteins, given the inherently modular character of viral evolution. Furthermore, diverse, widespread, capsidless selfish genetic elements, such as plasmids and various types of transposons, share hallmark genes with viruses. Viruses appear to have evolved from capsidless selfish elements, and vice versa, on multiple occasions during evolution. At the earliest, precellular stage of life's evolution, capsidless genetic parasites most likely emerged first and subsequently gave rise to different classes of viruses. In this review, we develop the concept of a greater virus world which forms an evolutionary network that is held together by shared conserved genes and includes both bona fide capsid-encoding viruses and different classes of capsidless replicons. Theoretical studies indicate that selfish replicons (genetic parasites) inevitably emerge in any sufficiently complex evolving ensemble of replicators. Therefore, the key signature of the greater virus world is not the presence of a capsid but rather genetic, informational parasitism itself, i.e., various degrees of reliance on the information processing systems of the host. PMID:24847023

Virus world as an evolutionary network of viruses and capsidless selfish elements.

PubMed

Koonin, Eugene V; Dolja, Valerian V

2014-06-01

Viruses were defined as one of the two principal types of organisms in the biosphere, namely, as capsid-encoding organisms in contrast to ribosome-encoding organisms, i.e., all cellular life forms. Structurally similar, apparently homologous capsids are present in a huge variety of icosahedral viruses that infect bacteria, archaea, and eukaryotes. These findings prompted the concept of the capsid as the virus "self" that defines the identity of deep, ancient viral lineages. However, several other widespread viral "hallmark genes" encode key components of the viral replication apparatus (such as polymerases and helicases) and combine with different capsid proteins, given the inherently modular character of viral evolution. Furthermore, diverse, widespread, capsidless selfish genetic elements, such as plasmids and various types of transposons, share hallmark genes with viruses. Viruses appear to have evolved from capsidless selfish elements, and vice versa, on multiple occasions during evolution. At the earliest, precellular stage of life's evolution, capsidless genetic parasites most likely emerged first and subsequently gave rise to different classes of viruses. In this review, we develop the concept of a greater virus world which forms an evolutionary network that is held together by shared conserved genes and includes both bona fide capsid-encoding viruses and different classes of capsidless replicons. Theoretical studies indicate that selfish replicons (genetic parasites) inevitably emerge in any sufficiently complex evolving ensemble of replicators. Therefore, the key signature of the greater virus world is not the presence of a capsid but rather genetic, informational parasitism itself, i.e., various degrees of reliance on the information processing systems of the host. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Tunable Protease-Activatable Virus Nanonodes

PubMed Central

2015-01-01

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery. PMID:24796495

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

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

Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072; Giolitti, Fabián

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cellmore » periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.« less

Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus.

PubMed

Kusumawati, Asmarani; Tampubolon, Issabellina Dwades; Hendarta, Narendra Yoga; Salasia, Siti Isrina Oktavia; Wanahari, Tenri Ashari; Mappakaya, Basofi Ashari; Hartati, Sri

2015-09-01

Jembrana disease virus (JDV) is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. An easy and rapid diagnostic method is essential for further control this disease. We used a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with lateral flow dipstick (LFD), based on conserved tm subunit of Jembrana disease virus env gene. The RT-LAMP conditions were optimized by varying the concentration of MgSO4, betaine, dNTP, and temperature as well as the time and duration of reaction. The primers sensitivity for JDV was confirmed. The method was able to detect env-tm gene dilution which contained 2 × 10(-15) g of template. Comparatively, the sensitivity of RT-LAMP/LFD was 100-fold more sensitive than reverse transcription-polymerase chain reaction. The primers specificity for JDV was also confirmed using positive and negative controls. This work also showed that virus detection could be done not only on total RNA extracted from blood but various organs could also be analyzed for the presence of JDV using RT-LAMP/LFD method. The whole process, including the LAMP reaction and the LFD hybridization step only lasts approximately 75 min. Results of analysis can be easily observed with naked eyes without addition of any chemical or further analysis. The combination of RT-LAMP with LFD makes the method a more suitable diagnostic tool in conditions where sophisticated and expensive equipments are not available for field investigations on Jembrana disease in Bali cattle.

Cowpea viruses: Effect of single and mixed infections on symptomatology and virus concentration

PubMed Central

Taiwo, Moni A; Kareem, Kehinde T; Nsa, Imade Y; D'A Hughes, Jackies

2007-01-01

Natural multiple viral infections of cultivated cowpeas have been reported in Nigeria. In this study, three Nigerian commercial cowpea cultivars ("Olo 11", "Oloyin" and "White") and two lines from the IITA (IT86D- 719 and TVU 76) were mechanically inoculated with Cowpea aphid-borne mosaic virus (CABMV), Bean southern mosaic virus (SBMV) and Cowpea mottle virus (CMeV) singly, as well as in all possible combinations at 10, 20 and 30 days after planting (DAP). Samples of leaves or stems were collected at 10, 20 and 30 days after inoculation (DAI) and analyzed for relative virus concentration by Enzyme-Linked Immunosrbent Assay. All the cultivars and lines {CVS/L} were susceptible to the viruses but the commercial CVS showed more severe symptoms and had relatively higher viral concentration. In single virus infections, CABMV which induced the most severe symptoms had absorbance values (at 405 nm) of 0.11 to 0.46 while SBMV and CMeV which induced moderate symptoms had virus titre of 0.74 to 1.99 and 0.11 to 0.90 respectively. Plants inoculated 10 DAP had significantly higher virus concentration than those inoculated 30 DAP. In mixed infections involving CABMV (10 DAP) apical necrosis and death were observed in commercial cultivars "Olo 11" and "White". Enhancement of CMeV titers were observed in plants infected with CMeV + CABMV. Multiple viral infections of cowpeas may result in complete yield loss, hence, the availability of seeds of cultivars with a high level of multiple virus resistance is recommended as a means of control. PMID:17900355

A virus-based biocatalyst

NASA Astrophysics Data System (ADS)

Carette, Noëlle; Engelkamp, Hans; Akpa, Eric; Pierre, Sebastien J.; Cameron, Neil R.; Christianen, Peter C. M.; Maan, Jan C.; Thies, Jens C.; Weberskirch, Ralf; Rowan, Alan E.; Nolte, Roeland J. M.; Michon, Thierry; van Hest, Jan C. M.

2007-04-01

Virus particles are probably the most precisely defined nanometre-sized objects that can be formed by protein self-assembly. Although their natural function is the storage and transport of genetic material, they have more recently been applied as scaffolds for mineralization and as containers for the encapsulation of inorganic compounds. The reproductive power of viruses has been used to develop versatile analytical methods, such as phage display, for the selection and identification of (bio)active compounds. To date, the combined use of self-assembly and reproduction has not been used for the construction of catalytic systems. Here we describe a self-assembled system based on a plant virus that has its coat protein genetically modified to provide it with a lipase enzyme. Using single-object and bulk catalytic studies, we prove that the virus-anchored lipase molecules are catalytically active. This anchored biocatalyst, unlike man-made supported catalysts, has the capability to reproduce itself in vivo, generating many independent catalytically active copies.

Efficacies of Gel Formulations Containing Foscarnet, Alone or Combined with Sodium Lauryl Sulfate, against Establishment and Reactivation of Latent Herpes Simplex Virus Type 1

PubMed Central

Piret, Jocelyne; Lamontagne, Julie; Désormeaux, André; Bergeron, Michel G.

2001-01-01

The influence of sodium lauryl sulfate (SLS) on the efficacies of gel formulations of foscarnet against herpes simplex virus type 1 (HSV-1) cutaneous lesions and on the establishment and reactivation of latent virus has been evaluated in a murine model of orofacial infection. Topical treatments were given twice daily for 3 days and were initiated at 6, 24, and 48 h after virus inoculation. The gel formulation that contained both 3% foscarnet and 5% SLS and that was administered within 48 h postinfection reduced the rate of development of herpetic skin lesions. This formulation also significantly decreased the viral content in skin tissues and in ipsilateral trigeminal ganglia when it was given within 24 and 6 h postinfection, respectively. A lower level of efficacy was observed for the gel formulation containing 3% foscarnet alone. Of prime interest, the gel formulation containing 5% SLS reduced significantly the mortality rate among mice in a zosteriform model of infection. Both formulations of foscarnet had no effect on the mean titers of reactivated virus in explant cultures of ipsilateral and contralateral trigeminal ganglia from latently infected mice. The use of a gel formulation containing combinations of foscarnet and SLS could represent an attractive approach for the treatment of herpetic mucocutaneous infections. PMID:11257012

Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine.

PubMed Central

Abad, F X; Pintó, R M; Diez, J M; Bosch, A

1994-01-01

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV. Images PMID:8074518

Lassa-Vesicular Stomatitis Chimeric Virus Safely Destroys Brain Tumors

PubMed Central

Wollmann, Guido; Drokhlyansky, Eugene; Davis, John N.; Cepko, Connie

2015-01-01

ABSTRACT High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We

Therapeutic vaccination to treat chronic infectious diseases

PubMed Central

Boukhebza, Houda; Bellon, Nadine; Limacher, Jean Marc; Inchauspé, Geneviève

2012-01-01

A famous milestone in the vaccine field has been the first successful vaccination against smallpox, in 1798, by Edward Jenner. Using the vaccinia cowpox virus, Jenner was able to protect vaccinees from variola or smallpox. The Modified Virus Ankara (MVA) poxvirus strain has been one of the vaccines subsequently developed to prevent smallpox infection and was selected by the US government in their Biodefense strategy. Progress in molecular biology and immunology associated with MVA infection has led to the development of MVA as vaccine platform, both in the field of preventive and therapeutic vaccines. This later class of therapeutics has witnessed growing interest that has translated into an increasing number of vaccine candidates reaching the clinics. Among those, MVA-based therapeutic vaccines have addressed four major chronic infections including viral hepatitis, AIDS, human papillomavirus-linked pathologies and tuberculosis. Clinical trials encompass phase 1 and 2 and have started to show significant results and promises. PMID:22894957

Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests.

PubMed

Tsai, Wen-Yang; Youn, Han Ha; Brites, Carlos; Tsai, Jih-Jin; Tyson, Jasmine; Pedroso, Celia; Drexler, Jan Felix; Stone, Mars; Simmons, Graham; Busch, Michael P; Lanteri, Marion; Stramer, Susan L; Balmaseda, Angel; Harris, Eva; Wang, Wei-Kung

2017-11-13

The explosive spread of Zika virus (ZIKV) and associated microcephaly present an urgent need for sensitive and specific serodiagnostic tests, particularly for pregnant women in dengue virus (DENV)-endemic regions. Recent reports of enhanced ZIKV replication by dengue-immune sera have raised concerns about the role of previous DENV infection on the risk and severity of microcephaly and other ZIKV complications. Enzyme-linked immunosorbent assays (ELISAs) based on ZIKV and DENV nonstructural protein 1 (NS1) were established to test acute, convalescent phase, and post-convalescent phase serum/plasma samples from reverse-transcription polymerase chain reaction-confirmed cases including 20 primary ZIKV, 25 ZIKV with previous DENV, 58 secondary DENV, and 16 primary DENV1 infections. ZIKV-NS1 immunoglobulin M (IgM) and immunoglobulin G (IgG) ELISAs combined can detect ZIKV infection with a sensitivity of 95% and specificity of 66.7%. The ZIKV-NS1 IgG cross-reactivity by samples from secondary DENV infection cases ranged from 66.7% to 28.1% (within 1 month to 1-2 years post-illness, respectively). Addition of DENV1-NS1 IgG ELISA can distinguish primary ZIKV infection; the ratio of absorbance of ZIKV-NS1 to DENV1-NS1 IgG ELISA can distinguish ZIKV with previous DENV and secondary DENV infections with a sensitivity of 87.5% and specificity of 81.3%. These findings were supported by analysis of sequential samples. An algorithm for ZIKV serodiagnosis based on 3 simple ELISAs is proposed to distinguish primary ZIKV, ZIKV with previous DENV, and secondary DENV infections; this could be applied to serodiagnosis for ZIKV, serosurveillance, and monitoring ZIKV infection during pregnancy to understand the epidemiology, pathogenesis, and complications of ZIKV in dengue-endemic regions. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of Herpes simplex virus types 1 and 2 and Varicella-zoster virus.

PubMed

Weidmann, Manfred; Armbruster, Katrin; Hufert, Frank T

2008-08-01

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.

A polyvalent Clade B virus-like particle HIV vaccine combined with partially protective oral preexposure prophylaxis prevents simian-human immunodeficiency virus Infection in macaques and primes for virus-amplified immunity.

PubMed

Ross, Ted M; Pereira, Lara E; Luckay, Amara; McNicholl, Janet M; García-Lerma, J Gerardo; Heneine, Walid; Eugene, Hermancia S; Pierce-Paul, Brooke R; Zhang, Jining; Hendry, R Michael; Smith, James M

2014-11-01

Vaccination and preexposure prophylaxis (PrEP) with antiretrovirals have shown only partial protection from HIV-1 infection in human trials. Oral Truvada (emtricitabine/tenofovir disoproxil fumarate) is FDA approved as PrEP but partial adherence reduces efficacy. If combined as biomedical preventions (CBP), an HIV vaccine could protect when PrEP adherence is low and PrEP could prevent vaccine breakthroughs. The efficacy of combining oral PrEP with an HIV vaccine has not been evaluated in humans. We determined the efficacy of combining a DNA/virus-like particle (VLP) vaccine with partially effective intermittent PrEP in Indian rhesus macaques (RM). Eight RM received intramuscular inoculations of five DNA plasmids encoding four HIV-1 Clade B primary isolate Envs and SIVmac239 Gag (at weeks 0 and 4), followed by intramuscular and intranasal inoculations of homologous Gag VLPs and four Env VLPs (at weeks 12, 16, and 53). At week 61, we initiated weekly rectal exposures with heterologous SHIV162p3 (10 TCID50) along with oral Truvada (TDF, 22 mg/kg; FTC 20 mg/kg) dosing 2 h before and 22 h after each exposure. This PrEP regimen previously demonstrated 50% efficacy. Five controls (no vaccine, no PrEP) received weekly SHIV162p3. All controls were infected after a median of four exposures; the mean peak plasma viral load (VL) was 3.9×10(7) vRNA copies/ml. CBP protected seven of eight (87.5%) RM. The one infected CBP RM had a reduced peak VL of 8.8×10(5) copies/ml. SHIV exposures during PrEP amplified Gag and Env antibody titers in protected RM. These results suggest that combining oral PrEP with HIV vaccines could enhance protection against HIV-1 infection.

Interim Report on SNP analysis and forensic microarray probe design for South American hemorrhagic fever viruses, tick-borne encephalitis virus, henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever viruses, Rift Valley fever

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

Jaing, C; Gardner, S

The goal of this project is to develop forensic genotyping assays for select agent viruses, enhancing the current capabilities for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the whole genomemore » wide SNP analysis and microarray probe design for forensics characterization of South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.« less

Induction of Mucosal Homing Virus-Specific CD8+ T Lymphocytes by Attenuated Simian Immunodeficiency Virus

PubMed Central

Cromwell, Mandy A.; Veazey, Ronald S.; Altman, John D.; Mansfield, Keith G.; Glickman, Rhona; Allen, Todd M.; Watkins, David I.; Lackner, Andrew A.; Johnson, R. Paul

2000-01-01

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8+ lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor α4β7 and traffic to the intestinal mucosa. SIV-specific CD8+ T cells expressing α4β7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express α4β7. These results demonstrate the selective induction of SIV-specific CD8+ T lymphocytes expressing α4β7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine. PMID:10954580

Combined use of the ASK and SHK-1 cell lines to enhance the detection of infectious salmon anemia virus

USGS Publications Warehouse

Rolland, J.B.; Bouchard, D.; Coll, J.; Winton, J.R.

2005-01-01

Infectious salmon anemia (ISA) is a severe disease primarily affecting commercially farmed Atlantic salmon (Salmo salar) in seawater. The disease has been reported in portions of Canada, the United Kingdom, the Faroe Islands, and the United States. Infectious salmon anemia virus (ISAV), the causative agent of ISA, has also been isolated from several asymptomatic marine and salmonid fish species. Diagnostic assays for the detection of ISAV include virus isolation in cell culture, a reverse transcriptase-PCR, an enzyme-linked immunosorbent assay, and an indirect fluorescent antibody test. Virus isolation is considered the gold standard, and 5 salmonid cell lines are known to support growth of ISAV. In this study, the relative performance of the salmon head kidney 1 (SHK-1), Atlantic salmon kidney (ASK), and CHSE-214 cell lines in detecting ISAV was evaluated using samples from both experimentally and naturally infected Atlantic salmon. Interlaboratory comparisons were conducted using a quality control-quality assurance ring test. Both the ASK and SHK-1 cell lines performed well in detecting ISAV, although the SHK-1 line was more variable in its sensitivity to infection and somewhat slower in the appearance of cytopathic effect. Relative to the SHK-1 and ASK lines, the CHSE-214 cell line performed poorly. Although the ASK line appeared to represent a good alternative to the more commonly used SHK-1 line, use of a single cell line for diagnostic assays may increase the potential for false-negative results. Thus, the SHK-1 and ASK cell lines can be used in combination to provide enhanced ability to detect ISAV.

Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

PubMed Central

VanSeggelen, Heather; Tantalo, Daniela GM; Afsahi, Arya; Hammill, Joanne A; Bramson, Jonathan L

2015-01-01

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments. PMID:27119109

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

The hepatitis C virus enigma.

PubMed

Myrmel, Helge; Ulvestad, Elling; Asjø, Birgitta

2009-05-01

Hepatitis C virus (HCV) has a high propensity to establish chronic infection with end-stage liver disease. The high turnover of virus particles and high transcription error rates due to lack of proof-reading function of the viral polymerase imply that HCV exists as quasispecies, thus enabling the virus to evade the host immune response. Clearance of the virus is characterized by a multispecific, vigorous and persistent T-cell response, whereas T-cell responses are weak, narrow and transient in patients who develop chronic infection. At present, standard treatment is a combination of pegylated interferon-alpha and ribavirin, with a sustained viral response rate of 40-80%, depending on genotype. The mechanisms for the observed synergistic effects of the two drugs are still not known in detail, but in addition to direct antiviral mechanisms, the immunomodulatory effects of both drugs seem to be important, with a shift from Th2- to Th1-cytokine profiles in successfully treated patients. This article describes virus-host relations in the natural course of HCV infection and during treatment.

Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

PubMed Central

Jazaeri Farsani, Seyed Mohammad; Deijs, Martin; Dijkman, Ronald; Molenkamp, Richard; Jeeninga, Rienk E; Ieven, Margareta; Goossens, Herman; van der Hoek, Lia

2015-01-01

Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates. PMID:25482367

Chimaera and its modern virus-like descendants.

PubMed

Ulrich, R; Gerlich, W H; Krüger, D H

1996-01-01

Chimaera was a monster in ancient Greek mythology combining elements from different animal species in its body. Modern molecular biology enabled the generation of harmless but useful chimaeras consisting of elements from different nonrelated viruses. The objective is that the resulting chimaeras form highly immunogenic virus-like particles (VLPs). Such chimaeric VLPs can be used as highly efficient carriers for sequential and conformational B cell epitopes and T cell epitopes. Most VLPs are readily produced in heterologous hosts and are easy to purify. This article deals with various systems of VLPs described in this topical issue of Intervirology and makes comparisons with chimaeric replication-competent viruses, recombinant virus vectors expressing foreign proteins, and DNA vaccines.

Effective virus inactivation and removal by steps of Biotest Pharmaceuticals IGIV production process

PubMed Central

Dichtelmüller, Herbert O.; Flechsig, Eckhard; Sananes, Frank; Kretschmar, Michael; Dougherty, Christopher J.

2012-01-01

The virus validation of three steps of Biotest Pharmaceuticals IGIV production process is described here. The steps validated are precipitation and removal of fraction III of the cold ethanol fractionation process, solvent/detergent treatment and 35 nm virus filtration. Virus validation was performed considering combined worst case conditions. By these validated steps sufficient virus inactivation/removal is achieved, resulting in a virus safe product. PMID:24371563

Virulence determinants of pandemic influenza viruses

PubMed Central

Tscherne, Donna M.; García-Sastre, Adolfo

2011-01-01

Influenza A viruses cause recurrent, seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. The ability of influenza A viruses to adapt to various hosts and undergo reassortment events ensures constant generation of new strains with unpredictable degrees of pathogenicity, transmissibility, and pandemic potential. Currently, the combination of factors that drives the emergence of pandemic influenza is unclear, making it impossible to foresee the details of a future outbreak. Identification and characterization of influenza A virus virulence determinants may provide insight into genotypic signatures of pathogenicity as well as a more thorough understanding of the factors that give rise to pandemics. PMID:21206092

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

Immunopathology of highly virulent pathogens: insights from Ebola virus.

PubMed

Zampieri, Carisa A; Sullivan, Nancy J; Nabel, Gary J

2007-11-01

Ebola virus is a highly virulent pathogen capable of inducing a frequently lethal hemorrhagic fever syndrome. Accumulating evidence indicates that the virus actively subverts both innate and adaptive immune responses and triggers harmful inflammatory responses as it inflicts direct tissue damage. The host immune system is ultimately overwhelmed by a combination of inflammatory factors and virus-induced cell damage, particularly in the liver and vasculature, often leading to death from septic shock. We summarize the mechanisms of immune dysregulation and virus-mediated cell damage in Ebola virus-infected patients. Future approaches to prevention and treatment of infection will be guided by answers to unresolved questions about interspecies transmission, molecular mechanisms of pathogenesis, and protective adaptive and innate immune responses to Ebola virus.

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

An Epidemiological Investigation of Hart Park and Turlock Viruses in California.

DTIC Science & Technology

1984-01-01

strains tested were truly HP and not FLA-like (unpublished). -p. Both HP and FLA viruses are classified as members of the family r Rhabdoviridae on...relationship. Investigations with sigma virus have shown thLt the genetics of both the virus and the insect play a role in viral replication in a...particular virus- insect combination (Brun and Plus, 1980). Alternative possibilities for maintenance of arboviruses have been reviewed by Reeves (1974) and

Electron microscopy and antigenic studies of uncharacterized viruses. I. Evidence suggesting the placement of viruses in families Arenaviridae, Paramyxoviridae, or Poxviridae.

PubMed

Zeller, H G; Karabatsos, N; Calisher, C H; Digoutte, J P; Murphy, F A; Shope, R E

1989-01-01

During approximately 35 years, investigators in various laboratories studying arbovirus ecology and epidemiology accumulated many virus isolates, more than 60 of which were not characterized or placed in taxa. By a combination of electron microscopic and antigenic studies we collected information sufficient to provisionally classify 60 isolates. Electron microscopic observations suggest that 20 are members of the virus family Bunyaviridae, 20 Rhabdoviridae, 14 Reoviridae, one Togaviridae, one Paramyxoviridae (Mapuera virus, from a bat), and one Poxviridae (Yoka virus, from mosquitoes). Serologic studies provided evidence sufficient to place some of these viruses in recognized antigenic groups, within families and genera, and to establish new antigenic groups and taxa for others. Three viruses were found to have morphologic and morphogenetic characteristics consistent with those of members of the family Arenaviridae: Quaranfil virus, a human pathogen, Johnston Atoll virus, isolated from birds and ticks, and Araguari virus, isolated from an opossum. This, the first in a series of three papers, described methods used for these investigations and also presents descriptions of viruses provisionally placed in the families Arenaviridae, Paramyxoviridae, or Poxviridae. Descriptions of viruses provisionally placed in families Bunyaviridae and Reoviridae are published in the second and third papers, respectively. Viruses of the family Rhabdoviridae have been described separately.

Pretreatment with propidium monoazide/sodium lauroyl sarcosinate improves discrimination of infectious waterborne virus by RT-qPCR combined with magnetic separation.

PubMed

Lee, Hae-Won; Lee, Hee-Min; Yoon, So-Ra; Kim, Sung Hyun; Ha, Ji-Hyoung

2018-02-01

RT-qPCR allows sensitive detection of viral particles of both infectious and noninfectious viruses in water environments, but cannot discriminate non-infectious from infectious viruses. In this study, we aimed to optimize RT-qPCR-based detection of chlorine-inactivated human norovirus (NoV) and pepper mild mottle virus (PMMoV) in suspension by pretreatment with an optimal combination of a monoazide and a detergent that can efficiently penetrate damaged viral capsids. Four methods were compared to determine the efficacy of chlorine disinfection (at 1, 3, and 5 min mg/L): (A) RT-qPCR alone, (B) RT-qPCR assay preceded by magnetic bead separation for enrichment of viral particles (MBS-RT-qPCR), (C) MBS-RT-qPCR assay with pretreatment with propidium monoazide (PMA-MBS-RT-qPCR), and (D) PMA-MBS-RT-qPCR assay with pretreatment with sodium lauroyl sarcosinate (INCI-PMA-MBS-RT-qPCR). On the basis of a PMA optimization assay, 200 and 300 μM PMA were used in subsequent experiments for NoV GII.4 and PMMoV, respectively. Optimal INCI concentrations, having minimal influence on NoV GII.4 and PMMoV, were found to be 0.5% and 0.2% INCI, respectively. For NoV GII.4, there were significant differences (P < 0.05) in log 10 genome copies between the PMA-treated and the INCI + PMA-treated samples (log 10 genome copies differed by 1.11 and 0.59 log 10 for 3 and 5 min mg/L of chlorine, respectively). For PMMoV, INCI induced differences in log 10 genome copies of 0.92, 1.18, and 1.86, for 1, 3, and 5 min mg/L of chlorine, respectively. Overall, the results of this study indicate that an optimal combination of PMA and INCI could be very useful for evaluating disinfection methods in water treatment strategies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

PubMed

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

A combination HIV reporter virus system for measuring post-entry event efficiency and viral outcome in primary CD4+ T cell subsets.

PubMed

Tilton, Carisa A; Tabler, Caroline O; Lucera, Mark B; Marek, Samantha L; Haqqani, Aiman A; Tilton, John C

2014-01-01

Fusion between the viral membrane of human immunodeficiency virus (HIV) and the host cell marks the end of the HIV entry process and the beginning of a series of post-entry events including uncoating, reverse transcription, integration, and viral gene expression. The efficiency of post-entry events can be modulated by cellular factors including viral restriction factors and can lead to several distinct outcomes: productive, latent, or abortive infection. Understanding host and viral proteins impacting post-entry event efficiency and viral outcome is critical for strategies to reduce HIV infectivity and to optimize transduction of HIV-based gene therapy vectors. Here, we report a combination reporter virus system measuring both membrane fusion and viral promoter-driven gene expression. This system enables precise determination of unstimulated primary CD4+ T cell subsets targeted by HIV, the efficiency of post-entry viral events, and viral outcome and is compatible with high-throughput screening and cell-sorting methods. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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

Herpes Simplex Virus 1 Tropism for Human Sensory Ganglion Neurons in the Severe Combined Immunodeficiency Mouse Model of Neuropathogenesis

PubMed Central

Che, Xibing; Reichelt, Mike; Qiao, Yanli; Gu, Haidong; Arvin, Ann

2013-01-01

The tropism of herpes simplex virus (HSV-1) for human sensory neurons infected in vivo was examined using dorsal root ganglion (DRG) xenografts maintained in mice with severe combined immunodeficiency (SCID). In contrast to the HSV-1 lytic infectious cycle in vitro, replication of the HSV-1 F strain was restricted in human DRG neurons despite the absence of adaptive immune responses in SCID mice, allowing the establishment of neuronal latency. At 12 days after DRG inoculation, 26.2% of human neurons expressed HSV-1 protein and 13.1% expressed latency-associated transcripts (LAT). Some infected neurons showed cytopathic changes, but HSV-1, unlike varicella-zoster virus (VZV), only rarely infected satellite cells and did not induce fusion of neuronal and satellite cell plasma membranes. Cell-free enveloped HSV-1 virions were observed, indicating productive infection. A recombinant HSV-1-expressing luciferase exhibited less virulence than HSV-1 F in the SCID mouse host, enabling analysis of infection in human DRG xenografts for a 61-day interval. At 12 days after inoculation, 4.2% of neurons expressed HSV-1 proteins; frequencies increased to 32.1% at 33 days but declined to 20.8% by 61 days. Frequencies of LAT-positive neurons were 1.2% at 12 days and increased to 40.2% at 33 days. LAT expression remained at 37% at 61 days, in contrast to the decline in neurons expressing viral proteins. These observations show that the progression of HSV-1 infection is highly restricted in human DRG, and HSV-1 genome silencing occurs in human neurons infected in vivo as a consequence of virus-host cell interactions and does not require adaptive immune control. PMID:23269807

Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

NASA Astrophysics Data System (ADS)

Liu, Jin

2012-11-01

Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

PubMed Central

Powers, D C; Fries, L F; Murphy, B R; Thumar, B; Clements, M L

1991-01-01

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group. PMID:2037667

A systematic approach to novel virus discovery in emerging infectious disease outbreaks.

PubMed

Sridhar, Siddharth; To, Kelvin K W; Chan, Jasper F W; Lau, Susanna K P; Woo, Patrick C Y; Yuen, Kwok-Yung

2015-05-01

The discovery of novel viruses is of great importance to human health-both in the setting of emerging infectious disease outbreaks and in disease syndromes of unknown etiology. Despite the recent proliferation of many efficient virus discovery methods, careful selection of a combination of methods is important to demonstrate a novel virus, its clinical associations, and its relevance in a timely manner. The identification of a patient or an outbreak with distinctive clinical features and negative routine microbiological workup is often the starting point for virus hunting. This review appraises the roles of culture, electron microscopy, and nucleic acid detection-based methods in optimizing virus discovery. Cell culture is generally slow but may yield viable virus. Although the choice of cell line often involves trial and error, it may be guided by the clinical syndrome. Electron microscopy is insensitive but fast, and may provide morphological clues to choice of cell line or consensus primers for nucleic acid detection. Consensus primer PCR can be used to detect viruses that are closely related to known virus families. Random primer amplification and high-throughput sequencing can catch any virus genome but cannot yield an infectious virion for testing Koch postulates. A systematic approach that incorporates carefully chosen combinations of virus detection techniques is required for successful virus discovery. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A manual and an automatic TERS based virus discrimination

NASA Astrophysics Data System (ADS)

Olschewski, Konstanze; Kämmer, Evelyn; Stöckel, Stephan; Bocklitz, Thomas; Deckert-Gaudig, Tanja; Zell, Roland; Cialla-May, Dana; Weber, Karina; Deckert, Volker; Popp, Jürgen

2015-02-01

Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%.Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses

Virus diseases of peppers (Capsicum spp.) and their control.

PubMed

Kenyon, Lawrence; Kumar, Sanjeet; Tsai, Wen-Shi; Hughes, Jacqueline d'A

2014-01-01

The number of virus species infecting pepper (Capsicum spp.) crops and their incidences has increased considerably over the past 30 years, particularly in tropical and subtropical pepper production systems. This is probably due to a combination of factors, including the expansion and intensification of pepper cultivation in these regions, the increased volume and speed of global trade of fresh produce (including peppers) carrying viruses and vectors to new locations, and perhaps climate change expanding the geographic range suitable for the viruses and vectors. With the increased incidences of diverse virus species comes increased incidences of coinfection with two or more virus species in the same plant. There is then greater chance of synergistic interactions between virus species, increasing symptom severity and weakening host resistance, as well as the opportunity for genetic recombination and component exchange and a possible increase in aggressiveness, virulence, and transmissibility. The main virus groups infecting peppers are transmitted by aphids, whiteflies, or thrips, and a feature of many populations of these vector groups is that they can develop resistance to some of the commonly used insecticides relatively quickly. This, coupled with the increasing concern over the impact of over- or misuse of insecticides on the environment, growers, and consumers, means that there should be less reliance on insecticides to control the vectors of viruses infecting pepper crops. To improve the durability of pepper crop protection measures, there should be a shift away from the broadscale use of insecticides and the use of single, major gene resistance to viruses. Instead, integrated and pragmatic virus control measures should be sought that combine (1) cultural practices that reduce sources of virus inoculum and decrease the rate of spread of viruliferous vectors into the pepper crop, (2) synthetic insecticides, which should be used judiciously and only when the

Evidence for genetic variation of Eurasian avian influenza viruses, subtype H15: The first report of an H15N7 virus

USDA-ARS?s Scientific Manuscript database

Since the first detection of H15 avian influenza viruses (AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against...

Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions.

PubMed

Meckes, David G

2014-01-01

The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

Avian Influenza A H7N9 Virus Induces Severe Pneumonia in Mice without Prior Adaptation and Responds to a Combination of Zanamivir and COX-2 Inhibitor

PubMed Central

Zhang, Anna J. X.; To, Kelvin K. W.; Lee, Andrew C. Y.; Zhu, Houshun; Wu, Hazel W. L.; Chan, Jasper F. W.; Chen, Honglin; Hung, Ivan F. N.; Li, Lanjuan; Yuen, Kwok-Yung

2014-01-01

Background Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed. Methods BALB/c mice were inoculated intranasally with an H7N9 virus isolated from a chicken in a wet market epidemiologically linked to a fatal human case, (A/chicken/Zhejiang/DTID-ZJU01/2013 [CK1]), and with an H7N9 virus isolated from a human (A/Anhui/01/2013 [AH1]). The pulmonary viral loads, cytokine/chemokine profiles and histopathological changes of the infected mice were compared. The therapeutic efficacy of a non-steroidal anti-inflammatory drug (NSAID), celecoxib, was assessed. Results Without prior adaptation, intranasal inoculation of 106 plaque forming units (PFUs) of CK1 caused a mortality rate of 82% (14/17) in mice. Viral nucleoprotein and RNA expression were limited to the respiratory system and no viral RNA could be detected from brain, liver and kidney tissues. CK1 caused heavy alveolar inflammatory exudation and pulmonary hemorrhage, associated with high pulmonary levels of proinflammatory cytokines. In the mouse lung cell line LA-4, CK1 also induced high levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA. Administration of the antiviral zanamivir did not significantly improve survival in mice infected with CK1, but co-administration of the non-steroidal anti-inflammatory drug (NSAID) celecoxib in combination with zanamivir improved survival and lung pathology. Conclusions Our findings suggested that H7N9 viruses isolated from chicken without preceding trans-species adaptation can cause lethal mammalian pulmonary infection. The severe proinflammatory responses might be a factor contributing to the mortality. Treatment with combination of antiviral and NSAID could ameliorate pulmonary inflammation and may improve survival. PMID:25232731

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction.

PubMed

Noronha, Jyothi M; Liu, Mengya; Squires, R Burke; Pickett, Brett E; Hale, Benjamin G; Air, Gillian M; Galloway, Summer E; Takimoto, Toru; Schmolke, Mirco; Hunt, Victoria; Klem, Edward; García-Sastre, Adolfo; McGee, Monnie; Scheuermann, Richard H

2012-05-01

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.

Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold.

PubMed

Aravapalli, Sridhar; Lai, Huiguo; Teramoto, Tadahisa; Alliston, Kevin R; Lushington, Gerald H; Ferguson, Eron L; Padmanabhan, R; Groutas, William C

2012-07-01

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening. Copyright © 2012 Elsevier Ltd. All rights reserved.

Quasispecies and virus.

PubMed

Domingo, Esteban; Perales, Celia

2018-05-01

Quasispecies theory has been instrumental in the understanding of RNA virus population dynamics because it considered for the first time mutation as an integral part of the replication process. The key influences of quasispecies theory on experimental virology have been: (1) to disclose the mutant spectrum nature of viral populations and to evaluate its consequences; (2) to unveil collective properties of genome ensembles that can render a mutant spectrum a unit of selection; and (3) to identify new vulnerability points of pathogenic RNA viruses on three fronts: the need to apply multiple selective constraints (in the form of drug combinations) to minimize selection of treatment-escape variants, to translate the error threshold concept into antiviral designs, and to construct attenuated vaccine viruses through alterations of viral polymerase copying fidelity or through displacements of viral genomes towards unfavorable regions of sequence space. These three major influences on the understanding of viral pathogens preceded extensions of quasispecies to non-viral systems such as bacterial and tumor cell collectivities and prions. These developments are summarized here.

Vaccine Efficacy against Malaria by the Combination of Porcine Parvovirus-Like Particles and Vaccinia Virus Vectors Expressing CS of Plasmodium

PubMed Central

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R.; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J. Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria. PMID:22529915

Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene

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

Pater, A.; Pater, M.M.

Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that the authors examined. However, cells isolated from foci were incapable of growth in soft agar. They then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and themore » activated form of Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.« less

H9N2 Influenza Whole Inactivated Virus Combined with Polyethyleneimine Strongly Enhances Mucosal and Systemic Immunity after Intranasal Immunization in Mice

PubMed Central

Qin, Tao; Yin, Yinyan; Huang, Lulu; Yu, Qinghua

2015-01-01

Influenza whole inactivated virus (WIV) is more immunogenic and induces protective antibody responses compared with other formulations, like split virus or subunit vaccines, after intranasal mucosal delivery. Polyethyleneimine (PEI), an organic polycation, is widely used as a reagent for gene transfection and DNA vaccine delivery. Although PEI recently has demonstrated potent mucosal adjuvant activity for viral subunit glycoprotein antigens, its immune activity with H9N2 WIV is not well demonstrated. Here, mice were immunized intranasally with H9N2 WIV combined with PEI, and the levels of local respiratory tract and systemic immune responses were measured. Compared to H9N2 WIV alone, antigen-specific IgA levels in the local nasal cavity, trachea, and lung, as well as levels of IgG and its subtypes (IgG1 and IgG2a) in the serum, were strongly enhanced with the combination. Similarly, the activation and proliferation of splenocytes were markedly increased. In addition, PEI is superior as an H9N2 WIV delivery system due to its ability to greatly increase the viral adhesion to mucosal epithelial cells and to enhance the cellular uptake and endosomal escape of antigens in dendritic cells (DCs) and further significantly activate DCs to mature. Taken together, these results provided more insights that PEI has potential as an adjuvant for H9N2 particle antigen intranasal vaccination. PMID:25673304

Burden of non-AIDS-defining and non-virus-related cancers among HIV-infected patients in the combined antiretroviral therapy era.

PubMed

Albini, Laura; Calabresi, Alessandra; Gotti, Daria; Ferraresi, Alice; Festa, Andrea; Donato, Francesco; Magoni, Michele; Castelli, Francesco; Quiros-Roldan, Eugenia

2013-08-01

The risk of cancer is substantially increased in HIV-infected patients. However, little is known about non-AIDS-defining cancers (NADCs) without an infectious etiology. A total of 5,090 HIV-infected patients registered in the Local Health Authority (LHA) of Brescia and receiving primary care at our clinic were included in a retrospective (1999-2009) analysis. The cancer diagnoses were obtained through a record-linkage procedure between our database and the LHA general database and population-based Cancer Registry of LHA. We compared risks of these malignancies with those of the general population living in the same health area by using age-standardized incidence ratios (SIRs). Poisson regression analysis was used to assess factors associated with non-virus-related NADCs. We recorded an increase in the SIR of non-virus-related NADCs over time, with 138 cancers diagnosed in 131 patients. The mean incidence rate was 42.6/10,000 person years and the median age at the diagnosis was 49 (range, 28-78) years old. Stratifying for gender, only HIV-infected males had an increased risk of non-virus-related NADCs [SIR=1.86; 95% confidence interval (CI), 1.55-2.26]. Risk was higher for lung (SIR=3.59; 95% CI, 2.36-5.45) and testis cancer (SIR=3.11; 95% CI, 1.48-6.52). However,, cancers of the prostate and breast in HIV-positive men and women were null (SIR=1.10; 95% CI, 0.53-2.32 and SIR=0.91; 95% CI, 0.47-1.74, respectively). The only predictors of non-virus-related NADCs included older age [incidence rate ratio (IRR)=1.10; 95% CI, 1.08-1.12 per each additional year, p<0.001] and a shorter or no exposition to combined antiretroviral therapy (cART) (IRR=2.31; 95% CI, 1.38-3.89, p=0.002). A CD4⁺ count lower than 50/mm³ was significantly associated with cancers only in the univariate model (IRR=1.40; 95% CI, 0.99-1.98, p=0.057). HIV-infected men showed a 2-fold increased risk of non-virus-related NADCs compared to the general population. However, the use of cART appeared to be

Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

USDA-ARS?s Scientific Manuscript database

In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

Transmission of Hepatitis A Virus through Combined Liver–Small Intestine–Pancreas Transplantation

PubMed Central

Weil, Lauren M.; Jin, Sherry; Johnson, Thomas; Hayden-Mixson, Tonya R.; Khudyakov, Yury; Annambhotla, Pallavi D.; Basavaraju, Sridhar V.; Kamili, Saleem; Ritter, Jana M.; Nelson, Noele; Mazariegos, George; Green, Michael; Himes, Ryan W.; Kuhar, David T.; Kuehnert, Matthew J.; Miller, Jeffrey A.; Wiseman, Rachel; Moorman, Anne C.

2017-01-01

Although transmission of hepatitis A virus (HAV) through blood transfusion has been documented, transmission through organ transplantation has not been reported. In August 2015, state health officials in Texas, USA, were notified of 2 home health nurses with HAV infection whose only common exposure was a child who had undergone multi–visceral organ transplantation 9 months earlier. Specimens from the nurses, organ donor, and all organ recipients were tested and medical records reviewed to determine a possible infection source. Identical HAV RNA sequences were detected from the serum of both nurses and the organ donor, as well as from the multi–visceral organ recipient’s serum and feces; this recipient’s posttransplant liver and intestine biopsy specimens also had detectable virus. The other organ recipients tested negative for HAV RNA. Vaccination of the donor might have prevented infection in the recipient and subsequent transmission to the healthcare workers. PMID:28322704

Transmission of Hepatitis A Virus through Combined Liver-Small Intestine-Pancreas Transplantation.

PubMed

Foster, Monique A; Weil, Lauren M; Jin, Sherry; Johnson, Thomas; Hayden-Mixson, Tonya R; Khudyakov, Yury; Annambhotla, Pallavi D; Basavaraju, Sridhar V; Kamili, Saleem; Ritter, Jana M; Nelson, Noele; Mazariegos, George; Green, Michael; Himes, Ryan W; Kuhar, David T; Kuehnert, Matthew J; Miller, Jeffrey A; Wiseman, Rachel; Moorman, Anne C

2017-04-01

Although transmission of hepatitis A virus (HAV) through blood transfusion has been documented, transmission through organ transplantation has not been reported. In August 2015, state health officials in Texas, USA, were notified of 2 home health nurses with HAV infection whose only common exposure was a child who had undergone multi-visceral organ transplantation 9 months earlier. Specimens from the nurses, organ donor, and all organ recipients were tested and medical records reviewed to determine a possible infection source. Identical HAV RNA sequences were detected from the serum of both nurses and the organ donor, as well as from the multi-visceral organ recipient's serum and feces; this recipient's posttransplant liver and intestine biopsy specimens also had detectable virus. The other organ recipients tested negative for HAV RNA. Vaccination of the donor might have prevented infection in the recipient and subsequent transmission to the healthcare workers.

Isolation of infectious chikungunya virus and dengue virus using anionic polymer-coated magnetic beads.

PubMed

Patramool, Sirilaksana; Bernard, Eric; Hamel, Rodolphe; Natthanej, Luplertlop; Chazal, Nathalie; Surasombatpattana, Pornapat; Ekchariyawat, Peeraya; Daoust, Simon; Thongrungkiat, Supatra; Thomas, Frédéric; Briant, Laurence; Missé, Dorothée

2013-10-01

Mosquitoes-borne viruses are a major threat for human populations. Among them, chikungunya virus (CHIKV) and dengue virus (DENV) cause thousands of cases worldwide. The recent propagation of mosquito vectors competent to transmit these viruses to temperate areas increases their potential impact on susceptible human populations. The development of sensitive methods allowing the detection and isolation of infectious viruses is of crucial interest for determination of virus contamination in humans and in competent mosquito vectors. However, simple and rapid method allowing the capture of infectious CHIKV and DENV from samples with low viral titers useful for further genetic and functional characterization of circulating strains is lacking. The present study reports a fast and sensitive isolation technique based on viral particles adsorption on magnetic beads coated with anionic polymer, poly(methyl vinyl ether-maleic anhydrate) and suitable for isolation of infectious CHIKV and DENV from the four serotypes. Starting from quite reduced biological material, this method was accurate to combine with conventional detection techniques, including qRT-PCR and immunoblotting and allowed isolation of infectious particles without resorting to a step of cultivation. The use of polymer-coated magnetic beads is therefore of high interest for rapid detection and isolation of CHIKV and DENV from samples with reduced viral loads and represents an accurate approach for the surveillance of mosquito vector in area at risk for arbovirus outbreaks. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Multiple oncogenic viruses are present in human breast tissues before development of virus associated breast cancer.

PubMed

Lawson, James S; Glenn, Wendy K

2017-01-01

Multiple oncogenic viruses including, mouse mammary tumor virus, bovine leukemia virus, human papilloma virus, and Epstein Barr virus, have been identified as separate infectious pathogens in human breast cancer. Here we demonstrate that these four viruses may be present in normal and benign breast tissues 1 to 11 years before the development of same virus breast cancer in the same patients. We combined the data we developed during investigations of the individual four oncogenic viruses and breast cancer. Patients who had benign breast biopsies 1-11 years prior to developing breast cancer were identified by pathology reports from a large Australian pathology service (Douglas Hanly Moir Pathology). Archival formalin fixed specimens from these patients were collected. The same archival specimens were used for (i) investigations of mouse mammary tumour virus (also known as human mammary tumour virus) conducted at the Icahn School of Medicine at Mount Sinai, New York and at the University of Pisa, Italy, (ii) bovine leukemia virus conducted at the University of California at Berkeley,(iii) human papilloma virus and Epstein Barr virus conducted at the University of New South Wales, Sydney, Australia. Seventeen normal breast tissues from cosmetic breast surgery conducted on Australian patients were used as controls. These patients were younger than those with benign and later breast cancer. Standard and in situ polymerase chain reaction (PCR) methods were used to identify the four viruses. The detailed methods are outlined in the separate publications.: mouse mammary tumor virus, human papilloma virus and Epstein Barr virus (Infect Agent Cancer 12:1, 2017, PLoS One 12:e0179367, 2017, Front Oncol 5:277, 2015, PLoS One 7:e48788, 2012). Epstein Barr virus and human papilloma virus were identified in the same breast cancer cells by in situ PCR. Mouse mammary tumour virus was identified in 6 (24%) of 25 benign breast specimens and in 9 (36%) of 25 breast cancer specimens

Treatment with the smallpox antiviral tecovirimat (ST-246) alone or in combination with ACAM2000 vaccination is effective as a postsymptomatic therapy for monkeypox virus infection.

PubMed

Berhanu, Aklile; Prigge, Jonathan T; Silvera, Peter M; Honeychurch, Kady M; Hruby, Dennis E; Grosenbach, Douglas W

2015-07-01

The therapeutic efficacies of smallpox vaccine ACAM2000 and antiviral tecovirimat given alone or in combination starting on day 3 postinfection were compared in a cynomolgus macaque model of lethal monkeypox virus infection. Postexposure administration of ACAM2000 alone did not provide any protection against severe monkeypox disease or mortality. In contrast, postexposure treatment with tecovirimat alone or in combination with ACAM2000 provided full protection. Additionally, tecovirimat treatment delayed until day 4, 5, or 6 postinfection was 83% (days 4 and 5) or 50% (day 6) effective. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The use of a handheld Raman system for virus detection

NASA Astrophysics Data System (ADS)

Song, Chunyuan; Driskell, Jeremy D.; Tripp, Ralph A.; Cui, Yiping; Zhao, Yiping

2012-06-01

The combination of surface enhanced Raman spectroscopy (SERS) with a handheld Raman system would lead to a powerful portable device for defense and security applications. The Thermo Scientific FirstDefender RM instrument is a 785-nm handheld Raman spectrometer intended for rapid field identification of unknown solid and liquid samples. Its sensitivity and effectiveness for SERS-based detection was initially confirmed by evaluating detection of 1,2-di(4- pyridyl)ethylene as a reporter molecule on a silver nanorod (AgNR) substrate, and the results are comparable to those from a confocal Bruker Raman system. As avian influenza A viruses (AIV) are recognized as an important emerging threat to public health, this portable handheld Raman spectrometer is used, for the first time, to detect and identify avian influenza A viruses using a multi-well AgNR SERS chip. The SERS spectra obtained had rich peaks which demonstrated that the instrument can be effectively used for SERS-based influenza virus detection. According to the SERS spectra, these different influenza viruses were distinguished from the negative control via the principal component analysis and by partial least squares-discriminate analysis. Together, these results show that the combination effective SERS substrates when combined with a portable Raman spectrometer provides a powerful field device for chemical and biological sensing.

Melon chlorotic leaf curl virus: characterization and differential reassortment with closest relatives reveal adaptive virulence in the squash leaf curl virus clade and host shifting by the host-restricted bean calico mosaic virus.

PubMed

Idris, A M; Mills-Lujan, K; Martin, K; Brown, J K

2008-02-01

The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at approximately 90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the "wild-type" (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.

Using epidemiological information to develop effective integrated virus disease management strategies.

PubMed

Jones, Roger A C

2004-03-01

Virus diseases cause serious losses in yield and quality of cultivated plants worldwide. These losses and the resulting financial damage can be limited by controlling epidemics using measures that minimise virus infection sources or suppress virus spread. For each combination of virus, cultivated plant and production system, there is an 'economic threshold' above which the financial damage is sufficient to justify using such measures. However, individual measures used alone may bring only small benefits and they may become ineffective, especially over the long term. When diverse control measures that act in different ways are combined and used together, their effects are complementary resulting in far more effective overall control. Such experiences have led to the development of integrated management concepts for virus diseases that combine available host resistance, cultural, chemical and biological control measures. Selecting the ideal mix of measures for each pathosystem and production situation requires detailed knowledge of the epidemiology of the causal virus and the mode of action of each individual control measure so that diverse responses can be devised to meet the unique features of each of the different scenarios considered. The strategies developed must be robust and necessitate minimal extra expense, labour demands and disruption to standard practices. Examples of how epidemiological information can be used to develop effective integrated disease management (IDM) strategies for diverse situations are described. They involve circumstances where virus transmission from plant-to-plant occurs in four different ways: by contact, non-persistently or persistently by insect vectors, and by root-infecting fungi. The examples are: Subterranean clover mottle virus (SCMoV) (contact-transmitted) and Bean yellow mosaic virus (BYMV) (non-persistently aphid-transmitted) in annually self-regenerating clover pasture; three seed-borne viruses (all non-persistently aphid

Protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency.

PubMed

Taylor, Sandra D; Leib, Steven R; Wu, Wuwei; Nelson, Robert; Carpenter, Susan; Mealey, Robert H

2011-07-01

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.

Virus-Vectored Influenza Virus Vaccines

PubMed Central

Tripp, Ralph A.; Tompkins, S. Mark

2014-01-01

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

The CD8 T Cell Response to Respiratory Virus Infections.

PubMed

Schmidt, Megan E; Varga, Steven M

2018-01-01

Humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. While some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. Despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. Nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. However, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. CD8 T cells are critical for mediating clearance following many acute viral infections in the lung. In addition, memory CD8 T cells are capable of providing protection against secondary infections. Therefore, the combined induction of virus-specific CD8 T cells and antibodies may provide optimal protective immunity. Herein, we review the current literature on CD8 T cell responses induced by respiratory virus infections. Additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on RSV vaccination.

Synergistic inhibition of influenza A virus replication by a plant polyphenol-rich extract and epsilon-aminocaproic acid in vitro and in vivo.

PubMed

Serkedjieva, J; Nikolova, E; Kirilov, N

2010-01-01

A combined antiviral effect of a polyphenol-rich extract of the medicinal plant Geranium sanguineum L. (PC) and a protease inhibitor, epsilon-aminocaproic acid (ACA) was examined in Influenza A virus (IAV)-infected MDCK cell cultures and mice. Synergistic, antagonistic, or indifferent antiviral effects were distinguished on the basis of virus yields, namely fractional yields of individual compounds and yields of both compounds in combination. Combinations of PC and ACA in particular concentrations proved synergistic in the inhibition of virus replication in MDCK cells and in protection of mice against virus infection as determined by virus titers, lung weight, mean survival time (MST), mortality rate, and protection rate (PR). Following the application of a combination of PC and ACA to the virus-infected mice, the levels of the lung protease and protease-inhibitory activity, which were increased due to the virus infection, were brought to normal. These results demonstrate the rationale for a combined application of viral inhibitors with different modes of action to the treatment of IAV infection, in particular PC as a natural inhibitor of early viral transcription and translation and ACA as a synthetic inhibitor of cellular proteases. Influenza A virus; antiviral effect; synergism; plant polyphenol extract; epsilon-aminocaproic acid; protease inhibitors.

Pandemic Threat Posed by Avian Influenza A Viruses

PubMed Central

Horimoto, Taisuke; Kawaoka, Yoshihiro

2001-01-01

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans. PMID:11148006

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides.

PubMed

Laassri, Majid; Chizhikov, Vladimir; Mikheev, Maxim; Shchelkunov, Sergei; Chumakov, Konstantin

2003-09-01

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

Oncolytic herpes viruses, chemotherapeutics, and other cancer drugs

PubMed Central

Braidwood, Lynne; Graham, Sheila V; Graham, Alex; Conner, Joe

2013-01-01

Oncolytic viruses are emerging as a potential new way of treating cancers. They are selectively replication-competent viruses that propagate only in actively dividing tumor cells but not in normal cells and, as a result, destroy the tumor cells by consequence of lytic infection. At least six different oncolytic herpes simplex viruses (oHSVs) have undergone clinical trials worldwide to date, and they have demonstrated an excellent safety profile and intimations of efficacy. The first pivotal Phase III trial with an oHSV, talimogene laherparepvec (T-Vec [OncoVexGM-CSF]), is almost complete, with extremely positive early results reported. Intuitively, therapeutically beneficial interactions between oHSV and chemotherapeutic and targeted therapeutic drugs would be limited as the virus requires actively dividing cells for maximum replication efficiency and most anticancer agents are cytotoxic or cytostatic. However, combinations of such agents display a range of responses, with antagonistic, additive, or, perhaps most surprisingly, synergistic enhancement of antitumor activity. When synergistic interactions in cancer cell killing are observed, chemotherapy dose reductions that achieve the same overall efficacy may be possible, resulting in a valuable reduction of adverse side effects. Therefore, the combination of an oHSV with “standard-of-care” drugs makes a logical and reasonable approach to improved therapy, and the addition of a targeted oncolytic therapy with “standard-of-care” drugs merits further investigation, both preclinically and in the clinic. Numerous publications report such studies of oncolytic HSV in combination with other drugs, and we review their findings here. Viral interactions with cellular hosts are complex and frequently involve intracellular signaling networks, thus creating diverse opportunities for synergistic or additive combinations with many anticancer drugs. We discuss potential mechanisms that may lead to synergistic interactions

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis

PubMed Central

Wilkinson, Michelle J.; Smith, Henry G.; McEntee, Gráinne; Kyula-Currie, Joan; Mansfield, David C.; Khan, Aadil A.; Roulstone, Victoria

2016-01-01

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies. PMID:27783991

Long term impacts of combined sewer overflow remediation on water quality and population dynamics of Culex quinquefasciatus, the main urban West Nile virus vector in Atlanta, GA.

PubMed

Lund, Andrea; McMillan, Joseph; Kelly, Rosmarie; Jabbarzadeh, Shirin; Mead, Daniel G; Burkot, Thomas R; Kitron, Uriel; Vazquez-Prokopec, Gonzalo M

2014-02-01

Combined sewers are a significant source of urban water pollution due to periodic discharges into natural streams. Such events (called combined sewer overflows, or CSOs) contribute to the impairment of natural waterways and are associated with increased mosquito productivity and elevated risk of West Nile virus transmission. We investigated the impact of CSOs on water quality and immature mosquito productivity in the city of Atlanta, Georgia, one year before and four years after CSO facility remediation. Water quality (ammonia, phosphate, nitrate and dissolved oxygen concentrations), immature mosquitoes (larvae and pupae), water temperature and rainfall were quantified biweekly between June-October at two urban creeks during 2008-2012. A before-after control-intervention design tested the impact of remediation on mosquito productivity and water quality, whereas generalized linear mixed-effect models quantified the factors explaining the long term impacts of remediation on mosquito productivity. Ammonia and phosphate concentrations and late immature (fourth-instar and pupae) mosquito populations were significantly higher in CSO than in non-CSO creeks, while dissolved oxygen concentrations were lower. Remediation significantly improved water quality estimates (particularly ammonia and dissolved oxygen) and reduced the number of overflows, mosquito productivity and the overall contribution of CSO-affected streams as sources of vectors of West Nile virus. The quality of water in CSOs provided a suitable habitat for immature mosquitoes. Remediation of the CSO facility through the construction of a deep storage tunnel improved water quality indices and reduced the productivity of mosquito species that can serve as vectors of West Nile virus. Copyright © 2013 Elsevier Inc. All rights reserved.

Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Virus evolution and transmission in an ever more connected world

PubMed Central

Pybus, Oliver G.; Tatem, Andrew J.; Lemey, Philippe

2015-01-01

The frequency and global impact of infectious disease outbreaks, particularly those caused by emerging viruses, demonstrate the need for a better understanding of how spatial ecology and pathogen evolution jointly shape epidemic dynamics. Advances in computational techniques and the increasing availability of genetic and geospatial data are helping to address this problem, particularly when both information sources are combined. Here, we review research at the intersection of evolutionary biology, human geography and epidemiology that is working towards an integrated view of spatial incidence, host mobility and viral genetic diversity. We first discuss how empirical studies have combined viral spatial and genetic data, focusing particularly on the contribution of evolutionary analyses to epidemiology and disease control. Second, we explore the interplay between virus evolution and global dispersal in more depth for two pathogens: human influenza A virus and chikungunya virus. We discuss the opportunities for future research arising from new analyses of human transportation and trade networks, as well as the associated challenges in accessing and sharing relevant spatial and genetic data. PMID:26702033

Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model.

PubMed

Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Gorshkova, Inna; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

2007-09-01

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

PubMed

Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

2015-09-01

Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative impact of immunomodulation versus oncolysis with cytokine-expressing virus therapeutics.

PubMed

Kim, Peter S; Crivelli, Joseph J; Choi, Il-Kyu; Yun, Chae-Ok; Wares, Joanna R

2015-08-01

The past century's description of oncolytic virotherapy as a cancer treatment involving specially-engineered viruses that exploit immune deficiencies to selectively lyse cancer cells is no longer adequate. Some of the most promising therapeutic candidates are now being engineered to produce immunostimulatory factors, such as cytokines and co-stimulatory molecules, which, in addition to viral oncolysis, initiate a cytotoxic immune attack against the tumor. This study addresses the combined effects of viral oncolysis and T-cell-mediated oncolysis. We employ a mathematical model of virotherapy that induces release of cytokine IL-12 and co-stimulatory molecule 4-1BB ligand. We found that the model closely matches previously published data, and while viral oncolysis is fundamental in reducing tumor burden, increased stimulation of cytotoxic T cells leads to a short-term reduction in tumor size, but a faster relapse. In addition, we found that combinations of specialist viruses that express either IL-12 or 4-1BBL might initially act more potently against tumors than a generalist virus that simultaneously expresses both, but the advantage is likely not large enough to replace treatment using the generalist virus. Finally, according to our model and its current assumptions, virotherapy appears to be optimizable through targeted design and treatment combinations to substantially improve therapeutic outcomes.

Novel, diverse RNA viruses from Mediterranean isolates of the phytopathogenic fungus, Rosellinia necatrix: insights into evolutionary biology of fungal viruses.

PubMed

Arjona-Lopez, Juan Manuel; Telengech, Paul; Jamal, Atif; Hisano, Sakae; Kondo, Hideki; Yelin, Mery Dafny; Arjona-Girona, Isabel; Kanematsu, Satoko; Lopez-Herrera, Carlos José; Suzuki, Nobuhiro

2018-04-01

To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

New vaccines against influenza virus

PubMed Central

Lee, Young-Tae; Kim, Ki-Hye; Ko, Eun-Ju; Lee, Yu-Na; Kim, Min-Chul; Kwon, Young-Man; Tang, Yinghua; Cho, Min-Kyoung; Lee, Youn-Jeong

2014-01-01

Vaccination is one of the most effective and cost-benefit interventions that prevent the mortality and reduce morbidity from infectious pathogens. However, the licensed influenza vaccine induces strain-specific immunity and must be updated annually based on predicted strains that will circulate in the upcoming season. Influenza virus still causes significant health problems worldwide due to the low vaccine efficacy from unexpected outbreaks of next epidemic strains or the emergence of pandemic viruses. Current influenza vaccines are based on immunity to the hemagglutinin antigen that is highly variable among different influenza viruses circulating in humans and animals. Several scientific advances have been endeavored to develop universal vaccines that will induce broad protection. Universal vaccines have been focused on regions of viral proteins that are highly conserved across different virus subtypes. The strategies of universal vaccines include the matrix 2 protein, the hemagglutinin HA2 stalk domain, and T cell-based multivalent antigens. Supplemented and/or adjuvanted vaccination in combination with universal target antigenic vaccines would have much promise. This review summarizes encouraging scientific advances in the field with a focus on novel vaccine designs. PMID:24427759

Animal models of disease shed light on Nipah virus pathogenesis and transmission

PubMed Central

de Wit, Emmie; Munster, Vincent J.

2014-01-01

Nipah virus is an emerging virus infection that causes yearly disease outbreaks with high case fatality rates in Bangladesh. Nipah virus causes encephalitis and systemic vasculitis, sometimes in combination with respiratory disease. Pteropus species fruit bats are the natural reservoir of Nipah virus and zoonotic transmission can occur directly or via an intermediate host; human-to-human transmission occurs regularly. In this review we discuss the current state of knowledge on the pathogenesis and transmission of Nipah virus, focusing on dissemination of the virus through its host, known determinants of pathogenicity and routes of zoonotic and human-to-human transmission. Since data from human cases are sparse, this knowledge is largely based on the results of studies performed in animal models that recapitulate Nipah virus disease in humans. PMID:25229234

Evaluation of the immunomodulatory and antiviral effects of the cytokine combination IFN-α and IL-7 in the lymphocytic choriomeningitis virus and Friend retrovirus mouse infection models.

PubMed

Audigé, Annette; Hofer, Ursula; Dittmer, Ulf; van den Broek, Maries; Speck, Roberto F

2011-10-01

Existing therapies for chronic viral infections are still suboptimal or have considerable side effects, so new therapeutic strategies need to be developed. One option is to boost the host's immune response with cytokines. We have recently shown in an acute ex vivo HIV infection model that co-administration of interferon (IFN)-α and interleukin (IL)-7 allows us to combine the potent anti-HIV activity of IFN-α with the beneficial effects of IL-7 on T-cell survival and function. Here we evaluated the effect of combining IFN-α and IL-7 on viral replication in vivo in the chronic lymphocytic choriomeningitis virus (LCMV) and acute Friend retrovirus (FV) infection models. In the chronic LCMV model, cytokine treatment was started during the early replication phase (i.e., on day 7 post-infection [pi]). Under the experimental conditions used, exogenous IFN-α inhibited FV replication, but had no effect on viral replication in the LCMV model. There was no therapeutic benefit of IL-7 either alone or in combination with IFN-α in either of the two infection models. In the LCMV model, dose-dependent effects of the cytokine combination on T-cell phenotype/function were observed. It is possible that these effects would translate into antiviral activity in re-challenged mice. It is also possible that another type of IFN-α/β or induction of endogenous IFN-α/β alone or in combination with IL-7 would have antiviral activity in the LCMV model. Furthermore, we cannot exclude that some effect on viral titers would have been seen at later time points not investigated here (i.e., beyond day 34 pi). Finally, IFN-α/IL-7 may inhibit the replication of other viruses. Thus it might be worth testing these cytokines in other in vivo models of chronic viral infections.

Horizon scanning for emergence of new viruses: from constructing complex scenarios to online games.

PubMed

Gale, P; Breed, A C

2013-10-01

Horizon scanning techniques can be developed to identify novel routes and sources for the emergence of viruses in the medium to long term. Central to horizon scanning is prediction of the complex scenarios through which viruses could emerge before they occur. One approach involves 'spidergrams' in which complex scenarios are generated by combining factors randomly selected from different categories of events. Spidergrams provide a framework for how different factors could interact, irrespective of the virus, and also enable testing of combinations not previously considered but which would be 'tested' in nature by a virus. The emergence of viruses through new routes is often related to changes, for example, in environmental and social factors, and the Internet will undoubtedly be used to identify long-term trends for consideration. In addition, online games may provide horizon scanners with suggestions for new routes and strategies that could be used by emerging viruses. © 2012 Crown copyright Reproduced with the permission of the Controller of Her Majesty's Stationery Office and Animal Health and Veterinary Laboratories Agency.

Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs

PubMed Central

Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

2015-01-01

ABSTRACT Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against

Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection.

PubMed

Azinas, S; Bano, F; Torca, I; Bamford, D H; Schwartz, G A; Esnaola, J; Oksanen, H M; Richter, R P; Abrescia, N G

2018-04-26

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

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

Infection of phytoplankton by aerosolized marine viruses

PubMed Central

Sharoni, Shlomit; Trainic, Miri; Schatz, Daniella; Lehahn, Yoav; Flores, Michel J.; Bidle, Kay D.; Ben-Dor, Shifra; Rudich, Yinon; Vardi, Assaf

2015-01-01

Marine viruses constitute a major ecological and evolutionary driving force in the marine ecosystems. However, their dispersal mechanisms remain underexplored. Here we follow the dynamics of Emiliania huxleyi viruses (EhV) that infect the ubiquitous, bloom-forming phytoplankton E. huxleyi and show that EhV are emitted to the atmosphere as primary marine aerosols. Using a laboratory-based setup, we showed that the dynamic of EhV aerial emission is strongly coupled to the host–virus dynamic in the culture media. In addition, we recovered EhV DNA from atmospheric samples collected over an E. huxleyi bloom in the North Atlantic, providing evidence for aerosolization of marine viruses in their natural environment. Decay rate analysis in the laboratory revealed that aerosolized viruses can remain infective under meteorological conditions prevailing during E. huxleyi blooms in the ocean, allowing potential dispersal and infectivity over hundreds of kilometers. Based on the combined laboratory and in situ findings, we propose that atmospheric transport of EhV is an effective transmission mechanism for spreading viral infection over large areas in the ocean. This transmission mechanism may also have an important ecological impact on the large-scale host–virus “arms race” during bloom succession and consequently the turnover of carbon in the ocean. PMID:25964340

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance

PubMed Central

Lin, Jing; Pramono, Zacharias Aloysius Dwi; Maurer-Stroh, Sebastian

2016-01-01

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for

Suppressing Aedes albopictus, an Emerging Vector of Dengue and Chikungunya Viruses, by a Novel Combination of a Monomolecular Film and an Insect-Growth Regulator

PubMed Central

Nelder, Mark; Kesavaraju, Banugopan; Farajollahi, Ary; Healy, Sean; Unlu, Isik; Crepeau, Taryn; Ragavendran, Ashok; Fonseca, Dina; Gaugler, Randy

2010-01-01

The Asian tiger mosquito Aedes albopictus (Skuse) is rapidly increasing its global range and importance in transmission of chikungunya and dengue viruses. We tested pellet formulations of a monomolecular film (Agnique) and (S)-methoprene (Altosid) under laboratory and field conditions. In the laboratory, Agnique provided 80% control for 20 days, whereas Altosid, in combination with Agnique, provided 80% control for > 60 days. During field trials, the 1:1 pellet ratio of combined products provided > 95% control for at least 32 days and 50% control for at least 50 days. Altosid remained effective after a 107-day laboratory-induced drought, suggesting that the product serves as a means of control during drought conditions and against spring broods in temperate regions. Agnique and Altosid, when used in tandem for cryptic, difficult-to-treat locations, can provide long-term control of Ae. albopictus larvae and pupae. The possible additive or synergistic effects of the combined products deserve further investigation. PMID:20439963

Squirrelpox virus: assessing prevalence, transmission and environmental degradation.

PubMed

Collins, Lisa M; Warnock, Neil D; Tosh, David G; McInnes, Colin; Everest, David; Montgomery, W Ian; Scantlebury, Mike; Marks, Nikki; Dick, Jaimie T A; Reid, Neil

2014-01-01

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25 °C) and dry conditions than in cooler (5 and 15 °C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Poxviruses and the Evolution of Host Range and Virulence

PubMed Central

Haller, Sherry L.; Peng, Chen; McFadden, Grant; Rothenburg, Stefan

2013-01-01

Poxviruses as a group can infect a large number of animals. However, at the level of individual viruses, even closely related poxviruses display highly diverse host ranges and virulence. For example, variola virus, the causative agent of smallpox, is human-specific and highly virulent only to humans, whereas related cowpox viruses naturally infect a broad spectrum of animals and only cause relatively mild disease in humans. The successful replication of poxviruses depends on their effective manipulation of the host antiviral responses, at the cellular-, tissue- and species-specific levels, which constitutes a molecular basis for differences in poxvirus host range and virulence. A number of poxvirus genes have been identified that possess host range function in experimental settings, and many of these host range genes target specific antiviral host pathways. Herein, we review the biology of poxviruses with a focus on host range, zoonotic infections, virulence, genomics and host range genes as well as the current knowledge about the function of poxvirus host range factors and how their interaction with the host innate immune system contributes to poxvirus host range and virulence. We further discuss the evolution of host range and virulence in poxviruses as well as host switches and potential poxvirus threats for human and animal health. PMID:24161410

Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections.

PubMed

Iversen, Patrick L; Warren, Travis K; Wells, Jay B; Garza, Nicole L; Mourich, Dan V; Welch, Lisa S; Panchal, Rekha G; Bavari, Sina

2012-11-06

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

The in vitro and in vivo antiviral properties of combined monoterpene alcohols against West Nile virus infection.

PubMed

Pliego Zamora, Adriana; Edmonds, Judith H; Reynolds, Maxwell J; Khromykh, Alexander A; Ralph, Stephen J

2016-08-01

West Nile Virus (WNV) is a mosquito-borne flavivirus that can cause neuroinvasive disease in humans and animals for which no therapies are currently available. We studied an established combination of monoterpene alcohols (CMA) derived from Melaleuca alternifolia, against WNV infection. The in vitro results show that CMA exhibits virucidal activity, as well as reduces the viral titres and percentage of infected cells. The antiviral mechanism of action of CMA was studied. We found that CMA did not alter the intracellular pH, neither induced apoptosis, but did induce cell cycle arrest in the G0/G1-phase although that was not the antiviral mechanism. Furthermore, we tested CMA in vivo using IRF 3(-)(/)(-)/7(-/-)mice and it was found that CMA treatment significantly delayed morbidity due to WNV infection, reduced the loss of body weight and reduced the viral titres in brain. These findings suggest that CMA could be a therapeutic agent against WNV infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Stability of pseudorabies virus during freeze-drying and storage: effect of suspending media.

PubMed Central

Scott, E M; Woodside, W

1976-01-01

The effect of suspending media on the stability of pseudorabies virus upon freeze-drying and subsequent storage was studied. A variety of media was tested, including: sodium glutamate; sucrose; lactose; lactalbumin hydrolysate; peptone; a combination of sucrose, dextran, and glutamate; and various combinations of sucrose, glutamate, and potassium phosphates. Suspending media containing glutamate, either alone or in combination with sucrose and either dextran or phosphates, afforded the greatest degree of protection during the freeze-drying process and upon storage. Some possible functions of these additives in preventing injury to the virus during freezing and drying have been suggested. PMID:182713

Isolation of sochi virus from a fatal case of hantavirus disease with fulminant clinical course.

PubMed

Dzagurova, Tamara K; Witkowski, Peter T; Tkachenko, Evgeniy A; Klempa, Boris; Morozov, Vyacheslav G; Auste, Brita; Zavora, Dmitriy L; Iunicheva, Iulia V; Mutnih, Elena S; Kruger, Detlev H

2012-01-01

Sochi virus, a novel genetic variant of Dobrava-Belgrade virus, was isolated in cell culture from a fulminant lethal case of hantavirus disease presenting with shock and combined kidney and lung failure. Sochi virus is transmitted to humans from host reservoir Apodemus ponticus and must be considered a life-threatening emerging agent.

Progress in Developing Virus-like Particle Influenza Vaccines

PubMed Central

Quan, Fu-Shi; Lee, Young-Tae; Kim, Ki-Hye; Kim, Min-Chul; Kang, Sang-Moo

2016-01-01

Summary Recombinant vaccines based on virus-like particles (VLPs) or nanoparticles have been successful in their safety and efficacy in preclinical and clinical studies. The technology of expressing enveloped VLP vaccines has combined with molecular engineering of proteins in membrane-anchor and immunogenic forms mimicking the native conformation of surface proteins on the enveloped viruses. This review summarizes recent developments in influenza VLP vaccines against seasonal, pandemic, and avian influenza viruses from the perspective of use in humans. The immunogenicity and efficacies of influenza VLP vaccine in the homologous and cross-protection were reviewed. Discussions include limitations of current influenza vaccination strategies and future directions to confer broadly cross protective new influenza vaccines as well as vaccination. PMID:27058302

Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

PubMed

Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2015-03-02

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba. Copyright © 2015 Elsevier B.V. All rights reserved.

Imaging and tracking HIV viruses in human cervical mucus

NASA Astrophysics Data System (ADS)

Boukari, Fatima; Makrogiannis, Sokratis; Nossal, Ralph; Boukari, Hacène

2016-09-01

We describe a systematic approach to image, track, and quantify the movements of HIV viruses embedded in human cervical mucus. The underlying motivation for this study is that, in HIV-infected adults, women account for more than half of all new cases and most of these women acquire the infection through heterosexual contact. The endocervix is believed to be a susceptible site for HIV entry. Cervical mucus, which coats the endocervix, should play a protective role against the viruses. Thus, we developed a methodology to apply time-resolved confocal microscopy to examine the motion of HIV viruses that were added to samples of untreated cervical mucus. From the images, we identified the viruses, tracked them over time, and calculated changes of the statistical mean-squared displacement (MSD) of each virus. Approximately half of tracked viruses appear constrained while the others show mobility with MSDs that are proportional to τα+ν2τ2, over time range τ, depicting a combination of anomalous diffusion (0<α<0.4) and flow-like behavior. The MSD data also reveal plateaus attributable to possible stalling of the viruses. Although a more extensive study is warranted, these results support the assumption of mucus being a barrier against the motion of these viruses.

A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases.

PubMed

Chen, Xilin; Han, Jianfeng; Chu, Jianhong; Zhang, Lingling; Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E Antonio; Yu, Jianhua

2016-05-10

Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.

Viruses, Artificial Viruses and Virus-Based Structures for Biomedical Applications.

PubMed

van Rijn, Patrick; Schirhagl, Romana

2016-06-01

Nanobiomaterials such as virus particles and artificial virus particles offer tremendous opportunities to develop new biomedical applications such as drug- or gene-delivery, imaging and sensing but also improve understanding of biological mechanisms. Recent advances within the field of virus-based systems give insights in how to mimic viral structures and virus assembly processes as well as understanding biodistribution, cell/tissue targeting, controlled and triggered disassembly or release and circulation times. All these factors are of high importance for virus-based functional systems. This review illustrates advances in mimicking and enhancing or controlling these aspects to a high degree toward delivery and imaging applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polyomavirus-Specific Cellular Immunity: From BK-Virus-Specific Cellular Immunity to BK-Virus-Associated Nephropathy?

PubMed Central

Dekeyser, Manon; François, Hélène; Beaudreuil, Séverine; Durrbach, Antoine

2015-01-01

In renal transplantation, BK-virus (BKV)-associated nephropathy has emerged as a major complication, with a prevalence of 1–10% and graft loss in >50% of cases. BKV is a member of the polyomavirus family and rarely induces apparent clinical disease in the general population. However, replication of polyomaviruses, associated with significant organ disease, is observed in patients with acquired immunosuppression. Monitoring of specific immunity combined with viral load could be used to individually assess the risk of viral reactivation and virus control. We review the current knowledge on BKV-specific cellular immunity and, more specifically, in immunocompromised patients. In the future, immune-based therapies could allow us to treat and prevent BKV-associated nephropathy. PMID:26136745

First detection of bluetongue virus serotype 14 in Poland.

PubMed

Orłowska, Anna; Trębas, Paweł; Smreczak, Marcin; Marzec, Anna; Żmudziński, Jan F

2016-07-01

Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical.

Data-driven identification of potential Zika virus vectors

PubMed Central

Evans, Michelle V; Dallas, Tad A; Han, Barbara A; Murdock, Courtney C; Drake, John M

2017-01-01

Zika is an emerging virus whose rapid spread is of great public health concern. Knowledge about transmission remains incomplete, especially concerning potential transmission in geographic areas in which it has not yet been introduced. To identify unknown vectors of Zika, we developed a data-driven model linking vector species and the Zika virus via vector-virus trait combinations that confer a propensity toward associations in an ecological network connecting flaviviruses and their mosquito vectors. Our model predicts that thirty-five species may be able to transmit the virus, seven of which are found in the continental United States, including Culex quinquefasciatus and Cx. pipiens. We suggest that empirical studies prioritize these species to confirm predictions of vector competence, enabling the correct identification of populations at risk for transmission within the United States. DOI: http://dx.doi.org/10.7554/eLife.22053.001 PMID:28244371

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

PubMed

Fox, Candace R; Parks, Griffith D

2018-04-01

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked

Inactivation of Viruses and Bacteriophages as Models for Swine Hepatitis E Virus in Food Matrices.

PubMed

Emmoth, Eva; Rovira, Jordi; Rajkovic, Andreja; Corcuera, Elena; Wilches Pérez, Diego; Dergel, Irene; Ottoson, Jakob R; Widén, Frederik

2017-03-01

Hepatitis E virus has been recognised as a food-borne virus hazard in pork products, due to its zoonotic properties. This risk can be reduced by adequate treatment of the food to inactivate food-borne viruses. We used a spectrum of viruses and bacteriophages to evaluate the effect of three food treatments: high pressure processing (HPP), lactic acid (LA) and intense light pulse (ILP) treatments. On swine liver at 400 MPa for 10 min, HPP gave log 10 reductions of ≥4.2, ≥5.0 and 3.4 for feline calicivirus (FCV) 2280, FCV wildtype (wt) and murine norovirus 1 (MNV 1), respectively. Escherichia coli coliphage ϕX174 displayed a lower reduction of 1.1, while Escherichia coli coliphage MS2 was unaffected. For ham at 600 MPa, the corresponding reductions were 4.1, 4.4, 2.9, 1.7 and 1.3 log 10 . LA treatment at 2.2 M gave log 10 reductions in the viral spectrum of 0.29-2.1 for swine liver and 0.87-3.1 for ham, with ϕX174 and MNV 1, respectively, as the most stable microorganisms. The ILP treatment gave log 10 reductions of 1.6-2.8 for swine liver, 0.97-2.2 for ham and 1.3-2.3 for sausage, at 15-60 J cm -2 , with MS2 as the most stable microorganism. The HPP treatment gave significantly (p < 0.05) greater virus reduction on swine liver than ham for the viruses at equivalent pressure/time combinations. For ILP treatment, reductions on swine liver were significantly (p < 0.05) greater than on ham for all microorganisms. The results presented here could be used in assessments of different strategies to protect consumers against virus contamination and in advice to food producers. Conservative model indicators for the pathogenic viruses could be suggested.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase of coat protein subgroup II gene from Cucumber mosaic virus

USDA-ARS?s Scientific Manuscript database

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These...

Diversity and Ecology of Viruses in Hyperarid Desert Soils

PubMed Central

Zablocki, Olivier; Adriaenssens, Evelien M.

2015-01-01

In recent years, remarkable progress has been made in the field of virus environmental ecology. In marine ecosystems, for example, viruses are now thought to play pivotal roles in the biogeochemical cycling of nutrients and to be mediators of microbial evolution through horizontal gene transfer. The diversity and ecology of viruses in soils are poorly understood, but evidence supports the view that the diversity and ecology of viruses in soils differ substantially from those in aquatic systems. Desert biomes cover ∼33% of global land masses, and yet the diversity and roles of viruses in these dominant ecosystems remain poorly understood. There is evidence that hot hyperarid desert soils are characterized by high levels of bacterial lysogens and low extracellular virus counts. In contrast, cold desert soils contain high extracellular virus titers. We suggest that the prevalence of microbial biofilms in hyperarid soils, combined with extreme thermal regimens, exerts strong selection pressures on both temperate and virulent viruses. Many desert soil virus sequences show low values of identity to virus genomes in public databases, suggesting the existence of distinct and as-yet-uncharacterized soil phylogenetic lineages (e.g., cyanophages). We strongly advocate for amplification-free metavirome analyses while encouraging the classical isolation of phages from dominant and culturable microbial isolates in order to populate sequence databases. This review provides an overview of recent advances in the study of viruses in hyperarid soils and of the factors that contribute to viral abundance and diversity in hot and cold deserts and offers technical recommendations for future studies. PMID:26590289

Grapevine fleck virus-like viruses in Vitis.

PubMed

Sabanadzovic, S; Abou-Ghanem, N; Castellano, M A; Digiaro, M; Martelli, G P

2000-01-01

Two sets of degenerate primers for the specific amplification of 572-575 nt and 386 nt segments of the methyltransferase and RNA- dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers.

Novel reassortant of swine influenza H1N2 virus in Germany.

PubMed

Zell, Roland; Motzke, Susann; Krumbholz, Andi; Wutzler, Peter; Herwig, Volker; Dürrwald, Ralf

2008-01-01

European porcine H1N2 influenza viruses arose after multiple reassortment steps involving a porcine influenza virus with avian-influenza-like internal segments and human H1N1 and H3N2 viruses in 1994. In Germany, H1N2 swine influenza viruses first appeared in 2000. Two German H1N2 swine influenza virus strains isolated from pigs with clinical symptoms of influenza are described. They were characterized by the neutralization test, haemagglutination inhibition (HI) test and complete sequencing of the viral genomes. The data demonstrate that these viruses represent a novel H1N2 reassortant. The viruses showed limited neutralization by sera raised against heterologous A/sw/Bakum/1,832/00-like H1N2 viruses. Sera pools from recovered pigs showed a considerably lower HI reaction, indicative of diagnostic difficulties in using the HI test to detect these viruses with A/sw/Bakum/1,832/00-like H1N2 antigens. Genome sequencing revealed the novel combination of the human-like HAH1 gene of European porcine H1N2 influenza viruses and the NAN2 gene of European porcine H3N2 viruses.

Histology, immunohistochemistry, and in situ hybridization reveal overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model.

PubMed

Cooper, Timothy K; Huzella, Louis; Johnson, Joshua C; Rojas, Oscar; Yellayi, Sri; Sun, Mei G; Bavari, Sina; Bonilla, Amanda; Hart, Randy; Jahrling, Peter B; Kuhn, Jens H; Zeng, Xiankun

2018-01-19

Survivors of Ebola virus infection may become subclinically infected, but whether animal models recapitulate this complication is unclear. Using histology in combination with immunohistochemistry and in situ hybridization in a retrospective review of a guinea pig confirmation-of-virulence study, we demonstrate for the first time Ebola virus infection in hepatic oval cells, the endocardium and stroma of the atrioventricular valves and chordae tendinae, satellite cells of peripheral ganglia, neurofibroblasts and Schwann cells of peripheral nerves and ganglia, smooth muscle cells of the uterine myometrium and vaginal wall, acini of the parotid salivary glands, thyroid follicular cells, adrenal medullary cells, pancreatic islet cells, endometrial glandular and surface epithelium, and the epithelium of the vagina, penis and, prepuce. These findings indicate that standard animal models for Ebola virus disease are not as well-described as previously thought and may serve as a stepping stone for future identification of potential sites of virus persistence.

Animal models of disease shed light on Nipah virus pathogenesis and transmission.

PubMed

de Wit, Emmie; Munster, Vincent J

2015-01-01

Nipah virus is an emerging virus infection that causes yearly disease outbreaks with high case fatality rates in Bangladesh. Nipah virus causes encephalitis and systemic vasculitis, sometimes in combination with respiratory disease. Pteropus species fruit bats are the natural reservoir of Nipah virus and zoonotic transmission can occur directly or via an intermediate host; human-to-human transmission occurs regularly. In this review we discuss the current state of knowledge on the pathogenesis and transmission of Nipah virus, focusing on dissemination of the virus through its host, known determinants of pathogenicity and routes of zoonotic and human-to-human transmission. Since data from human cases are sparse, this knowledge is largely based on the results of studies performed in animal models that recapitulate Nipah virus disease in humans. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Annulate lamellae in phloem cells of virus-infected Sonchus plants.

PubMed

Steinkamp, M P; Hoefert, L L

1977-07-01

The occurrence of annulate lamellae (AL) in differentiating phloem of Sonchus oleraceus (Compositae) singly infected with sowthistle yellow vein virus (SYVV) and doubly infected with a combination of SYVV and beet yellow stunt virus is documented by electron microscopy. Cell types in which AL were found were immature sieve elements and phloem parenchyma cells. AL were found only in cells that also contained SYVV particles although a direct association between the virus and AL was not apparent. The substructure of the AL and the relationships between the AL and the nuclear envelope and endoplasmic reticulum are similar to those reported in other descriptions of this organelle in the literature. This report appears to be the first one concerning the association of AL with a plant virus disease.

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors

PubMed Central

Lomonosova, Elena; Zlotnick, Adam

2016-01-01

ABSTRACT Combination therapies are standard for management of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections; however, no such therapies are established for human hepatitis B virus (HBV). Recently, we identified several promising inhibitors of HBV RNase H (here simply RNase H) activity that have significant activity against viral replication in vitro. Here, we investigated the in vitro antiviral efficacy of combinations of two RNase H inhibitors with the current anti-HBV drug nucleoside analog lamivudine, with HAP12, an experimental core protein allosteric modulator, and with each other. Anti-HBV activities of the compounds were tested in a HepG2-derived cell line by monitoring intracellular core particle DNA levels, and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The antiviral efficiencies of the drug combinations were evaluated using the median-effect equation derived from the mass-action law principle and combination index theorem of Chou and Talalay. We found that combinations of two RNase H inhibitors from different chemical classes were synergistic with lamivudine against HBV DNA synthesis. Significant synergism was also observed for the combination of the two RNase H inhibitors. Combinations of RNase H inhibitors with HAP12 had additive antiviral effects. Enhanced cytotoxicity was not observed in the combination experiments. Because of these synergistic and additive effects, the antiviral activity of combinations of RNase H inhibitors with drugs that act by two different mechanisms and with each other can be achieved by administering the compounds in combination at doses below the respective single drug doses. PMID:27956427

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors.

PubMed

Lomonosova, Elena; Zlotnick, Adam; Tavis, John E

2017-03-01

Combination therapies are standard for management of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections; however, no such therapies are established for human hepatitis B virus (HBV). Recently, we identified several promising inhibitors of HBV RNase H (here simply RNase H) activity that have significant activity against viral replication in vitro Here, we investigated the in vitro antiviral efficacy of combinations of two RNase H inhibitors with the current anti-HBV drug nucleoside analog lamivudine, with HAP12, an experimental core protein allosteric modulator, and with each other. Anti-HBV activities of the compounds were tested in a HepG2-derived cell line by monitoring intracellular core particle DNA levels, and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The antiviral efficiencies of the drug combinations were evaluated using the median-effect equation derived from the mass-action law principle and combination index theorem of Chou and Talalay. We found that combinations of two RNase H inhibitors from different chemical classes were synergistic with lamivudine against HBV DNA synthesis. Significant synergism was also observed for the combination of the two RNase H inhibitors. Combinations of RNase H inhibitors with HAP12 had additive antiviral effects. Enhanced cytotoxicity was not observed in the combination experiments. Because of these synergistic and additive effects, the antiviral activity of combinations of RNase H inhibitors with drugs that act by two different mechanisms and with each other can be achieved by administering the compounds in combination at doses below the respective single drug doses. Copyright © 2017 American Society for Microbiology.

Three-year rabies duration of immunity in dogs following vaccination with a core combination vaccine against canine distemper virus, canine adenovirus type-1, canine parvovirus, and rabies virus.

PubMed

Lakshmanan, Nallakannu; Gore, Thomas C; Duncan, Karen L; Coyne, Michael J; Lum, Melissa A; Sterner, Frank J

2006-01-01

Thirty-two seronegative pups were vaccinated at 8 weeks of age with modified-live canine distemper virus (CDV), canine adenovirus type-2 (CAV-2), and canine parvovirus (CPV) vaccine and at 12 weeks with a modified-live CDV, CAV-2, CPV, and killed rabies virus vaccine. An additional 31 seronegative pups served as age-matched, nonvaccinated controls. All test dogs were strictly isolated for 3 years after receiving the second vaccination and then were challenged with virulent rabies virus. Clinical signs of rabies were prevented in 28 (88%) of the 32 vaccinated dogs. In contrast, 97% (30 of 31) of the control dogs died of rabies infection. These study results indicated that no immunogenic interference occurred between the modified-live vaccine components and the killed rabies virus component. Furthermore, these results indicated that the rabies component in the test vaccine provided protection against virulent rabies challenge in dogs 12 weeks of age or older for a minimum of 3 years following vaccination.

Susceptibility of Vaccinia Virus to Chemical Disinfectants

PubMed Central

de Oliveira, Tércia Moreira Ludolfo; Rehfeld, Izabelle Silva; Coelho Guedes, Maria Isabel Maldonado; Ferreira, Jaqueline Maria Siqueira; Kroon, Erna Geessien; Lobato, Zélia Inês Portela

2011-01-01

Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV. PMID:21734141

CRISPR-Cas9 Genetic Analysis of Virus-Host Interactions.

PubMed

Gebre, Makda; Nomburg, Jason L; Gewurz, Benjamin E

2018-01-30

Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus-host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR-Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein-Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus-host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens.

[History of vaccination: from empiricism towards recombinant vaccines].

PubMed

Guérin, N

2007-01-01

Two hundreds years after the discovery of the smallpox vaccine, immunization remains one of the most powerful tools of preventive medicine. Immunization was born with Jenner, then Pasteur and expanded during the 19th and 20th century. It started with the empirical observation of cross-immunity between two diseases, cowpox and smallpox. It became a real science, with pathogen isolation, culture and attenuation or inactivation, to prepare a vaccine. Together with clinical and biological efficacy studies and adverse events assessments, it constructed the concept of "vaccinology". Protein conjugation of polyosidic vaccines has made possible early immunisation of infants. Nowadays, recombinant, reassortant, or virus-like particles technologies open the road for new vaccines. Ongoing research opens the way for the development of new vaccines that will help to control transmittable diseases for which we are lacking antimicrobial agents.

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bovine Virus Diarrhea Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.215 Bovine Virus Diarrhea Vaccine, Killed Virus. Bovine Virus Diarrhea Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed virus...

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Bovine Virus Diarrhea Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.215 Bovine Virus Diarrhea Vaccine, Killed Virus. Bovine Virus Diarrhea Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed virus...

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Bovine Virus Diarrhea Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.215 Bovine Virus Diarrhea Vaccine, Killed Virus. Bovine Virus Diarrhea Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed virus...

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Bovine Virus Diarrhea Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.215 Bovine Virus Diarrhea Vaccine, Killed Virus. Bovine Virus Diarrhea Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed virus...

9 CFR 113.215 - Bovine Virus Diarrhea Vaccine, Killed Virus.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Bovine Virus Diarrhea Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.215 Bovine Virus Diarrhea Vaccine, Killed Virus. Bovine Virus Diarrhea Vaccine, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Only Master Seed virus...

Nairobi sheep disease virus/Ganjam virus.

PubMed

M D, Baron; B, Holzer

2015-08-01

Nairobi sheep disease virus (NSDV) is a tick-borne virus which causes a severe disease in sheep and goats, and has been responsible for several outbreaks of disease in East Africa. The virus is also found in the Indian subcontinent, where it is known as Ganjam virus. The virus only spreads through the feeding of competent infected ticks, and is therefore limited in its geographic distribution by the distribution of those ticks, Rhipicephalus appendiculata in Africa and Haemaphysalis intermedia in India. Animals bred in endemic areas do not normally develop disease, and the impact is therefore primarily on animals being moved for trade or breeding purposes. The disease caused by NSDV has similarities to several other ruminant diseases, and laboratory diagnosis is necessary for confirmation. There are published methods for diagnosis based on polymerase chain reaction, for virus growth in cell culture and for other simple diagnostic tests, though none has been commercialised. There is no established vaccine against NSDV, although cell-culture attenuated strains have been developed which show promise and could be put into field trials if it were deemed necessary. The virus is closely related to Crimean-Congo haemorrhagic fever virus, and studies on NSDV may therefore be useful in understanding this important human pathogen.

A new reassortment of influenza A (H7N9) virus causing human infection in Beijing, 2014.

PubMed

Bi, Yuhai; Liu, Jingyuan; Xiong, Haofeng; Zhang, Yue; Liu, Di; Liu, Yingxia; Gao, George F; Wang, Beibei

2016-05-27

A 73-year-old man was confirmed to have an influenza A (H7N9) virus infection, and the causative agent A/Beijing/02/2014(H7N9) virus was isolated. Genetic and phylogenetic analyses revealed that the virus belonged to a novel genotype, which probably emerged and further reassorted with other H9 or H7 viruses in poultry before transmitting to humans. This virus caused a severe infection with high levels of cytokines and neutralizing antibodies. Eventually, the patient was cured after serially combined treatments. Taken together, our findings indicated that this novel genotype of the human H7N9 virus did not evolve directly from the first Beijing isolate A/Beijing/01/2013(H7N9), suggesting that the H7N9 virus has not obtained the ability for human-to-human transmissibility and the virus only evolves in poultry and then infects human by direct contact. Hence, the major measures to prevent human H7N9 virus infection are still to control and standardize the live poultry trade. Early antiviral treatment with combination therapies, including mechanical ventilation, nutrition support and symptomatic treatment, are effective for H7N9 infection.

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice.

PubMed Central

Sidwell, R W; Huffman, J H; Bailey, K W; Wong, M H; Nimrod, A; Panet, A

1996-01-01

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone. PMID:8913477

Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections

PubMed Central

Iversen, Patrick L.; Warren, Travis K.; Wells, Jay B.; Garza, Nicole L.; Mourich, Dan V.; Welch, Lisa S.; Panchal, Rekha G.; Bavari, Sina

2012-01-01

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates. PMID:23202506

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

PubMed

Schweneker, Marc; Laimbacher, Andrea S; Zimmer, Gert; Wagner, Susanne; Schraner, Elisabeth M; Wolferstätter, Michael; Klingenberg, Marieken; Dirmeier, Ulrike; Steigerwald, Robin; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2017-06-01

vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines. Copyright © 2017 American Society for Microbiology.

Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

NASA Astrophysics Data System (ADS)

Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

Function of bacterial cells and their exuded extracellular polymeric substances (EPS) in virus removal by red soils.

PubMed

Zhao, Bingzi; Jiang, Yan; Jin, Yan; Zhang, Jiabao

2014-01-01

The potential influence of autochthonous microorganisms on virus fate in soil is usually determined through extreme conditions of sterilization vs. nonsterilization; however, the relative importance of microbial cells and their exudates remains unclear. In this study, bacterial cells (cell) were harvested, and their exuded extracellular polymeric substances (EPS) were extracted from three strains of bacteria, namely, Gram-negative bacteria Pseudomonas putida and Pseudomonas aeruginosa as well as Gram-positive bacterium Bacillus subtilis. This study aimed to evaluate virus removal in solutions in the presence of cell, EPS, and their combination (cell/EPS), as well as to investigate how their presence affects virus removal efficiencies by four red soils based on batch experiments. Results showed that virus removal percentage in solutions ranged from 11 to 23 in the presence of cells only and from 12 to 15 in the presence of EPS only. The removal percentage in the combined cell/EPS treatment can be estimated by summing the results achieved by the cell and EPS treatments, separately. Meanwhile, cell presence had a negligible effect on virus removal by red soils. EPS and combined cell/EPS significantly reduced virus removal by 20 to 69% and 16 to 50%, respectively, which indicated that EPS served a dominant function in reducing virus removal. This study clearly demonstrated that the prediction of virus removal by red soils must consider the effect of bacteria, especially those producing large quantities of EPS, which can be responsible for the underestimation of viral load in certain studies.

Successful topical respiratory tract immunization of primates against Ebola virus.

PubMed

Bukreyev, Alexander; Rollin, Pierre E; Tate, Mallory K; Yang, Lijuan; Zaki, Sherif R; Shieh, Wun-Ju; Murphy, Brian R; Collins, Peter L; Sanchez, Anthony

2007-06-01

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

Accelerated detection of viral particles by combining AC electric field effects and micro-Raman spectroscopy.

PubMed

Tomkins, Matthew Robert; Liao, David Shiqi; Docoslis, Aristides

2015-01-08

A detection method that combines electric field-assisted virus capture on antibody-decorated surfaces with the "fingerprinting" capabilities of micro-Raman spectroscopy is demonstrated for the case of M13 virus in water. The proof-of-principle surface mapping of model bioparticles (protein coated polystyrene spheres) captured by an AC electric field between planar microelectrodes is presented with a methodology for analyzing the resulting spectra by comparing relative peak intensities. The same principle is applied to dielectrophoretically captured M13 phage particles whose presence is indirectly confirmed with micro-Raman spectroscopy using NeutrAvidin-Cy3 as a labeling molecule. It is concluded that the combination of electrokinetically driven virus sampling and micro-Raman based signal transduction provides a promising approach for time-efficient and in situ detection of viruses.

Accelerated Detection of Viral Particles by Combining AC Electric Field Effects and Micro-Raman Spectroscopy

PubMed Central

Tomkins, Matthew Robert; Liao, David Shiqi; Docoslis, Aristides

2015-01-01

A detection method that combines electric field-assisted virus capture on antibody-decorated surfaces with the “fingerprinting” capabilities of micro-Raman spectroscopy is demonstrated for the case of M13 virus in water. The proof-of-principle surface mapping of model bioparticles (protein coated polystyrene spheres) captured by an AC electric field between planar microelectrodes is presented with a methodology for analyzing the resulting spectra by comparing relative peak intensities. The same principle is applied to dielectrophoretically captured M13 phage particles whose presence is indirectly confirmed with micro-Raman spectroscopy using NeutrAvidin-Cy3 as a labeling molecule. It is concluded that the combination of electrokinetically driven virus sampling and micro-Raman based signal transduction provides a promising approach for time-efficient and in situ detection of viruses. PMID:25580902

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

PubMed Central

Aleshin, SE; Timofeev, AV; Khoretonenko, MV; Zakharova, LG; Pashvykina, GV; Stephenson, JR; Shneider, AM; Altstein, AD

2005-01-01

Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines. PMID:16076390

The ability to cause infection in a pathogenic fungus uncovers a new biological feature of honey bee viruses.

PubMed

Li, Zhiguo; Su, Songkun; Hamilton, Michele; Yan, Limin; Chen, Yanping

2014-07-01

We demonstrated that honey bee viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV) and Israeli acute paralysis virus (IAPV) could infect and replicate in the fungal pathogen Ascosphaera apis that causes honey bee chalkbrood disease, revealing a novel biological feature of honey bee viruses. The phylogenetic analysis show that viruses of fungal and honey bee origins form two clusters in the phylogenetic trees distinctly and that host range of honey bee viruses is dynamic. Further studies are warranted to investigate the impact of the viruses on the fitness of their fungal host and phenotypic effects the virus-fungus combination has on honey bee hosts. Published by Elsevier Inc.

Investigation of Influenza Virus Polymerase Activity in Pig Cells

PubMed Central

Moncorgé, Olivier; Long, Jason S.; Cauldwell, Anna V.; Zhou, Hongbo; Lycett, Samantha J.

2013-01-01

Reassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a “mixing vessel” for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs. PMID:23077313

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

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

Meador, Lydia R.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viralmore » vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.« less

MicroRNA-mediated species-specific attenuation of influenza A virus.

PubMed

Perez, Jasmine T; Pham, Alissa M; Lorini, Maria H; Chua, Mark A; Steel, John; tenOever, Benjamin R

2009-06-01

Influenza A virus leads to yearly epidemics and sporadic pandemics. Present prophylactic strategies focus on egg-grown, live, attenuated influenza vaccines (LAIVs), in which attenuation is generated by conferring temperature sensitivity onto the virus. Here we describe an alternative approach to attenuating influenza A virus based on microRNA-mediated gene silencing. By incorporating nonavian microRNA response elements (MREs) into the open-reading frame of the viral nucleoprotein, we generate reassortant LAIVs for H1N1 and H5N1 that are attenuated in mice but not in eggs. MRE-based LAIVs show a greater than two-log reduction in mortality compared with control viruses lacking MREs and elicit a diverse antibody response. This approach might be combined with existing LAIVs to increase attenuation and improve vaccine safety.

Annulate lamellae in phloem cells of virus-infected Sonchus plants

PubMed Central

1977-01-01

The occurrence of annulate lamellae (AL) in differentiating phloem of Sonchus oleraceus (Compositae) singly infected with sowthistle yellow vein virus (SYVV) and doubly infected with a combination of SYVV and beet yellow stunt virus is documented by electron microscopy. Cell types in which AL were found were immature sieve elements and phloem parenchyma cells. AL were found only in cells that also contained SYVV particles although a direct association between the virus and AL was not apparent. The substructure of the AL and the relationships between the AL and the nuclear envelope and endoplasmic reticulum are similar to those reported in other descriptions of this organelle in the literature. This report appears to be the first one concerning the association of AL with a plant virus disease. PMID:873998

Protective Effect of Dietary Xylitol on Influenza A Virus Infection

PubMed Central

Yin, Sun Young; Kim, Hyoung Jin; Kim, Hong-Jin

2014-01-01

Xylitol has been used as a substitute for sugar to prevent cavity-causing bacteria, and most studies have focused on its benefits in dental care. Meanwhile, the constituents of red ginseng (RG) are known to be effective in ameliorating the symptoms of influenza virus infection when they are administered orally for 14 days. In this study, we investigated the effect of dietary xylitol on influenza A virus infection (H1N1). We designed regimens containing various fractions of RG (RGs: whole extract, water soluble fraction, saponin and polysaccharide) and xylitol, and combination of xylitol with the RG fractions. Mice received the various combinations orally for 5 days prior to lethal influenza A virus infection. Almost all the mice died post challenge when xylitol or RGs were administered separately. Survival was markedly enhanced when xylitol was administered along with RGs, pointing to a synergistic effect. The effect of xylitol plus RG fractions increased with increasing dose of xylitol. Moreover, dietary xylitol along with the RG water soluble fraction significantly reduced lung virus titers after infection. Therefore, we suggest that dietary xylitol is effective in ameliorating influenza-induced symptoms when it is administered with RG fractions, and this protective effect of xylitol should be considered in relation to other diseases. PMID:24392148

Zoonotic Poxviruses Associated with Companion Animals

PubMed Central

Tack, Danielle M.; Reynolds, Mary G.

2011-01-01

Simple Summary Contemporary enthusiasm for the ownership of exotic animals and hobby livestock has created an opportunity for the movement of poxviruses—such as monkeypox, cowpox, and orf—outside their traditional geographic range bringing them into contact with atypical animal hosts and groups of people not normally considered at risk. It is important that pet owners and practitioners of human and animal medicine develop a heightened awareness for poxvirus infections and understand the risks that can be associated with companion animals and livestock. This article reviews the epidemiology and clinical features of zoonotic poxviruses that are most likely to affect companion animals. Abstract Understanding the zoonotic risk posed by poxviruses in companion animals is important for protecting both human and animal health. The outbreak of monkeypox in the United States, as well as current reports of cowpox in Europe, point to the fact that companion animals are increasingly serving as sources of poxvirus transmission to people. In addition, the trend among hobbyists to keep livestock (such as goats) in urban and semi-urban areas has contributed to increased parapoxvirus exposures among people not traditionally considered at high risk. Despite the historic notoriety of poxviruses and the diseases they cause, poxvirus infections are often missed. Delays in diagnosing poxvirus-associated infections in companion animals can lead to inadvertent human exposures. Delays in confirming human infections can result in inappropriate treatment or prolonged recovery. Early recognition of poxvirus-associated infections and application of appropriate preventive measures can reduce the spread of virus between companion animals and their owners. This review will discuss the epidemiology and clinical features associated with the zoonotic poxvirus infections most commonly associated with companion animals. PMID:26486622

Thunder and lightning: immunotherapy and oncolytic viruses collide.

PubMed

Melcher, Alan; Parato, Kelley; Rooney, Cliona M; Bell, John C

2011-06-01

For the last several decades, the development of antitumor immune-based strategies and the engineering and testing of oncolytic viruses (OVs) has occurred largely in parallel tracks. Indeed, the immune system is often thought of as an impediment to successful oncolytic virus delivery and efficacy. More recently, however, both preclinical and clinical results have revealed potential synergy between these two promising therapeutic strategies. Here, we summarize some of the evidence that supports combining OVs with immuno-therapeutics and suggest new ways to mount a multipronged biological attack against cancers.

Thunder and Lightning: Immunotherapy and Oncolytic Viruses Collide

PubMed Central

Melcher, Alan; Parato, Kelley; Rooney, Cliona M; Bell, John C

2011-01-01

For the last several decades, the development of antitumor immune-based strategies and the engineering and testing of oncolytic viruses (OVs) has occurred largely in parallel tracks. Indeed, the immune system is often thought of as an impediment to successful oncolytic virus delivery and efficacy. More recently, however, both preclinical and clinical results have revealed potential synergy between these two promising therapeutic strategies. Here, we summarize some of the evidence that supports combining OVs with immuno-therapeutics and suggest new ways to mount a multipronged biological attack against cancers. PMID:21505424

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus.

PubMed

Roush, David J; Myrold, Adam; Burnham, Michael S; And, Joseph V; Hughes, Joseph V

2015-01-01

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10(10) PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4-5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3-101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79-85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic-Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of

Squirrelpox Virus: Assessing Prevalence, Transmission and Environmental Degradation

PubMed Central

Tosh, David G.; McInnes, Colin; Everest, David; Montgomery, W. Ian; Scantlebury, Mike; Marks, Nikki; Dick, Jaimie T. A.

2014-01-01

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species. PMID

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

PubMed

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Relative insignificance of virus inactivation during aluminum electrocoagulation of saline waters.

PubMed

Tanneru, Charan Tej; Jothikumar, N; Hill, Vincent R; Chellam, Shankararaman

2014-12-16

Combined removal and inactivation of the MS2 bacteriophage from model saline (0-100 mM NaCl) waters by electrochemical treatment using a sacrificial aluminum anode was evaluated. Both chemical and electrodissolution contributed to coagulant dosing since measured aluminum concentrations were statistically higher than purely electrochemical predictions using Faraday's law. Electrocoagulation generated only small amounts of free chlorine in situ but effectively destabilized viruses and incorporated them into Al(OH)3(s) flocs during electrolysis. Low chlorine concentrations combined with virus shielding and aggregation within flocs resulted in very slow disinfection rates necessitating extended flocculation/contact times to achieve significant log-inactivation. Therefore, the dominant virus control mechanism during aluminum electrocoagulation of saline waters is "physical" removal by uptake onto flocs rather than "chemical" inactivation by chlorine. Attenuated total reflectance-Fourier transform infrared spectroscopy provided evidence for oxidative transformations of capsid proteins including formation of oxyacids, aldehydes, and ketones. Electrocoagulation significantly altered protein secondary structures decreasing peak areas associated with turns, bends, α-helices, β-structures, and random coils for inactivated viruses compared with the MS2 stock. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements showed rapid initial RNA damage following a similar trend as plaque assay measurements of infectious viruses. However, ssRNA cleavage measured by qRT-PCR underestimated inactivation over longer durations. Although aluminum electrocoagulation of saline waters disorders virus capsids and damages RNA, inactivation occurs at a sufficiently low rate so as to only play a secondary role to floc-encapsulation during residence times typical of electrochemical treatment.

Dispersed gold nanoparticles potentially ruin gold barley yellow dwarf virus and eliminate virus infectivity hazards

NASA Astrophysics Data System (ADS)

Alkubaisi, Noorah A.; Aref, Nagwa M. A.

2017-02-01

Gold nanoparticles (AuNPs) application melted barley yellow dwarf virus-PAV (BYDV-PAV) spherical nanoparticle capsids. Synergistic therapeutic effects for plant virus resistance were induced by interaction with binding units of prepared AuNPs in a water solution which was characterized and evaluated by zeta sizer, zeta potential and transmission electron microscopy (TEM). The yield of purified nanoparticles of BYDV-PAV was obtained from Hordeum vulgare (Barley) cultivars, local and Giza 121/Justo. It was 0.62 mg/ml from 27.30 g of infected leaves at an A260/A280 ratio. Virus nanoparticle has a spherical shape 30 nm in size by TEM. BYDV-PAV combined with AuNPs to challenge virus function in vivo and in vitro. Dual AuNPs existence in vivo and in vitro affected compacted configuration of viral capsid protein in the interior surface of capsomers, the outer surface, or between the interface of coat protein subunits for 24 and 48 h incubation period in vitro at room temperature. The sizes of AuNPs that had a potentially dramatic deteriorated effect are 3.151 and 31.67 nm with a different intensity of 75.3% for the former and 24.7% for the latter, which enhances optical sensing applications to eliminate virus infectivity. Damages of capsid protein due to AuNPs on the surface of virus subunits caused variable performance in four different types of TEM named puffed, deteriorated and decorated, ruined and vanished. Viral yield showed remarkably high-intensity degree of particle symmetry and uniformity in the local cultivar greater than in Giza 121/Justo cultivar. A high yield of ruined VLPs in the local cultivar than Justo cultivar was noticed. AuNPs indicated complete lysed VLPs and some deteriorated VLPs at 48 h.

Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery.

PubMed

Hall, Richard J; Wang, Jing; Todd, Angela K; Bissielo, Ange B; Yen, Seiha; Strydom, Hugo; Moore, Nicole E; Ren, Xiaoyun; Huang, Q Sue; Carter, Philip E; Peacey, Matthew

2014-01-01

The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample

Effect of Suberoylanilide Hydroxamic Acid (SAHA) Administration on the Residual Virus Pool in a Model of Combination Antiretroviral Therapy-Mediated Suppression in SIVmac239-Infected Indian Rhesus Macaques

PubMed Central

Del Prete, Gregory Q.; Shoemaker, Rebecca; Oswald, Kelli; Lara, Abigail; Trubey, Charles M.; Fast, Randy; Schneider, Douglas K.; Kiser, Rebecca; Coalter, Vicky; Wiles, Adam; Wiles, Rodney; Freemire, Brandi; Keele, Brandon F.; Estes, Jacob D.; Quiñones, Octavio A.; Smedley, Jeremy; Macallister, Rhonda; Sanchez, Rosa I.; Wai, John S.; Tan, Christopher M.; Alvord, W. Gregory; Hazuda, Daria J.; Piatak, Michael

2014-01-01

Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4+ T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches. PMID:25182644

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors*

PubMed Central

Pontejo, Sergio M.; Alejo, Ali; Alcami, Antonio

2015-01-01

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. PMID:25940088

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV), Bovine Leukemia Virus (BLV), Human Papilloma Virus (HPV), and Epstein-Barr Virus (EBV).

PubMed

Lawson, James S; Salmons, Brian; Glenn, Wendy K

2018-01-01

Although the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV), bovine leukemia virus (BLV), human papilloma viruses (HPVs), and Epstein-Barr virus (EBV-also known as human herpes virus type 4). Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence. MMTV and human breast cancer-the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer-the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer-the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer-the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal. The influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer.

Risk of Progressive Multifocal Leukoencephalopathy in the Combination Antiretroviral Therapy Era in the French Hospital Database on Human Immunodeficiency Virus (ANRS-C4).

PubMed

Melliez, Hugues; Mary-Krause, Murielle; Bocket, Laurence; Guiguet, Marguerite; Abgrall, Sophie; De Truchis, Pierre; Katlama, Christine; Martin-Blondel, Guillaume; Henn, Aurelia; Revest, Matthieu; Robineau, Olivier; Khuong-Josses, Marie-Aude; Canestri, Anna; De Castro, Nathalie; Joly, Véronique; Mokhtari, Saadia; Risso, Karine; Gasnault, Jacques; Costagliola, Dominique

2018-04-04

Risk factors for progressive multifocal leukoencephalopathy (PML) in individuals with human immunodeficiency virus (HIV) infection are poorly documented in the era of combination antiretroviral therapy (cART). We studied HIV-1-infected individuals aged ≥15 years who had no history of PML and were prospectively followed up between 1997 and 2011 in the French Hospital Database on HIV (FHDH-ANRS CO4) cohort. Cox models were used to calculate adjusted hazard ratios (HRs), focusing on sub-Saharan origin, suggested to be protective, and recent cART initiation, potentially associated with an increased risk of PML. PML developed in 555 individuals, in 57 during the first 6 months of cART. From 1997-2000 to 2009-2011, the incidence fell from 1.15 (95% confidence interval [CI], .98-1.31) to 0.49 (.37-.61) per 1000 person-years. Sub-Saharan African origin had no clear influence (HR, 0.80; 95% CI, .58-1.11). Compared with men who have sex with men, injection drug users (IDUs) were at higher risk (HR, 1.80 [95% CI, 1.32-2.45] for male and 1.68 [1.13-2.48] for female IDUs). When IDUs were excluded, hepatitis C virus seropositivity was associated with an increased risk (HR, 1.40; 95% CI, 1.02-1.93). Compared with no cART initiation, initiation <6 months previously was associated with PML onset (HR, 4.91; 95% CI, 2.42-9.95). Recent cART initiation is associated with an increased risk of PML, as are injection drug use and hepatitis C virus seropositivity. Sub-Saharan African origin had no protective effect.

Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

PubMed

Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

2017-02-22

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

Influenza Virus Database (IVDB): an integrated information resource and analysis platform for influenza virus research.

PubMed

Chang, Suhua; Zhang, Jiajie; Liao, Xiaoyun; Zhu, Xinxing; Wang, Dahai; Zhu, Jiang; Feng, Tao; Zhu, Baoli; Gao, George F; Wang, Jian; Yang, Huanming; Yu, Jun; Wang, Jing

2007-01-01

Frequent outbreaks of highly pathogenic avian influenza and the increasing data available for comparative analysis require a central database specialized in influenza viruses (IVs). We have established the Influenza Virus Database (IVDB) to integrate information and create an analysis platform for genetic, genomic, and phylogenetic studies of the virus. IVDB hosts complete genome sequences of influenza A virus generated by Beijing Institute of Genomics (BIG) and curates all other published IV sequences after expert annotation. Our Q-Filter system classifies and ranks all nucleotide sequences into seven categories according to sequence content and integrity. IVDB provides a series of tools and viewers for comparative analysis of the viral genomes, genes, genetic polymorphisms and phylogenetic relationships. A search system has been developed for users to retrieve a combination of different data types by setting search options. To facilitate analysis of global viral transmission and evolution, the IV Sequence Distribution Tool (IVDT) has been developed to display the worldwide geographic distribution of chosen viral genotypes and to couple genomic data with epidemiological data. The BLAST, multiple sequence alignment and phylogenetic analysis tools were integrated for online data analysis. Furthermore, IVDB offers instant access to pre-computed alignments and polymorphisms of IV genes and proteins, and presents the results as SNP distribution plots and minor allele distributions. IVDB is publicly available at http://influenza.genomics.org.cn.

Cordycepin enhances Epstein-Barr virus lytic infection and Epstein-Barr virus-positive tumor treatment efficacy by doxorubicin.

PubMed

Du, Yinping; Yu, Jieshi; Du, Li; Tang, Jun; Feng, Wen-Hai

2016-07-01

The consistent latent presence of Epstein-Barr virus (EBV) in tumor cells offers potential for virus-targeted therapies. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. In this study, we report that a natural small molecule compound, cordycepin, can induce lytic EBV infection in tumor cells. Subsequently, we demonstrate that cordycepin can enhance EBV reactivating capacity and EBV-positive tumor cell killing ability of low dose doxorubicin. The combination of cordycepin and doxorubicin phosphorylates CCAAT/enhancer binding protein β (C/EBPβ) through protein kinase C (PKC)-p38 mitogen activated protein kinases (p38 MAPK) signaling pathway, and C/EBPβ is required for the activation of lytic EBV infection. Most importantly, an in vivo experiment demonstrates that the combination of cordycepin and doxorubicin is more effective in inhibiting tumor growth in SCID mice than is doxorubicin alone. Our findings establish that cordycepin can enhance the efficacy of conventional chemotherapy for treatment of EBV-positive tumors. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Personal historical chronicle of six decades of basic and applied research in virology, immunology, and vaccinology.

PubMed

Hilleman, M R

1999-08-01

The sciences of vaccinology and of immunology were created just two centuries ago by Jenner's studies of prevention of smallpox by inoculation with cowpox virus. This rudimentary beginning was expanded greatly by the giants of late 19th and early 20th centuries biomedical sciences. The period from 1930 to 1950 was a transitional era with the introduction of chick embryos and minced tissues for propagating viruses and rickettsiae in vitro for vaccines. Modern era vaccinology began about 1950 as a continuum of notable advances made during the 1940s and World War II. Present vaccinology is based largely on breakthroughs in cell culture, bacterial polysaccharide chemistry, molecular biology, and immunology. By invitation, the author, who is a microbe hunter in fact, was asked to chronicle his six decades of pioneering achievements in basic and applied virology, bacteriology, immunology, molecular biology, epidemiology, and cancer, with special reference to the pioneering creation of most of the present day vaccines. Knowledge of the past may guide the present and future. This chronicle will have achieved its legacy if it helps others to understand the why and how of the past that may help to create the substance of the future.

Lack of involvement of strand s1'A of the viral serpin CrmA in anti-apoptotic or caspase-inhibitory functions

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

Simonovic, Miljan; Denault, Jean-Bernard; Salvesen, Guy S.

2010-11-30

CrmA is a cowpox virus serpin required for full host infectivity and virulence. Residues 51-56 (DKNKDD), the only region that differs significantly from related viral serpins, were investigated for functional importance. A 1.6 {angstrom} X-ray structure reported here showed that this region can adopt either structured or unstructured conformations. Three variants were expressed, one with the region 51-56 deleted, one substituted by alanines, and one in which this region was replaced by the sequence encoded in smallpox virus. NMR showed that the region is an exposed, flexible loop that can be deleted without perturbing the serpin. The region is alsomore » very susceptible to proteolysis. Significantly, inhibition of caspases 1 and 8 was unaffected by the mutations, and each of the variants was as effective as wild-type CrmA in promoting survival from apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, although the 51-56 region of CrmA is unique, and is exposed and highly susceptible to proteolysis, any in vivo role must involve a function other than proteinase inhibition or cell sparing.« less

Exploring interaction of TNF and orthopoxviral CrmB protein by surface plasmon resonance and free energy calculation.

PubMed

Ivanisenko, Nikita V; Tregubchak, Tatiana V; Saik, Olga V; Ivanisenko, Vladimir A; Shchelkunov, Sergei N

2014-01-01

Inhibition of the activity of the tumor necrosis factor (TNF) has become the main strategy for treating inflammatory diseases. The orthopoxvirus TNF-binding proteins can bind and efficiently neutralize TNF. To analyze the mechanisms of the interaction between human (hTNF) or mouse (mTNF) TNF and the cowpox virus N-terminal binding domain (TNFBD-CPXV), also the variola virus N-terminal binding domain (TNFBD-VARV) and to define the amino acids most importantly involved in the formation of complexes, computer models, derived from the X-ray structure of a homologous hTNF/TNFRII complex, were used together with experiments. The hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV, and mTNF/TNFBD-VARV complexes were used in the molecular dynamics (MD) simulations and MM/GBSA free energy calculations. The complexes were ordered as hTNF/TNFBD-CPXV, hTNF/TNFBD-VARV, mTNF/TNFBD-CPXV and mTNF/TNFBD-VARV according to increase in the binding affinity. The calculations were in agreement with surface plasmon resonance (SPR) measurements of the binding constants. Key residues involved in complex formation were identified.

Possible Increased Pathogenicity of Pandemic (H1N1) 2009 Influenza Virus upon Reassortment

PubMed Central

Schrauwen, Eefje J.A.; Herfst, Sander; Chutinimitkul, Salin; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Kuiken, Thijs

2011-01-01

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses—seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009—have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets. PMID:21291589

Global circulation patterns of seasonal influenza viruses vary with antigenic drift

PubMed Central

Bedford, Trevor; Riley, Steven; Barr, Ian G.; Broor, Shobha; Chadha, Mandeep; Cox, Nancy J.; Daniels, Rodney S.; Gunasekaran, C. Palani; Hurt, Aeron C.; Kelso, Anne; Lewis, Nicola S.; Li, Xiyan; McCauley, John W.; Odagiri, Takato; Potdar, Varsha; Rambaut, Andrew; Shu, Yuelong; Skepner, Eugene; Smith, Derek J.; Suchard, Marc A.; Tashiro, Masato; Wang, Dayan; Xu, Xiyan; Lemey, Philippe; Russell, Colin A.

2015-01-01

Understanding the spatio-temporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well-characterized1-7 but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here, based on analyses of 9,604 hemagglutinin sequences of human seasonal influenza viruses from 2000–2012, we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses. While genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast (E-SE) Asia, genetic variants of A/H1N1 and B viruses persisted across multiple seasons and exhibited complex global dynamics with E-SE Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as likely drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology and human behavior. PMID:26053121

Global circulation patterns of seasonal influenza viruses vary with antigenic drift

NASA Astrophysics Data System (ADS)

Bedford, Trevor; Riley, Steven; Barr, Ian G.; Broor, Shobha; Chadha, Mandeep; Cox, Nancy J.; Daniels, Rodney S.; Gunasekaran, C. Palani; Hurt, Aeron C.; Kelso, Anne; Klimov, Alexander; Lewis, Nicola S.; Li, Xiyan; McCauley, John W.; Odagiri, Takato; Potdar, Varsha; Rambaut, Andrew; Shu, Yuelong; Skepner, Eugene; Smith, Derek J.; Suchard, Marc A.; Tashiro, Masato; Wang, Dayan; Xu, Xiyan; Lemey, Philippe; Russell, Colin A.

2015-07-01

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

Viruses of invasive Argentine ants from the European Main supercolony: characterization, interactions and evolution.

PubMed

Viljakainen, Lumi; Holmberg, Ida; Abril, Sílvia; Jurvansuu, Jaana

2018-06-25

The Argentine ant (Linepithema humile) is a highly invasive pest, yet very little is known about its viruses. We analysed individual RNA-sequencing data from 48 Argentine ant queens to identify and characterisze their viruses. We discovered eight complete RNA virus genomes - all from different virus families - and one putative partial entomopoxvirus genome. Seven of the nine virus sequences were found from ant samples spanning 7 years, suggesting that these viruses may cause long-term infections within the super-colony. Although all nine viruses successfully infect Argentine ants, they have very different characteristics, such as genome organization, prevalence, loads, activation frequencies and rates of evolution. The eight RNA viruses constituted in total 23 different virus combinations which, based on statistical analysis, were non-random, suggesting that virus compatibility is a factor in infections. We also searched for virus sequences from New Zealand and Californian Argentine ant RNA-sequencing data and discovered that many of the viruses are found on different continents, yet some viruses are prevalent only in certain colonies. The viral loads described here most probably present a normal asymptomatic level of infection; nevertheless, detailed knowledge of Argentine ant viruses may enable the design of viral biocontrol methods against this pest.

[Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

PubMed

Tokuno, Osamu; Fujiwara, Miki; Nakajoh, Yoshimi; Yamanouchi, Sumika; Adachi, Masayo; Ikeda, Akiko; Kitayama, Shigeo; Takahashi, Toshio; Kase, Tetsuo; Kinoshita, Shouhiro; Kumagai, Shunichi

2009-09-01

Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

Background review for diagnostic test development for Zika virus infection.

PubMed

Charrel, Rémi N; Leparc-Goffart, Isabelle; Pas, Suzan; de Lamballerie, Xavier; Koopmans, Marion; Reusken, Chantal

2016-08-01

To review the state of knowledge about diagnostic testing for Zika virus infection and identify areas of research needed to address the current gaps in knowledge. We made a non-systematic review of the published literature about Zika virus and supplemented this with information from commercial diagnostic test kits and personal communications with researchers in European preparedness networks. The review covered current knowledge about the geographical spread, pathogen characteristics, life cycle and infection kinetics of the virus. The available molecular and serological tests and biosafety issues are described and discussed in the context of the current outbreak strain. We identified the following areas of research to address current knowledge gaps: (i) an urgent assessment of the laboratory capacity and capability of countries to detect Zika virus; (ii) rapid and extensive field validation of the available molecular and serological tests in areas with and without Zika virus transmission, with a focus on pregnant women; (iii) monitoring the genomic diversity of circulating Zika virus strains; (iv) prospective studies into the virus infection kinetics, focusing on diagnostic sampling (specimen types, combinations and timings); and (v) developing external quality assessments for molecular and serological testing, including differential diagnosis for similar viruses and symptom clusters. The availability of reagents for diagnostic development (virus strains and antigens, quantified viral ribonucleic acid) needs to be facilitated. An international laboratory response is needed, including preparation of protocols for prospective studies to address the most pressing information needs.

Concentration of infectious hematopoietic necrosis virus from water samples by tangential flow filtration and polyethylene glycol precipitation

USGS Publications Warehouse

Batts, W.N.; Winton, J.R.

1989-01-01

Infectious hematopoietic necrosis virus (IHNV) was concentrated from water samples by polyethylene glycol (PEG) precipitation, tangential flow filtration (TFF), and by a combination of TFF followed by PEG precipitation of the retentate. Used alone, PEG increased virus titers more than 200-fold, and the efficiency of recovery was as great as 100%. Used alone, TFF concentrated IHNV more than 20-fold, and average recovery was 70%. When the two techniques were combined, 10-L water samples were reduced to about 300 mL by TFF and the virus was precipitated with PEG into a 1 to 2 g pellet; total recovery was as great as 100%. The combined techniques were used to isolate IHNV from water samples taken from a river containing adult sockeye salmon (Oncorhynchus nerka) and from a hatchery pond containing adult spring chinook salmon (O. tshawytscha). The combination of these methods was effective in concentrating and detecting IHNV from water containing only three infectious particles per 10-L sample.

Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

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

Zheng Min; Guangxi Center for Animal Disease Control and Prevention, Nanning 530001; College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062

Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AALmore » and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.« less

Virus inactivation by nucleic acid extraction reagents.

PubMed

Blow, Jamie A; Dohm, David J; Negley, Diane L; Mores, Christopher N

2004-08-01

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

Oncolytic Viruses: Therapeutics With an Identity Crisis.

PubMed

Breitbach, Caroline J; Lichty, Brian D; Bell, John C

2016-07-01

Oncolytic viruses (OV) are replicating viral therapeutics for the treatment of cancer and have been in laboratory development for about twenty years. Recently, the FDA approved Imlygic, a herpes virus based therapeutic for the treatment of melanoma and thus OVs have entered a new era where they are a weapon in the armament of the oncologist. OVs are unique therapeutics with multiple mechanisms of therapeutic activity. The exact path for their development and eventual uptake by pharmaceutical companies is somewhat clouded by an uncertain identity. Are they vaccines, tumour lysing therapeutics, inducers of innate immunity, gene therapy vectors, anti-vascular agents or all of the above? Should they be developed as stand-alone loco-regional therapeutics, systemically delivered tumour hunters or immune modulators best tested as combination therapeutics? We summarize data here supporting the idea, depending upon the virus, that OVs can be any or all of these things. Pursuing a "one-size fits all" approach is counter-productive to their clinical development and instead as a field we should build on the strengths of individual virus platforms. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Carrot yellow leaf virus is associated with carrot internal necrosis.

PubMed

Adams, Ian P; Skelton, Anna; Macarthur, Roy; Hodges, Tobias; Hinds, Howard; Flint, Laura; Nath, Palash Deb; Boonham, Neil; Fox, Adrian

2014-01-01

Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots.

Carrot yellow leaf virus Is Associated with Carrot Internal Necrosis

PubMed Central

Adams, Ian P.; Skelton, Anna; Macarthur, Roy; Hodges, Tobias; Hinds, Howard; Flint, Laura; Nath, Palash Deb; Boonham, Neil; Fox, Adrian

2014-01-01

Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots. PMID:25365290

Virus Neutralisation: New Insights from Kinetic Neutralisation Curves

PubMed Central

Magnus, Carsten

2013-01-01

Antibodies binding to the surface of virions can lead to virus neutralisation. Different theories have been proposed to determine the number of antibodies that must bind to a virion for neutralisation. Early models are based on chemical binding kinetics. Applying these models lead to very low estimates of the number of antibodies needed for neutralisation. In contrast, according to the more conceptual approach of stoichiometries in virology a much higher number of antibodies is required for virus neutralisation by antibodies. Here, we combine chemical binding kinetics with (virological) stoichiometries to better explain virus neutralisation by antibody binding. This framework is in agreement with published data on the neutralisation of the human immunodeficiency virus. Knowing antibody reaction constants, our model allows us to estimate stoichiometrical parameters from kinetic neutralisation curves. In addition, we can identify important parameters that will make further analysis of kinetic neutralisation curves more valuable in the context of estimating stoichiometries. Our model gives a more subtle explanation of kinetic neutralisation curves in terms of single-hit and multi-hit kinetics. PMID:23468602

Landscape Utilisation, Animal Behaviour and Hendra Virus Risk.

PubMed

Field, H E; Smith, C S; de Jong, C E; Melville, D; Broos, A; Kung, N; Thompson, J; Dechmann, D K N

2016-03-01

Hendra virus causes sporadic fatal disease in horses and humans in eastern Australia. Pteropid bats (flying-foxes) are the natural host of the virus. The mode of flying-fox to horse transmission remains unclear, but oro-nasal contact with flying-fox urine, faeces or saliva is the most plausible. We used GPS data logger technology to explore the landscape utilisation of black flying-foxes and horses to gain new insight into equine exposure risk. Flying-fox foraging was repetitious, with individuals returning night after night to the same location. There was a preference for fragmented arboreal landscape and non-native plant species, resulting in increased flying-fox activity around rural infrastructure. Our preliminary equine data logger study identified significant variation between diurnal and nocturnal grazing behaviour that, combined with the observed flying-fox foraging behaviour, could contribute to Hendra virus exposure risk. While we found no significant risk-exposing difference in individual horse movement behaviour in this study, the prospect warrants further investigation, as does the broader role of animal behaviour and landscape utilisation on the transmission dynamics of Hendra virus.

Effective inactivation of a wide range of viruses by pasteurization.

PubMed

Gröner, Albrecht; Broumis, Connie; Fang, Randel; Nowak, Thomas; Popp, Birgit; Schäfer, Wolfram; Roth, Nathan J

2018-01-01

Careful selection and testing of plasma reduces the risk of blood-borne viruses in the starting material for plasma-derived products. Furthermore, effective measures such as pasteurization at 60°C for 10 hours have been implemented in the manufacturing process of therapeutic plasma proteins such as human albumin, coagulation factors, immunoglobulins, and enzyme inhibitors to inactivate blood-borne viruses of concern. A comprehensive compilation of the virus reduction capacity of pasteurization is presented including the effect of stabilizers used to protect the therapeutic protein from modifications during heat treatment. The virus inactivation kinetics of pasteurization for a broad range of viruses were evaluated in the relevant intermediates from more than 15 different plasma manufacturing processes. Studies were carried out under the routine manufacturing target variables, such as temperature and product-specific stabilizer composition. Additional studies were also performed under robustness conditions, that is, outside production specifications. The data demonstrate that pasteurization inactivates a wide range of enveloped and nonenveloped viruses of diverse physicochemical characteristics. After a maximum of 6 hours' incubation, no residual infectivity could be detected for the majority of enveloped viruses. Effective inactivation of a range of nonenveloped viruses, with the exception of nonhuman parvoviruses, was documented. Pasteurization is a very robust and reliable virus inactivation method with a broad effectiveness against known blood-borne pathogens and emerging or potentially emerging viruses. Pasteurization has proven itself to be a highly effective step, in combination with other complementary safety measures, toward assuring the virus safety of final product. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Virus Removal by Biogenic Cerium

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

De Gusseme, B.; Du Laing, G; Hennebel, T

2010-01-01

The rare earth element cerium has been known to exert antifungal and antibacterial properties in the oxidation states +III and +IV. This study reports on an innovative strategy for virus removal in drinking water by the combination of Ce(III) on a bacterial carrier matrix. The biogenic cerium (bio-Ce) was produced by addition of aqueous Ce(III) to actively growing cultures of either freshwater manganese-oxidizing bacteria (MOB) Leptothrix discophora or Pseudomonas putida MnB29. X-ray absorption spectroscopy results indicated that Ce remained in its trivalent state on the bacterial surface. The spectra were consistent with Ce(III) ions associated with the phosphoryl groups ofmore » the bacterial cell wall. In disinfection assays using a bacteriophage as model, it was demonstrated that bio-Ce exhibited antiviral properties. A 4.4 log decrease of the phage was observed after 2 h of contact with 50 mg L{sup -1} bio-Ce. Given the fact that virus removal with 50 mg L{sup -1} Ce(III) as CeNO{sub 3} was lower, the presence of the bacterial carrier matrix in bio-Ce significantly enhanced virus removal.« less

An inquiry into the causes and effects of the variolae (or Cow-pox. 1798).

PubMed

Jenson, Alfred B; Ghim, Shin-Je; Sundberg, John P

2016-03-01

Few papers have had a greater impact on the health of the human species than the simple, yet elegant, observations and clinical trials of Edward Jenner with what was at the time called the Cow Pox. In fact, this was a naturally attenuated rodent (probably rat) pox that could infect horses and, through farriers and farm hands, dairy cattle. While commonly called the Cow Pox at the time, Jenner's transmission studies between humans used infectious materials from horses. His methods provided protection from the serious effects of smallpox infections. In 1977, smallpox was considered to be eradicated, although people continue to be infected by pox viruses from other mammalian species. We consider this to be our 'favorite historical paper' because it emphasizes careful clinical observation followed by relatively simple clinical testing can have a profound influence on human health, even when almost nothing is known about the underlying molecular mechanisms. Continued follow-up with strict attention to detail resulted in a crude but effective way to deal with an epidemic, methods still used today for containing infectious diseases. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of Liposome, Heat-Killed Mycobacterium w, and Alum Adjuvants in the Protection Offered by Different Combinations of Recombinant HA, NP proteins, and M2e Against Homologous H5N1 Virus.

PubMed

Ingle, Nilesh B; Virkar, Rashmi G; Agnihotri, Kalpana; Sharma, Kapil S; Lole, Kavita S; Arankalle, Vidya A

2016-10-01

Continued evolution of highly pathogenic H5N1 viruses causing high mortality in humans obviates need for broadly cross-reactive vaccines. For this, hemagglutinin (HA) inducing specific protective antibodies, highly conserved nucleoprotein (NP), and ectodomain of matrix (M2e) protein, either singly or in combination, were evaluated in BALB/c mice. Recombinant HA and NP (baculovirus system) and M2e (synthetic peptide) and 3 adjuvants, that is, liposomes, Mw (heat killed Mycobacterium w), and alum were utilized for the homologous virus challenge. Additional immunogens included liposome-encapsulated HA/NP proteins and corresponding DNAs. Mice groups received two doses of respective formulations given at 3-week intervals and challenged intranasally with 100LD50 of H5N1 virus strain. Dynamics of weight loss, lung viral load, titres of IgG-anti-HA, NP, and M2e antibodies (ELISA), and IgG-subtype analysis was done. Two doses of all the formulations led to 100% seroconversion against the immunogens evaluated (100% seroconversion after the first dose in majority). Antibody titres against the components were dependent on the adjuvant and combination. HA-driven Th2 response with all the adjuvants, balanced Th1/Th2 response to NP protein, and Th2-bias with alum were noted. Low anti-M2e antibody titres did not allow subtype analysis. On challenge, complete protection was observed with Mw-HA, alum-HA+NP, Lipo-HA+NP+M2e, alum-HA+NP+M2e, and HA-DP formulations with 12-fold, 8-fold, 720-fold, 17-fold, and no reduction, respectively, in lung viral load. In conclusion, the results identify several adjuvant-immunogen combinations conferring 100% protection in mice that need further evaluation in higher animals.

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

PubMed

Grant-Klein, Rebecca J; Altamura, Louis A; Badger, Catherine V; Bounds, Callie E; Van Deusen, Nicole M; Kwilas, Steven A; Vu, Hong A; Warfield, Kelly L; Hooper, Jay W; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

2015-01-01

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

Combined semi-empirical screening and design of experiments (DOE) approach to identify candidate formulations of a lyophilized live attenuated tetravalent viral vaccine candidate.

PubMed

Patel, Ashaben; Erb, Steven M; Strange, Linda; Shukla, Ravi S; Kumru, Ozan S; Smith, Lee; Nelson, Paul; Joshi, Sangeeta B; Livengood, Jill A; Volkin, David B

2018-05-24

A combination experimental approach, utilizing semi-empirical excipient screening followed by statistical modeling using design of experiments (DOE), was undertaken to identify stabilizing candidate formulations for a lyophilized live attenuated Flavivirus vaccine candidate. Various potential pharmaceutical compounds used in either marketed or investigative live attenuated viral vaccine formulations were first identified. The ability of additives from different categories of excipients, either alone or in combination, were then evaluated for their ability to stabilize virus against freeze-thaw, freeze-drying, and accelerated storage (25°C) stresses by measuring infectious virus titer. An exploratory data analysis and predictive DOE modeling approach was subsequently undertaken to gain a better understanding of the interplay between the key excipients and stability of virus as well as to determine which combinations were interacting to improve virus stability. The lead excipient combinations were identified and tested for stabilizing effects using a tetravalent mixture of viruses in accelerated and real time (2-8°C) stability studies. This work demonstrates the utility of combining semi-empirical excipient screening and DOE experimental design strategies in the formulation development of lyophilized live attenuated viral vaccine candidates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

PubMed

Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

2015-06-01

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

A Combined NMR-Computational Study of the Interaction between Influenza Virus Hemagglutinin and Sialic Derivatives from Human and Avian Receptors on the Surface of Transfected Cells.

PubMed

Vasile, Francesca; Panigada, Maddalena; Siccardi, Antonio; Potenza, Donatella; Tiana, Guido

2018-04-24

The development of small-molecule inhibitors of influenza virus Hemagglutinin could be relevant to the opposition of the diffusion of new pandemic viruses. In this work, we made use of Nuclear Magnetic Resonance (NMR) spectroscopy to study the interaction between two derivatives of sialic acid, Neu5Ac-α-(2,6)-Gal-β-(1⁻4)-GlcNAc and Neu5Ac-α-(2,3)-Gal-β-(1⁻4)-GlcNAc, and hemagglutinin directly expressed on the surface of recombinant human cells. We analyzed the interaction of these trisaccharides with 293T cells transfected with the H5 and H1 variants of hemagglutinin, which thus retain their native trimeric conformation in such a realistic environment. By exploiting the magnetization transfer between the protein and the ligand, we obtained evidence of the binding event, and identified the epitope. We analyzed the conformational features of the glycans with an approach combining NMR spectroscopy and data-driven molecular dynamics simulations, thus obtaining useful information for an efficient drug design.

Phylogeny, integration and expression of sigma virus-like genes in Drosophila.

PubMed

Ballinger, Matthew J; Bruenn, Jeremy A; Taylor, Derek J

2012-10-01

The recent and surprising discovery of widespread NIRVs (non-retroviral integrated RNA viruses) has highlighted the importance of genomic interactions between non-retroviral RNA viruses and their eukaryotic hosts. Among the viruses with integrated representatives are the rhabdoviruses, a family of negative sense single-stranded RNA viruses. We identify sigma virus-like NIRVs of Drosophila spp. that represent unique cases where NIRVs are closely related to exogenous RNA viruses in a model host organism. We have used a combination of bioinformatics and laboratory methods to explore the evolution and expression of sigma virus-like NIRVs in Drosophila. Recent integrations in Drosophila provide a promising experimental system to study functionality of NIRVs. Moreover, the genomic architecture of recent NIRVs provides an unusual evolutionary window on the integration mechanism. For example, we found that a sigma virus-like polymerase associated protein (P) gene appears to have been integrated by template switching of the blastopia-like LTR retrotransposon. The sigma virus P-like NIRV is present in multiple retroelement fused open reading frames on the X and 3R chromosomes of Drosophila yakuba - the X-linked copy is transcribed to produce an RNA product in adult flies. We present the first account of sigma virus-like NIRVs and the first example of NIRV expression in a model animal system, and therefore provide a platform for further study of the possible functions of NIRVs in animal hosts. Copyright © 2012 Elsevier Inc. All rights reserved.

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

Safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies.

PubMed

Heald, Alison E; Iversen, Patrick L; Saoud, Jay B; Sazani, Peter; Charleston, Jay S; Axtelle, Tim; Wong, Michael; Smith, William B; Vutikullird, Apinya; Kaye, Edward

2014-11-01

Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Viral Inhibition of the Transporter Associated with Antigen Processing (TAP): A Striking Example of Functional Convergent Evolution

PubMed Central

Verweij, Marieke C.; Horst, Daniëlle; Griffin, Bryan D.; Luteijn, Rutger D.; Davison, Andrew J.; Ressing, Maaike E.; Wiertz, Emmanuel J. H. J.

2015-01-01

Herpesviruses are large DNA viruses that are highly abundant within their host populations. Even in the presence of a healthy immune system, these viruses manage to cause lifelong infections. This persistence is partially mediated by the virus entering latency, a phase of infection characterized by limited viral protein expression. Moreover, herpesviruses have devoted a significant part of their coding capacity to immune evasion strategies. It is believed that the close coexistence of herpesviruses and their hosts has resulted in the evolution of viral proteins that specifically attack multiple arms of the host immune system. Cytotoxic T lymphocytes (CTLs) play an important role in antiviral immunity. CTLs recognize their target through viral peptides presented in the context of MHC molecules at the cell surface. Every herpesvirus studied to date encodes multiple immune evasion molecules that effectively interfere with specific steps of the MHC class I antigen presentation pathway. The transporter associated with antigen processing (TAP) plays a key role in the loading of viral peptides onto MHC class I molecules. This is reflected by the numerous ways herpesviruses have developed to block TAP function. In this review, we describe the characteristics and mechanisms of action of all known virus-encoded TAP inhibitors. Orthologs of these proteins encoded by related viruses are identified, and the conservation of TAP inhibition is discussed. A phylogenetic analysis of members of the family Herpesviridae is included to study the origin of these molecules. In addition, we discuss the characteristics of the first TAP inhibitor identified outside the herpesvirus family, namely, in cowpox virus. The strategies of TAP inhibition employed by viruses are very distinct and are likely to have been acquired independently during evolution. These findings and the recent discovery of a non-herpesvirus TAP inhibitor represent a striking example of functional convergent evolution

The Incidence and Genetic Diversity of Apple Mosaic Virus (ApMV) and Prune Dwarf Virus (PDV) in Prunus Species in Australia

PubMed Central

Constable, Fiona E.; Nancarrow, Narelle; Rodoni, Brendan

2018-01-01

Apple mosaic virus (ApMV) and prune dwarf virus (PDV) are amongst the most common viruses infecting Prunus species worldwide but their incidence and genetic diversity in Australia is not known. In a survey of 127 Prunus tree samples collected from five states in Australia, ApMV and PDV occurred in 4 (3%) and 13 (10%) of the trees respectively. High-throughput sequencing (HTS) of amplicons from partial conserved regions of RNA1, RNA2, and RNA3, encoding the methyltransferase (MT), RNA-dependent RNA polymerase (RdRp), and the coat protein (CP) genes respectively, of ApMV and PDV was used to determine the genetic diversity of the Australian isolates of each virus. Phylogenetic comparison of Australian ApMV and PDV amplicon HTS variants and full length genomes of both viruses with isolates occurring in other countries identified genetic strains of each virus occurring in Australia. A single Australian Prunus infecting ApMV genetic strain was identified as all ApMV isolates sequence variants formed a single phylogenetic group in each of RNA1, RNA2, and RNA3. Two Australian PDV genetic strains were identified based on the combination of observed phylogenetic groups in each of RNA1, RNA2, and RNA3 and one Prunus tree had both strains. The accuracy of amplicon sequence variants phylogenetic analysis based on segments of each virus RNA were confirmed by phylogenetic analysis of full length genome sequences of Australian ApMV and PDV isolates and all published ApMV and PDV genomes from other countries. PMID:29562672

Foodborne viruses

USDA-ARS?s Scientific Manuscript database

Testing for human pathogenic viruses in foods represents a formidable task requiring the extraction, concentration, and assay of a host of viruses from a wide range of food matrices. The enteric viruses, particularly genogroup I and II (GI and GII) noroviruses and hepatitis A virus, are the princip...

Anti-rotavirus effects by combination therapy of stevioside and Sophora flavescens extract.

PubMed

Alfajaro, Mia Madel; Rho, Mun-Chual; Kim, Hyun-Jeong; Park, Jun-Gyu; Kim, Deok-Song; Hosmillo, Myra; Son, Kyu-Yeol; Lee, Ju-Hwan; Park, Sang-Ik; Kang, Mun-Il; Ryu, Young Bae; Park, Ki Hun; Oh, Hyun-Mee; Lee, Seung Woong; Park, Su-Jin; Lee, Woo Song; Cho, Kyoung-Oh

2014-06-01

Anti-rotaviral activities of Sophora flavescens extract (SFE) and stevioside (SV) from Stevia rebaudiana Bertoni either singly or in various combinations were examined in vitro and in vivo using a porcine rotavirus G5[P7] strain. Combination of SFE and SV inhibited in vitro virus replication more efficiently than each single treatment. In the piglet model, SV had no effect on rotavirus enteritis, whereas SFE improved but did not completely cure rotaviral enteritis. Interestingly, combination therapy of SFE and SV alleviated diarrhea, and markedly improved small intestinal lesion score and fecal virus shedding. Acute toxicity tests including the piglet lethal dose 50, and body weight, organ weight and pathological changes for the combination therapy did not show any adverse effect on the piglets. These preliminary data suggest that the combination therapy of SV and SFE is a potential curative medication for rotaviral diarrhea in pigs. Determination of the efficacy of this combination therapy in other species including humans needs to be addressed in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus.

PubMed

Yang, Yang; Qin, Xiaodong; Zhang, Xiangle; Zhao, Zhixun; Zhang, Wei; Zhu, Xueliang; Cong, Guozheng; Li, Yanmin; Zhang, Zhidong

2017-07-17

Goatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases. Recombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively. The sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 10 2 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood. This study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

Discrete virus infection model of hepatitis B virus.

PubMed

Zhang, Pengfei; Min, Lequan; Pian, Jianwei

2015-01-01

In 1996 Nowak and his colleagues proposed a differential equation virus infection model, which has been widely applied in the study for the dynamics of hepatitis B virus (HBV) infection. Biological dynamics may be described more practically by discrete events rather than continuous ones. Using discrete systems to describe biological dynamics should be reasonable. Based on one revised Nowak et al's virus infection model, this study introduces a discrete virus infection model (DVIM). Two equilibriums of this model, E1 and E2, represents infection free and infection persistent, respectively. Similar to the case of the basic virus infection model, this study deduces a basic virus reproductive number R0 independing on the number of total cells of an infected target organ. A proposed theorem proves that if the basic virus reproductive number R0<1 then the virus free equilibrium E1 is locally stable. The DVIM is more reasonable than an abstract discrete susceptible-infected-recovered model (SIRS) whose basic virus reproductive number R0 is relevant to the number of total cells of the infected target organ. As an application, this study models the clinic HBV DNA data of a patient who was accepted via anti-HBV infection therapy with drug lamivudine. The results show that the numerical simulation is good in agreement with the clinic data.

Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection.

PubMed

Nelson, Jacob; Roe, Kelsey; Orillo, Beverly; Shi, Pei-Yong; Verma, Saguna

2015-10-01

West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. Previous studies demonstrated NITD008 as a potent antiviral for dengue virus, however this drug was associated with preclinical toxicity. The ability of NITD008 to block WNV replication is only shown in Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25mg/kg at days 1-6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3-6 and days 5-9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis. Copyright © 2015 Elsevier B.V. All rights reserved.

A quantitative risk-analysis for introduction of Bovine Viral Diarrhoea Virus in the Netherlands through cattle imports.

PubMed

Santman-Berends, I M G A; Mars, M H; Van Duijn, L; Van den Broek, K W H; Van Schaik, G

2017-10-01

Many countries have implemented control programmes aiming to eradicate Bovine Viral Diarrhoea Virus (BVDV). After obtaining the free status, a risk of re-introduction of the virus through import may remain. Therefore the risk of introduction of BVDV through cattle imports in the Netherlands was quantified and the effectiveness of subsequent intervention measures was assessed. Data, literature and expert opinion were used to estimate values for input parameters to feed a stochastic simulation model. The probability that BVDV was imported was differentiated into persistently infected (PI) cattle, trojan cows that transmitted the virus vertically resulting in a PI foetus (TR) and transient infected cattle (TI). The import risk was stratified to beef, dairy, small scale, suckler, trade, veal and young stock herds. The intervention scenarios that were evaluated consisted of virus testing, a combination of virus testing and antibody testing in pregnant cows, abolishment of imports from high risk countries (i.e. countries with a BVDV prevalence >15%) and a combination of import restrictions and testing prior to import. Each year, 334 (5th and 95th percentile: 65-902) Dutch cattle herds were estimated to be infected with BVDV through import. Veal herds account for most infections associated with import (87%), whereas in the other herd types, only 9 beef, 6 dairy, 2 small scale, 16 suckler, 10 trade and 2 young stock herds are infected through imports per year. Import of PI cattle is the most important risk for introduction in veal herds, while import of TR cows is the main source of BVDV introduction in dairy, small scale and suckler herds. With the intervention scenarios, the number of BVDV infected herds in the Netherlands could be reduced to 81 and 58 herds per year when respectively virus testing or a combination of virus and antibody testing was applied or to 108 herds when import from high risk countries was abolished. With the scenario in which both import from high

Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine.

PubMed

Ferrier-Rembert, Audrey; Drillien, Robert; Tournier, Jean-Nicolas; Garin, Daniel; Crance, Jean-Marc

2008-03-25

This study assessed three non-replicating smallpox vaccine candidates (modified vaccinia Ankara (MVA), NYVAC and HR) for their immunogenicity and ability to protect mice against an intranasal cowpox virus challenge and compared them with the traditional replicating vaccine. A single immunisation with the non-replicating vaccines induced a complete protection from death at short-term, but was not fully protective when mice were challenged 150 days post-vaccination with protection correlated with the specific neutralizing antibodies and CD4(+) T-cells responses. Prime-boost vaccination enabled effective long-term protection from death for mice vaccinated with MVA, but protection from disease and CD4(+) T-cell level were lower than the ones induced by the traditional vaccine over the long-term period. Further investigations are necessary with MVA to determine the optimal conditions of immunisation to induce at long-term immunogenicity and protection observed with the 1st generation smallpox vaccine.

Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

PubMed Central

Kolodkin-Gal, Dror; Eslamizar, Leila; Owuor, Joshua O.; Mazzola, Emanuele; Gonzalez, Ana M.; Korioth-Schmitz, Birgit; Gelman, Rebecca S.; Montefiori, David C.; Haynes, Barton F.; Schmitz, Joern E.

2015-01-01

ABSTRACT To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies

Prokaryotic expression of CP gene of Fritillary virus Y infecting Thunberg fritillary and antiserum preparation.

PubMed

Wei, Chuan-Bao; Wei, Yang-Yang; Yang, Yu; Liu, Shi-Liang; Hu, Hao-Yu; He, Yue

2011-10-01

To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses. Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis. It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate). The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.

The natural history of West Nile virus infection presenting with West Nile virus meningoencephalitis in a man with a prolonged illness: a case report.

PubMed

Mainali, Shraddha; Afshani, Mansoor; Wood, James B; Levin, Michael C

2011-05-25

Estimates indicate that West Nile virus infects approximately one and a half million people in the United States of America. Up to 1% may develop West Nile virus neuroinvasive disease, in which infected patients develop any combination of meningitis, encephalitis, or acute paralysis. A 56-year-old African-American man presented to our hospital with headache, restlessness, fever, myalgias, decreased appetite, and progressive confusion. A cerebrospinal fluid examination showed mild leukocytosis and an elevated protein level. Testing for routine infections was negative. Brain T2-weighted magnetic resonance imaging scans showed marked enlargement of caudate nuclei and increased intensity within the basal ganglia and thalami. A West Nile virus titer was positive, and serial brain magnetic resonance imaging scans showed resolving abnormalities that paralleled his neurological examination. This report is unusual as it portrays the natural history and long-term consequences of West Nile virus meningoencephalitis diagnosed on the basis of serial brain images.

Background review for diagnostic test development for Zika virus infection

PubMed Central

Charrel, Rémi N; Leparc-Goffart, Isabelle; Pas, Suzan; de Lamballerie, Xavier; Koopmans, Marion; Reusken, Chantal

2016-01-01

Abstract Objective To review the state of knowledge about diagnostic testing for Zika virus infection and identify areas of research needed to address the current gaps in knowledge. Methods We made a non-systematic review of the published literature about Zika virus and supplemented this with information from commercial diagnostic test kits and personal communications with researchers in European preparedness networks. The review covered current knowledge about the geographical spread, pathogen characteristics, life cycle and infection kinetics of the virus. The available molecular and serological tests and biosafety issues are described and discussed in the context of the current outbreak strain. Findings We identified the following areas of research to address current knowledge gaps: (i) an urgent assessment of the laboratory capacity and capability of countries to detect Zika virus; (ii) rapid and extensive field validation of the available molecular and serological tests in areas with and without Zika virus transmission, with a focus on pregnant women; (iii) monitoring the genomic diversity of circulating Zika virus strains; (iv) prospective studies into the virus infection kinetics, focusing on diagnostic sampling (specimen types, combinations and timings); and (v) developing external quality assessments for molecular and serological testing, including differential diagnosis for similar viruses and symptom clusters. The availability of reagents for diagnostic development (virus strains and antigens, quantified viral ribonucleic acid) needs to be facilitated. Conclusion An international laboratory response is needed, including preparation of protocols for prospective studies to address the most pressing information needs. PMID:27516635

Aedes-Borne Virus-Mosquito Interactions: Mass Spectrometry Strategies and Findings.

PubMed

Pando-Robles, Victoria; Batista, Cesar V

2017-06-01

Aedes-borne viruses are responsible for high-impact neglected tropical diseases and unpredictable outbreaks such as the ongoing Zika epidemics. Aedes mosquitoes spread different arboviruses such as Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus, among others, and are responsible for the continuous emergence and reemergence of these pathogens. These viruses have complex transmission cycles that include two hosts, namely the Aedes mosquito as a vector and susceptible vertebrate hosts. Human infection with arboviruses causes diseases that range from subclinical or mild to febrile diseases, encephalitis, and hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The infection of the Aedes mosquito by viruses involves a molecular crosstalk between cell and viral proteins. An understanding of how mosquito vectors and viruses interact is of fundamental interest, and it also offers novel perspectives for disease control. In recent years, mass spectrometry (MS)-based strategies in combination with bioinformatics have been successfully applied to identify and quantify global changes in cellular proteins, lipids, peptides, and metabolites in response to viral infection. Although the information about proteomics in the Aedes mosquito is limited, the information that has been reported can set up the basis for future studies. This review reflects how MS-based approaches have extended our understanding of Aedes mosquito biology and the development of DENV and CHIKV infection in the vector. Finally, this review discusses future challenges in the field.

Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.

PubMed

Courtin, Noémie; Fotso, Aurélien Fotso; Fautrad, Pierre; Mas, Floriane; Alessi, Marie-Christine; Riteau, Béatrice

2017-07-01

Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza. Copyright © 2017 Elsevier B.V. All rights reserved.

Virus Characterization by FFF-MALS Assay

NASA Astrophysics Data System (ADS)

Razinkov, Vladimer

2009-03-01

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

PubMed

Aghaie, A; Pourfatollah, A A; Bathaie, S Z; Moazzeni, S M; Khorsand Mohammad Pour, H; Sharifi, Z

2008-01-01

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

PubMed

Beljanski, Vladimir; Chiang, Cindy; Kirchenbaum, Greg A; Olagnier, David; Bloom, Chalise E; Wong, Terianne; Haddad, Elias K; Trautmann, Lydie; Ross, Ted M; Hiscott, John

2015-10-01

The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens

Epstein-Barr virus infection induces lupus autoimmunity.

PubMed

Harley, John B; James, Judith A

2006-01-01

Systemic lupus erythematosus (SLE or lupus) is a systemic autoimmune disease characterized by a constellation of varied clinical presentations, although the nearly universal presence of autoantibodies is a salient unifying feature. Ongoing research efforts focus on understanding the complex combination of genetic and environmental factors that lead to SLE in select individuals. Our previous work has demonstrated that years before diagnosis abnormal autoantibody responses are present in the sera of patients who will subsequently develop lupus and, further, that the initial targets of two of these key responses (anti-Sm B' and anti-60 kD Ro alone) have been identified for some patients. Indeed, our results suggest that the first lupus-specific autoantibodies arise from particular antibodies directed against Epstein-Barr virus Nuclear Antigen-1 (EBNA-1) and that infection with Epstein-Barr virus (EBV) is an environmental risk factor for lupus. The predicted sequence of events is normal immunity, followed by Epstein- Barr virus infection, the generation of anti-EBNA-1 antibodies, then followed by those particular anti-EBNA-1 antibodies that also bind lupus-specific autoantigens (Sm or Ro), followed by the development of more complex autoimmune responses, and, finally, culminating in clinical disease. Studies from others and those underway suggest that lupus patients have unusual immune responses to Epstein-Barr virus. In aggregate, these results are consistent with an immune response against Epstein-Barr virus being important in at least some patients for the initiation of lupus autoimmunity.

Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy.

PubMed

Hoffmann, B; Freuling, C M; Wakeley, P R; Rasmussen, T B; Leech, S; Fooks, A R; Beer, M; Müller, T

2010-11-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

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

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

PubMed Central

Borchardt, Mark A.; Bertz, Phil D.; Spencer, Susan K.; Battigelli, David A.

2003-01-01

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses. PMID:12571044

Detection of West Nile Virus and other common equine viruses in three locations from the Leeward Islands, West Indies.

PubMed

Bolfa, Pompei; Jeon, Isaac; Loftis, Amanda; Leslie, Teresa; Marchi, Silvia; Sithole, Fortune; Beck, Cecile; Lecollinet, Sylvie; Zientara, Stephan; Hans, Aymeric; Issel, Charles J

2017-10-01

Equines in the West Indies are used for recreational purposes, tourism industry, racing and agriculture or can be found in feral populations. Little is known in the Caribbean basin about the prevalence of some major equine infectious diseases, some with zoonotic potential, listed as reportable by the OIE. Our objective was to study the prevalence of antibodies for West Nile Virus (WNV), Equine Herpes Virus-1 and 4 (EHV-1 and EHV-4), Equine Influenza (EI), Equine Viral Arteritis (EVA) and Equine Infectious Anemia Virus (EIAV) using a retrospective serological convenience study. We used 180 equine serum samples, 140 from horses and 40 from donkeys in St. Kitts, Nevis, and Sint Eustatius, collected between 2006 and 2015 that were tested with ELISA kits and virus neutralization (for WNV and EVA). Combining ELISA with virus neutralization testing, 25 (13.8%) equine sera were WNV positive (a mixture of indigenous and imported equines) and 3 sera (1.6%) showed doubtful results. For EHV-1, 41 equines (23.7%), mean age 6.7 years, were seropositive. For EHV-4, 138 equines were found seropositive (82.8%), mean age 6.3 years. For EI, 49 equines (27.2%), mean age 7.5 years, were seropositive on ELISA, some previously vaccinated horses. No antibodies against EAV were found on virus neutralization testing, although one animal (0.6%), was EAV positive on ELISA. All samples were EIAV negative. The seroprevalence for EHV-1 and EHV-4 is similar to other parts of the world. For the first time in the study location serologic evidence of antibodies against WNV and EI is reported. This was found in both indigenous and imported animals, highlighting the need for developing proper surveillance plans based on complementary methods of virus detection. Further studies will be needed to define the prevalence, rates of transmission, characterize local virus strains, and study their impact on these populations. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of Favipiravir Alone and in Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa Fever.

PubMed

Oestereich, Lisa; Rieger, Toni; Lüdtke, Anja; Ruibal, Paula; Wurr, Stephanie; Pallasch, Elisa; Bockholt, Sabrina; Krasemann, Susanne; Muñoz-Fontela, César; Günther, Stephan

2016-03-15

We studied the therapeutic potential of favipiravir (T-705) for Lassa fever, both alone and in combination with ribavirin. Favipiravir suppressed Lassa virus replication in cell culture by 5 log10 units. In a novel lethal mouse model, it lowered the viremia level and the virus load in organs and normalized levels of cell-damage markers. Treatment with 300 mg/kg per day, commenced 4 days after infection, when the viremia level had reached 4 log10 virus particles/mL, rescued 100% of Lassa virus-infected mice. We found a synergistic interaction between favipiravir and ribavirin in vitro and an increased survival rate and extended survival time when combining suboptimal doses in vivo. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

Virus characterization and discovery in formalin-fixed paraffin-embedded tissues.

PubMed

Bodewes, Rogier; van Run, Peter R W A; Schürch, Anita C; Koopmans, Marion P G; Osterhaus, Albert D M E; Baumgärtner, Wolfgang; Kuiken, Thijs; Smits, Saskia L

2015-03-01

Detection and characterization of novel viruses is hampered frequently by the lack of properly stored materials. Especially for the retrospective identification of viruses responsible for past disease outbreaks, often only formalin-fixed paraffin-embedded (FFPE) tissue samples are available. Although FFPE tissues can be used to detect known viral sequences, the application of FFPE tissues for detection of novel viruses is currently unclear. In the present study it was shown that sequence-independent amplification in combination with next-generation sequencing can be used to detect sequences of known and unknown viruses, although with relatively low sensitivity. These findings indicate that this technique could be useful for detecting novel viral sequences in FFPE tissues collected from humans and animals with disease of unknown origin, when other samples are not available. In addition, application of this method to FFPE tissues allows to correlate with the presence of histopathological changes in the corresponding tissue sections. Copyright © 2015 Elsevier B.V. All rights reserved.

Activation thermodynamics of virus adsorption to solids.

PubMed Central

Preston, D R; Farrah, S R

1988-01-01

The kinetics of bacteriophage MS2, T2, and f2 adsorption to powdered nitrocellulose and disrupted Seitz S1 filters at pH 7 were determined as a function of temperature. Data from these studies were combined with data produced in a previous study on MS2 adsorption to clay by Stagg et al. (Appl. Environ. Microbiol. 33:385-391, 1977). These workers studied the adsorption of MS2 to bentonite clay as a function of temperature. Data from both this previous study and the current one were used to calculate the thermodynamic parameters of virus adsorption. The results show that adsorption of bacteriophages to the solids tested is a physical process (energy of activation, less than 40 kcal [168 J]/mol) rather than a chemical process (energy of activation, greater than 40 kcal/mol). The free energy of activation showed a high negative correlation (r = -0.904, r2 = 0.817) with the percentage of virus adsorption to the solids tested. The energy of activation was highly negatively correlated with the percentage of virus adsorption to nitrocellulose and clay (r = -0.913, r2 = 0.834) but poorly correlated with the percentage of virus adsorption to disrupted Seitz S1 filters (r = -0.348, r2 = 0.121). In general, under conditions in which the percentage of virus adsorption was low, the energy of activation, the free energy of activation, and the entropy of activation were high. Increasing the percentage of virus adsorbed by changing the adsorbing conditions or changing the adsorbing solid decreased the energy of activation, the free energy of activation, and the entropy of activation. PMID:3214152

Inactivation of the Hutchinson strain of non-A, non-B hepatitis virus by combined use of beta-propiolactone and ultraviolet irradiation

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

Prince, A.M.; Stephan, W.; Dichtelmueller, H.B.

1985-06-01

A beta-propiolactone/ultraviolet irradiation procedure (beta PL/UV) has been evaluated for its ability to inactivate 30,000 chimpanzee infectious doses of the Hutchinson strain of non-A, non-B (NANB) virus. The chimpanzees were inoculated with plasma to which this dose of the titrated virus had been added prior to application of the beta PL/UV process in accordance with a procedure used for licensed blood derivatives in Germany. Neither animal developed hepatitis. When subsequently challenged with the same contaminated plasma, which had not been sterilized, both animals promptly developed typical NANB hepatitis. This study extends the high (approximately 10(7)-fold) process efficiency of the betamore » PL/UV procedure previously reported for hepatitis B virus to a blood-borne NANB virus.« less

Immunovirotherapy with measles virus strains in combination with anti-PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment.

PubMed

Hardcastle, Jayson; Mills, Lisa; Malo, Courtney S; Jin, Fang; Kurokawa, Cheyne; Geekiyanage, Hirosha; Schroeder, Mark; Sarkaria, Jann; Johnson, Aaron J; Galanis, Evanthia

2017-04-01

Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment. © The Author(s) 2016. Published by Oxford University Press

Current concerns and perspectives on Zika virus co-infection with arboviruses and HIV.

PubMed

Rothan, Hussin A; Bidokhti, Mehdi R M; Byrareddy, Siddappa N

2018-05-01

Dissemination of vector-borne viruses, such as Zika virus (ZIKV), in tropical and sub-tropical regions has a complicated impact on the immunopathogenesis of other endemic viruses such as dengue virus (DENV), chikungunya virus (CHIKV) and human immunodeficiency virus (HIV). The consequences of the possible co-infections with these viruses have specifically shown significant impact on the treatment and vaccination strategies. ZIKV is a mosquito-borne flavivirus from African and Asian lineages that causes neurological complications in infected humans. Many of DENV and CHIKV endemic regions have been experiencing outbreaks of ZIKV infection. Intriguingly, the mosquitoes, Aedes Aegypti and Aedes Albopictus, can simultaneously transmit all the combinations of ZIKV, DENV, and CHIKV to the humans. The co-circulation of these viruses leads to a complicated immune response due to the pre-existence or co-existence of ZIKV infection with DENV and CHIKV infections. The non-vector transmission of ZIKV, especially, via sexual intercourse and placenta represents an additional burden that may hander the treatment strategies of other sexually transmitted diseases such as HIV. Collectively, ZIKV co-circulation and co-infection with other viruses have inevitable impact on the host immune response, diagnosis techniques, and vaccine development strategies for the control of these co-infections. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rapid diagnostic methods for influenza virus in clinical specimens - A comparative study

NASA Technical Reports Server (NTRS)

Evans, A. S.; Olson, B.

1982-01-01

A comparison of five rapid viral diagnostic techniques for identifying influenza virus in nasopharyngeal aspirates has been made on patients with influenza-like illnesses. Initial results with immune electron microscopy were positive in only one of 11 specimens from which virus was isolated and further work abandoned. Four other rapid tests were carried out on 39 specimens from which influenza virus had been isolated in tissue culture in 28. Of these 28 specimens yielding virus, 24 (85.7 percent) were positive by an indirect fluorescent antibody test (IFAT) on nasopharyngeal cells, 18 (64.3 percent) by enzyme-linked immunosorbent assay (ELISA), 19 (67.8 percent) by enzyme-linked fluorescent assay (ELFA), and 26 (92.8 percent) by a rapid tissue culture amplification method (TCA) in a continuous Rhesus monkey kidney line (LLC-MK2) with identification of virus by fluorescent antibody. In terms of sensitivity, simplicity, and rapidity, a combination of the IFAT and TCA methods seems to be very useful.

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.

PubMed

Marissen, Wilfred E; Kramer, R Arjen; Rice, Amy; Weldon, William C; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W; Meloen, Rob H; Clijsters-van der Horst, Marieke; Visser, Therese J; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B H

2005-04-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

PubMed Central

Wang, Wenling; Li, Renqing; Deng, Yao; Lu, Ning; Chen, Hong; Meng, Xin; Wang, Wen; Wang, Xiuping; Yan, Kexia; Qi, Xiangrong; Zhang, Xiangmin; Xin, Wei; Lu, Zhenhua; Li, Xueren; Bian, Tao; Gao, Yingying; Tan, Wenjie

2015-01-01

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics. PMID:25834017

Threshold virus dynamics with impulsive antiretroviral drug effects

PubMed Central

Lou, Jie; Lou, Yijun; Wu, Jianhong

2013-01-01

The purposes of this paper are twofold: to develop a rigorous approach to analyze the threshold behaviors of nonlinear virus dynamics models with impulsive drug effects and to examine the feasibility of virus clearance following the Manuals of National AIDS Free Antiviral Treatment in China. An impulsive system of differential equations is developed to describe the within-host virus dynamics of both wild-type and drug-resistant strains when a combination of antiretroviral drugs is used to induce instantaneous drug effects at a sequence of dosing times equally spaced while drug concentrations decay exponentially after the dosing time. Threshold parameters are derived using the basic reproduction number of periodic epidemic models, and are used to depict virus clearance/persistence scenarios using the theory of asymptotic periodic systems and the persistence theory of discrete dynamical systems. Numerical simulations using model systems parametrized in terms of the antiretroviral therapy recommended in the aforementioned Manuals illustrate the theoretical threshold virus dynamics, and examine conditions under which the impulsive antiretroviral therapy leads to treatment success. In particular, our results show that only the drug-resistant strain can dominate (the first-line treatment program guided by the Manuals) or both strains may be rapidly eliminated (the second-line treatment program), thus the work indicates the importance of implementing the second-line treatment program as soon as possible. PMID:21987085

Identification, sequence analysis, and infectivity of H9N2 avian influenza viruses isolated from geese.

PubMed

Zhu, Rui; Yang, Xueqin; Zhang, Jianjun; Xu, Danwen; Fan, Jiawen; Shi, Huoying; Wang, Shifeng; Liu, Xiufan

2018-05-31

The subtype H9N2 avian influenza virus greatly threatens the Chinese poultry industry, even with annual vaccination. Waterfowl can be asymptomatically infected with the H9N2 virus. In this study, three H9N2 virus strains, designated A/Goose/Jiangsu/YZ527/2011 (H9N2, Gs/JS/YZ527/11), A/Goose/Jiangsu/SQ119/2012 (H9N2, Gs/JS/SQ119/12), and A/Goose/Jiangsu/JD564/2012 (H9N2, Gs/JS/JD564/12), were isolated from domestic geese. Molecular characterization of the three isolates showed that the Gs/JS/YZ527/11 virus is a double-reassortant virus, combining genes of A/Quail/Hong Kong/G1/97 (H9N2, G1/97)-like and A/Chicken/Shanghai/F/98 (H9N2, F/98)-like; the Gs/JS/SQ119/12 virus is a triple-reassortant virus combining genes of G1/97-like, F/98-like, and A/Duck/Shantou/163/2004 (H9N2, ST/163/04)-like. The sequences of Gs/JS/JD564/12 share high homology with those of the F/98 virus, except for the neuraminidase gene, whereas the internal genes of Gs/JS/YZ527/11 and Gs/JS/SQ119/12 are closely related to those of the H7N9 viruses. An infectivity analysis of the three isolates showed that Gs/JS/SQ119/12 and Gs/JS/YZ527/11 replicated well, with seroconversion, in geese and chickens, the Gs/JS/JD564/12 did not infect well in geese or chickens, and the F/98 virus only infected chickens, with seroconversion. Emergence of these new reassortant H9N2 avian influenza viruses indicates that these viruses can infect both chicken and goose and can produce different types of lesions in each species.

Identification, sequence analysis, and infectivity of H9N2 avian influenza viruses isolated from geese

PubMed Central

Zhu, Rui; Yang, Xueqin; Zhang, Jianjun; Xu, Danwen; Fan, Jiawen; Wang, Shifeng; Liu, Xiufan

2018-01-01

The subtype H9N2 avian influenza virus greatly threatens the Chinese poultry industry, even with annual vaccination. Waterfowl can be asymptomatically infected with the H9N2 virus. In this study, three H9N2 virus strains, designated A/Goose/Jiangsu/YZ527/2011 (H9N2, Gs/JS/YZ527/11), A/Goose/Jiangsu/SQ119/2012 (H9N2, Gs/JS/SQ119/12), and A/Goose/Jiangsu/JD564/2012 (H9N2, Gs/JS/JD564/12), were isolated from domestic geese. Molecular characterization of the three isolates showed that the Gs/JS/YZ527/11 virus is a double-reassortant virus, combining genes of A/Quail/Hong Kong/G1/97 (H9N2, G1/97)-like and A/Chicken/Shanghai/F/98 (H9N2, F/98)-like; the Gs/JS/SQ119/12 virus is a triple-reassortant virus combining genes of G1/97-like, F/98-like, and A/Duck/Shantou/163/2004 (H9N2, ST/163/04)-like. The sequences of Gs/JS/JD564/12 share high homology with those of the F/98 virus, except for the neuraminidase gene, whereas the internal genes of Gs/JS/YZ527/11 and Gs/JS/SQ119/12 are closely related to those of the H7N9 viruses. An infectivity analysis of the three isolates showed that Gs/JS/SQ119/12 and Gs/JS/YZ527/11 replicated well, with seroconversion, in geese and chickens, the Gs/JS/JD564/12 did not infect well in geese or chickens, and the F/98 virus only infected chickens, with seroconversion. Emergence of these new reassortant H9N2 avian influenza viruses indicates that these viruses can infect both chicken and goose and can produce different types of lesions in each species. PMID:29366299

Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

PubMed Central

Zheng, Fei-qun; Xu, Yin; Yang, Ren-jie; Wu, Bin; Tan, Xiao-hua; Qin, Yi-de; Zhang, Qun-wei

2009-01-01

Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting. Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo. Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma. PMID:19363518

Opsoclonus myoclonus ataxia associated with West Nile virus infection: A dramatic presentation with benign prognosis?

PubMed

Zaltzman, Roy; Klein, Colin; Gordon, Carlos R

2017-05-15

Opsoclonus myoclonus and ataxia is a combination of severe neurological signs associated with several pathologic agents and conditions. Only few cases of opsoclonus have been related to West Nile virus infection. We report on a 61-year-old woman and on a 55-year-old man who had history of recent fever, who were hospitalized because of acute severe truncal ataxia, opsoclonus and tremor with minimal myoclonic jerks. A through work-up revealed the presence of both IgM and IgG antibodies against West Nile virus both in the serum and Cerebrospinal Fluid and excluded other causes known to be associated with this combination of neurological signs. The first case was treated with corticosteroids, followed by significant improvement, and the second recovered spontaneously. The acute combination of opsoclonus, severe truncal ataxia and tremor with a history of recent fever requires, during the relevant season and in the relevant geographic area, a search for a recent infection with West Nile virus. Though initially suffering from a devastating sickness, our patients eventually recovered. Copyright © 2017 Elsevier B.V. All rights reserved.

ECHO virus

MedlinePlus

... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that can lead ...

Avian influenza virus with Hemagglutinin-Neuraminidase combination H8N8, isolated in Russia

USDA-ARS?s Scientific Manuscript database

This study reports the genome sequence of an avian influenza virus (AIV) subtype H8N8 isolated in Russia. The genome analysis shows that all genes belong to AIV Eurasian lineages. The PB2 gene was similar to a Mongolian low pathogenic (LP) AIV H7N1 and a Chinese high pathogenic (HP) AIV H5N2....

Epidemiology, surveillance and control of Nipah virus infections in Malaysia.

PubMed

Chua, K B

2010-12-01

The outbreak of Nipah virus, affecting pigs and pig-farm workers, was first noted in September 1998 in the north-western part of peninsular Malaysia. By March 1999, the outbreak had spread to other pig-farming areas of the country, inclusive of the neighbouring country, Singapore. A total of 283 human cases of viral encephalitis with 109 deaths were recorded in Malaysia from 29 September 1998 to December 1999. During the outbreak period, a number of surveillances under three broad groups; Surveillance in Human Health Sector, Surveillance in Animal Health Sector, and Surveillance for the Reservoir Hosts, were carried out to determine the prevalence, risk of virus infections and transmission in human and swine populations as well as the source and reservoir hosts of Nipah virus. Surveillance data showed that the virus spread rapidly among pigs within infected farms and transmission was attributed to direct contact with infective excretions and secretions. The spread of the virus among pig farms within and between states of peninsular Malaysia was due to movement of pigs. The transmission of the virus to humans was through close contact with infected pigs. Human to human transmission was considered a rare event though the Nipah virus could be isolated from saliva, urine, nasal and pharyngeal secretions of patients. Field investigations identified fruitbats of the Pteropid species as the natural reservoir hosts of the viruses. The outbreak was effectively brought under control following the discovery of the virus and institution of correct control measures through a combined effort of multi-ministerial and multidisciplinary teams working in close co-operation and collaboration with other international agencies.

Post-epizootic surveys of waterfowl for duck plague (duck virus enteritis)

USGS Publications Warehouse

Brand, C.J.; Docherty, D.E.

1988-01-01

Surviving birds from nine duck plague outbreaks in urban and confined waterfowl were sampled for duck plague (DP) virus and DP antibody during 1979-86. Duck plague virus was found in combined oral and cloacal swabs of birds from three outbreaks, and DP-neutralizing antibody was demonstrated in some birds from all nine outbreaks. Greater prevalence of DP antibody and higher titers were found in survivors from confined populations than from free-flying urban populations. Free-flying waterfowl from within 52 km of four DP outbreak sites were also sampled; virus was not found in any birds, but DP antibody was found in urban waterfowl in the vicinity of an outbreak in Potterville, Michigan. No evidence of exposure to or shedding of DP virus in migratory waterfowl was found in two regions where DP appears enzootic in urban and confined waterfowl (Eastern Shore of Maryland and the vicinity of Sacramento, California).

Development of Potential Small Molecule Therapeutics for Treatment of Ebola Virus.

PubMed

Schafer, Adam Michael; Cheng, Han; Lee, Charles; Du, Ruikun; Han, Julianna; Perez, Jasmine; Peet, Norton; Manicassamy, Balaji; Rong, Lijun

2017-10-10

Ebola virus has caused 26 outbreaks in 10 different countries since its identification in 1976, making it one of the deadliest emerging viral pathogens. The most recent outbreak in West Africa from 2014-16 was the deadliest yet and culminated in 11,310 deaths out of 28,616 confirmed cases. Currently there are no FDA-approved therapeutics or vaccines to treat Ebola virus infections. The slow development of effective vaccines combined with the severity of past outbreaks emphasizes the need to accelerate research into understanding the virus lifecycle and the development of therapeutics for post exposure treatment. Here we present a summary of the major findings on the Ebola virus replication cycle and the therapeutic approaches explored to treat this devastating disease. The major focus of this review is on small molecule inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Daclatasvir and asunaprevir combination therapy for patients with chronic hepatitis C virus genotype 1b infection in real world.

PubMed

Oh, Jae Young; Kim, Byung Seok; Lee, Chang Hyeong; Song, Jeong Eun; Lee, Heon Ju; Park, Jung Gil; Hwang, Jae Seok; Chung, Woo Jin; Jang, Byoung Kuk; Kweon, Young Oh; Tak, Won Young; Park, Soo Young; Jang, Se Young; Suh, Jeong Ill; Kwak, Sang Gyu

2018-05-25

Previous studies have reported a high rate of sustained virologic response (SVR) and a low rate of serious adverse events with the use of daclatasvir (DCV) and asunaprevir (ASV) combination therapy. We evaluated the efficacy and safety of DCV and ASV combination therapy for patients with chronic hepatitis C virus (HCV) genotype 1b infection in real world. We enrolled 278 patients (184 treatment-naïve patients) from five hospitals in Daegu and Gyeongsangbuk-do. We evaluated the rates of rapid virologic response (RVR), end-of-treatment response (ETR), and SVR at 12 weeks after completion of treatment (SVR12). Furthermore, we investigated the rate of adverse events and predictive factors of SVR12 failure. The mean age of patients was 59.5 ± 10.6 years, and 140 patients (50.2%) were men. Seventy-seven patients had cirrhosis. Baseline information regarding nonstructural protein 5A (NS5A) sequences was available in 268 patients. Six patients presented with pretreatment NS5A resistance-associated variants. The RVR and the ETR rates were 96.6% (258/267) and 95.2% (223/232), respectively. The overall SVR12 rate was 91.6% (197/215). Adverse events occurred in 17 patients (7.9%). Six patients discontinued treatment because of liver enzyme elevation (n = 4) and severe nausea (n = 2). Among these, four achieved SVR12. Other adverse events observed were fatigue, headache, diarrhea, dizziness, loss of appetite, skin rash, and dyspnea. Univariate analysis did not show significant predictive factors of SVR12 failure. DCV and ASV combination therapy showed high rates of RVR, ETR, and SVR12 in chronic HCV genotype 1b-infected patients in real world and was well tolerated without serious adverse events.

Identification of Multiple Novel Viruses, Including a Parvovirus and a Hepevirus, in Feces of Red Foxes

PubMed Central

van der Giessen, Joke; Haagmans, Bart L.; Osterhaus, Albert D. M. E.; Smits, Saskia L.

2013-01-01

Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified. PMID:23616657

MIV-150 and zinc acetate combination provides potent and broad activity against HIV-1.

PubMed

Mizenina, Olga; Hsu, Mayla; Jean-Pierre, Ninochka; Aravantinou, Meropi; Levendosky, Keith; Paglini, Gabriela; Zydowsky, Thomas M; Robbiani, Melissa; Fernández-Romero, José A

2017-12-01

We previously showed that the combination of the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 with zinc acetate (ZA) formulated in a carrageenan (CG; MZC) gel provided macaques significant protection against vaginal simian-human immunodeficiency virus-RT (SHIV-RT) challenge, better than either MIV-150/CG or ZA/CG. The MZC gel was shown to be safe in a phase 1 clinical trial. Herein, we used in vitro approaches to study the antiviral properties of ZA and the MIV-150/ZA combination, compared to other NNRTIs. Like other NNRTIs, MIV-150 has EC 50 values in the subnanomolar to nanomolar range against wild type and NNRTI or RT-resistant HIVs. While less potent than NNRTIs, ZA was shown to be active in primary cells against laboratory-adapted and primary HIV-1 isolates and HIV-1 isolates/clones with NNRTI and RT resistance mutations, with EC 50 values between 20 and 110 μM. The MIV-150/ZA combination had a potent and broad antiviral activity in primary cells. In vitro resistance selection studies revealed that previously described NNRTI-resistant mutations were selected by MIV-150. ZA-resistant virus retained susceptibility to MIV-150 (and other RTIs) and MIV-150-selected virus remained sensitive to ZA. Notably, resistant virus was not selected when cultured in the presence of both ZA and MIV-150. This underscores the potency and breadth of the MIV-150/ZA combination, supporting preclinical macaque studies and the advancement of MZC microbicides into clinical testing.

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

PubMed

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-06-26

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

PubMed

Milrot, Elad; Mutsafi, Yael; Fridmann-Sirkis, Yael; Shimoni, Eyal; Rechav, Katya; Gurnon, James R; Van Etten, James L; Minsky, Abraham

2016-01-01

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway. © 2015 John Wiley & Sons Ltd.

CHLORELLA VIRUSES

PubMed Central

Yamada, Takashi; Onimatsu, Hideki; Van Etten, James L.

2007-01-01

Chlorella viruses or chloroviruses are large, icosahedral, plaque‐forming, double‐stranded‐DNA—containing viruses that replicate in certain strains of the unicellular green alga Chlorella. DNA sequence analysis of the 330‐kbp genome of Paramecium bursaria chlorella virus 1 (PBCV‐1), the prototype of this virus family (Phycodnaviridae), predict ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼50% of these genes resemble proteins of known function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site‐specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus‐encoded K+ channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV‐1 has three types of introns; a self‐splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV‐1 as well as other related viruses. PMID:16877063

Ross River virus and Barmah Forest virus infection. Commonly asked questions.

PubMed

Hills, S

1996-12-01

Ross River virus infection and Barmah Forest virus infection are two commonly reported arboviral diseases in Australia. Ross River virus has long been recognised as a cause of epidemic polyarthritis and polyarticular disease. Clinical disease as a result of Barmah Forest virus infection has only been identified since 1988 and Australia is the only country in which this virus has been detected. Severe and prolonged symptoms can occur as a result of infection with either virus and may result in significant distress to the patient. This article reviews some of the issues that patients raise in relation to both Ross River virus and Barmah Forest virus disease including the source of infection, the duration of symptoms and measures to prevent infection.

Viruses Infecting Reptiles

PubMed Central

Marschang, Rachel E.

2011-01-01

A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch’s postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions. PMID:22163336

Viruses infecting reptiles.

PubMed

Marschang, Rachel E

2011-11-01

A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch's postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions.

Canine Distemper Virus (CDV) in Another Big Cat: Should CDV Be Renamed Carnivore Distemper Virus?

PubMed Central

Terio, Karen A.; Craft, Meggan E.

2013-01-01

ABSTRACT One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or “spillover” hosts but, rather, a normal part of the complex ecology of this infectious disease. PMID:24045642

Canine distemper virus (CDV) in another big cat: should CDV be renamed carnivore distemper virus?

PubMed

Terio, Karen A; Craft, Meggan E

2013-09-17

One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or "spillover" hosts but, rather, a normal part of the complex ecology of this infectious disease.

Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

PubMed

Zhou, Bin; Ma, Jingjiao; Liu, Qinfang; Bawa, Bhupinder; Wang, Wei; Shabman, Reed S; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B; Richt, Juergen A; Wentworth, David E; Ma, Wenjun

2014-10-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

PubMed Central

Bawa, Bhupinder; Wang, Wei; Shabman, Reed S.; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B.; Richt, Juergen A.; Wentworth, David E.; Ma, Wenjun

2014-01-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. PMID:25275541

Ebola virus encodes a miR-155 analog to regulate importin-α5 expression.

PubMed

Liu, Yuanwu; Sun, Jing; Zhang, Hongwen; Wang, Mingming; Gao, George Fu; Li, Xiangdong

2016-10-01

The 2014 outbreak of Ebola virus caused more than 10,000 human deaths. Current knowledge of suitable drugs, clinical diagnostic biomarkers and molecular mechanisms of Ebola virus infection is either absent or insufficient. By screening stem-loop structures from the viral genomes of four virulence species, we identified a novel, putative viral microRNA precursor that is specifically expressed by the Ebola virus. The sequence of the microRNA precursor was further confirmed by mining the existing RNA-Seq database. Two putative mature microRNAs were predicted and subsequently validated in human cell lines. Combined with this prediction of the microRNA target, we identified importin-α5, which is a key regulator of interferon signaling following Ebola virus infection, as one putative target. We speculate that this microRNA could facilitate the evasion of the host immune system by the virus. Moreover, this microRNA might be a potential clinical therapeutic target or a diagnostic biomarker for Ebola virus.

Imported pigs may have introduced the first classical swine influenza viruses into Mainland China.

PubMed

Zhu, Wenfei; Yang, Shuai; Guo, Yuanji; Yang, Lei; Bai, Tian; Yu, Zaijiang; Li, Xiaodan; Li, Ming; Guo, Junfeng; Wang, Dayan; Gao, Rongbao; Dong, Libo; Zou, Shumei; Li, Zi; Wang, Min; Shu, Yuelong

2013-07-01

The first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. We sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. All 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. Classical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong. Copyright © 2013 Elsevier B.V. All rights reserved.

Modelling the impact of a combined varicella and zoster vaccination programme on the epidemiology of varicella zoster virus in England.

PubMed

van Hoek, Albert Jan; Melegaro, Alessia; Zagheni, Emelio; Edmunds, W John; Gay, Nigel

2011-03-16

This study updates previous work on modelling the incidence of varicella and Herpes Zoster (HZ) following the introduction of childhood vaccination. The updated model includes new data on age-specific contact patterns, as well as data on the efficacy of zoster vaccination in the elderly and allows for HZ among vaccinees. The current study also looks at two-dose varicella childhood programmes, and assesses the combined impact of varicella vaccination in childhood and zoster vaccination of the elderly. The results suggest that a two-dose schedule is likely to reduce the incidence of varicella to very low levels, provided first dose coverage is around 90% and second dose coverage is in excess of 70%. Single dose varicella vaccination programmes are expected to result in large numbers of breakthrough cases. Childhood vaccination is expected to increase the incidence of zoster for more than 40 years after introduction of the programme, the magnitude of this increase being influenced primarily by the duration of boosting following exposure to the varicella zoster virus. Though this increase in zoster incidence can be partly offset by vaccination of the elderly, the effectiveness of this combined strategy is limited, as much of the increase occurs in those adults too young to be vaccinated. Childhood vaccination at intermediate levels of coverage (70% and 60% for first and second dose coverage respectively) is expected to lead to an increase in adult varicella. At high coverage (90% and 80% coverage) this is unlikely to be the case. These results will be used to inform a cost-effectiveness analysis of combined varicella and zoster vaccination programmes. Copyright © 2011 Elsevier Ltd. All rights reserved.

THE FEATURES OF THE COURSE OF CERTAIN VIRUS INFECTIONS AGAINST A BACKGROUND OF RADIATION AFFLICTION