Centrosomal Latency of Incoming Foamy Viruses in Resting Cells

PubMed Central

Giron, Marie Lou; Roingeard, Philippe; Clave, Emmanuel; Tobaly-Tapiero, Joelle; Bittoun, Patricia; Toubert, Antoine; de Thé, Hugues; Saïb, Ali

2007-01-01

Completion of early stages of retrovirus infection depends on the cell cycle. While gammaretroviruses require mitosis for proviral integration, lentiviruses are able to replicate in post-mitotic non-dividing cells. Resting cells such as naive resting T lymphocytes from peripheral blood cannot be productively infected by retroviruses, including lentiviruses, but the molecular basis of this restriction remains poorly understood. We demonstrate that in G0 resting cells (primary fibroblasts or peripheral T cells), incoming foamy retroviruses accumulate in close proximity to the centrosome, where they lie as structured and assembled capsids for several weeks. Under these settings, virus uncoating is impaired, but upon cell stimulation, Gag proteolysis and capsid disassembly occur, which allows viral infection to proceed. The data imply that foamy virus uncoating is the rate-limiting step for productive infection of primary G0 cells. Incoming foamy retroviruses can stably persist at the centrosome, awaiting cell stimulation to initiate capsid cleavage, nuclear import, and viral gene expression. PMID:17530924

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

PubMed Central

Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V.; Kern, Tobias; Gerresheim, Gesche K.; Serrao, Erik; Lesbats, Paul; Engelman, Alan N.; Cherepanov, Peter; Lindemann, Dirk

2016-01-01

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

Isolation and Characterization of an Equine Foamy Virus

PubMed Central

Tobaly-Tapiero, Joelle; Bittoun, Patricia; Neves, Manuel; Guillemin, Marie-Claude; Lecellier, Charles-Henri; Puvion-Dutilleul, Francine; Gicquel, Bernard; Zientara, Stephan; Giron, Marie-Louise; de Thé, Hugues; Saïb, Ali

2000-01-01

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway. PMID:10756018

Identification of novel, highly expressed retroviral microRNAs in cells infected by bovine foamy virus.

PubMed

Whisnant, Adam W; Kehl, Timo; Bao, Qiuying; Materniak, Magdalena; Kuzmak, Jacek; Löchelt, Martin; Cullen, Bryan R

2014-05-01

While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼ 122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼ 70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼ 122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo.

Identification of Novel, Highly Expressed Retroviral MicroRNAs in Cells Infected by Bovine Foamy Virus

PubMed Central

Whisnant, Adam W.; Kehl, Timo; Bao, Qiuying; Materniak, Magdalena; Kuzmak, Jacek; Löchelt, Martin

2014-01-01

ABSTRACT While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. IMPORTANCE Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo. PMID:24522910

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

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

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity.

PubMed

Duda, Anja; Stange, Annett; Lüftenegger, Daniel; Stanke, Nicole; Westphal, Dana; Pietschmann, Thomas; Eastman, Scott W; Linial, Maxine L; Rethwilm, Axel; Lindemann, Dirk

2004-12-01

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.

Tenth International Foamy Virus Conference 2014--achievements and perspectives.

PubMed

Materniak, Magdalena; Kubiś, Piotr; Rola-Łuszczak, Marzena; Khan, Arifa S; Buseyne, Florence; Lindemann, Dirk; Löchelt, Martin; Kuźmak, Jacek

2015-03-31

For the past two decades, scientists from around the world, working on different aspects of foamy virus (FV) research, have gathered in different research institutions almost every two years to present their recent results in formal talks, to discuss their ongoing studies informally, and to initiate fruitful collaborations. In this report we review the 2014 anniversary conference to share the meeting summary with the virology community and hope to arouse interest by other researchers to join this exciting field. The topics covered included epidemiology, virus molecular biology, and immunology of FV infection in non-human primates, cattle, and humans with zoonotic FV infections, as well as recent findings on endogenous FVs. Several topics focused on virus replication and interactions between viral and cellular proteins. Use of FV in biomedical research was highlighted with presentations on using FV vectors for gene therapy and FV proteins as scaffold for vaccine antigen presentation. On behalf of the FV community, this report also includes a short tribute to commemorate Prof. Axel Rethwilm, one of the leading experts in the field of retrovirology and foamy viruses, who passed away 29 July 2014.

Tenth International Foamy Virus Conference 2014–Achievements and Perspectives

PubMed Central

Materniak, Magdalena; Kubiś, Piotr; Rola–Łuszczak, Marzena; Khan, Arifa S.; Buseyne, Florence; Lindemann, Dirk; Löchelt, Martin; Kuźmak, Jacek

2015-01-01

For the past two decades, scientists from around the world, working on different aspects of foamy virus (FV) research, have gathered in different research institutions almost every two years to present their recent results in formal talks, to discuss their ongoing studies informally, and to initiate fruitful collaborations. In this report we review the 2014 anniversary conference to share the meeting summary with the virology community and hope to arouse interest by other researchers to join this exciting field. The topics covered included epidemiology, virus molecular biology, and immunology of FV infection in non-human primates, cattle, and humans with zoonotic FV infections, as well as recent findings on endogenous FVs. Several topics focused on virus replication and interactions between viral and cellular proteins. Use of FV in biomedical research was highlighted with presentations on using FV vectors for gene therapy and FV proteins as scaffold for vaccine antigen presentation. On behalf of the FV community, this report also includes a short tribute to commemorate Prof. Axel Rethwilm, one of the leading experts in the field of retrovirology and foamy viruses, who passed away 29 July 2014. PMID:25835535

Functional analysis of human foamy virus accessory reading frames.

PubMed Central

Baunach, G; Maurer, B; Hahn, H; Kranz, M; Rethwilm, A

1993-01-01

Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses. Images PMID:8394455

Complete Genome Sequence of a Naturally Occurring Simian Foamy Virus Isolate from Rhesus Macaque (SFVmmu_K3T).

PubMed

Nandakumar, Subhiksha; Bae, Eunhae H; Khan, Arifa S

2017-08-17

The full-length genome sequence of a simian foamy virus (SFVmmu_K3T), isolated from a rhesus macaque ( Macaca mulatta ), was obtained using high-throughput sequencing. SFVmmu_K3T consisted of 12,983 bp and had a genomic organization similar to that of other SFVs, with long terminal repeats (LTRs) and open reading frames for Gag, Pol, Env, Tas, and Bet.

IFP35 Is Involved in the Antiviral Function of Interferon by Association with the Viral Tas Transactivator of Bovine Foamy Virus▿

PubMed Central

Tan, Juan; Qiao, Wentao; Wang, Jian; Xu, Fengwen; Li, Yue; Zhou, Jun; Chen, Qimin; Geng, Yunqi

2008-01-01

Interferon-induced proteins (IFPs) exert multiple functions corresponding to diverse interferon signals. However, the intracellular functions of many IFPs are not fully characterized. Here, we report that IFP35, a member of the IFP family with a molecular mass of 35 kDa, can interact with the bovine Tas (BTas) regulatory protein of bovine foamy virus (BFV). The interaction involves NID2 (IFP35/Nmi homology domain) of IFP35 and the central domain of BTas. The overexpression of IFP35 disturbs the ability of BTas to activate viral-gene transcription and inhibits viral replication. The depletion of endogenous IFP35 by interfering RNA can promote the activation of BFV, suggesting an inhibitory function of IFP35 in viral-gene expression. In addition, IFP35 can interact with the homologous regulatory protein of prototype FV and arrest viral replication and repress viral transcription. Our study suggests that IFP35 may represent a novel pathway of interferon-mediated antiviral activity in host organisms that plays a role in the maintenance of FV latency. PMID:18305040

Complete genome sequences of two novel European clade bovine foamy viruses from Germany and Poland.

PubMed

Hechler, Torsten; Materniak, Magdalena; Kehl, Timo; Kuzmak, Jacek; Löchelt, Martin

2012-10-01

Bovine foamy virus (BFV), or bovine spumaretrovirus, is an infectious agent of cattle with no obvious disease association but high prevalence in its host. Here, we report two complete BFV sequences, BFV-Riems, isolated in 1978 in East Germany, and BFV100, isolated in 2005 in Poland. Both new BFV isolates share the overall genetic makeup of other foamy viruses (FV). Although isolated almost 25 years apart and propagated in either bovine (BFV-Riems) or nonbovine (BFV100) cells, both viruses are highly related, forming the European BFV clade. Despite clear differences, BFV-Riems and BFV100 are still very similar to BFV isolates from China and the United States, comprising the non-European BFV clade. The genomic sequences presented here confirm the concept of high sequence conservation across most of the FV genome. Analyses of cell culture-derived genomes reveal that proviral DNA may specifically lack introns in the env-bel coding region. The spacing of the splice sites in this region suggests that BFV has developed a novel mode to express a secretory but nonfunctional Env protein.

Complete Genome Sequences of Two Novel European Clade Bovine Foamy Viruses from Germany and Poland

PubMed Central

Hechler, Torsten; Materniak, Magdalena; Kehl, Timo; Kuzmak, Jacek

2012-01-01

Bovine foamy virus (BFV), or bovine spumaretrovirus, is an infectious agent of cattle with no obvious disease association but high prevalence in its host. Here, we report two complete BFV sequences, BFV-Riems, isolated in 1978 in East Germany, and BFV100, isolated in 2005 in Poland. Both new BFV isolates share the overall genetic makeup of other foamy viruses (FV). Although isolated almost 25 years apart and propagated in either bovine (BFV-Riems) or nonbovine (BFV100) cells, both viruses are highly related, forming the European BFV clade. Despite clear differences, BFV-Riems and BFV100 are still very similar to BFV isolates from China and the United States, comprising the non-European BFV clade. The genomic sequences presented here confirm the concept of high sequence conservation across most of the FV genome. Analyses of cell culture-derived genomes reveal that proviral DNA may specifically lack introns in the env-bel coding region. The spacing of the splice sites in this region suggests that BFV has developed a novel mode to express a secretory but nonfunctional Env protein. PMID:22966195

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

PubMed

Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

2015-11-11

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

PubMed Central

Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

2015-01-01

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies. PMID:26569290

Serological detection systems for identification of cows shedding bovine foamy virus via milk.

PubMed

Romen, Fabian; Backes, Perdita; Materniak, Magda; Sting, Reinhard; Vahlenkamp, Thomas W; Riebe, Roland; Pawlita, Michael; Kuzmak, Jacek; Löchelt, Martin

2007-07-20

The biology of foamy viruses, their mode of transmission and disease potential in their natural host and after interspecies transmission are largely unknown. To gain insights into the prevalence of bovine foamy virus (BFV) and its zoonotic potential, enzyme-linked immunosorbent assays (ELISAs) were established to determine antibody responses against Gag, Env, and the non-structural protein Bet in bovine serum and milk. In Polish cattle, strong Gag reactivity was most frequent (41.5%) and strongly associated with Bet antibodies, Env antibodies were less frequent. German cattle showed a low overall BFV antibody prevalence of 6.8%. Besides clearly BFV-positive animals, a substantial number of weakly reacting cattle were identified. BFV-specific antibodies were also detectable in milk. BFV was isolated from PBLs and milk cells of BFV-positive cattle but not from antibody-negative or weakly reacting animals. The implications of these findings for the potential interspecies transmission of BFV to humans will be discussed.

Evolution of Foamy Viruses: The Most Ancient of All Retroviruses †

PubMed Central

Rethwilm, Axel; Bodem, Jochen

2013-01-01

Recent evidence indicates that foamy viruses (FVs) are the oldest retroviruses (RVs) that we know and coevolved with their hosts for several hundred million years. This coevolution may have contributed to the non-pathogenicity of FVs, an important factor in development of foamy viral vectors in gene therapy. However, various questions on the molecular evolution of FVs remain still unanswered. The analysis of the spectrum of animal species infected by exogenous FVs or harboring endogenous FV elements in their genome is pivotal. Furthermore, animal studies might reveal important issues, such as the identification of the FV in vivo target cells, which than require a detailed characterization, to resolve the molecular basis of the accuracy with which FVs copy their genome. The issues of the extent of FV viremia and of the nature of the virion genome (RNA vs. DNA) also need to be experimentally addressed. PMID:24072062

A comparative analysis of the foamy and ortho virus capsid structures reveals an ancient domain duplication.

PubMed

Taylor, William R; Stoye, Jonathan P; Taylor, Ian A

2017-04-04

The Spumaretrovirinae (foamy viruses) and the Orthoretrovirinae (e.g. HIV) share many similarities both in genome structure and the sequences of the core viral encoded proteins, such as the aspartyl protease and reverse transcriptase. Similarity in the gag region of the genome is less obvious at the sequence level but has been illuminated by the recent solution of the foamy virus capsid (CA) structure. This revealed a clear structural similarity to the orthoretrovirus capsids but with marked differences that left uncertainty in the relationship between the two domains that comprise the structure. We have applied protein structure comparison methods in order to try and resolve this ambiguous relationship. These included both the DALI method and the SAP method, with rigorous statistical tests applied to the results of both methods. For this, we employed collections of artificial fold 'decoys' (generated from the pair of native structures being compared) to provide a customised background distribution for each comparison, thus allowing significance levels to be estimated. We have shown that the relationship of the two domains conforms to a simple linear correspondence rather than a domain transposition. These similarities suggest that the origin of both viral capsids was a common ancestor with a double domain structure. In addition, we show that there is also a significant structural similarity between the amino and carboxy domains in both the foamy and ortho viruses. These results indicate that, as well as the duplication of the double domain capsid, there may have been an even more ancient gene-duplication that preceded the double domain structure. In addition, our structure comparison methodology demonstrates a general approach to problems where the components have a high intrinsic level of similarity.

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

PubMed Central

2012-01-01

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

Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

PubMed Central

Tobaly-Tapiero, Joelle; Bittoun, Patricia; Giron, Marie-Lou; Neves, Manuel; Koken, Marcel; Saïb, Ali; de Thé, Hugues

2001-01-01

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly. PMID:11287585

Diverse Contexts of Zoonotic Transmission of Simian Foamy Viruses in Asia

PubMed Central

May, Cynthia C.; Engel, Gregory A.; Steinkraus, Katherine A.; Schillaci, Michael A.; Fuentes, Agustin; Rompis, Aida; Chalise, Mukesh K.; Aggimarangsee, Nantiya; Feeroz, Mohammed M.; Grant, Richard; Allan, Jonathan S.; Putra, Arta; Wandia, I. Nengah; Watanabe, Robin; Kuller, LaRene; Thongsawat, Satawat; Chaiwarith, Romanee; Kyes, Randall C.; Linial, Maxine L.

2008-01-01

In Asia, contact between persons and nonhuman primates is widespread in multiple occupational and nonoccupational contexts. Simian foamy viruses (SFVs) are retroviruses that are prevalent in all species of nonhuman primates. To determine SFV prevalence in humans, we tested 305 persons who lived or worked around nonhuman primates in several South and Southeast Asian countries; 8 (2.6%) were confirmed SFV positive by Western blot and, for some, by PCR. The interspecies interactions that likely resulted in virus transmission were diverse; 5 macaque taxa were implicated as a potential source of infection. Phylogenetic analysis showed that SFV from 3 infected persons was similar to that from the nonhuman primate populations with which the infected persons reported contact. Thus, SFV infections are likely to be prevalent among persons who live or work near nonhuman primates in Asia. PMID:18680642

Isolation and sequence analysis of a novel rhesus macaque foamy virus isolate with a serotype-1-like env.

PubMed

Ensser, Armin; Großkopf, Anna K; Mätz-Rensing, Kerstin; Roos, Christian; Hahn, Alexander S

2018-06-02

SFVmmu-DPZ9524 represents the third completely sequenced rhesus macaque simian foamy virus (SFV) isolate, alongside SFVmmu_K3T with a similar SFV-1-type env, and R289HybAGM with a SFV-2-like env. Sequence analysis demonstrates that, in gag and pol, SFVmmu-DPZ9524 is more closely related to R289HybAGM than to SFVmmu_K3T, which, outside of env, is more similar to a Japanese macaque isolate than to the other two rhesus macaque isolates SFVmmu-DPZ9524 and R289HybAGM. Further, we identify bel as another recombinant locus in R289HybAGM, confirming that recombination contributes to sequence diversity in SFV.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

PubMed Central

Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin

2015-01-01

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy. PMID:26397953

Population dynamics of rhesus macaques and associated foamy virus in Bangladesh

PubMed Central

Feeroz, Mostafa M; Soliven, Khanh; Small, Christopher T; Engel, Gregory A; Andreina Pacheco, M; Yee, JoAnn L; Wang, Xiaoxing; Kamrul Hasan, M; Oh, Gunwha; Levine, Kathryn L; Rabiul Alam, SM; Craig, Karen L; Jackson, Dana L; Lee, Eun-Gyung; Barry, Peter A; Lerche, Nicholas W; Escalante, Ananias A; Matsen IV, Frederick A; Linial, Maxine L; Jones-Engel, Lisa

2013-01-01

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses. PMID:26038465

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.

PubMed

Leo, Berit; Schweimer, Kristian; Rösch, Paul; Hartl, Maximilian J; Wöhrl, Birgitta M

2012-09-10

The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

Helper-Free Foamy Virus Vectors

PubMed Central

TROBRIDGE, GRANT D.; RUSSELL, DAVID W.

2010-01-01

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV–LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 105 transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR. OVERVIEW SUMMARY Vectors based on human foamy virus have been developed but low titers and the presence of replication-competent retrovirus (RCR) in vector stocks have prevented their use in preclinical animal experiments. We have developed a transient transfection method that can be used to produce replication-incompetent HFV vector stocks at titers greater than 105/ml, and that does not produce contaminating RCR. The use of CMV-HFV LTR fusion promoters in the helper and vector constructs has circumvented the requirement for the HFV Bel-1 trans-activator protein. Consequently, the potential for generating wild-type HFV by recombination between helper and vector constructs during vector production has been eliminated. Here we describe HFV vector production using this Bel-independent system. PMID:9853518

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction.

PubMed

Chareza, Sarah; Slavkovic Lukic, Dragana; Liu, Yang; Räthe, Ann-Mareen; Münk, Carsten; Zabogli, Elisa; Pistello, Mauro; Löchelt, Martin

2012-03-15

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable. Copyright © 2012 Elsevier Inc. All rights reserved.

HTLV-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, cross-species transmission and molecular virology.

PubMed

Gessain, Antoine; Rua, Réjane; Betsem, Edouard; Turpin, Jocelyn; Mahieux, Renaud

2013-01-05

Non-human primates are considered to be likely sources of viruses that can infect humans and thus pose a significant threat to human population. This is well illustrated by some retroviruses, as the simian immunodeficiency viruses and the simian T lymphotropic viruses, which have the ability to cross-species, adapt to a new host and sometimes spread. This leads to a pandemic situation for HIV-1 or an endemic one for HTLV-1. Here, we present the available data on the discovery, epidemiology, cross-species transmission and molecular virology of the recently discovered HTLV-3 and HTLV-4 deltaretroviruses, as well as the simian foamy retroviruses present in different human populations at risk, especially in central African hunters. We discuss also the natural history in humans of these retroviruses of zoonotic origin (magnitude and geographical distribution, possible inter-human transmission). In Central Africa, the increase of the bushmeat trade during the last decades has opened new possibilities for retroviral emergence in humans, especially in immuno-compromised persons. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Chen, Qi; Cheng, Xiaolin; Univ. of Tennessee, Knoxville, TN

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was usedmore » to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.« less

Molecular Ecology and Natural History of Simian Foamy Virus Infection in Wild-Living Chimpanzees

PubMed Central

Liu, Weimin; Worobey, Michael; Li, Yingying; Keele, Brandon F.; Bibollet-Ruche, Frederic; Guo, Yuanyuan; Goepfert, Paul A.; Santiago, Mario L.; Ndjango, Jean-Bosco N.; Neel, Cecile; Clifford, Stephen L.; Sanz, Crickette; Kamenya, Shadrack; Wilson, Michael L.; Pusey, Anne E.; Gross-Camp, Nicole; Boesch, Christophe; Smith, Vince; Zamma, Koichiro; Huffman, Michael A.; Mitani, John C.; Watts, David P.; Peeters, Martine; Shaw, George M.; Switzer, William M.; Sharp, Paul M.; Hahn, Beatrice H.

2008-01-01

Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies. PMID:18604273

Detection and analysis of bovine foamy virus infection by an indicator cell line.

PubMed

Ma, Zhe; Qiao, Wen-tao; Xuan, Cheng-hao; Xie, Jin-hui; Chen, Qi-min; Geng, Yun-qi

2007-07-01

To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. BFV infection was induced by the co-culture method or the transient transfection of the infectious BFV plasmid [pCMV (cytomegalovirus) - BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein-positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. The titer of BFV in fetal bovine lung cells was 4-5-folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co-culture method, and in the meantime was significantly higher than that produced by the infectious clone pCMV-BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. BICL can be used for the titration of the BFV viral infection in non-cytopathic condition. In addition, we provide important evidence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

Dimerization of BTas is required for the transactivational activity of bovine foamy virus

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

Tan Juan; Qiao Wentao; Xu Fengwen

2008-06-20

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activitymore » on the LTR and IP. Furthermore, BTas ({delta}46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.« less

Lysine acetylation sites in bovine foamy virus transactivator BTas are important for its DNA binding activity.

PubMed

Chang, Rui; Tan, Juan; Xu, Fengwen; Han, Hongqi; Geng, Yunqi; Li, Yue; Qiao, Wentao

2011-09-15

Cellular acetylation signaling is important for viral gene regulation, particularly during the transactivation of retroviruses. The regulatory protein of bovine foamy virus (BFV), BTas, is a transactivator that augments viral gene transcription from both the long terminal repeat (LTR) promoter and the internal promoter (IP). In this study, we report that the histone acetyltransferase (HAT), p300, specifically acetylates BTas both in vivo and in vitro. Further studies demonstrated that BTas acetylation markedly enhances its transactivation activity. Mutagenesis analysis identified three lysines at positions 66, 109 and 110 in BTas that are acetylated by p300. The K110R mutant lost its binding to BFV promoter as well as its ability to activate BFV promoter. The acetylation of K66 and K109 may contribute to increased BTas binding ability. These results suggest that the p300-acetylated lysines of BTas are important for transactivation of BFV promoters and therefore have an important role in BFV replication. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification and functional characterization of BTas transactivator as a DNA-binding protein.

PubMed

Tan, Juan; Hao, Peng; Jia, Rui; Yang, Wei; Liu, Ruichang; Wang, Jinzhong; Xi, Zhen; Geng, Yunqi; Qiao, Wentao

2010-09-30

The genome of bovine foamy virus (BFV) encodes a transcriptional transactivator, namely BTas, that remarkably enhances gene expression by binding to the viral long-terminal repeat promoter (LTR) and internal promoter (IP). In this report, we characterized the functional domains of BFV BTas. BTas contains two major functional domains: the N-terminal DNA-binding domain (residues 1-133) and the C-terminal activation domain (residues 198-249). The complete BTas responsive regions were mapped to the positions -380/-140 of LTR and 9205/9276 of IP. Four BTas responsive elements were identified at the positions -368/-346, -327/-307, -306/-285 and -186/-165 of the BFV LTR, and one element was identified at the position 9243/9264 of the BFV IP. Unlike other foamy viruses, the five BTas responsive elements in BFV shared obvious sequence homology. These data suggest that among the complex retroviruses, BFV appears to have a unique transactivation mechanism. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

PubMed Central

Enssle, J; Fischer, N; Moebes, A; Mauer, B; Smola, U; Rethwilm, A

1997-01-01

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked. PMID:9311808

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase.

PubMed

Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

2015-03-01

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we herein use the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

Detection of viruses using discarded plants from wild mountain gorillas and golden monkeys.

PubMed

Smiley Evans, Tierra; Gilardi, Kirsten V K; Barry, Peter A; Ssebide, Benard Jasper; Kinani, Jean Felix; Nizeyimana, Fred; Noheri, Jean Bosco; Byarugaba, Denis K; Mudakikwa, Antoine; Cranfield, Michael R; Mazet, Jonna A K; Johnson, Christine K

2016-11-01

Infectious diseases pose one of the most significant threats to the survival of great apes in the wild. The critically endangered mountain gorilla (Gorilla beringei beringei) is at high risk for contracting human pathogens because approximately 60% of the population is habituated to humans to support a thriving ecotourism program. Disease surveillance for human and non-human primate pathogens is important for population health and management of protected primate species. Here, we evaluate discarded plants from mountain gorillas and sympatric golden monkeys (Cercopithecus mitis kandti), as a novel biological sample to detect viruses that are shed orally. Discarded plant samples were tested for the presence of mammalian-specific genetic material and two ubiquitous DNA and RNA primate viruses, herpesviruses, and simian foamy virus. We collected discarded plant samples from 383 wild human-habituated mountain gorillas and from 18 habituated golden monkeys. Mammalian-specific genetic material was recovered from all plant species and portions of plant bitten or chewed by gorillas and golden monkeys. Gorilla herpesviral DNA was most consistently recovered from plants in which leafy portions were eaten by gorillas. Simian foamy virus nucleic acid was recovered from plants discarded by golden monkeys, indicating that it is also possible to detect RNA viruses from bitten or chewed plants. Our findings show that discarded plants are a useful non-invasive sampling method for detection of viruses that are shed orally in mountain gorillas, sympatric golden monkeys, and potentially other species. This method of collecting specimens from discarded plants is a new non-invasive sampling protocol that can be combined with collection of feces and urine to evaluate the most common routes of viral shedding in wild primates. Am. J. Primatol. 78:1222-1234, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

PubMed Central

Denner, Joachim

2013-01-01

The induction of neutralizing antibodies is a promising way to prevent retrovirus infections. Neutralizing antibodies are mainly directed against the envelope proteins, which consist of two molecules, the surface envelope (SU) protein and the transmembrane envelope (TM) protein. Antibodies broadly neutralizing the human immunodeficiencvy virus-1 (HIV-1) and binding to the TM protein gp41 of the virus have been isolated from infected individuals. Their epitopes are located in the membrane proximal external region (MPER). Since there are difficulties to induce such neutralizing antibodies as basis for an effective AIDS vaccine, we performed a comparative analysis immunising with the TM proteins of different viruses from the family Retroviridae. Both subfamilies, the Orthoretrovirinae and the Spumaretrovirinae were included. In this study, the TM proteins of three gammaretroviruses including (1) the porcine endogenous retrovirus (PERV), (2) the Koala retrovirus (KoRV), (3) the feline leukemia virus (FeLV), of two lentiviruses, HIV-1, HIV-2, and of two spumaviruses, the feline foamy virus (FFV) and the primate foamy virus (PFV) were used for immunisation. Whereas in all immunisation studies binding antibodies were induced, neutralizing antibodies were only found in the case of the gammaretroviruses. The induced antibodies were directed against the MPER and the fusion peptide proximal region (FPPR) of their TM proteins; however only the antibodies against the MPER were neutralizing. Most importantly, the epitopes in the MPER were localized in the same position as the epitopes of the antibodies broadly neutralizing HIV-1 in the TM protein gp41 of HIV-1, indicating that the MPER is an effective target for the neutralization of retroviruses. PMID:23249763

A new indicator cell line established to monitor bovine foamy virus infection.

PubMed

Guo, Hong-Yan; Liang, Zhi-Bin; Li, Yue; Tan, Juan; Chen, Qi-Min; Qiao, Wen-Tao

2011-10-01

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Establishment of an indicator cell line to quantify bovine foamy virus infection.

PubMed

Ma, Zhe; Hao, Peng; Yao, Xue; Liu, Chang; Tan, Juan; Liu, Li; Yang, Rongge; Geng, Yunqi; Chen, Qimin; Qiao, Wentao

2008-08-01

A cell line derived from baby hamster kidney (BHK-21) cells was transfected with the enhanced green fluorescent protein gene driven by the bovine foamy virus (BFV) long terminal repeat (LTR) to establish a BFV indicator cell line (BICL). Among 48 clones, one clone was chosen for its little constitutive enhanced green fluorescent protein (EGFP) expression and high level of EGFP expression after activation by BFV infection. By detecting the EGFP expression of the BFV indicator cell line, the titers of BFV were quantified by the end point method. As a result, the titer determined by the EGFP based assay 5-6 days post infection (d.p.i.) was 100 fold higher than traditional assays measuring cytopathic effects 8-9 d.p.i.. Moreover, the EGFP based assay was also used to determine the titer of those cells infected by BFV without inducing cytopathic effects. Using this simple and rapid assay, we examined the in vitro host range of BFV. It was found that BFV can productively infect various cell lines derived from bovine, human, rat and monkey. (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

PubMed Central

Uchiyama, Toru; Adriani, Marsilio; Jagadeesh, G Jayashree; Paine, Adam; Candotti, Fabio

2012-01-01

The Wiskott–Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS. PMID:22215016

Biochemical Characterization of Novel Retroviral Integrase Proteins

PubMed Central

Ballandras-Colas, Allison; Naraharisetty, Hema; Li, Xiang; Serrao, Erik; Engelman, Alan

2013-01-01

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies. PMID:24124581

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Clinical Signs and Blood Test Results Among Humans Infected With Zoonotic Simian Foamy Virus: A Case-Control Study.

PubMed

Buseyne, Florence; Betsem, Edouard; Montange, Thomas; Njouom, Richard; Bilounga Ndongo, Chanceline; Hermine, Olivier; Gessain, Antoine

2018-06-05

A spillover of simian foamy virus (SFV) to humans, following bites from infected nonhuman primates (NHPs), is ongoing in exposed populations. These retroviruses establish persistent infections of unknown physiological consequences to the human host. We performed a case-control study to compare 24 Cameroonian hunters infected with gorilla SFV and 24 controls matched for age and ethnicity. A complete physical examination and blood test were performed for all participants. Logistic regression and Wilcoxon signed rank tests were used to compare cases and controls. The cases had significantly lower levels of hemoglobin than the controls (median, 12.7 vs 14.4 g/dL; P = .01). Basophil levels were also significantly lower in cases than controls, with no differences for other leukocyte subsets. Cases had significantly higher urea, creatinine, protein, creatinine phosphokinase, and lactate dehydrogenase levels and lower bilirubin levels than controls. Cases and controls had similar frequencies of general, cutaneous, gastrointestinal, neurological, and cardiorespiratory signs. The first case-control study of apparently healthy SFV-infected Cameroonian hunters showed the presence of hematological abnormalities. A thorough clinical and laboratory workup is now needed to establish the medical relevance of these observations because more than half of cases had mild or moderate anemia. NCT03225794.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

Frequent Simian Foamy Virus Infection in Persons Occupationally Exposed to Nonhuman Primates

PubMed Central

Switzer, William M.; Bhullar, Vinod; Shanmugam, Vedapuri; Cong, Mian-er; Parekh, Bharat; Lerche, Nicholas W.; Yee, JoAnn L.; Ely, John J.; Boneva, Roumiana; Chapman, Louisa E.; Folks, Thomas M.; Heneine, Walid

2004-01-01

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections. PMID:14990698

Antibody responses to Herpesvirus papio antigens in baboons with lymphoma.

PubMed

Neubauer, R H; Rabin, H; Strnad, B C; Lapin, B A; Yakovleva, L A; Indzie, E

1979-02-01

An Epstein-Barr virus-related herpesvirus, termed Herpesvirus papio (HVP), was isolated from baboons (Papio hamadryas) at the Institute of Experimental Pathology and Therapy, Sukhumi, USSR, where there is a continuing outbreak of lymphoma. In the present study sera from diseased baboons and from age- and sex-matched control animals were examined for antibodies to HVP antigens. Results showed that animals with lymphoid disease had antibodies to HVP virus capsid, early, soluble, and nuclear antigens at higher frequencies and at higher titers than did control animals. Antibody titers were not age- or sex-related. No concordancy was detected for antibodies to soluble and nuclear antigens. The sera were also examined for antibodies to two other widely distributed viruses of hamadryas baboons, cytomegalovirus and foamy virus. The results of these studies did not indicate a disease-related role for either of these viruses.

Similar Patterns of Infection with Bovine Foamy Virus in Experimentally Inoculated Calves and Sheep

PubMed Central

Hechler, Torsten; Löchelt, Martin; Kuźmak, Jacek

2013-01-01

Foamy viruses (FVs) are the least known retroviruses commonly found in primates, cats, horses, and cattle. Although FVs are considered apathogenic, simian and feline FVs have been shown to be associated with some transient health abnormalities in animal models. Currently, data regarding the course of infection with bovine FV (BFV) are not available. In this study, we conducted experimental infections of natural (cattle) and heterologous (sheep) hosts with the BFV100 isolate and monitored infection patterns in both hosts during the early phase postinoculation as well as after long-term infection. Four calves and six sheep inoculated with BFV100 showed no signs of pathology but developed persistent infection, as confirmed by virus rescue, consistent detection of BFV-specific antibodies, and presence of viral DNA. In both hosts, antibodies against BFV Gag and Bet appeared early after infection and persisted at high and stable levels while seroreactivity toward Env was consistently detectable only in BFV-infected sheep. Interestingly, the BFV proviral DNA load was highest in lung, spleen, and liver and moderate in leukocytes, while salivary glands contained either low or undetectable DNA loads in calves or sheep, respectively. Additionally, comparison of partial BFV sequences from inoculum and infected animals demonstrated very limited changes after long-term infection in the heterologous host, clearly less than those found in BFV field isolates. The persistence of BFV infection in both hosts suggests full replication competence of the BFV100 isolate with no requirement of genetic adaptation for productive replication in the authentic and even in a heterologous host. PMID:23325680

Similar patterns of infection with bovine foamy virus in experimentally inoculated calves and sheep.

PubMed

Materniak, Magdalena; Hechler, Torsten; Löchelt, Martin; Kuzmak, Jacek

2013-03-01

Foamy viruses (FVs) are the least known retroviruses commonly found in primates, cats, horses, and cattle. Although FVs are considered apathogenic, simian and feline FVs have been shown to be associated with some transient health abnormalities in animal models. Currently, data regarding the course of infection with bovine FV (BFV) are not available. In this study, we conducted experimental infections of natural (cattle) and heterologous (sheep) hosts with the BFV(100) isolate and monitored infection patterns in both hosts during the early phase postinoculation as well as after long-term infection. Four calves and six sheep inoculated with BFV(100) showed no signs of pathology but developed persistent infection, as confirmed by virus rescue, consistent detection of BFV-specific antibodies, and presence of viral DNA. In both hosts, antibodies against BFV Gag and Bet appeared early after infection and persisted at high and stable levels while seroreactivity toward Env was consistently detectable only in BFV-infected sheep. Interestingly, the BFV proviral DNA load was highest in lung, spleen, and liver and moderate in leukocytes, while salivary glands contained either low or undetectable DNA loads in calves or sheep, respectively. Additionally, comparison of partial BFV sequences from inoculum and infected animals demonstrated very limited changes after long-term infection in the heterologous host, clearly less than those found in BFV field isolates. The persistence of BFV infection in both hosts suggests full replication competence of the BFV(100) isolate with no requirement of genetic adaptation for productive replication in the authentic and even in a heterologous host.

RNAi and retroviruses: are they in RISC?

PubMed

Vasselon, Thierry; Bouttier, Manuella; Saumet, Anne; Lecellier, Charles-Henri

2013-02-01

RNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

PubMed

Bing, Tiejun; Zhang, Suzhen; Liu, Xiaojuan; Liang, Zhibin; Shao, Peng; Zhang, Song; Qiao, Wentao; Tan, Juan

2016-06-30

Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

Large Animal Models for Foamy Virus Vector Gene Therapy

PubMed Central

Trobridge, Grant D.; Horn, Peter A.; Beard, Brian C.; Kiem, Hans-Peter

2012-01-01

Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. PMID:23223198

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

PubMed Central

Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

2016-01-01

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

Bovine Foamy Virus Transactivator BTas Interacts with Cellular RelB To Enhance Viral Transcription▿

PubMed Central

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-01-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription. PMID:20844054

Bovine foamy virus transactivator BTas interacts with cellular RelB to enhance viral transcription.

PubMed

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-11-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

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

Guan, Rongjin; Aiyer, Sriram; Cote, Marie L.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structuresmore » of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.« less

Primate-to-Human Retroviral Transmission in Asia

PubMed Central

Engel, Gregory A.; Schillaci, Michael A.; Rompis, Aida; Putra, Artha; Suaryana, Komang Gde; Fuentes, Agustin; Beer, Brigitte; Hicks, Sarah; White, Robert; Wilson, Brenda; Allan, Jonathan S.

2005-01-01

We describe the first reported transmission to a human of simian foamy virus (SFV) from a free-ranging population of nonhuman primates in Asia. The transmission of an exogenous retrovirus, SFV, from macaques (Macaca fascicularis) to a human at a monkey temple in Bali, Indonesia, was investigated with molecular and serologic techniques. Antibodies to SFV were detected by Western blotting of serum from 1 of 82 humans tested. SFV DNA was detected by nested polymerase chain reaction (PCR) from the blood of the same person. Cloning and sequencing of PCR products confirmed the virus's close phylogenetic relationship to SFV isolated from macaques at the same temple. This study raises concerns that persons who work at or live around monkey temples are at risk for infection with SFV. PMID:16022776

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

PubMed

Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

2016-04-01

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Two distinct variants of simian foamy virus in naturally infected mandrills (Mandrillus sphinx) and cross-species transmission to humans.

PubMed

Mouinga-Ondémé, Augustin; Betsem, Edouard; Caron, Mélanie; Makuwa, Maria; Sallé, Bettina; Renault, Noemie; Saib, Ali; Telfer, Paul; Marx, Preston; Gessain, Antoine; Kazanji, Mirdad

2010-12-14

Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans. We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time. Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.

[Mechanisms of viral emergence and interspecies transmission: the exemple of simian foamy viruses in Central Africa].

PubMed

Gessain, Antoine

2013-12-01

A large proportion of viral pathogens that have emerged during the last decades in humans are considered to have originated from various animal species. This is well exemplified by several recent epidemics such as those of Nipah, Severe Acute Respiratory Syndrome, Avian flu, Ebola, Monkeypox, and Hantaviruses. After the initial interspecies transmission per se, the viruses can disseminate into the human population through various and distinct mechanisms. Some of them are well characterized and understood, thus allowing a certain level of risk control and prevention. Surprisingly and in contrast, the initial steps that lead to the emergence of several viruses, and of their associated diseases, remain still poorly understood. Epidemiological field studies conducted in certain specific high-risk populations are thus necessary to obtain new insights into the early events of this emergence process. Human infections by simian viruses represent increasing public health concerns. Indeed, by virtue of their genetic andphysiological similarities, non-human primates (NHPs) are considered to be likely the sources of viruses that can infect humans and thus may pose a significant threat to human population. This is well illustrated by retroviruses, which have the ability to cross species, adapt to a new host and sometimes spread within these new species. Sequence comparison and phylogenetic studies have thus clearly showed that the emergence of human immunodeficiency virus type 1 (HIV-1) and HIV-2 in humans have resulted from several independent interspecies transmissions of different SIV types from Chimpanzees and African monkeys (including sooty mangabeys), respectively, probably during the first part of the last century. The situation for Human T cell Lymphotropic virus type 1 (HTLV-1) is, for certain aspects, quite comparable. Indeed, the origin of most HTLV-1 subtypes appears to be linked to interspecies transmission between STLV-1-infected monkeys and humans, followed by variable periods of evolution in the human host. In this review, after an introduction on emerging viruses, we will briefly present the results of a large epidemiological study performed in groups of Bantus and Pygmies living in villages and settlements located in the rain forest of the South region of Cameroon. These populations are living nearby the habitats of several monkeys and apes, often naturally infected by different retroviruses including SIV, STLV and simianfoamy virus. Most of the persons included in this study were hunters of such NHPs, thus at high risk of contact with infected body fluids (blood, saliva,...) during hunting activities. After reviewing the current available data on the discovery, cross-species transmission from monkeys and apes to humans of the simian foamy retroviruses, we will report the results of our study. Such infection is a unique natural model to study the different mechanisms of restriction of retroviral emergence in Humans.

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains.

PubMed

Chen, Qi; Thomas, Joseph T; Giménez-Lirola, Luis G; Hardham, John M; Gao, Qinshan; Gerber, Priscilla F; Opriessnig, Tanja; Zheng, Ying; Li, Ganwu; Gauger, Phillip C; Madson, Darin M; Magstadt, Drew R; Zhang, Jianqiang

2016-04-05

At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.

Genetic Characterization of Simian Foamy Viruses Infecting Humans

PubMed Central

Rua, Réjane; Betsem, Edouard; Calattini, Sara; Saib, Ali

2012-01-01

Simian foamy viruses (SFVs) are retroviruses that are widespread among nonhuman primates (NHPs). SFVs actively replicate in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data on the genetic structure of such SFVs found in humans are available. In the framework of ongoing studies searching for SFV-infected humans in south Cameroon rainforest villages, we studied 38 SFV-infected hunters whose times of infection had presumably been determined. By long-term cocultures of peripheral blood mononuclear cells with BHK-21 cells, we isolated five new SFV strains and obtained complete genomes of SFV strains from chimpanzee (Pan troglodytes troglodytes; strains BAD327 and AG15), monkey (Cercopithecus nictitans; strain AG16), and gorilla (Gorilla gorilla; strains BAK74 and BAD468). These zoonotic strains share a very high degree of similarity with their NHP counterparts and have a high degree of conservation of the genetic elements important for viral replication. Interestingly, analysis of FV DNA sequences obtained before cultivation revealed variants with deletions in both the U3 region and tas that may correlate with in vivo chronicity in humans. Genomic changes in bet (a premature stop codon) and gag were also observed. To determine if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes. Our study reveals that natural polymorphism of SFV strains does exist at both the intersubspecies level (gag, bet) and the intrasubspecies (U3, tas) levels but does not seem to reflect a viral adaptation specific to zoonotic SFV strains. PMID:23015714

Foamy virus–mediated gene transfer to canine repopulating cells

PubMed Central

Kiem, Hans-Peter; Allen, James; Trobridge, Grant; Olson, Erik; Keyser, Kirsten; Peterson, Laura; Russell, David W.

2007-01-01

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)–expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/μL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical. PMID:16968897

Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces.

PubMed

Wu, Zhiqiang; Ren, Xianwen; Yang, Li; Hu, Yongfeng; Yang, Jian; He, Guimei; Zhang, Junpeng; Dong, Jie; Sun, Lilian; Du, Jiang; Liu, Liguo; Xue, Ying; Wang, Jianmin; Yang, Fan; Zhang, Shuyi; Jin, Qi

2012-10-01

Bats are natural hosts for a large variety of zoonotic viruses. This study aimed to describe the range of bat viromes, including viruses from mammals, insects, fungi, plants, and phages, in 11 insectivorous bat species (216 bats in total) common in six provinces of China. To analyze viromes, we used sequence-independent PCR amplification and next-generation sequencing technology (Solexa Genome Analyzer II; Illumina). The viromes were identified by sequence similarity comparisons to known viruses. The mammalian viruses included those of the Adenoviridae, Herpesviridae, Papillomaviridae, Retroviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviridae, Coronaviridae, Picornaviridae, and Parvovirinae; insect viruses included those of the Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae; fungal viruses included those of the Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages included those of the Caudovirales, Inoviridae, and Microviridae and unclassified phages. In addition to the viruses and phages associated with the insects, plants, and bacterial flora related to the diet and habitation of bats, we identified the complete or partial genome sequences of 13 novel mammalian viruses. These included herpesviruses, papillomaviruses, a circovirus, a bocavirus, picornaviruses, a pestivirus, and a foamy virus. Pairwise alignments and phylogenetic analyses indicated that these novel viruses showed little genetic similarity with previously reported viruses. This study also revealed a high prevalence and diversity of bat astroviruses and coronaviruses in some provinces. These findings have expanded our understanding of the viromes of bats in China and hinted at the presence of a large variety of unknown mammalian viruses in many common bat species of mainland China.

Virome Analysis for Identification of Novel Mammalian Viruses in Bat Species from Chinese Provinces

PubMed Central

Wu, Zhiqiang; Ren, Xianwen; Yang, Li; Hu, Yongfeng; Yang, Jian; He, Guimei; Zhang, Junpeng; Dong, Jie; Sun, Lilian; Du, Jiang; Liu, Liguo; Xue, Ying; Wang, Jianmin; Yang, Fan

2012-01-01

Bats are natural hosts for a large variety of zoonotic viruses. This study aimed to describe the range of bat viromes, including viruses from mammals, insects, fungi, plants, and phages, in 11 insectivorous bat species (216 bats in total) common in six provinces of China. To analyze viromes, we used sequence-independent PCR amplification and next-generation sequencing technology (Solexa Genome Analyzer II; Illumina). The viromes were identified by sequence similarity comparisons to known viruses. The mammalian viruses included those of the Adenoviridae, Herpesviridae, Papillomaviridae, Retroviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviridae, Coronaviridae, Picornaviridae, and Parvovirinae; insect viruses included those of the Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae; fungal viruses included those of the Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages included those of the Caudovirales, Inoviridae, and Microviridae and unclassified phages. In addition to the viruses and phages associated with the insects, plants, and bacterial flora related to the diet and habitation of bats, we identified the complete or partial genome sequences of 13 novel mammalian viruses. These included herpesviruses, papillomaviruses, a circovirus, a bocavirus, picornaviruses, a pestivirus, and a foamy virus. Pairwise alignments and phylogenetic analyses indicated that these novel viruses showed little genetic similarity with previously reported viruses. This study also revealed a high prevalence and diversity of bat astroviruses and coronaviruses in some provinces. These findings have expanded our understanding of the viromes of bats in China and hinted at the presence of a large variety of unknown mammalian viruses in many common bat species of mainland China. PMID:22855479

Zoonotic viruses associated with illegally imported wildlife products

USGS Publications Warehouse

Smith, Kristine M.; Anthony, Simon J.; Switzer, William M.; Epstein, Jonathan H.; Seimon, Tracie; Jia, Hongwei; Sanchez, Maria D.; Huynh, Thanh Thao; Galland, G. Gale; Shapiro, Sheryl E.; Sleeman, Jonathan M.; McAloose, Denise; Stuchin, Margot; Amato, George; Kolokotronis, Sergios-Orestis; Lipkin, W. Ian; Karesh, William B.; Daszak, Peter; Marano, Nina

2012-01-01

The global trade in wildlife has historically contributed to the emergence and spread of infectious diseases. The United States is the world's largest importer of wildlife and wildlife products, yet minimal pathogen surveillance has precluded assessment of the health risks posed by this practice. This report details the findings of a pilot project to establish surveillance methodology for zoonotic agents in confiscated wildlife products. Initial findings from samples collected at several international airports identified parts originating from nonhuman primate (NHP) and rodent species, including baboon, chimpanzee, mangabey, guenon, green monkey, cane rat and rat. Pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the NHP samples. These results are the first demonstration that illegal bushmeat importation into the United States could act as a conduit for pathogen spread, and suggest that implementation of disease surveillance of the wildlife trade will help facilitate prevention of disease emergence.

Zoonotic Viruses Associated with Illegally Imported Wildlife Products

PubMed Central

Switzer, William M.; Epstein, Jonathan H.; Seimon, Tracie; Jia, Hongwei; Sanchez, Maria D.; Huynh, Thanh Thao; Galland, G. Gale; Shapiro, Sheryl E.; Sleeman, Jonathan M.; McAloose, Denise; Stuchin, Margot; Amato, George; Kolokotronis, Sergios-Orestis; Lipkin, W. Ian; Karesh, William B.; Daszak, Peter; Marano, Nina

2012-01-01

The global trade in wildlife has historically contributed to the emergence and spread of infectious diseases. The United States is the world's largest importer of wildlife and wildlife products, yet minimal pathogen surveillance has precluded assessment of the health risks posed by this practice. This report details the findings of a pilot project to establish surveillance methodology for zoonotic agents in confiscated wildlife products. Initial findings from samples collected at several international airports identified parts originating from nonhuman primate (NHP) and rodent species, including baboon, chimpanzee, mangabey, guenon, green monkey, cane rat and rat. Pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the NHP samples. These results are the first demonstration that illegal bushmeat importation into the United States could act as a conduit for pathogen spread, and suggest that implementation of disease surveillance of the wildlife trade will help facilitate prevention of disease emergence. PMID:22253731

Foaming in simulated radioactive waste.

PubMed

Bindal, S K; Nikolov, A D; Wasan, D T; Lambert, D P; Koopman, D C

2001-10-01

Radioactive waste treatment process usually involves concentration of radionuclides before waste can be immobilized by storing it in stable solid form. Foaming is observed at various stages of waste processing like SRAT (sludge receipt and adjustment tank) and melter operations. This kind of foaming greatly limits the process efficiency. The foam encountered can be characterized as a three-phase foam that incorporates finely divided solids (colloidal particles). The solid particles stabilize foaminess in two ways: by adsorption of biphilic particles at the surfaces of foam lamella and by layering of particles trapped inside the foam lamella. During bubble generation and rise, solid particles organize themselves into a layered structure due to confinement inside the foam lamella, and this structure provides a barrier against the coalescence of the bubbles, thereby causing foaming. Our novel capillary force balance apparatus was used to examine the particle-particle interactions, which affect particle layer formation in the foam lamella. Moreover, foaminess shows a maximum with increasing solid particle concentration. To explain the maximum in foaminess, a study was carried out on the simulated sludge, a non-radioactive simulant of the radioactive waste sludge at SRS, to identify the parameters that affect the foaming in a system characterized by the absence of surface-active agents. This three-phase foam does not show any foam stability unlike surfactant-stabilized foam. The parameters investigated were solid particle concentration, heating flux, and electrolyte concentration. The maximum in foaminess was found to be a net result of two countereffects that arise due to particle-particle interactions: structural stabilization and depletion destabilization. It was found that higher electrolyte concentration causes a reduction in foaminess and leads to a smaller bubble size. Higher heating fluxes lead to greater foaminess due to an increased rate of foam lamella generation in the sludge system.

PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator

PubMed Central

Regad, Tarik; Saib, Ali; Lallemand-Breitenbach, Valérie; Pandolfi, Pier Paolo; de Thé, Hugues; Chelbi-Alix, Mounira K.

2001-01-01

The promyelocytic leukaemia (PML) protein localizes in the nucleus both in the nucleoplasm and in matrix-associated multiprotein complexes known as nuclear bodies (NBs). The number and the intensity of PML NBs increase in response to interferon (IFN). Overexpression of PML affects the replication of vesicular stomatitis virus and influenza virus. However, PML has a less powerful antiviral activity against these viruses than the IFN mediator MxA. Here, we show that overexpression of PML, but not that of Mx1 or MxA, leads to a drastic decrease of a complex retrovirus, the human foamy virus (HFV), gene expression. PML represses HFV transcription by complexing the HFV transactivator, Tas, preventing its direct binding to viral DNA. This physical interaction requires the N-terminal region of Tas and the RING finger of PML, but does not necessitate PML localization in NBs. Finally, we show that IFN treatment inhibits HFV replication in wild-type but not in PML–/– cells. These findings point to a role for PML in transcriptional repression and suggest that PML could play a key role in mediating an IFN-induced antiviral state against a complex retrovirus. PMID:11432836

Pathogenesis comparison between the United States porcine epidemic diarrhoea virus prototype and S-INDEL-variant strains in conventional neonatal piglets.

PubMed

Chen, Qi; Gauger, Phillip C; Stafne, Molly R; Thomas, Joseph T; Madson, Darin M; Huang, Haiyan; Zheng, Ying; Li, Ganwu; Zhang, Jianqiang

2016-05-01

At least two genetically different porcine epidemic diarrhoea virus (PEDV) strains have been identified in the USA: US PEDV prototype and S-INDEL-variant strains. The objective of this study was to compare the pathogenicity differences of the US PEDV prototype and S-INDEL-variant strains in conventional neonatal piglets under experimental infections. Fifty PEDV-negative 5-day-old pigs were divided into five groups of ten pigs each and were inoculated orogastrically with three US PEDV prototype isolates (IN19338/2013, NC35140/2013 and NC49469/2013), an S-INDEL-variant isolate (IL20697/2014), and virus-negative culture medium, respectively, with virus titres of 104 TCID50 ml- 1, 10 ml per pig. All three PEDV prototype isolates tested in this study, regardless of their phylogenetic clades, had similar pathogenicity and caused severe enteric disease in 5-day-old pigs as evidenced by clinical signs, faecal virus shedding, and gross and histopathological lesions. Compared with pigs inoculated with the three US PEDV prototype isolates, pigs inoculated with the S-INDEL-variant isolate had significantly diminished clinical signs, virus shedding in faeces, gross lesions in small intestines, caeca and colons, histopathological lesions in small intestines, and immunohistochemistry staining in ileum. However, the US PEDV prototype and the S-INDEL-variant strains induced similar viraemia levels in inoculated pigs. Whole genome sequences of the PEDV prototype and S-INDEL-variant strains were determined, but the molecular basis of virulence differences between these PEDV strains remains to be elucidated using a reverse genetics approach.

BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription

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

Wang Jian; Tan Juan; Zhang Xihui

2010-05-10

Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may bemore » responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription.« less

BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription.

PubMed

Wang, Jian; Tan, Juan; Zhang, Xihui; Guo, Hongyan; Zhang, Qicheng; Guo, Tingting; Geng, Yunqi; Qiao, Wentao

2010-05-10

Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may be responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription. Copyright 2010 Elsevier Inc. All rights reserved.

Identification of a broad-spectrum inhibitor of virus RNA synthesis: validation of a prototype virus-based approach

PubMed Central

Filone, Claire Marie; Hodges, Erin N.; Honeyman, Brian; Bushkin, G. Guy; Boyd, Karla; Platt, Andrew; Ni, Feng; Strom, Kyle; Hensley, Lisa; Snyder, John K.; Connor, John H.

2013-01-01

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify new chemical scaffolds for development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity, and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses. PMID:23521799

Foamy Monocytes Are Enriched in cis-7-Hexadecenoic Fatty Acid (16:1n-9), a Possible Biomarker for Early Detection of Cardiovascular Disease.

PubMed

Guijas, Carlos; Meana, Clara; Astudillo, Alma M; Balboa, María A; Balsinde, Jesús

2016-06-23

Human monocytes respond to arachidonic acid, a secretory product of endothelial cells, by activating the de novo pathway of fatty acid biosynthesis, resulting in the acquisition of a foamy phenotype due to accumulation of cytoplasmic lipid droplets. Recruitment of foamy monocytes to endothelium is a key step in the formation of atherosclerotic plaques. Here we describe that lipid droplets of foamy monocytes are enriched in a rather uncommon fatty acid, cis-7-hexadecenoic acid (16:1n-9), a positional isomer of palmitoleic acid. 16:1n-9 was found to possess an anti-inflammatory activity both in vitro and in vivo that is comparable with that of omega-3 fatty acids and clearly distinguishable from the effects of palmitoleic acid. Selective accumulation in neutral lipids of phagocytic cells of an uncommon fatty acid reveals an early phenotypic change that may provide a biomarker of proatherogenicity, and a potential target for intervention in the early stages of cardiovascular disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Construction of three-dimensional graphene interfaces into carbon fiber textiles for increasing deposition of nickel nanoparticles: flexible hierarchical magnetic textile composites for strong electromagnetic shielding

NASA Astrophysics Data System (ADS)

Bian, Xing-Ming; Liu, Lin; Li, Hai-Bing; Wang, Chan-Yuan; Xie, Qing; Zhao, Quan-Liang; Bi, Song; Hou, Zhi-Ling

2017-01-01

Since manipulating electromagnetic waves with electromagnetic active materials for environmental and electric engineering is a significant task, here a novel prototype is reported by introducing reduced graphene oxide (RGO) interfaces in carbon fiber (CF) networks for a hierarchical carbon fiber/reduced graphene oxide/nickel (CF-RGO-Ni) composite textile. Upon charaterizations of the microscopic morphologies, electrical and magnetic properties, the presence of three-dimensional RGO interfaces and bifunctional nickel nanoparticles substantially influences the related physical properties in the resulting hierarchical composite textiles. Eletromagnetic interference (EMI) shielding performance suggests that the hierarchical composite textiles hold a strong shielding effectiveness greater than 61 dB, showing greater advantages than conventional polymeric and foamy shielding composites. As a polymer-free lightweight structure, flexible CF-RGO-Ni composites of all electromagnetic active components offer unique understanding of the multi-scale and multiple mechanisms in electromagnetic energy consumption. Such a novel prototype of shielding structures along with convenient technology highlight a strategy to achieve high-performance EMI shielding, coupled with a universal approach for preparing advanced lightweight composites with graphene interfaces.

Construction of three-dimensional graphene interfaces into carbon fiber textiles for increasing deposition of nickel nanoparticles: flexible hierarchical magnetic textile composites for strong electromagnetic shielding.

PubMed

Bian, Xing-Ming; Liu, Lin; Li, Hai-Bing; Wang, Chan-Yuan; Xie, Qing; Zhao, Quan-Liang; Bi, Song; Hou, Zhi-Ling

2017-01-27

Since manipulating electromagnetic waves with electromagnetic active materials for environmental and electric engineering is a significant task, here a novel prototype is reported by introducing reduced graphene oxide (RGO) interfaces in carbon fiber (CF) networks for a hierarchical carbon fiber/reduced graphene oxide/nickel (CF-RGO-Ni) composite textile. Upon charaterizations of the microscopic morphologies, electrical and magnetic properties, the presence of three-dimensional RGO interfaces and bifunctional nickel nanoparticles substantially influences the related physical properties in the resulting hierarchical composite textiles. Eletromagnetic interference (EMI) shielding performance suggests that the hierarchical composite textiles hold a strong shielding effectiveness greater than 61 dB, showing greater advantages than conventional polymeric and foamy shielding composites. As a polymer-free lightweight structure, flexible CF-RGO-Ni composites of all electromagnetic active components offer unique understanding of the multi-scale and multiple mechanisms in electromagnetic energy consumption. Such a novel prototype of shielding structures along with convenient technology highlight a strategy to achieve high-performance EMI shielding, coupled with a universal approach for preparing advanced lightweight composites with graphene interfaces.

INITIAL SYMPTOMATIC PITUITARY METASTASIS IN A PATIENT WITH PROSTATE FOAMY GLAND CARCINOMA: TAILORING SAFE AND EFFECTIVE THERAPY.

PubMed

Prpić, Marin; Fröbe, Ana; Zadravec, Dijana; Pažanin, Leo; Jakšić, Blanka; Bolanča, Ante; Kusić, Zvonko

2015-06-01

Metastases to pituitary gland are unusual and mostly asymptomatic, presenting with local symptoms in one of ten patients, and only 3%-5% of them are of prostate origin. Here we report and evaluate the effectiveness and safety of multimodal treatment in a patient with pituitary metastasis of a prostate foamy gland carcinoma. A 78-year-old male patient presented with blurred vision and headache without a previous history of malignancy. Magnetic resonance imaging scans revealed a large sellar mass, with infiltration of the surrounding structures. Maximal transsphenoidal reduction of pituitary metastasis was performed, with a histologic finding of metastatic prostate foamy gland adenocarcinoma. Evaluation of the prostate specific antigen revealed a very high level (1461 ng/mL) and foamy gland carcinoma was found on prostate needle biopsy. The patient received 3D conformal external beam radiotherapy with 6 MV photons to the sellar and parasellar region with a tumor dose of 44 Gy, followed by androgen deprivation therapy. Follow up magnetic resonance imaging done after radiotherapy showed shrinkage of the tumor process, with rapid prostate specific antigen decline to 0.3 ng/mL. The visual function was fully established and headache resolved. On the last follow up 14 months after the diagnosis, the patient was alive and free from clinical signs of disease. Tailored treatment, including limited radiotherapy in a higher palliative dose, in a patient with foamy gland symptomatic pituitary metastatic disease resulted in good local and systemic control of the disease. In older male patients with clinical and/or radiologic characteristics suggestive of metastatic pituitary disease, the prostate specific antigen test should be included as part of the work-up.

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins.

PubMed

Wang, Jiawen; Zhang, Wenyan; Lv, Mingyu; Zuo, Tao; Kong, Wei; Yu, Xianghui

2011-12-01

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

Coinfection of Ugandan Red Colobus (Procolobus [Piliocolobus] rufomitratus tephrosceles) with Novel, Divergent Delta-, Lenti-, and Spumaretroviruses ▿

PubMed Central

Goldberg, Tony L.; Sintasath, David M.; Chapman, Colin A.; Cameron, Kenneth M.; Karesh, William B.; Tang, Shaohua; Wolfe, Nathan D.; Rwego, Innocent B.; Ting, Nelson; Switzer, William M.

2009-01-01

Nonhuman primates host a plethora of potentially zoonotic microbes, with simian retroviruses receiving heightened attention due to their roles in the origins of human immunodeficiency viruses type 1 (HIV-1) and HIV-2. However, incomplete taxonomic and geographic sampling of potential hosts, especially the African colobines, has left the full range of primate retrovirus diversity unexplored. Blood samples collected from 31 wild-living red colobus monkeys (Procolobus [Piliocolobus] rufomitratus tephrosceles) from Kibale National Park, Uganda, were tested for antibodies to simian immunodeficiency virus (SIV), simian T-cell lymphotrophic virus (STLV), and simian foamy virus (SFV) and for nucleic acids of these same viruses using genus-specific PCRs. Of 31 red colobus tested, 22.6% were seroreactive to SIV, 6.4% were seroreactive to STLV, and 97% were seroreactive to SFV. Phylogenetic analyses of SIV polymerase (pol), STLV tax and long terminal repeat (LTR), and SFV pol and LTR sequences revealed unique SIV and SFV strains and a novel STLV lineage, each divergent from corresponding retroviral lineages previously described in Western red colobus (Procolobus badius badius) or black-and-white colobus (Colobus guereza). Phylogenetic analyses of host mitochondrial DNA sequences revealed that red colobus populations in East and West Africa diverged from one another approximately 4.25 million years ago. These results indicate that geographic subdivisions within the red colobus taxonomic complex exert a strong influence on retroviral phylogeny and that studying retroviral diversity in closely related primate taxa should be particularly informative for understanding host-virus coevolution. PMID:19692478

Coatings and Biodegradable and Bioabsorbable Films

DTIC Science & Technology

2006-09-01

Properties Properties (Latex) Properties (Paint) M1. 726-39 Sodium lauryl sulfate 2.27 phi Control APS Viscous caossy, some Acceptablecracks, fo a y" l...SS 726-49 Sodium laufyl sulfate 2.17 phr Control APS Viscous, foamy Cracks in film Foam SIS 726-51 Sodium lauryl sulfate 2.17 phi Control APS Crashed...Not Formulated Not Formulated SS 726-35 Sodium lauryl sulfate 2.17 phr Control APS Acceptable Glossy with some Unusually foamy ________ _______fo am

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

PubMed

Dietrich, Isabelle; McMonagle, Elizabeth L; Petit, Sarah J; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N; Towers, Greg J; Hosie, Margaret J; Willett, Brian J

2011-06-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

Feline Tetherin Efficiently Restricts Release of Feline Immunodeficiency Virus but Not Spreading of Infection▿

PubMed Central

Dietrich, Isabelle; McMonagle, Elizabeth L.; Petit, Sarah J.; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N.; Towers, Greg J.; Hosie, Margaret J.; Willett, Brian J.

2011-01-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and “OrfA” proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread. PMID:21490095

Isolation of measles virus in primary rhesus monkey cells from a child with acute interstitial pneumonia who cytologically had giant-cell pneumonia without a rash.

PubMed

Siegel, C; Johnston, S; Adair, S

1990-10-01

The isolation of measles virus in primary Rhesus monkey kidney cells (PRMK) in patients with documented giant-cell pneumonia who have presented without a rash is limited. The diagnosis usually is made by cytologic examination of nasal or bronchial secretions in which characteristic multinucleated giant cells with intranuclear and intracytoplasmic inclusion bodies are observed. The diagnosis of giant-cell pneumonia has been associated with measles virus but not exclusively. Canine distemper, herpes group viruses, and parainfluenza infections have been associated with these cells. In addition, vitamin A deficiency also has been cytologically associated with multinucleated giant cells. The authors describe the isolation of measles virus from bronchial washing and sputum in PRMK cells at 4 days from an 11-year-old child with acute interstitial pneumonia who was in remission for acute lymphocytic leukemia. Classic cytopathologic effect (CPE) consisting of syncytial and hole formation on the PRMK monolayer was apparent. In addition, a foamy appearance of the monolayer was noted in an otherwise clean lot of monkey cells. Confirmatory testing with measles antibody of the infected areas of the monolayer by indirect immunofluorescence (IFA) was positive for measles antigen and negative for mumps, parainfluenza (types I, II, and III) and influenza A and B virus. Serologic studies for measles antibody revealed an IFA IgG titer of greater than 1:10,240, and an IgM titer of 1:128. Cytologic examination of the same bronchial fluid revealed the typical giant cells with characteristic inclusions associated with measles virus. Because this disease usually is severe, and often fatal, prompt recognition of this virus is essential, not only to the patient, who can be treated with immunoglobulin and/or antiviral therapy, but also to prevent the spread of the virus to other patients and medical personnel. These findings also support direct evidence for the etiologic role of measles virus in giant-cell pneumonia that has been detected either histologically or cytologically and in tissue culture at autopsy.

Secondary sea-blue histiocytosis derived from Niemann-Pick disease.

PubMed

Suzuki, Osamu; Abe, Masafumi

2007-04-01

Sea-blue histiocytosis is a rare disorder seen in patients with lipid metabolic or ceroid storage diseases. Sea-blue histiocytes are ceroid-laden macrophages detectable by May-Giemsa staining. We report a case of a 28-year-old woman diagnosed with Niemann-Pick disease at 2 or 3 years of age. To confirm this diagnosis, we examined her bone marrow, which revealed scattered foci containing aggregates of foamy macrophages. May-Giemsa staining identified blue-staining foamy macrophages, referred to as sea-blue histiocytes. In summary, we report the detection of sea-blue histiocytosis in an adult with Niemann-Pick disease.

Full genome sequence of Rocio virus reveal substantial variations from the prototype Rocio virus SPH 34675 sequence.

PubMed

Setoh, Yin Xiang; Amarilla, Alberto A; Peng, Nias Y; Slonchak, Andrii; Periasamy, Parthiban; Figueiredo, Luiz T M; Aquino, Victor H; Khromykh, Alexander A

2018-01-01

Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3'UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639).

Wide distribution and ancient evolutionary history of simian foamy viruses in New World primates.

PubMed

Ghersi, Bruno M; Jia, Hongwei; Aiewsakun, Pakorn; Katzourakis, Aris; Mendoza, Patricia; Bausch, Daniel G; Kasper, Matthew R; Montgomery, Joel M; Switzer, William M

2015-10-29

Although simian foamy viruses (SFV) are the only exogenous retroviruses to infect New World monkeys (NWMs), little is known about their evolutionary history and epidemiology. Previous reports show distinct SFVs among NWMs but were limited to small numbers of captive or wild monkeys from five (Cebus, Saimiri, Ateles, Alouatta, and Callithrix) of the 15 NWM genera. Other studies also used only PCR testing or serological assays with limited validation and may have missed infection in some species. We developed and validated new serological and PCR assays to determine the prevalence of SFV in blood specimens from a large number of captive NWMs in the US (n = 274) and in captive and wild-caught NWMs (n = 236) in Peruvian zoos, rescue centers, and illegal trade markets. Phylogenetic and co-speciation reconciliation analyses of new SFV polymerase (pol) and host mitochondrial cytochrome B sequences, were performed to infer SFV and host co-evolutionary histories. 124/274 (45.2 %) of NWMs captive in the US and 59/157 (37.5 %) of captive and wild-caught NWMs in Peru were SFV WB-positive representing 11 different genera (Alouatta, Aotus, Ateles, Cacajao, Callithrix, Cebus, Lagothrix, Leontopithecus, Pithecia, Saguinus and Saimiri). Seroprevalences were lower at rescue centers (10/53, 18.9 %) compared to zoos (46/97, 47.4 %) and illegal trade markets (3/7, 8/19, 42.9 %) in Peru. Analyses showed that the trees of NWM hosts and SFVs have remarkably similar topologies at the level of species and sub-populations suggestive of co-speciation. Phylogenetic reconciliation confirmed 12 co-speciation events (p < 0.002) which was further supported by obtaining highly similar divergence dates for SFV and host genera and correlated SFV-host branch times. However, four ancient cross-genus transmission events were also inferred for Pitheciinae to Atelidae, Cacajao to ancestral Callithrix or Cebus monkeys, between Callithrix and Cebus monkeys, and Lagothrix to Alouatta. We demonstrate a broad distribution and stable co-speciation history of SFV in NWMs at the species level. Additional studies are necessary to further explore the epidemiology and natural history of SFV infection of NWMs and to determine the zoonotic potential for persons exposed to infected monkeys in captivity and in the wild.

Novel simian foamy virus infections from multiple monkey species in women from the Democratic Republic of Congo

PubMed Central

2012-01-01

Background Zoonotic transmission of simian retroviruses in Central Africa is ongoing and can result in pandemic human infection. While simian foamy virus (SFV) infection was reported in primate hunters in Cameroon and Gabon, little is known about the distribution of SFV in Africa and whether human-to-human transmission and disease occur. We screened 3,334 plasmas from persons living in rural villages in central Democratic Republic of Congo (DRC) using SFV-specific EIA and Western blot (WB) tests. PCR amplification of SFV polymerase sequences from DNA extracted from buffy coats was used to measure proviral loads. Phylogenetic analysis was used to define the NHP species origin of SFV. Participants completed questionnaires to capture NHP exposure information. Results Sixteen (0.5%) samples were WB-positive; 12 of 16 were from women (75%, 95% confidence limits 47.6%, 92.7%). Sequence analysis detected SFV in three women originating from Angolan colobus or red-tailed monkeys; both monkeys are hunted frequently in DRC. NHP exposure varied and infected women lived in distant villages suggesting a wide and potentially diverse distribution of SFV infections across DRC. Plasmas from 22 contacts of 8 WB-positive participants were all WB negative suggesting no secondary viral transmission. Proviral loads in the three women ranged from 14 – 1,755 copies/105 cells. Conclusions Our study documents SFV infection in rural DRC for the first time and identifies infections with novel SFV variants from Colobus and red-tailed monkeys. Unlike previous studies, women were not at lower risk for SFV infection in our population, providing opportunities for spread of SFV both horizontally and vertically. However, limited testing of close contacts of WB-positive persons did not identify human-to-human transmission. Combined with the broad behavioral risk and distribution of NHPs across DRC, our results suggest that SFV infection may have a wider geographic distribution within DRC. These results also reinforce the potential for an increased SFV prevalence throughout the forested regions of Africa where humans and simians co-exist. Our finding of endemic foci of SFV infection in DRC will facilitate longitudinal studies to determine the potential for person-to-person transmissibility and pathogenicity of these zoonotic retroviral infections. PMID:23217108

Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen?

PubMed Central

Allison, Andrew B.; Keel, M. Kevin; Philips, Jamie E.; Cartoceti, Andrew N.; Munk, Brandon A.; Nemeth, Nicole M.; Welsh, Trista I.; Thomas, Jesse M.; Crum, James M.; Lichtenwalner, Anne B.; Fadly, Aly M.; Zavala, Guillermo; Holmes, Edward C.; Brown, Justin D.

2014-01-01

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown. PMID:24503062

Maguari Virus Associated with Human Disease

PubMed Central

Groseth, Allison; Vine, Veronica; Weisend, Carla; Guevara, Carolina; Watts, Douglas; Russell, Brandy; Tesh, Robert B.

2017-01-01

Despite the lack of evidence for symptomatic human infection with Maguari virus (MAGV), its close relation to Cache Valley virus (CVV), which does infect humans, remains a concern. We sequenced the complete genome of a MAGV-like isolate (OBS6657) obtained from a febrile patient in Pucallpa, Ucayali, Peru, in 1998. To facilitate its classification, we generated additional full-length sequences for the MAGV prototype strain, 3 additional MAGV-like isolates, and the closely related CVV (7 strains), Tlacotalpan (1 strain), Playas (3 strains), and Fort Sherman (1 strain) viruses. The OBS6657 isolate is similar to the MAGV prototype, whereas 2 of the other MAGV-like isolates are located on a distinct branch and most likely warrant classification as a separate virus species and 1 is, in fact, a misclassified CVV strain. Our findings provide clear evidence that MAGV can cause human disease. PMID:28726602

Muju Virus, Harbored by Myodes regulus in Korea, Might Represent a Genetic Variant of Puumala Virus, the Prototype Arvicolid Rodent-Borne Hantavirus

PubMed Central

Lee, Jin Goo; Gu, Se Hun; Baek, Luck Ju; Shin, Ok Sarah; Park, Kwang Sook; Kim, Heung-Chul; Klein, Terry A.; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture. PMID:24736214

West Nile Virus: Using Adapted Primary Literature in Mathematical Biology to Teach Scientific and Mathematical Reasoning in High School

NASA Astrophysics Data System (ADS)

Norris, Stephen P.; Macnab, John S.; Wonham, Marjorie; de Vries, Gerda

2009-05-01

This paper promotes the use of adapted primary literature as a curriculum and instruction innovation for use in high school. Adapted primary literature is useful for promoting an understanding of scientific and mathematical reasoning and argument and for introducing modern science into the schools. We describe a prototype adapted from a published article on a mathematical model of the spread of the West Nile virus in North America. The prototype is available as a web-based resource that includes supplemental pedagogical units. Preliminary feedback from use of the prototype in two classrooms is described and a sketch of an ongoing formal evaluation is provided.

Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens.

PubMed

Kwon, Hyuk Moo; LeRoith, Tanya; Pudupakam, R S; Pierson, F William; Huang, Yao-Wei; Dryman, Barbara A; Meng, Xiang-Jin

2011-01-27

A genetically distinct strain of avian hepatitis E virus (avian HEV-VA strain) was isolated from a healthy chicken in Virginia, and thus it is important to characterize and compare its pathogenicity with the prototype strain (avian HEV-prototype) isolated from a diseased chicken. Here we first constructed an infectious clone of the avian HEV-VA strain. Capped RNA transcripts from the avian HEV-VA clone were replication-competent after transfection of LMH chicken liver cells. Chickens inoculated intrahepatically with RNA transcripts of avian HEV-VA clone developed active infection as evidenced by fecal virus shedding, viremia, and seroconversion. To characterize the pathogenicity, RNA transcripts of both avian HEV-VA and avian HEV-prototype clones were intrahepatically inoculated into the livers of chickens. Avian HEV RNA was detected in feces, serum and bile samples from 10/10 avian HEV-VA-inoculated and 9/9 avian HEV-prototype-inoculated chickens although seroconversion occurred only in some chickens during the experimental period. The histopathological lesion scores were lower for avian HEV-VA group than avian HEV-prototype group in the liver at 3 and 5 weeks post-inoculation (wpi) and in the spleen at 3 wpi, although the differences were not statistically significant. The liver/body weight ratio, indicative of liver enlargement, of both avian HEV-VA and avian HEV-prototype groups were significantly higher than that of the control group at 5 wpi. Overall, the avian HEV-VA strain still induces histological liver lesions even though it was isolated from a healthy chicken. The results also showed that intrahepatic inoculation of chickens with RNA transcripts of avian HEV infectious clone may serve as an alternative for live virus in animal pathogenicity studies. Copyright © 2010 Elsevier B.V. All rights reserved.

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12.

PubMed

Brzezinski, Jonathon D; Modi, Apexa; Liu, Mengdan; Roth, Monica J

2016-12-15

Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60 PSPMA 65 ), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25 DLLTEDPPPY 34 , which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70 RREPP 74 ) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70 AAAAA 74 ) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p12 70-74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61 SPIASRLRGRR 71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25 DLLTEDPPPY 34 motif in the N terminus suppresses the ability to tether. This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p12 61-71 Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen?

PubMed

Allison, Andrew B; Kevin Keel, M; Philips, Jamie E; Cartoceti, Andrew N; Munk, Brandon A; Nemeth, Nicole M; Welsh, Trista I; Thomas, Jesse M; Crum, James M; Lichtenwalner, Anne B; Fadly, Aly M; Zavala, Guillermo; Holmes, Edward C; Brown, Justin D

2014-02-01

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown. © 2013 Published by Elsevier Inc.

Characterization of African Human Retroviruses Related to HTLV-III/LAV

DTIC Science & Technology

1988-06-15

the prototype AIDS virus , HIV -1 (4,5). This new human retrovirus has now been termed Human Immunodeficiency Virus Type...lImmunodeficiency Virus and its Relationship to the Human Immunodeficiency Viruses . Nature 32:539-543, 1987. (12) Guyader, M., Emerman, M., Sonigo, P...populations the number of AIDS cases is still quite low suggesting a distinct pathobiology to this new human retrovirus . Cohort studies of

Sensory descriptive quantitative analysis of unpasteurized and pasteurized juçara pulp (Euterpe edulis) during long-term storage

PubMed Central

da Silva, Paula Porrelli Moreira; Casemiro, Renata Cristina; Zillo, Rafaela Rebessi; de Camargo, Adriano Costa; Prospero, Evanilda Teresinha Perissinotto; Spoto, Marta Helena Fillet

2014-01-01

This study evaluated the effect of pasteurization followed by storage under different conditions on the sensory attributes of frozen juçara pulp using quantitative descriptive analysis (QDA). Pasteurization of packed frozen pulp was performed by its immersion in stainless steel tank containing water (80°C) for 5 min, followed by storage under refrigerated and frozen conditions. A trained sensory panel evaluated the samples (6°C) on day 1, 15, 30, 45, 60, 75, and 90. Sensory attributes were separated as follows: appearance (foamy, heterogeneous, purple, brown, oily, and creamy), aroma (sweet and fermented), taste (astringent, bitter, and sweet), and texture (oily and consistent), and compared to a reference material. In general, unpasteurized frozen pulp showed the highest score for foamy appearance, and pasteurized samples showed highest scores to creamy appearance. Pasteurized samples remained stable regarding brown color development while unpasteurized counterparts presented increase. Color is an important attribute related to the product identity. All attributes related to taste and texture remained constant during storage for all samples. Pasteurization followed by storage under frozen conditions has shown to be the best conservation method as samples submitted to such process received the best sensory evaluation, described as foamy, slightly heterogeneous, slightly bitter, and slightly astringent. PMID:25473489

Sensory descriptive quantitative analysis of unpasteurized and pasteurized juçara pulp (Euterpe edulis) during long-term storage.

PubMed

da Silva, Paula Porrelli Moreira; Casemiro, Renata Cristina; Zillo, Rafaela Rebessi; de Camargo, Adriano Costa; Prospero, Evanilda Teresinha Perissinotto; Spoto, Marta Helena Fillet

2014-07-01

This study evaluated the effect of pasteurization followed by storage under different conditions on the sensory attributes of frozen juçara pulp using quantitative descriptive analysis (QDA). Pasteurization of packed frozen pulp was performed by its immersion in stainless steel tank containing water (80°C) for 5 min, followed by storage under refrigerated and frozen conditions. A trained sensory panel evaluated the samples (6°C) on day 1, 15, 30, 45, 60, 75, and 90. Sensory attributes were separated as follows: appearance (foamy, heterogeneous, purple, brown, oily, and creamy), aroma (sweet and fermented), taste (astringent, bitter, and sweet), and texture (oily and consistent), and compared to a reference material. In general, unpasteurized frozen pulp showed the highest score for foamy appearance, and pasteurized samples showed highest scores to creamy appearance. Pasteurized samples remained stable regarding brown color development while unpasteurized counterparts presented increase. Color is an important attribute related to the product identity. All attributes related to taste and texture remained constant during storage for all samples. Pasteurization followed by storage under frozen conditions has shown to be the best conservation method as samples submitted to such process received the best sensory evaluation, described as foamy, slightly heterogeneous, slightly bitter, and slightly astringent.

The Influence of Sulfur on Dephosphorization Kinetics Between Bloated Metal Droplets and Slag Containing FeO

NASA Astrophysics Data System (ADS)

Gu, Kezhuan; Dogan, Neslihan; Coley, Kenneth S.

2017-10-01

The bloating behavior of metal droplets and the dephosphorization behavior of bloated droplets at 1853 K (1580 °C) were investigated using X-ray fluoroscopy coupled with constant volume pressure change measurements and chemical analysis of quenched samples. The effect of sulfur content on dephosphorization kinetics was studied during the decarburization period. The slag foamed during the reaction forming a foamy layer over a dense layer. After a short incubation period, the droplets became bloated due to internal decarburization. The bloated droplets floated from the dense slag into the foamy slag. The behavioral changes are directly related to the effect of sulfur on the incubation time for swelling. The dephosphorization reaction was very fast; droplets with low sulfur contents experienced phosphorus reversion shortly after entering the foamy slag, while those with higher sulfur content took a longer time to swell and went through reversion before they entered the foam. The dephosphorization rate and maximum phosphorus partition were higher at lower CO evolution rates because the dynamic interfacial oxygen potential increased with the decreasing oxygen consumption rate. The rate controlling step for dephosphorization was initially a combination of mass transport in both the metal and the slag. As the iron oxide in the slag was depleted, the rate control shifted to mass transport in slag.

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation

PubMed Central

Genoula, Melanie; Marín Franco, José Luis; Dupont, Maeva; Kviatcovsky, Denise; Milillo, Ayelén; Schierloh, Pablo; Moraña, Eduardo Jose; Poggi, Susana; Palmero, Domingo; Mata-Espinosa, Dulce; González-Domínguez, Erika; León Contreras, Juan Carlos; Barrionuevo, Paula; Rearte, Bárbara; Córdoba Moreno, Marlina Olyissa; Fontanals, Adriana; Crotta Asis, Agostina; Gago, Gabriela; Cougoule, Céline; Neyrolles, Olivier; Maridonneau-Parini, Isabelle; Sánchez-Torres, Carmen; Hernández-Pando, Rogelio; Vérollet, Christel; Lugo-Villarino, Geanncarlo; Sasiain, María del Carmen; Balboa, Luciana

2018-01-01

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10−/− mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence. PMID:29593722

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation.

PubMed

Genoula, Melanie; Marín Franco, José Luis; Dupont, Maeva; Kviatcovsky, Denise; Milillo, Ayelén; Schierloh, Pablo; Moraña, Eduardo Jose; Poggi, Susana; Palmero, Domingo; Mata-Espinosa, Dulce; González-Domínguez, Erika; León Contreras, Juan Carlos; Barrionuevo, Paula; Rearte, Bárbara; Córdoba Moreno, Marlina Olyissa; Fontanals, Adriana; Crotta Asis, Agostina; Gago, Gabriela; Cougoule, Céline; Neyrolles, Olivier; Maridonneau-Parini, Isabelle; Sánchez-Torres, Carmen; Hernández-Pando, Rogelio; Vérollet, Christel; Lugo-Villarino, Geanncarlo; Sasiain, María Del Carmen; Balboa, Luciana

2018-01-01

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14 + cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10 -/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform

PubMed Central

Cnops, Lieselotte; Van den Eede, Peter; Pettitt, James; Heyndrickx, Leo; De Smet, Birgit; Coppens, Sandra; Andries, Ilse; Pattery, Theresa; Van Hove, Luc; Meersseman, Geert; Van Den Herrewegen, Sari; Vergauwe, Nicolas; Thijs, Rein; Jahrling, Peter B.; Nauwelaers, David; Ariën, Kevin K.

2016-01-01

Background. The 2013–2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV). Methods. The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus. Results. The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%. Conclusions. The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola. PMID:27247341

Characterization of an endogenous retrovirus class in elephants and their relatives

PubMed Central

Greenwood, Alex D; Englbrecht, Claudia C; MacPhee, Ross DE

2004-01-01

Background Endogenous retrovirus-like elements (ERV-Ls, primed with tRNA leucine) are a diverse group of reiterated sequences related to foamy viruses and widely distributed among mammals. As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa. Results Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria (Asian and African elephants, manatees, and hyraxes). The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity. Conclusions In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria. PMID:15476555

Greek Goat Encephalitis Virus Strain Isolated from Ixodes ricinus, Greece

PubMed Central

Pavlidou, Vasiliki; Antoniadis, Antonis

2008-01-01

A strain of Greek goat encephaltitis virus was isolated from engorged Ixodes ricinus ticks that had fed on goats in northern Greece. The strain was almost identical to the prototype strain isolated 35 years ago. PMID:18258134

Foamy macrophages and the progression of the human TB granuloma

PubMed Central

Russell, David G.; Cardona, Pere-Joan; Kim, Mi-Jeong; Allain, Sophie; Altare, Frédéric

2009-01-01

The progression of tuberculosis from a latent, sub-clinical infection to active disease that culminates in transmission of infectious bacilli is determined locally at the level of the granuloma. This progression takes place even in the face of a robust immune response that, while it contains infection, is unable to eliminate the bacterium. The factors or environmental conditions that influence this progression remain to be determined. Recent advances have indicated that pathogen-induced dysregulation of host lipid synthesis and sequestration plays a critical role in this transition. The foamy macrophage appears to be a key player in both sustaining persistent bacteria and contributing to the tissue pathology that leads to cavitation and release of infectious bacilli. PMID:19692995

Norwalk virus: How infectious is it?

EPA Science Inventory

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in No...

Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform.

PubMed

Cnops, Lieselotte; Van den Eede, Peter; Pettitt, James; Heyndrickx, Leo; De Smet, Birgit; Coppens, Sandra; Andries, Ilse; Pattery, Theresa; Van Hove, Luc; Meersseman, Geert; Van Den Herrewegen, Sari; Vergauwe, Nicolas; Thijs, Rein; Jahrling, Peter B; Nauwelaers, David; Ariën, Kevin K

2016-10-15

 The 2013-2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV).  The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus.  The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%.  The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola. © 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.

Calcium lignosulphonate: re-evaluation of relevant endpoints to re-confirm validity and NOAEL of a 90-day feeding study in rats.

PubMed

Thiel, Anette; Braun, William; Cary, Maurice G; Engelhardt, Jeffery A; Goodman, Dawn G; Hall, William C; Romeike, Annette; Ward, Jerrold M

2013-08-01

A 90-day feeding study in Han/Wistar rats with calcium lignosulphonate was evaluated by the EFSA. The study was considered to be inadequate due to potentially impaired health status of the animals based upon a high incidence of minimal lymphoid hyperplasia in mesenteric/mandibular lymph nodes and Peyer's patches, and minimal lymphoid cell infiltration in the liver in all animals. The EFSA Panel further disagreed with the conclusion that the treatment-related observation of foamy histiocytosis in mesenteric lymph nodes was non-adverse and asked whether this observation would progress to something more adverse over time. A PWG was convened to assess the sections of lymph nodes, Peyer's patches and liver. In addition, all lymphoid tissues were re-examined. The clinical pathology and animal colony health screening data were re-evaluated. The question whether the foamy histiocytosis could progress to an adverse finding with increasing exposure duration was addressed by read-across. In conclusion, the animals on the 90-day feeding study were in good health, the study was adequate for safety evaluation, and the foamy histiocytes in the mesenteric lymph nodes were not considered adverse, but rather an adaptive response that was considered unlikely to progress to an adverse condition with time. The NOAEL was re-affirmed to be 2000 mg/kgbw/d. Copyright © 2013 Elsevier Inc. All rights reserved.

Genomic Sequences of Australian Bluetongue Virus Prototype Serotypes Reveal Global Relationships and Possible Routes of Entry into Australia

PubMed Central

Bulach, Dieter M.; Amos-Ritchie, Rachel; Adams, Mathew M.; Walker, Peter J.; Weir, Richard

2012-01-01

Bluetongue virus (BTV) is transmitted by biting midges (Culicoides spp.). It causes disease mainly in sheep and occasionally in cattle and other species. BTV has spread into northern Europe, causing disease in sheep and cattle. The introduction of new serotypes, changes in vector species, and climate change have contributed to these changes. Ten BTV serotypes have been isolated in Australia without apparent associated disease. Simplified methods for preferential isolation of double-stranded RNA (dsRNA) and template preparation enabled high-throughput sequencing of the 10 genome segments of all Australian BTV prototype serotypes. Phylogenetic analysis reinforced the Western and Eastern topotypes previously characterized but revealed unique features of several Australian BTVs. Many of the Australian BTV genome segments (Seg-) were closely related, clustering together within the Eastern topotypes. A novel Australian topotype for Seg-5 (NS1) was identified, with taxa spread across several serotypes and over time. Seg-1, -2, -3, -4, -6, -7, -9, and -10 of BTV_2_AUS_2008 were most closely related to the cognate segments of viruses from Taiwan and Asia and not other Australian viruses, supporting the conclusion that BTV_2 entered Australia recently. The Australian BTV_15_AUS_1982 prototype was revealed to be unusual among the Australian BTV isolates, with Seg-3 and -8 distantly related to other BTV sequences from all serotypes. PMID:22514341

Comparative pathogenesis in specific-pathogen-free chickens of two strains of avian hepatitis E virus recovered from a chicken with Hepatitis-Splenomegaly syndrome and from a clinically healthy chicken.

PubMed

Billam, P; LeRoith, T; Pudupakam, R S; Pierson, F W; Duncan, R B; Meng, X J

2009-11-18

Avian hepatitis E virus (avian HEV) is the primary causative agent of Hepatitis-Splenomegaly (HS) syndrome in chickens. Recently, a genetically unique strain of avian HEV, designated avian HEV-VA, was recovered from healthy chickens in Virginia. The objective of this study was to experimentally compare the pathogenicity of the prototype strain recovered from a chicken with HS syndrome and the avian HEV-VA strain in specific-pathogen-free chickens. An infectious stock of the avian HEV-VA strain was first generated and its infectivity titer determined in chickens. For the comparative pathogenesis study, 54 chickens of 6-week-old were assigned to 3 groups of 18 chickens each. The group 1 chickens were each intravenously inoculated with 5x10(2.5) 50% chicken infectious dose of the prototype strain. The group 2 received the same dose of the avian HEV-VA strain, and the group 3 served as negative controls. Six chickens from each group were necropsied at 2, 3 and 4 weeks post-inoculation (wpi). Most chickens in both inoculated groups seroconverted by 3wpi, and the mean anti-avian HEV antibody titers were higher for the prototype strain group than the avian HEV-VA strain group. There was no significant difference in the patterns of viremia and fecal virus shedding. Blood analyte profiles did not differ between treatment groups except for serum creatine phosphokinase levels which were higher for prototype avian HEV group than avian HEV-VA group. The hepatic lesion score was higher for the prototype strain group than the other two groups. The results indicated that the avian HEV-VA strain is only slightly attenuated compared to the prototype strain, suggesting that the full spectrum of HS syndrome is likely associated with other co-factors.

Vesicular disease in 9-week-old pigs experimentally infected with Senecavirus A

USDA-ARS?s Scientific Manuscript database

Senecavirus A (SVA), formerly known as Seneca Valley Virus, is a non-enveloped, single-stranded, positive-sense RNA virus that belongs to the family Picornaviridae and has recently been proposed to be the prototype species of the Senecavirus genus. Although SVA was first identified as a contaminant ...

Phylogenetic analyses indicate little variation among reticuloendotheliosis viruses infecting avian species, including the endangered Attwater's prairie chicken.

PubMed

Bohls, Ryan L; Linares, Jose A; Gross, Shannon L; Ferro, Pam J; Silvy, Nova J; Collisson, Ellen W

2006-08-01

Reticuloendotheliosis virus infection, which typically causes systemic lymphomas and high mortality in the endangered Attwater's prairie chicken, has been described as a major obstacle in repopulation efforts of captive breeding facilities in Texas. Although antigenic relationships among reticuloendotheliosis virus (REV) strains have been previously determined, phylogenetic relationships have not been reported. The pol and env of REV proviral DNA from prairie chickens (PC-R92 and PC-2404), from poxvirus lesions in domestic chickens, the prototype poultry derived REV-A and chick syncytial virus (CSV), and duck derived spleen necrosis virus (SNV) were PCR amplified and sequenced. The 5032bp, that included the pol and most of env genes, of the PC-R92 and REV-A were 98% identical, and nucleotide sequence identities of smaller regions within the pol and env from REV strains examined ranged from 95 to 99% and 93 to 99%, respectively. The putative amino acid sequences were 97-99% identical in the polymerase and 90-98% in the envelope. Phylogenetic analyses of the nucleotide and amino acid sequences indicated the closest relationship among the recent fowl pox-associated chicken isolates, the prairie chicken isolates and the prototype CSV while only the SNV appeared to be distinctly divergent. While the origin of the naturally occurring viruses is not known, the avian poxvirus may be a critical component of transmission of these ubiquitous oncogenic viruses.

Characterization of virulent West Nile virus Kunjin strain, Australia, 2011.

PubMed

Frost, Melinda J; Zhang, Jing; Edmonds, Judith H; Prow, Natalie A; Gu, Xingnian; Davis, Rodney; Hornitzky, Christine; Arzey, Kathleen E; Finlaison, Deborah; Hick, Paul; Read, Andrew; Hobson-Peters, Jody; May, Fiona J; Doggett, Stephen L; Haniotis, John; Russell, Richard C; Hall, Roy A; Khromykh, Alexander A; Kirkland, Peter D

2012-05-01

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.

Vaginal Infections

MedlinePlus

... possible problems include discharge that is: Green or gray Smelly Foamy or lumpy Problems with your discharge ... fluid) than you usually have and that is gray or white Itching around your vagina It’s important ...

Involvement of Escherichia coli in pathogenesis of xanthogranulomatous cholecystitis with scavenger receptor class A and CXCL16-CXCR6 interaction.

PubMed

Sawada, Seiko; Harada, Kenichi; Isse, Kumiko; Sato, Yasunori; Sasaki, Motoko; Kaizaki, Yasuharu; Nakanuma, Yasuni

2007-10-01

Xanthogranulomatous cholecystitis (XGC) is characterized by the infiltration of numerous foamy macrophages. Bacterial infection is thought to be involved in the pathogenesis of XGC. Using XGC and cultured murine biliary epithelial cells (BEC), the participation of E. coli and the role of the scavenger receptor class A (SCARA), as well as chemokine(C-X-C motif) ligand 16 (CXCL16) and its receptor chemokine(C-X-C motif) receptor 6 (CXCR6), were examined in the pathogenesis of XGC. E. coli components and genes were detected in XGC on immunohistochemistry and polymerase chain reaction (PCR), respectively. SCARA-recognizing E. coli was found in foamy macrophages aggregated in xanthogranulomatous lesions. CXCL16, which functions as a membrane-bound molecule and soluble chemokine to induce adhesion and migration of CXCR6(+) cells, was detected on gallbladder epithelia, and CXCR6(+)/CD8(+) T cells and CXCR6(+)/CD68(+) macrophages were also accumulated. In cultured BEC, CXCL16 mRNA and secreted soluble CXCL16 were constantly detected and upregulated by treatment with E. coli and lipopolysaccharide through Toll-like receptor 4. These suggest that SCARA in macrophages is involved in the phagocytosis of E. coli followed by foamy changes and that bacterial infection causes the upregulation of CXCL16 in gallbladder epithelia, leading to the chemoattraction of macrophages via CXCL16-CXCR6 interaction and formation of the characteristic histology of XGC.

Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis

PubMed Central

Caire-Brändli, Irène; Papadopoulos, Alexia; Malaga, Wladimir; Marais, David; Canaan, Stéphane; Thilo, Lutz

2014-01-01

During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis. PMID:24478064

GM1-gangliosidosis in Alaskan huskies: clinical and pathologic findings.

PubMed

Müller, G; Alldinger, S; Moritz, A; Zurbriggen, A; Kirchhof, N; Sewell, A; Baumgärtner, W

2001-05-01

Three Alaskan Huskies, two females and one male, were diagnosed with GM1-gangliosidosis. Clinically, diseased animals exhibited proportional dwarfism and developed progressive neurologic impairment with signs of cerebellar dysfunction at the age of 5-7 months. Skeletal lesions characterized by retarded enchondral ossification of vertebral epiphyses were revealed by radiographs of the male dog at 5.5 months of age. Histologic examination of the central nervous system (CNS) revealed that most neurons were enlarged with a foamy to granular cytoplasm due to tightly packed vacuoles that displaced the Nissl substance. Vacuoles in paraffin-embedded sections stained positively with Luxol fast blue and Grocott's method, and in frozen sections vacuoles were periodic acid-Schiff positive. Foamy vacuolation also occurred within neurons of the autonomic ganglia. Extracerebral cells such as macrophages and peripheral lymphocytes also displayed foamy cytoplasm and vacuolation. In the CNS of diseased animals, a mild demyelination and axonal degeneration was accompanied by a significant astrogliosis (P < 0.05) in the gray matter as compared with age- and sex-matched control dogs. There was also a significant loss (P < 0.05) of oligodendrocytes in the gray and white matter of affected animals as compared with controls. Ultrastructurally, the neuronal storage material consisted of numerous circular to concentric whorls of lamellated membranes or stacks of membranes in parallel arrays. GM1-gangliosidosis in Alaskan Huskies resembles beta-galactosidase deficiency in other canine breeds, and these CNS disorders may be a consequence of neuronal storage and disturbed myelin processing.

Lupus nephritis

MedlinePlus

... time, kidney failure can result. Symptoms Symptoms of lupus nephritis include: Blood in the urine Foamy appearance to urine Swelling (edema) of any area of the body High blood pressure Exams and Tests The health care provider will perform a physical ...

H13 influenza viruses in wild birds have undergone genetic and antigenic diversification in nature.

PubMed

Wang, Zu-Jyun; Kikutani, Yuto; Nguyen, Lam Thanh; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Krauss, Scott; Webby, Richard; Lee, Youn-Jeong; Kida, Hiroshi; Sakoda, Yoshihiro

2018-05-23

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.

Keratomalacia

MedlinePlus

... affected, resulting in an inadequate tear film and dry eyes. People with extreme eye dryness can develop foamy ... keratomalacia is based on the presence of a dry or ulcerated cornea in an undernourished person. ... eye drops or ointments Treatment of vitamin A deficiency ...

Autographa caljfornica nuclear polyhedrosis virus replication in non-permissive Lymantria dispar cell lines

Treesearch

Edward M. Dougherty; David Guzo; Kathleen S. Shields; Dwight E. Lynn; Susan K. Braun

1991-01-01

Autographa californica nuclear polyhedrosis virus (AcNPV) the prototypic group A baculovirus, has the widest reported host range of the baculoviruses and is considered to be one of the most virulent baculoviruses studied. The gypsy moth Lymantria dispar is not considered a natural host of AcNPV, however. To determine the factors...

Molecular Characterization of the Kamese Virus, an Unassigned Rhabdovirus, Isolated from Culex pruina in the Central African Republic.

PubMed

Simo Tchetgna, Huguette Dorine; Nakoune, Emmanuel; Selekon, Benjamin; Gessain, Antoine; Manuguerra, Jean-Claude; Kazanji, Mirdad; Berthet, Nicolas

2017-06-01

Rhabdoviridae is one of the most diversified families of RNA viruses whose members infect a wide range of plants, animals, and arthropods. The members of this family are classified into 13 genera and >150 unassigned viruses. Here, we sequenced the complete genome of a rhabdovirus belonging to the Hart Park serogroup, the Kamese virus (KAMV), isolated in 1977 from Culex pruina in the Central African Republic. The genomic sequence showed an organization typical of rhabdoviruses with additional genes in the P-M and G-L intergenic regions, as already reported for the Hart Park serogroup. Our Kamese strain (ArB9074) had 98% and 78.8% nucleotide sequence similarity with the prototypes of the KAMV and Mossuril virus isolated in Uganda and Mozambique in two different Culex species, respectively. Moreover, the protein sequences had 98-100% amino acid similarity with the prototype of the KAMV, except for an additional gene (U3) that showed a divergence of 6%. These molecular data show that our strain of the KAMV is genetically close to the Culex annuliorus strain that was circulating in Uganda in 1967. However, this study suggests the need to improve our knowledge of the KAMV to better understand its behavior, its life cycle, and its potential reservoirs.

Characterization of tick-borne encephalitis (TBE) foci in Germany and Latvia (1997-2000).

PubMed

Süss, Jochen; Schrader, Christina; Abel, Ulrich; Bormane, Antra; Duks, Arnis; Kalnina, Vaira

2002-06-01

Knowledge concerning the prevalence of the tick-borne encephalitis virus (TBEV) in wild living tick populations is very important for understanding the epidemiology of the disease and for immuno prophylactic strategy. In Germany high and low risk areas of TBE exist. In the years 1997-2000, 533 autochthonous clinical TBE cases were recorded, in the high-risk areas of Bavaria and Baden-Wuerttemberg 140 and 363, and in the low risk areas in Hesse (Odenwald) and Rhineland-Palatinate 22 and 8, respectively. Corresponding to these case reports we have measured the virus prevalence in free living ticks in these four risk areas and compared these findings with the situation in high-risk areas in Latvia. In the years 1997-2000, 2,797 clinical TBE cases were recorded in Latvia. For the studies in Germany, a total of 17,398 Ixodesricinus ticks (14,860 nymphs and 2,538 adults) were collected by flagging and examined for TBEV, in Latvia the corresponding numbers were 525 I. ricinus ticks (350 adults and 175 nymphs) and 281 I. persulcatus ticks (adults only). Information concerning annual and seasonal differences of the TBEV prevalence in natural TBE foci is not available in Germany. This paper is a continuation of the study (Süss et al., 1999), starting in 1997. We investigated every year, in May and September, the virus prevalence in ticks in high risk areas of Bavaria (8 foci) and Baden-Wuerttemberg (5 foci). A total of 15,400 ticks (13,100 nymphs and 2,300 adults) were examined for TBEV. The ticks were tested for the presence of TBEV-RNA using a sensitive, nested-RT-PCR. The virus prevalence in the Bavarian foci of the whole tick population ranged from 0.3 to 2.0% during these four years, in adults between 1.2 and 5.3% and in nymphs between 0.1 and 1.4%. In the high-risk areas of Baden-Wuerttemberg, in the Black Forest, the estimated virus prevalence rates of investigated ticks varied from 0.2 to 3.4%, in adults from 0 to 4.8%, and in nymphs from 0.2 to 3.4%. Using the same methods, we have also tested the low risk areas in the Odenwald (840 nymphs, 160 adults) and in Rhineland-Palatinate (920 nymphs, 78 adults). Ticks were collected in those areas where most TBE cases were registered. The virus prevalence in the Odenwald was 0% in adults and 0.5% in nymphs, whereas in ticks from Rhineland-Palatinate we have not found any positive PCR signal. Sequence data of the PCR products have shown that all strains in Germany were closely related to the central European virus prototype Neudoerfl. In I. ricinus ticks, collected in Riga county, the following virus prevalence rates were found: in females 2.4%, in males 3.7%, and in all adults 3.0%, in nymphs 2.4% and in the I. ricinus tick population examined 2.8%. The virus prevalence in I. persulcatus, collected in the eastern parts of Latvia was 6% in females, 4% in males and 5% in all adults. All the PCR products were sequenced and a phylogenetic tree was constructed. Studies in natural foci of TBE in Latvia have shown that I. ricinus carried the central European virus subtype (prototype Neudoerfl) whereas in I. persulcatus two strains have been found, the central European virus subtype (prototype Neudoerfl) and the Siberian virus subtype (prototype Vasilchenko). Sequences of the Far Eastern subtype have not been detected yet.

Widespread bronchogenic dissemination makes DBA/2 mice more susceptible than C57BL/6 mice to experimental aerosol infection with Mycobacterium tuberculosis.

PubMed

Cardona, Pere-Joan; Gordillo, Sergi; Díaz, Jorge; Tapia, Gustavo; Amat, Isabel; Pallarés, Angeles; Vilaplana, Cristina; Ariza, Aurelio; Ausina, Vicenç

2003-10-01

We have used the murine model of aerosol-induced experimental tuberculosis to assess the effects of four clinical isolates and a reference strain of Mycobacterium tuberculosis on resistant C57BL/6 mice and susceptible DBA/2 mice. Histological studies and detection of 25 cytokines potentially involved in the infection were carried out. DBA/2 mice showed higher concentrations of bacilli in bronchoalveolar lavage fluid and lung tissue. Furthermore, these mice evidenced a larger granulomatous infiltration in the parenchyma due to an increased rate of emigration of infected foamy macrophages from the granulomas to the neighboring pulmonary alveolar spaces. The better control of bacillary concentrations and pulmonary infiltration observed in C57BL/6 mice from week 3 postinfection could result from their higher RANTES, ICAM-1, and gamma interferon (IFN-gamma) mRNA levels. On the other hand, the higher MIP-2 and MCP-3 mRNA levels seen in DBA/2 mice would result in stronger lung recruitment of macrophages and neutrophils. Additionally, DBA/2 mice showed increased inducible nitric oxide synthase expression, induced by the larger number of foamy macrophages, at weeks 18 and 22. This increment was a consequence of phagocytosed bacillary debris, was independent of IFN-gamma expression, and could exert only a bacteriostatic effect. The results of the study suggest that DBA/2 mice are more susceptible than C57BL/6 mice to M. tuberculosis infection due to a higher bronchial dissemination of bacilli inside poorly activated foamy macrophages.

Numerical modeling of the simulated gas hydrate production test at Mallik 2L-38 in the pilot scale pressure reservoir LARS - Applying the "foamy oil" model

NASA Astrophysics Data System (ADS)

Abendroth, Sven; Thaler, Jan; Klump, Jens; Schicks, Judith; Uddin, Mafiz

2014-05-01

In the context of the German joint project SUGAR (Submarine Gas Hydrate Reservoirs: exploration, extraction and transport) we conducted a series of experiments in the LArge Reservoir Simulator (LARS) at the German Research Centre of Geosciences Potsdam. These experiments allow us to investigate the formation and dissociation of hydrates at large scale laboratory conditions. We performed an experiment similar to the field-test conditions of the production test in the Mallik gas hydrate field (Mallik 2L-38) in the Beaufort Mackenzie Delta of the Canadian Arctic. The aim of this experiment was to study the transport behavior of fluids in gas hydrate reservoirs during depressurization (see also Heeschen et al. and Priegnitz et al., this volume). The experimental results from LARS are used to provide details about processes inside the pressure vessel, to validate the models through history matching, and to feed back into the design of future experiments. In experiments in LARS the amount of methane produced from gas hydrates was much lower than expected. Previously published models predict a methane production rate higher than the one observed in experiments and field studies (Uddin et al. 2010; Wright et al. 2011). The authors of the aforementioned studies point out that the current modeling approach overestimates the gas production rate when modeling gas production by depressurization. They suggest that trapping of gas bubbles inside the porous medium is responsible for the reduced gas production rate. They point out that this behavior of multi-phase flow is not well explained by a "residual oil" model, but rather resembles a "foamy oil" model. Our study applies Uddin's (2010) "foamy oil" model and combines it with history matches of our experiments in LARS. Our results indicate a better agreement between experimental and model results when using the "foamy oil" model instead of conventional models of gas flow in water. References Uddin M., Wright J.F. and Coombe D. (2010) - Numerical Study of gas evolution and transport behaviors in natural gas hydrate reservoirs; CSUG/SPE 137439. Wright J.F., Uddin M., Dallimore S.R. and Coombe D. (2011) - Mechanisms of gas evolution and transport in a producing gas hydrate reservoir: an unconventional basis for successful history matching of observed production flow data; International Conference on Gas Hydrates (ICGH 2011).

Production of Antigens and Antibodies for Diagnosis of Arbovirus Diseases

DTIC Science & Technology

1994-05-20

ISS Phl-3 Vlsm2 7 314 VS-Indiana Indiana Lab sm9 5 271 VS-New Jersey Hazelhurst CEl8V4sml 4 364 Zika prototype smlSi 11 501 5 Additionally, 8 viruses ...residual infectivity was inactivated with beta-propiolactone. An additional 8 viruses were passaged in mice and the mice were stored frozen awaiting...beta- propiolactone. An additional 8 viruses were passaged in mice and the mice were stored frozen awaiting sucrose-acetone extraction of the brains

A Retrospective Analysis of Sera Collected by the Hemorrhagic Fever Commission during the Korean Conflict

DTIC Science & Technology

1990-05-01

of leptospirosis ; no diagnosis could be made for the other 14. virus-specific pooled mouse monoclonal antibodies or control fluids: Seropositive...day 7 of disease. Further, 1184 Concise Communications JID 1990:162 INovember) 100,00o - tion of a single case of leptospirosis , the etiology of the...prototype Hantaan tibodies to Rift Valley fever virus in ovine and bovine sera. Am JVet Res 1987:48:1138-1141virus as the causative agent of hemorrhagic

Utilization of feline ELISPOT for mapping vaccine epitopes.

PubMed

Abbott, Jeffrey R; Pu, Ruiyu; Coleman, James K; Yamamoto, Janet K

2012-01-01

A commercial feline immunodeficiency virus (FIV) vaccine consisting of inactivated dual-subtype viruses was released in the USA in 2002 and released subsequently over the next 6 years in Canada, Australia, New Zealand, and Japan. Based on the genetic, morphologic, and biochemical similarities between FIV and human immunodeficiency virus-1 (HIV-1), FIV infection of domestic cats is being used as a small animal model of HIV/AIDS vaccine. Studies on prototype and commercial FIV vaccines provide new insights to the types of immunity and the vaccine epitopes required for an effective human HIV-1 vaccine. ELISPOT assays to detect cytokines, chemokines, and cytolytic mediators are widely used to measure the magnitude and the types of cellular immunity produced by vaccination. Moreover, such approach has identified regions on both HIV-1 and FIV proteins that induce robust antiviral cellular immunity in infected hosts. Using the same strategy, cats immunized with prototype and commercial FIV vaccines are being analyzed by feline interferon-γ and IL-2 ELISPOT systems to identify the vaccine epitope repertoire for prophylaxis.

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.

Johnston Atoll virus (Quaranfil group) from Ornithodoros capensis (Ixodoidea: Argasidae) infesting a gannet colony in New Zealand.

PubMed

Austin, F J

1978-09-01

Ten strains of Johnston Atoll (JA) virus were isolated from Ornithodoros capensis collected in a Gannet (Sula bassana serrator) colony in New Zealand. Its sensitivity to ether and sodium deoxycholate were confirmed and it was shown to have an RNA genome. It multiplied in day-old chicks but, unlike the prototype virus, it was not pathogenic for them. Transmission experiments and the high incidence of birds with neutralizing antibody indicate that the virus is maintained in the colony by a cycle involving ticks and Gannets. This is the first recorded tickborne arbovirus in New Zealand and extends the known range of JA virus from the tropics into the temperate zone.

Improving Virus Taxonomy by Recontextualizing Sequence-Based Classification with Biologically Relevant Data: the Case of the Alphacoronavirus 1 Species

PubMed Central

André, Nicole M.

2018-01-01

ABSTRACT The difficulties related to virus taxonomy have been amplified by recent advances in next-generation sequencing and metagenomics, prompting the field to revisit the question of what constitutes a useful viral classification. Here, taking a challenging classification found in coronaviruses, we argue that consideration of biological properties in addition to sequence-based demarcations is critical for generating useful taxonomy that recapitulates complex evolutionary histories. Within the Alphacoronavirus genus, the Alphacoronavirus 1 species encompasses several biologically distinct viruses. We carried out functionally based phylogenetic analysis, centered on the spike gene, which encodes the main surface antigen and primary driver of tropism and pathogenesis. Within the Alphacoronavirus 1 species, we identify clade A (encompassing serotype I feline coronavirus [FCoV] and canine coronavirus [CCoV]) and clade B (grouping serotype II FCoV and CCoV and transmissible gastroenteritis virus [TGEV]-like viruses). We propose this clade designation, along with the newly proposed Alphacoronavirus 2 species, as an improved way to classify the Alphacoronavirus genus. IMPORTANCE Our work focuses on improving the classification of the Alphacoronavirus genus. The Alphacoronavirus 1 species groups viruses of veterinary importance that infect distinct mammalian hosts and includes canine and feline coronaviruses and transmissible gastroenteritis virus. It is the prototype species of the Alphacoronavirus genus; however, it encompasses biologically distinct viruses. To better characterize this prototypical species, we performed phylogenetic analyses based on the sequences of the spike protein, one of the main determinants of tropism and pathogenesis, and reveal the existence of two subgroups or clades that fit with previously established serotype demarcations. We propose a new clade designation to better classify Alphacoronavirus 1 members. PMID:29299531

Improving Virus Taxonomy by Recontextualizing Sequence-Based Classification with Biologically Relevant Data: the Case of the Alphacoronavirus 1 Species.

PubMed

Whittaker, Gary R; André, Nicole M; Millet, Jean Kaoru

2018-01-01

The difficulties related to virus taxonomy have been amplified by recent advances in next-generation sequencing and metagenomics, prompting the field to revisit the question of what constitutes a useful viral classification. Here, taking a challenging classification found in coronaviruses, we argue that consideration of biological properties in addition to sequence-based demarcations is critical for generating useful taxonomy that recapitulates complex evolutionary histories. Within the Alphacoronavirus genus, the Alphacoronavirus 1 species encompasses several biologically distinct viruses. We carried out functionally based phylogenetic analysis, centered on the spike gene, which encodes the main surface antigen and primary driver of tropism and pathogenesis. Within the Alphacoronavirus 1 species, we identify clade A (encompassing serotype I feline coronavirus [FCoV] and canine coronavirus [CCoV]) and clade B (grouping serotype II FCoV and CCoV and transmissible gastroenteritis virus [TGEV]-like viruses). We propose this clade designation, along with the newly proposed Alphacoronavirus 2 species, as an improved way to classify the Alphacoronavirus genus. IMPORTANCE Our work focuses on improving the classification of the Alphacoronavirus genus. The Alphacoronavirus 1 species groups viruses of veterinary importance that infect distinct mammalian hosts and includes canine and feline coronaviruses and transmissible gastroenteritis virus. It is the prototype species of the Alphacoronavirus genus; however, it encompasses biologically distinct viruses. To better characterize this prototypical species, we performed phylogenetic analyses based on the sequences of the spike protein, one of the main determinants of tropism and pathogenesis, and reveal the existence of two subgroups or clades that fit with previously established serotype demarcations. We propose a new clade designation to better classify Alphacoronavirus 1 members.

An infectious bat chimeric influenza virus harboring the entry machinery of a influenza A virus

PubMed Central

Juozapaitis, Mindaugas; Moreira, Étori Aguiar; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

2017-01-01

In 2012 the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the HA and NA proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event. PMID:25055345

Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

NASA Astrophysics Data System (ADS)

Panicali, Dennis; Paoletti, Enzo

1982-08-01

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

PubMed

Juozapaitis, Mindaugas; Aguiar Moreira, Étori; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

2014-07-23

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

Fast Dissemination of New HIV-1 CRF02/A1 Recombinants in Pakistan

PubMed Central

Chen, Yue; Hora, Bhavna; DeMarco, Todd; Shah, Sharaf Ali; Ahmed, Manzoor; Sanchez, Ana M.; Su, Chang; Carter, Meredith; Stone, Mars; Hasan, Rumina; Hasan, Zahra; Busch, Michael P.; Denny, Thomas N.; Gao, Feng

2016-01-01

A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan. PMID:27973597

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru.

PubMed

Hontz, Robert D; Guevara, Carolina; Halsey, Eric S; Silvas, Jesus; Santiago, Felix W; Widen, Steven G; Wood, Thomas G; Casanova, Wilma; Vasilakis, Nikos; Watts, Douglas M; Kochel, Tadeusz J; Ebihara, Hideki; Aguilar, Patricia V

2015-05-01

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru

PubMed Central

Hontz, Robert D.; Guevara, Carolina; Halsey, Eric S.; Silvas, Jesus; Santiago, Felix W.; Widen, Steven G.; Wood, Thomas G.; Casanova, Wilma; Vasilakis, Nikos; Watts, Douglas M.; Kochel, Tadeusz J.; Ebihara, Hideki

2015-01-01

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America. PMID:25898901

Lipid Droplets and Mycobacterium leprae Infection

PubMed Central

Elamin, Ayssar A.; Stehr, Matthias; Singh, Mahavir

2012-01-01

Leprosy is a chronic infectious disease and is a major source of morbidity in developing countries. Leprosy is caused by the obligate intracellular bacterium Mycobacterium leprae, which infects as primary target Schwann cells. Lepromatous leprosy exhibits multiple lesions of the skin, eyes, nerves, and lymph nodes. The sites of infection are characterized by the presence of foamy macrophages, fully packed with lipid droplets (LDs), which are induced by M. leprae. In the last years, it has become evident that M. tuberculosis imports lipids from foamy macrophages and is dependent on fatty acids for growth in infected macrophages. M. leprae seems to have similar mechanisms for scavenging lipids from the host. But due to the inability to culture M. leprae on laboratory media, research progresses only slowly. However, in the last years, substantial progress has been made in the field of lipid metabolism in M. leprae. Herein, we will present and summarize the lipid droplets formation and the metabolism of lipids during M. leprae infection. PMID:23209912

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

PubMed

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

2015-01-01

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA sequence of the lymphotropic variant of minute virus of mice, MVM(i), and comparison with the DNA sequence of the fibrotropic prototype strain.

PubMed

Astell, C R; Gardiner, E M; Tattersall, P

1986-02-01

The sequence of molecular clones of the genome of MVM(i), a lymphotropic variant of minute virus of mice, was determined and compared with that of MVM(p), the fibrotropic prototype strain. At the nucleotide level there are 163 base changes: 129 transitions and 34 transversions. Most nucleotide changes are silent, with only 27 amino acids changes predicted, of which 22 are conservative. Notable differences between the MVM(i) and MVM(p) genomes which may account for the cell specificities of these viruses occur within the 3' nontranslated regions. The differences discussed include the absence of a 65-base-pair direct in MVM(i), the presence of only two polyadenylation sites in MVM(i) compared with four in MVM(p), and sequences that bear a resemblance to enhancer sequences. Also included in this paper is an important correction to the MVM(p) sequence (C.R. Astell, M. Thomson, M. Merchlinsky, and D. C. Ward, Nucleic Acids Res. 11:999-1018, 1983).

Disabled infectious single animal (DISA) vaccine against Bluetongue by deletion of viroporin-like NS3/NS3a expression is effective, safe, and enables differentiation of infected from vaccinated animals (DIVA)

USDA-ARS?s Scientific Manuscript database

The prototype virus species of the genus Orbivirus (family Reoviridae) is bluetongue virus (BTV) consisting of at least 27 serotypes. Bluetongue is a noncontagious haemorrhagic disease of ruminants spread by competent species of Culicoides biting midges in large parts of the world leading to huge ec...

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana)

PubMed Central

Cajimat, Maria N. B.; Milazzo, Mary Louise; Borchert, Jeff N.; Abbott, Ken D.; Bradley, Robert D.; Fulhorst, Charles F.

2008-01-01

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus. PMID:18304671

Liquid/Gas Flow Mixers

NASA Technical Reports Server (NTRS)

Fabris, Gracio

1994-01-01

Improved devices mix gases and liquids into bubbly or foamy flows. Generates flowing, homogeneous foams or homogeneous dispersions of small, noncoalescing bubbles entrained in flowing liquids. Mixers useful in liquid-metal magnetohydrodynamic electric-power generator, froth flotation in mining industry, wastewater treatment, aerobic digestion, and stripping hydrocarbon contaminants from ground water.

An improved Abbott ARCHITECT assay for the detection of hepatitis B virus surface antigen (HBsAg).

PubMed

Lou, Sheng C; Pearce, Sandra K; Lukaszewska, Teresa X; Taylor, Russell E; Williams, Gregg T; Leary, Thomas P

2011-05-01

The sensitive and accurate detection of hepatitis B virus surface antigen (HBsAg) is critical to the identification of infection and the prevention of transfusion transmitted disease. Improvement in HBsAg assay sensitivity is essential to reduce the window to detect an acute HBV infection. Additionally, the sensitive detection of HBsAg mutants that continue to evolve due to vaccine escape, immune selection and an error prone reverse transcriptase is a necessity. A fully automated HBsAg prototype assay on the Abbott ARCHITECT instrument was developed to improve sensitivity and mutant detection. This magnetic microparticle-based assay utilizes anti-HBsAg monoclonal antibodies to capture antigen present in serum or plasma. Captured antigen is then detected using anti-HBsAg antibody conjugated with the chemiluminescent compound, acridinium. The sensitivity of the ARCHITECT HBsAg prototype assay was improved as compared to the current ARCHITECT, PRISM, and competitor HBsAg assays. The enhancement in assay sensitivity was demonstrated by the use of commercially available HBV seroconversion panels. The prototype assay detected more panel members (185 of 383) vs. the current ARCHITECT (171), PRISM (181), or competitor HBsAg assays (73/140 vs. 62/140, respectively). The ARCHITECT prototype assay also efficiently detected all mutants evaluated. Finally, the sensitivity improvement did not compromise the specificity of the assay (99.94%). An improved Abbott ARCHITECT HBsAg prototype assay with enhanced detection of HBsAg and HBsAg mutants, as well as equivalent specificity was developed for the detection, diagnosis, and management of HBV infection. Copyright © 2011 Elsevier B.V. All rights reserved.

Demonstration & Testing of ClimaStat for Improved DX Air-Conditioning Efficiency

DTIC Science & Technology

2013-04-01

impaired productivity and increased transmission of viruses and bacteria. Allowing indoor RH to rise above an average of 60%rh or a peak of 70%rh can...testing of an engineering prototype culminated in issuance of US Patent 6,427,454 in 2002. Then, development, testing and refinement of a production ...field tests on four Trane (American Standard) systems at a university site were concluded in 2009. A production prototype was constructed based on

Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

PubMed Central

Münk, Carsten; Beck, Thomas; Zielonka, Jörg; Hotz-Wagenblatt, Agnes; Chareza, Sarah; Battenberg, Marion; Thielebein, Jens; Cichutek, Klaus; Bravo, Ignacio G; O'Brien, Stephen J; Lochelt, Martin; Yuhki, Naoya

2008-01-01

Background Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Results Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Conclusion Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher. PMID:18315870

Development of a preliminary design of a method to measure the effectiveness of virus exclusion during water process reclamation at zero-G

NASA Technical Reports Server (NTRS)

Fraser, A. S.; Wells, A. F.; Tenoso, H. J.; Linnecke, C. B.

1976-01-01

Organon Diagnostics has developed, under NASA sponsorship, a monitoring system to test the capability of a water recovery system to reject the passage of viruses into the recovered water. In this system, a non-pathogenic marker virus, bacteriophage F2, is fed into the process stream before the recovery unit and the reclaimed water is assayed for its presence. An engineering preliminary design has been performed as a parallel effort to the laboratory development of the marker virus test system. Engineering schematics and drawings present a preliminary instrument design of a fully functional laboratory prototype capable of zero-G operation.

Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection

PubMed Central

Perez, Daniel Roberto; Sorrell, Erin; Angel, Matthew; Ye, Jianqiang; Hickman, Danielle; Pena, Lindomar; Ramirez-Nieto, Gloria; Kimble, Brian; Araya, Yonas

2009-01-01

On June 11, 2009 the World Health Organization (WHO) declared a new H1N1 influenza pandemic. This pandemic strain is as transmissible as seasonal H1N1 and H3N2 influenza A viruses. Major concerns facing this pandemic are whether the new virus will replace, co-circulate and/or reassort with seasonal H1N1 and/or H3N2 human strains. Using the ferret model, we investigated which of these three possibilities were most likely favored. Our studies showed that the current pandemic virus is more transmissible than, and has a biological advantage over, prototypical seasonal H1 or H3 strains. PMID:20029606

Characterization of a prototype strain of hepatitis E virus.

PubMed

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-15

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia.

76 FR 81496 - Product Cancellation Order for Certain Pesticide Registrations

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-28

... ). 010807-00123 Misty Flea Killer... Phenothrin. 010807-00151 One Shot ``foamy'' Alkyl*dimethylbenzyl... Programs (OPP) Regulatory Public Docket in Rm. S- 4400, One Potomac Yard (South Bldg.), 2777 S. Crystal Dr... list of registrations for which companies paying at one of the maintenance fee caps requested...

Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts.

PubMed

Rose, Patrick P; Hanna, Sheri L; Spiridigliozzi, Anna; Wannissorn, Nattha; Beiting, Daniel P; Ross, Susan R; Hardy, Richard W; Bambina, Shelly A; Heise, Mark T; Cherry, Sara

2011-08-18

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection. Copyright © 2011 Elsevier Inc. All rights reserved.

[Experimental monkey encephalitis caused by Powassan virus].

PubMed

Frolova, M P; Isachkova, L M; Shestopalova, N M; Pogodina, V V

1981-01-01

A comparative study of the experimental infection of monkeys caused by brain P-40 of Powassan virus isolated in the Primorye Territory of the USSR and by the prototype Canadian strain LB was carried out. Powassan virus was found to be pathogenic for Macaca rhesus. Clinical and pathomorphological picture of the experimental encephalitis was studied. Full identity of the infection caused in the monkeys by the strain P-40 and the Canadian strain LB of Powassan virus has been proved. On electronmicroscopic examination of the central nervous system the virus was detected in the neurons, glial cells and intercellular spaces. The virions of the strains studied have identical morphological parameters, being 37 to 45 nm in diameter and having spherical shape. The data obtained point to a marked neurotropism of the virus. They will contribute to elucidation of the virus role in the infectious pathology of man, and namely, in verification of encephalitis cases not associated etiologically with the virus of the spring-summer tick-borne encephalitis.

Antigenic characterization of intermediate adenovirus 14-11 strains associated with upper respiratory illness in a military camp.

PubMed Central

Hierholzer, J C; Pumarola, A

1976-01-01

An unusual variant of adenovirus (AV) 11 was isolated from throat and rectal swabs from six persons with upper respiratory illness in a Spanish military camp in March 1969. The same strain was serologically related to the upper respiratory illness of seven other men among 25 sample cases studied in detail. After strain purification, the virus was grouped as an AV by standard biological tests; it possessed the usual titers of group-specific hexon antigen but only low hemagglutinin titers (1:4 to 1:8) with erythrocytes from selected rhesus monkeys. The virus gave little reaction in hemagglutination inhibition (HI) tests with antisera to AV 1 through 35, but was neutralized to homologous titers by AV 11 antiserum. Reciprocally, rabbit and guinea pig antisera to the isolates possessed high HI antibody titers to prototype AV 14 and high serum neutralization (SN) antibody titers to prototype AV 11. On this basis, the variants were classified as AV 14-11 intermediates. Sequential serum specimens from the patients with and without positive cultures showed diagnostic rises in HI and SN antibody levels to the AV 14-11 intermediate and to prototype AV 11, but little response to AV 14. PMID:177365

Cell-free synthetic biology for in vitro prototype engineering.

PubMed

Moore, Simon J; MacDonald, James T; Freemont, Paul S

2017-06-15

Cell-free transcription-translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. © 2017 The Author(s).

Cell-free synthetic biology for in vitro prototype engineering

PubMed Central

Moore, Simon J.; MacDonald, James T.

2017-01-01

Cell-free transcription–translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. PMID:28620040

Water system virus detection

NASA Technical Reports Server (NTRS)

Fraser, A. S.; Wells, A. F.; Tenoso, H. J.

1975-01-01

A monitoring system developed to test the capability of a water recovery system to reject the passage of viruses into the recovered water is described. A nonpathogenic marker virus, bacteriophage F2, is fed into the process stream before the recovery unit and the reclaimed water is assayed for its presence. Detection of the marker virus consists of two major components, concentration and isolation of the marker virus, and detection of the marker virus. The concentration system involves adsorption of virus to cellulose acetate filters in the presence of trivalent cations and low pH with subsequent desorption of the virus using volumes of high pH buffer. The detection of the virus is performed by a passive immune agglutination test utilizing specially prepared polystyrene particles. An engineering preliminary design was performed as a parallel effort to the laboratory development of the marker virus test system. Engineering schematics and drawings of a fully functional laboratory prototype capable of zero-G operation are presented. The instrument consists of reagent pump/metering system, reagent storage containers, a filter concentrator, an incubation/detector system, and an electronic readout and control system.

Hantavirus pulmonary syndrome.

PubMed

Macneil, Adam; Nichol, Stuart T; Spiropoulou, Christina F

2011-12-01

Hantavirus pulmonary syndrome (HPS) is a severe disease characterized by a rapid onset of pulmonary edema followed by respiratory failure and cardiogenic shock. The HPS associated viruses are members of the genus Hantavirus, family Bunyaviridae. Hantaviruses have a worldwide distribution and are broadly split into the New World hantaviruses, which includes those causing HPS, and the Old World hantaviruses [including the prototype Hantaan virus (HTNV)], which are associated with a different disease, hemorrhagic fever with renal syndrome (HFRS). Sin Nombre virus (SNV) and Andes virus (ANDV) are the most common causes of HPS in North and South America, respectively. Case fatality of HPS is approximately 40%. Pathogenic New World hantaviruses infect the lung microvascular endothelium without causing any virus induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of HPS. This article briefly reviews the knowledge on HPS-associated hantaviruses accumulated since their discovery, less than 20 years ago. Published by Elsevier B.V.

[Electron microscopic study of the An-750 strain of Powassan virus isolated in the Soviet Union].

PubMed

Sobolev, S G; Shestopalova, N M; Linev, M B; Rubin, S G

1978-01-01

Electron microscopic examinations of brains of white mice inoculated with the An 750 strain isolated for the first time from adult mosquitoes and with the prototype LB strain of Powassan virus were carried out. The method of combination of light and electron microscopy used in the study permitted to compare ultrastructural changes in one cell with the results of light microscopy. Sizes of virions and their localizations in the brain cells were determined. Virus particles were found in large and small neurons as well as in glial elements. Subcellular changes in neurons associated with virus multiplication are described. The causes of differences in sizes of virions measured in ultrathin sections are discussed.

Studies of the Suitability of Fowlpox as Decontamination and Thermal Stability Simulant for Variola Major

DTIC Science & Technology

2009-01-01

that 77.6% of cases are still protected against severe disease 70 years after vaccination [5]. Vaccina virus, long considered the prototype of variola...major, has been used as both a simulant and vaccine for variola major. They show remarkable sequence similarity; however, vaccinia virus does not...Another gene shows similar homology to a protein found in yeast, human, tomato and fruit fly. Secondly, the fowlpox genome contains host range genes

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

[Hepatitis C virus: sequence homology of a European isolate and divergence from the prototype].

PubMed

Seelig, R; Seelig, H P; Renz, M

1991-08-01

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in liver biopsies from two patients with chronic hepatitis, in the tissue of a liver implantate, in plasma from four chronic non-A, non-B hepatitis (NANBH) patients and, for the first time, in an infectious anti-D-immunoglobulin preparation. A comparison of the viral sequences coding for a region for the nonstructural NS3 protein from the liver tissues revealed only a very small degree of sequence divergence on the cDNA as well as on the amino acid level (between 0 and 5%). The sequence similarities of the RNA isolated from plasma of the four chronic NANBH patients and the anti-D-immunoglobulin preparation were partly somewhat lower but altogether also high (between 90 and 100%). In contrast, all eight cDNA and amino acid sequences exhibited a significantly higher degree of divergence in comparison with the HCV prototype sequence (between 29 and 32%) than among themselves (between 0 and 10%). This unexpected high sequence similarity of the eight European isolates and their low homology to the Northamerican prototype sequence is indicative for the existence of different types of HCV. This will be important not only for epidemiological studies but also for the development of effective diagnostic procedures and vaccines. Concerning the pathogenesis of NANBH, a double infection or a helper mechanism has to be considered: in addition to the C virus, sequences of an other virus particle were found in the infectious IgG preparation as well as in the liver biopsies.

Characterization of a prototype strain of hepatitis E virus.

PubMed Central

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-01

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia. Images PMID:1731327

Coltiviruses and Seadornaviruses in North America, Europe, and Asia

PubMed Central

Jaafar, Fauziah Mohd; de Micco, Philippe; de Lamballerie, Xavier

2005-01-01

Coltiviruses are tickborne viruses of the genus Coltivirus. The type species, Colorado tick fever virus (from North America), has been isolated from patients with flulike syndromes, meningitis, encephalitis, and other severe complications. Another coltivirus, Eyach virus, has been isolated from ticks in France and Germany and incriminated in febrile illnesses and neurologic syndromes. Seadornaviruses are endemic in Southeast Asia, particularly Indonesia and China. The prototype virus of the genus, Banna virus (BAV), has been isolated from many mosquito species, humans with encephalitis, pigs, and cattle. Two other seadornaviruses, Kadipiro and Liao Ning, were isolated only from mosquitoes. The epidemiology of seadornaviruses remains poorly documented. Evidence suggests that BAV is responsible for encephalitis in humans. Infection with BAV may be underreported because it circulates in regions with a high incidence of Japanese encephalitis and could be misdiagnosed as this disease. PMID:16318717

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.

Six Month Oral Toxicity Study of WR238605 Succinate in Rats. Volume 2

DTIC Science & Technology

1996-02-02

chronic, per {vascular Accumulation, foamy macrophage KIDNEY Mineralization Inf laomat ion, chronic Nephropathy Hydronephrosis Pyelonephritis...KIDNEY # EX Mineralization Inflammation, chronic Nephropathy Hydronephrosis Py»lonaphrItIs Hyperplasia, pelvic epithelium Pigmentation, cortex...Mineralization Inf Lajaaat ion, chronic Nephropathy Hydronephrosis Pigmentation, cor ten (1) - - - ɚ> (1) - ə> - (1) - ə> - 2L

76 FR 23588 - Notice of Receipt of Requests To Voluntarily Cancel Certain Pesticide Registrations

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-27

...-00123 Misty Flea Killer Phenothrin. 010807-00151 One Shot ``foamy'' Alkyl*dimethylbenzylammoniumchloride... EPA-HQ-OPP-2010-0014, by one of the following methods: Federal eRulemaking Portal: http://www... (7502P), Environmental Protection Agency, Rm. S-4400, One Potomac Yard (South Bldg.), 2777 S. Crystal Dr...

Detection and molecular characterization of J subgroup avian leukosis virus in wild ducks in China.

PubMed

Zeng, Xiangwei; Liu, Lanlan; Hao, Ruijun; Han, Chunyan

2014-01-01

To assess the status of avian leukosis virus subgroup J (ALV-J) in wild ducks in China, we examined samples from 528 wild ducks, representing 17 species, which were collected in China over the past 3 years. Virus isolation and PCR showed that 7 ALV-J strains were isolated from wild ducks. The env genes and the 3'UTRs from these isolates were cloned and sequenced. The env genes of all 7 wild duck isolates were significantly different from those in the prototype strain HPRS-103, American strains, broiler ALV-J isolates and Chinese local chicken isolates, but showed close homology with those found in some layer chicken ALV-J isolates and belonged to the same group. The 3'UTRs of 7 ALV-J wild ducks isolates showed close homology with the prototype strain HPRS-103 and no obvious deletion was found in the 3'UTR except for a 1 bp deletion in the E element that introduced a binding site for c-Ets-1. Our study demonstrated the presence of ALV-J in wild ducks and investigated the molecular characterization of ALV-J in wild ducks isolates.

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

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

Arthur, L.O.; Pyle, S.W.; Nara, P.L.

1987-12-01

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/more » cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.« less

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

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

Lamb, Kristen; Lokesh, G.L.; Sherman, Michael

2010-10-25

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to themore » mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.« less

A survey of endogenous retrovirus (ERV) sequences in the vicinity of multiple sclerosis (MS)-associated single nucleotide polymorphisms (SNPs).

PubMed

Brütting, Christine; Emmer, Alexander; Kornhuber, Malte; Staege, Martin S

2016-08-01

Although multiple sclerosis (MS) is one of the most common central nervous system diseases in young adults, little is known about its etiology. Several human endogenous retroviruses (ERVs) are considered to play a role in MS. We are interested in which ERVs can be identified in the vicinity of MS associated genetic marker to find potential initiators of MS. We analysed the chromosomal regions surrounding 58 single nucleotide polymorphisms (SNPs) that are associated with MS identified in one of the last major genome wide association studies. We scanned these regions for putative endogenous retrovirus sequences with large open reading frames (ORFs). We observed that more retrovirus-related putative ORFs exist in the relatively close vicinity of SNP marker indices in multiple sclerosis compared to control SNPs. We found very high homologies to HERV-K, HCML-ARV, XMRV, Galidia ERV, HERV-H/env62 and XMRV-like mouse endogenous retrovirus mERV-XL. The associated genes (CYP27B1, CD6, CD58, MPV17L2, IL12RB1, CXCR5, PTGER4, TAGAP, TYK2, ICAM3, CD86, GALC, GPR65 as well as the HLA DRB1*1501) are mainly involved in the immune system, but also in vitamin D regulation. The most frequently detected ERV sequences are related to the multiple sclerosis-associated retrovirus, the human immunodeficiency virus 1, HERV-K, and the Simian foamy virus. Our data shows that there is a relation between MS associated SNPs and the number of retroviral elements compared to control. Our data identifies new ERV sequences that have not been associated with MS, so far.

Study methods for disinfection water for injection

NASA Astrophysics Data System (ADS)

Grishkanich, Alexander; Zhevlakov, Alexander; Kascheev, Sergey; Polyakov, Vladimir; Sidorov, Igor; Ruzankina, Julia; Yakovlev, Alexey; Mak, Andrey

2016-04-01

Experimental results presented in this study tends to explore viruses in the water for their further decontamination under the influence of laser radiation (λ=220-390 nm). Conducted a series of experiments to study the dependence of water quality from the effects of laser radiation. Correlation between degree of survival of viruses and power density. The results showed that all the analyzed samples of water is clearing from bacteria to 98%. Preliminary tests of the prototype laboratory system UFOVI has opened up new opportunities for water sterilizing.

Experimental encephalitis in monkeys caused by the Powassan virus.

PubMed

Frolova, M P; Isachkova, L M; Shestopalova, N M; Pogodina, V V

1985-01-01

We have carried out a comparative study of the experimental infection of monkeys with the P-40 strain of the Powassen virus, isolated in the Primor'e Territory of the USSR, and with the Canadian prototype LB strain. The Powassan virus was found to be pathogenic for Macaca rhesus. The clinical and pathomorphological picture of the experimental encephalitis was studied, and the full identity of the infection produced in the monkeys by the P-40 strain and the Canadian LB strain of the Powassan virus was demonstrated. On electron microscopic examination of the central nervous system the virus was detected in the neurons, glial cells, and intercellular spaces. The virions of the strains studied have identical morphological parameters, being 37-45 nm in diameter and of spherical shape. The data obtained indicated a marked neurotropism of the virus. They will contribute to the elucidation of the role of the virus in the infection pathology of humans, i.e., in the differentiation of encephalitis cases not associated etiologically with the virus of the spring-summer tickborne encephalitis.

Vaccination against porcine parvovirus protects against disease, but does not prevent infection and virus shedding after challenge infection with a heterologous virus strain.

PubMed

Jóźwik, A; Manteufel, J; Selbitz, H-J; Truyen, U

2009-10-01

The demonstration of field isolates of porcine parvovirus (PPV) that differ genetically and antigenically from vaccine strains of PPV raises the question of whether the broadly used inactivated vaccines can still protect sows against the novel viruses. Ten specific-pathogen-free primiparous sows were assigned to three groups and were vaccinated with one of two vaccines based on the old vaccine strains, or served as non-vaccinated controls. After insemination, all sows were challenged with the prototype genotype 2 virus, PPV-27a, on gestation day 41; fetuses were delivered on gestation day 90 and examined for virus infection. The fetuses of the vaccinated sows were protected against disease, but both the vaccinated and the non-vaccinated sows showed a marked increase in antibody titres after challenge infection, indicating replication of the challenge virus. All sows (vaccinated and non-vaccinated) shed the challenge virus for at least 10 days after infection, with no difference in the pattern or duration of virus shedding.

Norwalk virus: how infectious is it?

PubMed

Teunis, Peter F M; Moe, Christine L; Liu, Pengbo; Miller, Sara E; Lindesmith, Lisa; Baric, Ralph S; Le Pendu, Jacques; Calderon, Rebecca L

2008-08-01

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose-dependent probability of becoming ill, ranging from 0.1 (at a dose of 10(3) NV genomes) to 0.7 (at 10(8) virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models.

Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection: Evidence of competitive advantage of pandemic H1N1 influenza versus seasonal influenza.

PubMed

Perez, Daniel Roberto; Sorrell, Erin; Angel, Matthew; Ye, Jianqiang; Hickman, Danielle; Pena, Lindomar; Ramirez-Nieto, Gloria; Kimble, Brian; Araya, Yonas

2009-08-24

On June 11, 2009 the World Health Organization (WHO) declared a new H1N1 influenza pandemic. This pandemic strain is as transmissible as seasonal H1N1 and H3N2 influenza A viruses. Major concerns facing this pandemic are whether the new virus will replace, co-circulate and/or reassort with seasonal H1N1 and/or H3N2 human strains. Using the ferret model, we investigated which of these three possibilities were most likely favored. Our studies showed that the current pandemic virus is more transmissible than, and has a biological advantage over, prototypical seasonal H1 or H3 strains.

Varicella zoster virus latency

PubMed Central

Eshleman, Emily; Shahzad, Aamir; Cohrs, Randall J

2011-01-01

Primary infection by varicella zoster virus (VZV) typically results in childhood chickenpox, at which time latency is established in the neurons of the cranial nerve, dorsal root and autonomic ganglia along the entire neuraxis. During latency, the histone-associated virus genome assumes a circular episomal configuration from which transcription is epigenetically regulated. The lack of an animal model in which VZV latency and reactivation can be studied, along with the difficulty in obtaining high-titer cell-free virus, has limited much of our understanding of VZV latency to descriptive studies of ganglia removed at autopsy and analogy to HSV-1, the prototype alphaherpesvirus. However, the lack of miRNA, detectable latency-associated transcript and T-cell surveillance during VZV latency highlight basic differences between the two neurotropic herpesviruses. This article focuses on VZV latency: establishment, maintenance and reactivation. Comparisons are made with HSV-1, with specific attention to differences that make these viruses unique human pathogens. PMID:21695042

Identification of novel anelloviruses with broad diversity in UK rodents

PubMed Central

Nishiyama, Shoko; Dutia, Bernadette M.; Stewart, James P.; Meredith, Anna L.; Shaw, Darren J.; Simmonds, Peter

2014-01-01

Anelloviruses are a family of small circular ssDNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small-animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus), but absent from house mice (Mus musculus). The viruses identified have a genomic organization consistent with other anelloviruses, but form two clear phylogenetic groups that are as distinct from each other as from defined genera. PMID:24744300

Molecular phylogeny of edge hill virus supports its position in the yellow Fever virus group and identifies a new genetic variant.

PubMed

Macdonald, Joanne; Poidinger, Michael; Mackenzie, John S; Russell, Richard C; Doggett, Stephen; Broom, Annette K; Phillips, Debra; Potamski, Joseph; Gard, Geoff; Whelan, Peter; Weir, Richard; Young, Paul R; Gendle, Debra; Maher, Sheryl; Barnard, Ross T; Hall, Roy A

2010-06-15

Edge Hill virus (EHV) is a mosquito-borne flavivirus isolated throughout Australia during mosquito surveillance programs. While not posing an immediate threat to the human population, EHV is a taxonomically interesting flavivirus since it remains the only member of the yellow fever virus (YFV) sub-group to be detected within Australia. Here we present both an antigenic and genetic investigation of collected isolates, and confirm taxonomic classification of the virus within the YFV-group. Isolates were not clustered based on geographical origin or time of isolation, suggesting that minimal genetic evolution of EHV has occurred over geographic distance or time within the EHV cluster. However, two isolates showed significant differences in antigenic reactivity patterns, and had a much larger divergence from the EHV prototype (19% nucleotide and 6% amino acid divergence), indicating a distinct subtype or variant within the EHV subgroup.

African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses.

PubMed

Gallardo, Carmina; Sánchez, Elena G; Pérez-Núñez, Daniel; Nogal, Marisa; de León, Patricia; Carrascosa, Ángel L; Nieto, Raquel; Soler, Alejandro; Arias, María Luisa; Revilla, Yolanda

2018-05-03

The risk of spread of African swine fever virus (ASFV) from Russia and Caucasian areas to several EU countries has recently emerged, making it imperative to improve our knowledge and defensive tools against this important pathogen. The ASFV genome encodes many genes which are not essential for virus replication but are known to control host immune evasion, such as NFκB and the NFAT regulator A238L, the apoptosis inhibitor A224L, the MHC-I antigen presenting modulator EP153R, and the A276R gene, involved in modulating type I IFN. These genes are hypothesized to be involved in virulence of the genotype I parental ASFV NH/P68. We here describe the generation of putative live attenuated vaccines (LAV) prototypes by constructing recombinant NH/P68 viruses lacking these specific genes and containing specific markers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency

PubMed Central

Lungu, Octavian; Panagiotidis, Christos A.; Annunziato, Paula W.; Gershon, Anne A.; Silverstein, Saul J.

1998-01-01

Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus. PMID:9618542

Effectiveness of different avian influenza (H5) vaccination regimens in layer chickens on the humoral immune response and interferon-alpha signalling immune marker.

PubMed

Hamad, Mustafa; Amen, Omar; Mahmoud, Mohamed; Hassanin, Ola; Saif-Edin, Mostafa

2018-06-01

Avian influenza (AI) vaccines are widely used to control and eliminate the ongoing avian influenza virus epidemic in Egypt. A strict vaccination policy with inactivated AI vaccines has been widely applied, however the virus still circulating, evolving and causing great negative impact to the poultry sector in Egypt. Therefore, an updated poultry vaccination policy using different vaccine technologies might be valuable as an innovative additional control strategy of AIV in Egypt. In the present study, the effectiveness of different avian influenza (AI) vaccination schedules was evaluated in 300 commercial layer chicks (ISA White) using either the oil-emulsion baculovirus-H5-prototype vaccine (baculovirus-H5 prototype) or turkey herpesvirus (HVT) vector vaccine containing the hemagglutinin (HA) gene from H5N1 strain (rHVT-H5), applied alone or in combination and in different settings. Vaccination with either two injections of the baculovirus-H5 prototype, a single injection of rHVT-H5 or priming with rHVT-H5 at 1 day old followed by boosting with the baculovirus-H5 prototype induced AI-HI protective antibody responses starting as early as 3 to 4 weeks of age and lasting up to the end of the rearing period (16 weeks). A single vaccination with the baculovirus-H5 prototype did not generate a protective antibody titre for the entire rearing period. Furthermore, the present study elucidated that vaccination once or twice with the baculovirus-H5 vaccine prototype activated the chicken interferon-alpha (Ch-IFN-alpha) signalling pathway via transduction of antiviral components, e.g., Mx1 and IRF7. Birds immunized once with rHVT-H5 at 1 day old did not show activation of the Mx1 and IRF7 transcripts; however, following boosting with the baculovirus-H5 prototype vaccine, up-regulation of Mx1 and IRF7 was observed. Based on our findings, it can be concluded that either reinforcement with two injections of the baculovirus-H5 prototype or prime-boost vaccination (rHVT-H5 at 1 day old followed by the baculovirus-H5 prototype vaccine at 8 days old) is a successful strategy to induce both innate and humoral immune responses and could be recommended for the layer production sector over the entire rearing period, especially in AI-endemic areas.

Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine.

PubMed

Berg, Michael G; Adams, Robert J; Gambhira, Ratish; Siracusa, Mark C; Scott, Alan L; Roden, Richard B S; Ketner, Gary

2014-09-01

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Characterization of Farmington virus, a novel virus from birds that is distantly related to members of the family Rhabdoviridae.

PubMed

Palacios, Gustavo; Forrester, Naomi L; Savji, Nazir; Travassos da Rosa, Amelia P A; Guzman, Hilda; Detoy, Kelly; Popov, Vsevolod L; Walker, Peter J; Lipkin, W Ian; Vasilakis, Nikos; Tesh, Robert B

2013-07-01

Farmington virus (FARV) is a rhabdovirus that was isolated from a wild bird during an outbreak of epizootic eastern equine encephalitis on a pheasant farm in Connecticut, USA. Analysis of the nearly complete genome sequence of the prototype CT AN 114 strain indicates that it encodes the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (> 180 nt) in the N and G genes. Phenotypic and genetic characterization of FARV has confirmed that it is a novel rhabdovirus and probably represents a new species within the family Rhabdoviridae. In sum, our analysis indicates that FARV represents a new species within the family Rhabdoviridae.

On the general theory of the origins of retroviruses

PubMed Central

2010-01-01

Background The order retroviridae comprises viruses based on ribonucleic acids (RNA). Some, such as HIV and HTLV, are human pathogens. Newly emerged human retroviruses have zoonotic origins. As far as has been established, both repeated infections (themselves possibly responsible for the evolution of viral mutations (Vm) and host adaptability (Ha)); along with interplay between inhibitors and promoters of cell tropism, are needed to effect retroviral cross-species transmissions. However, the exact modus operadi of intertwine between these factors at molecular level remains to be established. Knowledge of such intertwine could lead to a better understanding of retrovirology and possibly other infectious processes. This study was conducted to derive the mathematical equation of a general theory of the origins of retroviruses. Methods and results On the basis of an arbitrarily non-Euclidian geometrical "thought experiment" involving the cross-species transmission of simian foamy virus (sfv) from a non-primate species Xy to Homo sapiens (Hs), initially excluding all social factors, the following was derived. At the port of exit from Xy (where the species barrier, SB, is defined by the Index of Origin, IO), sfv shedding is (1) enhanced by two transmitting tensors (Tt), (i) virus-specific immunity (VSI) and (ii) evolutionary defenses such as APOBEC, RNA interference pathways, and (when present) expedited therapeutics (denoted e2D); and (2) opposed by the five accepting scalars (At): (a) genomic integration hot spots, gIHS, (b) nuclear envelope transit (NMt) vectors, (c) virus-specific cellular biochemistry, VSCB, (d) virus-specific cellular receptor repertoire, VSCR, and (e) pH-mediated cell membrane transit, (↓pH CMat). Assuming As and Tt to be independent variables, IO = Tt/As. The same forces acting in an opposing manner determine SB at the port of sfv entry (defined here by the Index of Entry, IE = As/Tt). Overall, If sfv encounters no unforeseen effects on transit between Xy and Hs, then the square root of the combined index of sfv transmissibility (√|RTI|) is proportional to the product IO* IE (or ~Vm* Ha* ∑Tt*∑As*Ω), where Ω is the retrovirological constant and ∑ is a function of the ratio Tt/As or As/Tt for sfv transmission from Xy to Hs. Conclusions I present a mathematical formalism encapsulating the general theory of the origins of retroviruses. It summarizes the choreography for the intertwined interplay of factors influencing the probability of retroviral cross-species transmission: Vm, Ha, Tt, As, and Ω. PMID:20158888

A focusing reflectarray and its application in microwave virus sanitizer

NASA Astrophysics Data System (ADS)

Hung, Wan-Ting; Tung, Jen-Jung; Chen, Shih-Yuan

2014-10-01

In this paper, a focusing reflectarray based on the conductor-backed strip dipole unit cell is proposed and designed for use in the microwave virus sanitizer. Unlike traditional far-field antennas that form a planar phase front in a specified far-field direction, the focusing reflectarray is designed to coherently add the fields radiated from the feeding antenna at a predetermined focal point, typically within its radiating near-field region and to ensure adequate power density to inactivate the H3N2 virus sample. Furthermore, the focusing reflectarray has a simple and planar structure compared with conventional focusing antennas. Since the microwave resonant absorption frequency of the H3N2 virus is at about 8 GHz, an 8 × 8 focusing reflectarray is designed for operation at 8 GHz. A prototype antenna is then fabricated and used for H3N2 virus sanitization. It is demonstrated experimentally that the death rate of the H3N2 virus sample is up to 93%, verifying the feasibility of the microwave virus sanitizer as well as the proposed focusing reflectarray.

Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

PubMed

Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

2010-12-01

Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Rapid Detection of Human Immunodeficiency Virus Types 1 and 2 by Use of an Improved Piezoelectric Biosensor

PubMed Central

Severns, Virginia; Branch, Darren W.; Edwards, Thayne L.; Larson, Richard S.

2013-01-01

Disasters can create situations in which blood donations can save lives. However, in emergency situations and when resources are depleted, on-site blood donations require the rapid and accurate detection of blood-borne pathogens, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Techniques such as PCR and antibody capture by an enzyme-linked immunosorbent assay (ELISA) for HIV-1 and HIV-2 are precise but time-consuming and require sophisticated equipment that is not compatible with emergency point-of-care requirements. We describe here a prototype biosensor based on piezoelectric materials functionalized with specific antibodies against HIV-1 and HIV-2. We show the rapid and accurate detection of HIV-1 and HIV-2 in both simple and complex solutions, including human serum, and in the presence of a cross-confounding virus. We report detection limits of 12 50% tissue culture infective doses (TCID50s) for HIV-1 and 87 TCID50s for HIV-2. The accuracy, precision of measurements, and operation of the prototype biosensor compared favorably to those for nucleic acid amplification. We conclude that the biosensor has significant promise as a successful point-of-care diagnostic device for use in emergency field applications requiring rapid and reliable testing for blood-borne pathogens. PMID:23515541

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

PubMed

Ghedin, Elodie; Rogers, Matthew B; Widen, Steven G; Guzman, Hilda; Travassos da Rosa, Amelia P A; Wood, Thomas G; Fitch, Adam; Popov, Vsevolod; Holmes, Edward C; Walker, Peter J; Vasilakis, Nikos; Tesh, Robert B

2013-12-01

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.

Experimental therapies for yellow fever

PubMed Central

Julander, Justin G.

2013-01-01

A number of viruses in the family Flaviviridae are the focus of efforts to develop effective antiviral therapies. Success has been achieved with inhibitors for the treatment of hepatitis C, and there is interest in clinical trials of drugs against dengue fever. Antiviral therapies have also been evaluated in patients with Japanese encephalitis and West Nile encephalitis. However, no treatment has been developed against the prototype flavivirus, yellow fever virus (YFV). Despite the availability of the live, attenuated 17D vaccine, thousands of cases of YF continue to occur each year in Africa and South America, with a significant mortality rate. In addition, a small number of vaccinees develop severe systemic infections with the 17D virus. This paper reviews current efforts to develop antiviral therapies, either directly targeting the virus or blocking detrimental host responses to infection. PMID:23237991

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

Xanthogranulomatous Inflammation of the Female Genital Tract: Report of Three Cases

PubMed Central

Zhang, Xiang-sheng; Dong, Hong-yan; Zhang, Lei-lei; Desouki, Mohamed Mokhtar; Zhao, Chengquan

2012-01-01

Purpose and Methods: This is a series of three cases diagnosed with xanthogranulomatous inflammation of the female genital with emphasis on the etiology, clinical-pathologic features and biological behavior. Clinical, pathologic, radiologic and follow up data are reported. Results: The three cases of Xanthogranulomatous inflammation of the female genital tract are the followings: 1) one case affecting the endometrium, 2) one case affecting the fallopian tube, and 3) one case confined to the ovary. The patient's age was 37, 22 and 62 year-old, respectively. Histologic examination revealed extensive infiltration of foamy histiocytes admixed with variable amount of inflammatory cells. The later include plasma cells, lymphocytes, and occasional multinucleated giant cells. Immunohistochemistry showed positive staining for CD68, a histiocytic marker, in foamy histiocytes, CD3, a T cell marker, and CD20, a B cell marker, in the background lymphocytes. The plasma cells were polyclonal with expression of both κ and λ light chains. Conclusion: Xanthogranulomatous inflammation of the female genital tract is an unusual lesion, and clinically forms mass- like lesion in the pelvic cavity that invades the surrounding tissues, which may mimic the tumor clinically and by imaging. PMID:22393333

Isolation of a new herpes virus from human CD4 sup + T cells

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

Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.

1990-01-01

A new human herpes virus has been isolated from CD4{sup +} T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpesmore » virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date.« less

VizieR Online Data Catalog: Emission-line galaxies from HETDEX pilot survey (Adams+, 2011)

NASA Astrophysics Data System (ADS)

Adams, J. J.; Blanc, G. A.; Hill, G. J.; Gebhardt, K.; Drory, N.; Hao, L.; Bender, R.; Byun, J.; Ciardullo, R.; Cornell, M. E.; Finkelstein, S. L.; Fry, A.; Gawiser, E.; Gronwall, C.; Hopp, U.; Jeong, D.; Kelz, A.; Kelzenberg, R.; Komatsu, E.; MacQueen, P. J.; Murphy, J.; Odoms, P. S.; Roth, M.; Schneider, D. P.; Tufts, J. R.; Wilkinson, C. P.

2011-03-01

We obtained regular fall/winter/spring dark time observations from 2007 September to 2010 February on the McDonald 2.7m Harlan J. Smith telescope with the Visible Integral-field Replicable Unit Spectrograph Prototype (VIRUS-P). (3 data files).

A treatment for and vaccine against the deadly Hendra and Nipah viruses.

PubMed

Broder, Christopher C; Xu, Kai; Nikolov, Dimitar B; Zhu, Zhongyu; Dimitrov, Dimiter S; Middleton, Deborah; Pallister, Jackie; Geisbert, Thomas W; Bossart, Katharine N; Wang, Lin-Fa

2013-10-01

Hendra virus and Nipah virus are bat-borne paramyxoviruses that are the prototypic members of the genus Henipavirus. The henipaviruses emerged in the 1990s, spilling over from their natural bat hosts and causing serious disease outbreaks in humans and livestock. Hendra virus emerged in Australia and since 1994 there have been 7 human infections with 4 case fatalities. Nipah virus first appeared in Malaysia and subsequent outbreaks have occurred in Bangladesh and India. In total, there have been an estimated 582 human cases of Nipah virus and of these, 54% were fatal. Their broad species tropism and ability to cause fatal respiratory and/or neurologic disease in humans and animals make them important transboundary biological threats. Recent experimental findings in animals have demonstrated that a human monoclonal antibody targeting the viral G glycoprotein is an effective post-exposure treatment against Hendra and Nipah virus infection. In addition, a subunit vaccine based on the G glycoprotein of Hendra virus affords protection against Hendra and Nipah virus challenge. The vaccine has been developed for use in horses in Australia and is the first vaccine against a Biosafety Level-4 (BSL-4) agent to be licensed and commercially deployed. Together, these advances offer viable approaches to address Hendra and Nipah virus infection of livestock and people. Published by Elsevier B.V.

Antigenic variation of European haemorrhagic fever with renal syndrome virus strains characterized using bank vole monoclonal antibodies.

PubMed

Lundkvist, A; Fatouros, A; Niklasson, B

1991-09-01

Monoclonal antibodies (MAbs) against Puumala (PUU) virus, the aetiological agent of nephropathia epidemica, were produced by fusing activated spleen cells from a bank vole (Clethrionomys glareolus) with the mouse myeloma cell line SP2/0. This novel approach, utilizing the natural vector of PUU virus for hybridoma production, proved to be highly efficient, and eight stable PUU virus-specific heterohybridomas were isolated and characterized. The bank vole MAbs were all specific for the nucleocapsid protein (N) of PUU virus, as determined by immunoprecipitation. When evaluated by additivity immunoassays, the MAbs were found to recognize several different, distinct or overlapping, epitopes on N. The MAbs were used in immunofluorescence assays to compare eight PUU-related virus isolates, and the prototype Hantaan, Urban rat and Prospect Hill viruses. The reactivity varied among the different MAbs and could be classified into five groups. One MAb reacted exclusively with PUU-related viruses; two MAbs reacted with all PUU-related virus strains tested, as well as Prospect Hill virus, but did not react with Urban rat virus and Hantaan virus; one MAb reacted with all PUU-related virus strains tested and weakly with Hantaan virus, but not with Urban rat and Prospect Hill viruses; two MAbs reacted with all the virus strains tested. Two virus strains, K-27 and CG-1820, isolated in the western U.S.S.R., were distinguished from the other PUU-related virus strains by two MAbs, suggesting that the large group of independently isolated PUU-related viruses may be more heterogeneous than previously believed.

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Epidemic Keratoconjunctivitis Due to the Novel Hexon-Chimeric-Intermediate 22,37/H8 Human Adenovirus ▿

PubMed Central

Aoki, Koki; Ishiko, Hiroaki; Konno, Tsunetada; Shimada, Yasushi; Hayashi, Akio; Kaneko, Hisatoshi; Ohguchi, Takeshi; Tagawa, Yoshitsugu; Ohno, Shigeaki; Yamazaki, Shudo

2008-01-01

In a 2-month period in 2003, we encountered an outbreak of epidemic keratoconjunctivitis (EKC) in Japan. We detected 67 human adenoviruses (HAdVs) by PCR from eye swabs of patients with EKC at five eye clinics in different parts of Japan. Forty-one of the 67 HAdV DNAs from the swabs were identified as HAdV-37 by phylogenetic analysis using a partial hexon gene sequence. When the restriction patterns of these viral genomes were compared with that of the HAdV-37 prototype strain, one isolate showed a never-before-seen restriction pattern. Within 1 year, we encountered three more EKC cases caused by a genetically identical virus: two nosocomial infections at two different university hospitals and a sporadic infection at an eye clinic. We determined the nucleotide sequences of the full-length hexon and fiber genes of these isolates and compared them to those of the 51 prototype strains. Surprisingly, the sequence of the hexon (ɛ determinant) loop-1 and -2 regions showed the highest nucleotide identity with HAdV-22, a rare EKC isolate. However, the nucleotide sequence of the fiber gene was identical to that of the HAdV-8 prototype strain. 22 We propose that this virus is a new hexon-chimeric intermediate HAdV-22,37/H8, and may be an etiological agent of EKC. PMID:18701656

Japanese Encephalitis Virus Immunoglobulin M Antibodies in Porcine Sera

DTIC Science & Technology

1985-10-01

units of other prototype flavivirus suckling mouse brain antigens were used: 2.0 0 0 00 Wesselsbron, Langat , Tembusu, and Dengue type 2. which are o all...strongly reactive :. sentinel pig sera and all 7 positive abattoir sera were Discussion • . retested, using Wesselsbron, Langat , Tembusu, or Den

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

USDA-ARS?s Scientific Manuscript database

Genomes from fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolates were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200...

Characterization of Farmington virus, a novel virus from birds that is distantly related to members of the family Rhabdoviridae

PubMed Central

2013-01-01

Background Farmington virus (FARV) is a rhabdovirus that was isolated from a wild bird during an outbreak of epizootic eastern equine encephalitis on a pheasant farm in Connecticut, USA. Findings Analysis of the nearly complete genome sequence of the prototype CT AN 114 strain indicates that it encodes the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (> 180 nt) in the N and G genes. Phenotypic and genetic characterization of FARV has confirmed that it is a novel rhabdovirus and probably represents a new species within the family Rhabdoviridae. Conclusions In sum, our analysis indicates that FARV represents a new species within the family Rhabdoviridae. PMID:23816310

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor.

PubMed

Rubio, Mari-Paz; López-Bueno, Alberto; Almendral, José M

2005-09-01

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.

Virulent Variants Emerging in Mice Infected with the Apathogenic Prototype Strain of the Parvovirus Minute Virus of Mice Exhibit a Capsid with Low Avidity for a Primary Receptor

PubMed Central

Rubio, Mari-Paz; López-Bueno, Alberto; Almendral, José M.

2005-01-01

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease. PMID:16103180

How Does Vaccinia Virus Interfere With Interferon?

PubMed

Smith, Geoffrey L; Talbot-Cooper, Callum; Lu, Yongxu

2018-01-01

Interferons (IFNs) are secreted glycoproteins that are produced by cells in response to virus infection and other stimuli and induce an antiviral state in cells bearing IFN receptors. In this way, IFNs restrict virus replication and spread before an adaptive immune response is developed. Viruses are very sensitive to the effects of IFNs and consequently have evolved many strategies to interfere with interferon. This is particularly well illustrated by poxviruses, which have large dsDNA genomes and encode hundreds of proteins. Vaccinia virus is the prototypic poxvirus and expresses many proteins that interfere with IFN and are considered in this review. These proteins act either inside or outside the cell and within the cytoplasm or nucleus. They function by restricting the production of IFN by blocking the signaling pathways leading to transcription of IFN genes, stopping IFNs binding to their receptors, blocking IFN-induced signal transduction leading to expression of interferon-stimulated genes (ISGs), or inhibiting the antiviral activity of ISG products. © 2018 Elsevier Inc. All rights reserved.

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.

Development of fire-resistant, low smoke generating, thermally stable end items for aircraft and spacecraft

NASA Technical Reports Server (NTRS)

Gagliani, J.

1978-01-01

A new approach to the problem of flammability by the use of materials obtained from foamy polyimide resins is developed. The ability of these materials to provide fire protection is demonstrated. The development of processes for producing resilient cell foam for use in aircraft seating, thermal acoustical insulation, floor and wall panels, coated glass fabrics, and molded hardware.

Interferon in lyssavirus infection.

PubMed

Rieder, Martina; Finke, Stefan; Conzelmann, Karl-Klaus

2012-01-01

Rabies is a zoonosis still claiming more than 50 000 human deaths per year. Typically, human cases are due to infection with rabies virus, the prototype of the Lyssavirus genus, but sporadic cases of rabies-like encephalitis caused by other lyssaviruses have been reported. In contrast to rabies virus, which has an extremely broad host range including many terrestrial warm-blooded animals, rabies-related viruses are associated predominantly with bats and rarely infect terrestrial species. In spite of a very close genetic relationship of rabies and rabies-related viruses, the factors determining the limited host range of rabies-related viruses are not clear. In the past years the importance of viral countermeasures against the host type I interferon system for establishment of an infection became evident. The rabies virus phosphoprotein (P) has emerged as a critical factor required for paralysing the signalling cascades leading to transcriptional activation of interferon genes as well as interferon signalling pathways, thereby limiting expression of antiviral and immune stimulatory genes. Comparative studies would be of interest in order to determine whether differential abilities of the lyssavirus P proteins contribute to the restricted host range of lyssaviruses.

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

PubMed

Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

2016-11-29

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

Yellow fever virus: genetic and phenotypic diversity and implications for detection, prevention and therapy.

PubMed

Beasley, David W C; McAuley, Alexander J; Bente, Dennis A

2015-03-01

Yellow fever virus (YFV) is the prototypical hemorrhagic fever virus, yet our understanding of its phenotypic diversity and any molecular basis for observed differences in disease severity and epidemiology is lacking, when compared to other arthropod-borne and haemorrhagic fever viruses. This is, in part, due to the availability of safe and effective vaccines resulting in basic YFV research taking a back seat to those viruses for which no effective vaccine occurs. However, regular outbreaks occur in endemic areas, and the spread of the virus to new, previously unaffected, areas is possible. Analysis of isolates from endemic areas reveals a strong geographic association for major genotypes, and recent epidemics have demonstrated the emergence of novel sequence variants. This review aims to outline the current understanding of YFV genetic and phenotypic diversity and its sources, as well as the available animal models for characterizing these differences in vivo. The consequences of genetic diversity for detection and diagnosis of yellow fever and development of new vaccines and therapeutics are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice.

PubMed

Engers, H D; Louis, J A; Zubler, R H; Hirt, B

1981-12-01

A purified preparation of MVM(i), a murine parvovirus closely related to minute virus of mice (MVM), was found to inhibit various functions mediated by murine T cells in vitro. Addition of MVM(i) virus to secondary allogeneic mixed leukocyte cultures resulted in the inhibition of both lymphocyte proliferation (3H-thymidine incorporation) and the generation of cytolytic T lymphocyte activity but not interferon production. MVM(i) virus also inhibited the growth and cytolytic activity of several cloned, long-term Lyt-2+ cytolytic T cell lines. Furthermore, the antigen-induced proliferative responses of parasite- (Leishmania) specific Lyt-1+ T cells in vitro was abrogated by the addition of MVM(i) virus to the culture. Finally, the suppression of an in vitro antibody response to SRBC by MVM(i) virus was the result of the inhibition of T helper cells required for the B cell response. These suppressive effects were specific for MVM(i); parallel studies in which the prototype MVM parvovirus was used showed no significant inhibition in the various systems tested.

Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests.

PubMed

Paneth Iheozor-Ejiofor, Rommel; Levanov, Lev; Hepojoki, Jussi; Strandin, Tomas; Lundkvist, Åke; Plyusnin, Alexander; Vapalahti, Olli

2016-05-01

Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSVΔG*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 105-108, and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.

Murine Models for Viral Hemorrhagic Fever.

PubMed

Gonzalez-Quintial, Rosana; Baccala, Roberto

2018-01-01

Hemorrhagic fever (HF) viruses, such as Lassa, Ebola, and dengue viruses, represent major human health risks due to their highly contagious nature, the severity of the clinical manifestations induced, the lack of vaccines, and the very limited therapeutic options currently available. Appropriate animal models are obviously critical to study disease pathogenesis and develop efficient therapies. We recently reported that the clone 13 (Cl13) variant of the lymphocytic choriomeningitis virus (LCMV-Cl13), a prototype arenavirus closely related to Lassa virus, causes in some mouse strains endothelial damage, vascular leakage, platelet loss, and death, mimicking pathological aspects typically observed in Lassa and other HF syndromes. This model has the advantage that the mice used are fully immunocompetent, allowing studies on the contribution of the immune response to disease progression. Moreover, LCMV is very well characterized and exhibits limited pathogenicity in humans, allowing handling in convenient BSL-2 facilities. In this chapter we outline protocols for the induction and analysis of arenavirus-mediated pathogenesis in the NZB/LCMV model, including mouse infection, virus titer determination, platelet counting, phenotypic analysis of virus-specific T cells, and assessment of vascular permeability.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

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

Kononchik, Joseph P.; Vancini, Ricardo; Brown, Dennis T., E-mail: dennis_brown@ncsu.edu

Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4 h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction ofmore » the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor.« less

Suppression of Poxvirus Replication by Resveratrol.

PubMed

Cao, Shuai; Realegeno, Susan; Pant, Anil; Satheshkumar, Panayampalli S; Yang, Zhilong

2017-01-01

Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV), the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea

PubMed Central

Ghedin, Elodie; Rogers, Matthew B.; Widen, Steven G.; Guzman, Hilda; Travassos da Rosa, Amelia P. A.; Wood, Thomas G.; Fitch, Adam; Popov, Vsevolod; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2013-01-01

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae. PMID:24062532

Provenance and geographic spread of St. Louis encephalitis virus.

PubMed

Kopp, Anne; Gillespie, Thomas R; Hobelsberger, Daniel; Estrada, Alejandro; Harper, James M; Miller, Richard A; Eckerle, Isabella; Müller, Marcel A; Podsiadlowski, Lars; Leendertz, Fabian H; Drosten, Christian; Junglen, Sandra

2013-06-11

St. Louis encephalitis virus (SLEV) is the prototypic mosquito-borne flavivirus in the Americas. Birds are its primary vertebrate hosts, but amplification in certain mammals has also been suggested. The place and time of SLEV emergence remain unknown. In an ecological investigation in a tropical rainforest in Palenque National Park, Mexico, we discovered an ancestral variant of SLEV in Culex nigripalpus mosquitoes. Those SLEV-Palenque strains form a highly distinct phylogenetic clade within the SLEV species. Cell culture studies of SLEV-Palenque versus epidemic SLEV (MSI-7) revealed no growth differences in insect cells but a clear inability of SLEV-Palenque to replicate in cells from birds, cotton rats, and free-tailed bats permissive for MSI-7 replication. Only cells from nonhuman primates and neotropical fruit bats were moderately permissive. Phylogeographic reconstruction identified the common ancestor of all epidemic SLEV strains to have existed in an area between southern Mexico and Panama ca. 330 years ago. Expansion of the epidemic lineage occurred in two waves, the first representing emergence near the area of origin and the second involving almost parallel appearances of the virus in the lower Mississippi and Amazon delta regions. Early diversification events overlapped human habitat invasion during the post-Columbian era. Several documented SLEV outbreaks, such as the 1964 Houston epidemic or the 1990 Tampa epidemic, were predated by the arrival of novel strains between 1 and 4 years before the outbreaks. Collectively, our data provide insight into the putative origins of SLEV, suggesting that virus emergence was driven by human invasion of primary rainforests. IMPORTANCE St. Louis encephalitis virus (SLEV) is the prototypic mosquito-transmitted flavivirus of the Americas. Unlike the West Nile virus, which we know was recently introduced into North America from the Old World, the provenience of SLEV is obscure. In an ecological investigation in a primary rainforest area of Palenque National Park, Mexico, we have discovered an ancestral variant of SLEV. The ancestral virus was much less active than the epidemic virus in cell cultures, reflecting its incomplete adaptation to hosts encountered outside primary rainforests. Knowledge of this virus enabled a spatiotemporal reconstruction of the common ancestor of all SLEVs and how the virus spread from there. We can infer that the cosmopolitan SLEV lineage emerged from Central America in the 17th century, a period of post-Columbian colonial history marked by intense human invasion of primary rainforests. Further spread followed major bird migration pathways over North and South America.

A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

PubMed Central

Déjardin, Jérôme; Bompard-Maréchal, Guillaume; Audit, Muriel; Hope, Thomas J.; Sitbon, Marc; Mougel, Marylène

2000-01-01

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID:10729146

Genetic and antigenic relationships of vesicular stomatitis viruses from South America.

PubMed

Pauszek, Steven J; Barrera, Jose Del C; Goldberg, Tony; Allende, Rossana; Rodriguez, Luis L

2011-11-01

Vesicular stomatitis (VS) viruses have been classified into two serotypes: New Jersey (VSNJV) and Indiana (VSIV). Here, we have characterized field isolates causing vesicular stomatitis in Brazil and Argentina over a 35-year span. Cluster analysis based on either serological relatedness, as inferred from virus neutralization and complement fixation assays, or nucleotide sequences of two separate genes (phosphoprotein or glycoprotein) grouped the field isolates into two distinct monophyletic groups within the Indiana serogroup. One group included seven viruses from Brazil and Argentina that were serologically classified as Indiana-2 and Cocal virus (COCV). The other group contained three viruses from Brazil that were serologically classified as Indiana-3 and the prototype of this group, Alagoas virus (VSAV). Interestingly, two vesiculoviruses that were isolated from insects but do not cause disease in animals, one from Brazil (Maraba virus; MARAV) and the other from Colombia (CoAr 171638), grouped into two separate genetic lineages within the Indiana serotype. Our data provide support for the classification of viruses causing clinical VS in livestock in Brazil and Argentina into two distinct groups: Indiana-2 (VSIV-2) and Indiana-3 (VSIV-3). We suggest using nomenclature for these viruses that includes the serotype, year and place of occurrence, and affected host. This nomenclature is consistent with that currently utilized to describe field isolates of VSNJV or VSIV in scientific literature.

[Genetic characterisation of Powassan virus (POWV) isolated from Haemophysalis longicornis ticks in Primorye and two strains of Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus): Alma-Arasan virus (AAV) isolated from Ixodes persulcatus ticks in Kazakhstan and Malyshevo virus isolated from Aedes vexans nipponii mosquitoes in Khabarovsk kray].

PubMed

L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Deriabin, P G; Gitel'man, A K; Botikov, A G; Aristova, V A

2014-01-01

The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).

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

Molecular identification of enteroviruses including two new types (EV-98 and EV-107) isolated from Japanese travellers from Asian countries.

PubMed

Yamashita, Teruo; Ito, Miyabi; Tsuzuki, Hideaki; Sakae, Kenji; Minagawa, Hiroko

2010-04-01

Of 58 enterovirus strains isolated from Japanese travellers returning from Asian countries, eight were non-serotypable with existing antisera. By sequencing a part of the VP1 region, six of these strains were typed as echovirus 9, enterovirus (EV)-73, EV-79 or EV-97. The nucleotide identity of the VP1 region of isolate T92-1499 to all enterovirus prototypes was <70 %. The VP1 sequence of isolate TN94-0349 was closely related to coxsackievirus (CV)-A9 (73.3 % nucleotide identity), but the virus could not be neutralized with a serum raised against the prototype CV-A9 strain. On the basis of complete molecular comparisons, T92-1499 and TN94-0349 were identified as EV-98 and EV-107, respectively, by the ICTV Picornavirus Study Group. Serum neutralization tests of Japanese individuals revealed a seroprevalence rate of 11 % for EV-73, and even lower seroprevalence rates, 1.0-3.8 %, were found for the other new enteroviruses, suggesting that prior circulation of these viruses in Japan was unlikely.

The impact of calibration and clock-model choice on molecular estimates of divergence times.

PubMed

Duchêne, Sebastián; Lanfear, Robert; Ho, Simon Y W

2014-09-01

Phylogenetic estimates of evolutionary timescales can be obtained from nucleotide sequence data using the molecular clock. These estimates are important for our understanding of evolutionary processes across all taxonomic levels. The molecular clock needs to be calibrated with an independent source of information, such as fossil evidence, to allow absolute ages to be inferred. Calibration typically involves fixing or constraining the age of at least one node in the phylogeny, enabling the ages of the remaining nodes to be estimated. We conducted an extensive simulation study to investigate the effects of the position and number of calibrations on the resulting estimate of the timescale. Our analyses focused on Bayesian estimates obtained using relaxed molecular clocks. Our findings suggest that an effective strategy is to include multiple calibrations and to prefer those that are close to the root of the phylogeny. Under these conditions, we found that evolutionary timescales could be estimated accurately even when the relaxed-clock model was misspecified and when the sequence data were relatively uninformative. We tested these findings in a case study of simian foamy virus, where we found that shallow calibrations caused the overall timescale to be underestimated by up to three orders of magnitude. Finally, we provide some recommendations for improving the practice of molecular-clock calibration. Copyright © 2014 Elsevier Inc. All rights reserved.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

PubMed Central

Humbert, Olivier; Chan, Frieda; Rajawat, Yogendra S.; Torgerson, Troy R.; Burtner, Christopher R.; Hubbard, Nicholas W.; Humphrys, Daniel; Norgaard, Zachary K.; O’Donnell, Patricia; Adair, Jennifer E.; Trobridge, Grant D.; Scharenberg, Andrew M.; Felsburg, Peter J.; Rawlings, David J.

2018-01-01

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases. PMID:29720491

The discovery of the enteroviruses and the classification of poliovirus among them.

PubMed

Melnick, J L

1993-12-01

The history of the enteroviruses is described, and how poliovirus came to be recognized as the prototype species of the genus, a subdivision of the family Picornaviridae. Albert Sabin was one of the main contributors. He isolated several enterovirus types and established them as causative agents of human disease. The enteroviruses were discovered only after new methods were introduced for working with viruses. They are now recognized as constituting one of the genera of the picornavirus family. Pico-rna-virus stands for viruses which are small (pico), and have an RNA genome. The enterovirus genus includes the polioviruses, the coxsackieviruses and the echoviruses of humans, plus a number of enteroviruses of lower animals (e.g., monkeys, cattle, pigs, mice). Over 100 serotypes are now recognized, the first having been the polioviruses.

Lubricant Foaming and Aeration Study. Part 2.

DTIC Science & Technology

1985-12-01

phosphate. The blend 0-77-10, composed of tmp-heptanoate plus neopentyl glycol esters, tested in the same way and with the same combination of solutes at...the same concentrations, showed about half the foaminess of the unblended tmp-heptanoate. The neopentyl glycol esters are, therefore, less...substituent methyl groups in a solute confer profoaming activity in these neopentyl glycol esters as solvents. Also, not forgotten, is that the

Manual Fire Suppression Methods on Typical Machinery Space Spray Fires

DTIC Science & Technology

1990-07-31

Aqueous Film Forming Foam Manuscnpt approved April 25, 1990. ( AFFF ), has been incorporated in machinery space fire protection systems to...distribution unlimited. 13. ABSTRACT (Maximum 200 words) A series of tests was conducted to evaluate the effectiveness of Aqueous Film Forming Foami ( AFFF ...machinery space fire protection systems to control running fuel and fuel spray fires (PKP side of TAFES), and bilge fires ( aqueous film forming foam

Insulated Foamy Viral Vectors

PubMed Central

Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

2016-01-01

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

Efficacy and safety of a clinically relevant foamy vector design in human hematopoietic repopulating cells.

PubMed

Everson, Elizabeth M; Hocum, Jonah D; Trobridge, Grant D

2018-06-23

Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. Here we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1α and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). Human CD34 + cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. FV-EGW-A1 resulted in high-marking, multi-lineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. An FV vector with an elongation factor-1α promoter and an A1 insulator is a promising vector design for use in the clinic. This article is protected by copyright. All rights reserved.

Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

PubMed Central

Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

2014-01-01

ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development. PMID:25008922

Evidence in Gabon for an intrafamilial clustering with mother-to-child and sexual transmission of a new molecular variant of human T-lymphotropic virus type-II subtype B.

PubMed

Tuppin, P; Gessain, A; Kazanji, M; Mahieux, R; Cosnefroy, J Y; Tekaia, F; Georges-Courbot, M C; Georges, A; de Thé, G

1996-01-01

Following the observation of an HTLV-II seropositive 60-year-old woman living in Gabon (Central Africa), a serologic and molecular study of her family members was conducted in an attempt to determine the duration of the HTLV-II infection and the modes of transmission of the virus. Among 41 family members, five were HTLV-I seropositive and 7 exhibited specific HTLV-II antibodies in their sera as demonstrated by high immunofluorescence titers on C19 cells and/or specific Western-blot pattern. The second husband of the index case and two of his sisters were infected by the virus, suggesting the presence of HTLV-II in this family over two generations. Sequence analysis of an amplified fragment of 172 nucleotides within the gp21 of the env region (6469-6640) of four HTLV-II infected individuals revealed a new HTLV-II molecular variant of the subtype b diverging from the prototypes NRA and G12 by seven (4.1%) and five (2.9%) bases substitutions, respectively. Molecular analysis of the total env gene (1462 bp) and fragments of the pol and pX regions confirmed that this new African variant was the most divergent HTLV-II subtype b yet described, exhibiting 2.3% of nucleotide substitutions in the env gene (33 bases) as compared to the two HTLV-II b prototypes. These data demonstrate, for the first time in Africa, intrafamilial both mother-to-child transmission and sexual transmission between spouses of an HTLV-II b molecular variant, and also suggest that this virus has been present in Gabon for a long period of time.

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

PubMed Central

Jansen van Vuren, Petrus; Grobbelaar, Antoinette; Storm, Nadia; Conteh, Ousman; Konneh, Kelfala; Kamara, Abdul; Sanne, Ian

2015-01-01

The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks. PMID:26637383

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015.

PubMed

Wen, X; Zhu, D; Cheng, A; Wang, M; Chen, S; Jia, R; Liu, M; Sun, K; Zhao, X; Yang, Q; Wu, Y; Chen, X

2018-02-01

Duck hepatitis A virus (DHAV) is the most common aetiologic agent of duck virus hepatitis (DVH), causing substantial economic losses in the duck industry worldwide. In China, officially approved DHAV-1 live-attenuated vaccines have been used widely to vaccinate breeder ducks since 2013. However, following the reports of DVH outbreaks, it has become necessary to assess the epidemiological situation of this virus in China. We conducted molecular epidemiological analyses of 32 DHAV field isolates while analysing the samples from ducks suspected of having hepatitis collected from commercial duck farms in China between May 2010 and December 2015. Considerable changes were observed in the epidemiology of DHAV-1 and DHAV-3 in China over time. A higher number of DHAV-1 strains were isolated during 2010-2012, coinciding with the widespread use of officially approved DHAV-1 live vaccine strains beginning in 2013. In contrast, a higher rate of DHAV-3 causing DHAV infections was observed between 2013 and 2015. Phylogenetic analyses based on the full-length VP1 gene were performed on these field isolates and using reference strains available in GenBank. DHAV-1 field isolates were evaluated in two groups: one group closely related to prototype strains and circulating in China between 2010 and 2012 and another group exhibiting genetic and serological differences from prototype strains. All DHAV-3 strains isolated in this study were grouped as monophyletic, which has become the predominant viral type, particularly in Shandong and Sichuan provinces, since 2013. In conclusion, these data provide updated information on the genetic and serological diversity of DHAV-1 and DHAV-3, and our findings may serve as a foundation for the prevention of, and vaccine development for, DHAV in China. © 2017 Blackwell Verlag GmbH.

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

Eukaryotic-Like Virus Budding in Archaea

PubMed Central

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

2016-01-01

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

Focal ratio degradation and transmission in VIRUS-P optical fibers

NASA Astrophysics Data System (ADS)

Murphy, Jeremy D.; MacQueen, Phillip J.; Hill, Gary J.; Grupp, Frank; Kelz, Andreas; Palunas, Povilas; Roth, Martin; Fry, Alexander

2008-07-01

We have conducted extensive tests of both transmission and focal ratio degradation (FRD) on two integral field units currently in use on the VIRUS-P integral field spectrograph. VIRUS-P is a prototype for the VIRUS instrument proposed for the Hobby-Eberly Telescope at McDonald Observatory. All tests have been conducted at an input f-ratio of F/3.65 and with an 18% central obscuration in order to simulate optical conditions on the HET. Transmission measurements were conducted with narrow-band interference filters (FWHM: 10 nm) at 10 discrete wavelengths (337 to 600 nm), while FRD tests were made at 365 nm, 400 nm and 600 nm. The influence of wavelength, end immersion, fiber type and length on both FRD and transmission is explored. Most notably, we find no wavelength dependence on FRD down to 365 nm. All fibers tested are within the VIRUS instrument specifications for both FRD and transmission. We present the details of our differential FRD testing method and explain a simple and robust technique of aligning the test bench and optical fiber axes to within +/-0.1 degrees.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

PubMed Central

Stohr, Thomas; Palomo, Concepción; Piedra, Pedro A.; Gilbert, Brian E.; Mas, Vicente; Millar, Andrena; Power, Ultan F.; Stortelers, Catelijne; Allosery, Koen; Melero, José A.; Depla, Erik

2015-01-01

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease. PMID:26438495

Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver–Chimeric Mouse Models

PubMed Central

Bukh, Jens; Meuleman, Philip; Tellier, Raymond; Engle, Ronald E.; Feinstone, Stephen M.; Eder, Gerald; Satterfield, William C.; Govindarajan, Sugantha; Krawczynski, Krzysztof; Miller, Roger H.; Leroux-Roels, Geert; Purcell, Robert H.

2010-01-01

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1–6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >104.7 IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 103–105 chimpanzee infectious doses/mL. Human liver–chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1–6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo. PMID:20353362

Variation in susceptibility to oral infection with dengue viruses among geographic strains of Aedes aegypti.

PubMed

Gubler, D J; Nalim, S; Tan, R; Saipan, H; Sulianti Saroso, J

1979-11-01

The comparative susceptibility of 13 geographic strains of Aedes aegypti to oral infection with dengue viruses was studied by feeding the mosquitoes on a virus-erythrocyte-sugar suspension. Significant variation in susceptibility to four dengue serotypes was observed among the geographic strains tested. Mosquito strains which were more susceptible to one serotype were also more susceptible to the other serotypes, suggesting that the factors controlling susceptibility were the same for all types. The amount of virus required to infect mosquitoes orally varied inversely with the susceptibility of the geographic strain. Thresholds of infection were not the same for dengue types 1, 2, 3 and 4. There was no apparent difference in infectivity between prototype and recently isolated strains of dengue types 1 and 3. Crossing experimentibility as the resistant parent. No difference was observed between resistant and susceptible mosquito strains in the rate or the amount of viral replication after infection by the parenteral route, or in their ability to transmit dengue 2 virus after infection by the oral route.

Feline fecal virome reveals novel and prevalent enteric viruses

PubMed Central

Ng, Terry Fei Fan; Mesquita, João Rodrigo; Nascimento, Maria São José; Kondov, Nikola O.; Wong, Walt; Reuter, Gábor; Knowles, Nick J.; Vega, Everardo; Esona, Mathew D.; Deng, Xutao; Vinjé, Jan; Delwart, Eric

2014-01-01

Humans keep more than 80 million cats worldwide, ensuring frequent contacts with their viruses. Despite such interactions the enteric virome of cats remains poorly understood. We analyzed a fecal sample from a single healthy cat from Portugal using viral metagenomics and detected five eukaryotic viral genomes. These viruses included a novel picornavirus (proposed genus “Sakobuvirus”) and bocavirus (feline bocavirus 2), a variant of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus. Feline astroviruses (mamastrovirus 2) are the closest relatives of the classic human astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7% (astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal samples. Co-infections were frequent with 40% (4/10) of cats shedding more than one of these viruses. Our study provides an initial unbiased description of the feline fecal virome indicating a high level of asymptomatic infections. Availability of the genome sequences of these viruses will facilitate future tropism and disease association studies. PMID:24793097

Designing for Learner Engagement in Middle School Science: Technology, Inquiry, and the Hierarchies of Engagement

ERIC Educational Resources Information Center

Harmer, Andrea J.; Cates, Ward Mitchell

2007-01-01

Engaging middle-school students in scientific inquiry is typically recognized as important, but difficult. Designed to foster learner engagement, this method used an online, problem-based, science inquiry that investigated the West Nile virus during four weeks of collaborative classroom sessions. The inquiry prototype was authored in WISE, the…

[The Alkhurma virus (family Flaviviridae, genus Flavivirus): an emerging pathogen responsible for hemorrhage fever in the Middle East].

PubMed

Charrel, R N; de Lamballerie, X

2003-01-01

To date tick-borne flaviviruses causing hemorrhagic fevers in humans have been isolated in Siberia (Omsk hemorrhagic fever virus), India (Kyasanur Forest disease virus), and Saudi Arabia (Akhurma virus). Because of their potential use as biological weapons for bioterrorism, these 3 viruses require level 4 biosafety handling facilities and have been listed as hypervirulent pathogens by the Center for Disease Control and Prevention. Alkhurma virus was isolated in 1995 from patients with hemorrhagic fever in Saudi Arabia. Current evidence suggests that transmission to humans can occur either transcutaneously either by contamination of a skin wound with the blood of an infected vertebrate or bites of an infected tick or orally by drinking unpasteurized contaminated milk. To date a total of 24 symptomatic human cases have been recorded with a mortality rate at 25% (6/24). Pauci-symptomatic or asymptomatic cases are likely but epidemiologic data are currently unavailable. The complete coding sequence of the prototype strain of Alkhurma virus was determined and published in 2001 based on international research project involving investigators from France, Great Britain, and Saudi Arabia. Phylogenetic studies demonstrate that closest known relative of Alkhurma virus is Kyasanur Forest disease virus and that both viruses share a common ancestor. Genetic analysis of several human strains sequentially isolated over a 5-year period showed a very low diversity. This finding has important potential implications for diagnosis and vaccination.

Polymorphisms affecting the gE and gI proteins partly contribute to the virulence of a newly-emergent highly virulent Chinese pseudorabies virus.

PubMed

Dong, Jing; Gu, Zhenqing; Jin, Ling; Lv, Lin; Wang, Jichun; Sun, Tao; Bai, Juan; Sun, Haifeng; Wang, Xianwei; Jiang, Ping

2018-06-01

An outbreak of a highly virulent pseudorabies virus strain, ZJ01, occurred in PRV-vaccinated pigs in China in 2011. In this study, ZJ01 caused fatal diseases, while the Chinese prototypic PRV strain LA caused mild respiratory disorders. Full-genome sequencing results indicate the two viruses can be classified into two sub-clusters that distinct from traditional European and US strains. To examine the potential role of the gE and gI proteins in ZJ01 virulence, we generated several recombinant viruses. In two chimeric viruses (rZJ01-LA/gEI and rLA-ZJ01/gEI), the gE and gI genes were swapped using corresponding genes from ZJ01 and LA. rZJ01-LA/gEI and the parental virus rZJ01 retained high virulence in piglets, although the survival time for rZJ01-LA/gEI infected piglets was obviously prolonged. In contrast, rLA-ZJ01/gEI exhibited higher virulence than its parental virus rLA. We conclude that changes in gE and gI proteins partly contribute to the enhanced virulence of ZJ01 strain. Copyright © 2018 Elsevier Inc. All rights reserved.

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

PubMed

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

2012-11-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

Longshore Sediment Transport Rate Calculated Incorporating Wave Orbital Velocity Fluctuations

DTIC Science & Technology

2006-09-01

distribution of longshore sediment transport in the surf zone is necessary in the design and planning of groins, jetties, weirs and pipeline landfalls...transported by any current. Breaker height is defined as the vertical distance between the wave crest and the preceding wave trough at incipient...terminology; spilling breakers occur if the wave crest becomes unstable and flows down the front face of the wave producing a foamy water surface; plunging

Complete genome analysis of 33 ecologically and biologically diverse Rift Valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry.

PubMed

Bird, Brian H; Khristova, Marina L; Rollin, Pierre E; Ksiazek, Thomas G; Nichol, Stuart T

2007-03-01

Rift Valley fever (RVF) virus is a mosquito-borne RNA virus responsible for large explosive outbreaks of acute febrile disease in humans and livestock in Africa with significant mortality and economic impact. The successful high-throughput generation of the complete genome sequence was achieved for 33 diverse RVF virus strains collected from throughout Africa and Saudi Arabia from 1944 to 2000, including strains differing in pathogenicity in disease models. While several distinct virus genetic lineages were determined, which approximately correlate with geographic origin, multiple exceptions indicative of long-distance virus movement have been found. Virus strains isolated within an epidemic (e.g., Mauritania, 1987, or Egypt, 1977 to 1978) exhibit little diversity, while those in enzootic settings (e.g., 1970s Zimbabwe) can be highly diverse. In addition, the large Saudi Arabian RVF outbreak in 2000 appears to have involved virus introduction from East Africa, based on the close ancestral relationship of a 1998 East African virus. Virus genetic diversity was low (approximately 5%) and primarily involved accumulation of mutations at an average of 2.9 x 10(-4) substitutions/site/year, although some evidence of RNA segment reassortment was found. Bayesian analysis of current RVF virus genetic diversity places the most recent common ancestor of these viruses in the late 1800s, the colonial period in Africa, a time of dramatic changes in agricultural practices and introduction of nonindigenous livestock breeds. In addition to insights into the evolution and ecology of RVF virus, these genomic data also provide a foundation for the design of molecular detection assays and prototype vaccines useful in combating this important disease.

Potential role of deer tick virus in Powassan encephalitis cases in Lyme disease-endemic areas of New York, U.S.A.

PubMed

El Khoury, Marc Y; Camargo, Jose F; White, Jennifer L; Backenson, Bryon P; Dupuis, Alan P; Escuyer, Kay L; Kramer, Laura; St George, Kirsten; Chatterjee, Debarati; Prusinski, Melissa; Wormser, Gary P; Wong, Susan J

2013-12-01

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004-2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease-endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.

Potential Role of Deer Tick Virus in Powassan Encephalitis Cases in Lyme Disease–endemic Areas of New York, USA

PubMed Central

Camargo, Jose F.; White, Jennifer L.; Backenson, Bryon P.; Dupuis, Alan P.; Escuyer, Kay L.; Kramer, Laura; St. George, Kirsten; Chatterjee, Debarati; Prusinski, Melissa; Wormser, Gary P.; Wong, Susan J.

2013-01-01

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004–2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease–endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage. PMID:24274334

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αβ/γ) receptor double knockout mice.

PubMed

Patterson, Michael; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Smith, Jennifer; Miller, Milagros; Smith, Jeanon; Yun, Nadezhda; Poussard, Allison; Grant, Ashley; Tigabu, Bersabeh; Walker, Aida; Paessler, Slobodan

2014-02-01

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

Quasispecies evolution of the prototypical genotype 1 porcine reproductive and respiratory syndrome virus early during in vivo infection is rapid and tissue specific.

PubMed

Lu, Zen H; Wang, Xinglong; Wilson, Alison D; Dorey-Robinson, Daniel L W; Archibald, Alan L; Ait-Ali, Tahar; Frossard, Jean-Pierre

2017-08-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.

West Nile Virus: Using Adapted Primary Literature in Mathematical Biology to Teach Scientific and Mathematical Reasoning in High School

ERIC Educational Resources Information Center

Norris, Stephen P.; Macnab, John S.; Wonham, Marjorie; de Vries, Gerda

2009-01-01

This paper promotes the use of adapted primary literature as a curriculum and instruction innovation for use in high school. Adapted primary literature is useful for promoting an understanding of scientific and mathematical reasoning and argument and for introducing modern science into the schools. We describe a prototype adapted from a published…

[The comprehensive prevention of suppurative wound infection in the postoperative period in diabetic patients].

PubMed

Veligotskiĭ, N N; Florikian, A K; Bashura, G S; Dubenko, V V

1998-01-01

The strict acceptance of the radicalism principles while the purulent inflammation origin eliminate, an accurate sanation and early placement of suture on the operation wound, its adequate and active drainage in combination with expedient local with the help of foamy aerosol "Dioxizol" and stable compensation of hyperglycemia, correction of the homeostasis disorders are necessary in the treatment of diabetes mellitus patients with local purulent-necrotic disease of the soft tissues.

Influence of pressurized carbon dioxide on ketoprofen-incorporated hot-melt extruded low molecular weight hydroxypropylcellulose.

PubMed

A Ashour, Eman; Kulkarni, Vijay; Almutairy, Bjad; Park, Jun-Bom; Shah, Sejal P; Majumdar, Soumyajit; Lian, Zhuoyang; Pinto, Elanor; Bi, Vivian; Durig, Thomas; Martin, Scott T; Repka, Michael A

2016-01-01

The aim of the current research project was to investigate the effect of pressurized carbon dioxide (P-CO 2 ) on the physico-mechanical properties of ketoprofen (KTP)-incorporated hydroxypropylcellulose (HPC) (Klucel™ ELF, EF, and LF) produced using hot-melt extrusion (HME) techniques and to assess the plasticization effect of P-CO 2 on the various polymers tested. The physico-mechanical properties of extrudates with and without injection of P-CO 2 were examined and compared with extrudates with the addition of 5% liquid plasticizer of propylene glycol (PG). The extrudates were milled and compressed into tablets. Tablet characteristics of the extrudates with and without injection of P-CO 2 were evaluated. P-CO 2 acted as a plasticizer for tested polymers, which allowed for the reduction in extrusion processing temperature. The microscopic morphology of the extrudates was changed to a foam-like structure due to the expansion of the CO 2 at the extrusion die. The foamy extrudates demonstrated enhanced KTP release compared with the extrudates processed without P-CO 2 due to the increase of porosity and surface area of those extrudates. Furthermore, the hardness of the tablets prepared by foamy extrudates was increased and the percent friability was decreased. Thus, the good binding properties and compressibility of the extrudates were positively influenced by utilizing P-CO 2 processing.

Influence of Pressurized Carbon Dioxide on Ketoprofen-Incorporated Hot-Melt Extruded Low Molecular Weight Hydroxypropylcellulose

PubMed Central

Ashour, Eman A.; Kulkarni, Vijay; Almutairy, Bjad; Park, Jun-Bom; Shah, Sejal; Majumdar, Soumyajit; Lian, Zhuoyang; Pinto, Elanor; Bi, Yunxia; Durig, Thomas; Martin, Scott T.; Repka, Michael A.

2017-01-01

Objectives The aim of the current research project was to investigate the effect of pressurized carbon dioxide (P-CO2) on the physico-mechanical properties of Ketoprofen (KTP)-incorporated hydroxypropylcellulose (HPC) (Klucel™ ELF, EF and LF) produced using hot melt extrusion (HME) techniques and to assess the plasticization effect of P-CO2 on the various polymers tested. Methods The physico-mechanical properties of extrudates with and without injection of P-CO2 were examined and compared to extrudates with the addition of 5% liquid plasticizer of propylene glycol (PG). The extrudates were milled and compressed into tablets. Tablet characteristics of the extrudates with and without injection of P-CO2 were evaluated. Results & conclusion P-CO2 acted as a plasticizer for tested polymers, which allowed for the reduction in extrusion processing temperature. The microscopic morphology of the extrudates were changed to a foam-like structure due to expansion of the CO2 at the extrusion die. The foamy extrudates demonstrated enhanced KTP release compared to the extrudates processed without P-CO2 due to the increase of porosity and surface area of those extrudates. Furthermore, the hardness of the tablets prepared by foamy extrudates was increased and the percent friability was decreased. Thus, the good binding properties and compressibility of the extrudates were positively influenced by utilizing P-CO2 processing. PMID:25997363

Novel Human Adenovirus Causing Nosocomial Epidemic Keratoconjunctivitis▿

PubMed Central

Ishiko, Hiroaki; Shimada, Yasushi; Konno, Tsunetada; Hayashi, Akio; Ohguchi, Takeshi; Tagawa, Yoshitsugu; Aoki, Koki; Ohno, Shigeaki; Yamazaki, Shudo

2008-01-01

In 2000, we encountered cases of nosocomial infections with epidemic keratoconjunctivitis (EKC) at a university hospital in Kobe, in the western part of Japan. Two human adenovirus (HAdV) strains, Kobe-H and Kobe-S, were isolated from patients with nosocomial EKC infection. They were untypeable by existing neutralizing antisera; however, the isolate was neutralized with homologous antisera. We then encountered several cases of EKC due to nosocomial infections in eye clinics in different parts of Japan. A total of 80 HAdVs were isolated from patients with EKC at eight different hospitals. The partial hexon gene sequences of the isolates were determined and compared to those of the prototype strains of 51 serotypes. All isolates had identical partial hexon nucleotide sequences. Phylogenetic analysis classified these isolates into species of HAdV-D. The isolates showed 93.9 to 96.7% nucleotide identity with HAdV-D prototype strains, while all 32 HAdV-D prototype strains ranged from 93.2 to 99.2% identity. The sequences of the loop 2 and fiber knob regions from the representative strain, Kobe-H, were dissimilar in all prototype strains of 51 serotypes. We believe that this virus is a novel serotype of HAdV that causes EKC. PMID:18385435

Upolu virus and Aransas Bay virus, Two Presumptive Bunyaviruses, Are Novel Members of the Family Orthomyxoviridae

PubMed Central

Chowdhary, Rashmi; Travassos da Rosa, Amelia; Hutchison, Stephen K.; Popov, Vsevolod; Street, Craig; Tesh, Robert B.; Lipkin, W. Ian

2014-01-01

ABSTRACT Emerging and zoonotic pathogens pose continuing threats to human health and ongoing challenges to diagnostics. As nucleic acid tests are playing increasingly prominent roles in diagnostics, the genetic characterization of molecularly uncharacterized agents is expected to significantly enhance detection and surveillance capabilities. We report the identification of two previously unrecognized members of the family Orthomyxoviridae, which includes the influenza viruses and the tick-transmitted Thogoto and Dhori viruses. We provide morphological, serologic, and genetic evidence that Upolu virus (UPOV) from Australia and Aransas Bay virus (ABV) from North America, both previously considered potential bunyaviruses based on electron microscopy and physicochemical features, are orthomyxoviruses instead. Their genomes show up to 68% nucleotide sequence identity to Thogoto virus (segment 2; ∼74% at the amino acid level) and a more distant relationship to Dhori virus, the two prototype viruses of the recognized species of the genus Thogotovirus. Despite sequence similarity, the coding potentials of UPOV and ABV differed from that of Thogoto virus, instead being like that of Dhori virus. Our findings suggest that the tick-transmitted viruses UPOV and ABV represent geographically distinct viruses in the genus Thogotovirus of the family Orthomyxoviridae that do not fit in the two currently recognized species of this genus. IMPORTANCE Upolu virus (UPOV) and Aransas Bay virus (ABV) are shown to be orthomyxoviruses instead of bunyaviruses, as previously thought. Genetic characterization and adequate classification of agents are paramount in this molecular age to devise appropriate surveillance and diagnostics. Although more closely related to Thogoto virus by sequence, UPOV and ABV differ in their coding potentials by lacking a proposed pathogenicity factor. In this respect, they are similar to Dhori virus, which, despite the lack of a pathogenicity factor, can cause disease. These findings enable further studies into the evolution and pathogenicity of orthomyxoviruses. PMID:24574415

Carrot cells: a pioneering platform for biopharmaceuticals production.

PubMed

Rosales-Mendoza, Sergio; Tello-Olea, Marlene Anahí

2015-03-01

Carrot (Daucus carota L.) is of importance in the molecular farming field as it constitutes the first plant species approved to produce biopharmaceuticals for human use. In this review, features that make carrot an advantageous species in the molecular farming field are analyzed and a description of the developments achieved with this crop thus far is presented. A guide for genetic transformation procedures is also included. The state of the art comprises ten vaccine prototypes against Measles virus, Hepatitis B virus, Human immunodeficiency virus, Yersinia pestis, Chlamydia trachomatis, Mycobacterium tuberculosis, enterotoxigenic Escherichia coli, Corynebacterium diphtheria/Clostridium tetani/Bordetella pertussis, and Helicobacter pylori; as well as the case of the glucocerebrosidase, an enzyme used for replacement therapy, and other therapeutics. Perspectives for these developments are envisioned and innovations are proposed such as the use of transplastomic technologies-, hairy roots-, and viral expression-based systems to improve yields and develop new products derived from this advantageous plant species.

Murine Leukemia Viruses: Objects and Organisms

PubMed Central

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera. PMID:22312342

Murine leukemia viruses: objects and organisms.

PubMed

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome

PubMed Central

Douam, Florian; Hrebikova, Gabriela; Albrecht, Yentli E. Soto; Sellau, Julie; Sharon, Yael; Ding, Qiang; Ploss, Alexander

2017-01-01

Positive-sense RNA viruses pose increasing health and economic concerns worldwide. Our limited understanding of how these viruses interact with their host and how these processes lead to virulence and disease seriously hampers the development of anti-viral strategies. Here, we demonstrate the tracking of (+) and (−) sense viral RNA at single-cell resolution within complex subsets of the human and murine immune system in different mouse models. Our results provide insights into how a prototypic flavivirus, yellow fever virus (YFV-17D), differentially interacts with murine and human hematopoietic cells in these mouse models and how these dynamics influence distinct outcomes of infection. We detect (−) YFV-17D RNA in specific secondary lymphoid compartments and cell subsets not previously recognized as permissive for YFV replication, and we highlight potential virus–host interaction events that could be pivotal in regulating flavivirus virulence and attenuation. PMID:28290449

Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

PubMed

Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

2011-04-01

Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles.

Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.

PubMed

Hurrelbrink, R J; Nestorowicz, A; McMinn, P C

1999-12-01

An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.

Development of replication-competent viral vectors for HIV vaccine delivery

PubMed Central

Parks, Christopher L.; Picker, Louis J.; King, C. Richter

2014-01-01

Purpose of review Briefly describe some of the replication-competent (RC) vectors being investigated for development of candidate HIV vaccines focusing primarily on technologies that have advanced to testing in macaques or have entered clinical trials. Recent findings RC viral vectors have advanced to the stage were decisions can be made regarding future development of HIV vaccines. The viruses being used as RC vector platforms vary considerably, and their unique attributes make it possible to test multiple vaccine design concepts and also mimic various aspects of an HIV infection. RC viral vectors encoding SIV or HIV proteins can be used to safely immunize macaques, and in some cases, there is evidence of significant vaccine efficacy in challenge protection studies. Several live HIV vaccine vectors are in clinical trials to evaluate immunogenicity, safety, the effect of mucosal delivery, and potential effects of pre-existing immunity. Summary A variety of DNA and RNA viruses are being used to develop RC viral vectors for HIV vaccine delivery. Multiple viral vector platforms have proven to be safe and immunogenic with evidence of efficacy in macaques. Some of the more advanced HIV vaccine prototypes based on vesicular stomatitis virus, vaccinia virus, measles virus, and Sendai virus are in clinical trials. PMID:23925000

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

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

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin

2012-01-01

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

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

PubMed Central

Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

1978-01-01

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein.

PubMed

Spehner, D; De Carlo, S; Drillien, R; Weiland, F; Mildner, K; Hanau, D; Rziha, H-J

2004-08-01

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

Chinese and Vietnamese strains of HP-PRRSV cause different pathogenic outcomes in United States high health swine

USDA-ARS?s Scientific Manuscript database

An infectious clone of a highly pathogenic PRRSV strain from Vietnam (rSRV07) was prepared, analyzed and compared to Chinese highly pathogenic PRRSV rJXwn06 and US Type 2 prototype VR-2332 in order to examine the effects of virus phenotype and genotype on growth in MARC-145 cells, as well as the imp...

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

PubMed Central

Ringe, Rajesh P.; Ozorowski, Gabriel; Yasmeen, Anila; Cupo, Albert; Cruz Portillo, Victor M.; Pugach, Pavel; Golabek, Michael; Rantalainen, Kimmo; Holden, Lauren G.; Cottrell, Christopher A.; Wilson, Ian A.; Sanders, Rogier W.; Ward, Andrew B.; Klasse, P. J.

2017-01-01

ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design. PMID:28381572

Analysis of the Highly Diverse Gene Borders in Ebola Virus Reveals a Distinct Mechanism of Transcriptional Regulation

PubMed Central

Brauburger, Kristina; Boehmann, Yannik; Tsuda, Yoshimi; Hoenen, Thomas; Olejnik, Judith; Schümann, Michael; Ebihara, Hideki

2014-01-01

ABSTRACT Ebola virus (EBOV) belongs to the group of nonsegmented negative-sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4- or 5-nucleotide) intergenic regions (IRs), a single long (144-nucleotide) IR, and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24-L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for but regulates transcription reinitiation in a length-dependent but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates but demonstrated that specific IR lengths not found naturally in filoviruses profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144-nucleotide-long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth. IMPORTANCE Our current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3′ end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle. PMID:25142600

Analysis of the highly diverse gene borders in Ebola virus reveals a distinct mechanism of transcriptional regulation.

PubMed

Brauburger, Kristina; Boehmann, Yannik; Tsuda, Yoshimi; Hoenen, Thomas; Olejnik, Judith; Schümann, Michael; Ebihara, Hideki; Mühlberger, Elke

2014-11-01

Ebola virus (EBOV) belongs to the group of nonsegmented negative-sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4- or 5-nucleotide) intergenic regions (IRs), a single long (144-nucleotide) IR, and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24-L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for but regulates transcription reinitiation in a length-dependent but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates but demonstrated that specific IR lengths not found naturally in filoviruses profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144-nucleotide-long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth. Our current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3' end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A case of sebaceous carcinoma of the parotid gland.

PubMed

Siriwardena, B S M S; Tilakaratne, W M; Rajapakshe, R M S K

2003-02-01

Sebaceous carcinoma of salivary gland origin is an extremely rare malignancy. It occurs mainly in the parotid gland. This is a case report of a sebaceous carcinoma in a 57-year-old woman who had a lump over the right parotid region for 8-9 months. The tumour was composed of small basaloid cells and large foamy cells. Sebaceous differentiation was evident in some tumour islands. This is the first case reported in the Department of Oral Pathology, Faculty of Dental Sciences, University of Peradeniya.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

PubMed

Lukashevich, I S; Maryankova, R; Vladyko, A S; Nashkevich, N; Koleda, S; Djavani, M; Horejsh, D; Voitenok, N N; Salvato, M S

1999-12-01

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. Copyright 1999 Wiley-Liss, Inc.

N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

PubMed Central

Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

2015-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

Tropical arthritogenic alphaviruses.

PubMed

Mejía, Carla-Ruth; López-Vélez, Rogelio

Tropical alphaviruses have special tropism for bone and joint tissue. Patients can develop chronic rheumatic disorders similar to rheumatoid arthritis and ankylosing spondylitis. The prototype is Chikungunya virus, although other lesser known viruses in our environment such as Sindbis, Ross River, Mayaro, O'nyong nyong and Barmah Forest viruses have the potential to be sped through vectors and cause chronic rheumatic disease. International population movements have increased the numbers of patients diagnosed with these tropical viruses in areas in which they are not endemic. Since they can leave persistent symptoms and affect the quality of life of the patients, it is important that we be aware of them. Changes in ecosystems have favored the expansion of competent mosquitoes, making fears of local transmission in southern Europe a reality. The objective of this review is to provide a clinical approach to the different arthritogenic tropical alphaviruses, especially those in which chronic rheumatic disease is more frequent. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Pathogenesis of varicelloviruses in primates.

PubMed

Ouwendijk, Werner J D; Verjans, Georges M G M

2015-01-01

Varicelloviruses in primates comprise the prototypic human varicella-zoster virus (VZV) and its non-human primate homologue, simian varicella virus (SVV). Both viruses cause varicella as a primary infection, establish latency in ganglionic neurons and reactivate later in life to cause herpes zoster in their respective hosts. VZV is endemic worldwide and, although varicella is usually a benign disease in childhood, VZV reactivation is a significant cause of neurological disease in the elderly and in immunocompromised individuals. The pathogenesis of VZV infection remains ill-defined, mostly due to the species restriction of VZV that impedes studies in experimental animal models. SVV infection of non-human primates parallels virological, clinical, pathological and immunological features of human VZV infection, thereby providing an excellent model to study the pathogenesis of varicella and herpes zoster in its natural host. In this review, we discuss recent studies that provided novel insight in both the virus and host factors involved in the three elementary stages of Varicellovirus infection in primates: primary infection, latency and reactivation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy

PubMed Central

Vogt, Volker M.; Simon, Martha N.

1999-01-01

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 × 108 Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation. PMID:10400808

Detection of virus-like particles in the liver of black and white ruffed lemurs with hepatitis.

PubMed

Worley, Michael B; Stalis, Ilse H

2002-04-01

Two young black and white ruffed lemurs (Varecia variegata variegata) died at the San Diego Zoo (San Diego, California, USA) with extensive liver lesions suggestive of acute viral infection. Immunoassays performed to detect hepatitis B virus (HBV) markers were negative. Polymerase chain reaction (PCR) primers overlapping the HBV core gene produced an amplicon of approximately 411 base pairs (bp) from serum DNA of a HBV-positive western lowland gorilla (Gorilla gorilla gorilla) but not from serum DNA of either lemur. Cesium chloride gradient fractions of liver homogenates from both lemurs contained a peak protein fraction with a density of 1.18 g/cm3. Electron microscopic analysis of fraction contents, concentrated by ultracentrifulgation, revealed numerous pleomorphic, spherical particles varying in diameter from 16-25 nm. In one of the lemurs, this peak fraction also contained a double-shelled virus-like particle 47-50 nm in diameter. The size, morphology, and density of these particles suggest they are members of the Hepadnaviridae, a group of hepatotropic DNA-genome viruses for which HBV is the prototype.

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Phosphorylation of paramyxovirus phosphoprotein and its role in viral gene expression.

PubMed

Fuentes, Sandra M; Sun, Dengyun; Schmitt, Anthony P; He, Biao

2010-01-01

Paramyxoviruses include many important human and animal pathogens such as measles virus, mumps virus, human parainfluenza viruses, and respiratory syncytial virus, as well as emerging viruses such as Nipah virus and Hendra virus. The paramyxovirus RNA-dependent RNA polymerase consists of the phosphoprotein (P) and the large protein. Both of these proteins are essential for viral RNA synthesis. The P protein is phosphorylated at multiple sites, probably by more than one host kinase. While it is thought that the phosphorylation of P is important for its role in viral RNA synthesis, the precise role of P protein phosphorylation remains an enigma. For instance, it was demonstrated that the putative CKII phosphorylation sites of the P protein of respiratory syncytial virus play a role in viral RNA synthesis using a minigenome replicon system; however, mutating these putative CKII phosphorylation sites within a viral genome had no effect on viral RNA synthesis, leading to the hypothesis that P protein phosphorylation, at least by CKII, does not play a role in viral RNA synthesis. Recently, it has been reported that the phosphorylation state of the P protein of parainfluenza virus 5, a prototypical paramyxovirus, correlates with the ability of P protein to synthesize viral RNA, indicating that P protein phosphorylation does in fact play a role in viral RNA synthesis. Furthermore, host kinases PLK1, as well as AKT1 have been found to play critical roles in paramyxovirus RNA synthesis through regulation of P protein phosphorylation status. Beyond furthering our understanding of paramyxovirus RNA replication, these recent discoveries may also result in a new paradigm in treating infections caused by these viruses, as host kinases that regulate paramyxovirus replication are investigated as potential targets of therapeutic intervention.

Field Testing of a Prototype Filter System for the Removal of the Human Pathogen Giardia intestinales from Ground Water

NASA Astrophysics Data System (ADS)

Rust, C.; Schulze-Makuch, D.; Bowman, R.; Meier, D.

2005-12-01

Pathogenic bacteria, viruses, and protozoans tend to be negatively charged in the pH range of most ground waters. Thus, naturally occurring and modified materials such as surfactant-modified zeolites (SMZ), which have net positive surface charges and hydrophobic properties, are suitable as barriers to impede pathogen migration in aquifer systems. In our experiments SMZ has been used to remove E. coli and the bacteriophage MS-2 from sewage water with a high success rate ( E. coli 100%, MS-2 > 90%). Testing was conducted both in the laboratory and the field. Laboratory experiments were conducted to test the removal efficiency of SMZ for Giardia intestinales using the Giardia cysts and microsphere analogs. The SMZ was effective at removing Giardia intestinales cysts from the groundwater, but removal rates were not as high as for bacteria and viruses in the earlier experiments. The removal efficiency varied with the particular formulation of the SMZ used. The most effective SMZ formulation is being further tested at our field site using water amended with microspheres to simulate Giardia behavior. The field site is an existing multiple well site at the University of Idaho in Moscow. The wells are completed in the Lolo Basalt Formation; a highly heterogeneous and anisotropic fractured basalt aquifer system typical of the subsurface of most of eastern Washington and northeastern Oregon. The SMZ pathogen filter is installed directly in the well bore and the concentrations of microsphere-amended ground water are measured before and after filtration. Pumping over an extended period is continuing in order to test the lifetime of our prototype filter system. Our tests and results are targeted at developing a prototype filter system for removing a multitude of human pathogens in drinking water.

Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

PubMed

Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

2012-01-01

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

PubMed Central

Poquet, Yannick; Levillain, Florence; Botanch, Catherine; Bardou, Fabienne; Daffé, Mamadou; Emile, Jean-François; Marchou, Bruno; Cardona, Pere-Joan; de Chastellier, Chantal; Altare, Frédéric

2008-01-01

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria. PMID:19002241

Delayed Disease Progression in Cynomolgus Macaques Infected with Ebola Virus Makona Strain.

PubMed

Marzi, Andrea; Feldmann, Friederike; Hanley, Patrick W; Scott, Dana P; Günther, Stephan; Feldmann, Heinz

2015-10-01

In late 2013, the largest documented outbreak of Ebola hemorrhagic fever started in Guinea and has since spread to neighboring countries, resulting in almost 27,000 cases and >11,000 deaths in humans. In March 2014, Ebola virus (EBOV) was identified as the causative agent. This study compares the pathogenesis of a new EBOV strain, Makona, which was isolated in Guinea in 2014 with the prototype strain from the 1976 EBOV outbreak in the former Zaire. Both strains cause lethal disease in cynomolgus macaques with similar pathologic changes and hallmark features of Ebola hemorrhagic fever. However, disease progression was delayed in EBOV-Makona-infected animals, suggesting decreased rather than increased virulence of this most recent EBOV strain.

Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses.

PubMed

Guy, Bruno; Guirakhoo, Farshad; Barban, Veronique; Higgs, Stephen; Monath, Thomas P; Lang, Jean

2010-01-08

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

PubMed Central

Reyes-del Valle, Jorge; de la Fuente, Cynthia; Turner, Mallory A.; Springfeld, Christoph; Apte-Sengupta, Swapna; Frenzke, Marie E.; Forest, Amelie; Whidby, Jillian; Marcotrigiano, Joseph; Rice, Charles M.

2012-01-01

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination. PMID:22896607

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

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

Geller, A.I.; Keyomarsi, K.; Bryan, J.

1990-11-01

The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; tsmore » mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.« less

A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae▿

PubMed Central

Woolford, Lucy; Rector, Annabel; Van Ranst, Marc; Ducki, Andrea; Bennett, Mark D.; Nicholls, Philip K.; Warren, Kristin S.; Swan, Ralph A.; Wilcox, Graham E.; O'Hara, Amanda J.

2007-01-01

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families. PMID:17898069

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii.

PubMed Central

Herwaldt, B L; Lew, J F; Moe, C L; Lewis, D C; Humphrey, C D; Monroe, S S; Pon, E W; Glass, R I

1994-01-01

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents. Images PMID:8027335

Recombinant Canine Distemper Virus Strain Snyder Hill Expressing Green or Red Fluorescent Proteins Causes Meningoencephalitis in the Ferret

PubMed Central

Ludlow, M.; Nguyen, D. T.; Silin, D.; Lyubomska, O.; de Vries, R. D.; von Messling, V.; McQuaid, S.; De Swart, R. L.

2012-01-01

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDVSH) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDVSH (rCDVSH) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV5804P and the prototypic wild-type CDVR252 showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDVSH-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis. PMID:22553334

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

PubMed

Ba Abdullah, Mohammed M; Palermo, Richard D; Palser, Anne L; Grayson, Nicholas E; Kellam, Paul; Correia, Samantha; Szymula, Agnieszka; White, Robert E

2017-12-01

Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1-and, by extension, EBV-diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC). IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage. Copyright © 2017 Ba abdullah et al.

Influenza A Virus Polymerase Is a Site for Adaptive Changes during Experimental Evolution in Bat Cells

PubMed Central

Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.

2014-01-01

ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity. PMID:25142579

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

PubMed Central

Baker, Steven F.; Nogales, Aitor; Finch, Courtney; Tuffy, Kevin M.; Domm, William; Perez, Daniel R.; Topham, David J.

2014-01-01

ABSTRACT Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions. IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or gene exchange between influenza A and B viruses is not well understood. Nucleotides comprising the coding termini of each influenza A virus gene segment are required for specific segment incorporation during budding. Whether influenza B virus shares a similar selective packaging strategy or if packaging signals prevent intertypic reassortment remains unknown. Here, we provide evidence suggesting a similar mechanism of influenza B virus genome packaging. Furthermore, by appending influenza A virus packaging signals onto influenza B virus segments, we rescued recombinant influenza A/B viruses that could reassort in vitro with another influenza A virus. These findings suggest that the divergent evolution of packaging signals aids with the speciation of influenza A and B viruses and is in part responsible for the lack of intertypic viral reassortment. PMID:25008914

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

PubMed

Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L

2008-12-01

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

Genetics-Based Classification of Filoviruses Calls for Expanded Sampling of Genomic Sequences

PubMed Central

Lauber, Chris; Gorbalenya, Alexander E.

2012-01-01

We have recently developed a computational approach for hierarchical, genome-based classification of viruses of a family (DEmARC). In DEmARC, virus clusters are delimited objectively by devising a universal family-wide threshold on intra-cluster genetic divergence of viruses that is specific for each level of the classification. Here, we apply DEmARC to a set of 56 filoviruses with complete genome sequences and compare the resulting classification to the ICTV taxonomy of the family Filoviridae. We find in total six candidate taxon levels two of which correspond to the species and genus ranks of the family. At these two levels, the six filovirus species and two genera officially recognized by ICTV, as well as a seventh tentative species for Lloviu virus and prototyping a third genus, are reproduced. DEmARC lends the highest possible support for these two as well as the four other levels, implying that the actual number of valid taxon levels remains uncertain and the choice of levels for filovirus species and genera is arbitrary. Based on our experience with other virus families, we conclude that the current sampling of filovirus genomic sequences needs to be considerably expanded in order to resolve these uncertainties in the framework of genetics-based classification. PMID:23170166

Genetics-based classification of filoviruses calls for expanded sampling of genomic sequences.

PubMed

Lauber, Chris; Gorbalenya, Alexander E

2012-09-01

We have recently developed a computational approach for hierarchical, genome-based classification of viruses of a family (DEmARC). In DEmARC, virus clusters are delimited objectively by devising a universal family-wide threshold on intra-cluster genetic divergence of viruses that is specific for each level of the classification. Here, we apply DEmARC to a set of 56 filoviruses with complete genome sequences and compare the resulting classification to the ICTV taxonomy of the family Filoviridae. We find in total six candidate taxon levels two of which correspond to the species and genus ranks of the family. At these two levels, the six filovirus species and two genera officially recognized by ICTV, as well as a seventh tentative species for Lloviu virus and prototyping a third genus, are reproduced. DEmARC lends the highest possible support for these two as well as the four other levels, implying that the actual number of valid taxon levels remains uncertain and the choice of levels for filovirus species and genera is arbitrary. Based on our experience with other virus families, we conclude that the current sampling of filovirus genomic sequences needs to be considerably expanded in order to resolve these uncertainties in the framework of genetics-based classification.

Outer Membrane Vesicles from Neisseria Meningitidis (Proteossome) Used for Nanostructured Zika Virus Vaccine Production.

PubMed

Martins, Paula; Machado, Daisy; Theizen, Thais Holtz; Guarnieri, João Paulo Oliveira; Bernardes, Bruno Gaia; Gomide, Gabriel Piccirillo; Corat, Marcus Alexandre Finzi; Abbehausen, Camilla; Módena, José Luiz Proença; Melo, Carlos Fernando Odir Rodrigues; Morishita, Karen Noda; Catharino, Rodrigo Ramos; Arns, Clarice Weis; Lancellotti, Marcelo

2018-05-29

The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

Chlorovirus-Mediated Membrane Depolarization of Chlorella Alters Secondary Active Transport of Solutes▿

PubMed Central

Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L.

2008-01-01

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake. PMID:18842725

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

Hepatitis B virus molecular biology and pathogenesis.

PubMed

Lamontagne, R Jason; Bagga, Sumedha; Bouchard, Michael J

2016-01-01

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350-500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae . In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC.

Efficiency of different air filter types for pig facilities at laboratory scale

PubMed Central

Wenke, Cindy; Pospiech, Janina; Reutter, Tobias; Truyen, Uwe

2017-01-01

Air filtration has been shown to be efficient in reducing pathogen burden in circulating air. We determined at laboratory scale the retention efficiency of different air filter types either composed of a prefilter (EU class G4) and a secondary fiberglass filter (EU class F9) or consisting of a filter mat (EU class M6 and F8-9). Four filter prototypes were tested for their capability to remove aerosol containing equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), bovine enterovirus 1 (BEV), Actinobacillus pleuropneumoniae (APP), and Staphylococcus (S.) aureus from air. Depending on the filter prototype and utilisation, the airflow was set at 1,800 m3/h (combination of upstream prefilter and fiberglass filter) or 80 m3/h (filter mat). The pathogens were aerosolized and their concentration was determined in front of and behind the filter by culture or quantitative real-time RT-PCR. Furthermore, survival of the pathogens over time in the filter material was determined. Bacteria were most efficiently filtered with a reduction rate of up to 99.9% depending on the filter used. An approximately 98% reduction was achieved for the viruses tested. Viability or infectivity of APP or PRRSV in the filter material decreased below the detection limit after 4 h and 24 h, respectively, whereas S. aureus was still culturable after 4 weeks. Our results demonstrate that pathogens can efficiently be reduced by air filtration. Consequently, air filtration combined with other strict biosecurity measures markedly reduces the risk of introducing airborne transmitted pathogens to animal facilities. In addition, air filtration might be useful in reducing bioaerosols within a pig barn, hence improving respiratory health of pigs. PMID:29028843

Efficiency of different air filter types for pig facilities at laboratory scale.

PubMed

Wenke, Cindy; Pospiech, Janina; Reutter, Tobias; Truyen, Uwe; Speck, Stephanie

2017-01-01

Air filtration has been shown to be efficient in reducing pathogen burden in circulating air. We determined at laboratory scale the retention efficiency of different air filter types either composed of a prefilter (EU class G4) and a secondary fiberglass filter (EU class F9) or consisting of a filter mat (EU class M6 and F8-9). Four filter prototypes were tested for their capability to remove aerosol containing equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), bovine enterovirus 1 (BEV), Actinobacillus pleuropneumoniae (APP), and Staphylococcus (S.) aureus from air. Depending on the filter prototype and utilisation, the airflow was set at 1,800 m3/h (combination of upstream prefilter and fiberglass filter) or 80 m3/h (filter mat). The pathogens were aerosolized and their concentration was determined in front of and behind the filter by culture or quantitative real-time RT-PCR. Furthermore, survival of the pathogens over time in the filter material was determined. Bacteria were most efficiently filtered with a reduction rate of up to 99.9% depending on the filter used. An approximately 98% reduction was achieved for the viruses tested. Viability or infectivity of APP or PRRSV in the filter material decreased below the detection limit after 4 h and 24 h, respectively, whereas S. aureus was still culturable after 4 weeks. Our results demonstrate that pathogens can efficiently be reduced by air filtration. Consequently, air filtration combined with other strict biosecurity measures markedly reduces the risk of introducing airborne transmitted pathogens to animal facilities. In addition, air filtration might be useful in reducing bioaerosols within a pig barn, hence improving respiratory health of pigs.

Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans

PubMed Central

van Boheemen, Sander; de Graaf, Miranda; Lauber, Chris; Bestebroer, Theo M.; Raj, V. Stalin; Zaki, Ali Moh; Osterhaus, Albert D. M. E.; Haagmans, Bart L.; Gorbalenya, Alexander E.; Snijder, Eric J.; Fouchier, Ron A. M.

2012-01-01

ABSTRACT A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genome was reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C. PMID:23170002

Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

PubMed

Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

2016-04-01

Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

Persistence of vaccine-derived polioviruses among immunodeficient persons with vaccine-associated paralytic poliomyelitis.

PubMed

Khetsuriani, Nino; Prevots, D Rebecca; Quick, Linda; Elder, Melissa E; Pallansch, Mark; Kew, Olen; Sutter, Roland W

2003-12-15

To estimate long-term poliovaccine virus persistence among immunodeficient patients with vaccine-associated paralytic poliomyelitis (iVAPP), cases reported in the United States during 1975-1997 were reviewed, with subsequent follow-up and virological testing. Six (16.2%) of 37 subjects excreted poliovaccine viruses for > or =6 months. Partial genomic sequencing of their available poliovirus isolates showed considerable divergence from the prototype Sabin strain in all cases. Poliovirus persistence declined over time since the last oral poliovaccine dose: at 6 months, 19.4%; 1 year, 14.3%; 5 years, 4%; and 10 years, 0% (P<.05) of patients. Despite the high prevalence of poliovaccine virus persistence among patients with iVAPP, this group appears to be an unlikely source of poliovirus reintroduction in developed countries because of the rarity and high fatality rate of iVAPP and the possible spontaneous clearance of polioviruses. These results are important for developing "endgame" strategies for the Global Poliomyelitis Eradication Program.

Lenticular fibroxanthomatous nodule.

PubMed

Lee, Seok J; Ling, Jun X; Aaberg, Thomas M; Grossniklaus, Hans E

2003-02-01

To describe two patients with unique lenticular nodular proliferations. Observational case reports. The clinical histories and pathologic findings of two patients with lenticular nodular proliferations were reviewed. One patient with persistent hyperplastic primary vitreous and another patient with trauma developed lenticular nodular proliferations. The nodules were vascularized collections of foamy histiocytes, multinucleated cells, lens capsule, and lens epithelium that had undergone fibrous metaplasia. The lesions were classified as lenticular fibroxanthomatous nodules. A lenticular fibroxanthomatous nodule is a unique clinicopathologic entity that should be differentiated from Soemmerring ring, Elschnig pearl, and other simulating entities such as juvenile xanthogranuloma.

Cutaneous angiosarcoma mimicking xanthoma: a challenging histopathologic diagnosis with important consequences.

PubMed

Llamas-Velasco, Mar; Kutzner, Heinz; Requena, Luis

2016-09-01

Cutaneous angiosarcoma may show protean histopathologic features. Rare or uncommon variants include epithelioid, clear cell, granular cell, verrucous, pseudolymphomatous and signet-ring cell types. Perhaps the rarest type consists of cutaneous angiosarcoma with xanthomization of neoplastic cells. We report an extraordinary case with almost all neoplastic cells exhibiting a xanthomatous appearance that was studied both histopathologically and immunohistochemically. We discuss the histopathologic differential diagnosis of foamy cell angiosarcoma with other neoplasms that may show similar histopathology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of Influenza A (H7N9) Viruses Isolated from Human Cases Imported into Taiwan

PubMed Central

Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Wu, Ho-Sheng; Liu, Ming-Tsan

2015-01-01

A novel avian influenza A (H7N9) virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave) was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave). The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses. PMID:25748033

Comparative reduction of Norwalk virus, poliovirus type 1, F+ RNA coliphage MS2 and Escherichia coli in miniature soil columns.

PubMed

Meschke, J S; Sobsey, M D

2003-01-01

Norwalk-like viruses (NLVs) are important agents of waterborne illness and have been linked to several groundwater-related outbreaks. The presence of human enteric viruses, in particular the presence of NLVs, is difficult to detect in the environment. Consequently, surrogate organisms are typically used as indicators of viruses from faecal contamination. Whether traditional bacterial indicators are reliable indicators for viral pathogens remains uncertain. Few studies have directly compared mobility and reduction of bacterial indicators (e.g. coliforms, Escherichia coli) and other surrogate indicators (coliphages) with pathogenic human viruses in soil systems. In this study the mobility and comparative reduction of the prototype NLV, Norwalk Virus (NV), was compared to poliovirus 1 (PV1), a bacterial indicator (E coli, EC) and a viral indicator (coliphage MS2) through miniature soil columns. Replicate, 10 cm deep, miniature columns were prepared using three soils representing a range of soil textures (sand, organic muck, and clay). Columns were initially conditioned, then incubated at 10-14 degrees C, dosed twice weekly for 8 weeks with one column pore volume of virus-seeded groundwater per dose, followed by 8 weeks of dosing with one column pore volume per dose of unseeded, simulated rainwater. Columns were allowed to drain after each dosing until an effluent volume equivalent to an applied dose was collected. Column effluents and doses were assayed for all viruses and EC. Rapid mobility with minimal reduction was observed for all organisms in the sand. Similar reductions were observed in organic muck for most organisms but NV showed a greater reduction. No organisms were shown to pass through the clay columns. Elution of viruses, in particular PV1, from the columns was gradual. After cessation of microbe dosing, E. coli was less detectable than viruses in column effluents and, therefore, unreliable as a virus indicator.

Development of a Commercial Prototype of the Autonomous Pathogen Detection System Final Report CRADA No. TC-02077-04

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

Dzenitis, J. M.; Haigh, P.

This was a collaborative effort between The Regents of the University of California, Lawrence Livermore National Laboratory (LLNL), and GE Ion Track, Inc. (GEIT) to develop a commercial prototype of the Autonomous Pathogen Detection System (APDS), an instrument that monitors the air for all three biological threat agents (bacteria, viruses and toxins). This was originally a one year CRADA project, with the cost of the work at LLNL being funded by the Department of Homeland Security's Office of National Laboratories. The original project consisted of five major tasks and deliverables. The CRADA was then amended, converting the CRADA from amore » programmatically funded CRADA to a funds-in CRADA, extending the project for an additional 14 months, and adding four new tasks and deliverable to the project.« less

Advances in three-dimensional rapid prototyping of microfluidic devices for biological applications

PubMed Central

O'Neill, P. F.; Ben Azouz, A.; Vázquez, M.; Liu, J.; Marczak, S.; Slouka, Z.; Chang, H. C.; Diamond, D.; Brabazon, D.

2014-01-01

The capability of 3D printing technologies for direct production of complex 3D structures in a single step has recently attracted an ever increasing interest within the field of microfluidics. Recently, ultrafast lasers have also allowed developing new methods for production of internal microfluidic channels within the bulk of glass and polymer materials by direct internal 3D laser writing. This review critically summarizes the latest advances in the production of microfluidic 3D structures by using 3D printing technologies and direct internal 3D laser writing fabrication methods. Current applications of these rapid prototyped microfluidic platforms in biology will be also discussed. These include imaging of cells and living organisms, electrochemical detection of viruses and neurotransmitters, and studies in drug transport and induced-release of adenosine triphosphate from erythrocytes. PMID:25538804

A Highly Conserved Leucine in Mammarenavirus Matrix Z Protein Is Required for Z Interaction with the Virus L Polymerase and Z Stability in Cells Harboring an Active Viral Ribonucleoprotein.

PubMed

Iwasaki, Masaharu; de la Torre, Juan C

2018-06-01

Mammarenaviruses cause chronic infections in their natural rodent hosts. Infected rodents shed infectious virus into excreta. Humans are infected through mucosal exposure to aerosols or direct contact of abraded skin with fomites, resulting in a wide range of manifestations from asymptomatic or mild febrile illness to severe life-threatening hemorrhagic fever. The mammarenavirus matrix Z protein has been shown to be a main driving force of virus budding and to act as a negative regulator of viral RNA synthesis. To gain a better understanding of how the Z protein exerts its several different functions, we investigated the interaction between Z and viral polymerase L protein using the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV). We found that in the presence of an active viral ribonucleoprotein (vRNP), the Z protein translocated from nonionic detergent-resistant, membrane-rich structures to a subcellular compartment with a different membrane composition susceptible to disruption by nonionic detergents. Alanine (A) substitution of a highly conserved leucine (L) at position 72 in LCMV Z protein abrogated Z-L interaction. The L72A mutation did not affect the stability or budding activity of Z when expressed alone, but in the presence of an active vRNP, mutation L72A promoted rapid degradation of Z via a proteasome- and lysosome-independent pathway. Accordingly, L72A mutation in the Z protein resulted in nonviable LCMV. Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required. IMPORTANCE Several mammarenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose important public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. The mammarenavirus matrix Z protein plays critical roles in different steps of the viral life cycle by interacting with viral and host cellular components. Here we report that alanine substitution of a highly conserved leucine residue, located at position 72 in LCMV Z protein, abrogated Z-L interaction. The L72A mutation did not affect Z budding activity but promoted its rapid degradation in the presence of an active viral ribonucleoprotein (vRNP). Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required. Copyright © 2018 American Society for Microbiology.

Why Are Nigeria-Cameroon Chimpanzees (Pan troglodytes ellioti) Free of SIVcpz Infection?

PubMed

Locatelli, Sabrina; Harrigan, Ryan J; Sesink Clee, Paul R; Mitchell, Matthew W; McKean, Kurt A; Smith, Thomas B; Gonder, Mary Katherine

2016-01-01

Simian immunodeficiency virus (SIV) naturally infects two subspecies of chimpanzee: Pan troglodytes troglodytes from Central Africa (SIVcpzPtt) and P. t. schweinfurtii from East Africa (SIVcpzPts), but is absent in P. t. verus from West Africa and appears to be absent in P. t. ellioti inhabiting Nigeria and western Cameroon. One explanation for this pattern is that P. t. troglodytes and P. t schweinfurthii may have acquired SIVcpz after their divergence from P. t. verus and P. t. ellioti. However, all of the subspecies, except P. t. verus, still occasionally exchange migrants making the absence of SIVcpz in P. t. ellioti puzzling. Sampling of P. t. ellioti has been minimal to date, particularly along the banks of the Sanaga River, where its range abuts that of P. t. troglodytes. This study had three objectives. First, we extended the sampling of SIVcpz across the range of chimpanzees north of the Sanaga River to address whether under-sampling might account for the absence of evidence for SIVcpz infection in P. t. ellioti. Second, we investigated how environmental variation is associated with the spread and prevalence of SIVcpz in the two chimpanzee subspecies inhabiting Cameroon since environmental variation has been shown to contribute to their divergence from one another. Finally, we compared the prevalence and distribution of SIVcpz with that of Simian Foamy Virus (SFV) to examine the role of ecology and behavior in shaping the distribution of diseases in wild host populations. The dataset includes previously published results on SIVcpz infection and SFVcpz as well as newly collected data, and represents over 1000 chimpanzee fecal samples from 41 locations across Cameroon. Results revealed that none of the 181 P. t. ellioti fecal samples collected across the range of P. t. ellioti tested positive for SIVcpz. In addition, species distribution models suggest that environmental variation contributes to differences in the distribution and prevalence of SIVcpz and SFVcpz. The ecological niches of these two viruses are largely non-overlapping, although stronger statistical support for this conclusion will require more sampling. Overall this study demonstrates that SIVcpz infection is absent or very rare in P. t. ellioti, despite multiple opportunities for transmission. The reasons for its absence remain unclear, but might be explained by one or more factors, including environmental variation, viral competition, and/or local adaptation-all of which should be explored in greater detail through continued surveillance of this region.

Why Are Nigeria-Cameroon Chimpanzees (Pan troglodytes ellioti) Free of SIVcpz Infection?

PubMed Central

Locatelli, Sabrina; Harrigan, Ryan J.; Sesink Clee, Paul R.; Mitchell, Matthew W; McKean, Kurt A.; Smith, Thomas B.; Gonder, Mary Katherine

2016-01-01

Simian immunodeficiency virus (SIV) naturally infects two subspecies of chimpanzee: Pan troglodytes troglodytes from Central Africa (SIVcpzPtt) and P. t. schweinfurtii from East Africa (SIVcpzPts), but is absent in P. t. verus from West Africa and appears to be absent in P. t. ellioti inhabiting Nigeria and western Cameroon. One explanation for this pattern is that P. t. troglodytes and P. t schweinfurthii may have acquired SIVcpz after their divergence from P. t. verus and P. t. ellioti. However, all of the subspecies, except P. t. verus, still occasionally exchange migrants making the absence of SIVcpz in P. t. ellioti puzzling. Sampling of P. t. ellioti has been minimal to date, particularly along the banks of the Sanaga River, where its range abuts that of P. t. troglodytes. This study had three objectives. First, we extended the sampling of SIVcpz across the range of chimpanzees north of the Sanaga River to address whether under-sampling might account for the absence of evidence for SIVcpz infection in P. t. ellioti. Second, we investigated how environmental variation is associated with the spread and prevalence of SIVcpz in the two chimpanzee subspecies inhabiting Cameroon since environmental variation has been shown to contribute to their divergence from one another. Finally, we compared the prevalence and distribution of SIVcpz with that of Simian Foamy Virus (SFV) to examine the role of ecology and behavior in shaping the distribution of diseases in wild host populations. The dataset includes previously published results on SIVcpz infection and SFVcpz as well as newly collected data, and represents over 1000 chimpanzee fecal samples from 41 locations across Cameroon. Results revealed that none of the 181 P. t. ellioti fecal samples collected across the range of P. t. ellioti tested positive for SIVcpz. In addition, species distribution models suggest that environmental variation contributes to differences in the distribution and prevalence of SIVcpz and SFVcpz. The ecological niches of these two viruses are largely non-overlapping, although stronger statistical support for this conclusion will require more sampling. Overall this study demonstrates that SIVcpz infection is absent or very rare in P. t. ellioti, despite multiple opportunities for transmission. The reasons for its absence remain unclear, but might be explained by one or more factors, including environmental variation, viral competition, and/or local adaptation—all of which should be explored in greater detail through continued surveillance of this region. PMID:27505066

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

PubMed

Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

2015-10-01

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

PubMed Central

Iwasaki, Masaharu; Caì, Yíngyún; de la Torre, Juan C.

2018-01-01

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. PMID:29462184

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

PubMed

Ergünay, Koray; Brinkmann, Annika; Litzba, Nadine; Günay, Filiz; Kar, Sırrı; Öter, Kerem; Örsten, Serra; Sarıkaya, Yasemen; Alten, Bülent; Nitsche, Andreas; Linton, Yvonne-Marie

2017-07-01

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Amalga-like virus infecting Antonospora locustae, a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods.

PubMed

Pyle, Jesse D; Keeling, Patrick J; Nibert, Max L

2017-04-02

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens. Copyright © 2017 Elsevier B.V. All rights reserved.

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology

PubMed Central

Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

2014-01-01

The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine–thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine–cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health. PMID:24591590

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

PubMed

Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

2014-03-18

The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

PubMed

Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J; Farness, Peggy; Avanzini, Jenny B; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J; Mayall, Tim

2012-01-01

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

PubMed

Ludlow, M; Nguyen, D T; Silin, D; Lyubomska, O; de Vries, R D; von Messling, V; McQuaid, S; De Swart, R L; Duprex, W P

2012-07-01

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

PubMed

Kim, Shin-Hee; Samal, Siba K

2017-07-24

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro

PubMed Central

Papafragkou, Efstathia; Weaver, James C.; Ferrante, Thomas C.; Bahinski, Anthony; Elkins, Christopher A.; Kulka, Michael; Ingber, Donald E.

2017-01-01

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis. PMID:28146569

A Reverse Genetics Platform That Spans the Zika Virus Family Tree

PubMed Central

Widman, Douglas G.; Young, Ellen; Yount, Boyd L.; Plante, Kenneth S.; Gallichotte, Emily N.; Carbaugh, Derek L.; Plante, Jessica; Swanstrom, Jesica; Heise, Mark T.; Lazear, Helen M.

2017-01-01

ABSTRACT Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics. PMID:28270583

Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology.

PubMed

Pyle, Chloe J; Uwadiae, Faith I; Swieboda, David P; Harker, James A

2017-09-01

Interleukin-6 is a pleiotropic, pro-inflammatory cytokine that can promote both innate and adaptive immune responses. In humans with respiratory virus infections, such as Respiratory Syncytial Virus (RSV), elevated concentrations of IL-6 are associated with more severe disease. In contrast the polymorphisms in the Il6 promoter which favour lower IL-6 production are associated with increased risk of both RSV and Rhinovirus infections. To determine the precise contribution of IL-6 to protection and pathology we used murine models of respiratory virus infection. RSV infection resulted in increased IL-6 production both in the airways and systemically which remained heightened for at least 2 weeks. IL-6 depletion early, but not late, during RSV or Influenza A virus infection resulted in significantly increased disease associated with an influx of virus specific TH1 and cytotoxic CD8+ T cells, whilst not affecting viral clearance. IL-6 acted by driving production of the immunoregulatory cytokine IL-27 by macrophages and monocytes, which in turn promoted the local maturation of regulatory T cells. Concordantly IL-27 was necessary to regulate TH1 responses in the lungs, and sufficient to limit RSV induced disease. Overall we found that during respiratory virus infection the prototypic inflammatory cytokine IL-6 is a critical anti-inflammatory regulator of viral induced immunopathology in the respiratory tract through its induction of IL-27.

Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

PubMed Central

Swieboda, David P.

2017-01-01

Interleukin-6 is a pleiotropic, pro-inflammatory cytokine that can promote both innate and adaptive immune responses. In humans with respiratory virus infections, such as Respiratory Syncytial Virus (RSV), elevated concentrations of IL-6 are associated with more severe disease. In contrast the polymorphisms in the Il6 promoter which favour lower IL-6 production are associated with increased risk of both RSV and Rhinovirus infections. To determine the precise contribution of IL-6 to protection and pathology we used murine models of respiratory virus infection. RSV infection resulted in increased IL-6 production both in the airways and systemically which remained heightened for at least 2 weeks. IL-6 depletion early, but not late, during RSV or Influenza A virus infection resulted in significantly increased disease associated with an influx of virus specific TH1 and cytotoxic CD8+ T cells, whilst not affecting viral clearance. IL-6 acted by driving production of the immunoregulatory cytokine IL-27 by macrophages and monocytes, which in turn promoted the local maturation of regulatory T cells. Concordantly IL-27 was necessary to regulate TH1 responses in the lungs, and sufficient to limit RSV induced disease. Overall we found that during respiratory virus infection the prototypic inflammatory cytokine IL-6 is a critical anti-inflammatory regulator of viral induced immunopathology in the respiratory tract through its induction of IL-27. PMID:28953978

Development of Prototype Filovirus Recombinant Antigen Immunoassays

PubMed Central

Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

2015-01-01

Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

Walter Reed Army Institute of Research Annual Progress Report Fiscal Year 1982.

DTIC Science & Technology

1982-10-01

antibacterial activity . Fed. Proc. 4787, 1981. 11. Collins, H.H., D.F. Keren, P. Gemski, S.B. Formal, -.--| W.D. Zollinger, and G.H. Lowell...murlne myeloma cells. The fused cells will be subjected to specific selection by growth in selective media. The survivors from actively growing cell...prototype dengue virus strains. DEN-1 (Hawaiian), DEN-2 (New Guinea C), ÜEN-3 ( Philippines H-87), and DEN-4 ( Philippines H-241). Lymphocyte

The ROWPU Prefiltration System: Removal of Microorganisms

DTIC Science & Technology

1982-03-01

Pressman4 studied the performance of a prototype Rt(PTT filter rated at 360 gallons per hour. Raw river water was seeded with f2 coliphage virus prior to...block umaiber) Bacillus slobgii Water treatment Easheiebia foilr Cartridge filter Pouioviru Total aerobic bacteriaPalioirusTotal enteric bacteria 246...TTIACT ~ndo aEm. tom ebNi nmoveo -d hoioN 67 asoft aowl6) The Army has developed a Pleverse Osmosis Water Purification Unit (ROWPIT) to provide potable

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

PubMed Central

Suttiprapa, Sutas; Rinaldi, Gabriel; Brindley, Paul J.

2011-01-01

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5′- and 3′-Long Terminal Repeats (LTRs) flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3′-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3′-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed two to 20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference. PMID:21918820

Immunohistochemistry of LAMP-2 and adipophilin for phospholipidosis in liver and kidney in ketoconazole-treated mice.

PubMed

Asaoka, Yoshiji; Togashi, Yuko; Imura, Naoko; Sai, Takafumi; Miyoshi, Tomoya; Miyamoto, Yohei

2013-09-01

Drug-induced phospholipidosis is an abnormal accumulation of phospholipids in the lysosomes following repeated administration of cationic amphiphilic drugs. Phospholipidosis is detected histopathologically as cytoplasmic vacuolation; however, it is difficult to distinguish from lipid accumulation since their morphological features are similar. In this study, we investigated the usefulness of immunohistochemistry for lysosome-associated membrane protein-2 (LAMP-2) and adipophilin, a membrane protein of cytosolic non-lysosomal lipid droplets, in the liver and kidneys of mice orally administered ketoconazole, an inducer of hepatic phospholipidosis. In 7-week-old mice administered ketoconazole (300 mg/kg/day) for 7 days, cytoplasmic vacuolation was histopathologically observed in centrilobular hepatocytes and proximal tubular epithelial cells under the fasted condition. The cytoplasmic vacuolation consisted of foamy vacuoles, which were revealed to be phospholipidosis-characteristic lamellar bodies by electron microscopy. Furthermore, lipid-like vacuoles were observed in the perilobular hepatocytes, and revealed to be lipid droplets by electron microscopy. In immunohistochemistry, the foamy vacuoles and lipid-like vacuoles were positive for LAMP-2 and adipophilin, respectively. These results indicate that immunohistochemistry for LAMP-2 and adipophilin could distinguish between phospholipidosis and lipid accumulation. Additionally, it could detect ketoconazole-induced phospholipidosis in the glycogen-rich livers of non-fasted mice. In conclusion, ketoconazole induced phospholipidosis in not only the liver but also the kidneys, and immunohistochemistry for LAMP-2 and adipophilin could be useful for the pathological evaluation of drug-induced phospholipidosis in mice. Crown Copyright © 2012. Published by Elsevier GmbH. All rights reserved.

Rhabdoviruses as vaccine platforms for infectious disease and cancer.

PubMed

Zemp, Franz; Rajwani, Jahanara; Mahoney, Douglas J

2018-05-21

The family Rhabdoviridae (RV) comprises a large, genetically diverse collection of single-stranded, negative sense RNA viruses from the order Mononegavirales. Several RV members are being developed as live-attenuated vaccine vectors for the prevention or treatment of infectious disease and cancer. These include the prototype recombinant Vesicular Stomatitis Virus (rVSV) and the more recently developed recombinant Maraba Virus, both species within the genus Vesiculoviridae. A relatively strong safety profile in humans, robust immunogenicity and genetic malleability are key features that make the RV family attractive vaccine platforms. Currently, the rVSV vector is in preclinical development for vaccination against numerous high-priority infectious diseases, with clinical evaluation underway for HIV/AIDS and Ebola virus disease. Indeed, the success of the rVSV-ZEBOV vaccine during the 2014-15 Ebola virus outbreak in West Africa highlights the therapeutic potential of rVSV as a vaccine vector for acute, life-threatening viral illnesses. The rVSV and rMaraba platforms are also being tested as 'oncolytic' cancer vaccines in a series of phase 1-2 clinical trials, after being proven effective at eliciting immune-mediated tumour regression in preclinical mouse models. In this review, we discuss the biological and genetic features that make RVs attractive vaccine platforms and the development and ongoing testing of rVSV and rMaraba strains as vaccine vectors for infectious disease and cancer.

Genomic analysis reveals Nairobi sheep disease virus to be highly diverse and present in both Africa, and in India in the form of the Ganjam virus variant.

PubMed

Yadav, Pragya D; Vincent, Martin J; Khristova, Marina; Kale, Charuta; Nichol, Stuart T; Mishra, Akhilesh C; Mourya, Devendra T

2011-07-01

Nairobi sheep disease (NSD) virus, the prototype tick-borne virus of the genus Nairovirus, family Bunyaviridae is associated with acute hemorrhagic gastroenteritis in sheep and goats in East and Central Africa. The closely related Ganjam virus found in India is associated with febrile illness in humans and disease in livestock. The complete S, M and L segment sequences of Ganjam and NSD virus and partial sequence analysis of Ganjam viral RNA genome S, M and L segments encoding regions (396 bp, 701 bp and 425 bp) of the viral nucleocapsid (N), glycoprotein precursor (GPC) and L polymerase (L) proteins, respectively, was carried out for multiple Ganjam virus isolates obtained from 1954 to 2002 and from various regions of India. M segments of NSD and Ganjam virus encode a large ORF for the glycoprotein precursor (GPC), (1627 and 1624 amino acids in length, respectively) and their L segments encode a very large L polymerase (3991 amino acids). The complete S, M and L segments of NSD and Ganjam viruses were more closely related to one another than to other characterized nairoviruses, and no evidence of reassortment was found. However, the NSD and Ganjam virus complete M segment differed by 22.90% and 14.70%, for nucleotide and amino acid respectively, and the complete L segment nucleotide and protein differing by 9.90% and 2.70%, respectively among themselves. Ganjam and NSD virus, complete S segment differed by 9.40-10.40% and 3.2-4.10 for nucleotide and proteins while among Ganjam viruses 0.0-6.20% and 0.0-1.4%, variation was found for nucleotide and amino acids. Ganjam virus isolates differed by up to 17% and 11% at the nucleotide level for the partial S and L gene fragments, respectively, with less variation observed at the deduced amino acid level (10.5 and 2%, S and L, respectively). However, the virus partial M gene fragment (which encodes the hypervariable mucin-like domain) of these viruses differed by as much as 56% at the nucleotide level. Phylogenetic analysis of partial sequence differences suggests considerable mixing and movement of Ganjam virus strains within India, with no clear relationship between genetic lineages and virus geographic origin or year of isolation. Surprisingly, NSD virus does not represent a distinct lineage, but appears as a variant with other Ganjam virus among NSD virus group. Copyright © 2011 Elsevier B.V. All rights reserved.

Resource Requirements Planning for Hospitals Treating Serious Infectious Disease Cases

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

Vugrin, Eric D.; Verzi, Stephen Joseph; Finley, Patrick D.

This report presents a mathematical model of the way in which a hospital uses a variety of resources, utilities and consumables to provide care to a set of in-patients, and how that hospital might adapt to provide treatment to a few patients with a serious infectious disease, like the Ebola virus. The intended purpose of the model is to support requirements planning studies, so that hospitals may be better prepared for situations that are likely to strain their available resources. The current model is a prototype designed to present the basic structural elements of a requirements planning analysis. Some simplemore » illustrati ve experiments establish the mo del's general capabilities. With additional inve stment in model enhancement a nd calibration, this prototype could be developed into a useful planning tool for ho spital administrators and health care policy makers.« less

Antiviral Information Management System (AIMS): a prototype for operational innovation in drug development.

PubMed

Jadhav, Pravin R; Neal, Lauren; Florian, Jeff; Chen, Ying; Naeger, Lisa; Robertson, Sarah; Soon, Guoxing; Birnkrant, Debra

2010-09-01

This article presents a prototype for an operational innovation in knowledge management (KM). These operational innovations are geared toward managing knowledge efficiently and accessing all available information by embracing advances in bioinformatics and allied fields. The specific components of the proposed KM system are (1) a database to archive hepatitis C virus (HCV) treatment data in a structured format and retrieve information in a query-capable manner and (2) an automated analysis tool to inform trial design elements for HCV drug development. The proposed framework is intended to benefit drug development by increasing efficiency of dose selection and improving the consistency of advice from US Food and Drug Administration (FDA). It is also hoped that the framework will encourage collaboration among FDA, industry, and academic scientists to guide the HCV drug development process using model-based quantitative analysis techniques.

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

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

Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, wasmore » found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.« less

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

PubMed

Gumpper, Ryan H; Li, Weike; Castañeda, Carlos H; Scuderi, M José; Bashkin, James K; Luo, Ming

2018-04-15

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs. Copyright © 2018 American Society for Microbiology.

Genetic characterization of a Coxsackie A9 virus associated with aseptic meningitis in Alberta, Canada in 2010

PubMed Central

2013-01-01

Background An unusually high incidence of aseptic meningitis caused by enteroviruses was noted in Alberta, Canada between March and October 2010. Sequence based typing was performed on the enterovirus positive samples to gain a better understanding of the molecular characteristics of the Coxsackie A9 (CVA-9) strain responsible for most cases in this outbreak. Methods Molecular typing was performed by amplification and sequencing of the VP2 region. The genomic sequence of one of the 2010 outbreak isolates was compared to a CVA-9 isolate from 2003 and the prototype sequence to study genetic drift and recombination. Results Of the 4323 samples tested, 213 were positive for enteroviruses (4.93%). The majority of the positives were detected in CSF samples (n = 157, 73.71%) and 81.94% of the sequenced isolates were typed as CVA-9. The sequenced CVA-9 positives were predominantly (94.16%) detected in patients ranging in age from 15 to 29 years and the peak months for detection were between March and October. Full genome sequence comparisons revealed that the CVA-9 viruses isolated in Alberta in 2003 and 2010 were highly homologous to the prototype CVA-9 in the structural VP1, VP2 and VP3 regions but divergent in the VP4, non-structural and non-coding regions. Conclusion The increase in cases of aseptic meningitis was associated with enterovirus CVA-9. Sequence divergence between the prototype strain of CVA-9 and the Alberta isolates suggests genetic drifting and/or recombination events, however the sequence was conserved in the antigenic regions determined by the VP1, VP2 and VP3 genes. These results suggest that the increase in CVA-9 cases likely did not result from the emergence of a radically different immune escape mutant. PMID:23521862

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

PubMed

Wiltzer, L; Larrous, F; Oksayan, S; Ito, N; Marsh, G A; Wang, L F; Blondel, D; Bourhy, H; Jans, D A; Moseley, G W

2012-09-01

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

Characterization of Reaerosolization in an Effort to Improve Sampling of Airborne Viruses

DTIC Science & Technology

2008-04-01

financial support which helped me get through graduate school: Camp Dresser McKee for the CDM Fellowship; the UF Environmental Engineering Department...reservoir H um id ifi er /S at ur at or C ondenser THot TCold RH Figure A-1. BAU prototype schematic. A) Overview of system. B) Cross -sectional view of...degree in environmental engineering in August 2008 and entered the environmental engineering consulting industry with Camp Dresser McKee as an Engineer II in the Water/Wastewater Services Group.

Conservation of a Unique Mechanism of Immune Evasion across the Lyssavirus Genus

PubMed Central

Wiltzer, L.; Larrous, F.; Oksayan, S.; Ito, N.; Marsh, G. A.; Wang, L. F.; Blondel, D.; Bourhy, H.; Jans, D. A.

2012-01-01

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses. PMID:22740405

Esophageal xanthoma--report of two new cases and review of the literature.

PubMed

Becheanu, Gabriel; Dumbrava, Mona; Arbanas, Tudor; Diculescu, Mircea; Hoyeau-Idrissi, Nadia; Fléjou, Jean-François

2011-12-01

Esophageal xanthoma is a very rare lesion which can be incidentally discovered during endoscopy. Only eleven cases have been reported, including ours. We present two new cases of esophageal xanthoma localized in the lower esophagus in a 56-year-old woman and a 62-year-old man. Endoscopically, esophageal xanthoma appears as yellowish granular spots or a slightly elevated lesion. Microscopically, it consists of fat accumulation in foamy histiocytes beneath the squamous epithelium. The clinical and pathological importance of these lesions and what they mean in patients is discussed, along with a review of the literature.

Grid-based International Network for Flu observation (g-INFO).

PubMed

Doan, Trung-Tung; Bernard, Aurélien; Da-Costa, Ana Lucia; Bloch, Vincent; Le, Thanh-Hoa; Legre, Yannick; Maigne, Lydia; Salzemann, Jean; Sarramia, David; Nguyen, Hong-Quang; Breton, Vincent

2010-01-01

The 2009 H1N1 outbreak has demonstrated that continuing vigilance, planning, and strong public health research capability are essential defenses against emerging health threats. Molecular epidemiology of influenza virus strains provides scientists with clues about the temporal and geographic evolution of the virus. In the present paper, researchers from France and Vietnam are proposing a global surveillance network based on grid technology: the goal is to federate influenza data servers and deploy automatically molecular epidemiology studies. A first prototype based on AMGA and the WISDOM Production Environment extracts daily from NCBI influenza H1N1 sequence data which are processed through a phylogenetic analysis pipeline deployed on EGEE and AuverGrid e-infrastructures. The analysis results are displayed on a web portal (http://g-info.healthgrid.org) for epidemiologists to monitor H1N1 pandemics.

Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

PubMed

Al-Qahtani, Ahmed Ali; Mubin, Muhammad; Dela Cruz, Damian M; Althawadi, Sahar Isa; Ul Rehman, Muhammad Shah Nawaz; Bohol, Marie Fe F; Al-Ahdal, Mohammed N

2017-01-30

In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

PubMed

Chou, Ai-Hsiang; Liu, Chia-Chyi; Chang, Cheng-Peng; Guo, Meng-Shin; Hsieh, Shih-Yang; Yang, Wen-Hsueh; Chao, Hsin-Ju; Wu, Chien-Long; Huang, Ju-Lan; Lee, Min-Shi; Hu, Alan Yung-Chi; Lin, Sue-Chen; Huang, Yu-Yun; Hu, Mei-Hua; Chow, Yen-Hung; Chiang, Jen-Ron; Chang, Jui-Yuan; Chong, Pele

2012-01-01

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Hepatitis B virus molecular biology and pathogenesis

PubMed Central

Lamontagne, R. Jason; Bagga, Sumedha; Bouchard, Michael J.

2016-01-01

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350–500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae. In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC. PMID:28042609

Pharmacological intervention of HIV-1 maturation.

PubMed

Wang, Dan; Lu, Wuxun; Li, Feng

2015-11-01

Despite significant advances in antiretroviral therapy, increasing drug resistance and toxicities observed among many of the current approved human immunodeficiency virus (HIV) drugs indicate a need for discovery and development of potent and safe antivirals with a novel mechanism of action. Maturation inhibitors (MIs) represent one such new class of HIV therapies. MIs inhibit a late step in the HIV-1 Gag processing cascade, causing defective core condensation and the release of non-infectious virus particles from infected cells, thus blocking the spread of the infection to new cells. Clinical proof-of-concept for the MIs was established with betulinic acid derived bevirimat, the prototype HIV-1 MI. Despite the discontinuation of its further clinical development in 2010 due to a lack of uniform patient response caused by naturally occurring drug resistance Gag polymorphisms, several second-generation MIs with improved activity against viruses exhibiting Gag polymorphism mediated resistance have been recently discovered and are under clinical evaluation in HIV/AID patients. In this review, current understanding of HIV-1 MIs is described and recent progress made toward elucidating the mechanism of action, target identification and development of second-generation MIs is reviewed.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies.

PubMed

Laborde, Juan M; Sguazza, Guillermo H; Fuentealba, Nadia A; Corva, Santiago G; Carbone, Cecilia; Galosi, Cecilia M

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

PubMed Central

Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

1993-01-01

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

PubMed Central

Dobrikova, Elena Y.; Goetz, Christian; Walters, Robert W.; Lawson, Sarah K.; Peggins, James O.; Muszynski, Karen; Ruppel, Sheryl; Poole, Karyol; Giardina, Steven L.; Vela, Eric M.; Estep, James E.

2012-01-01

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied. PMID:22171271

Respiratory Syncytial Virus: Virology, Reverse Genetics, and Pathogenesis of Disease

PubMed Central

Fearns, Rachel; Graham, Barney S.

2016-01-01

Human respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus of family Paramyxoviridae. RSV is the most complex member of the family in terms of the number of genes and proteins. It is also relatively divergent and distinct from the prototype members of the family. In the past 30 years, we have seen a tremendous increase in our understanding of the molecular biology of RSV based on a succession of advances involving molecular cloning, reverse genetics, and detailed studies of protein function and structure. Much remains to be learned. RSV disease is complex and variable, and the host and viral factors that determine tropism and disease are poorly understood. RSV is notable for a historic vaccine failure in the 1960s involving a formalin-inactivated vaccine that primed for enhanced disease in RSV naïve recipients. Live vaccine candidates have been shown to be free of this complication. However, development of subunit or other protein-based vaccines for pediatric use is hampered by the possibility of enhanced disease and the difficulty of reliably demonstrating its absence in preclinical studies. PMID:24362682

Towards ambient temperature-stable vaccines: the identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay.

PubMed

Schlehuber, Lisa D; McFadyen, Iain J; Shu, Yu; Carignan, James; Duprex, W Paul; Forsyth, William R; Ho, Jason H; Kitsos, Christine M; Lee, George Y; Levinson, Douglas A; Lucier, Sarah C; Moore, Christopher B; Nguyen, Niem T; Ramos, Josephine; Weinstock, B André; Zhang, Junhong; Monagle, Julie A; Gardner, Colin R; Alvarez, Juan C

2011-07-12

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products. Copyright © 2011 Elsevier Ltd. All rights reserved.

Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease

PubMed Central

Williams, Marshall V.; Cox, Brandon; Ariza, Maria Eugenia

2016-01-01

The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person’s lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein–Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer. PMID:28036046

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles.

PubMed

Melegari, Irene; Marsilio, Fulvio; Di Profio, Federica; Sarchese, Vittorio; Massirio, Ivano; Palombieri, Andrea; D'Angelo, Anna Rita; Lanave, Gianvito; Diakoudi, Georgia; Cavalli, Alessandra; Martella, Vito; Di Martino, Barbara

2018-06-02

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age. Copyright © 2018 Elsevier B.V. All rights reserved.

Thiazolides as Novel Antiviral Agents: I. Inhibition of Hepatitis B Virus Replication

PubMed Central

Stachulski, Andrew V.; Pidathala, Chandrakala; Row, Eleanor C.; Sharma, Raman; Berry, Neil G.; Iqbal, Mazhar; Bentley, Joanne; Allman, Sarah A.; Edwards, Geoffrey; Helm, Alison; Hellier, Jennifer; Korba, Brent E.; Semple, J. Edward; Rossignol, Jean-Francois

2011-01-01

We report the syntheses and activities of a wide range of thiazolides [viz. 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide; NTZ] 1 is a broad spectrum antiinfective agent, effective against anaerobic bacteria, viruses and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent and selective inhibitor of hepatitis B replication (EC50 = 0.33 μm) but is inactive against anaerobes. Several 4′- and 5′-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1, viz. the O-acetate is an effective prodrug and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC90 s for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results. PMID:21553812

Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

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

Li, Jun; Shen, Wei; Liao, Ming, E-mail: mliao@scau.edu.cn

The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed inmore » Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.« less

Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity.

PubMed Central

Hoshino, Y; Sereno, M M; Midthun, K; Flores, J; Kapikian, A Z; Chanock, R M

1985-01-01

Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene-substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified. Images PMID:3001716

Crystal Structures of Beta- and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry

PubMed Central

Aydin, Halil; Cook, Jonathan D.

2014-01-01

Membrane fusion is a key step in the life cycle of all envelope viruses, but this process is energetically unfavorable; the transmembrane fusion subunit (TM) of the virion-attached glycoprotein actively catalyzes the membrane merger process. Retroviral glycoproteins are the prototypical system to study pH-independent viral entry. In this study, we determined crystal structures of extramembrane regions of the TMs from Mason-Pfizer monkey virus (MPMV) and xenotropic murine leukemia virus-related virus (XMRV) at 1.7-Å and 2.2-Å resolution, respectively. The structures are comprised of a trimer of hairpins that is characteristic of class I viral fusion proteins and now completes a structural library of retroviral fusion proteins. Our results allowed us to identify a series of intra- and interchain electrostatic interactions in the heptad repeat and chain reversal regions. Mutagenesis reveals that charge-neutralizing salt bridge mutations significantly destabilize the postfusion six-helix bundle and abrogate retroviral infection, demonstrating that electrostatic stapling of the fusion subunit is essential for viral entry. Our data indicate that salt bridges are a major stabilizing force on the MPMV and XMRV retroviral TMs and likely provide the key energetics for viral and host membrane fusion. PMID:24131724

Seroprevalence of yellow fever virus in selected health facilities in Western Kenya from 2010 to 2012.

PubMed

Kwallah, Allan ole; Inoue, Shingo; Thairu-Muigai, Anne Wangari; Kuttoh, Nancy; Morita, Kouichi; Mwau, Matilu

2015-01-01

Yellow fever (YF), which is caused by a mosquito-borne virus, is an important viral hemorrhagic fever endemic in equatorial Africa and South America. Yellow fever virus (YFV) is the prototype of the family Flaviviridae and genus Flavivirus. The aim of this study was to determine the seroprevalence of YFV in selected health facilities in Western Kenya during the period 2010-2012. A total of 469 serum samples from febrile patients were tested for YFV antibodies using in-house IgM-capture ELISA, in-house indirect IgG ELISA, and 50% focus reduction neutralization test (FRNT50). The present study did not identify any IgM ELISA-positive cases, indicating absence of recent YFV infection in the area. Twenty-eight samples (6%) tested positive for YFV IgG, because of either YFV vaccination or past exposure to various flaviviruses including YFV. Five cases were confirmed by FRNT50; of these, 4 were either vaccination or natural infection during the YF outbreak in 1992-1993 or another period and 1 case was confirmed as a West Nile virus infection. Domestication and routine performance of arboviral differential diagnosis will help to address the phenomenon of pyrexia of unknown origin, contribute to arboviral research in developing countries, and enhance regular surveillance.

Bovine Viral Diarrhea Virus (BVDV) in White-Tailed Deer (Odocoileus virginianus)

PubMed Central

Passler, Thomas; Ditchkoff, Stephen S.; Walz, Paul H.

2016-01-01

Bovine viral diarrhea virus (BVDV) is the prototypic member of the genus Pestivirus in the family Flaviviridae. Infections with BVDV cause substantial economic losses to the cattle industries, prompting various organized control programs in several countries. In North America, these control programs are focused on the identification and removal of persistently infected (PI) cattle, enhancement of BVDV-specific immunity through vaccination, and the implementation of biosecure farming practices. To be successful, control measures must be based on complete knowledge of the epidemiology of BVDV, including the recognition of other potential sources of the virus. BVDV does not possess strict host-specificity, and infections of over 50 species in the mammalian order Artiodactyla have been reported. Over 50 years ago, serologic surveys first suggested the susceptibility of white-tailed deer (Odocoileus virginianus), the most abundant free-ranging ruminant in North America, to BVDV. However, susceptibility of white-tailed deer to BVDV infection does not alone imply a role in the epidemiology of the virus. To be a potential wildlife reservoir, white-tailed deer must: (1) be susceptible to BVDV, (2) shed BVDV, (3) maintain BVDV in the population, and (4) have sufficient contact with cattle that allow spillback infections. Based on the current literature, this review discusses the potential of white-tailed deer to be a reservoir for BVDV. PMID:27379074

Norovirus-like VP1 particles exhibit isolate dependent stability profiles

NASA Astrophysics Data System (ADS)

Pogan, Ronja; Schneider, Carola; Reimer, Rudolph; Hansman, Grant; Uetrecht, Charlotte

2018-02-01

Noroviruses are the main cause of viral gastroenteritis with new variants emerging frequently. There are three norovirus genogroups infecting humans. These genogroups are divided based on the sequence of their major capsid protein, which is able to form virus-like particles (VLPs) when expressed recombinantly. VLPs of the prototypical GI.1 Norwalk virus are known to disassemble into specific capsid protein oligomers upon alkaline treatment. Here, native mass spectrometry and electron microscopy on variants of GI.1 and of GII.17 were performed, revealing differences in terms of stability between these groups. Beyond that, these experiments indicate differences even between variants within a genotype. The capsid stability was monitored in different ammonium acetate solutions varying both in ionic strength and pH. The investigated GI.1 West Chester isolate showed comparable disassembly profiles to the previously studied GI.1 Norwalk virus isolate. However, differences were observed with the West Chester being more sensitive to alkaline pH. In stark contrast to that, capsids of the variant belonging to the currently prevalent genogroup GII were stable in all tested conditions. Both variants formed smaller capsid particles already at neutral pH. Certain amino acid substitutions in the S domain of West Chester relative to the Norwalk virus potentially result in the formation of these T  =  1 capsids.

Nucleotide sequence and phylogenetic analysis of Cucurbit yellow stunting disorder virus RNA 2.

PubMed

Livieratos, Ioannis C; Coutts, Robert H A

2002-06-01

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5' to 3' direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.

From DCPD to NTCP: The long journey towards identifying a functional hepatitis B virus receptor

PubMed Central

2015-01-01

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection. PMID:26523264

From DCPD to NTCP: the long journey towards identifying a functional hepatitis B virus receptor.

PubMed

Li, Jisu; Tong, Shuping

2015-09-01

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.

Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries.

PubMed

Mattioli, S; Imberti, L; Stellini, R; Primi, D

1995-09-01

Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure.

Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs.

PubMed

Tilston-Lunel, Natasha L; Acrani, Gustavo Olszanski; Randall, Richard E; Elliott, Richard M

2015-12-23

Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

PubMed Central

Randall, Richard E.; Elliott, Richard M.

2015-01-01

ABSTRACT Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. IMPORTANCE Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen. PMID:26699638

Ozone-Induced Vascular Contractility and Pulmonary Injury Are Differentially Impacted by Diets Enriched With Coconut Oil, Fish Oil, and Olive Oil.

PubMed

Snow, Samantha J; Cheng, Wan-Yun; Henriquez, Andres; Hodge, Myles; Bass, Virgina; Nelson, Gail M; Carswell, Gleta; Richards, Judy E; Schladweiler, Mette C; Ledbetter, Allen D; Chorley, Brian; Gowdy, Kymberly M; Tong, Haiyan; Kodavanti, Urmila P

2018-05-01

Fish, olive, and coconut oil dietary supplementation have several cardioprotective benefits, but it is not established if they protect against air pollution-induced adverse effects. We hypothesized that these dietary supplements would attenuate ozone-induced systemic and pulmonary effects. Male Wistar Kyoto rats were fed either a normal diet, or a diet supplemented with fish, olive, or coconut oil for 8 weeks. Animals were then exposed to air or ozone (0.8 ppm), 4 h/day for 2 days. Ozone exposure increased phenylephrine-induced aortic vasocontraction, which was completely abolished in rats fed the fish oil diet. Despite this cardioprotective effect, the fish oil diet increased baseline levels of bronchoalveolar lavage fluid (BALF) markers of lung injury and inflammation. Ozone-induced pulmonary injury/inflammation were comparable in rats on normal, coconut oil, and olive oil diets with altered expression of markers in animals fed the fish oil diet. Fish oil, regardless of exposure, led to enlarged, foamy macrophages in the BALF that coincided with decreased pulmonary mRNA expression of cholesterol transporters, cholesterol receptors, and nuclear receptors. Serum microRNA profile was assessed and demonstrated marked depletion of a variety of microRNAs in animals fed the fish oil diet, several of which were of splenic origin. No ozone-specific changes were noted. Collectively, these data indicate that although fish oil offered vascular protection from ozone exposure, it increased pulmonary injury/inflammation and impaired lipid transport mechanisms resulting in foamy macrophage accumulation, demonstrating the need to be cognizant of potential off-target pulmonary effects that might offset the overall benefit of this vasoprotective supplement.

Postprandial Monocyte Activation in Individuals With Metabolic Syndrome

PubMed Central

Khan, Ilvira M.; Pokharel, Yashashwi; Dadu, Razvan T.; Lewis, Dorothy E.; Hoogeveen, Ron C.; Wu, Huaizhu

2016-01-01

Context: Postprandial hyperlipidemia has been suggested to contribute to atherogenesis by inducing proinflammatory changes in monocytes. Individuals with metabolic syndrome (MS), shown to have higher blood triglyceride concentration and delayed triglyceride clearance, may thus have increased risk for development of atherosclerosis. Objective: Our objective was to examine fasting levels and effects of a high-fat meal on phenotypes of monocyte subsets in individuals with obesity and MS and in healthy controls. Design, Setting, Participants, Intervention: Individuals with obesity and MS and gender- and age-matched healthy controls were recruited. Blood was collected from participants after an overnight fast (baseline) and at 3 and 5 hours after ingestion of a high-fat meal. At each time point, monocyte phenotypes were examined by multiparameter flow cytometry. Main Outcome Measures: Baseline levels of activation markers and postprandial inflammatory response in each of the three monocyte subsets were measured. Results: At baseline, individuals with obesity and MS had higher proportions of circulating lipid-laden foamy monocytes than controls, which were positively correlated with fasting triglyceride levels. Additionally, the MS group had increased counts of nonclassical monocytes, higher CD11c, CX3CR1, and human leukocyte antigen-DR levels on intermediate monocytes, and higher CCR5 and tumor necrosis factor-α levels on classical monocytes in the circulation. Postprandial triglyceride increases in both groups were paralleled by upregulation of lipid-laden foamy monocytes. MS, but not control, subjects had significant postprandial increases of CD11c and percentages of IL-1β+ and tumor necrosis factor-α+ cells in nonclassical monocytes. Conclusions: Compared to controls, individuals with obesity and MS had increased fasting and postprandial monocyte lipid accumulation and activation. PMID:27575945

Epidemiological aspects of astrovirus and coronavirus in poults in the South Eastern Region of Brazil

PubMed Central

da Silva, S.E.L.; Bonetti, A.M.; Petrocelli, A.; Ferrari, H.F.; Luvizotto, M.C.R.; Cardoso, T.C.

2009-01-01

A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileum-caeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries. PMID:24031353

Resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses

PubMed Central

Wang, Zheng; Malanoski, Anthony P; Lin, Baochuan; Kidd, Carolyn; Long, Nina C; Blaney, Kate M; Thach, Dzung C; Tibbetts, Clark; Stenger, David A

2008-01-01

Background Febrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. Results Using HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (Tessarae RPM-Flu). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level. Conclusion This study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses. PMID:19046445

Malpais spring virus is a new species in the genus vesiculovirus.

PubMed

Vasilakis, Nikos; Widen, Steven; Travassos da Rosa, Amelia Pa; Wood, Thomas G; Walker, Peter J; Holmes, Edward C; Tesh, Robert B

2013-03-04

Malpais Spring virus (MSPV) is a mosquito-borne rhabdovirus that infects a variety of wild and feral ungulates in New Mexico, including horses and deer. Although, initial serologic tests and electron microscopy at the time of isolation nearly 25 years ago provided evidence that MSPV is a novel virus, possibly related to vesiculoviruses, the virus still has not been approved as a new species. Use of the illumina platform allowed us to obtain the complete genome of MSPV. Analysis of the complete 11019 nt genome sequence of the prototype 85-488NM strain of MSPV indicates that it encodes the five common rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (> 180 nt) in the N, M and G genes, including a 249 nt ORF in the G gene predicted to encode a 9.26 kDa highly basic transmembrane protein. Although antigenically very distant, phylogenetic analysis of the L gene indicates that MSPV is most closely related to Jurona virus, also isolated from mosquitoes in Brazil, as well as a number of other vesiculoviruses. In sum, our analysis indicates MSPV should be classified as a member of the genus Vesiculovirus, family Rhabdoviridae. The complete genome sequence of MSPV will be helpful in the development of a reverse genetics system to study the unique aspects of this vesiculovirus in vivo and in vitro, and will assist development of specific diagnostic tests to study the epidemiology of MSPV infection.

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma

PubMed Central

2010-01-01

Background BamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing. Results Most NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence. Conclusion The sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence. PMID:20849661

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma.

PubMed

Hutajulu, Susanna H; Hoebe, Eveline K; Verkuijlen, Sandra Awm; Fachiroh, Jajah; Hariwijanto, Bambang; Haryana, Sofia M; Stevens, Servi Jc; Greijer, Astrid E; Middeldorp, Jaap M

2010-09-19

BamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing. Most NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence. The sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence.

Genetic diversity of human respiratory syncytial virus circulating among children in Ibadan, Nigeria.

PubMed

Ogunsemowo, Olukunle; Olaleye, David O; Odaibo, Georgina N

2018-01-01

Human respiratory syncytial virus (HRSV) is the most common viral cause of acute lower respiratory tract infections (LRTIs) in infants and young children however, without an effective vaccine licensed for human use till date. Information on the circulating genotypes of HRSV from regions with high-burden of infection is vital in the global efforts towards the development of protective vaccine. We report here the genotypes of HRSV circulating among children in Ibadan, the first of such from Nigeria.Nasopharyngeal and oropharyngeal swabs collected from 231 children presenting with respiratory infections in some health facilities for care as well as those attending immunization centers for routine vaccination in Ibadan, Nigeria were used for the study. The 2nd hypervariable (HVR2) region of the glycoprotein (G) gene of HRSV was amplified and sequenced using HRSV group specific primers. HRSV was detected in 41 out of the 231 samples. Thirty-three of the isolates were successfully subtyped(22 subtype A and 11 subtype B). Fourteen of the subtype A and all the subtype B were successfully sequenced and genotyped. Phylogenetic analysis showed that genotype ON1 with 72 nucleotide (nt) duplication was the major subgroup A virus (11 of 14) detected together with genotype NA2. All the HRSV subtype B detected belong to the BA genotype with characteristic 60nt duplication. The ON1 genotypes vary considerably from the prototype strain due to amino acid substitutions including T292I which has not been reported elsewhere. The NA2 genotypes have mutations on four antigenic sites within the HVR2relative to the prototype A2. In conclusion, three genotypes of HRSV were found circulating in Ibadan, Nigeria. Additional study that will include isolates from other parts of the country will be done to determine the extent of genotype diversity of HRSV circulating in Nigeria.

Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses.

PubMed

Konstantoulas, Constantine James; Indik, Stanislav

2014-04-30

Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and then spreads to T and B lymphocytes and finally to the mammary gland. It is not clear how the prototypic betaretrovirus infects mucosal DCs and naïve lymphocytes as these cells are considered to be non-proliferative. Studies of MMTV biology have been hampered by the difficulty of obtaining sufficient virus/vector titers after transfection of a molecular clone in cultured cells. To surmount this barrier we developed a novel MMTV-based vector system with a split genome design containing potent posttranscriptional regulatory functions. Using this system, vector particles were produced to markedly greater titers (>1000-fold) than those obtained previously. The titers (>106 transduction units /ml) were comparable to those achieved with lentiviral or gammaretroviral vectors. Importantly, the vector transduced the enhanced green fluorescence protein gene into the chromosomes of non-dividing cells, such as cells arrested at the G2/M phase of the cell cycle and unstimulated hematopoietic progenitor cells, at an efficiency similar to that obtained with the HIV-1-based vector. In contrast to HIV-1, MMTV transductions were not affected by knocking down the expression of a factor involved in nuclear import of the HIV-1 pre-integration complexes, TNPO3. In contrast to HIV-1, the MMTV-based vector did not preferentially integrate in transcription units. Additionally, no preference for integration near transcription start sites, the regions preferentially targeted by gammaretroviral vectors, was observed. The vector derived from MMTV exhibits a random integration pattern. Overall, the betaretroviral vector system should facilitate molecular virology studies of the prototypic betaretrovirus as well as studies attempting to elucidate fundamental cellular processes such as nuclear import pathways. Random integration in cycling and non-cycling cells may be applicable in unbiased gene delivery.

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication.

PubMed

Schuchman, Ryan; Kilianski, Andy; Piper, Amanda; Vancini, Ricardo; Ribeiro, José M C; Sprague, Thomas R; Nasar, Farooq; Boyd, Gabrielle; Hernandez, Raquel; Glaros, Trevor

2018-05-09

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. IMPORTANCE Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are no vaccines or prophylactics for these agents leaving one third of the world population at risk of infection. Identifying effective antivirals has been a long term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses. Copyright © 2018 Schuchman et al.

Prevention and control of seasonal influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices--United States, 2013-2014.

PubMed

2013-09-20

This report updates the 2012 recommendations by CDC's Advisory Committee on Immunization Practices (ACIP) regarding the use of influenza vaccines for the prevention and control of seasonal influenza (CDC. Prevention and control of influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2012;61:613-8). Routine annual influenza vaccination is recommended for all persons aged ≥ 6 months. For the 2013-14 influenza season, it is expected that trivalent live attenuated influenza vaccine (LAIV3) will be replaced by a quadrivalent LAIV formulation (LAIV4). Inactivated influenza vaccines (IIVs) will be available in both trivalent (IIV3) and quadrivalent (IIV4) formulations. Vaccine virus strains included in the 2013-14 U.S. trivalent influenza vaccines will be an A/California/7/2009 (H1N1)-like virus, an H3N2 virus antigenically like the cell-propagated prototype virus A/Victoria/361/2011, and a B/Massachusetts/2/2012-like virus. Quadrivalent vaccines will include an additional influenza B virus strain, a B/Brisbane/60/2008-like virus, intended to ensure that both influenza B virus antigenic lineages (Victoria and Yamagata) are included in the vaccine. This report describes recently approved vaccines, including LAIV4, IIV4, trivalent cell culture-based inactivated influenza vaccine (ccIIV3), and trivalent recombinant influenza vaccine (RIV3). No preferential recommendation is made for one influenza vaccine product over another for persons for whom more than one product is otherwise appropriate. This information is intended for vaccination providers, immunization program personnel, and public health personnel. These recommendations and other information are available at CDC's influenza website (http://www.cdc.gov/flu); any updates also will be found at this website. Vaccination and health-care providers should check the CDC influenza website periodically for additional information.

Nucleotide sequences of Herpes Simplex Virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7)

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

DeLuca, N.; Bzik, D.J.; Bond, V.C.

1982-10-30

The tsB5 strain of Herpes Simplex Virus type 1 (HSV-1) contains at least two mutations; one mutation specifies the syncytial phenotype and the other confers temperature sensitivity for virus growth. These functions are known to be located between the prototypic map coordinates 0.30 and 0.42. In this study it was demonstrated that tsB5 enters human embryonic lung (HEL) cells more rapidly than KOS, another strain of HSV-1. The EcoRI restriction fragment F from the KOS strain (map coordinates 0.315 to 0.421) was mapped with eight restriction endonucleases, and 16 recombinant plasmids were constructed which contained varying portions of the KOSmore » genome. Recombinant viruses were generated by marker-rescue and marker-transfer cotransfection procedures, using intact DNA from one strain and a recombinant plasmid containing DNA from the other strain. The region of the crossover between the two nonisogenic strains was inferred by the identification of restriction sites in the recombinants that were characteristic of the parental strains. The recombinants were subjected to phenotypic analysis. Syncytium formation, rate of virus entry, and the production of gB were all separable by the crossovers that produced the recombinants. The KOS sequences which rescue the syncytial phenotype of tsB5 were localized to 1.5 kb (map coordinates 0.345 to 0.355), and the temperature-sensitive mutation was localized to 1.2 kb (0.360 to 0.368), giving an average separation between the mutations of 2.5 kb on the 150-kb genome. DNA sequences that specify a functional domain for virus entry were localized to the nucleotide sequences between the two mutations. All three functions could be encoded by the virus gene specifying the gB glycoprotein.« less

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12).

PubMed

Terregino, C; Aldous, E W; Heidari, A; Fuller, C M; De Nardi, R; Manvell, R J; Beato, M S; Shell, W M; Monne, I; Brown, I H; Alexander, D J; Capua, I

2013-11-01

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

Biofabrication of Tobacco mosaic virus-nanoscaffolded supercapacitors via temporal capillary microfluidics

NASA Astrophysics Data System (ADS)

Zang, Faheng; Chu, Sangwook; Gerasopoulos, Konstantinos; Culver, James N.; Ghodssi, Reza

2017-06-01

This paper reports the implementation of temporal capillary microfluidic patterns and biological nanoscaffolds in autonomous microfabrication of nanostructured symmetric electrochemical supercapacitors. A photoresist layer was first patterned on the substrate, forming a capillary microfluidics layer with two separated interdigitated microchannels. Tobacco mosaic virus (TMV) macromolecules suspended in solution are autonomously delivered into the microfluidics, and form a dense bio-nanoscaffolds layer within an hour. This TMV layer is utilized in the electroless plating and thermal oxidation for creating nanostructured NiO supercapacitor. The galvanostatic charge/discharge cycle showed a 3.6-fold increase in areal capacitance for the nanostructured electrode compared to planar structures. The rapid creation of nanostructure-textured microdevices with only simple photolithography and bionanostructure self-assembly can completely eliminate the needs for sophisticated synthesis or deposition processes. This method will contribute to rapid prototyping of wide range of nano-/micro-devices with enhanced performance.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

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

Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin

2009-10-10

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibersmore » is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.« less

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

PubMed Central

Iverson, Eric A.; Goodman, David A.; Gorchels, Madeline E.

2017-01-01

ABSTRACT Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses. PMID:28148789

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

PubMed

Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

2017-05-15

Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses. Copyright © 2017 American Society for Microbiology.

Investigation of Multiscale and Multiphase Flow, Transport and Reaction in Heavy Oil Recovery Processes

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

Yortsos, Yanis C.

In this report, the thrust areas include the following: Internal drives, vapor-liquid flows, combustion and reaction processes, fluid displacements and the effect of instabilities and heterogeneities and the flow of fluids with yield stress. These find respective applications in foamy oils, the evolution of dissolved gas, internal steam drives, the mechanics of concurrent and countercurrent vapor-liquid flows, associated with thermal methods and steam injection, such as SAGD, the in-situ combustion, the upscaling of displacements in heterogeneous media and the flow of foams, Bingham plastics and heavy oils in porous media and the development of wormholes during cold production.

ELECTRON MICROSCOPIC OBSERVATIONS OF AMOEBA PROTEUS IN GROWTH AND INANITION

PubMed Central

Cohen, Adolph I.

1957-01-01

Electron microscopic observations have been made on growing and dividing specimens of Amoeba proteus and also on starving animals. Structures presumably corresponding to the mitochondria, alpha particles, vacuoles, and Golgi material are described. A new entity, designated as a foamy particle, is noted. Descriptions are given of the cytoplasmic and nuclear membranes. During division the inner, thick nuclear membrane component is seen to vanish and the outer membrane persist. Measurements suggest a gradual reappearance of the inner component with growth. Starving animals show a loss of cytoplasmic granularity and an increase in the electron density of mitochondria, presumably due to lipide accumulation. PMID:13481020

Electron microscopic observations of amoeba proteus in growth and inanition.

PubMed

COHEN, A I

1957-11-25

Electron microscopic observations have been made on growing and dividing specimens of Amoeba proteus and also on starving animals. Structures presumably corresponding to the mitochondria, alpha particles, vacuoles, and Golgi material are described. A new entity, designated as a foamy particle, is noted. Descriptions are given of the cytoplasmic and nuclear membranes. During division the inner, thick nuclear membrane component is seen to vanish and the outer membrane persist. Measurements suggest a gradual reappearance of the inner component with growth. Starving animals show a loss of cytoplasmic granularity and an increase in the electron density of mitochondria, presumably due to lipide accumulation.

Water-clear cell adenoma of the parathyroid. A case report with immunohistochemistry and electron microscopy.

PubMed

Grenko, R T; Anderson, K M; Kauffman, G; Abt, A B

1995-11-01

We report a water-clear cell adenoma of the parathyroid gland, a lesion which to our knowledge has not been described previously. Like its rare but well-described hyperplastic counterpart, water-clear cell hyperplasia, this adenoma is composed of cells with abundant foamy-to-granular cytoplasm and mild nuclear pleomorphism. The cells form glandular structures and cell nests separated by fine fibrovascular septae. The tumor cells stain positively with anti-parathyroid hormone and show characteristic glassy and flocculate material by electron microscopy. Unlike water-clear cell hyperplasia, water-clear cell adenoma is a solitary lesion that compresses the residual nonneoplastic parathyroid gland.

Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes.

PubMed

Fujino, Kan; Yamamoto, Yusuke; Daito, Takuji; Makino, Akiko; Honda, Tomoyuki; Tomonaga, Keizo

2017-09-01

Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia.

PubMed

Hall-Mendelin, Sonja; Pyke, Alyssa T; Moore, Peter R; Mackay, Ian M; McMahon, Jamie L; Ritchie, Scott A; Taylor, Carmel T; Moore, Frederick A J; van den Hurk, Andrew F

2016-09-01

Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. After 14 days incubation at 28°C and high relative humidity, infection, dissemination and transmission rates were assessed. Infection in Culex annulirostris and Cx. sitiens could not be detected. 8% of Cx. quinquefasciatus were infected, but the virus did not disseminate in this species. Despite having infection rates > 50%, Aedes notoscriptus and Ae. vigilax did not transmit ZIKV. In contrast, Ae. aegypti had infection and transmission rates of 57% and 27%, respectively. In susceptibility trials, the virus dose required to infect 50% (ID50) of Ae. aegypti was106.4 tissue culture infectious dose50 (TCID50)/mL. Additionally, a threshold viral load within the mosquito of at least 105.1 TCID50 equivalents/mL had to be reached before virus transmission occurred. We confirmed Ae. aegypti to be the most likely mosquito vector of ZIKV in Australia, although the restricted distribution of this species will limit the receptive zone to northern Queensland where this species occurs. Importantly, the role in ZIKV transmission of Culex and other Aedes spp. tested will be negligible. Despite being the implicated vector, the relatively high ID50 and need for a high titer disseminated infection in Ae. aegypti suggest that high mosquito population densities will be required to facilitate epidemic ZIKV transmission among the currently immunologically naïve human population in Australia.

Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus.

PubMed

Tiwari, Mugdha; Parida, Manmohan; Santhosh, S R; Khan, Mohsin; Dash, Paban Kumar; Rao, P V Lakshmana

2009-04-21

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6 -- 8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.

Human T Cell Lymphotropic Virus Type 2a Strains Among HIV Type 1-Coinfected Patients from Brazil Have Originated Mostly from Brazilian Amerindians

PubMed Central

Magri, Mariana Cavalheiro; Brigido, Luis Fernando de Macedo; Morimoto, Helena Kaminami

2013-01-01

Abstract The human T cell lymphotropic virus type 2 (HTLV-2) is found mainly in Amerindians and in intravenous drug users (IDUs) from urban areas of the United States, Europe, and Latin America. Worldwide, HTLV-2a and HTLV-2b subtypes are the most prevalent. Phylogenetic analysis of HTLV-2 isolates from Brazil showed the HTLV-2a subtype, variant -2c, which spread from Indians to the general population and IDUs. The present study searched for the types of HTLV-2 that predominate among HIV-1-coinfected patients from southern and southeastern Brazil. Molecular characterization of the LTR, env, and tax regions of 38 isolates confirmed the HTLV-2c variant in 37 patients, and one HTLV-2b in a patient from Paraguay. Phylogenetic analysis of sequences showed different clades of HTLV-2 associated with risk factors and geographic region. These clades could represent different routes of virus transmission and/or little diverse evolutionary rates of virus. Taking into account the results obtained in the present study and the lack of the prototypic North American HTLV-2a strain and HTLV-2b subtypes commonly detected among HIV-coinfected individuals worldwide, we could speculate on the introduction of Brazilian HTLV-2 strains in such populations before the introduction of HIV. PMID:23484539

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat.

PubMed

Lee, Hee Suk; Sobsey, Mark D

2011-06-01

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23-25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall. Copyright © 2011 Elsevier Ltd. All rights reserved.

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

PubMed Central

Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

1996-01-01

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

[Human Papilloma virus in Quechua women from Jujuy with high frequency of cervical cancer: viral types and HPV-16 variants].

PubMed

Picconi, Maria Alejandra; Gronda, Jorge; Alonio, Lidia V; Villa, Luisa L; Sichero, Laura; Miranda, Sergio; Barcena, Martin; Teyssie, Angelica

2002-01-01

Human Papillomaviruses (HPVs) are etiologically associated to cervical carcinoma. In order to evaluate HPV infection and its relationship with the high frequency of this neoplasia in Quechua women from Jujuy (Argentina), 271 cervical samples from preneoplastic and neoplastic lesions (biopsies) and normal controls (cytologies) were studied. Detection and typing were performed using PCR-RFLP or PCR-hybridization and the HPV-16 variability in L1 and E6 genes (by PCR-hybridization) was analysed. HPV was detected in 52% of controls, 91% of low-grade lesions, 97% of high-grade lesions and 100% of invasive carcinomas, corresponding 55% to HPV-16. HPV-16 European variants were predominant, most of them being non-prototypic strains. The high frequency of high risk infection types and the raised proportion of HPV-16 non-prototypic variants related to a greater oncogenic potential could explain, in part, the high cervical cancer frequency of this native population. These data may contribute to disease control and vaccinal formulation.

Dengue virus infection-enhancing antibody activities against Indonesian strains in inhabitants of central Thailand.

PubMed

Yamanaka, Atsushi; Oddgun, Duangjai; Chantawat, Nantarat; Okabayashi, Tamaki; Ramasoota, Pongrama; Churrotin, Siti; Kotaki, Tomohiro; Kameoka, Masanori; Soegijanto, Soegeng; Konishi, Eiji

2016-04-01

Dengue virus (DENV) infection-enhancing antibodies are a hypothetic factor to increase the dengue disease severity. In this study, we investigated the enhancing antibodies against Indonesian strains of DENV-1-4 in 50 healthy inhabitants of central Thailand (Bangkok and Uthai Thani). Indonesia and Thailand have seen the highest dengue incidence in Southeast Asia. The infection history of each subject was estimated by comparing his/her neutralizing antibody titers against prototype DENV-1-4 strains. To resolve the difficulty in obtaining foreign live viruses for use as assay antigens, we used a recombinant system to prepare single-round infectious dengue viral particles based on viral sequence information. Irrespective of the previously infecting serotype(s), most serum samples showed significantly higher enhancement titers against Indonesian DENV-2 strains than against Thai DENV-2 strains, whereas the opposite effect was observed for the DENV-3 strains. Equivalent enhancing activities were observed against both DENV-1 and DENV-4. These results suggest that the genotype has an impact on enhancing antibody activities against DENV-2 and DENV-3, because the predominant circulating genotypes of each serotype differ between Indonesia and Thailand. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys.

PubMed

Tsarev, S A; Tsareva, T S; Emerson, S U; Yarbough, P O; Legters, L J; Moskal, T; Purcell, R H

1994-06-01

The infectivity titer of a standard stock of the SAR-55 strain of hepatitis E virus (HEV) was determined in cynomolgus macaques (Macaca fascicularis) and the effect of dose on the course of the infection was examined by weekly monitoring of alanine aminotransferase (ALT) and anti-HEV levels. Antibody to HEV (anti-HEV) was measured with ELISAs based on ORF-2 recombinant antigens consisting of either a 55 kDa region expressed in insect cells or shorter regions expressed as fusion proteins in bacteria. The ELISA based on the 55 kDa antigen was generally more sensitive. The infectivity titer of SAR-55 was 10(6) cynomolgus 50% infectious doses per gram of feces. The infectivity titer corresponded to the HEV genome titer of the inoculum as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Anti-HEV IgM was detected in only a portion of the animals that had an anti-HEV IgG response. Biochemical evidence of hepatitis was most prominent in animals that were inoculated with the higher concentrations of virus and the incubation period to seroconversion was prolonged in animals that received the lower doses.

Studies on manifestations of canine distemper virus infection in an urban dog population.

PubMed

Blixenkrone-Møller, M; Svansson, V; Have, P; Orvell, C; Appel, M; Pedersen, I R; Dietz, H H; Henriksen, P

1993-10-01

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.

The minute virus of mice exploits different endocytic pathways for cellular uptake

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

Garcin, Pierre O.; Panté, Nelly, E-mail: pante@zoology.ubc.ca

The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy andmore » flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. - Highlights: • MVMp uptake takes place at the leading edge of migrating cells. • MVMp exploits a variety of endocytic pathways. • MVMp could use clathrin- and caveolin-mediated endocytosis. • MVMp could also use clathrin-independent carriers for cellular uptake.« less

Genomic and Biological Characterization of Aggressive and Docile Strains of LCMV Rescued from a Plasmid-Based Reverse Genetics System

PubMed Central

Chen, Minjie; Lan, Shuiyun; Ou, Rong; Price, Graeme E.; Jiang, Hong; de la Torre, Juan Carlos; Moskophidis, Demetrius

2008-01-01

Arenaviruses include several causative agents of hemorrhagic fever disease in humans. In addition, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a superb model for the study of virus-host interactions, including the basis of viral persistence and associated diseases. The molecular mechanisms concerning the regulation and specific role of viral proteins in modulating arenavirus-host cell interactions associated either with an acute or persistent infection and associated disease remain little understood. Here we report the genomic and biological characterization of LCMV strains Docile (persistent) and Aggressive (not persistent) recovered from cloned cDNA via reverse genetics. Our results confirmed that the cloned viruses accurately recreated the in vivo phenotypes associated with the corresponding natural Docile and Aggressive viral isolates. In addition, we provide evidence that the ability of the Docile strain to persist is determined by the nature of both S and L RNA segments. Thus, our findings provide the foundation for studies aimed at gaining a detailed understanding of viral determinants of LCMV persistence in its natural host that may aid in the development of vaccines to prevent or treat the diseases caused by arenaviruses in humans. PMID:18474558

Molecular epidemiology of porcine reproductive and respiratory syndrome viruses isolated from 1991 to 2013 in Taiwan.

PubMed

Deng, Ming-Chung; Chang, Chia-Yi; Huang, Tien-Shine; Tsai, Hsiang-Jung; Chang, Chieh; Wang, Fun-In; Huang, Yu-Liang

2015-11-01

Porcine reproductive and respiratory syndrome virus (PRRSV) was first identified in Taiwan in 1991, but the genetic diversity and evolution of PRRSV has not been thoroughly investigated over the past 20 years. The aim of this study was to bridge the gap in understanding of its molecular epidemiology. A total of 31 PRRSV strains were collected and sequenced. The sequences were aligned using the MUSCLE program, and phylogenetic analysis were performed by the maximum-likelihood method and the neighbor-joining method using MEGA 5.2 software. In the early 1990s, two prototype strains, WSV and MD001 of the North American genotype, were first identified. Over the years, both viruses evolved separately. The population dynamics of PRRSV revealed that the strains of the MD001 group were predominant in Taiwan. Evolution was manifested in changes in the nsp2 and ORF5 genes. In addition, a suspected newly invading exotic strain was recovered in 2013, suggesting that international spread is still taking place and that it is affecting the population dynamics. Overall, the results provide an important basis for vaccine development for the control and prevention of PRRS.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens

PubMed Central

Martínez, Isidoro; Oliveros, Juan C.; Cuesta, Isabel; de la Barrera, Jorge; Ausina, Vicente; Casals, Cristina; de Lorenzo, Alba; García, Ernesto; García-Fojeda, Belén; Garmendia, Junkal; González-Nicolau, Mar; Lacoma, Alicia; Menéndez, Margarita; Moranta, David; Nieto, Amelia; Ortín, Juan; Pérez-González, Alicia; Prat, Cristina; Ramos-Sevillano, Elisa; Regueiro, Verónica; Rodriguez-Frandsen, Ariel; Solís, Dolores; Yuste, José; Bengoechea, José A.; Melero, José A.

2017-01-01

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here. PMID:28298903

A Reverse Genetics Platform That Spans the Zika Virus Family Tree.

PubMed

Widman, Douglas G; Young, Ellen; Yount, Boyd L; Plante, Kenneth S; Gallichotte, Emily N; Carbaugh, Derek L; Peck, Kayla M; Plante, Jessica; Swanstrom, Jesica; Heise, Mark T; Lazear, Helen M; Baric, Ralph S

2017-03-07

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics. IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design. Copyright © 2017 Widman et al.

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

PubMed

Dietzel, Erik; Schudt, Gordian; Krähling, Verena; Matrosovich, Mikhail; Becker, Stephan

2017-01-15

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity. Copyright © 2017 American Society for Microbiology.

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

PubMed

Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

2010-05-01

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

High-Throughput Dissection of AAV-Host Interactions: The Fast and the Curious.

PubMed

Herrmann, Anne-Kathrin; Grimm, Dirk

2018-05-18

Over fifty years after its initial description, Adeno-associated virus (AAV) remains a most exciting but also most elusive study object in basic or applied virology. On the one hand, its simple structure not only facilitates investigations into virus biology, but combined with the availability of numerous natural AAV variants with distinct infection efficiency and specificity also makes AAV a preferred substrate for engineering of gene delivery vectors. On the other hand, it is striking to witness a recent flurry of reports that highlight and partially close persistent gaps in our understanding of AAV virus and vector biology. This is all the more perplexing considering that recombinant AAVs have already been used in >160 clinical trials and recently been commercialized as gene therapeutics. Here, we discuss a reason for these advances in AAV research, namely, the advent and application of powerful high-throughput technology for dissection of AAV-host interactions and optimization of AAV gene therapy vectors. As relevant examples, we focus on the discovery of (i) a "new" cellular AAV receptor, AAVR, (ii) host restriction factors for AAV entry, and (iii) AAV capsid determinants that mediate trafficking through the blood-brain barrier. While (i)/(ii) are prototypes of extra- or intracellular AAV host factors that were identified via high-throughput screenings, (iii) exemplifies the power of molecular evolution to investigate the virus itself. In the future, we anticipate that these and other key technologies will continue to accelerate the dissection of AAV biology and will yield a wealth of new designer viruses for clinical use. Copyright © 2018. Published by Elsevier Ltd.

On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

PubMed Central

Carvalho, Carlos A.M.; Silva, Jerson L.; Oliveira, Andréa C.

2017-01-01

Mayaro virus (MAYV) is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell. PMID:28462045

West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia.

PubMed

Öncü, Ceren; Brinkmann, Annika; Günay, Filiz; Kar, Sırrı; Öter, Kerem; Sarıkaya, Yasemen; Nitsche, Andreas; Linton, Yvonne-Marie; Alten, Bülent; Ergünay, Koray

2018-01-01

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n=3), Cx. pipiens s.l. (n=1) and Aedes caspius (n=1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s.l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate. Copyright © 2017 Elsevier B.V. All rights reserved.

Endogenous lipid (cholesterol) pneumonia in three captive Siberian tigers (Panthera tigris altaica).

PubMed

Bollo, Enrico; Scaglione, Frine Eleonora; Chiappino, Laura; Sereno, Alessandra; Triberti, Orfeo; Schröder, Cathrin

2012-05-01

During the years 2009-2011, 7 Siberian tigers (Panthera tigris altaica), aged between 2 and 14 years, from the Safaripark of Pombia were referred for necropsy to the Department of Animal Pathology of the University of Turin (Italy). Three tigers, aged 10 (2 animals) and 14 years, had multifocal, irregularly distributed, white, soft, subpleural, 3-mm nodules scattered throughout the lungs. Histologically, there was a marked infiltration of macrophages, with foamy cytoplasm, and multinucleate giant cells interspersed with numerous clusters of cholesterol clefts. A mild lymphocytic infiltration was localized around the lesion. The findings were consistent with endogenous lipid pneumonia, which was considered an incidental finding of no clinical significance.

Novel foamy origin for singlet fermion masses

NASA Astrophysics Data System (ADS)

Ellis, John; Mavromatos, Nick E.; Nanopoulos, Dimitri V.

2017-10-01

We show how masses for singlet fermions can be generated by interactions with a D-particle model of space-time foam inspired by brane theory. It has been shown previously by one of the authors (N. E. M.) that such interactions may generate dynamically small masses for charged fermions via the recoils of D-particle defects interacting with photons. In this work we consider the direct interactions of D-particle with uncharged singlet fermions such as right-handed neutrinos. Quantum fluctuations of the lattice of D-particles have massless vector (spin-one) excitations that are analogues of phonons. These mediate forces with the singlet fermions, generating large dynamical masses that may be communicated to light neutrinos via the seesaw mechanism.

Antigenically Diverse Swine Origin H1N1 Variant Influenza Viruses Exhibit Differential Ferret Pathogenesis and Transmission Phenotypes.

PubMed

Pulit-Penaloza, Joanna A; Jones, Joyce; Sun, Xiangjie; Jang, Yunho; Thor, Sharmi; Belser, Jessica A; Zanders, Natosha; Creager, Hannah M; Ridenour, Callie; Wang, Li; Stark, Thomas J; Garten, Rebecca; Chen, Li-Mei; Barnes, John; Tumpey, Terrence M; Wentworth, David E; Maines, Taronna R; Davis, C Todd

2018-06-01

Influenza A(H1) viruses circulating in swine represent an emerging virus threat, as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from the classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus, A/Iowa/39/2015, as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Preexisting immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015. IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a nonfatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey.

PubMed

Ergünay, Koray; Litzba, Nadine; Brinkmann, Annika; Günay, Filiz; Sarıkaya, Yasemen; Kar, Sırrı; Örsten, Serra; Öter, Kerem; Domingo, Cristina; Erisoz Kasap, Özge; Özkul, Aykut; Mitchell, Luke; Nitsche, Andreas; Alten, Bülent; Linton, Yvonne-Marie

2017-03-20

Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as "Ochlerotatus caspius flavivirus Turkey", was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.

NK cells and poxvirus infection

PubMed Central

Burshtyn, Deborah N.

2013-01-01

In recent years, our understanding of the role of natural killer (NK) cells in the response to viral infection has grown rapidly. Not only do we realize viruses have many immune-evasion strategies to escape NK cell responses, but that stimulation of NK cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that NK cells can provide a memory response to viral pathogens. Tremendous knowledge has been gained in this area through the study of herpes viruses, but appreciation for the significance of NK cells in the response to other types of viral infections is growing. The function of NK cells in defense against poxviruses has emerged over several decades beginning with the early seminal studies showing the role of NK cells and the NK gene complex in susceptibility of mouse strains to ectromelia, a poxvirus pathogen of mice. More recently, greater understanding has emerged of the molecular details of the response. Given that human diseases caused by poxviruses can be as lethal as smallpox or as benign as Molluscum contagiosum, and that vaccinia virus, the prototypic member of the pox family, persists as a mainstay of vaccine design and has potential as an oncolytic virus for tumor therapy, further research in this area remains important. This review focuses on recent advances in understanding the role of NK cells in the immune response to poxviruses, the receptors involved in activation of NK cells during poxvirus infection, and the viral evasion strategies poxviruses employ to avoid the NK response. PMID:23372568

The microtubule motor protein KIF13A is involved in intracellular trafficking of the Lassa virus matrix protein Z.

PubMed

Fehling, Sarah Katharina; Noda, Takeshi; Maisner, Andrea; Lamp, Boris; Conzelmann, Karl-Klaus; Kawaoka, Yoshihiro; Klenk, Hans-Dieter; Garten, Wolfgang; Strecker, Thomas

2013-02-01

The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins. © 2012 Blackwell Publishing Ltd.

Characterization of a novel hepadnavirus in the white sucker (Catostomus commersonii) from the Great Lakes Region of the USA

USGS Publications Warehouse

Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Conway, Carla M.; Winton, James R.; Blazer, Vicki S.

2015-01-01

The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the USA. Dideoxysequencing confirmed the white sucker hepatitis B virus (WSHBV) has a circular genome (3542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase and surface proteins was low and ranged from 16-27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively non-coding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV other hepadnaviruses, and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, of which only 2.4% were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker.

Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells

PubMed Central

Kemble, George W.; Annunziato, Paula; Lungu, Octavian; Winter, Ruth E.; Cha, Tai-An; Silverstein, Saul J.; Spaete, Richard R.

2000-01-01

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3′ untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation. PMID:11070031

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1

PubMed Central

Varghese, Vici; Mitsuya, Yumi; Fessel, W. Jeffrey; Liu, Tommy F.; Melikian, George L.; Katzenstein, David A.; Schiffer, Celia A.; Holmes, Susan P.

2013-01-01

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses. PMID:23796938

[Acute inflammatory polyradiculoneuropathy and membranous glomerulonephritis following Epbstein-Barr virus primary infection in a 12-year-old girl].

PubMed

Meyer, P; Soëte, S; Raynaud, P; Henry, V; Morin, D; Rodière, M; Rivier, F; Roubertie, A

2010-11-01

Acute inflammatory polyradiculoneuropathy, or Guillain-Barré syndrome (GBS), is characterized by peripheral nerve demyelination, which leads to rapidly progressive weakness, loss of sensation, and loss of deep tendon reflexes. It is a prototype of postinfectious autoimmune disease, whose pathophysiology is well described in the forms provoked by certain bacteria (molecular mimicry with Campylobacter jejuni), but remains unclear for the forms related to other organisms (cytomegalovirus, Epstein-Barr virus and other herpes group viruses, Mycoplasma pneumoniae). Glomerular lesions can be associated with the neurological symptoms and have also been described after various infections, independently of any signs of polyradiculoneuropathy. We report the observation of a 12-year-old girl who presented with Guillain-Barré syndrome with facial diplegia, ataxia, and intracranial hypertension following Epstein-Barr virus (EBV) primary infection. During the course of the neurological disease, membranous glomerulonephritis (MGN) was diagnosed. The neurological impairment was regressive within 6 months after intravenous immunoglobulin treatment followed by intravenous then oral corticosteroid administration. Viremia remained high more than 6 months after the onset of symptoms. Glomerulopathy progressed independently and finally required immunosuppressant medication with cyclosporine. EBV might be the factor that triggered the autoimmune disorders, as previously reported for systemic lupus erythematosus and multiple sclerosis in children. To the best of our knowledge, this association of 3 conditions (GBS, MGN, and EBV primary infection) has never been reported in the literature. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

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

Leong, JoAnn Ching

A prototype subunit vaccine to IHN virus is being developed by recombinant DNA techniques. The techniques involve the isolation and characterization of the glycoprotein gene, which encodes the viral protein responsible for inducing a protective immune response in fish. The viral glycoprotein gene has been cloned and a restriction map of the cloned gene has been prepared. Preliminary DNA sequence analysis of the cloned gene has been initiated so that manipulation of the gene for maximum expression in appropriate plasmid vectors is possible. A recombinant plasmid containing the viral gene inserted in the proper orientation adjacent to a very strongmore » lambda promoter and ribosome binding site has been constructed. Evaluation of this recombinant plasmid for gene expression is being conducted. Immunization trials with purified viral glycoprotein indicate that fish are protected against lethal doses of IHNV after immersion and intraperitoneal methods of immunization. In addition, cross protection immunization trials indicate that Type 2 and Type 1 IHN virus produce glycoproteins that are cross-protective.« less

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies.

PubMed

Xu, Gelin; Weber, Patrick; Hu, Qiaoling; Xue, Honggang; Audry, Laurent; Li, Chengping; Wu, Jie; Bourhy, Herve

2007-10-01

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

PubMed

Meyer, D; Fritsche, S; Luo, Y; Engemann, C; Blome, S; Beyerbach, M; Chang, C-Y; Qiu, H-J; Becher, P; Postel, A

2017-12-01

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of E rns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel E rns -specific prototype ELISA (pigtype CSFV E rns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect E rns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other E rns -based antibody ELISAs were identified correctly by the novel prototype E rns ELISA and vice versa. In conclusion, the pigtype CSFV E rns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional E rns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine. © 2017 Blackwell Verlag GmbH.

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

PubMed Central

Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie; Smith, David F.; Cummings, Richard D.; Chen, Xi; Trollope, Alana J.; North, Simon J.; Haslam, Stuart M.; Dell, Anne; Tattersall, Peter; McKenna, Robert; Agbandje-McKenna, Mavis

2014-01-01

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen. PMID:24475195

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug.

PubMed

Mastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M; Frick, David N; Bolognesi, Martino; Milani, Mario

2012-08-01

Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC₅₀ values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug

PubMed Central

Mastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M.; Frick, David N.; Bolognesi, Martino; Milani, Mario

2012-01-01

Objectives Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Methods Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Results Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. Conclusions The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV. PMID:22535622

A Novel Avian Paramyxovirus (Putative Serotype 15) Isolated from Wild Birds.

PubMed

Lee, Hyun-Jeong; Kim, Ji-Ye; Lee, Youn-Jeong; Lee, Eun-Kyung; Song, Byoung-Min; Lee, Hee-Soo; Choi, Kang-Seuk

2017-01-01

In January 2014, a viral hemagglutinating agent named UPO216 was isolated from fecal droppings of wild birds at the UPO wetland in South Korea during an avian influenza surveillance program. Electron microscopy identified the UPO216 virus as an avian paramyxovirus (APMV). Pathogenicity tests and molecular pathotyping revealed that the virus was avirulent in chickens. The UPO216 virus was assigned to a serological group antigenically distinct from known serotypes of APMV (-1, -2, -3, -4, -6, -7, -8, and -9) by hemagglutination inhibition test, despite showing weak cross-reactivity with APMV-1 and APMV-9. The UPO216 virus RNA genome is 15,180 nucleotides (nts) in length, encodes 3'-N-P(V/W)-M-F-HN-L-5' in that order, and shows unique genetic characteristics in terms of genomic composition and evolutionary divergence (0.43 or greater from known serotypes of APMV). Phylogenetic analysis revealed that the UPO216 occupies a branch separate from APMV-1, -9, -12, and -13. Serologic surveillance of wild birds ( n = 880; 15 species, five Orders) detected UPO216-reactive antibodies in 4% (20/494) of serum samples taken from five species of wild duck belonging to the Order Anseriformes . In particular, UPO216-specific antibodies showing no cross-reaction with other serotypes of APMV were detected in four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). These results indicate that the UPO216 virus has antigenically and genetically unique characteristics distinct from known serotypes of APMV and likely has been circulating widely in wild duck species of the Order Anseriformes . Thus, we propose the UPO216 isolate as a prototype strain of a novel APMV serotype (putative APMV-15).

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

PubMed Central

Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

2010-01-01

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic “virion factory,” we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5′ and 3′ extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus “virophage.” These results—validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)—will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

Curious discoveries in antiviral drug development: the role of serendipity.

PubMed

De Clercq, Erik

2015-07-01

Antiviral drug development has often followed a curious meandrous route, guided by serendipity rather than rationality. This will be illustrated by ten examples. The polyanionic compounds (i) polyethylene alanine (PEA) and (ii) suramin were designed as an antiviral agent (PEA) or known as an antitrypanosomal agent (suramin), before they emerged as, respectively, a depilatory agent, or reverse transcriptase inhibitor. The 2',3'-dideoxynucleosides (ddNs analogues) (iii) have been (and are still) used in the "Sanger" DNA sequencing technique, although they are now commercialized as nucleoside reverse transcriptase inhibitors (NRTIs) in the treatment of HIV infections. (E)-5-(2-Bromovinyl)-2'-deoxyuridine (iv) was discovered as a selective anti-herpes simplex virus compound and is now primarily used for the treatment of varicella-zoster virus infections. The prototype of the acyclic nucleoside phosphonates (ANPs), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], (v) was never commercialized, although it gave rise to several marketed products (cidofovir, adefovir, and tenofovir). 1-[2-(Hydroxyethoxy)methyl]-6-(phenylthio)thymine (vi) and TIBO (tetrahydroimidazo[4,5,1-jk][1,4-benzodiazepin-2(1H)]-one and -thione) (vii) paved the way to a number of compounds (i.e., nevirapine, delavirdine, etravirine, and rilpivirine), which are now collectively called non-NRTIs. The bicyclam AMD3100 (viii) was originally described as an anti-HIV agent before it became later marketed as a stem cell mobilizer. The S-adenosylhomocysteine hydrolase inhibitors (ix), while active against a broad range of (-)RNA viruses and poxviruses may be particularly effective against Ebola virus, and for (x) the O-ANP derivatives, the potential application range encompasses virtually all DNA viruses. © 2015 Wiley Periodicals, Inc.

Human endogenous retroviruses: nature, occurrence, and clinical implications in human disease.

PubMed Central

Urnovitz, H B; Murphy, W H

1996-01-01

Retroviral diagnostics have become standard in human laboratory medicine. While current emphasis is placed on the human exogenous viruses (human immunodeficiency virus and human T-cell leukemia virus), evidence implicating human endogenous retroviruses (HERVs) in various human disease entities continues to mount. Literature on the occurrence of HERVs in human tissues and cells was analyzed. Substantial evidence documents that retrovirus particles were clearly demonstrable in various tissues and cells in both health and disease and were abundant in the placenta and that their occurrence could be implicated in some of the reproductive diseases. The characteristics of HERVs are summarized, mechanisms of replication and regulation are outlined, and the consistent hormonal responsiveness of HERVs is noted. Clear evidence implicating HERV gene products as participants in glomerulonephritis in some cases of systemic lupus erythematosus is adduced. Data implicating HERVs as etiologic factors in reproductive diseases, in some of the autoimmune diseases, in some forms of rheumatoid arthritis and connective tissue disease, in psoriasis, and in some of the inflammatory neurologic diseases are reviewed. The current major needs are to improve methods for HERV detection, to identify the most appropriate HERV prototypes, and to develop diagnostic reagents so that the putative biologic and pathologic roles of HERVs can be better evaluated. PMID:8665478

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

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

Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less

Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing.

PubMed

Legendre, Matthieu; Santini, Sébastien; Rico, Alain; Abergel, Chantal; Claverie, Jean-Michel

2011-03-04

Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes. We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

DOE PAGES

Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; ...

2016-03-14

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less

Expanding the histologic spectrum of mucinous tubular and spindle cell carcinoma of the kidney.

PubMed

Fine, Samson W; Argani, Pedram; DeMarzo, Angelo M; Delahunt, Brett; Sebo, Thomas J; Reuter, Victor E; Epstein, Jonathan I

2006-12-01

Mucinous tubular and spindle cell carcinomas (MTSCs) are polymorphic neoplasms characterized by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by pale mucinous stroma. Nonclassic morphologic variants and features of MTSC have not been well studied. We identified 17 previously unreported MTSCs from Surgical Pathology and consultative files of the authors and their respective institutions and studied their morphologic features. A total of 10/17 cases were considered "classic," as described above, with 5/10 showing at least focal (20% to 50%) tubular predominance without apparent mucinous matrix. Alcian blue staining revealed abundant (>50%) mucin in all classic cases. Seven of 17 MTSCs were classified as "mucin-poor," with little to no extracellular mucin appreciable by hematoxylin and eosin. Four of these cases showed equal tubular and spindled morphology, 2 cases showed spindle cell predominance (70%; 95%), and 1 case showed tubular predominance (90%). In 5/7 mucin-poor cases, staining for Alcian blue revealed scant (<10%) mucin in cellular areas with the other 2 cases having 30% mucin. Unusual histologic features identified in the 17 cases were: foamy macrophages (n=8), papillations/well formed papillae (n=6/n=1), focal clear cells in tubules (n=3), necrosis (n=3), oncocytic tubules (n=2; 40%, 5%), numerous small vacuoles (n=2), heterotopic bone (n=1), psammomatous calcification (n=1), and nodular growth with lymphocytic cuffing (n=1). An exceptional case contained a well-circumscribed, HMB45-positive angiomyolipoma within the MTSC. MTSCs may be "mucin-poor" and show a marked predominance of either of its principal morphologic components, which coupled with the presence of other unusual features such as clear cells, papillations, foamy macrophages, and necrosis, may mimic other forms of renal cell carcinoma. Pathologists must be aware of the spectrum of histologic findings within MTSCs to ensure their accurate diagnosis.

Sialidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype.

PubMed

Walz, Lisa; Kays, Sarah-Katharina; Zimmer, Gert; von Messling, Veronika

2018-06-20

Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the level of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of a subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strain and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with similar efficacy as the matched HA protein, illustrating the potential of the NA protein as vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive sialidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype. IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen not only conferred protection of mice and ferrets to the matched influenza strains, but also against viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype. Copyright © 2018 American Society for Microbiology.

Cyclic Avian Mass Mortality in the Northeastern United States Is Associated with a Novel Orthomyxovirus

PubMed Central

Ballard, Jennifer R.; Tesh, Robert B.; Brown, Justin D.; Ruder, Mark G.; Keel, M. Kevin; Munk, Brandon A.; Mickley, Randall M.; Gibbs, Samantha E. J.; Travassos da Rosa, Amelia P. A.; Ellis, Julie C.; Ip, Hon S.; Shearn-Bochsler, Valerie I.; Rogers, Matthew B.; Ghedin, Elodie; Holmes, Edward C.; Parrish, Colin R.; Dwyer, Chris

2014-01-01

ABSTRACT Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health. PMID:25392223

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

PubMed

Allison, Andrew B; Ballard, Jennifer R; Tesh, Robert B; Brown, Justin D; Ruder, Mark G; Keel, M Kevin; Munk, Brandon A; Mickley, Randall M; Gibbs, Samantha E J; Travassos da Rosa, Amelia P A; Ellis, Julie C; Ip, Hon S; Shearn-Bochsler, Valerie I; Rogers, Matthew B; Ghedin, Elodie; Holmes, Edward C; Parrish, Colin R; Dwyer, Chris

2015-01-15

Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Isolation of deer tick virus (Powassan virus, lineage II) from Ixodes scapularis and detection of antibody in vertebrate hosts sampled in the Hudson Valley, New York State

PubMed Central

2013-01-01

Background Deer tick virus, DTV, is a genetically and ecologically distinct lineage of Powassan virus (POWV) also known as lineage II POWV. Human incidence of POW encephalitis has increased in the last 15 years potentially due to the emergence of DTV, particularly in the Hudson Valley of New York State. We initiated an extensive sampling campaign to determine whether POWV was extant throughout the Hudson Valley in tick vectors and/or vertebrate hosts. Methods More than 13,000 ticks were collected from hosts or vegetation and tested for the presence of DTV using molecular and virus isolation techniques. Vertebrate hosts of Ixodes scapularis (black-legged tick) were trapped (mammals) or netted (birds) and blood samples analyzed for the presence of neutralizing antibodies to POWV. Maximum likelihood estimates (MLE) were calculated to determine infection rates in ticks at each study site. Results Evidence of DTV was identified each year from 2007 to 2012, in nymphal and adult I. scapularis collected from the Hudson Valley. 58 tick pools were positive for virus and/or RNA. Infection rates were higher in adult ticks collected from areas east of the Hudson River. MLE limits ranged from 0.2-6.0 infected adults per 100 at sites where DTV was detected. Virginia opossums, striped skunks and raccoons were the source of infected nymphal ticks collected as replete larvae. Serologic evidence of POWV infection was detected in woodchucks (4/6), an opossum (1/6), and birds (4/727). Lineage I, prototype POWV, was not detected. Conclusions These data demonstrate widespread enzootic transmission of DTV throughout the Hudson Valley, in particular areas east of the river. High infection rates were detected in counties where recent POW encephalitis cases have been identified, supporting the hypothesis that lineage II POWV, DTV, is responsible for these human infections. PMID:24016533

Isolation of deer tick virus (Powassan virus, lineage II) from Ixodes scapularis and detection of antibody in vertebrate hosts sampled in the Hudson Valley, New York State.

PubMed

Dupuis, Alan P; Peters, Ryan J; Prusinski, Melissa A; Falco, Richard C; Ostfeld, Richard S; Kramer, Laura D

2013-07-15

Deer tick virus, DTV, is a genetically and ecologically distinct lineage of Powassan virus (POWV) also known as lineage II POWV. Human incidence of POW encephalitis has increased in the last 15 years potentially due to the emergence of DTV, particularly in the Hudson Valley of New York State. We initiated an extensive sampling campaign to determine whether POWV was extant throughout the Hudson Valley in tick vectors and/or vertebrate hosts. More than 13,000 ticks were collected from hosts or vegetation and tested for the presence of DTV using molecular and virus isolation techniques. Vertebrate hosts of Ixodes scapularis (black-legged tick) were trapped (mammals) or netted (birds) and blood samples analyzed for the presence of neutralizing antibodies to POWV. Maximum likelihood estimates (MLE) were calculated to determine infection rates in ticks at each study site. Evidence of DTV was identified each year from 2007 to 2012, in nymphal and adult I. scapularis collected from the Hudson Valley. 58 tick pools were positive for virus and/or RNA. Infection rates were higher in adult ticks collected from areas east of the Hudson River. MLE limits ranged from 0.2-6.0 infected adults per 100 at sites where DTV was detected. Virginia opossums, striped skunks and raccoons were the source of infected nymphal ticks collected as replete larvae. Serologic evidence of POWV infection was detected in woodchucks (4/6), an opossum (1/6), and birds (4/727). Lineage I, prototype POWV, was not detected. These data demonstrate widespread enzootic transmission of DTV throughout the Hudson Valley, in particular areas east of the river. High infection rates were detected in counties where recent POW encephalitis cases have been identified, supporting the hypothesis that lineage II POWV, DTV, is responsible for these human infections.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes▿

PubMed Central

Damen, Marjolein; Minnaar, René; Glasius, Patricia; van der Ham, Alwin; Koen, Gerrit; Wertheim, Pauline; Beld, Marcel

2008-01-01

The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed. PMID:18923006

Characterization of Epstein-Barr virus (EBV) BZLF1 gene promoter variants and comparison of cellular gene expression profiles in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis.

PubMed

Imajoh, Masayuki; Hashida, Yumiko; Murakami, Masanao; Maeda, Akihiko; Sato, Tetsuya; Fujieda, Mikiya; Wakiguchi, Hiroshi; Daibata, Masanori

2012-06-01

Epstein-Barr virus (EBV) genotypes can be distinguished based on gene sequence differences in EBV nuclear antigens 2, 3A, 3B, and 3C, and the BZLF1 promoter zone (Zp). EBV subtypes and BZLF1 Zp variants were examined in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis. The results of EBV typing showed that samples of infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis all belonged to EBV type 1. However, sequencing analysis of BZLF1 Zp found three polymorphic Zp variants in the same samples. The Zp-P prototype and the Zp-V3 variant were both detected in infectious mononucleosis and chronic active EBV infection. Furthermore, a novel variant previously identified in Chinese children with infectious mononucleosis, Zp-V1, was also found in 3 of 18 samples of infectious mononucleosis, where it coexisted with the Zp-P prototype. This is the first evidence that the EBV variant distribution in Japanese patients resembles that found in other Asian patients. The expression levels of 29 chronic active EBV infection-associated cellular genes were also compared in the three EBV-related disorders, using quantitative real-time reverse transcription polymerase chain reaction analysis. Two upregulated genes, RIPK2 and CDH9, were identified as common specific markers for chronic active EBV infection in both in vitro and in vivo studies. RIPK2 activates apoptosis and autophagy, and could be responsible for the pathogenesis of chronic active EBV infection. Copyright © 2012 Wiley Periodicals, Inc.

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients.

PubMed

Caliendo, A M; St George, K; Kao, S Y; Allega, J; Tan, B H; LaFontaine, R; Bui, L; Rinaldo, C R

2000-06-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.

Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients

PubMed Central

Caliendo, Angela M.; St. George, Kirsten; Kao, Shaw-Yi; Allega, Jessica; Tan, Ban-Hock; LaFontaine, Robert; Bui, Larry; Rinaldo, Charles R.

2000-01-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients. PMID:10834964

Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the United States.

PubMed

Hahn, Cassidy M; Iwanowicz, Luke R; Cornman, Robert S; Conway, Carla M; Winton, James R; Blazer, Vicki S

2015-12-01

The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker. We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research. Copyright © 2015 Hahn et al.

Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the United States

PubMed Central

Hahn, Cassidy M.; Cornman, Robert S.; Conway, Carla M.; Winton, James R.; Blazer, Vicki S.

2015-01-01

ABSTRACT The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker. IMPORTANCE We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research. PMID:26378165

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

PubMed Central

Soboleski, Mark R.; Gabbard, Jon D.; Price, Graeme E.; Misplon, Julia A.; Lo, Chia-Yun; Perez, Daniel R.; Ye, Jianqiang; Tompkins, S. Mark; Epstein, Suzanne L.

2011-01-01

Background The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus. Methodology/Principal Findings BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus. Conclusion/Significance Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic. PMID:21789196

Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

PubMed Central

Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

2015-01-01

ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs. PMID:26378167

Sphingomyelin lipidosis (Niemann-Pick disease) in a juvenile raccoon (Procyon lotor).

PubMed

Vapniarsky, N; Wenger, D A; Scheenstra, D; Mete, A

2013-01-01

A wild caught juvenile male raccoon with neurological disease was humanely destroyed due to poor prognosis. Necropsy examination revealed hepatomegaly, splenomegaly and multicentric lymphadenomegaly with diffuse hepatic pallor and pulmonary consolidation with pinpoint pale subpleural foci. Microscopically, there was marked pale cytoplasmic swelling of the central and peripheral neurons as well as the glial cells in the brain, accompanied by multiorgan infiltration by abundant foamy macrophages. Ultrastructural investigation revealed accumulation of concentrically arranged lamellar material within lysosomes of the affected neurons, macrophages and endothelial cells. Biochemical enzymatic analysis detected sphingomyelinase deficiency and lysosomal storage disease consistent with sphingomyelin lipidosis (Niemann-Pick disease [NPD]) was diagnosed. This is the first report of NPD in a raccoon. Copyright © 2013 Elsevier Ltd. All rights reserved.

Malakoplakia mimics urinary bladder cancer: a case report.

PubMed

Ristić-Petrović, Ana; Stojnev, Slavica; Janković-Velicković, Ljubinka; Marjanović, Goran

2013-06-01

Malakoplakia is an unusual and very rare chronic inflammatory disease. In bladder especially it can mimic malignancy and lead to serious misdiagnosis. We presented a case of a middle-aged woman with persistent macrohematuria and cystoscopically polypoid bladder mass that resembled a neoplastic process. The final diagnosis was based on cystoscopic biopsy and microscopic findings of acidophilic, foamy histiocytes with the presence of Michaelis-Gutmann inclusions which are characteristic for diagnosis of malakoplakia. Immunohistochemistry confirmed diagnosis by demonstrating CD68-positive macrophages. Urinary bladder malakoplakia should be considered in patients with persistent urinary tract infections and tumor mass at cystoscopy. Early identification with prompt antibiotic treatment can be helpful in avoiding unnecessary surgical interventions and in preventing development of possible complications.

Performance Testing of Thermal Cutting Systems for Sweet Pepper Harvesting Robot in Greenhouse Horticulture

NASA Astrophysics Data System (ADS)

Bachche, Shivaji; Oka, Koichi

2013-03-01

This paper proposes design of end-effector and prototype of thermal cutting system for harvesting sweet peppers. The design consists of two parallel gripper bars mounted on a frame connected by specially designed notch plate and operated by servo motor. Based on voltage and current, two different types of thermal cutting system prototypes; electric arc and temperature arc respectively were developed and tested for performance. In electric arc, a special electric device was developed to obtain high voltage to perform cutting operation. At higher voltage, electrodes generate thermal arc which helps to cut stem of sweet pepper. In temperature arc, nichrome wire was mounted between two electrodes and current was provided directly to electrodes which results in generation of high temperature arc between two electrodes that help to perform cutting operation. In both prototypes, diameters of basic elements were varied and the effect of this variation on cutting operation was investigated. The temperature arc thermal system was found significantly suitable for cutting operation than electric arc thermal system. In temperature arc thermal cutting system, 0.5 mm nichrome wire shows significant results by accomplishing harvesting operation in 1.5 seconds. Also, thermal cutting system found suitable to increase shelf life of fruits by avoiding virus and fungal transformation during cutting process and sealing the fruit stem. The harvested sweet peppers by thermal cutting system can be preserved at normal room temperature for more than 15 days without any contamination.

An ac electroosmosis device for the detection of bioparticles with piezoresistive microcantilever sensors

NASA Astrophysics Data System (ADS)

Arefin, Md Shamsul; Porter, Timothy L.

2012-03-01

This work reports on the behavior of piezoresistive microcantilever sensors under optimizing conditions of ac electroosmotic enhancement. Piezoresistive microcantilevers are used as sensor elements for detection of concentrated bio-particles. Without preconcentrating the samples, using ac electroosmosis, these bio-particles have been manipulated onto the piezoresistive microcantilever. A piezoresistive microcantilever senses the dimensional changes upon particle exposure as a resistance change. This paper represents the integration of ac electroosmosis with a piezoresistive micro-cantilever sensor for the detection of bio-particles. A working prototype is presented here, and the experiments are conducted on Herpes Simplex type-1 virus (HSV-1) and Escherichia Coli (E. coli) bacteria.

Sequence variation of functional HTLV-II tax alleles among isolates from an endemic population: lack of evidence for oncogenic determinant in tax.

PubMed

Hjelle, B; Chaney, R

1992-02-01

Human T-cell leukemia-lymphoma virus type II (HTLV-II) has been isolated from patients with hairy cell leukemia (HCL). We previously described a population with longstanding endemic HTLV-II infection, and showed that there is no increased risk for HCL in the affected groups. We thus have direct evidence that the endemic form(s) of HTLV-II cause HCL infrequently, if at all. By comparison, there is reason to suspect that the viruses isolated from patients with HCL had an etiologic role in the disease in those patients. One way to reconcile these conflicting observations is to consider that isolates of HTLV-II might differ in oncogenic potential. To determine whether the structure of the putative oncogenic determinant of HTLV-II, tax2, might differ in the new isolates compared to the tax of the prototype HCL isolate, MO, four new functional tax cDNAs were cloned from new isolates. Sequence analysis showed only minor (0.9-2.0%) amino acid variation compared to the published sequence of MO tax2. Some codons were consistently different from published sequences of the MO virus, but in most cases, such variations were also found in each of two tax2 clones we isolated from the MO T-cell line. These variations rendered the new clones more similar to the tax1 of the pathogenic virus HTLV-I. Thus we find no evidence that pathologic determinants of HTLV-II can be assigned to the tax gene.

Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

PubMed

Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-06-25

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement

PubMed Central

Rosengard, Ariella M.; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-01-01

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges. PMID:12034872

Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism

PubMed Central

Yi, Yanjie; Isaacs, Stuart N.; Williams, Darlisha A.; Frank, Ian; Schols, Dominique; De Clercq, Erik; Kolson, Dennis L.; Collman, Ronald G.

1999-01-01

Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4). PMID:10438797

Comparative Genomics of Chrysochromulina Ericina Virus and Other Microalga-Infecting Large DNA Viruses Highlights Their Intricate Evolutionary Relationship with the Established Mimiviridae Family.

PubMed

Gallot-Lavallée, Lucie; Blanc, Guillaume; Claverie, Jean-Michel

2017-07-15

Chrysochromulina ericina virus CeV-01B (CeV) was isolated from Norwegian coastal waters in 1998. Its icosahedral particle is 160 nm in diameter and encloses a 474-kb double-stranded DNA (dsDNA) genome. This virus, although infecting a microalga (the haptophyceae Haptolina ericina , formerly Chrysochromulina ericina ), is phylogenetically related to members of the Mimiviridae family, initially established with the acanthamoeba-infecting mimivirus and megavirus as prototypes. This family was later split into two genera ( Mimivirus and Cafeteriavirus ) following the characterization of a virus infecting the heterotrophic stramenopile Cafeteria roenbergensis (CroV). CeV, as well as two of its close relatives, which infect the unicellular photosynthetic eukaryotes Phaeocystis globosa (Phaeocystis globosa virus [PgV]) and Aureococcus anophagefferens (Aureococcus anophagefferens virus [AaV]), are currently unclassified by the International Committee on Viral Taxonomy (ICTV). The detailed comparative analysis of the CeV genome presented here confirms the phylogenetic affinity of this emerging group of microalga-infecting viruses with the Mimiviridae but argues in favor of their classification inside a distinct clade within the family. Although CeV, PgV, and AaV share more common features among them than with the larger Mimiviridae , they also exhibit a large complement of unique genes, attesting to their complex evolutionary history. We identified several gene fusion events and cases of convergent evolution involving independent lateral gene acquisitions. Finally, CeV possesses an unusual number of inteins, some of which are closely related despite being inserted in nonhomologous genes. This appears to contradict the paradigm of allele-specific inteins and suggests that the Mimiviridae are especially efficient in spreading inteins while enlarging their repertoire of homing genes. IMPORTANCE Although it infects the microalga Chrysochromulina ericina , CeV is more closely related to acanthamoeba-infecting viruses of the Mimiviridae family than to any member of the Phycodnaviridae , the ICTV-approved family historically including all alga-infecting large dsDNA viruses. CeV, as well as its relatives that infect the microalgae Phaeocystic globosa (PgV) and Aureococcus anophagefferens (AaV), remains officially unclassified and a source of confusion in the literature. Our comparative analysis of the CeV genome in the context of this emerging group of alga-infecting viruses suggests that they belong to a distinct clade within the established Mimiviridae family. The presence of a large number of unique genes as well as specific gene fusion events, evolutionary convergences, and inteins integrated at unusual locations document the complex evolutionary history of the CeV lineage. Copyright © 2017 American Society for Microbiology.

Preventative Vaccines for Zika Virus Outbreak: Preliminary Evaluation.

PubMed

Kim, Eun; Erdos, Geza; Huang, Shaohua; Kenniston, Thomas; Falo, Louis D; Gambotto, Andrea

2016-11-01

Since it emerged in Brazil in May 2015, the mosquito-borne Zika virus (ZIKV) has raised global concern due to its association with a significant rise in the number of infants born with microcephaly and neurological disorders such as Guillain-Barré syndrome. We developed prototype subunit and adenoviral-based Zika vaccines encoding the extracellular portion of the ZIKV envelope gene (E) fused to the T4 fibritin foldon trimerization domain (Efl). The subunit vaccine was delivered intradermally through carboxymethyl cellulose microneedle array (MNA). The immunogenicity of these two vaccines, named Ad5.ZIKV-Efl and ZIKV-rEfl, was tested in C57BL/6 mice. Prime/boost immunization regimen was associated with induction of a ZIKV-specific antibody response, which provided neutralizing immunity. Moreover, protection was evaluated in seven-day-old pups after virulent ZIKV intraperitoneal challenge. Pups born to mice immunized with Ad5.ZIKV-Efl were all protected against lethal challenge infection without weight loss or neurological signs, while pups born to dams immunized with MNA-ZIKV-rEfl were partially protected (50%). No protection was seen in pups born to phosphate buffered saline-immunized mice. This study illustrates the preliminary efficacy of the E ZIKV antigen vaccination in controlling ZIKV infectivity, providing a promising candidate vaccine and antigen format for the prevention of Zika virus disease. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Aryl hydrocarbon receptor signaling modulates antiviral immune responses: ligand metabolism rather than chemical source is the stronger predictor of outcome.

PubMed

Boule, Lisbeth A; Burke, Catherine G; Jin, Guang-Bi; Lawrence, B Paige

2018-01-29

The aryl hydrocarbon receptor (AHR) offers a compelling target to modulate the immune system. AHR agonists alter adaptive immune responses, but the consequences differ across studies. We report here the comparison of four agents representing different sources of AHR ligands in mice infected with influenza A virus (IAV): TCDD, prototype exogenous AHR agonist; PCB126, pollutant with documented human exposure; ITE, novel pharmaceutical; and FICZ, degradation product of tryptophan. All four compounds diminished virus-specific IgM levels and increased the proportion of regulatory T cells. TCDD, PCB126 and ITE, but not FICZ, reduced virus-specific IgG levels and CD8 + T cell responses. Similarly, ITE, PCB126, and TCDD reduced Th1 and Tfh cells, whereas FICZ increased their frequency. In Cyp1a1-deficient mice, all compounds, including FICZ, reduced the response to IAV. Conditional Ahr knockout mice revealed that all four compounds require AHR within hematopoietic cells. Thus, differences in the immune response to IAV likely reflect variances in quality, magnitude, and duration of AHR signaling. This indicates that binding affinity and metabolism may be stronger predictors of immune effects than a compound's source of origin, and that harnessing AHR will require finding a balance between dampening immune-mediated pathologies and maintaining sufficient host defenses against infection.

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.

PubMed

Dash, Paban Kumar; Boutonnier, Alain; Prina, Eric; Sharma, Shashi; Reiter, Paul

2012-01-22

Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.

West Nile Virus Temperature Sensitivity and Avian Virulence Are Modulated by NS1-2B Polymorphisms.

PubMed

Dietrich, Elizabeth A; Langevin, Stanley A; Huang, Claire Y-H; Maharaj, Payal D; Delorey, Mark J; Bowen, Richard A; Kinney, Richard M; Brault, Aaron C

2016-08-01

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.

Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.

PubMed

Dang, Jason; Tiwari, Shashi Kant; Lichinchi, Gianluigi; Qin, Yue; Patil, Veena S; Eroshkin, Alexey M; Rana, Tariq M

2016-08-04

Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

Reconsidering the classification of tick-borne encephalitis virus within the Siberian subtype gives new insights into its evolutionary history.

PubMed

Kovalev, S Y; Mukhacheva, T A

2017-11-01

Tick-borne encephalitis is widespread in Eurasia and transmitted by Ixodes ticks. Classification of its causative agent, tick-borne encephalitis virus (TBEV), includes three subtypes, namely Far-Eastern, European, and Siberian (TBEV-Sib), as well as a group of 886-84-like strains with uncertain taxonomic status. TBEV-Sib is subdivided into three phylogenetic lineages: Baltic, Asian, and South-Siberian. A reason to reconsider TBEV-Sib classification was the analysis of 186 nucleotide sequences of an E gene fragment submitted to GenBank during the last two years. Within the South-Siberian lineage, we have identified a distinct group with prototype strains Aina and Vasilchenko as an individual lineage named East-Siberian. The analysis of reclassified lineages has promoted a new model of the evolutionary history of TBEV-Sib lineages and TBEV-Sib as a whole. Moreover, we present arguments supporting separation of 886-84-like strains into an individual TBEV subtype, which we propose to name Baikalian (TBEV-Bkl). Copyright © 2017 Elsevier B.V. All rights reserved.

Immunity and immune response, pathology and pathologic changes: progress and challenges in the immunopathology of yellow fever.

PubMed

Quaresma, Juarez A S; Pagliari, Carla; Medeiros, Daniele B A; Duarte, Maria I S; Vasconcelos, Pedro F C

2013-09-01

Yellow fever is a viral hemorrhagic fever, which affects people living in Africa and South America and is caused by the yellow fever virus, the prototype species in the Flavivirus genus (Flaviviridae family). Yellow fever virus infection can produce a wide spectrum of symptoms, ranging from asymptomatic infection or oligosymptomatic illness to severe disease with a high fatality rate. In this review, we focus in the mechanisms associated with the physiopathology of yellow fever in humans and animal models. It has been demonstrated that several factors play a role in the pathological outcome of the severe form of the disease including direct viral cytopathic effect, necrosis and apoptosis of hepatocyte cells in the midzone, and a minimal inflammatory response as well as low-flow hypoxia and cytokine overproduction. New information has filled several gaps in the understanding of yellow fever pathogenesis and helped comprehend the course of illness. Finally, we discuss prospects for an immune therapy in the light of new immunologic, viral, and pathologic tools. Copyright © 2013 John Wiley & Sons, Ltd.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

PubMed

Gardner, Thomas J; Stein, Kathryn R; Duty, J Andrew; Schwarz, Toni M; Noriega, Vanessa M; Kraus, Thomas; Moran, Thomas M; Tortorella, Domenico

2016-12-14

The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

PubMed

Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

2017-12-01

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Resequencing Pathogen Microarray (RPM) for prospective detection and identification of emergent pathogen strains and variants

NASA Astrophysics Data System (ADS)

Tibbetts, Clark; Lichanska, Agnieszka M.; Borsuk, Lisa A.; Weslowski, Brian; Morris, Leah M.; Lorence, Matthew C.; Schafer, Klaus O.; Campos, Joseph; Sene, Mohamadou; Myers, Christopher A.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Metzgar, David

2010-04-01

High-density resequencing microarrays support simultaneous detection and identification of multiple viral and bacterial pathogens. Because detection and identification using RPM is based upon multiple specimen-specific target pathogen gene sequences generated in the individual test, the test results enable both a differential diagnostic analysis and epidemiological tracking of detected pathogen strains and variants from one specimen to the next. The RPM assay enables detection and identification of pathogen sequences that share as little as 80% sequence similarity to prototype target gene sequences represented as detector tiles on the array. This capability enables the RPM to detect and identify previously unknown strains and variants of a detected pathogen, as in sentinel cases associated with an infectious disease outbreak. We illustrate this capability using assay results from testing influenza A virus vaccines configured with strains that were first defined years after the design of the RPM microarray. Results are also presented from RPM-Flu testing of three specimens independently confirmed to the positive for the 2009 Novel H1N1 outbreak strain of influenza virus.

A “building block” approach to the new influenza A virus entry inhibitors with reduced cellular toxicities

NASA Astrophysics Data System (ADS)

Lin, Dongguo; Li, Fangfang; Wu, Qiuyi; Xie, Xiangkun; Wu, Wenjiao; Wu, Jie; Chen, Qing; Liu, Shuwen; He, Jian

2016-03-01

Influenza A virus (IAV) is a severe worldwide threat to public health and economic development that results in the emergence of drug-resistant or highly virulent strains. Therefore, it is imperative to develop potent anti-IAV drugs with different modes of action to currently available drugs. Herein, we show a new class of antiviral peptides generated by conjugating two known short antiviral peptides: part-1 (named Jp with the sequence of ARLPR) and part-2 (named Hp with the sequence of KKWK). The new peptides were thus created by hybridization of these two domains at C- and N- termini, respectively. The anti-IAV screening results identified that C20-Jp-Hp was the most potent peptide with IC50 value of 0.53 μM against A/Puerto Rico/8/34 (H1N1) strain. Interestingly, these new peptides display lower toxicities toward mammalian cells and higher therapeutic indices than their prototypes. In addition, the mechanism of action of C20-Jp-Hp was extensively investigated.

Inactivated coxsackievirus A10 experimental vaccines protect mice against lethal viral challenge.

PubMed

Shen, Chaoyun; Liu, Qingwei; Zhou, Yu; Ku, Zhiqiang; Wang, Lili; Lan, Ke; Ye, Xiaohua; Huang, Zhong

2016-09-22

Coxsackievirus A10 (CVA10) has become one of the major causative agents of hand, foot and mouth disease (HFMD). It is now recognized that CVA10 should be targeted for vaccine development. We report here that β-propiolactone inactivated whole-virus based CVA10 vaccines can elicit protective immunity in mice. We prepared two inactivated CVA10 experimental vaccines derived from the prototype strain CVA10/Kowalik and from a clinical isolate CVA10/S0148b, respectively. Immunization with the experimental vaccines elicited CVA10-specific serum antibodies in mice. The antisera from vaccinated mice could potently neutralize in vitro infection with either homologous or heterologous CVA10 strains. Importantly, passive transfer of the anti-CVA10 sera protected recipient mice against CVA10/Kowalik or CVA10/S0148b infections. Moreover, active immunization with the inactivated vaccines also conferred protection against homologous and heterologous infections in mice. Collectively, our results demonstrate the proof-of-concept for inactivated whole-virus based CVA10 vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates

PubMed Central

Wang, Yimeng; O'Dell, Sijy; Turner, Hannah L.; Chiang, Chi-I; Lei, Lin; Guenaga, Javier; Wilson, Richard; Martinez-Murillo, Paola; Doria-Rose, Nicole; Ward, Andrew B.; Mascola, John R.; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

2017-01-01

ABSTRACT Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates elicit predominantly tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic, as a sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with predominant VH4 or VH5 gene family usage and Env V3 specificity. Of these vaccine-elicited V3-specific MAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation. IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated broadly neutralizing antibodies [bNAbs]) remains a high priority for the vaccine field. bNAb responses were so far observed only in response to natural infection within a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses, including at the monoclonal Ab level, is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing MAbs targeting the Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between the vaccine- and infection-induced responses to V3 and support the feasibility of exploring the V3 epitope as a HIV-1 vaccine target in nonhuman primates. PMID:28835491

[Current wound care in patients with elephantiasis--third-stage lymphedema].

PubMed

Rucigaj, Tanja Planinsek; Slana, Ana; Leskovec, Nada Kecelj

2012-10-01

Lymphedema resulting from fluid accumulation due to impairment in the lymphatic system drainage leads to enlargement of the body part involved. If left untreated, in its third stage it results in elephantiasis. Elephantiasis is frequently accompanied by papillomatosis and lymphocutaneous fistulas with lymphorrhoea, erosions and ulcers, frequently with the loss of function in the respective part of the body. Unlike other chronic wounds, wound healing in lymphedema is highly dependent on the use of combined therapies because local treatment with modern supportive dressings and compression therapy with adhesive and non-adhesive short-stretch systems is only part of the complete treatment. This treatment also includes sub-bandage foamy materials, kinesitherapy with tapes (kinesiotaping), intermittent local application of high-pressure oxygen, breathing exercise, and manual lymph drainage and exercises.

Indeterminate cell histiocytosis in a pediatric patient: successful treatment with thalidomide.

PubMed

Tóth, Béla; Katona, Mária; Hársing, Judit; Szepesi, Agota; Kárpáti, Sarolta

2012-04-01

The 15-year-old male patient presented several 2-6 mm large livid reddish-yellowish, shiny, compact papules on the head, trunk and extremities, which had developed within the last 4 months. Histology showed normal epidermis with dense dermal infiltrate of histiocytes accompanied by few eosinophils, Touton or foamy giant cells. The histiocytes were S100 positive, CD1a negative and did not contain Birbeck granules ultrastructurally. Chest X ray, EEG, skull MRI did not show pathology. Opthalmology, neurology, oto-rhino-laryngology did not reveal alterations. Based upon the clinical symptoms and the histopathology, the diagnosis of indeterminate cell histiocytosis was confirmed. Cryotherapy and cauterization did not stop the progression of the disease, however, under thalidomide treatment no new symptoms developed and the lesions healed with pigmentation.

Chewing the fat: lipid metabolism and homeostasis during M. tuberculosis infection.

PubMed

Lovewell, Rustin R; Sassetti, Christopher M; VanderVen, Brian C

2016-02-01

The interplay between Mycobacterium tuberculosis lipid metabolism, the immune response and lipid homeostasis in the host creates a complex and dynamic pathogen-host interaction. Advances in imaging and metabolic analysis techniques indicate that M. tuberculosis preferentially associates with foamy cells and employs multiple physiological systems to utilize exogenously derived fatty-acids and cholesterol. Moreover, novel insights into specific host pathways that control lipid accumulation during infection, such as the PPARγ and LXR transcriptional regulators, have begun to reveal mechanisms by which host immunity alters the bacterial micro-environment. As bacterial lipid metabolism and host lipid regulatory pathways are both important, yet inherently complex, components of active tuberculosis, delineating the heterogeneity in lipid trafficking within disease states remains a major challenge for therapeutic design. Copyright © 2015. Published by Elsevier Ltd.

Piezoelectric-assisted removal of a benign fibrous histiocytoma of the mandible: An innovative technique for prevention of dentoalveolar nerve injury

PubMed Central

2011-01-01

In this article, we present our experience with a piezoelectric-assisted surgical device by resection of a benign fibrous histiocytoma of the mandible. A 41 year-old male was admitted to our hospital because of slowly progressive right buccal swelling. After further radiographic diagnosis surgical removal of the yellowish-white mass was performed. Histologic analysis showed proliferating histiocytic cells with foamy, granular cytoplasm and no signs of malignancy. The tumor was positive for CD68 and vimentin in immunohistochemical staining. Therefore the tumor was diagnosed as primary benign fibrous histiocytoma. This work provides a new treatment device for benign mandibular tumour disease. By using a novel piezoelectric-assisted cutting device, protection of the dentoalveolar nerve could be achieved. PMID:22040611

Foams and antifoams.

PubMed

Karakashev, Stoyan I; Grozdanova, Michaela V

2012-01-01

Foams and antifoams are two entities with completely different natures. For example, the foams are structures of bubbles in contact, while the antifoams are emulsions containing hydrophobic particles. The interaction between them makes the foam decay faster and in the same time exhausts the antifoam. The mechanism of such an effect is complex of many phenomena taking place in the foam. Thus the antifoams are known as powerful foam suppressors. For these reasons, they are very important from fundamental and practical viewpoints. This paper summarizes the knowledge on antifoams since their very creation till nowadays. In this regard, the review discloses the scientific interpretations on antifoams in chronological order in accord with the literature. Thus, for example it begins with description of the first antifoams (oils) from the 1940s and the pioneering studies of S. Ross and his group. The first physical methods for studying antifoams were presented along with the concepts of spreading and entering coefficients of oils (W. Harkins, 1941, J. Robinson and W. Woods, 1948). The further development of the antifoams (oils+hydrophobic particles) was described by means of the works of R. Kulkarni et al., A. Dippenaar and P. Garrett in the late 1970s and the early 1980s. The theoretical models on the antifoam performance of R. Pelton and P. Garrett, developed in 1980s and 1990s, were presented and analyzed as well in regard with their limits of applicability. Substantial advance on the experimental techniques for studying antifoams has been achieved by introducing different variants of the film trapping technique (FTT) developed by D. Wasan et al., I. Ivanov et al. and T. Tamura et al. in the middle and the late 1990s. An assessment of these techniques was carried out in regard with their capacity for detailed studying the antifoam action within the thin liquid films. Finally, the latest knowledge on the antifoams was achieved due to N. Denkov and his group, who harnessed both the most successful type of FTT and the interferometric thin film setup of Scheludko to conduct innovative experiments on the antifoam's action in the foam films under different conditions. They derived new more detailed understanding on the antifoam's action. For this reason, we must acknowledge the series of works under the supervision of N. Denkov performed between 1996 and 2004 as the lately ones in the field. The present work contains in addition a subchapter devoted to describing alternative methods for design and control of the foam stability. As far as the foaminess and the rate of foam decay depends on the states of the surfactant adsorption layers situated on the bubble surfaces, both foaminess and foam durability can be designed by means of proper choices of surfactants, concentrations and methods of foam generation. Therefore, this paper scrutinized the very mechanism of foam generation whose product is initial foam. Afterwards it was pointed out that the elastic modulus of the foam bubbles is responsible for the further "life" of the already generated foam. A compilation between foaminess and average rate of foam decay named foam production was shown as more successful way to describe the foaming capacity of the frothers. In addition, the properties of tenacious famous under various conditions were exhibited as well. This subchapter does not give any formula for precise design of foams with entailed durability but rather outlines new ways to achieve such recipe. Copyright © 2012 Elsevier B.V. All rights reserved.

Regions of conservation and divergence in the 3' untranslated sequences of genomic RNA from Ross River virus isolates.

PubMed

Faragher, S G; Dalgarno, L

1986-07-20

The 3' untranslated (UT) sequences of the genomic RNAs of five geographic variants of the alphavirus Ross River virus (RRV) were determined and compared with the 3' UT sequence of RRV T48, the prototype strain. Part of the 3' UT region of Getah virus, a close serological relative of RRV, was also sequenced. The RRV 3' UT region varies markedly in length between variants. Large deletions or insertions, sequence rearrangements and single nucleotide substitutions are observed. A sequence tract of 49 to 58 nucleotides, which is repeated as four blocks in the RRV T48 3' UT region, occurs only once in the 3' UT region of one RRV strain (NB5092), indicating that the existence of repeat sequence blocks is not essential for RRV replication. However, the precise sequence of the 3' proximal copy of the repeat block and its position relative to the poly(A) tail were identical in all RRV isolates examined, suggesting that it has an important role in RRV replication. Nucleotide substitutions between RRV variants are distributed non-randomly along the length of the 3' UT region. The sequence of 120 to 130 nucleotides adjacent to the poly(A) tail is strongly conserved. Getah virus RNA contains three repeat sequence blocks in the 3' UT region. These are similar in sequence to those in RRV RNA but differ in their arrangement. Homology between the RRV and Getah 3' UT sequences is greatest in the 3' proximal repeat sequence block that shows three differences in 49 nucleotides. The 3' proximal repeat in Getah RNA occurs at the same position, relative to the poly(A) tail, as in all RRV variants. The RRV and Getah virus 3' UT sequences show extensive homology in the region between the 3' proximal repeat and the poly(A) tail but, apart from the repeat blocks themselves, they show no significant homology elsewhere.

Environmental factor and inflammation-driven alteration of the total peripheral T-cell compartment in granulomatosis with polyangiitis.

PubMed

Kerstein, Anja; Schüler, Silke; Cabral-Marques, Otávio; Fazio, Juliane; Häsler, Robert; Müller, Antje; Pitann, Silke; Moosig, Frank; Klapa, Sebastian; Haas, Christian; Kabelitz, Dieter; Riemekasten, Gabriela; Wolters, Steffen; Lamprecht, Peter

2017-03-01

Autoimmune diseases are initiated by a combination of predisposing genetic and environmental factors resulting in self-perpetuating chronic inflammation and tissue damage. Autoantibody production and an imbalance of effector and regulatory T-cells are hallmarks of autoimmune dysregulation. While expansion of circulating effector memory T-cells is linked to disease pathogenesis and progression, the causes driving alterations of the peripheral T-cell compartment have remained poorly understood so far. In granulomatosis with polyangiitis (GPA), a prototypical autoimmune disorder of unknown aetiology, we performed for the first time a combined approach using phenotyping, transcriptome and functional analyses of T-cell populations to evaluate triggers of memory T-cell expansion. In more detail, we found increased percentages of circulating CD4+CD28-, CD8+CD28- and CD4+CD161+ single-positive and CD4+CD8+ double-positive T-cells in GPA. Transcriptomic profiling of sorted T-cell populations showed major differences between GPA and healthy controls reflecting antigen- (bacteria, viruses, fungi) and cytokine-driven impact on T-cell populations in GPA. Concomitant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) - positivity was associated with a significant increase in the percentage of CD28- T-cells in GPA-patients compared to sole CMV- or EBV-positivity or CMV- and EBV-negativity. T-cells specific for other viruses (influenza A virus, metapneumovirus, respiratory syncytial virus) and the autoantigen proteinase 3 (PR3) were infrequently detected in GPA. Antigen-specific T-cells were not specifically enriched in any of the T-cell subsets. Altogether, on a genetic and cellular basis, here we show that alterations of the peripheral T-cell compartment are driven by inflammation and various environmental factors including concomitant CMV and EBV infection. Our study provides novel insights into mechanisms driving autoimmune disease and on potential therapeutic targets. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Lack of transmission of mouse minute virus (MMV) from in vitro-produced embryos to recipients and pups due to the presence of cumulus cells during the in vitro fertilization process.

PubMed

Mahabir, E; Bulian, D; Needham, J; Schmidt, J

2009-09-01

The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (10(4) TCID(50) [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 10(5) cumulus cells were analyzed 5 h after exposure to 10(4) TCID(50)/ml MMVp, cells had an average titer of 10(4) TCID(50)/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

PubMed

Tapia, Lorena I; Shaw, Chad A; Aideyan, Letisha O; Jewell, Alan M; Dawson, Brian C; Haq, Taha R; Piedra, Pedro A

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.