Vesicular Stomatitis Virus.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by vesicular stomatitis New Jersey virus (VSNJV) or vesicular stomatitis Indiana virus (VSIV). The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. Over the past 20 years, VS outbreaks in the southwestern and Rocky Mountain regions occurred periodically with incursion years followed by virus overwintering and subsequent expansion outbreak years. The regulatory response by animal health officials prevents the spread of disease by animals with lesions and manages trade impacts. Recent US outbreaks highlight potential climate change impacts on insect vectors or other transmission-related variables.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Recombinant adenovirus expressing vesicular stomatitis virus G proteins induce both humoral and cell-mediated immune responses in mice and goats.

BACKGROUND: Vesicular stomatitis (VS) is an acute, highly contagious and economically important zoonotic disease caused by the vesicular stomatitis virus (VSV). There is a need for effective and safe stable recombinant vaccine for the control of the disease. The human type 5 replication-defective adenovirus expression vector is a good way to construct recombinant vaccines. RESULTS: Three recombinant adenoviruses (rAd) were successfully constructed that expressed the VSV Indiana serotype glycoprotein (VSV-IN-G), VSV New Jersey serotype glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]) with potentiality to induce protective immunity. G proteins were successfully expressed with good immunogenicity. The rAds could induce the production of VSV antibodies in mice, and VSV neutralizing antibodies in goats, respectively. The neutralizing antibody titers could reach 1:32 in mice and 1:64 in goats. The rAds induced strong lymphocyte proliferation in mice and goats, which was significantly higher compared to the negative control groups. CONCLUSIONS: The three rAds constructed in the study expressed VSV-G proteins and induced both humoral and cellular immune responses in mice and goats. These results lay the foundation for further studies on the use of rAds in vaccines expressing VSV-G.

Recombinant vesicular stomatitis virus glycoprotein carrying a foot-and-mouth disease virus epitope as a vaccine candidate.

Foot-and-mouth disease (FMD) is one of the most highly contagious animal diseases. In an effort to overcome the drawbacks of the currently used inactivated foot-and-mouth disease virus vaccine, a novel recombinant protein carrying foot-and-mouth disease virus VP1 GH loop epitope linked to vesicular stomatitis virus glycoprotein was expressed in a baculovirus system. Its antigenicity was confirmed with ELISA using monoclonal antibody against foot-and-mouth disease virus. Twice immunizations one month apart in field pigs resulted in a significant antibody increase compared to the glutathione S-transferase carrier containing the same epitope and the commercial vaccine. To my knowledge, this is the first report that the recombinant protein vaccine was superior to the current vaccine. Although further studies are required to examine their immunogenicity in a large number of animals, this study sheds light on the development of a novel recombinant protein vaccine that could be easily produced in a general laboratory as an alternative to the current FMD vaccine, which requires a biosafety level 3 containment facility for vaccine production.

Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

Validation of a site-specific recombination cloning technique for the rapid development of a full-length cDNA clone of a virulent field strain of vesicular stomatitis New Jersey virus.

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.

The methyltransferase PRMT6 attenuates antiviral innate immunity by blocking TBK1-IRF3 signaling.

Protein arginine methyltransferases (PRMTs) play diverse biological roles and are specifically involved in immune cell development and inflammation. However, their role in antiviral innate immunity has not been elucidated. Viral infection triggers the TBK1-IRF3 signaling pathway to stimulate the production of type-I interferon, which mediates antiviral immunity. We performed a functional screen of the nine mammalian PRMTs for regulators of IFN-ß expression and found that PRMT6 inhibits the antiviral innate immune response. Viral infection also upregulated PRMT6 protein levels. We generated PRMT6-deficient mice and found that they exhibited enhanced antiviral innate immunity. PRMT6 deficiency promoted the TBK1-IRF3 interaction and subsequently enhanced IRF3 activation and type-I interferon production. Mechanistically, viral infection enhanced the binding of PRMT6 to IRF3 and inhibited the interaction between IRF3 and TBK1; this mechanism was independent of PRMT6 methyltransferase activity. Thus, PRMT6 inhibits antiviral innate immunity by sequestering IRF3, thereby blocking TBK1-IRF3 signaling. Our work demonstrates a methyltransferase-independent role for PRMTs. It also identifies a negative regulator of the antiviral immune response, which may protect the host from the damaging effects of an overactive immune system and/or be exploited by viruses to escape immune detection.

Epidemiological situation of vesicular stomatitis virus infection in cattle in the state of Paraíba, semiarid region of Brazil.

The aim of this survey was to estimate the apparent herd-level and animal-level prevalences, as well as to identify risk factors and spatial clustering of vesicular stomatitis virus (VSV) positive herds in the state of Paraíba, semiarid of Brazil. The state was divided into three sampling strata: Sertão, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd, a pre-established number of cows aged ≥ 24 months were systematically selected (secondary sampling units). In total, 2279 animals were sampled from 468 herds. Serum samples were submitted to virus neutralization (VN) test for detection of antibodies to VSV using three viral strains: VSIV-3 2013SaoBento/Paraiba E, strain Indiana (VSIV-1) and VSNJV. A herd was considered positive for VSV if it included at least one positive animal in herds of up to 10 females, two positive animals in herds of 11-99 females, and three positive in herds with more than 99 females. The spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. The apparent herd-level prevalence in the state of Paraíba was 38.5% (95% CI = 35.5-41.6%), 80.6% (95% CI = 73.6-86.2%) in the region of Sertão, 7.0% (95% CI = 3.9-12.2%) in Borborema, and 2.6% (95% CI = 1.0-6.7%) in Agreste/Zona da Mata. The apparent animal-level prevalence was 26.2% (95% CI = 20.6-32.8%) in the state of Paraíba, 48.2% (95% CI = 41.5-54.9%) in Sertão, 6.3% (95% CI = 2.7-14%) in Borborema, and 3.2% 1.9% (95% CI = 0.4-8.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: mixed production (milk/beef) (OR = 4.54), herd size > 23 animals (OR = 3.57), presence of cervids (OR = 15.24), rental of pastures (OR = 3.02), sharing of water sources (OR = 2.57) and presence of horses (OR = 1.69). Two significant clusters of positive herds were detected: the primary cluster covered the Sertão region and the secondary cluster covered part of the Sertão and Borborema regions. Our results suggest high VSV circulation in the bovine population of the state of Paraíba, semiarid region of Brazil, mainly in the Sertão mesoregion, and based on risk factor analysis it was possible to identify important associations that deserve more investigation on causal factors.

Oncolytic Recombinant Vesicular Stomatitis Virus (VSV) Is Nonpathogenic and Nontransmissible in Pigs, a Natural Host of VSV.

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus that naturally causes disease in livestock including horses, cattle and pigs. The two main identified VSV serotypes are New Jersey (VSNJV) and Indiana (VSIV). VSV is a rapidly replicating, potently immunogenic virus that has been engineered to develop novel oncolytic therapies for cancer treatment. Swine are a natural host for VSV and provide a relevant and well-established model, amenable to biological sampling to monitor virus shedding and neutralizing antibodies. Previous reports have documented the pathogenicity and transmissibility of wild-type isolates and recombinant strains of VSIV and VSNJV using the swine model. Oncolytic VSV engineered to express interferon-beta (IFNß) and the sodium iodide symporter (NIS), VSV-IFNß-NIS, has been shown to be a potent new therapeutic agent inducing rapid and durable tumor remission following systemic therapy in preclinical mouse models. VSV-IFNß-NIS is currently undergoing clinical evaluation for the treatment of advanced cancer in human and canine patients. To support clinical studies and comprehensively assess the risk of transmission to susceptible species, we tested the pathogenicity and transmissibility of oncolytic VSV-IFNß-NIS using the swine model. Following previously established protocols to evaluate VSV pathogenicity, intradermal inoculation with 107 TCID50 VSV-IFNß-NIS caused no observable symptoms in pigs. There was no detectable shedding of infectious virus in VSV-IFNß-NIS in biological excreta of inoculated pigs or exposed naive pigs kept in direct contact throughout the experiment. VSV-IFNß-NIS inoculated pigs became seropositive for VSV antibodies, while contact pigs displayed no symptoms of VSV infection, and importantly did not seroconvert. These data indicate that oncolytic VSV is both nonpathogenic and not transmissible in pigs, a natural host. These findings support further clinical development of oncolytic VSV-IFNß-NIS as a safe therapeutic for human and canine cancer.

Pathogenesis of Vesicular Stomatitis New Jersey Virus Infection in Deer Mice ( Peromyscus maniculatus) Transmitted by Black Flies ( Simulium vittatum).

The natural transmission of vesicular stomatitis New Jersey virus (VSNJV), an arthropod-borne virus, is not completely understood. Rodents may have a role as reservoir or amplifying hosts. In this study, juvenile and nestling deer mice ( Peromyscus maniculatus) were exposed to VSNJV-infected black fly ( Simulium vittatum) bites followed by a second exposure to naive black flies on the nestling mice. Severe neurological signs were observed in some juvenile mice by 6 to 8 days postinoculation (DPI); viremia was not detected in 25 juvenile deer mice following exposure to VSNJV-infected fly bites. Both juvenile and nestling mice had lesions and viral antigen in the central nervous system (CNS); in juveniles, their distribution suggested that the sensory pathway was the most likely route to the CNS. In contrast, a hematogenous route was probably involved in nestling mice, since all of these mice developed viremia and had widespread antigen distribution in the CNS and other tissues on 2 DPI. VSNJV was recovered from naive flies that fed on viremic nestling mice. This is the first report of viremia in a potential natural host following infection with VSNJV via insect bite and conversely of an insect becoming infected with VSNJV by feeding on a viremic host. These results, along with histopathology and immunohistochemistry, show that nestling mice have widespread dissemination of VSNJV following VSNJV-infected black fly bite and are a potential reservoir or amplifying host for VSNJV.

Expression Kinetics of RANTES and MCP-1 in the Brain of Deer Mice (Peromyscus maniculatus) Infected with Vesicular Stomatitis New Jersey Virus.

The vesicular stomatitis virus (VSV) causes encephalitis in mice when inoculated intranasally. The deer mouse (Peromyscus maniculatus), a native New World rodent, is also susceptible to VSV infection and develops similar central nervous system (CNS) lesions to those observed in other rodent species. Chemokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES; CCL-5) and monocyte chemoattractant protein (MCP)-1 (CCL-2), which are important for chemotaxis and activation of inflammatory cells, are expressed during the course of VSV encephalitis. However, the role of CNS resident cells in chemokine expression is poorly characterized. Here, we show that during vesicular stomatitis New Jersey virus (VSNJV) encephalitis in deer mice, RANTES and MCP-1 are expressed only in the olfactory bulb (OB), where the virus was localized. This chemokine expression was followed by the influx of inflammatory cells to the OB later in the course of acute disease. Neurons, astrocytes and microglia expressed RANTES, while MCP-1 was expressed by neurons and astrocytes. Although astrocytes and microglia responded to VSNJV infection by expressing chemokines, neurons were the cell type that was predominantly infected. Therefore, infected neurons may have a critical role in initiating an immune response in the OB. The signalling between neurons and other CNS resident cells is most likely the mechanism by which astrocytes and microglia are activated during the course of VSV encephalitis.

Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: Use of rapid molecular assays to differentiate between vesicular disease viruses.

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these 'look-a-like' diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV's and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.

Investigação epidemiológica de estomatite vesicular por achados clínicos em bovinos e equinos no Estado do Maranhão / Stomatitis vesicular in cattle and horse in the Maranhão state

A Estomatite Vesicular (EV) é uma doença infecciosa que acomete equinos, bovinos, suínos, mamíferos silvestres e humanos. Por apresentar sinais clínicos semelhantes a outras doenças vesiculares, principalmente, febre aftosa, sua presença em determinadas regiões pode interferir no intercâmbio comercial internacional dos animais, seus produtos e subprodutos. Apesar de sua importância, a epidemiologia e a manutenção do vírus no ambiente não estão totalmente esclarecidas dificultando a aplicação de medidas de controle efetivas. A doença já foi diagnosticada em todas as regiões brasileiras. Bovinos com sialorréia, perda do epitélio lingual, lesões abertas com bordas amareladas nas gengivas, lábios, língua e mucosa oral e equinos com sialorréia e lesões abertas na mucosa oral e lábios foram observados e notificados ao Serviço Veterinário Oficial do Estado do Maranhão, Agência Estadual de Defesa Agropecuária do Maranhão (AGRD/MA). Amostras de soro de equinos e bovinos com sintomas de EV foram coletadas para investigação por ELISA e por neutralização viral, além do diagnóstico diferencial para Febre Aftosa (FA). Fragmentos epiteliais de bovinos com lesões na língua foram coletados para identificação molecular do agente. Todos os animais foram negativos para FA. Todos os bovinos e equinos foram reativos para EV nos testes sorológicos. A partir dos fragmentos epiteliais de bovinos enviados ao Instituto Biológico de São Paulo para PCR, foi possível caracterizar o agente como VesiculovirusIndiana III (Alagoas/VSAV).

Vesicular stomatitis (VS) is an infectious viral disease that affects bovines, equines, swine, wild animals and humans. As it is indistinguishable from other vesicular diseases, mainly Foot and Mouth Disease (FMD), it causes restrictions in commercial livestock trade at national and international levels and also significant economic losses. As the epidemiology and maintenance of VS virus in nature are not clearly understood it is difficult to take effective control measures. VS was diagnosed in some regions of Brazil, such as Minas Gerais, Santa Catarina, São Paulo and Alagoas. Cattle and horses with clinical symptoms of drooling, shedding of the lingual epithelium, presence of vesicles on the oral mucosa were observed and reported to the National Animal Health Office health of Maranhão State, Brazil. Samples of serum of these animals were collected and sent to Laboratório Nacional de Agropecuaria for ELISA and virus neutralization and differential diagnosis for Foot and Mouth Disease (FMD). The results of ELISA confirmed the VS. In the differential diagnosis, the results were negative for FMD. Samples of bovine epithelial tissue for VS by PCR confirmation of diagnosis were collected and sent to Biological Institute of São Paulo. Molecular results confirmed the VesiculovirusIndiana III (Alagoas/VSAV) infection.

Phylogeographic characteristics of vesicular stomatitis New Jersey viruses circulating in Mexico from 2005 to 2011 and their relationship to epidemics in the United States.

We analyzed the phylogenetic and time-space relationships (phylodynamics) of 181 isolates of vesicular stomatitis New Jersey virus (VSNJV) causing disease in Mexico and the United States (US) from 2005 through 2012. We detail the emergence of a genetic lineage in southern Mexico causing outbreaks in central Mexico spreading into northern Mexico and eventually into the US. That emerging lineage showed higher nucleotide sequence identity (99.5%) than that observed for multiple lineages circulating concurrently in southern Mexico (96.8%). Additionally, we identified 58 isolates from Mexico that, unlike previous isolates from Mexico, grouped with northern Central America clade II viruses. This study provides the first direct evidence for the emergence and northward migration of a specific VSNJV genetic lineage from endemic areas in Mexico causing VS outbreaks in the US. In addition we document the emergence of a Central American VSNJV genetic lineage moving northward and causing outbreaks in central Mexico.

Vesicular stomatitis outbreak in the southwestern United States, 2012.

Vesicular stomatitis is a viral disease primarily affecting horses and cattle when it occurs in the United States. Outbreaks in the southwestern United States occur sporadically, with initial cases typically occurring in Texas, New Mexico, or Arizona and subsequent cases occurring in a northward progression. The viruses causing vesicular stomatitis can be transmitted by direct contact of lesioned animals with other susceptible animals, but transmission is primarily through arthropod vectors. In 2012, an outbreak of vesicular stomatitis in the United States occurred that was caused by Vesicular stomatitis New Jersey virus serotype. Overall, 51 horses on 36 premises in 2 states were confirmed positive. Phylogenetic analysis of the virus indicated that it was most closely related to viruses detected in the state of Veracruz, Mexico, in 2000.

Genetically modified VSV(NJ) vector is capable of accommodating a large foreign gene insert and allows high level gene expression.

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSV(Ind)), but less with New Jersey serotype (VSV(NJ)). Here, we report constructions of genetically modified rVSV(NJ) vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSV(NJ). We modified the M gene of VSV(NJ) by changing methionine to arginine at positions 48 and 51 (rVSV(NJ)-M) (Kim and Kang, 2007) for construction of rVSV(NJ) with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSV(NJ)-M vector, and recombinant VSV(NJ)-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSV(NJ)-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSV(NJ)-M without gene of interests, VSV(NJ)-M with a gene of HCV NS3 and NS4A (VSV(NJ)-M-NS3/4A), VSV(NJ)-M with a gene of HCV NS4AB plus NS5AB (VSV(NJ)-M-NS4AB/5AB), and VSV(NJ)-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSV(NJ)-M-NS3/4AB/5AB) were 172±10.5 nm, 201±12.5 nm, 226±12.9 nm, and 247±18.2 nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSV(NJ)-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSV(NJ) wild type and rVSV(NJ) M mutant viruses and show that the rVSV(NJ) M mutant virus infection induced a higher level of the IFN-ß compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSV(NJ)-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSV(NJ)-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site.

Host predilection and transmissibility of vesicular stomatitis New Jersey virus strains in domestic cattle (Bos taurus) and swine (Sus scrofa).

BACKGROUND: Epidemiologic data collected during epidemics in the western United States combined with limited experimental studies involving swine and cattle suggest that host predilection of epidemic vesicular stomatitis New Jersey virus (VSNJV) strains results in variations in clinical response, extent and duration of virus shedding and transmissibility following infection in different hosts. Laboratory challenge of livestock with heterologous VSNJV strains to investigate potential viral predilections for these hosts has not been thoroughly investigated. In separate trials, homologous VSNJV strains (NJ82COB and NJ82AZB), and heterologous strains (NJ06WYE and NJOSF [Ossabaw Island, sand fly]) were inoculated into cattle via infected black fly bite. NJ82AZB and NJ06WYE were similarly inoculated into swine. RESULTS: Clinical scores among viruses infecting cattle were significantly different and indicated that infection with a homologous virus resulted in more severe clinical presentation and greater extent and duration of viral shedding. No differences in clinical severity or extent and duration of viral shedding were detected in swine. CONCLUSIONS: Differences in clinical presentation and extent and duration of viral shedding may have direct impacts on viral spread during epidemics. Viral transmission via animal-to-animal contact and insect vectored transmission are likely to occur at higher rates when affected animals are presenting severe clinical signs and shedding high concentrations of virus. More virulent viral strains resulting in more severe disease in livestock hosts are expected to spread more rapidly and greater distances during epidemics than those causing mild or inapparent signs.

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila.

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways.