Vesicular Stomatitis Virus Serotypes Research Articles

Comparative analysis of oncolytic potential of vesicular stomatitis virus serotypes Indiana and New Jersey in cancer cell lines

Aim. Compare the lytic efficiency and the kinetics of accumulation of vesicular stomatitis virus serotypes Indiana (VSV-IND) and New Jersey (VSV-NJ) on cell lines of mouse melanoma B16F10, human hepatocellular carcinoma HepG2 and human mammary adenocarcinoma MCF7.Materials and methods. The viability of mouse melanoma B16F10, human hepatocellular carcinoma HepG2 and human mammary adenocarcinoma MCF7 cell lines infected with VSV-IND and VSV-NJ viruses at different multiplicity of infection (10 MOI; 1 MOI; 0.1 MOI) was assessed after 24, 48 and 72 hours, and the half maximal inhibitory concentration (IC50) values were measured using the methyl tetrazolium test. The relationship with virus accumulation in cell culture was determined using reverse transcription – quantitative polymerase chain reaction; 50% tissue culture infectious dose (TCID50) of VSV-IND and VSV-NJ for B16F10, HepG2, MCF7 were calculated using the Reed-Muench method.Results. The most susceptible cell line for both viruses was B16F10: cell viability 72 hours after infection at 10 MOI was only 10.4% and 5.7% for VSV-IND and VSV-NJ, respectively. HepG2 cell viability at 72 hours post-infection at 10 MOI was 10.8% and 9.8% for VSV-IND and VSV-NJ, and for MCF7 adenocarcinoma it was 46.6% and 36.2%, respectively. Moreover, only in the B16F10 culture was a positive statistically significant correlation of medium strength established between the inhibition of cell viability and the accumulation of viral RNA: for VSV-IND r = 0.601 (p < 0.05); for VSV-NJ r = 0.668 (p < 0.05). HepG2 and MCF7 showed no significant correlation.Conclusion. The research results indicate the potential of using oncolytic viruses of the VSV-IND and VSV-NJ as a platform for the development of new recombinant viruses for virotherapy of solid tumors in combination with other types of treatment.

Near-Complete Genome Sequences of Vesicular Stomatitis Virus Isolates from the 2020 Outbreak in Kansas.

Here, we report the near-complete genome sequences of vesicular stomatitis virus (VSV) serotype Indiana isolates from the 2020 U.S. outbreak. The sequences were obtained from swabs collected from Kansas horses in July and August. The four genome sequences help improve our understanding of VSV outbreak dynamics in the United States.

Comparative evaluation of pathogenicity of three isolates of vesicular stomatitis virus (Indiana serotype) in pigs.

Vesicular stomatitis (VS) is a notifiable disease of livestock affecting cattle, horses, pigs and humans. Vesicular stomatitis virus (VSV) serotypes Indiana and New Jersey are endemic to Central America; however, they also cause sporadic and scattered outbreaks in various countries in South and North America, including the USA. In order to develop an effective experimental challenge model for VSV, we compared the pathogenicity of three VSV serotype Indiana isolates in 36 4-5 week-old pigs. Two bovine isolates of Central American origin and one equine isolate from the USA were used for the experimental infections. Each pig was inoculated with a single isolate by both the intradermal and intranasal routes. Clinical signs of VSV infection were recorded daily for 10 days post-inoculation (days p.i.). Nasal and tonsillar swab samples and blood were collected to monitor immune responses, virus replication and shedding. Post-challenge, characteristic signs of VS were observed, including vesicles on the nasal planum and coronary bands, lameness, loss of hoof walls and pyrexia. Pigs inoculated with the Central American isolates showed consistently more severe clinical signs in comparison to the pigs infected with the USA isolate. Genomic RNA was isolated from the original challenge virus stocks, sequenced and compared to VSV genomes available in GenBank. Comparative genome analysis demonstrated significant differences between the VSV isolate from the USA and the two Central American isolates. Our results indicate that the Central American isolates of VSV serotype Indiana used in this study are more virulent in swine than the USA VSV serotype Indiana isolate and represent good candidate challenge strains for future VSV studies.

Vaccine induction of antibodies and tissue-resident CD8+ T cells enhances protection against mucosal SHIV-infection in young macaques.

Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

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 (IFNβ) and the sodium iodide symporter (NIS), VSV-IFNβ-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-IFNβ-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-IFNβ-NIS using the swine model. Following previously established protocols to evaluate VSV pathogenicity, intradermal inoculation with 107 TCID50 VSV-IFNβ-NIS caused no observable symptoms in pigs. There was no detectable shedding of infectious virus in VSV-IFNβ-NIS in biological excreta of inoculated pigs or exposed naive pigs kept in direct contact throughout the experiment. VSV-IFNβ-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-IFNβ-NIS as a safe therapeutic for human and canine cancer.

Antibodies against vesicular stomatitis virus in horses from southern, midwestern and northeastern Brazilian States

ABSTRACT: Vesicular stomatitis virus (VSV) is the agent of a vesicular disease that affects many animal species and may be clinically confounded with foot-and-mouth disease in ruminant and swine. Horses are especially susceptible to VSV and may serve as sentinels for virus circulation. The present study investigated the presence of neutralizing antibodies against VSV Indiana III (VSIV-3) in serum samples of 3,626 horses from six states in three Brazilian regions: Southern (RS, n = 1,011), Midwest (GO/DF, n = 1,767) and Northeast (PB, PE, RN and CE, n = 848) collected between 2013 and 2014. Neutralizing antibodies against VSIV-3 (titers ≥40) were detected in 641 samples (positivity of 17.7%; CI95%:16.5-19.0%), being 317 samples from CE (87.3%; CI95%: 83.4-90.5 %); 109 from RN (65.7%; CI95%: 57.8 -72.7%); 124 from PB (45.4%; CI95%: 39.4-51.5%); 78 from GO/DF (4.4%; CI95%: 3.5-5.5%) and nine samples of RS (0.9%; CI95%: 0.4-1.7%). Several samples from the Northeast and Midwest harbored high neutralizing titers, indicating a recent exposure to the virus. In contrast, samples from RS had low titers, possibly due to a past remote exposure. Several positive samples presented neutralizing activity against other VSV serotypes (Indiana I and New Jersey), yet in lower titers, indicating the specificity of the response to VSIV-3. These results demonstrated a relatively recent circulation of VSIV-3 in northeastern Brazilian States, confirming clinical findings and demonstrating the sanitary importance of this infection.

Creation of matrix protein gene variants of two serotypes of vesicular stomatitis virus as prime-boost vaccine vectors.

To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSVInd) with a temperature-sensitive mutation (tsO23) of the VSVInd Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSVNJ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E M48R M51R) [rVSVNJ(GMM)] and VSVNJ(G22E M48R M51R L110F) [rVSVNJ(GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSVInd significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK21 cells and SH-SY5Y human neuroblastoma cells infected with rVSVInd(GML), rVSVNJ(GMM), and rVSVNJ(GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperature-sensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of rVSVInd(GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene. Recombinant vesicular stomatitis viruses as live vaccine vectors are very effective in expressing foreign genes and inducing adaptive T cell and B cell immune responses. As with any other live viruses in humans or animals, the use of live rVSVs as vaccine vectors demands the utmost safety. Our strategy to attenuate rVSVInd by utilizing a temperature-sensitive assembly-defective mutation of L111A and combining it with an M51R mutation in the M protein of rVSVInd significantly reduced the pathogenicity of the virus while maintaining highly effective virus production. We believe our new temperature-sensitive M gene mutant of rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and rVSVNJ(GMML) add excellent vaccine vectors to the pool of live viral vectors.

Putative domain-domain interactions in the vesicular stomatitis virus L polymerase protein appendage region.

The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an "unstructured" linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in the pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

Development of a convenient immunochromatographic strip for the diagnosis of vesicular stomatitis virus serotype Indiana infections

A rapid and simple immunochromatography strip (ICS) test for the specific detection of vesicular stomatitis virus serotype Indiana (VSV-IND) using two distinct monoclonal antibodies MAbs (1A2 and 4C3) against the G protein of VSV-IND was developed. The MAb 1A2 was conjugated with colloidal gold, and the MAb 4C3 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated T and C, respectively. The results showed that samples of VSV-IND combined with CG-MAb 1A2, and that these complexes were captured by MAb 4C3 at the test line (T), resulting in the appearance of a purple band. When samples did not contain VSV-IND or when they contained a quantity of VSV-IND below the detection limit of the test, only the control line (C) was visible. The analysis of the sensitivity of the test demonstrated that the lowest detected quantity of VSV-IND was 1.85×10(3.0)TCID50/ml. Storage of the ICS test at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. In clinical trials using RT-PCR as a reference test, the relative specificity and sensitivity of the ICS were determined to be 98.9% and 91.4%, respectively. Based on these results, the ICS test developed may be a suitable tool for rapid on-site testing for VSV-IND infection.

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes

Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5′ or 3′ end of N that significantly reduced N, P, and L viral transcripts (p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells (p<0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts (p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.

ANTICUERPOS CONTRA EL VIRUS DE LA ESTOMATITIS VESICULAR EN SAJINOS (Tayassu tajacu) DE ZOOCRIADEROS DE IQUITOS Y PUCALLPA

The objetive of this study was to detect antibodies against vesicular stomatitis virus (VSV) in collared peccaries (Tayassu tajacu) from farms of Iquitos and Pucallpa, Peru. Blood samples were collected in animals olders than 6 months of age (pregnant females were not included) from a farm in Iquitos (n = 21) and from four farms in Pucallpa (n = 47), for detection of antibodies against Indiana 1 and New Jersey serotype of VSV by viral neutralization test. No specific antibodies against both serotypes of VSV were detected, indicating that the peccaries were not exposed to VSV, and suggesting absence of the infection in those animals.

Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples

Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3 of subtype 3 [VSV-IND-3]) and 189 positive for VSV-NJ (162 vesicular epithelial suspensions and 27 cell culture antigens) from suspected cases of vesicular disease in cattle and horses collected from 11 countries between 1937 and 2008 or else were derived from experimental infection and 777 samples that were either shown to be positive or negative for foot-and-mouth disease (FMD) virus (FMDV) and swine vesicular disease virus (SVDV) or else collected from healthy cattle or pigs and collected from 68 countries between 1965 and 2011. The diagnostic sensitivity of the VSV-IND (for reaction with VSV-IND-1) and VSV-NJ LFDs was either similar or identical at 94.6% (VSV-IND) and 97.4% (VSV-NJ) compared to 92.5% and 97.4% obtained by the reference method of antigen ELISA. The VSV-IND LFD failed to react with viruses of VSV-IND-2 and 3, while the VSV-NJ device recognized all VSV-NJ virus strains. The diagnostic specificities of the VSV-IND and VSV-NJ LFDs were 99.1% and 100, respectively, compared to 99.6% and 99.8% for the ELISA. Reactions with FMDV which can produce indistinguishable syndromes clinically in cattle, pigs and sheep and SVDV (vesicular disease in pigs) did not occur. These data illustrate the potential for the LFDs to be used next to the animal for providing rapid and objective support to veterinarians in their clinical judgment of vesicular disease and for the subtype (VSV-IND-1) and type-specific (VSV-NJ) pen-side diagnosis of VS and differential diagnosis from FMD.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6 TCID 50/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Specific Detection of Rinderpest Virus by Real-Time Reverse Transcription-PCR in Preclinical and Clinical Samples from Experimentally Infected Cattle

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID(50)) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.

Spatial and phylogenetic analysis of vesicular stomatitis virus over-wintering in the United States

From 2004 through 2006, 751 vesicular stomatitis (VS) outbreaks caused by vesicular stomatitis virus serotype New Jersey (VSNJV) were reported in nine states of the southwestern United States. The normal model of the spatial scan statistic and phylogenetic techniques were used to assess whether the spatial and genetic relations among VSNJV outbreaks were consistent with the hypothesis that VSNJV over-wintered in specific regions of the southwestern United States infected in 2004 and 2005, respectively. Use of the spatial scan statistic led to the identification of two clusters of outbreaks for which the Euclidean distance to the nearest outbreak reported in the previous or following year, whichever was shorter, was significantly ( P < 0.01) shorter than the epidemic's (2004–2006) mean. Clusters were centered at Colorado and Wyoming and included 375 and 21 outbreaks, respectively. Results were supported by the phylogenetic analysis of 49 VSV samples collected from 2004 through 2006 in the United States and 10 VSV samples originated from Mexico. These findings, which were displayed using a publicly accessible web-based system referred to as the FMD BioPortal, were consistent with over-wintering of specific sub-lineages of VSNJV in a limited geographical region of the United States affected by a VS epidemic in 2005 and 2006.

Lentiviral Vector-Based Prime/Boost Vaccination against AIDS: Pilot Study Shows Protection against Simian Immunodeficiency Virus SIVmac251 Challenge in Macaques

AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log(10) fold reduction) and by the full preservation of the CD28(+) CD95(+) memory CD4(+) T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.

Enzyme-Linked Immunosorbent Assay Using Glycoprotein and Monoclonal Antibody for Detecting Antibodies to Vesicular Stomatitis Virus Serotype New Jersey

In this study, an enzyme-linked immunosorbent assay (ELISA) using glycoprotein and a monoclonal antibody (MAb) was developed for the detection of antibodies to vesicular stomatitis virus (VSV) serotype New Jersey (NJ). The glycoprotein to be used as a diagnostic antigen was extracted from partially purified VSV-NJ, and a neutralizing MAb specific to VSV-NJ was incorporated to compete with antibodies in a blocking ELISA using glycoprotein (GP ELISA). The cutoff of the GP ELISA was set at 40% inhibition, which corresponded to a virus neutralization test (VNT) titer of 32. With this threshold, the GP ELISA exhibited 99.6% specificity for naïve sera (n = 3,005) from cattle (n = 1,040), pigs (n = 1,120), and horses (n = 845) from domestic farms. The GP ELISA did not cross-react with sera positive for foot-and-mouth disease virus, swine vesicular disease virus, or VSV serotype Indiana. The GP ELISA was more compatible with the VNT than was the nucleocapsid-based ELISA for VSV-NJ-positive sera (n = 19). Taken together, this GP ELISA could be a useful tool as an alternative to the VNT for detecting antibodies specific to VSV-NJ.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus. Evidence in primary bovine fibroblast cultures

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/susceptibility and production of factors with antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.