Isolates Of Vesicular Stomatitis Virus Research Articles

Culicoides-Specific Fitness Increase of Vesicular Stomatitis Virus in Insect-to-Insect Infections.

Vesicular stomatitis virus (VSV) is an arthropod-borne virus affecting livestock. In the United States, sporadic outbreaks result in significant economic losses. During epizootics, Culicoides biting midges are biological vectors and key to the geographic expansion of outbreaks. Additionally, Culicoides may play a role in VSV overwintering because females and males are capable of highly efficient venereal transmission, despite their relatively low virus titers. We hypothesized that VSV propagated within a midge has increased fitness for subsequent midge infections. To evaluate the potential host-specific fitness increase, we propagated three viral isolates of VSV in porcine skin fibroblasts and Culicoides cell lines. We then evaluated the viral infection dynamics of the different cell-source groups in Culicoides sonorensis. Our results indicate that both mammalian- and insect-derived VSV replicate well in midges inoculated via intrathoracic injection, thereby bypassing the midgut barriers. However, when the virus was required to infect and escape the midgut barrier to disseminate after oral acquisition, the insect-derived viruses had significantly higher titers, infection, and dissemination rates than mammalian-derived viruses. Our research suggests that VSV replication in Culicoides cells increases viral fitness, facilitating midge-to-midge transmission and subsequent replication, and further highlights the significance of Culicoides midges in VSV maintenance and transmission dynamics.

Exploring the Molecular Basis of Vesicular Stomatitis Virus Pathogenesis in Swine: Insights from Expression Profiling of Primary Macrophages Infected with M51R Mutant Virus.

Vesicular stomatitis virus (VSV) is an emergent virus affecting livestock in the US. Previously, using a recombinant VSV carrying the M51R mutation in the matrix protein (rNJ0612NME6-M51R), we evaluated the pathogenesis of this virus in pigs. Our results indicated that rNJ0612NME6-M51R represented an attenuated phenotype in in-vivo and in ex-vivo in pig macrophages, resembling certain clinical features observed in field VSV isolates. In order to gain more insight into the molecular basis leading to the attenuation of rNJ0612NME6-M51R in pigs, we conducted a microarray analysis to assess the gene expression profiles of primary porcine macrophages infected with rNJ0612NME6-M51R compared to its parental virus (rNJ0612NME6). Our results showed an overall higher gene expression in macrophages infected with rNJ0612NME6-M51R. Specifically, we observed that the pathways related with immune cytokine signaling and interferon (IFN)-related responses (including activation, signaling, induction, and antiviral mechanisms) were the ones comprising most of the relevant genes identified during this study. Collectively, the results presented herein highlight the relevance of type I interferon during the pathogenesis of VSV in pigs. The information generated from this study may represent a framework for future studies intended to understand the molecular bases of the pathogenesis of field strains in livestock.

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.

Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus strains occurring in Mexico and North and Central America. A comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America was tested by the real-time RT-PCR assay and virus isolation. Overall, clinical sensitivity and specificity of the real-time RT-PCR were 83% and 99%, respectively. Interestingly, VS virus isolates originating from a specific region of Costa Rica were not detected by real-time RT-PCR. Sequence comparisons of these viruses with the real-time RT-PCR probe and primers showed mismatches in the probe and forward and reverse primer regions. Additional lineage-specific primers and a probe corrected the lack of detection of the missing genetic lineage. Thus, this assay reliably identified existing Mexican and Central American VS viruses and proved readily adaptable as new VS viruses were encountered. An important secondary result of this research was the collection of hundreds of new VS virus isolates that provide a foundation from which many additional studies can arise.

Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus

Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5′ untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5′ nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak.

Full-length genome analysis of natural isolates of vesicular stomatitis virus (Indiana 1 serotype) from North, Central and South America.

Most studies on the molecular biology and functional analysis of vesicular stomatitis virus Indiana 1 serotype (VSV-IN1) are based on the only full-length genomic sequence currently deposited in GenBank. This sequence is a composite of several VSV-IN1 laboratory strains passaged extensively in tissue culture over the years and it is not certain that this sequence is representative of strains circulating in nature. We describe here the complete genomic sequence of three natural isolates, each representing a distinct genetic lineage and geographical origin: 98COE (North America), 94GUB (Central America) and 85CLB (South America). Genome structure and organization were conserved, with a 47 nucleotide 3' leader, five viral genes -- N, P, M, G and L -- and a 59 nucleotide 5' trailer. The most conserved gene was N, followed by M, L and G, with the most variable being P. Sequences containing the polyadenylation and transcription stop and start signals were completely conserved among all the viruses studied, but changes were found in the non-transcribed intergenic nucleotides, including the presence of a trinucleotide at the M-G junction of the South American lineage isolate. A 102-189 nucleotide insertion was present in the 5' non-coding region of the G gene only in the viruses within a genetic lineage from northern Central America. These full-length genomic sequences should be useful in designing diagnostic probes and in the interpretation of functional genomic analyses using reverse genetics.

Laboratory Vector Competence of Black Flies (Diptera: Simuliidæ) for the Indiana Serotype of Vesicular Stomatitis Virus

In previous experiments we have demonstrated that colonized and wild black flies are competent laboratory vectors of different Mexican and Western USA isolates of vesicular stomatitis virus, serotype New Jersey (VSV-NJ). We have recently demonstrated biological VSV-NJ transmission by black flies using animal models. In the study described here, we tested the vector competence of colonized and wild black flies for the vesicular stomatitis virus, serotype Indiana (VSV-IN). A 1998 equine isolate was used. After a 10 day incubation period, saliva from experimentally infected Simulium vittatum and S. notatum was individually collected and tested for the presence of infectious virus. Virus was detected in the saliva of both species following oral infection, indicating that they are competent laboratory vectors of VSV-IN. In addition, the results suggest that the black fly gut may exert evolutionary pressures on the virus.

Interferon induction as a quasispecies marker of vesicular stomatitis virus populations.

The interferon (IFN)-inducing capacity of different isolates of vesicular stomatitis virus (VSV) of the Indiana (IN) and New Jersey (NJ) serotypes were measured to assess the extent of variability of this phenotype. Over 200 preparations of wild-type field isolates, laboratory strains, and plaque-derived subpopulations were examined. Marked heterogeneity was found in the ability of these viruses to induce IFN, covering a 10,000-fold range. A good fit to a normal distribution for the log of the IFN yields suggests a continuum of incremental changes in the viral genome may govern the IFN-inducing capacity of consensus populations derived from independently arising infections. A broad range in the magnitude of these changes, skewed towards inducers of high IFN yields, is consistent with a comparable series of ribonucleotide changes in the VSV genome, a sine qua non of a quasispecies population. Plaque- or vesicle-derived populations displayed standard deviations less than the mean IFN yields, though skewed to higher yielders, whereas populations from field and laboratory samples which differed widely in time and origin of isolation gave standard deviations greater than the means. The plaque isolation of IFN-inducing particles of VSV-IN, normally masked in populations by the predominance of non-IFN-inducing particles that suppress IFN induction, and the isolation of potent wild-type IFN-inducing VSV-IN from cows during an outbreak of vesicular stomatitis in a region that had yielded only virus expressing the non-IFN-inducing phenotype in prior and subsequent years, supports the view that genetic bottlenecks are operative in the natural transmission of this disease.

Vector competence of select black fly species for vesicular stomatitis virus (New Jersey serotype).

Black flies collected from southern Arizona were evaluated for their vector competence to the Oaxaca and Camp Verde isolates of vesicular stomatitis virus (New Jersey serotype) (VSV-NJ). The Camp Verde isolate is the index isolate of the 1982-1983 VSV-NJ epizootic that infected humans and livestock in 14 western states. Previous experiments have shown that colonized Simulium vittatum females are competent laboratory vectors of both virus isolates. However, under controlled laboratory conditions, Simulium bivittatum and S. longithallum were found to be incompetent vectors of both virus isolates. After oral infections, the Oaxaca isolate replicated in 35% and 38% of S. bivittatum and S. longithallum, respectively, but did not disseminate to the salivary glands. Thus, virus was not detected in the saliva of either black fly species with either VSV-NJ isolate, indicating the presence of a midgut barrier. Simulium notatum was found to be a competent laboratory vector of both virus isolates. Infectious virions were detected in the saliva of 23% and 26% of S. notatum infected orally with the Oaxaca and Camp Verde VSV-NJ isolates, respectively. This study suggests that the black fly identified as S. bivittatum was probably not involved in virus dissemination during the 1982-1983 epizootic in the western United States. Because the geographic distribution of S. notatum is not known, its involvement in that epizootic remains obscure.

Analysis of the Nucleocapsid Gene of Lettuce Necrotic Yellows Rhabdovirus

The complete nucleotide sequence of the nucleocapsid (N) gene of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus of plant rhabdoviruses, has been determined from cDNA clones of both the viral genomic and messenger RNAs. The previously identified N gene was adjacent to the 3′ leader RNA and began at position 91 from the 3′ end of the LNYV genome. Analysis of the sequence showed that the N mRNA contained a 78-nt 5′ untranslated region, followed by a 1377-nt open reading frame (ORF) encoding a 459-amino-acid protein. This ORF was similar in size to the N protein ORFs of other rhabdoviruses. However, little if any direct sequence homology was found with N protein sequences of other rhabdoviruses. Short amino acid sequences were found to be conserved between the N proteins of LNYV and sonchus yellow net virus (SYNV), a member of the genus Nucleorhabdovirus of plant rhabdoviruses. Among them, a sequence of 24 amino acids which was similar between LNYV and SYNV N proteins corresponded to a region with high homology between isolates of vesicular stomatitis virus and rabies virus N protein sequences, the type members of the genera Vesiculovirus and Lyssavirus of animal rhabdoviruses, respectively.

Punctuated equilibrium and positive Darwinian evolution in vesicular stomatitis virus.

RNA viruses possess the potential for rapid evolution and serve as excellent models to test evolutionary theory. Molecular phylogenetic analysis of the P gene for a larger number of diverse natural isolates of vesicular stomatitis virus reveals no evidence for a molecular clock but instead shows a stepwise evolutionary pattern unlike that ever seen before. Each step out from the tree's ancestral root to terminal branch tips correlates not with time of virus isolation but with a south-to-north geographical progression from Panama to the United States. The grossly unequal rates of change within this single species imply an underlying mechanism at odds with the prevailing notion that neutral changes are the dominating feature of molecular evolution. This is also a demonstration of punctuated equilibrium at the molecular level.

Interferon induction by viruses. XXI. Vesicular stomatitis virus: interferon inducibility as a phylogenetic marker.

Forty-five vesiculovirus isolates were systematically compared for their capacity to induce interferon (IFN) in chick embryo cells under conditions such that the maximum (quantum) yield of IFN per cell and the titer of IFN-inducing particles (IFP) could validly be determined. Twelve isolates of the New Jersey (NJ) serotype of vesicular stomatitis virus (VSV) were good inducers, yielding amounts of IFN that ranged in a continuum from 300 to more than 8,000 units per 10(7) cells. These must reflect genetic differences between the closely related viruses. These differences were not reflected in the nucleotide sequence of the viral 3' leader RNA, for analysis of eight of the NJ isolates showed no correlation with the IFN yields. As found in previous smaller surveys, 28 out of 32 VSV isolates of the Indiana (IN) serotype produced little or no IFN, or even suppressed its induction. However, four exceptional IN strains were isolated during 1984 and 1985 from cattle within a relatively circumscribed geographical area in Costa Rica and Panama; all belonged to Indiana virus, type 1, subtype IV, in the proposed G-protein gene evolutionary tree. This is the first example of an IFN-inducing phenotype serving as a phylogenetic marker.

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.

The Nucleocapsid Protein of Vesicular Stomatitis Virus Isolated from the Brains of Nude Mice is Responsible for Abated Viral RNA Synthesis at the Normal Body Temperature of Mice

Six temperature-sensitive mutants of vesicular stomatitis virus (VSV) were isolated from the central nervous system (CNS) of athymic nude mice. The nude mice had been reconstituted with syngeneic T lymphocytes and then infected with a temperature-sensitive mutant of VSV, tsG31-KS5 VSV, for 20 days. In BHK-21 cells incubated at 38 degrees C, the normal body temperature of mice, all six CNS virus clones had diminished RNA synthesis, when compared to RNA production in BHK-21 cells incubated at 31 degrees C. In contrast, the original tsG31-KS5 VSV mutant synthesized more RNA at 38 degrees C than it did at 31 degrees C. In vitro transcription assays were exploited to discern which viral protein(s) was functionally accountable for the abated synthesis of RNA of the CNS VSV isolates. The ribonucleoprotein complexes from the CNS VSV isolates were disrupted and template (N protein and RNA) and enzyme (L and NS proteins) fractions were purified. In vitro transcription assays were performed with template fractions of the brain isolates, added to enzyme fractions either wild-type wt VSV or tsG31-KS5 VSV, or with template fractions of wt VSV or tsG31-KS5 VSV mixed with enzyme fractions of the CNS isolates. The template fraction was responsible for the decrease in RNA synthesis in all six of the brain-isolated clones. When the template fractions of wt or tsG31-KS5 VSV were mixed with enzyme fractions of all the CNS-derived VSV, except BP5A VSV, leader sequence RNA and large Mr transcripts were transcribed. One clone, BP5A VSV, did not synthesize RNA when mixed with either template or enzyme of wt VSV, and probably had more than one functional mutation that influenced viral RNA synthesis.

Epizoötic vesicular stomatitis in Colorado, 1982: isolation of virus from insects collected along the northern Colorado Rocky Mountain Front Range.

Field studies were conducted during an epizootic of vesicular stomatitis virus (VSV) in Colorado to further assess the possible role of insects in the transmission of VSV. Insects associated with domestic livestock were collected at 11 premises along the Front Range of the Rocky Mountains in Colorado during the 1982 epizootic of vesicular stomatitis. Insects were pooled by date, location, species, and sex and were processed for virus isolation in three cell culture systems. Thirty-four isolates of vesicular stomatitis virus, New Jersey serotype, were obtained from 51,036 insects. Of these, 27 isolates were from Musca domestica (126 pools/5,285 specimens), 5 from other nonhematophagous Diptera (56 pools/936 specimens), and 2 from unengorged black flies (Simuliidae) (55 pools/1,221 specimens). Results suggest that nonblood-feeding insects, such as houseflies, play a role in VSV transmission and that black flies also serve as vectors.

Characterization of virulent isolates of vesicular stomatitis virus in relation to interference by defective particles

To investigate the role that defective interfering (DI) particles might conceivably play in the epizootiology of vesicular stomatitis, two virulent New Jersey (NJ) isolates from the 1982–1983 epizootic in the United States (US) were compared with three laboratory adapted strains of vesicular stomatitis virus (VSV): NJ Hazelhurst, NJ Ogden and Indiana San Juan. Successive undiluted passages showed that the virulent isolates did not readily exhibit ‘autointerference’ because they did not readily generate and amplify DI particles. Viral RNA synthesis of isolates that were exposed to homotypic or heterotypic DI particles generated from the laboratory strains showed that the isolates were totally resistant to the heterotypic DI particle and partially resistant to the homotypic DI particle. In contrast, Indiana San Juan and NJ Ogden were inhibited by hetero- or homotypic DI particles. NJ Hazelhurst more closely resembled the isolates. This demonstrates that virulence of VSV in its natural setting may be related to a number of factors, including the slower generation and amplification of endogenous DI particles, as well as the increased resistance of the virus to some pre-existing DI particles.

Interaction between di particles and recent virulent isolates of vesicular stomatitis virus (VSV)

Interaction between di particles and recent virulent isolates of vesicular stomatitis virus (VSV)

Conserved peptides in the proteins of vesicular stomatitis virus

The structural proteins of several isolates of the Indiana serotype (Orsay, Glasgow, and San Juan) of vesicular stomatitis virus (VSV) were compared by sizing peptides generated by digestion with Staphylococcus aureus V8 protease or α-chymotrypsin. These digestions yielded nearly identical peptide patterns for all the VSV proteins except for the glycoprotein, indicating that the major surface antigen, even of these closely related isolates, is subject to change. When two serologically distinct VSV isolates (Indiana and New Jersey) were similarly examined, each protein had a peptide pattern characteristic of that serotype. The glycoprotein of the two serotypes had no peptides in common, while the internal viral proteins L, N, NS, and M showed various degrees of similarity. Digests of the L (polymerase) and M (membrane) protein, in particular, generated groups of small peptides with identical mobilities, suggesting that there may be core regions in the Indiana and New Jersey strain which are conserved. These conserved regions may in turn represent essential functions of rhabdovirus proteins and are promising targets for broad-spectrum antivirals.

Interference and RNA homologies of New Jersey serotype isolates of vesicular stomatitis virus and their defective particles

Three New Jersey serotype isolates of vesicular stomatitis virus (VSV) were found to produce a population of defective interfering (DI) particles heterogeneous in size and in their RNAs. Only Ogden and Glasgow isolates produced DI particles containing a well-defined major RNA component. Annealing of Ogden mRNAs to Glasgow virion RNA revealed that the two isolates had approximately 24% homologies in their nucleotide sequences. One short DI particle, generated by the Glasgow isolate, contained only approximately 230 nucleotides homologous to the Ogden virion RNA. In spite of this lack of homology, the particle fully interfered with infections by Ogden virions. A previously described Indiana serotype DI particle (HR), which was shown to interfere heterotypically with Prevec's New Jersey virion, also contained only approximately 260 nucleotides homologous to the RNA of this isolate. None of the New Jersey DI particles exhibited the ability to fully interfere heterotypically with Indiana serotype virion infections. DI particles, generated by a given viral isolate, contained nucleotide sequences complementary to a part of their 30 S mRNAs, and the sequences of shorter particle RNAs were always contained in the RNA of the larger particles. In this respect, they resembled the nonheterotypically interfering DI particles of the Indiana serotype.