Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype)

The nucleotide sequence of the mRNA encoding the nucleocapsid protein of the New Jersey serotype (Ogden strain) of vesicular stomatitis virus (VSV) was determined from two overlapping cDNA clones spanning almost entirely the coding region of the mRNA. The 5'-terminal noncoding sequence present in the mRNA but not in the cDNA clones was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1329 nucleotides long (excluding polyadenylic acid) and encodes a protein of 422 amino acids. The nucleotide sequence was compared with the previously determined nucleotide sequence of the nucleocapsid protein of the Indiana serotype. An overall identity of 67.7% was found between the two serotypes. The only place where insertions and/or deletions have occurred during the evolution of the two viral genes from their presumed common ancestor is in the untranslated region. The nonidentical nucleotides are distributed throughout the length of the mRNA although not in an entirely random manner. The predicted amino acid sequence demonstrates that both proteins are initiated from the initiator codon located at the same distance from the 5' end (nucleotides 14 to 16) and contain the same number of amino acids. An overall identity of more than 80% of the amino acid sequence was observed between the two proteins when conservative replacements of amino acids were considered.     

Complete nucleotide sequence of the matrix protein mRNA of mumps virus

The complete nucleotide sequence of the mumps virus membrane protein or matrix protein (M) has been determined by sequencing cDNA clones and confirmed by partially sequencing the M mRNA and the genome. The mRNA is 1248 nucleotides long excluding the poly(A) and encodes a protein of 375 amino acids. The molecular weight (38,670), deduced from the amino acid sequence, is in agreement with the molecular weight of the viral M protein estimated by polyacrylamide gel electrophoresis (39-40 kDa). The mumps virus M protein shows 23-27% homology with M proteins of Newcastle disease virus (NDV), measles virus, canine distemper virus (CDV), parainfluenza virus type 3, and Sendai virus, respectively. A comparison of the M protein sequences of the above six paramyxoviruses did not reveal any conserved area of homology common among all paramyxovirus M proteins.     

Functional analysis of hypomethylation variants of the New Jersey serotype of vesicular stomatitis virus

The ts mutant F1 of vesicular stomatitis virus, New Jersey serotype, directs the synthesis of undermethylated 5'-terminal cap structures in vitro. In order to determine the relationship between the ts and hypomethylation phenotypes, a spontaneous revertant rev(ts)F1 of the ts phenotype was analyzed. The revertant retained the hypomethylation phenotype. The four cap structures (GpppA, 7mGpppA, GpppAm, and 7mGpppAm) synthesized in mutant and revertant-directed reactions in the presence of low as well as high concentrations of AdoMet were resolved by HPLC. Quantitation of the data and analysis of cap substrate to product ratios revealed that despite apparent similarities between the two hypomethylation phenotypes, the functional lesions in F1 and rev(ts)F1 were different. F1 displayed an AdoMet concentration-dependent alteration in the GpppA----GpppAm reaction and an AdoMet concentration-independent alteration in the GpppA----7mGpppA reaction. In contrast, rev(ts)F1 displayed AdoMet concentration-dependent alterations in the reactions GpppA----7mGpppA and GpppAm----7mGpppAm.     

Glycoprotein evolution of vesicular stomatitis virus New Jersey

A T1 ribonuclease fingerprinting study of a large number of virus isolates had previously demonstrated that considerable genetic variability existed among natural isolates of the vesicular stomatitis virus (VSV) New Jersey (NJ) serotype [S.T. Nichol (1988) J. Virol. 62, 572-579]. Based on these results, 34 virus isolates were chosen as representing the extent of genetic diversity within the VSV NJ serotype. We report the entire glycoprotein (G) gene nucleotide sequence and the deduced amino acid sequence for each of these viruses. Up to 19.8% G gene sequence differences could be seen among NJ serotype isolates. Analysis of the distribution of nucleotide substitutions relative to nucleotide codon position revealed that third position changes were distributed randomly throughout the gene. Third base changes constituted 84% of the observed nucleotide substitutions and affected 89% of the third base positions located in the G gene. Only three short oligonucleotide stretches of complete sequence conservation were observed. The remaining nucleotide changes located in the first and second positions were not distributed randomly, indicating that most of the amino acids coded by the G gene cannot be altered without reducing the fitness of the VSV NJ serotype viruses. Despite these constraints, up to 8.5% amino acid differences were observed between virus isolates. These differences were located throughout the G protein including regions adjacent to defined major antibody neutralization epitopes. Apparent clusters of amino acid substitutions were present in the hydrophobic signal sequence, transmembrane domain, and within the cytoplasmic domain of the G protein. A maximum parsimony analysis of the G gene nucleotide sequences allowed construction of a phylogram indicating the evolutionary relationship of these viruses. The VSV NJ serotype appears to contain at least three distinct lineages or subtypes. All recent virus isolates from the United States and Mexico are within subtype I and appear to have evolved from an ancestor more closely related to the Hazelhurst historic strain than other older strains. The implications of these findings for the evolution, epizootiology, and classification of these viruses are discussed.     

RNA synthesis by temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype.

We report the results of studies using temperature-sensitive mutants of Rous sarcoma virus (ts RSV) to study the alterations in surface proteins occurring on transformation. A large external transformation-sensitive (LETS) protein is detected by lactoperoxidase-catalyzed iodination on normal chicken embryo fibroblasts (CEF) but is reduced on RSV-transformed cells. The LETS protein is temperature sensitive in ts RSV-infected CEF and the kinetics of the alterations occurring on shifts between permissive and restrictive temperature are reported. During reversion to normality on shift-up, the LETS protein appears at the surface quite rapidly and in parallel with the morphological changes, although the change is less rapid than that in glucose transport. On transformation during shift-down, the disappearance of the LETS protein lags behind the morphological change. Cycloheximide does not inhibit reappearance of iodinatable LETS protein on shift-up, suggesting that the LETS protein is synthesized in transformed cells, although it is not present at the surface. Hypotheses to explain the absence of the LETS protein at the surface are discussed and evidence is presented for an increased rate of turnover after transformation.     

Effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

The conformational epitopes reactive with neutralizing monoclonal antibodies (MAbs) appear to be clustered at the middle third of the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) and are flanked by two N-linked carbohydrate chains (W. Keil and R.R. Wagner, Virology 170, 392-407, 1989). We report here studies on the effect of glycosylation on the reactivity of VSV-NJ G protein derived from released virions or immunoprecipitated from pulse-labeled cells was not significantly affected in its reactivity with MAbs directed to epitope IV mapped toward the amino-terminus, nor to the centrally located conformational epitopes VI, VIII, and IX. However, there was a 5- to 15-fold decrease in the reactivity with MAb of epitopes VI, VIII, and IX on unglycosylated G protein either isolated from a ribosome-enriched membrane fraction or immunoprecipitated from whole VSV-infected cells labeled for 15 hr in the presence of tunicamycin. In sharp contrast, epitope V and to a somewhat lesser extent epitope VII exhibited decreased reactivity with their respective MAbs when unglycosylated G protein was isolated from released viral particles or from pulse-labeled cells infected with VSV-NJ in the presence of tunicamycin. Enzymatic removal of preformed carbohydrate chains with N-glycanase had little or no effect on the MAb-reactivity of epitopes V and VII, indicating that the carbohydrate chains per se do not influence the antigenic specificity of VSV-NJ G protein. These data suggest that the formation of N-linked carbohydrate chains influences the structure of the VSV-NJ G protein in such a way that epitopes V and VII are shielded from reactivity with their specific MAbs from an early stage of G-protein processing and to a much lesser extent epitopes VI, VIII, and IX at late stages of intracellular processing. These results are compatible with, but do not prove, the hypothesis that N-linked glycosylation plays a key role in promoting the formation and the stability of the disulfide bonds that determine the epitope-specific conformational integrity of the VSV-NJ glycoprotein.     

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (New Jersey serotype): a method for preliminary mapping of epitopes

Of the nine antigenic determinants on the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV) identified by competitive binding of 25 monoclonal antibodies (MAbs), those relegated to epitopes I, II, III, and IV exhibited no significant ability to neutralize virus infectivity but some nonneutralizing MAbs cross-reacted by ELISA with the G protein of VSV-Indiana. High-titered neutralization of homotypic virus was exhibited by epitope V, VI, and VII MAbs but quite variable neutralizing activity was found among MAbs of epitope "family" VIII and particularly the heterogeneous epitope "family" IX. Peptide mapping of the epitopes was not feasible because most MAbs would not bind by Western blotting to G protein under standard conditions of proteolysis or disulfide bond reduction. Therefore, a technique was devised for roughly locating epitopes by protease footprinting of G protein partially protected by individual MAbs complexed with staphylococcal protein A-Sepharose beads. Under these conditions, MAbs to all nine epitopes protected a similar 12-kDa fragment of the G protein from proteolysis by Staphylococcus aureus V8 protease. N-Terminal amino acid sequencing mapped two of these 12-kDa peptide footprints to a position on the G protein extending from amino acid 219 to about 100 amino acids downstream. Although MAbs to only one epitope bound to the 12-kDa fragment by Western blotting, these data suggest, but do not prove, that all nine epitopes of the undenatured VSV-New Jersey G protein are clustered at the middle 20% of a highly structured protein. This method may help to identify the general regions for epitopes on complex proteins of as yet unknown three-dimensional structure.     

Bite transmission of vesicular stomatitis virus (New Jersey serotype) to laboratory mice by Simulium vittatum (Diptera: Simuliidae).

Laboratory-reared female black flies (Simulium vittatum Zetterstedt) were infected experimentally with a 1997 vesicular stomatitis virus New Jersey serotype isolate and allowed to feed on susceptible laboratory mice. All mice exposed to black fly bite seroconverted by day 21 after infection, an indication of virus transmission. In addition, viral RNA was detected in the spleen of several mice. These findings are consistent with the hypothesis that black flies are involved in VSV-NJ transmission during epizootics in the western USA and represent the 1st confirmed example of biological transmission of an arbovirus by a member of the Simuliidae using an animal model.     

Interactions of plus and minus strand leader RNAs of the New Jersey serotype of vesicular stomatitis virus with the cellular La protein

The New Jersey serotype of vesicular stomatitis virus (VSV-NJ) was found to synthesize a minus strand leader RNA of 44-46 bases long and a plus strand leader RNA of 47-50 bases long in infected cells. The minus strand leader RNA of VSV-NJ was found associated with the host cell La protein in infected cells by immunoprecipitation with antisera from patients with systemic lupus erythematosus. These results differ from those reported previously (J. Wilusz , M. G. Kurilla , and J. D. Keene (1983). Proc. Natl. Acad. Sci. USA 80, 5827-5831) for the similarly sized species of minus strand leader RNA made by the Indiana serotype of VSV (VSV-IND). Despite sequence differences between the 3' ends of the plus strand leader RNAs of the two serotypes, the plus strand leader RNA of VSV-NJ was found to have a pattern of La protein accumulation similar to that reported previously for the plus strand leader RNA of VSV-IND. These results provide additional support for a role for La protein in VSV replication and help further delineate the sequence requirements for La protein binding to VSV leader RNAs.     

Interference and RNA homologies of New Jersey serotype isolates 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.     

1995 epizootic of vesicular stomatitis (New Jersey serotype) in the western United States: an entomologic perspective

Entomologic and epizootic data are reviewed concerning the potential for transmission of vesicular stomatitis (VS) virus by insects, including field data from case-positive premises in New Mexico and Colorado during the 1995 outbreak of the New Jersey serotype (VSNJ). As with previous outbreaks of VSNJ in the western United States, the 1995 epizootic illustrated that risk of exposure is seasonal, increasing during warm weather and decreasing with onset of cool weather; virus activity spread from south to north along river valleys of the southwestern and Rocky Mountain states; clinical disease was detected most commonly in horses, but also occurred in cattle and 1 llama; and most infections were subclinical. Overall, 367 case-positive premises were identified during the 1995 outbreak, with foci of virus activity along the Rio Grande River south of Albuquerque, NM, in southwestern Colorado, and along the Colorado River near Grand Junction, CO. The establishment of a 16-km (10-mile) radius zone of restricted animal movement around confirmed positive premises, along with imposition of state and international embargoes, created economic hardship for livestock owners and producers. The importance of defining the role of blood-feeding insects as biological vectors of VSNJ virus relative to risk factors that promote high levels of insect transmission, such as the presence of livestock along western river valleys, blood feeding activity, and frequent transport of animals for recreational purposes, is emphasized as a basis for developing effective disease management.     

Biological transmission of vesicular stomatitis virus (New Jersey serotype) by Simulium vittatum (Diptera: Simuliidae) to domestic swine (Sus scrofa)

The role of hematophagous arthropods in vesicular stomatitis virus (New Jersey serotype; VSV-NJ) transmission during epizootics has remained unclear for decades in part because it has never been shown that clinical or subclinical disease in a livestock host results from the bite of an infected insect. In this study, we investigated the ability of VSV-NJ-infected black flies (Simulium vittatum Zetterstedt) to transmit the virus to domestic swine, Sus scrofa L. Experimental evidence presented here clearly demonstrates that VSV-NJ was transmitted from black flies to the swine. Transmission was confirmed by seroconversion or by the presence of clinical vesicular stomatitis followed by seroconversion. Our results represent the first report of clinical vesicular stomatitis in a livestock host after virus transmission by an insect.