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Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells., (© 2020 Wiley Periodicals LLC.)

The phosphoprotein (P) of the nonsegmented negative-sense RNA viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (P MD ), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, P MD , for viral gene expression and virus growth. A truncated P protein lacking the central dimerization domain (P ΔMD ) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type virus, showing that the dimerization domain of P is dispensable for viral gene expression and virus replication in cell culture. Because RNA viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new viruses. IMPORTANCE The phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA viruses, including some major human pathogens (e.g., rabies virus, measles virus, respiratory syncytial virus [RSV], Ebola virus, and Nipah virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different viruses and, in some cases, hijacks cell components to the advantage of the virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA viruses., (Copyright © 2020 American Society for Microbiology.)

Viral vectors derived from vesicular stomatitis virus (VSV) are important vectors for the development of vaccines and for the treatment of cancer. The efficiency of therapy based on VSV is dependent on the dose of virus used. Therefore it is essential to measure accurately and reproducibly the amount of functional vectors in the samples to be tested. Two common methods used to measure the titer of VSV are TCID 50% and plaque assay. In the current study, we compared these two titration methods by using a recombinant VSV expressing the green fluorescent protein (VSV-GFP) as a model virus. Some culture media developed for suspension mammalian cells contain dextran sulfate. We observed that plaque assay, but not TCID 50% , can underestimate the virus titer up to 10 fold when VSV-GFP was produced in culture media containing dextran sulfate. Dextran sulfate is commonly used in serum-free culture media to reduce cell aggregation in suspension culture. The inhibitory effect of dextran sulfate on the titration of VSV-GFP was confirmed by supplementing the culture medium with this compound during virus production. Our results also demonstrated that extending the incubation time during plaque assay and TCID 50% increases virus titer., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Developments in genetic engineering have allowed researchers and clinicians to begin harnessing viruses to target and kill cancer cells, either through direct lysis or through recruitment of antiviral immune responses. Two powerful viruses in the fight against cancer are the single-stranded RNA viruses vesicular stomatitis virus and Zika virus. Here, we describe methods to propagate and titer these two viruses. We also describe a simple cell-killing assay to begin testing modified viruses for increased potential killing of glioblastoma cells.

During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif-Glu169, Phe171, and Leu174-to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid. IMPORTANCE During viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis., (Copyright © 2018 American Society for Microbiology.)

The matrix protein of vesicular stomatitis virus (VSV) performs multiple functions during viral genome replication and virion production and is involved in modulating multiple host signaling pathways that favor virus replication. To perform numerous functions within infected cells, the M protein needs to recruit cellular partners. To better understand the role of M during VSV replication, we looked for interacting partners by using the two-hybrid system. The eukaryotic translation initiation factor 3, subunit i (eIF3i) was identified to be an M-binding partner, and this interaction was validated by GST pull-down and laser confocal assays. Through a mutagenesis analysis, we found that some mutants of M between amino acids 122 and 181 impaired but did not completely abolish the M-eIF3i interaction. Furthermore, the knockdown of eIF3i by RNA interference decreased viral replication and transcription in the early stages but led to increase in later stages. VSV transcription was increased at 4h post-infection but was not changed at 8 and 12h post-infection after the over-expression of eIF3i. Finally, we also demonstrated that VSV could inhibit the activity of Akt1 and that the knockdown of eIF3i inhibited the expression of the ISGs regulated by phospho-Akt1. These results indicated that eIF3i may affect VSV growth by regulating the host antiviral response in HeLa cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Unlabelled: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high case fatality rate caused by SFTS virus (SFTSV). Effective vaccines and specific therapies for SFTS are urgently sought, and investigation into virus-host cell interactions is expected to contribute to the development of antiviral strategies. In this study, we have developed a pseudotype vesicular stomatitis virus (VSV) bearing the unmodified Gn/Gc glycoproteins (GPs) of SFTSV (SFTSVpv). We have analyzed the host cell entry of this pseudotype virus and native SFTSV. Both SFTSVpv and SFTSV exhibited high infectivity in various mammalian cell lines. The use of lysosomotropic agents indicated that virus entry occurred via pH-dependent endocytosis. SFTSVpv and SFTSV infectivity was neutralized by serial dilutions of convalescent-phase patient sera. Entry of SFTSVpv and growth of SFTSV were increased in Raji cells expressing not only the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) but also DC-SIGN-related (DC-SIGNR) and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). 25-Hydroxycholesterol (25HC), a soluble oxysterol metabolite, inhibited the cell entry of SFTSVpv and the membrane fusion of SFTSV. These results indicate that pH-dependent endocytosis of SFTSVpv and SFTSV is enhanced by attachment to certain C-type lectins. SFTSVpv is an appropriate model for the investigation of SFTSV-GP-mediated cell entry and virus neutralization at lower biosafety levels. Furthermore, 25HC may represent a potential antiviral agent against SFTS., Importance: SFTSV is a recently discovered bunyavirus associated with SFTS, a viral hemorrhagic fever with a high case fatality rate endemic to China, South Korea, and Japan. Because little is known about the characteristics of the envelope protein and entry mechanisms of SFTSV, further studies will be required for the development of a vaccine or effective therapies. In this study, we investigated the mechanism of SFTSV cell entry using SFTSVpv and the native virus. SFTSV can grow in nonsusceptible cell lines in the presence of certain C-type lectins. Moreover, 25HC, an oxysterol metabolite, may represent a potential therapeutic inhibitor of SFTSV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-β and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-β (boIFN-β; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-β and demonstrate the activation of a general antiviral state after EHMT2 inhibition.

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface., (Copyright © 2015, Drokhlyansky et al.)

Non-segmented negative strand RNA viruses belonging to the Mononegavirales order possess RNA-dependent RNA polymerase L proteins within viral particles. The L protein is a multifunctional enzyme catalyzing viral RNA synthesis and processing (i.e., mRNA capping, cap methylation, and polyadenylation). Using vesicular stomatitis virus (VSV) as a prototypic model virus, we have shown that the L protein catalyzes the unconventional mRNA capping reaction, which is strikingly different from the eukaryotic reaction. Furthermore, co-transcriptional pre-mRNA capping with the VSV L protein was found to be required for accurate stop?start transcription to synthesize full-length mRNAs in vitro and virus propagation in host cells. This article provides a review of historical and present studies leading to the elucidation of the molecular mechanism of VSV mRNA capping.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Technology for producing biologically active recombinant alpha2b-interferon is based on creating a bacterial producer strain containing the cDNA of human interferon alpha. The authors have obtained two producers of recombinant alpha2b-interferon, the synthesis of the target protein in them occurs in the inclusion bodies. The schemes of isolation and purification of biologically active recombinant alpha2b-interferon have been developed. The drug purity was approximately 97-98%. Biological activity in the culture of sensory cells in the cytopathic test was 4.2*10(8)ME/mg.

Innate immunity to viruses involves receptors such as Retinoic Acid Induced Gene-1 (RIG-I), which senses viral RNA and triggers a signaling pathway involving the outer mitochondrial membrane protein mitochondrial antiviral signaling (MAVS). Recent work has identified that NLRX1, a member of another class of innate immune receptors, sequesters MAVS away from RIG-I and thereby prevents mitochondrial antiviral immunity. In this study, we demonstrate that the proteasome PSMA7 (alpha4) subunit associates with MAVS in vivo and in vitro. Expression of PSMA7 results in a potent inhibition of RIG-1 and MAVS-mediated IFN-beta promoter activity; conversely, depletion of PSMA7 with small interference RNA enhances virus-induced type I IFN production, with consequent reduction of virus replication. Furthermore, a striking reduction in the abundance of endogenous MAVS with overexpressed PSMA7 was found and virus infection leads to transient increase in the endogenous PSMA7 protein level. Cumulatively, these results suggest that PSMA7 is a negative regulator of the MAVS-mediated innate immunity that probably serves to attenuate the establishment of an antiviral state during viral infection, highlighting the biological significance of PSMA7-MAVS association as an important cellular regulatory control.

Overexpression of myeloid cell leukemia 1 protein (Mcl-1), an anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member, contributes to chemotherapy resistance of tumors. The short half-life of Mcl-1 makes it an interesting target for therapeutic agents that negatively interfere with cellular protein biosynthesis, such as oncolytic viruses. Vesicular Stomatitis Virus (VSV) has been established as the oncolytic virus that efficiently disrupts de novo protein biosynthesis of infected cells. Here, we show that after VSV infection, Mcl-1 protein levels rapidly declined, whereas the expression of other members of the Bcl-2 family remained unchanged. Mcl-1 elimination was a consequence of proteasomal degradation, as overexpression of a degradation-resistant Mcl-1 mutant restored Mcl-1 levels. Mcl-1 rescue inhibited apoptosis and thereby confirmed that Mcl-1 downregulation contributes to VSV-induced apoptosis. In vitro, VSV virotherapy in combination with chemotherapy revealed an enhanced therapeutic effect compared with the single treatments, which could be reverted by Mcl-1 rescue or RNA interference (RNAi)-mediated knockdown of pro-apoptotic Bax and Bak proteins. Finally, in a tumor mouse model, combinations of doxorubicin and VSV showed a superior therapeutic efficacy compared with VSV or doxorubicin alone. In summary, our data indicate that VSV virotherapy is an attractive strategy to overcome tumor resistance against conventional chemotherapy by elimination of Mcl-1.

The plaque assay has long served as the "gold standard" to measure virus infectivity and test antiviral drugs, but the assay is labor-intensive, lacks sensitivity, uses excessive reagents, and is hard to automate. Recent modification of the assay to exploit flow-enhanced virus spread with quantitative imaging has increased its sensitivity. Here we performed flow-enhanced infection assays in microscale channels, employing passive fluid pumping to inoculate cell monolayers with virus and drive infection spread. Our test of an antiviral drug (5-fluorouracil) against vesicular stomatitis virus infections of BHK cell monolayers yielded a two-fold improvement in sensitivity, relative to the standard assay based on plaque counting. The reduction in scale, simplified fluid handling, image-based quantification, and higher assay sensitivity will enable infection measurements for high-throughput drug screening, sero-conversion testing, and patient-specific diagnosis of viral infections.

Defective interfering particles (DIPs) are virus-like particles that arise during virus growth, fail to grow in the absence of virus, and replicate at the expense of virus during co-infections. The inhibitory effects of DIPs on virus growth are well established, but little is known about how DIPs influence their own growth. Here vesicular stomatitis virus (VSV) and its DIPs were used to co-infect BHK cells, and the effect of DIP dose on virus and DIP production was measured using a yield-reduction assay. The resulting dose-response data were used to fit and evaluate mathematical models that employed different assumptions. Our analysis supports a multiple-hit process where DIPs inhibit or promote virus and DIP production, depending on dose. Specifically, three regimes of co-infection were apparent: (i) low DIP - where both virus and DIPs are amplified, (ii) medium DIP - where amplification of both virus and DIPs is inhibited, and (iii) high DIP - with limited recovery of virus production and further inhibition of DIP growth. In addition, serial-passage infections enabled us to estimate the frequency of de novo DIP generation during virus amplification. Our combined experiments and models provide a means to understand better how DIPs quantitatively impact the growth of viruses and the spread of their infections.

Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.

Given the lethality of H5N1 avian influenza viruses (AIV) and the recurring spread from poultry to humans, an effective vaccine against H5N1 viruses may be needed to prevent a pandemic. We generated experimental vaccine vectors based on recombinant vesicular stomatitis virus (VSV) expressing the H5 hemagglutinin (HA) from an H5N1 virus isolated in 1997. The HA gene was expressed either from an attenuated wild-type VSV vector or from a single-cycle vector containing a deletion of the VSV G gene. We found that all of the vectors induced potent neutralizing antibody titers against the homologous and antigenically heterologous H5N1 viruses isolated in 2004 and 2005. Vaccination of mice with any combination of prime or prime/boost vectors provided long-lasting protection (>7 months) against challenge with AIV, even in animals receiving a single dose of single-cycle vaccine. Our data indicate that these recombinants are promising vaccine candidates for pandemic influenza.

Sinefungin (SIN), a natural S-adenosyl-L-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2'-O (2'-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2'-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.

A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD(50)) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD(50) of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

In a companion paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:492-502, 2007), we provided indirect evidence that cell-type-specific growth restriction of the vesicular stomatitis virus (VSV) polR mutants may be due to enhanced production of double-stranded RNA (dsRNA). We show here that polR growth in mouse L-929 cells was rescued by vaccinia virus coinfection and that sole expression of the vaccinia virus dsRNA-binding E3L protein, via coinfection with an engineered VSV minigenome, also restored polR growth. Expression of dsRNA-binding protein NS1A or NS1B from influenza virus, but not C protein from Sendai virus, which does not bind dsRNA, likewise effected polR rescue. The N-terminal dsRNA-binding domain of NS1A, only 73 amino acids in length, but not a full-size mutant NS1A lacking dsRNA-binding activity, restored polR growth. Both key aspects of polR growth restriction, namely inhibition of genome replication and release of low-infectivity virus particles, were countered by expression of the dsRNA-binding proteins. We tested the effects of overproducing dsRNA in wild-type VSV infections by coinfecting cells with a VSV recombinant expressing the sense strand of the enhanced green fluorescent protein gene (VSV-GFP) and one expressing the antisense strand (VSV-PFG). These coinfections mimicked all aspects of polR restriction, including host range, lack of effect on transcription, reduced virus particle infectivity, and insensitivity to inhibition of host gene transcription or dsRNA-activated protein kinase activity. We conclude that, for some cell types, overproduction of dsRNA during VSV infection triggers an immediate and constitutive host cell antiviral effector response independent of interferon induction or signaling.

Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.

Pharmacologic inhibition of the proteasome resulted in increased NOS-1 protein levels and increased NO production by neuronal cells. This correlated with an increased antiviral effect of IFN-gamma against the replication of vesicular stomatitis virus (VSV) replication in vitro. We also observed that a regulatory protein, Protein Inhibitor of NOS-1 (PIN) was down-regulated by IFN-gamma treatment, and more ubiquitinated PIN accumulated in IFN-gamma treated neurons. In cells of the reticuloendothelial system, IFN-gamma treatment induces the expression of a set of low molecular weight MHC-encoded proteins (LMPs), which replace the beta-subunit of the proteasome complex during the proteasome neosynthesis, resulting in a complex termed the immunoproteasome. LMP2, -7, and -10 were induced and the immunoproteasome was generated by IFN-gamma treatment in neuronal cells. Importantly, we observed that IFN-gamma induced inhibition of VSV protein synthesis was not dependent on ubiquitination.

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (approximately 30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.

Background: Virotherapy represents a novel therapeutic modality for the treatment of malignant diseases. Vesicular stomatitis virus (VSV) has been shown to exert antitumor effect in several tumor types. Since the potential oncolytic activity of VSV has not yet been evaluated in epithelial tumors of the conjunctiva, we set out to investigate the susceptibility of the immortalized Wong-Kilbourne derivative of the Chang conjunctival cell line (WK) to VSV and analyze the role of apoptosis in VSV-mediated induction of cell death., Methods: WK cells were infected with VSV at various multiplicities and maintained for different periods of time. VSV-infected cells were analyzed by inverted microscopy for the development of cytopathic effects (CPE). Virus replication was measured by indirect immunofluorescence assay, Western blot analysis and plaque titration. The apoptotic response of the infected cells was quantitated by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of Bcl-2 and Bax proteins., Results: The WK cell line was highly permissive to VSV replication and was highly susceptible for the CPE of this virus. VSV infection elicited the apoptotic death of WK cells. Mock-infected cells exhibited endogenous expression of Bcl-2 and p21 Bax proteins. VSV infection caused a significant decrease in the expression level of Bcl-2. Moreover, in parallel with a slight decrease in the level of p21 Bax, p18 Bax protein accumulated in VSV-infected WK cells., Conclusions: VSV is a powerful inducer of apoptosis in immortalized WK cells. The VSV-mediated alterations in the expressions of Bcl-2 and Bax proteins may play important roles in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. This virus may possess oncolytic activity in epithelial tumors of the conjunctiva.

We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.

A quantitative understanding of the innate immune response will enable its recruitment against emerging, poorly characterized, or weaponized viral pathogens. To gain insights into how the innate responses can limit viral spread, we used quantitative focal infections to study how the spread of recombinant vesicular stomatitis viruses (VSV) on baby hamster kidney (BHK) and delayed brain tumor (DBT) cell monolayers is affected by innate cellular antiviral responses. We observed that rates of infection spread correlated with one-step growth rankings for four ectopic VSV strains: N1, N2, N3, and N4. However, this correlation was lost for M51R, a recombinant VSV mutant that lacks the ability to shut-off host gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1, even though their one-step growth was comparable. In DBT cells, M51R infections failed to spread beyond the site of inoculation. Addition of anti-interferon antibody restored M51R spread and one-step growth to wild-type levels. Interestingly, the antibody enhanced the spread of wild-type virus but not its growth. These results suggest that while the rate of viral spread generally correlates with the rate of viral growth, the induction of cellular antiviral activities can be in some cases, the overriding factor in both spread and growth. In summary, focal infections enabled us to visualize and quantify how viral spread was inhibited by cellular antiviral activities. This study demonstrates a mechanism for quantifying how innate cellular responses can mitigate infection spread in vitro.

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

To determine the vector competence of Culicoides sonorensis Wirth & Jones midges for vesicular stomatitis virus (VSV)-New Jersey, insects were experimentally infected per os and sampled over time. Viral replication, as determined by in situ hybridization, was seen in epithelial, neural, and hemolymph cell types throughout the insect. Spatial and temporal distribution of virus was determined by immunohistochemical examination of sequentially sampled insects. Tissues of the alimentary canal were infected in a temporal pattern that paralleled the route of digestion/absorption: foregut and midgut by day 1, surrounding hemolymph and Malpighian tubules by day 3, and finally the midgut/ hindgut junction, hindgut, and rectal region by day 5. The circulation of virus in the hemolymph by day 3 coincided with infection of the dermis and fat bodies, the salivary glands, eyes, cerebral and subthoracic ganglia, and the ovaries. Oviduct epithelium and ovarial sheaths were infected by day 3, followed by infection of the developing oocytes by day 5. Interestingly, neural infections were seen in the subabdominal ganglia innervating the midgut in 33% of insects by 1 d postfeeding in the absence of positive staining in the hemolymph or surrounding tissues. A retrograde axonal transport infection route for these ganglia is discussed. The disseminated, productive, noncytolytic infection in Culicoides is consistent with that of an efficient biological vector for VSV. Virus readily replicated throughout the insect, passing both midgut and salivary gland infection barriers and reaching transmission-related organs in 3 d. Establishing the competence of this insect vector for VSV provides the foundation for animal transmission studies in the future. The possibility of horizontal, transovarial, and mechanical transmission is discussed.

Influenza C virus contains two envelope glycoproteins: CM2, a putative ion channel protein; and HEF, a unique multifunctional protein that performs receptor-binding, receptor-destroying and fusion activities. Here, it is demonstrated that expression of HEF is sufficient to pseudotype replication-incompetent vesicular stomatitis virus (VSV) that lacks the VSV glycoprotein (G) gene. The pseudotyped virus showed characteristic features of influenza C virus with respect to proteolytic activation, receptor usage and cell tropism. Chimeric glycoproteins composed of HEF ectodomain and VSV-G C-terminal domains were efficiently incorporated into VSV particles and showed receptor-binding and receptor-destroying activities but, unlike authentic HEF, did not mediate efficient infection, probably because of impaired fusion activity. HEF-pseudotyped VSV efficiently infected polarized Madin-Darby canine kidney cells via the apical plasma membrane, whereas entry of VSV-G-complemented virus was restricted to the basolateral membrane. These findings suggest that pseudotyping of viral vectors with HEF might be useful for efficient apical gene transfer into polarized epithelial cells and for targeting cells that express 9-O-acetylated sialic acids.

Due to their extremely high genetic diversity, which is a direct consequence of high mutation rates, RNA viruses are often described as molecular quasispecies. According to this theory, RNA virus populations cannot be understood in terms of individual viral clones, as they are clouds of interconnected mutants, but this prediction has not yet been demonstrated experimentally. The goal of this study was to determine the fitness of individual clones sampled from a given RNA virus population, a necessary previous step to test the above prediction. To do so, limiting dilutions of a vesicular stomatitis virus population were employed to isolate single viral clones and their initial growth dynamics were followed, corresponding to the release of the first few hundred viral particles. This technique is useful for estimating basic fitness parameters, such as intracellular growth rate, viral yield per cell, rate at which cells are infected and time spent in cell-to-cell transmission. A combination of these parameters allows estimation of the fitness of individual clones, which seems to be determined mainly by their ability to complete infection cycles more quickly. Interestingly, fitness was systematically higher for initial clones than for their derived populations. In addition to environmental changes, such as cellular defence mechanisms, these differences are attributable to high RNA virus mutation rates.

A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxy-terminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus recovery, kinetics of growth, and mRNA and protein synthesis were examined. We observed that virus recovery was completely abolished when all three phosphate acceptor sites in domain I or both sites in domain II were replaced with alanine. Single or double mutations in domain I (with the exception of P60/64) or single mutations in domain II had no adverse effect on virus recovery. VSVP227, carrying alanine at position 227, showed reduced kinetics of virus growth but increased kinetics of viral mRNA synthesis in infected cells. More interestingly, this particular virus exhibited a significantly reduced cytopathic effects and apoptosis in infected cells, implying that P may be involved in these processes. Furthermore, we found that DI RNAs of different sizes were generated by high-multiplicity passaging of various mutant VSVs, indicating that the viral RdRp may play a significant role in the process of DI particle generation. Taken together, our results suggest that the phosphorylation of residues in domains I and II of VSV P is indispensable for virus growth.

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.

Previously, it has been shown that 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a natural compound isolated from leaf extracts of Melia azedarach L., inhibits the vesicular stomatitis virus (VSV) multiplication cycle when added before or after infection. Here, we have established that the lack of VSV protein synthesis in CDM pre-treated Vero cells is ascribed to the inhibition of an initial step during virus multiplication, although indirect immunofluorescence (IFI) studies confirmed that the binding and uptake of [(35)S]methionine-labelled VSV was not affected by CDM pre-treatment. Instead, our findings revealed that this compound impedes the uncoating of VSV nucleocapsids in pre-treated Vero cells, since the antiviral action of CDM was partially reversed by inducing VSV direct fusion at the plasma membrane, and VSV M protein fluorescence was confined to the endosomes, even 2 h post-internalization. Furthermore, CDM induced cytoplasmic alkalinization, as shown by acridine orange staining, consistent with the inhibition of virus uncoating. Although VSV proteins are synthesized when CDM is added after infection, IFI studies revealed that G protein was absent from the surface of infected cells and co-localized with a Golgi marker. Therefore, CDM inhibits the transport of G protein to the plasma membrane. Taken together, these findings indicate that CDM exerts its antiviral action on the endocytic and exocytic pathways of VSV by pre- or post-treatment, respectively.

Vesicular stomatitis (VS) is an economically devastating disease of livestock in the Americas. Despite strong circumstantial evidence for the role of arthropods in epizootics, no hematophagous vector explains the field evidence. Based on the spatiotemporal association of grasshopper outbreaks and VS epizootics, we investigated the potential role of these insects as vectors and reservoirs of the disease. The critical steps in the grasshopper-bovine transmission cycle were demonstrated, including 1) 62% of grasshoppers [Melanoplus sanguinipes (F.)] fed vesicular stomatitis virus (VSV) from cell culture became infected, with titers reaching 40,000 times the inoculative dose; 2) 40% of grasshoppers that cannibalized VSV-infected grasshopper cadavers became infected, amplifying virus up to 1,000-fold; 3) one of three cattle consuming VSV-infected grasshopper cadavers contracted typical VS and shed virus in saliva; and 4) 15% of grasshoppers became infected when fed saliva from this infected cow. The ecological conditions and biological processes necessary for these transmissions to occur are present throughout much of the Americas. Field studies will be required to show these findings are relevant to the natural epidemiology of VSV.

This study examines apoptosis and viral neuropathogenesis in a murine model infected with vesicular stomatitis virus (VSV). VSV induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells may contribute to disease and mortality. To determine whether or not VSV induces apoptosis in neural tissues, mice were inoculated intranasally with VSV. At 24, 48, 72, 96, and 120 hours postinfection, brain tissues were assayed for the presence of viral RNA by in situ hybridization and viral antigen by immunohistochemistry. Apoptosis was identified by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and electron microscopy. Viral replication and lesions were observed predominantly in central nervous system neurons. Apoptotic cell death was restricted to the same regions of the brain in which infected cells and tissue injury were identified. Results suggest that VSV-induced apoptosis is a mechanism causing cell death, tissue injury, and mortality in VSV-infected mice.

The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components.

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes., (Copyright 2003 Wiley Periodicals Inc. Biotechnol Bioeng 81: 719-725, 2003.)

In this report, the role of STAT4 and local production of interleukin (IL)-12 in the central nervous system (CNS) were examined during experimental vesicular stomatitis virus (VSV) encephalitis. We have previously shown that IL-12 treatment is beneficial both in vitro and in vivo during experimental VSV infection. This inhibition of VSV replication was dependent on the production of nitric oxide (NO) by the neuronal isoform of nitric oxide synthase (NOS-1). In vitro, IL-12 induces the phosphorylation and nuclear localization of STAT4 in neuroblastoma cell lines. STAT4 expression was not required for host survival or clearance of virus during experimental VSV encephalitis. Taken together, these data suggest that while neurons can respond directly to IL-12 in vitro by signaling through STAT4, STAT4 is not required for survival. It is likely that redundant innate host inflammatory cytokine responses compensate for the absence of IL-12 signaling.

Alternative splicing leads to the expression of multiple isoforms of the subunits (IFNAR1 and IFNAR2) of the type I IFN receptor. Here we describe two transcripts representing extracellular forms of ovine IFNAR1 and show that soluble extracellular forms of both IFNAR2 and IFNAR1, prepared in recombinant form in Escherichia coli, have antiviral (AV) activity in the absence of IFN. Exposure of Madin-Darby bovine kidney cells to the extracellular domain (R2E) of IFNAR2 at concentrations as low as 10 nM afforded complete protection against vesicular stomatitis virus and led to the rapid activation of the transcription factors ISGF3 and GAF. Although R2E can bind IFN (K(d) approximately 70 nM), activity was observed irrespective of whether or not ligand was present. R2E was inactive on mouse L929 cells but active on L929 cells expressing a membraneanchored, ovine/human chimeric IFNAR2 with an ovine extracellular domain. The data suggest that AV activity is conferred by the ability of soluble R2E to associate with the transfected IFNAR2 subunit rather than resident murine IFNAR1. Soluble extracellular forms of IFNAR1 have lower AV activity than R2E on Madin-Darby bovine kidney cells but are less species-specific and protect wild-type L929 cells as efficiently as the transfected cell line, presumably by interacting with one of the murine receptor subunits.

Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3'-N-P-M-G-L-5', and transcription is obligatorily sequential from the single 3' promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3'-G-N-P-M-L-5' (G1N2), 3'-P-M-G-N-L-5' (G3N4), and 3'-G-P-M-N-L-5' (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

The effects of three representative disinfectants, chlorine (sodium hypochlorite), iodine (potassium tetraglicine triiodide), and quaternary ammonium compound (didecyldimethylammonium chloride), on several exotic disease viruses were examined. The viruses used were four enveloped viruses (vesicular stomatitis virus, African swine fever virus, equine viral arteritis virus, and porcine reproductive and respiratory syndrome virus) and two non-enveloped viruses (swine vesicular disease virus (SVDV) and African horse sickness virus (AHSV)). Chlorine was effective against all viruses except SVDV at concentrations of 0.03% to 0.0075%, and a dose response was observed. Iodine was very effective against all viruses at concentrations of 0.015% to 0.0075%, but a dose response was not observed. Quaternary ammonium compound was very effective in low concentration of 0.003% against four enveloped viruses and AHSV, but it was only effective against SVDV with 0.05% NaOH. Electron microscopic observation revealed the probable mechanism of each disinfectant. Chlorine caused complete degeneration of the viral particles and also destroyed the nucleic acid of the viruses. Iodine destroyed mainly the inner components including nucleic acid of the viruses. Quaternary ammonium compound induced detachment of the envelope of the enveloped viruses and formation of micelle in non-enveloped viruses. According to these results, chlorine and iodine disinfectants were quite effective against most of the viruses used at adequately high concentration. The effective concentration of quaternary ammonium compound was the lowest among the disinfectants examined.

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.

The role of nitric oxide synthase (NOS) in host defense and clearance of vesicular stomatitis virus (VSV) from the central nervous system (CNS) was examined. NOS-1, NOS-2, and NOS-3 knockout mice were infected with VSV and were treated with either IL-12 or medium. IL-12 treatment resulted in substantially decreased VSV titers in wildtype and NOS-3 knockout mice, but had a marginal effect in the NOS-1 and NOS-2 knockout mice. NOS-1 expression in neurons was associated with survival from VSV infection. The data indicate that the enzyme activity is local, since NOS-2 expression in microglia and inflammatory macrophages and NOS-3 expression in astrocytes, endothelial cells, and ependymal cells did not compensate., (Copyright 1999 Academic Press.)

Sheep thyroid cells in primary culture are highly sensitive to thyroid stimulating hormone (TSH). We infected thyroid cells with vesicular stomatitis virus (VSV) in the course of studies on cell polarity, and we found that TSH augmented the speed of the replicative cycle of VSV but did not affect the final yield of the virus. Three hours post-infection, at a multiplicity of infection of 10, the virus was detected in the cell layer of the cultures incubated with TSH but not in those without TSH. Five hours post-infection, there was a 100-fold increase in the medium in the yield of VSV and a 60-fold increase in the cell-associated virus in the TSH-treated cells compared with the cells without TSH. We found that the early stages of infection were accelerated by TSH. This effect appears to be due, at least in part, to increased processing in the lysosomes, thus allowing deposition of the transcriptionally-active nucleocapsid into the cytoplasm. These studies show that TSH is critically involved in the infectivity of VSV and that by manipulating cell culture conditions, an increased rate of virus production can be achieved.