Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136-370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

We identified a novel rhabdovirus, American bat vesiculovirus, from postmortem tissue samples from 120 rabies-negative big brown bats with a history of human contact. Five percent of the tested bats were infected with this virus. The extent of zoonotic exposure and possible health effects in humans from this virus are unknown.

Vesiculoviruses enter cells by membrane fusion, driven by a large, low-pH-induced, conformational change in the fusion glycoprotein G that involves transition from a trimeric pre-fusion toward a trimeric post-fusion state via monomeric intermediates. Here, we present the structure of the G fusion protein at intermediate pH for two vesiculoviruses, vesicular stomatitis virus (VSV) and Chandipura virus (CHAV), which is responsible for deadly encephalopathies. First, a CHAV G crystal structure shows two intermediate conformations forming a flat dimer of heterodimers. On virions, electron microscopy (EM) and tomography reveal monomeric spikes similar to one of the crystal conformations. In solution, mass spectrometry shows dimers of G. Finally, mutations at a dimer interface, involving fusion domains associated in an antiparallel manner to form an intermolecular β-sheet, affect G fusion properties. The location of the compensatory mutations restoring fusion activity strongly suggests that this interface is functionally relevant. This work reveals the range of G structural changes and suggests that G monomers can re-associate, through antiparallel interactions between fusion domains, into dimers that play a role at some early stage of the fusion process.

Malpais Spring virus (MSPV) is a mosquito-borne rhabdovirus that infects a variety of wild and feral ungulates in New Mexico, including horses and deer. Although, initial serologic tests and electron microscopy at the time of isolation nearly 25 years ago provided evidence that MSPV is a novel virus, possibly related to vesiculoviruses, the virus still has not been approved as a new species.

A Vero cell based vaccine candidate against Chandipura (CHP) virus (Rhabdoviridae: Vesiculovirus), was developed and evaluated for immunogenicity in mice. Virus was purified by ultracentrifugation on 30% glycerol cushion followed by differential centrifugation on 10-60% sucrose gradient and inactivated with β-propio lactone at a concentration of 1:3500. The inactivated product was blended with aluminium phosphate (3%) and immunized 4-week-old Swiss albino mice. Neutralizing antibodies in the range of 1:10 to 160 and 1:80 to 1:320 was detected with 85% and 100% sero-conversion after 2nd and 3rd dose, respectively. All the immunized mice with antibody titer above 1:20 survived live virus challenge. The vaccine candidate has potential to be an efficient vaccine against CHP virus.

Vesiculoviruses are attractive oncolytic virus platforms due to their rapid replication, appreciable transgene capacity, broad tropism, limited preexisting immunity, and tumor selectivity through type I interferon response defects in malignant cells. We developed a synthetic chimeric virus (VMG) expressing the glycoprotein (G) from Morreton virus (MorV) and utilizing the remaining structural genes from vesicular stomatitis virus (VSV). VMG exhibited in vitro efficacy by inducing oncolysis in a broad range of sarcoma subtypes across multiple species. Notably, all cell lines tested showed the ability of VMG to yield productive infection with rapid replication kinetics and induction of apoptosis. Furthermore, pilot safety evaluations of VMG in immunocompetent, non-tumor-bearing mice showed an absence of toxicity with intranasal doses as high as 1e10 50% tissue culture infectious dose (TCID50)/kg. Locoregional administration of VMG in vivo resulted in tumor reduction in an immunodeficient Ewing sarcoma xenograft at doses as low as 2e5 TCID50. In a murine syngeneic fibrosarcoma model, while no tumor inhibition was achieved with VMG, there was a robust induction of CD8+ T cells within the tumor. The studies described herein establish the promising potential for VMG to be used as a novel oncolytic virotherapy platform with anticancer effects in sarcoma.

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra s0 tate, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission.

Vesicular stomatitis caused by Alagoas vesiculovirus (VSAV) has generated disease outbreaks in Brazil, mainly in the northeast region. Phylogenetic studies divide the isolates into three distinct genotypes (A, B, and C). However, there is no description of how this genetic divergence reflects on the phenotype of VSAV isolates such as in vitro replication fitness. Therefore, the objective of this work was to evaluate the ability of three distinct genotypes of Brazilian isolates of VSAV to grow in different cell-culture lines (BHK-21, Vero, and NCI-H1299). Quantification of viral RNA was performed using RT-PCR digital droplet from supernatant of cell culture collected every 4 h for a period of 24 h of viral growth in three different cell lines (BHK-21, Vero, and NCI-H1299). It was observed that the genotype C isolate has the lowest replication efficiency among the three analyzed viruses, without major changes in the copies of viral RNA over the entire time of the study.

While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer.

Abstract： Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV) [...].

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays an important role in regulating the replication of many viruses. However, it remains elusive whether and how hnRNPA1 regulates fish virus replication. In this study, the effects of twelve hnRNPs on the replication of snakehead vesiculovirus (SHVV) were screened. Three hnRNPs, one of which was hnRNPA1, were identified as anti-SHVV factors. Further verification showed that knockdown of hnRNPA1 promoted, while overexpression of hnRNPA1 inhibited, SHVV replication. SHVV infection reduced the expression level of hnRNPA1 and induced the nucleocytoplasmic shuttling of hnRNPA1. Besides, we found that hnRNPA1 interacted with the viral phosphoprotein (P) via its glycine-rich domain, but not with the viral nucleoprotein (N) or large protein (L). The hnRNPA1-P interaction competitively disrupted the viral P-N interaction. Moreover, we found that overexpression of hnRNPA1 enhanced the polyubiquitination of the P protein and degraded it through proteasomal and lysosomal pathways. This study will help understanding the function of hnRNPA1 in the replication of single-stranded negative-sense RNA viruses and providing a novel antiviral target against fish rhabdoviruses.

Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example, Cocal virus, have been proposed as alternative LV envelopes with possible advantages over VSVind.G. Well-characterized antibodies that recognize VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here, we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralizes G proteins from three strains of VSV, as well as Cocal and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralizes only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.

Vesicular stomatitis virus Indiana strain glycoprotein (VSVind.G) mediates broad tissue tropism and efficient cellular uptake. Lentiviral vectors (LVs) are particularly promising, as they can efficiently transduce non-dividing cells and facilitate stable genomic transgene integration; therefore, LVs have an enormous untapped potential for gene therapy applications, but the development of humoral and cell-mediated anti-vector responses may restrict their efficacy. We hypothesized that G proteins from different members of the vesiculovirus genus might allow the generation of a panel of serotypically distinct LV pseudotypes with potential for repeated in vivo administration. We found that mice hyperimmunized with VSVind.G were not transduced to any significant degree following intravenous injection of LVs with VSVind.G envelopes, consistent with the thesis that multiple LV administrations would likely be blunted by an adaptive immune response. Excitingly, bioluminescence imaging studies demonstrated that the VSVind-neutralizing response could be evaded by LV pseudotyped with Piry and, to a lesser extent, Cocal virus glycoproteins. Heterologous dosing regimens using viral vectors and oncolytic viruses with Piry and Cocal envelopes could represent a novel strategy to achieve repeated vector-based interventions, unfettered by pre-existing anti-envelope antibodies.

Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates.

Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.

Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines.

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

The sequences of the nucleocapsid and glycoprotein genes and the gene junctions of the fish pathogenic rhabdovirus 903/87 were determined from cDNA and PCR clones. The mRNA of the nucleocapsid is most likely 1492 nucleotides long and encodes a protein of 426 amino acids, whereas the mRNA of the glycoprotein is likely to be 1682 nucleotides long and the protein 517 amino acids. When the nucleocapsid and glycoprotein genes of virus 903/87 were compared at amino acid level with other rhabdoviruses they showed the highest homology with the Vesiculovirus genus. By sequencing the junctions between the N, P, M, G and L genes it was determined that transcription start and stop codons were conserved between virus 903/87 and the vesiculoviruses. Virus 903/87 has no open reading frame coding for a non-virion gene between the glycoprotein and the polymerase gene. Phylogenetic studies based on rhabdovirus nucleocapsid and glycoprotein genes suggested that virus 903/87 is related to viruses in the Vesiculovirus genus.

Two novel rhabdoviruses were isolated from birds during surveillance for arboviral encephalitis in the northeastern United States. The first, designated Farmington virus, is a tentative new member of the Vesiculovirus genus. The second, designated Rhode Island virus, is unclassified antigenically, but its ultrastructure and size are more similar to those of some of the plant rhabdoviruses. Both viruses infect birds and mice, as well as monkey kidney cells in culture, but their importance for human health is unknown.