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Signal transducers and activators of transcription 6 (STAT6), an essential member of the STAT protein family, plays vital roles in innate immunity, however, its function in regulating innate immunity through the degradation of MAVS has not been described. In this study, we found that STAT6 suppresses the replication of both bovine ephemeral fever virus (BEFV) and vesicular stomatitis virus (VSV). Further investigations revealed that STAT6 promotes the type I IFN (IFN-I) signaling pathway in the context of BEFV and VSV infection. Moreover, the knockout of STAT6 leads to the degradation of MAVS through both the ubiquitin-proteasome and autophagolysosomal pathways. Mechanistically, STAT6 results in the downregulation of E3 ubiquitin ligase STIP1 homology and Ubox-containing protein 1 (STUB1), inhibits the interaction between STUB1 and MAVS, and reduces STUB1- mediated K48-linked MAVS ubiquitination, thereby inhibiting the MAVS degradation through the ubiquitin-proteasome pathway. Furthermore, STAT6 also suppresses MAVS degradation through the autophagy receptor Bcl2 interacting protein 3 like (NIX)-mediated autophagy pathway. Taken together, our study unveils a novel mechanism by which STAT6 acts as a positive regulator of the type I IFN signaling pathway during BEFV and VSV infection, predominantly by inhibiting MAVS degradation and ultimately suppressing BEFV and VSV infection. These findings provide valuable insights into the regulation of MAVS degradation by STAT6, which may serve as a basis for the design of novel antiviral agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vesicular stomatitis virus (VSV) is a prototype RNA virus that has been instrumental in advancing our understanding of viral molecular biology and has applications in vaccine development, cancer therapy, antiviral screening, and more. Current VSV genome plasmids for purchase or contract virus services provide limited options for modification, restricted to predefined cloning sites and insert locations. Improved methods and tools to engineer VSV will unlock further insights into long-standing virology questions and new opportunities for innovative therapies. Here, we report the design and construction of a full-length VSV genome. The 11,161 base pair synthetic VSV (synVSV) was assembled from four modularized DNA fragments. Following rescue and titration, phenotypic analysis showed no significant differences between natural and synthetic viruses. To demonstrate the utility of a synthetic virology platform, we then engineered VSV with a foreign glycoprotein, a common use case for studying viral entry and developing anti-virals. To show the freedom of design afforded by this platform, we then modified the genome of VSV by rearranging the gene order, switching the positions of VSV-P and VSV-M genes. This work represents a significant technical advance, providing a flexible, cost-efficient platform for the rapid construction of VSV genomes, facilitating the development of innovative therapies.

Vesicular stomatitis virus (VSV) outbreaks periodically occur in livestock in the western US and are thought to originate from outside this country. Feral swine (Sus scrofa) have been identified as an amplifying host for vesicular stomatitis New Jersey virus (VSNJV) and have been used to better understand the epidemiology of this virus through serosurveillance. This study aimed to determine if antibodies to vesicular stomatitis Indiana virus (VSIV) and VSNJV were present in feral swine in the western US and to determine if seropositive animals were associated with areas of previously detected VSV in livestock. A total of 4,541 feral swine samples was tested using virus neutralization (VN); samples exhibiting neutralizing activity against one or more of the viruses were confirmed using competitive ELISA (cELISA). Eight sera exhibited neutralizing activity by VN assay and a single serum sample from an animal from Kinney County, Texas sampled in December 2019 tested positive for antibodies to VSIV by cELISA. This finding is supported by a local outbreak of VSIV in horses in the same county in June 2019. The low prevalence of antibodies against VSNJV and VSIV was unexpected but indicates that feral swine in the western US do not represent an endemic reservoir for either of these viruses., (© Wildlife Disease Association 2024.)

Vesicular stomatitis (VS) is a viral disease that affects horses, cattle, and swine that is transmitted by direct contact and hematophagous insects. In 2023, a multi-state outbreak of vesicular stomatitis New Jersey virus (VSNJV) occurred in California, Nevada, and Texas, infecting horses, cattle, and rhinoceros. To identify possible insect vectors, we conducted insect surveillance at various locations in San Diego County, CA, including at a wildlife park. CO 2 baited traps set from mid-May to mid-August 2023 collected 2357 Culicoides biting midges and 1215 Simulium black flies, which are insect genera implicated in VSNJV transmission. Insects were pooled by species, location, and date, then tested for viral RNA. Nine RNA-positive pools of Culicoides spp. and sixteen RNA-positive pools of Simulium spp were detected. Infectious virus was detected by cytopathic effect in 96% of the RNA-positive pools. This is the first report of VSNJV in wild-caught C. bergi , C. freeborni , C. occidentalis , S. argus , S. hippovorum , and S. tescorum. The vector competency of these species for VSNJV has yet to be determined but warrants examination. Active vector surveillance and testing during disease outbreaks increases our understanding of the ecology and epidemiology of VS and informs vector control efforts.

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-β) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb , whereas later, oligodendrocytes were the main producers. IFNb + cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection., Competing Interests: Competing interests statement:The authors declare no competing interest.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by the vesicular stomatitis New Jersey virus (VSNJV). This study presents the first application of an SEIR-SEI compartmental model to analyze VSNJV transmission dynamics. Focusing on the 2014-2015 outbreak in the United States, the model integrates vertebrate hosts and insect vector demographics while accounting for heterogeneous competency within the populations and observation bias in documented disease cases. Key epidemiological parameters were estimated using Bayesian inference and Markov chain Monte Carlo (MCMC) methods, including the force of infection, effective reproduction number (Rt), and incubation periods. The model revealed significant underreporting, with only 10-24% of infections documented, 23% of which presented with clinical symptoms. These findings underscore the importance of including competence and imperfect detection in disease models to depict outbreak dynamics and inform effective control strategies accurately. As a baseline model, this SEIR-SEI implementation is intended to serve as a foundation for future refinements and expansions to improve our understanding of VS dynamics. Enhanced surveillance and targeted interventions are recommended to manage future VS outbreaks.

The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2 -/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo ., Competing Interests: The authors declare no conflict of interest.

We conducted an integrative analysis to elucidate the spatial epidemiological patterns of the Vesicular Stomatitis New Jersey virus (VSNJV) during the 2014-15 epizootic cycle in the United States (US). Using georeferenced VSNJV genomics data, confirmed vesicular stomatitis (VS) disease cases from surveillance, and a suite of environmental factors, our study assessed environmental and phylogenetic similarity to compare VS cases reported in 2014 and 2015. Despite uncertainties from incomplete virus sampling and cross-scale spatial processes, patterns suggested multiple independent re-invasion events concurrent with potential viral overwintering between sequential seasons. Our findings pointed to a geographically defined southern virus pool at the US-Mexico interface as the source of VSNJV invasions and overwintering sites. Phylodynamic analysis demonstrated an increase in virus diversity before a rise in case numbers and a pronounced reduction in virus diversity during the winter season, indicative of a genetic bottleneck and a significant narrowing of virus variation between the summer outbreak seasons. Environment-vector interactions underscored the central role of meta-population dynamics in driving disease spread. These insights emphasize the necessity for location- and time-specific management practices, including rapid response, movement restrictions, vector control, and other targeted interventions.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by either VS New Jersey virus or VS Indiana virus. The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. During the past 20 years, VS outbreaks in the southwestern and Rocky Mountain regions occurred periodically with incursion years followed by virus overwintering and subsequent expansion outbreak years. Regulatory response by animal health officials prevents spread from lesioned animals and manages trade impacts. Recent US outbreaks highlight potential climate change impacts on insect vectors or other transmission-related variables., Competing Interests: Disclosure The author has nothing to disclose., (Published by Elsevier Inc.)

We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.

Objective: To describe an outbreak of vesicular stomatitis virus (VSV) in southern white rhinoceros (SWR; Ceratotherium simum simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) at a safari park in San Diego, CA, from May to September 2023., Animals: 21 SWR and 5 GOHR in professionally managed care., Methods: Rhinoceros of both species presented with a range of clinical signs and severities. Lesion locations were categorized as cutaneous (coronary bands, heels and soles, limbs, ventrum, neck folds, and ears) and mucocutaneous (lips, nostrils, mucous membranes of the oral cavity, and vulva). Clinical signs included lethargy, lameness, difficulty with prehension, hyporexia to anorexia, and hypersalivation. Severely affected rhinoceros had clinical pathology findings consistent with systemic inflammation., Results: Vesicular stomatitis New Jersey virus was confirmed via PCR from swabs of lesions in 10/26 (38%) rhinoceros. Of these 10 confirmed cases, 9 (90%) were SWR and 1 (10%) was a GOHR. A further 6/26 (24%) were considered probable cases, and 10/26 (38%) were considered suspect cases based on clinical signs, but the inability to appropriately sample due to the housing environment precluded confirmation. Histopathology samples from 3 rhinoceros were consistent with VSV, and viral RNA was localized in histologic lesions via RNA in situ hybridization for 1 case. All rhinoceros survived infection despite severe systemic illness in 2 animals., Clinical Relevance: This case series describes the clinical appearance and progression of VSV in 2 rhinoceros species. To the authors' knowledge, this is the first report of VSV in a rhinoceros.

Background/aim: Attempts have been made to enhance treatment with vesicular stomatitis virus (VSV) for osteosarcoma. We have previously shown that VSV incorporated with miRNA143 enhanced the antitumor effect at some doses; however, the range of the doses was narrow. This has not been evaluated in vivo, and the synergistic effect of this antitumor effect in animals is unknown. The purpose of the study was to evaluate the oncolytic effect of VSV-miRNA on osteosarcoma cells in vivo., Materials and Methods: A novel oncolytic VSV was developed by incorporating the tumor-suppressor microRNA143 (rVSV-miR143). In order to compare the antitumor effects of administration methods (intravenous and intratumoral administration) of rVSV-miR143 with those of VSV, a comparative analysis of primary tumor volume, metastatic lesions and survival rate was performed in mouse models of osteosarcoma., Results: Following intratumoral injection, rVSV-miR143 showed a significant reduction in primary tumor volume, but no significant difference was observed in metastatic lesions and survival rate compared to VSV. Following intravenous injection, rVSV-miR143 revealed no significant difference in primary tumor volume, metastatic lesion and survival rate compared to VSV., Conclusion: VSV incorporating tumor-suppressor miRNA143 demonstrated a slightly synergistic antitumor effect on osteosarcoma in vivo., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (10 4 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 10 3 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping., Competing Interests: The authors have declared that no competing interests exist.

The matrix (M) protein of vesicular stomatitis virus (VSV) has a complex role in infection and immune evasion, particularly with respect to suppression of Type I interferon (IFN). Viral strains bearing the wild-type (wt) M protein are able to suppress Type I IFN responses. We recently reported that the 22-25 strain of VSV encodes a wt M protein, however its sister plaque isolate, strain 22-20, carries a M[MD52G] mutation that perturbs the ability of the M protein to block NFκB, but not M-mediated inhibition of host transcription. Therefore, although NFκB is activated in 22-20 infected murine L929 cells infected, no IFN mRNA or protein is produced. To investigate the impact of the M[D52G] mutation on immune evasion by VSV, we used transcriptomic data from L929 cells infected with wt, 22-25, or 22-20 to define parameters in a family of executable logical models with the aim of discovering direct targets of viruses encoding a wt or mutant M protein. After several generations of pruning or fixing hypothetical regulatory interactions, we identified specific predicted targets of each strain. We predict that wt and 22-25 VSV both have direct inhibitory actions on key elements of the NFκB signaling pathway, while 22-20 fails to inhibit this pathway., Competing Interests: The authors have declared that no competing interests exist.

ABSTRACT The recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

Interferon regulatory factor 3 (IRF3) is an essential transductor for initiation of many immune responses. Here, we show that lncRNA-ISIR directly binds IRF3 to promote its phosphorylation, dimerization, and nuclear translocation, along with enhanced target gene productions. In vivo lncRNA-ISIR deficiency results in reduced IFN production, uncontrolled viral replication, and increased mortality. The human homolog, AK131315, also binds IRF3 and promotes its activation. More important, AK131315 expression is positively correlated with type I interferon (IFN-I) level and severity in patients with lupus. Mechanistically, in resting cells, IRF3 is bound to suppressor protein Flightless-1 (Fli-1), which keeps its inactive state. Upon infection, IFN-I-induced lncRNA-ISIR binds IRF3 at DNA-binding domain in cytoplasm and removes Fli-1's association from IRF3, consequently facilitating IRF3 activation. Our results demonstrate that IFN-I-inducible lncRNA-ISIR feedback strengthens IRF3 activation by removing suppressive Fli-1 in immune responses, revealing a method of lncRNA-mediated modulation of transcription factor (TF) activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lassa virus (LASV)-a member of the family Arenaviridae -causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that CP100356 hydrochloride (CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 μM, respectively, without significant cytotoxicity. Although CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections over 60 years. Like other members of the family Peribunyaviridae , the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium-sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins, Gn and Gc, responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here, we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized by intramuscular injection with VSV-OROV specifically neutralized wild-type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild-type OROV shows that the VSV-OROV chimeras reduce wild-type viral infection and suggest that antibodies that recognize the variable N terminus of Gc afford less protection than those that target more conserved regions of Gc. IMPORTANCE Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wild-type virus. These data provide novel evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

The antiviral innate immunity is the first line of host defense against viral infection. Mitochondrial antiviral signaling protein (MAVS, also named Cardif/IPS-1/VISA) is a critical protein in RNA virus-induced antiviral signaling pathways. Our previous research suggested that E3 ubiquitin-protein ligases RING-finger protein (RNF90) negatively regulate cellular antiviral responses by targeting STING for degradation, though its role in RNA virus infection remains unknown. This study demonstrated that RNF90 negatively regulated RNA virus-triggered antiviral innate immune responses in RNF90-silenced PMA-THP1 cells, RNF90-deficient cells (including HaCaTs, MEFs, and BMDMs), and RNF90-deficient mice. However, RNF90 regulated RNA virus-triggered antiviral innate immune responses independent of STING. RNF90 promoted K48-linked ubiquitination of MAVS and its proteasome-dependent degradation, leading to the inhibition of innate immune responses. Altogether, our findings suggested a novel function and mechanism of RNF90 in antiviral innate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Zhang, Qin, Huang, Ren, Sun, Ma, Liu, Song, Liu, Cui, Wang and Wang.)

Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m 7 Gpppm 6 A m and define the substrate requirements for this modification. Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-β. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-β treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m 7 Gpppm 6 A m This work identifies a function of PCIF1 and cap-proximal m 6 A m in attenuation of the host response to VSV infection that likely extends to other viruses., Competing Interests: The authors declare no competing interest.

Vesicular stomatitis virus (VSV) is an archetypal member of Mononegavirales which causes important diseases in cattle, horses and pigs. The matrix protein (M) of VSV plays critical roles in the replication, assembly/budding and pathogenesis of VSV. To further investigate the role of M during viral growth, we used a two-hybrid system to screen for host factors that interact with the M protein. Here, NADH: ubiquinone oxidoreductase complex assembly factor 4 (Ndufaf4) was identified as an M-binding partner, and this interaction was confirmed by yeast cotransformation and GST pulldown assays. The globular domain of M was mapped and shown to be critical for the M-Ndufaf4 interaction. Two double mutations (E156A/H157A, D180A/E181A) in M impaired the M-Ndufaf4 interaction. Overexpression of Ndufaf4 inhibited VSV propagation, and knockdown of Ndufaf4 by short hairpin RNA (shRNA) markedly promoted VSV replication. Finally, we also demonstrate that the anti-VSV effect of Ndufaf4 is independent of activation of the type I IFN response. These results indicated that Ndufaf4 might exploit other mechanisms to affect VSV replication. In summary, we identify Ndufaf4 as a potential target for the inhibition of VSV propagation. These results provided further insight into the study of VSV pathogenesis.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Linder, Bothe, Linder, Schwarzlmueller, Dahlström, Bartenhagen, Dugas, Pandey, Thorn-Seshold, Boehmer, Koenig, Kobold, Schnurr, Raedler, Spielmann, Karimzadeh, Schmidt, Endres and Rothenfusser.)

The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV., (Copyright © 2020. Published by Elsevier Inc.)

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m 6 A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m 6 A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m 6 A and the m 6 A methyltransferase proteins METTL3 and METTL14. We further determine that the m 6 A reader YTHDF1 increases the expression of IFITM1 in an m 6 A-binding-dependent manner. Importantly, we find that the m 6 A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m 6 A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction., Competing Interests: Declaration of interests C.E.M. is a cofounder and board member for Biotia and Onegevity Health and an advisor or compensated speaker for Abbvie, Acuamark Diagnostics, ArcBio, Bio-Rad, DNA Genotek, Genialis, Genpro, Illumina, New England Biolabs, QIAGEN, Whole Biome, and Zymo Research., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E ( Itgae , CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duhan, Khairnar, Kitanovski, Hamdan, Klein, Lang, Ali, Adomati, Bhat, Friedrich, Li, Krebs, Futerman, Addo, Hardt, Hoffmann, Lang and Lang.)

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier Inc. All rights reserved.)

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV., (Published by Elsevier B.V.)

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.)

Neutrophils are innate leukocytes that mount a rapid response to invading pathogens and sites of inflammation. Although neutrophils were traditionally considered responders to bacterial infections, recent advances have demonstrated that they are interconnected with both viral infections and cancers. One promising treatment strategy for cancers is to administer an oncolytic virus to activate the immune system and directly lyse cancerous cells. A detailed characterization of how the innate immune system responds to a viral-based therapy is paramount in identifying its systemic effects. This study analyzed how administering the rhabdovirus vesicular stomatitis virus (VSV) intravenously at 1 × 10 9 PFU acutely influenced neutrophil populations. Bone marrow, blood, lungs, and spleen were acquired three- and 24-h after administration of VSV for analysis of neutrophils by flow cytometry. Infection with VSV caused neutrophils to rapidly egress from the bone marrow and accumulate in the lungs. A dramatic increase in immature neutrophils was observed in the lungs, as was an increase in the antigen presentation potential of these cells within the spleen. Furthermore, the potential for neutrophils to acquire viral transgene-encoded proteins was monitored using a variant of VSV that expressed enhanced green fluorescent protein (GFP). If an in vitro population of splenocytes were exposed to αCD3 and αCD28, a substantial proportion of the neutrophils would become GFP-positive. This suggested that the neutrophils could either acquire more virus-encoded antigens from infected splenocytes or were being directly infected. Five different dosing regimens were tested in mice, and it was determined that a single dose of VSV or two doses of VSV administered at a 24-h interval, resulted in a substantial proportion of neutrophils in the bone marrow becoming GFP-positive. This correlated with a decrease in the number of splenic neutrophils. Two doses administered at intervals longer than 24-h did not have these effects, suggesting that neutrophils became resistant to antigen uptake or direct infection with VSV beyond 24-h of activation. These findings implicated neutrophils as major contributors to oncolytic rhabdoviral therapies. They also provide several clear future directions for research and suggest that neutrophils should be carefully monitored during the development of all oncolytic virus-based treatment regimens.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.

Cocal virus (COCV) is one of the causative agents of vesicular stomatitis, presenting clinical signs indistinguishable from those caused by foot-and-mouth disease virus (FMDV). Therefore, the differentiation of these two viruses via laboratory diagnosis is essential. The objective of this study was to develop and validate a real-time quantitative PCR (RT-qPCR) protocol for the diagnosis of COCV directly from epithelial samples. The method developed had 97% accuracy at 3950 pfu and a repeatability error of 1.29%. RT-qPCR was able to distinguish COCV from other viruses that cause vesicular diseases, an important factor because seroneutralization may produce cross-reactivity between COCV and vesicular stomatitis Alagoas virus (VSAV). No epithelial sample originating from vesicular disease outbreaks between 2014 and 2018 in Brazil was positive for COCV.

Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the L RdRP , L PRNT , and L MT , an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (P ΔOD ) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro Recombinant virus VSV-P ΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that P ΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of P ΔOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication., (Copyright © 2020 Bloyet et al.)

The neuroepithelium is a nasal barrier surface populated by olfactory sensory neurons that detect odorants in the airway and convey this information directly to the brain via axon fibers. This barrier surface is especially vulnerable to infection, yet respiratory infections rarely cause fatal encephalitis, suggesting a highly evolved immunological defense. Here, using a mouse model, we sought to understand the mechanism by which innate and adaptive immune cells thwart neuroinvasion by vesicular stomatitis virus (VSV), a potentially lethal virus that uses olfactory sensory neurons to enter the brain after nasal infection. Fate-mapping studies demonstrated that infected central nervous system (CNS) neurons were cleared noncytolytically, yet specific deletion of major histocompatibility complex class I (MHC I) from these neurons unexpectedly had no effect on viral control. Intravital imaging studies of calcium signaling in virus-specific CD8 + T cells revealed instead that brain-resident microglia were the relevant source of viral peptide-MHC I complexes. Microglia were not infected by the virus but were found to cross-present antigen after acquisition from adjacent neurons. Microglia depletion interfered with T cell calcium signaling and antiviral control in the brain after nasal infection. Collectively, these data demonstrate that microglia provide a front-line defense against a neuroinvasive nasal infection by cross-presenting antigen to antiviral T cells that noncytolytically cleanse neurons. Disruptions in this innate defense likely render the brain susceptible to neurotropic viruses like VSV that attempt to enter the CNS via the nose., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Viral infection of cultured cells induces changes in the biophysical characteristics of the affected cells. Advanced microscopic cameras such as Ovizio's QMod, coupled with the appropriate software, can measure a variety of characteristics on a per-cell basis. We have employed this system to monitor the progression of vesicular stomatitis virus infection in Vero cells and to describe the cellular changes associated with advancing vesicular stomatitis virus infection. The measurements of cellular characteristics are operator-independent, and the goal is to establish a robust method to mathematically determine viral infection levels in a given sample. This will provide a means to measure viral titer in a faster and less subjective way than manual reading of plaque assays or tissue culture infectious dose 50 assays.

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor β (TGF-β). Lack of Mertk in Mertk -/- mice prevents induction of IL-10 and TGF-β, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The spread of viruses among cells and hosts often involves multi-virion structures. For instance, virions can form aggregates that allow for the co-delivery of multiple genome copies to the same cell from a single infectious unit. Previously, we showed that vesicular stomatitis virus (VSV), an enveloped, negative-strand RNA virus, undergoes strong aggregation in the presence of saliva from certain individuals. However, the molecular components responsible for such aggregation remain unknown. Here we show that saliva-driven aggregation is protein dependent, and we use comparative proteomics to analyze the protein content of strongly versus poorly aggregating saliva. Quantitative analysis of over 300 proteins led to the identification of 18 upregulated proteins in strongly aggregating saliva. One of these proteins, the fibrinogen gamma chain, was verified experimentally as a factor promoting VSV aggregation in a dose-dependent manner. This study hence identifies a protein responsible for saliva-driven VSV aggregation. Yet, the possible involvement of additional proteins or factors cannot be discarded.

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.)

Both PM 2.5 and respiratory viruses are part of the atmospheric constituents. Respiratory viruses are often associated with PM 2.5 exposure, but the mechanism of toxicity remains to be explored. The vitro models that adequately reproduce healthy cells or diseased cells exposing to PM 2.5 and infecting VSV can provide a useful tool for studying innate immune mechanisms and investigating new therapeutic focus. In the environment of PM 2.5 , an infection model in which VSV infected A549 cells was established, that mimics the state in which the antiviral innate immune pathways are activated after the respiratory system is infected with RNA viruses. Subsequently, the model was exposed to PM 2.5 for 24 h. PM 2.5 could be ingested by A549 cells and synergize with VSV to inhibit cell viability and promote apoptosis. The expression of VSV-G were more abundant after VSV-infected A549 cells were exposed to PM 2.5 . Furthermore, PM 2.5 inhibits VSV-induced IFN-β expression in A549 cells. ISG15, CCL-5, and CXCL-10 had the same expression tendency with IFN-β mRNA, consistently. Interestingly, when MG132 was applied, the expression of p-IRF-3 and IFN-β proteins reduced by PM 2.5 were refreshed. Conversely, the expression of VSV-G proteins were decreased. PM 2.5 could degrade p-IRF-3 proteins by ubiquitination pathway to inhibit VSV-induced IFN-β expression in A549 cells. Therefore, replication of the VSV viruses was promoted., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes. IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer., (Copyright © 2020 Seegers et al.)

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSV IND ), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSV IND inhibitor. TRIM69 potently inhibited VSV IND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSV IND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSV IND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSV IND RNA synthesis. IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs., (Copyright © 2019 Kueck et al.)

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

ISG20 is a broad spectrum antiviral protein thought to directly degrade viral RNA. However, this mechanism of inhibition remains controversial. Using the Vesicular Stomatitis Virus (VSV) as a model RNA virus, we show here that ISG20 interferes with viral replication by decreasing protein synthesis in the absence of RNA degradation. Importantly, we demonstrate that ISG20 exerts a translational control over a large panel of non-self RNA substrates including those originating from transfected DNA, while sparing endogenous transcripts. This activity correlates with the protein's ability to localize in cytoplasmic processing bodies. Finally, these functions are conserved in the ISG20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. Overall, our results posit ISG20 as an important defense factor able to discriminate the self/non-self origins of the RNA through translation modulation., Competing Interests: The authors have declared that no competing interests exist.

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSV IND ), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies. IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection., (Copyright © 2019 Rihn et al.)

Conventional type 1 dendritic cells (cDC1s) are inherently resistant to many viruses but, paradoxically, possess fewer acidic phagosomes that enable antigen retention and cross-presentation. We report that palmitoyl-protein thioesterase 1 (PPT1), which catabolizes lipid-modified proteins in neurons, is highly expressed in cDC1s. PPT1-deficient DCs are more susceptible to vesicular stomatitis virus (VSV) infection, and mice with PPT1 deficiency in cDC1s show impaired response to VSV. Conversely, PPT1-deficient cDC1s enhance the priming of naive CD8 + T cells into tissue-resident KLRG1 + effectors and memory T cells, resulting in rapid clearance of tumors and Listeria monocytogenes Mechanistically, PPT1 protects steady state DCs from viruses by promoting antigen degradation and endosomal acidification via V-ATPase recruitment. After DC activation, immediate down-regulation of PPT1 is likely to facilitate efficient cross-presentation, production of costimulatory molecules and inflammatory cytokines. Thus, PPT1 acts as a molecular rheostat that allows cDC1s to crossprime efficiently without compromising viral resistance. These results suggest potential therapeutics to enhance cDC1-dependent crosspriming., (© 2019 Ou et al.)

Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.

The transcription factor interferon regulatory factor 3 (IRF3) is essential for virus infection-triggered induction of type I interferons (IFN-I) and innate immune responses. IRF3 activity is tightly regulated by conventional posttranslational modifications (PTMs) such as phosphorylation and ubiquitination. Here, we identify an unconventional PTM of IRF3 that directly inhibits its transcriptional activity and attenuates antiviral immune response. We performed an RNA interference screen and found that lysine acetyltransferase 8 (KAT8), which is ubiquitously expressed in immune cells (particularly in macrophages), selectively inhibits RNA and DNA virus-triggered IFN-I production in macrophages and dendritic cells. KAT8 deficiency protects mice from viral challenge by enhancing IFN-I production. Mechanistically, KAT8 directly interacts with IRF3 and mediates IRF3 acetylation at lysine 359 via its MYST domain. KAT8 inhibits IRF3 recruitment to IFN-I gene promoters and decreases the transcriptional activity of IRF3. Our study reveals a critical role for KAT8 and IRF3 lysine acetylation in the suppression of antiviral innate immunity., (© 2019 Huai et al.)

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus., (Copyright © 2019. Published by Elsevier B.V.)