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Marine algae, a rich source of bioactive substances, have long been utilized in biomedical and veterinary sciences. This study breaks new ground by assessing the antiviral therapeutic potential of water extracts from four algal species – Colaconema formosanum, Caulerpa microphysa, Gelidium amansii and Sarcodia suae – against vesicular stomatitis virus (VSV) infection. The standout among these was Sarcodia suae water extract (SSWE), which not only significantly repressed VSV replication and enhanced cell survival without cytotoxic effects, but also demonstrated its antiviral activity through interference with viral attachment, entry, RNA replication, and egress, and reduced VSV-induced autophagy in Mv1Lu cells during the late stages of infection. The EC50 of SSWE was 0.422 ± 0.14 mg ml‒1. The cytotoxicity assay confirmed the high biocompatibility of SSWE. This study identifies SSWE as a promising natural antiviral agent and elucidates its mechanisms of action, highlighting the need for further research to optimize its use in controlling VSV-related diseases. Highlights: The bioactive water extract of Sarcodia suae was identified as a sustainable antiviral agent for the algal cultivation industry. The antiviral activity of S. suae against vesicular stomatitis virus (VSV) infection was confirmed. The mechanisms of action of the extract from Sarcodia suae on the autophagic pathway were demonstrated. [ABSTRACT FROM AUTHOR]

Borna disease virus 1 (BoDV‐1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV‐1 cell entry has been hindered by low infectious viral particle production in cells and the lack of cytopathic effects, which are typically useful for screening. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of members of the genus Orthobornavirus, including BoDV‐1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV‐1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the methionine at BoDV‐1–G residue 307 increases the infectivity titer of VSV pseudotyped with BoDV‐1–G (VSV–BoDV‐1–G). A cell‒cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV–BoDV‐1–G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV‐1–G and provides insight into G‐mediated cell entry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To discuss the damage effect of vesicular stomatitis virus (VSV) on the vascular endothelial (VE) barrier, and to clarify its mechanism. Methods: The canine kidney cells were used to amplify VSV. The half tissue culture infective dose (TCID50) of VSV was determined using mouse brain endothelial tumor bEnd. 3 cells, and subsequent experiment was conducted using 300 times the TCID50. The bEnd. 3 cells were divided into infection 0 h group, infection 4 h group, infection 8 h group, and infection 12 h group for VE barrier damage experiments due to VSV infection. The bEnd. 3 cells were also divided into control group, infection group, and correction group for experiments to inhibit the VSV replication and restore the VE barrier. The bEnd. 3 cells were inoculated into Transwell chambers to construct an in vitro VE barrier model. Cell voltage resistance meter was used to detect the transepithelial resistance (TER) in various groups after the bEnd. 3 cells were infected with VSV at different time points; fluorescein isothiocyanate-dextran leakage assay was used to detect the permeability coefficients of the cells in various groups; immunofluorescence staining was used to observe the localization changes of VE-cadherin, β-catenin, and phosphorylated β-catenin (p-β-catenin) in cytoskeleton and adherens junctions (AJs) of the bEnd. 3 cells after VSV infection; real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression levels of Wnt and β-catenin mRNA in the cells in various groups; Western blotting method was used to detect the expression levels of Wnt, β -catenin, and p- β -catenin proteins in the cells in various groups. Results: The TCID50 of VSV was 10-4. 5 ·100 μL-1. The Transwell chamber experiment results showed that compared with infection 0 h group, the TERs in the cells in the other groups were significantly decreased (P<0. 05), and the permeability coefficients were significantly increased (P<0. 05). The immunofluorescence staining results showed that compared with control group, the cytoskeleton of the bEnd. 3 cells in infection group was disordered, the cell gaps was increased, the linear index of AJs was significantly decreased (P<0. 05), and β-catenin and p-β-catenin translocated from the cell membrane to the perinuclear area. The RT-qPCR results showed that compared with infection 0 h group, the expression levels of Wnt mRNA in the cells in the other groups were significantly decreased (P<0. 05), while the expression levels of β -catenin mRNA showed no statistically significant difference (P>0. 05). The Western blotting results showed that compared with infection 0 h group, the expression levels of Wnt protein in the cells in the other groups were significantly decreased (P<0. 05), the expression levels of β -catenin showed no statistically significant differences (P>0. 05), and the expression levels of p-β-catenin were significantly increased (P<0. 05). After inhibiting the VSV replication and correcting the low density lipoprotein receptor (LDLR) abnormalities, the Transwell chamber experiment results showed that compared with infection group, the TER in the cells in correction group was significantly increased (P<0. 05), and the permeability coefficient was significantly decreased (P<0. 05). The immunofluorescence staining results showed that compared with infection group, the gaps in the cells in correction group were reduced, and the perinuclear aggregation of β -catenin and p- β-catenin in the cells was restrained. The RT-qPCR results showed that compared with infection group, the expression level of Wnt mRNA in the cells in correction group was significantly increased (P<0. 05). The Western blotting results showed that compared with infection group, the expression level of Wnt protein in the cells in correction group was significantly increased (P<0. 05), the expression level of β -catenin showed no statistically significant difference (P>0. 05), and the expression level of p- β -catenin was significantly decreased (P<0. 05). Conclusion: VSV infection can cause the LDLR inactivation, reduce the expression level of Wnt protein, increase the phosphorylation level of β-catenin and cause its internalization, disrupt the stability of AJs, and ultimately lead to VE barrier damage. [ABSTRACT FROM AUTHOR]

Intratumoural oncolytic virotherapy may have promise as a means to debulk and downstage inoperable tumours in preparation for successful surgery. Here, we describe the unique case of a 50-year-old self-experimenting female virologist with locally recurrent muscle-invasive breast cancer who was able to proceed to simple, non-invasive tumour resection after receiving multiple intratumoural injections of research-grade virus preparations, which first included an Edmonston-Zagreb measles vaccine strain (MeV) and then a vesicular stomatitis virus Indiana strain (VSV), both prepared in her own laboratory. The intratumoural virus therapy was well tolerated. Frequent imaging studies and regular clinical observations documenting size, consistency and mobility of the injected tumour demonstrate that both the MeV- and VSV-containing parts of the protocol contributed to the overall favourable response. Two months after the start of the virus injections, the shrunken tumour was no longer invading the skin or underlying muscle and was surgically excised. The excised tumour showed strong lymphocytic infiltration, with an increase in CD20-positive B cells, CD8-positive T cells and macrophages. PD-L1 expression was detected in contrast to the baseline PD-L1-negative phenotype. The patient completed one-year trastuzumab adjuvant therapy and remains well and recurrence-free 45 months post-surgery. Although an isolated case, it encourages consideration of oncolytic virotherapy as a neoadjuvant treatment modality. [ABSTRACT FROM AUTHOR]

Abstract Oncolytic virotherapy has shown efficacy in various animal models and a few human cancers. However, there are still significant limitations for the implementation of these therapies. One such limitation is the emergence of cellular resistances, which may appear rapidly considering the high genetic heterogeneity of most tumors. We previously showed that cellular resistance to an oncolytic virus can be mediated by the chronic activation of innate immunity. Here, we explored the existence of additional resistance mechanisms in murine colon cancer-derived cells. For this purpose, we isolated two cellular clones that were resistant to the oncolytic virus VSV-D51. While one of the clones showed a strong resistance profile associated with increased cytokine-mediated antiviral responses, the other clone showed a lower level of resistance that involves cytoskeletal reorganization, signaling by small GTPases, and cell structural changes. These results demonstrate the capacity of tumor cells to deploy heterogeneous mechanisms of resistance to oncolytic viruses.

Filoviruses, like the Marburg (MARV) and Ebola (EBOV) viruses, have caused outbreaks associated with significant hemorrhagic morbidity and high fatality rates. Vaccines offer one of the best countermeasures for fatal infection, but to date only the EBOV vaccine has received FDA licensure. Given the limited cross protection between the EBOV vaccine and Marburg hemorrhagic fever (MHF), we analyzed the protective efficacy of a similar vaccine, rVSV-MARV, in the lethal cynomolgus macaque model. NHPs vaccinated with a single dose (as little as 1.6 × 107 pfu) of rVSV-MARV seroconverted to MARV G-protein prior to challenge on day 42. Vaccinemia was measured in all vaccinated primates, self-resolved by day 14 post vaccination. Importantly, all vaccinated NHPs survived lethal MARV challenge, and showed no significant alterations in key markers of morbid disease, including clinical signs, and certain hematological and clinical chemistry parameters. Further, apart from one primate (from which tissues were not collected and no causal link was established), no pathology associated with Marburg disease was observed in vaccinated animals. Taken together, rVSV-MARV is a safe and efficacious vaccine against MHF in cynomolgus macaques. [ABSTRACT FROM AUTHOR]

Marburg virus (MARV), a filovirus, was first identified in 1967 in Marburg, Germany, and Belgrade, former Yugoslavia. Since then, MARV has caused sporadic outbreaks of human disease with high case fatality rates in parts of Africa, with the largest outbreak occurring in 2004/05 in Angola. From 2021 to 2023, MARV outbreaks occurred in Guinea, Ghana, New Guinea, and Tanzania, emphasizing the expansion of its endemic area into new geographical regions. There are currently no approved vaccines or therapeutics targeting MARV, but several vaccine candidates have shown promise in preclinical studies. We compared three vaccine platforms simultaneously by vaccinating hamsters with either a single dose of an adenovirus-based (ChAdOx-1 MARV) vaccine, an alphavirus replicon-based RNA (LION-MARV) vaccine, or a recombinant vesicular stomatitis virus-based (VSV-MARV) vaccine, all expressing the MARV glycoprotein as the antigen. Lethal challenge with hamster-adapted MARV 4 weeks after vaccination resulted in uniform protection of the VSV-MARV and LION-MARV groups and 83% of the ChAdOx-1 MARV group. Assessment of the antigen-specific humoral response and its functionality revealed vaccine-platform-dependent differences, particularly in the Fc effector functions. [ABSTRACT FROM AUTHOR]

There are currently no prophylactic vaccines licensed to protect against Lassa fever caused by Lassa virus (LASV) infection. The Emergent BioSolutions (EBS) vaccine candidate, EBS-LASV, is being developed for the prevention of Lassa fever. EBS-LASV is a live-attenuated recombinant Vesicular Stomatitis Virus (rVSV)-vectored vaccine encoding the surface glycoprotein complex (GPC) from LASV and has two attenuating vector modifications: a gene shuffle of the VSV N gene and a deletion of the VSV G gene. Preclinical studies were performed to evaluate EBS-LASV's neurovirulence potential following intracranial (IC) injection and to determine the biodistribution and vector replication following intramuscular (IM) inoculation in mice. In addition, the potential EBS-LASV toxicity was assessed using repeated-dose IM EBS-LASV administration to rabbits. All mice receiving the IC injection of EBS-LASV survived, while mice administered the unattenuated control vector did not. The vaccine was only detected in the muscle at the injection site, draining lymph nodes, and the spleen over the first week following IM EBS-LASV injection in mice, with no detectable plasma viremia. No toxicity was observed in rabbits receiving a three-dose regimen of EBS-LASV. These studies demonstrate that EBS-LASV is safe when administered to animals and supported a first-in-human dose-escalation, safety, and immunogenicity clinical study. [ABSTRACT FROM AUTHOR]

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3–6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2–8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system. [ABSTRACT FROM AUTHOR]

Abstract Background Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. Methods Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. Results Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. Conclusion Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

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.

Extracellular vesicles (EVs) are mediators of intercellular communication in the tumor microenvironment. Tumor EVs are commonly associated with metastasis, immunosuppression or drug resistance. Viral infections usually increase EV secretion, but little is known about the effect of oncolytic viruses (OVs) on tumor EVs. Here, we investigated the impact of oncolytic vesicular stomatitis virus (VSV) and vaccinia virus on EVs secreted by human melanoma and thoracic cancer cells. We found that OV infection increases the production of EVs by tumor cells. These EVs contain proteins of viral origin, such as VSV-G, thus creating a continuum of particles sharing markers of both canonical EVs and viruses. As such, the presence of VSV-G on EVs improves the transfer of their protein content to cell types commonly found in the tumor microenvironment. A proteomic analysis also revealed that EVs-OV secreted during VSV infection are enriched in immunity-related proteins. Finally, CD8+ T cells incubated with EVs-OV from infected cells display slightly enhanced cytotoxic functions. Taken together, these data suggest that OVs enhance the communication mediated by tumor EVs, which could participate in the therapeutic efficacy of OVs. These results also provide rationale for engineering OVs to exploit EVs and disseminate therapeutic proteins within the tumor microenvironment.

Background: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. Methods: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. Results: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. Conclusion: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To explore the influence of extracellular matrix protein ABI-interactor 3-binding protein (ABI3BP) on vesicular stomatitis virus (VSV) genome replication and innate immune signaling pathway. The small interfering RNA (siRNA) was transfected to knock down ABI3BP gene in human skin fibroblast BJ-Sta cells. VSV-green fluorescent protein (VSV-GFP)-infected cell model was established. The morphological changes and F-actin stress fiber formation were detected on ABI3BP knockdown cells by phalloidin immunofluorescence staining. The mRNA level of virus replication was detected by RT-qPCR in BJ-Sta cells after VSV-GFP infection; western blotting was performed to detect the changes in interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) phosphorylation levels. The VSV-GFP-infected BJ-Sta cell model was successfully established. Efficient knockdown of ABI3BP in BJ-Sta cells was achieved. Phalloidin immunofluorescence staining revealed structural rearrangement of intracellular F-actin after ABI3BP gene knockdown. Compared with the control group, the gene copy number of VSV-GFP in ABI3BP knockdown cells increased by 2.2 – 3.5 times (P <0.01) and 2.2 – 4.0 times (P <0.01) respectively when infected with VSV of multiplicity of infection 0.1 and 1. The expression of viral protein significantly increased in ABI3BP knockdown cells after virus infection. The activation of type-I interferon pathway, as determined by phosphorylated IRF3 and phosphorylated TBK1, was significantly decreased in ABI3BP knockdown cells after VSV-GFP infection. Extracellular matrix protein ABI3BP plays an important role in maintaining the formation and rearrangement of actin structure. ABI3BP gene deletion promotes RNA virus replication, and ABI3BP is an important molecule that maintains the integrity of type I interferon pathway. [ABSTRACT FROM AUTHOR]

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size-exclusion chromatography (SEC) separation using high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method’s performance and applicability to in-process samples were characterized. The assay’s repeatability variation was

Abstract Background Vesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies (Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel. Methods We randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification. Results DNA barcoding of adult black flies detected four species in 2020: Simulium meridionale (N = 158), S. mediovittatum (N = 83), S. robynae (N = 26) and S. griseum/notatum (N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum, 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections. Conclusions Our results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted. Graphical Abstract

Abstract Background Vesicular stomatitis virus (VSV) is a typical non-segmented negative-sense RNA virus of the genus Vesiculovirus in the family Rhabdoviridae. VSV can infect a wide range of animals, including humans, with oral blister epithelial lesions. VSV is an excellent model virus with a wide range of applications as a molecular tool, a vaccine vector, and an oncolytic vector. To further understand the interaction between VSV and host cells and to provide a theoretical basis for the application prospects of VSV, we analyzed the expression of host differentially expressed genes (DEGs) during VSV infection using RNA-Seq. Results Our analyses found a total of 1015 differentially expressed mRNAs and 161 differentially expressed LncRNAs in BHK-21 cells infected with VSV for 24 h compared with controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment showed that the differentially expressed lncRNAs and their target genes were mainly concentrated in pathways related to apoptosis, cancer, disease, and immune system activation, including the TNF, P53, MAPK, and NF-kappaB signaling pathways. The differentially expressed lncRNA can modulate immune processes by regulating genes involved in these signaling transmissions. Ten randomly selected DEGs, namely, Il12rb2, F2, Masp2, Mcl1, FGF18, Ripk1, Fas, BMF, POLK, and JAG1, were validated using RT-qPCR. As predicted through RNA-Seq analysis, these DEGs underwent either up- or downregulation, suggesting that they may play key regulatory roles in the pathways mentioned previously. Conclusions Our study showed that VSV infection alters the host metabolic network and activates immune-related pathways, such as MAPK and TNF. The above findings provide unique insights for further study of the mechanism of VSV–host interactions and, more importantly, provide a theoretical basis for VSV as an excellent vaccine carrier.

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.

Vesicular stomatitis virus (VSV), comprising vesicular stomatitis New Jersey virus (VSNJV) and vesicular stomatitis Indiana virus (VSIV), emerges from its focus of endemic transmission in Southern Mexico to cause sporadic livestock epizootics in the Western United States. A dearth of information on the role of potential arthropod vectors in the endemic region hampers efforts to identify factors that enable endemicity and predict outbreaks. In a two-year, longitudinal study at five cattle ranches in Chiapas, Mexico, insect taxa implicated as VSV vectors (blackflies, sandflies, biting midges, and mosquitoes) were collected and screened for VSV RNA, livestock vesicular stomatitis (VS) cases were monitored, and serum samples were screened for neutralizing antibodies. VS cases were reported during the rainy (n = 20) and post-rainy (n = 2) seasons. Seroprevalence against VSNJV in adult cattle was very high (75–100% per ranch) compared with VSIV (0.6%, all ranches). All four potential vector taxa were sampled, and VSNJV RNA was detected in each of them (11% VSNJV-positive of 874 total pools), while VSIV RNA was only detected in four pools of mosquitoes. Our findings indicate that VSNJV is the dominant serotype across our sampling sites with a variety of potential insect vectors involved in its transmission throughout the year. Although no livestock cases were reported in Chiapas during the dry season, VSNJV was detected in insects during this period, suggesting that mechanisms other than transmission from livestock support VSV endemicity.

For the first time, we describe phylogenomic signatures of an epidemic lineage of vesicular stomatitis Indiana virus (VSIV). We applied multiple evolutionary analyses to a dataset of 87 full-length genome sequences representing the circulation of an epidemic VSIV lineage in the US between 2019 and 2020. Based on phylogenetic analyses, we predicted the ancestral relationship of this lineage with a specific group of isolates circulating in the endemic zone of Chiapas, Mexico. Subsequently, our findings indicate that the lineage diversified into at least four different subpopulations during its circulation in the US. We identified single nucleotide polymorphisms (SNPs) that differentiate viral subpopulations and assessed their potential relevance using comparative phylogenetic methods, highlighting the preponderance of synonymous mutations during the differentiation of these populations. Purifying selection was the main evolutionary force favoring the conservation of this epidemic phenotype, with P and G genes as the main drivers of the evolution of this lineage. Our analyses identified multiple codon sites under positive selection and the association of these sites with specific functional domains at P, M, G, and L proteins. Based on ancestral reconstruction analyses, we showed the potential relevance of some of the sites identified under positive selection to the adaptation of the epidemic lineage at the population level. Finally, using a representative group of viruses from Colorado, we established a positive correlation between genetic and geographical distances, suggesting that positive selection on specific codon positions might have favored the adaptation of different subpopulations to circulation in specific geographical settings. Collectively, our study reveals the complex dynamics that accompany the evolution of an epidemic lineage of VSIV in nature. Our analytical framework provides a model for conducting future evolutionary analyses. The ultimate goal is to support the implementation of an early warning system for vesicular stomatitis virus in the US, enabling early detection of epidemic precursors from Mexico.

Background: Vesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies (Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel. Methods: We randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification. Results: DNA barcoding of adult black flies detected four species in 2020: Simulium meridionale (N = 158), S. mediovittatum (N = 83), S. robynae (N = 26) and S. griseum/notatum (N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum, 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections. Conclusions: Our results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted. [ABSTRACT FROM AUTHOR]

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Background: Vesicular stomatitis virus (VSV) is a typical non-segmented negative-sense RNA virus of the genus Vesiculovirus in the family Rhabdoviridae. VSV can infect a wide range of animals, including humans, with oral blister epithelial lesions. VSV is an excellent model virus with a wide range of applications as a molecular tool, a vaccine vector, and an oncolytic vector. To further understand the interaction between VSV and host cells and to provide a theoretical basis for the application prospects of VSV, we analyzed the expression of host differentially expressed genes (DEGs) during VSV infection using RNA-Seq. Results: Our analyses found a total of 1015 differentially expressed mRNAs and 161 differentially expressed LncRNAs in BHK-21 cells infected with VSV for 24 h compared with controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment showed that the differentially expressed lncRNAs and their target genes were mainly concentrated in pathways related to apoptosis, cancer, disease, and immune system activation, including the TNF, P53, MAPK, and NF-kappaB signaling pathways. The differentially expressed lncRNA can modulate immune processes by regulating genes involved in these signaling transmissions. Ten randomly selected DEGs, namely, Il12rb2, F2, Masp2, Mcl1, FGF18, Ripk1, Fas, BMF, POLK, and JAG1, were validated using RT-qPCR. As predicted through RNA-Seq analysis, these DEGs underwent either up- or downregulation, suggesting that they may play key regulatory roles in the pathways mentioned previously. Conclusions: Our study showed that VSV infection alters the host metabolic network and activates immune-related pathways, such as MAPK and TNF. The above findings provide unique insights for further study of the mechanism of VSV–host interactions and, more importantly, provide a theoretical basis for VSV as an excellent vaccine carrier. [ABSTRACT FROM AUTHOR]

Simple Summary: Vesicular stomatitis virus (VSV) infects cows, horses, and pigs, economically impacting livestock producers due to animal production losses, quarantines, and animal movement/trade restrictions. Typically, VSV is transmitted from animal to animal by direct contact, but it is also transmitted by insects such as Culicoides biting midges. These tiny flies can ingest virus particles when blood feeding on an infected animal, multiply them inside their bodies, and then transmit them to other animals the next time they feed. In addition, midges are also able to pass the virus from one to another with extremely high efficiency when they mate, even though they carry very little virus in their bodies. Through this mechanism, VSV may be maintained and overwinter in midges and appear again in livestock the next summer once the insects start feeding on blood again. Our research shows that one reason midges can transmit VSV to other midges so efficiently is because viruses that come from insect cells have an increased ability to infect more insect cells. This helps explain the midge-to-midge infection efficiency and highlights the importance of Culicoides midges in VSV maintenance and transmission. Vesicular stomatitis virus (VSV) is an arthropod-borne virus affecting livestock. In the United States, sporadic outbreaks result in significant economic losses. During epizootics, Culicoides biting midges are biological vectors and key to the geographic expansion of outbreaks. Additionally, Culicoides may play a role in VSV overwintering because females and males are capable of highly efficient venereal transmission, despite their relatively low virus titers. We hypothesized that VSV propagated within a midge has increased fitness for subsequent midge infections. To evaluate the potential host-specific fitness increase, we propagated three viral isolates of VSV in porcine skin fibroblasts and Culicoides cell lines. We then evaluated the viral infection dynamics of the different cell-source groups in Culicoides sonorensis. Our results indicate that both mammalian- and insect-derived VSV replicate well in midges inoculated via intrathoracic injection, thereby bypassing the midgut barriers. However, when the virus was required to infect and escape the midgut barrier to disseminate after oral acquisition, the insect-derived viruses had significantly higher titers, infection, and dissemination rates than mammalian-derived viruses. Our research suggests that VSV replication in Culicoides cells increases viral fitness, facilitating midge-to-midge transmission and subsequent replication, and further highlights the significance of Culicoides midges in VSV maintenance and transmission dynamics. [ABSTRACT FROM AUTHOR]

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSV MT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSV MT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSV ΔGMT -S Δ21 , potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding S Δ21 of Delta-variant can induce more potent immune responses compared with those encoding S Δ21 of Omicron- or WA1-strain. VSV MT is a promising platform to develop a mucosal vaccine for countering COVID-19. Attenuated VSV MT -based vaccines successfully induce fast, endurable humoral and cellular immune responses in hamsters and hACE2-mouse, thus serving as promising candidates to develop next-generation mucosal vaccines against COVID-19. [Display omitted] [ABSTRACT FROM AUTHOR]

ABSTRACT Rubella virus (RuV) is an enveloped plus-sense RNA virus and a member of the Rubivirus genus. RuV infection in pregnant women can lead to miscarriage or an array of severe birth defects known as congenital rubella syndrome. Novel rubiviruses were recently discovered in various mammals, highlighting the spillover potential of other rubiviruses to humans. Many features of the rubivirus infection cycle remain unexplored. To promote the study of rubivirus biology, here, we generated replication-competent recombinant VSV-RuV (rVSV-RuV) encoding the RuV transmembrane glycoproteins E2 and E1. Sequencing of rVSV-RuV showed that the RuV glycoproteins acquired a single-point mutation W448R in the E1 transmembrane domain. The E1 W448R mutation did not detectably alter the intracellular expression, processing, glycosylation, colocalization, or dimerization of the E2 and E1 glycoproteins. Nonetheless, the mutation enhanced the incorporation of RuV E2/E1 into VSV particles, which bud from the plasma membrane rather than the RuV budding site in the Golgi. Neutralization by E1 antibodies, calcium dependence, and cell tropism were comparable between WT-RuV and either rVSV-RuV or RuV containing the E1 W448R mutation. However, the E1 W448R mutation strongly shifted the threshold for the acid pH-triggered virus fusion reaction, from pH 6.2 for the WT RuV to pH 5.5 for the mutant. These results suggest that the increased resistance of the mutant RuV E1 to acidic pH promotes the ability of viral envelope proteins to generate infectious rVSV and provide insights into the regulation of RuV fusion during virus entry and exit.IMPORTANCERubella virus (RuV) infection in pregnant women can cause miscarriage or severe fetal birth defects. While a highly effective vaccine has been developed, RuV cases are still a significant problem in areas with inadequate vaccine coverage. In addition, related viruses have recently been discovered in mammals, such as bats and mice, leading to concerns about potential virus spillover to humans. To facilitate studies of RuV biology, here, we generated and characterized a replication-competent vesicular stomatitis virus encoding the RuV glycoproteins (rVSV-RuV). Sequence analysis of rVSV-RuV identified a single-point mutation in the transmembrane region of the E1 glycoprotein. While the overall properties of rVSV-RuV are similar to those of WT-RuV, the mutation caused a marked shift in the pH dependence of virus membrane fusion. Together, our studies of rVSV-RuV and the identified W448R mutation expand our understanding of rubivirus biology and provide new tools for its study.

The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.

Intratumoural oncolytic virotherapy may have promise as a means to debulk and downstage inoperable tumours in preparation for successful surgery. Here, we describe the unique case of a 50-year-old self-experimenting female virologist with locally recurrent muscle-invasive breast cancer who was able to proceed to simple, non-invasive tumour resection after receiving multiple intratumoural injections of research-grade virus preparations, which first included an Edmonston-Zagreb measles vaccine strain (MeV) and then a vesicular stomatitis virus Indiana strain (VSV), both prepared in her own laboratory. The intratumoural virus therapy was well tolerated. Frequent imaging studies and regular clinical observations documenting size, consistency and mobility of the injected tumour demonstrate that both the MeV- and VSV-containing parts of the protocol contributed to the overall favourable response. Two months after the start of the virus injections, the shrunken tumour was no longer invading the skin or underlying muscle and was surgically excised. The excised tumour showed strong lymphocytic infiltration, with an increase in CD20-positive B cells, CD8-positive T cells and macrophages. PD-L1 expression was detected in contrast to the baseline PD-L1-negative phenotype. The patient completed one-year trastuzumab adjuvant therapy and remains well and recurrence-free 45 months post-surgery. Although an isolated case, it encourages consideration of oncolytic virotherapy as a neoadjuvant treatment modality.

There are currently no prophylactic vaccines licensed to protect against Lassa fever caused by Lassa virus (LASV) infection. The Emergent BioSolutions (EBS) vaccine candidate, EBS-LASV, is being developed for the prevention of Lassa fever. EBS-LASV is a live-attenuated recombinant Vesicular Stomatitis Virus (rVSV)-vectored vaccine encoding the surface glycoprotein complex (GPC) from LASV and has two attenuating vector modifications: a gene shuffle of the VSV N gene and a deletion of the VSV G gene. Preclinical studies were performed to evaluate EBS-LASV’s neurovirulence potential following intracranial (IC) injection and to determine the biodistribution and vector replication following intramuscular (IM) inoculation in mice. In addition, the potential EBS-LASV toxicity was assessed using repeated-dose IM EBS-LASV administration to rabbits. All mice receiving the IC injection of EBS-LASV survived, while mice administered the unattenuated control vector did not. The vaccine was only detected in the muscle at the injection site, draining lymph nodes, and the spleen over the first week following IM EBS-LASV injection in mice, with no detectable plasma viremia. No toxicity was observed in rabbits receiving a three-dose regimen of EBS-LASV. These studies demonstrate that EBS-LASV is safe when administered to animals and supported a first-in-human dose-escalation, safety, and immunogenicity clinical study.

Filoviruses, like the Marburg (MARV) and Ebola (EBOV) viruses, have caused outbreaks associated with significant hemorrhagic morbidity and high fatality rates. Vaccines offer one of the best countermeasures for fatal infection, but to date only the EBOV vaccine has received FDA licensure. Given the limited cross protection between the EBOV vaccine and Marburg hemorrhagic fever (MHF), we analyzed the protective efficacy of a similar vaccine, rVSV-MARV, in the lethal cynomolgus macaque model. NHPs vaccinated with a single dose (as little as 1.6 × 107 pfu) of rVSV-MARV seroconverted to MARV G-protein prior to challenge on day 42. Vaccinemia was measured in all vaccinated primates, self-resolved by day 14 post vaccination. Importantly, all vaccinated NHPs survived lethal MARV challenge, and showed no significant alterations in key markers of morbid disease, including clinical signs, and certain hematological and clinical chemistry parameters. Further, apart from one primate (from which tissues were not collected and no causal link was established), no pathology associated with Marburg disease was observed in vaccinated animals. Taken together, rVSV-MARV is a safe and efficacious vaccine against MHF in cynomolgus macaques.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3–6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2–8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Objective To investigate the effect and mechanism of metabolite γ-aminobutyric acid (GABA) on viral infection to RAW264.7 cell lines and mouse primary peritoneal macrophages. Methods RAW264.7 cell lines were treated with 0,10,30,50 and 70 mmol/L GABA for 10 hours, and the cellular viability was detected by CCK-8 assay. RAW264.7 cell line was pre-treated with the indicated dose of GABA for 2 hours and then infected with recombinant green fluorescent protein-expressing vesicular stomatitis virus(GFP-VSV) or VSV for 8 hours. The level of GFP-VSV in RAW264.7 cell line was observed by fluorescence microscopy and was detected by flow cytometry. The level of VSV RNA, Ifnb1 and Il-6 mRNA was detected by RT-qPCR. Mouse primary peritoneal macrophages were pre-treated with 0,10, 30, 50 and 70 mmol/L GABA for 2 hours and then infected with VSV for 8 hours. The RNA level of VSV was detected by RT-qPCR. Results It was found that 10,30,50 and 70 mmol/L GABA failed to affect the viability of RAW264.7 cell line. GABA significantly increased the expression of GFP-VSV protein and the level of VSV RNA, Ifnb1 and Il-6 mRNA in RAW264.7 cell line in a dose-dependent manner (all P＜0.05). The level of VSV RNA in mouse primary peritoneal macrophages was also increased in a dose-dependent manner(P＜0.05). Conclusions Metabolite GABA promotes VSV infection to RAW264.7 cell line and mouse primary peritoneal macrophages in a dose-dependent manner, and the mRNA level of Ifnb1 and Il-6 is also increased.

In multiple models of oncolytic virotherapy, it is common to see an early anti-tumor response followed by recurrence. We have previously shown that frontline treatment with oncolytic VSV-IFN-β induces APOBEC proteins, promoting the selection of specific mutations that allow tumor escape. Of these mutations in B16 melanoma escape (ESC) cells, a C-T point mutation in the cold shock domain-containing E1 (CSDE1) gene was present at the highest frequency, which could be used to ambush ESC cells by vaccination with the mutant CSDE1 expressed within the virus. Here, we show that the evolution of viral ESC tumor cells harboring the escape-promoting CSDE1C-T mutation can also be exploited by a virological ambush. By sequential delivery of two oncolytic VSVs in vivo, tumors which would otherwise escape VSV-IFN-β oncolytic virotherapy could be cured. This also facilitated the priming of anti-tumor T cell responses, which could be further exploited using immune checkpoint blockade with the CD200 activation receptor ligand (CD200AR-L) peptide. Our findings here are significant in that they offer the possibility to develop oncolytic viruses as highly specific, escape-targeting viro-immunotherapeutic agents to be used in conjunction with recurrence of tumors following multiple different types of frontline cancer therapies.

IntroductionAfrican swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines.MethodsIn order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination.ResultsThe results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%.ConclusionOverall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

Infection with Marburg virus (MARV), the causative agent of Marburg virus disease (MVD), results in haemorrhagic disease and high case fatality rates (>40%) in humans. Despite its public health relevance, there are no licensed vaccines or therapeutics to prevent or treat MVD. A vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) is currently in clinical development. Previously, a single 10 million PFU dose of VSV-MARV administered 1–5 weeks before lethal MARV challenge conferred uniform protection in nonhuman primates (NHPs), demonstrating fast-acting potential. Additionally, our group recently demonstrated that even a low dose VSV-MARV (1000 PFU) protected NHPs when given 7 days before MARV challenge. In this study, we longitudinally profiled the transcriptional responses of NHPs vaccinated with this low dose of VSV-MARV either 14 or 7 days before lethal MARV challenge. NHPs vaccinated 14 days before challenge presented with transcriptional changes consistent with an antiviral response before challenge. Limited gene expression changes were observed in the group vaccinated 7 days before challenge. After challenge, genes related to lymphocyte-mediated immunity were only observed in the group vaccinated 14 days before challenge, indicating that the length of time between vaccination and challenge influenced gene expression. Our results indicate that a low dose VSV-MARV elicits distinct immune responses that correlate with protection against MVD. A low dose of VSV-MARV should be evaluated in clinical rails as it may be an option to deliver beneficial public health outcomes to more people in the event of future outbreaks.

Taï Forest virus (TAFV) is a lesser-known ebolavirus that causes lethal infections in chimpanzees and is responsible for a single human case. Limited research has been done on this human pathogen; however, with the recent emergence of filoviruses in West Africa, further investigation and countermeasure development against this virus is warranted. We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the TAFV glycoprotein as the viral antigen and assessed it for protective efficacy in nonhuman primates (NHPs). Following a single high-dose vaccination, NHPs developed antigen-specific binding and neutralizing antibodies as well as modest T cell responses. Importantly, all vaccinated NHPs were uniformly protected from disease after lethal TAFV challenge while the naïve control group succumbed to the disease. Histopathologic lesions consistent with filovirus disease were present in control NHPs but were not observed in vaccinated NHPs. Transcriptional analysis of whole blood samples obtained after vaccination and challenge was performed to gain insight into molecular underpinnings conferring protection. Differentially expressed genes (DEG) detected 7 days post-vaccination were enriched to processes associated with innate immunity and antiviral responses. Only a small number of DEG was detected in vaccinated NHPs post-challenge while over 1,000 DEG were detected in control NHPs at end-stage disease which mapped to gene ontology terms indicative of defense responses and inflammation. Taken together, this data demonstrates the effective single-dose protection of the VSV-TAFV vaccine, and its potential for use in outbreaks.

Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus. [ABSTRACT FROM AUTHOR]

Mat cells (MCs) are located in the skin and mucous membranes at points where the body meets the environment. When activated, MCs release inflammatory cytokines, which help the immune system to fight viruses. MCs produce, and have receptors for interferons (IFNs), which belong to a family of cytokines recognized for their antiviral properties. Previously, we reported that MCs produced proinflammatory cytokines in response to a recombinant vesicular stomatitis virus (rVSVΔm51) and that IFNAR signaling was required to down-modulate these responses. Here, we have demonstrated that UV-irradiated rVSVΔm51 did not cause any inflammatory cytokines in either in vitro cultured mouse IFNAR-intact (IFNAR+/+), or in IFNAR-knockout (IFNAR−/−) MCs. However, the non-irradiated virus was able to replicate more effectively in IFNAR−/− MCs and produced a higher level of inflammatory cytokines compared with the IFNAR+/+ MCs. Interestingly, MCs lacking IFNAR expression displayed reduced levels of reactive oxygen species (ROS) compared with IFNAR+/+ MCs. Additionally, upon the viral infection, these IFNAR−/− MCs were found to coexist with many dying cells within the cell population. Based on our findings, IFNAR-intact MCs exhibit a lower rate of rVSVΔm51 infectivity and lower levels of cytokines while demonstrating higher levels of ROS. This suggests that MCs with intact IFNAR signaling may survive viral infections by producing cell-protective ROS mechanisms and are less likely to die than IFNAR−/− cells. [ABSTRACT FROM AUTHOR]

Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies demonstrated that human PDAC cell lines are highly variable in their permissiveness to OVs. Mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during OV and other cancer therapies, have never been examined systematically for the impact of intertumoral heterogeneity (the difference s observed between tumors in different patients) on OV virus efficacy. Here, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines (C57BL6 genetic background): Panc02 (derived from chemically induced PDAC; also known as Pan02), and two cell lines originated from PDACs developed in two different KPC (KrasG12D, Trp53R172H, and PDX-1-Cre) mouse models. Our study (i) characterized the ability of a widely used attenuated oncolytic vesicular stomatitis virus VSV-△M51-GFP to infect, replicate in, and kill mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-△M51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-△M51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. The exome analysis demonstrated that more VSV-△M51-GFP-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. IMPORTANCE: Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies using various human PDAC cell lines demonstrated that they are highly variable in their permissiveness to OVs. In this study, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during cancer therapies. Mouse PDAC cell lines showed high divergence in their permissiveness to oncolytic vesicular stomatitis virus (VSV), which negatively correlated with their abilities to mount innate antiviral responses. Also, we discovered that more VSV-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. Our study provides essential information about three model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against different PDACs in immunocompetent mice. [ABSTRACT FROM AUTHOR]

Objective To evaluate the antiviral ability of cardiac glycosides of digoxin and oleandrin. Methods Human adenocarcinoma alveolar basal epithelial cell line （A549） and African green monkey kidney cell line （vero） were used for subsequent experiment. The cytotoxicity of cardiac glycosides of digoxin and oleandrin was assessed by CCK-8 assay. The effects of digoxin and oleandrin on the replication of vesicular stomatitis virus （VSV）， encephalomyocarditis virus （EMCV）， Sendai virus （SeV） and influenza A virus （H1N1） were determined by reverse transcription-real-time quantitative PCR （RT-qPCR） and Western blot. Vero cells with natural interferon （IFN） signaling pathway defects combined with fluorescence microscopy， RT-qPCR and Western blot were employed to analyze whether the antiviral mechanism of digoxin and oleandrin depended on IFN signaling pathway. In vero cells with ATP1A1 （sodium potassium ATPase protein 1） （ATP1A1-shRNA） knockdown， intracellular virus replication and antiviral IFN response were detected by RT-qPCR and Western blot. Results Digoxin and oleandrin could inhibit the expression levels of VSV， H1N1， SeV and EMCV mRNA. The combination of IFN-deficient vero cells demonstrated that digoxin and oleandrin played an antiviral role independent of IFN signaling pathway. Meantime， digoxin and oleandrin exerted no antiviral effect in vero cells with ATP1A1 knockdown. Conclusion Digoxin， oleandrin and other potent cardiac glycosides have a broad spectrum of antiviral capability mainly through the inhibition of Na+/K+ ion transporters intra- and extra-cell membrane.

The phospho- (P) protein, the co-factor of the RNA polymerase large (L) protein, of vesicular stomatitis virus (VSV, a prototype of nonsegmented negative-strand RNA viruses) plays pivotal roles in transcription and replication. However, the precise mechanism underlying the transcriptional transactivation by the P protein has remained elusive. Here, using an in vitro transcription system and a series of deletion mutants of the P protein, we mapped a region encompassing residues 51-104 as a transactivation domain (TAD) that is critical for terminal de novo initiation, the initial step of synthesis of the leader RNA and anti-genome/genome, with the L protein. Site-directed mutagenesis revealed that conserved amino acid residues in three discontinuous L-binding sites within the TAD are essential for the transactivation activity of the P protein or important for maintaining its full activity. Importantly, relative inhibitory effects of TAD point mutations on synthesis of the full-length leader RNA and mRNAs from the 3'-terminal leader region and internal genes, respectively, of the genome were similar to those on terminal de novo initiation. Furthermore, any of the examined TAD mutations did not alter the gradient pattern of mRNAs synthesized from internal genes, nor did they induce the production of readthrough transcripts. These results suggest that these TAD mutations impact mainly terminal de novo initiation but rarely other steps (e.g., elongation, termination, internal initiation) of single-entry stop-start transcription. Consistently, the mutations of the essential or important amino acid residues within the P TAD were lethal or deleterious to VSV replication in host cells. [ABSTRACT FROM AUTHOR]

Determination of the size, density, and mass of viral particles can provide valuable information to support process and formulation studies in clinical development. Analytical ultracentrifugation (AUC), as a first principal method, has been shown to be a beneficial tool for the characterization of the non-enveloped adeno associated virus (AAV). Here, we demonstrate the suitability of AUC for the challenging characterization of a representative for enveloped viruses, which usually are expected to exhibit higher dispersity than non-enveloped viruses. Specifically, the vesicular stomatitis virus (VSV)-based oncolytic virus VSV-GP was used to evaluate potential occurrence of non-ideal sedimentation by testing different rotor speeds and loading concentrations. The partial specific volume was determined via density gradients and density contrast experiments. Additionally, nanoparticle tracking analysis (NTA) was used to determine the hydrodynamic diameter of VSV-GP particles to calculate their molecular weight via the Svedberg equation. Overall, this study demonstrates the applicability of AUC and NTA for the characterization of size, density, and molar mass of an enveloped virus, namely VSV-GP. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect and mechanism of metabolite γ-aminobutyric acid (GABA) on viral infection to RAW264.7 cell lines and mouse primary peritoneal macrophages. Methods RAW264.7 cell lines were treated with 0,10,30,50 and 70 mmol/L GABA for 10 hours, and the cellular viability was detected by CCK-8 assay. RAW264.7 cell line was pre-treated with the indicated dose of GABA for 2 hours and then infected with recombinant green fluorescent protein-expressing vesicular stomatitis virus(GFP-VSV) or VSV for 8 hours. The level of GFP-VSV in RAW264.7 cell line was observed by fluorescence microscopy and was detected by flow cytometry. The level of VSV RNA, Ifnb1 and Il-6 mRNA was detected by RT-qPCR. Mouse primary peritoneal macrophages were pre-treated with 0,10, 30, 50 and 70 mmol/L GABA for 2 hours and then infected with VSV for 8 hours. The RNA level of VSV was detected by RT-qPCR. Results It was found that 10,30,50 and 70 mmol/L GABA failed to affect the viability of RAW264.7 cell line. GABA significantly increased the expression of GFP-VSV protein and the level of VSV RNA, Ifnb1 and Il-6 mRNA in RAW264.7 cell line in a dose-dependent manner (all P＜0.05). The level of VSV RNA in mouse primary peritoneal macrophages was also increased in a dose-dependent manner(P＜0.05). Conclusions Metabolite GABA promotes VSV infection to RAW264.7 cell line and mouse primary peritoneal macrophages in a dose-dependent manner, and the mRNA level of Ifnb1 and Il-6 is also increased. Objective To investigate the effect and mechanism of metabolite γ-aminobutyric acid (GABA) on viral infection to RAW264.7 cell lines and mouse primary peritoneal macrophages. Methods RAW264.7 cell lines were treated with 0,10,30,50 and 70 mmol/L GABA for 10 hours, and the cellular viability was detected by CCK-8 assay. RAW264.7 cell line was pre-treated with the indicated dose of GABA for 2 hours and then infected with recombinant green fluorescent protein-expressing vesicular stomatitis virus(GFP-VSV) or VSV for 8 hours. The level of GFP-VSV in RAW264.7 cell line was observed by fluorescence microscopy and was detected by flow cytometry. The level of VSV RNA, Ifnb1 and Il-6 mRNA was detected by RT-qPCR. Mouse primary peritoneal macrophages were pre-treated with 0,10, 30, 50 and 70 mmol/L GABA for 2 hours and then infected with VSV for 8 hours. The RNA level of VSV was detected by RT-qPCR. Results It was found that 10,30,50 and 70 mmol/L GABA failed to affect the viability of RAW264.7 cell line. GABA significantly increased the expression of GFP-VSV protein and the level of VSV RNA, Ifnb1 and Il-6 mRNA in RAW264.7 cell line in a dose-dependent manner (all P＜0.05). The level of VSV RNA in mouse primary peritoneal macrophages was also increased in a dose-dependent manner(P＜0.05). Conclusions Metabolite GABA promotes VSV infection to RAW264.7 cell line and mouse primary peritoneal macrophages in a dose-dependent manner, and the mRNA level of Ifnb1 and Il-6 is also increased. [ABSTRACT FROM AUTHOR]

Objective To evaluate the antiviral ability of cardiac glycosides of digoxin and oleandrin. Methods Human adenocarcinoma alveolar basal epithelial cell line (A549) and African green monkey kidney cell line (vero) were used for subsequent experiment. The cytotoxicity of cardiac glycosides of digoxin and oleandrin was assessed by CCK-8 assay. The effects of digoxin and oleandrin on the replication of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), Sendai virus (SeV) and influenza A virus (HlNl) were determined by reverse transcription-real-time quantitative PCR (RT-qPCR) and Western blot. Vero cells with natural interferon (IFN) signaling pathway defects combined with fluorescence microscopy, RT-qPCR and Western blot were employed to analyze whether the antiviral mechanism of digoxin and oleandrin depended on IFN signaling pathway. In vero cells with ATPlAl (sodium potassium ATPase protein 1) (ATPlAl-shRNA) knockdown, intracellular virus replication and antiviral IFN response were detected by RT-qPCR and Western blot. Results Digoxin and oleandrin could inhibit the expression levels of VSV, HlNl, Se V and EMCV mRNA. The combination of IFN-deficient vero cells demonstrated that digoxin and oleandrin played an antiviral role independent of IFN signaling pathway. Meantime, digoxin and oleandrin exerted no antiviral effect in vero cells with ATPlAl knockdown. Conclusion Digoxin, oleandrin and other potent cardiac glycosides have a broad spectrum of antiviral capability mainly through the inhibition of Na+/K+ ion transporters intra- and extra-cell membrane. [ABSTRACT FROM AUTHOR]

Viral-like particles (VLPs) because of their non-infectious and high immunogenic properties have important applications in diagnostics, drug delivery, and vaccine production. They also serve as an attractive model system to study virus assembly and fusion processes. Unlike other flaviviruses, Dengue virus (DENV) is not very efficient in the production of VLPs on the expression of DENV structural proteins. On the other hand, the stem region and transmembrane region (TM) of G protein of Vesicular Stomatitis virus (VSV) alone are sufficient for budding. Here we generated chimeric VLPs replacing regions of stem and transmembrane domain (STEM) or only transmembrane domain (TM) of E protein of DENV-2 with corresponding regions of VSV G protein. Both chimeric proteins secreted VLPs at higher levels than the wild type (2–4 folds) without any significant change in the expression in the cell. Chimeric VLPs could be recognized by a conformational monoclonal antibody, 4G2. They were also found to interact with dengue-infected patient sera effectively thus implying that their antigenic determinants are preserved. In addition, they were able to bind to its putative receptor, heparin with similar affinity as the parent counterpart thus retaining its functional property. However, cell-cell fusion revealed that there is no significant increase in the fusion ability of chimeras as compared to the parent clone, whereas VSV G protein displayed high cell-cell fusion activity. Overall, this study suggests that chimeric dengue VLPs can be taken forward for their likely potential as vaccine production and serodiagnosis. • Chimeric dengue E protein exhibit increased VLP secretion. • Chimeric dengue VLPs retain antigenicity and conformation. • Chimeric dengue VLPs show a slight increase in fusion capacity. [ABSTRACT FROM AUTHOR]

HIV subtypes B and C together account for around 60% of HIV-1 cases worldwide. We evaluated the safety and immunogenicity of a subtype B DNA vaccine prime followed by a subtype C viral vector boost. Fourteen healthy adults received DNA plasmid encoding HIV-1 subtype B nef/tat/vif and env (n = 11) or placebo (n = 3) intramuscularly (IM) via electroporation (EP) at 0, 1, and 3 months, followed by IM injection of recombinant vesicular stomatitis virus encoding subtype C Env or placebo at 6 and 9 months. Participants were assessed for safety, tolerability of EP, and Env-specific T-cell and antibody responses. EP was generally well tolerated, although some device-related adverse events did occur, and vaccine reactogenicity was mild to moderate. The vaccine stimulated Env-specific CD4 + T-cell responses in greater than 80% of recipients, and CD8 + T-cell responses in 30%. Subtype C Env-specific IgG binding antibodies (bAb) were elicited in all vaccine recipients, and antibody-dependent cell-mediated cytotoxicity (ADCC) responses to vaccine-matched subtype C targets in 80%. Negligible V1/V2 and neutralizing antibody (nAb) responses were detected. This prime/boost regimen was safe and tolerable, with some device-related events, and immunogenic. Although immunogenicity missed targets for an HIV vaccine, the DNA/rVSV platform may be useful for other applications. Trial registration. Clinicaltrials.gov : NCT02654080. [ABSTRACT FROM AUTHOR]