Abortive and productive infection of CNS cell types following in vivo delivery of VSV.

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.

Siah2- and LRSAM1-mediated K63-linked ubiquitination of snakehead vesiculovirus nucleoprotein facilitates viral replication.

Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses. IMPORTANCE: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.

Translational success of fundamental virology: a VSV-vectored Ebola vaccine.

Ebola virus disease (EVD) caused by Ebola virus (EBOV) is a severe, often fatal, hemorrhagic disease. A critical component of the public health response to curb EVD epidemics is the use of a replication-competent, recombinant vesicular stomatitis virus (rVSV)-vectored Ebola vaccine, rVSVΔG-ZEBOV-GP (ERVEBO). In this Gem, we will discuss the past and ongoing development of rVSVΔG-ZEBOV-GP, highlighting the importance of basic science and the strength of public-private partnerships to translate fundamental virology into a licensed VSV-vectored Ebola vaccine.

Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer.

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.

Protective Efficacy of Lyophilized Vesicular Stomatitis Virus-Based Vaccines in Animal Model.

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.

Investigating the effect of reduced temperatures on the efficacy of rhabdovirus-based viral vector platforms.

Rhabdoviral vectors can induce lysis of cancer cells. While studied almost exclusively at 37 °C, viruses are subject to a range of temperatures in vivo, including temperatures ≤31 °C. Despite potential implications, the effect of temperatures <37 °C on the performance of rhabdoviral vectors is unknown. We investigated the effect of low anatomical temperatures on two rhabdoviruses, vesicular stomatitis virus (VSV) and Maraba virus (MG1). Using a metabolic resazurin assay, VSV- and MG1-mediated oncolysis was characterized in a panel of cell lines at 28, 31, 34 and 37 °C. The oncolytic ability of both viruses was hindered at 31 and 28 °C. Cold adaptation of both viruses was attempted as a mitigation strategy. Viruses were serially passaged at decreasing temperatures in an attempt to induce mutations. Unfortunately, the cold-adaptation strategies failed to potentiate the oncolytic activity of the viruses at temperatures <37 °C. Interestingly, we discovered that viral replication was unaffected at low temperatures despite the abrogation of oncolytic activity. In contrast, the proliferation of cancer cells was reduced at low temperatures. Equivalent oncolytic effects could be achieved if cells at low temperatures were treated with viruses for longer times. This suggests that rhabdovirus-mediated oncolysis could be compromised at low temperatures in vivo where therapeutic windows are limited.

Natural IgG protects against early dissemination of vesicular stomatitis virus.

Neonatal Fc receptor (FcRn) recycles immunoglobulin G, and inhibition of FcRn is used clinically for treatment of autoimmune diseases. In this work, using the vesicular stomatitis virus (VSV) mouse infection model system, we determined the role of FcRn during virus infection. While induction of neutralizing antibodies and long-term protection of these antibodies was hardly affected in FcRn deficient mice, FcRn deficiency limited the amount of natural IgG (VSV-specific) antibodies. Lack of natural antibodies (nAbs) limited early control of VSV in macrophages, accelerated propagation of virus in several organs, led to the spread of VSV to the neural tissue resulting in fatal outcomes. Adoptive transfer of natural IgG into FcRn deficient mice limited early propagation of VSV in FcRn deficient mice and enhanced survival of FcRn knockout mice. In line with this, vaccination of FcRn mice with very low dose of VSV prior to infection similarly prevented death after infection. In conclusion we determined the importance of nAbs during VSV infection. Lack of FcRn limited nAbs and thereby enhanced the susceptibility to virus infection.

Snakehead vesiculovirus (SHVV) leader RNA interacts with host antiviral factors RPS8 and L13a and promotes virus replication.

To evade host antiviral response, viruses have evolved to take advantage of their noncoding RNAs (ncRNAs). Snakehead vesiculovirus (SHVV), a newly isolated fish rhabdovirus from diseased hybrid snakehead, has caused high mortality to the cultured snakehead fish during the past years in China. However, little is known about the mechanisms of its pathogenicity. Our study revealed that overexpression of the 30-nt leader RNA promoted SHVV replication. RNA-protein binding investigation revealed that SHVV leader RNA could interact with host 40S ribosomal protein S8 (RPS8) and 60S ribosomal protein L13a (L13a). Furthermore, we found that SHVV infection upregulated RPS8 and L13a, and in turn, overexpression of RPS8 or L13a inhibited, while knockdown of RPS8 or L13a promoted, SHVV replication, suggesting that RPS8 and L13a acted as host antiviral factors in response to SHVV infection. In addition, our study revealed that RPS8- or L13a-mediated inhibition of SHVV replication could be restored by co-transfection with leader RNA, suggesting that the interaction between leader RNA and RPS8 or L13a might affect the anti-SHVV effects of RPS8 and L13a. Taken together, these results suggest that SHVV leader RNA can interact with the host antiviral factors RPS8 and L13a, and promote SHVV replication. This study provides a better understanding of the molecular mechanism of the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

Genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc vaccine against SARS-CoV-2 in Sprague-Dawley rats and Beagle dogs.

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a pandemic, prompting rapid vaccine development. Although vaccines are effective, the occurrence of rare adverse events following vaccination highlights the necessity of determining whether the benefits outweigh the risks posed by the infection itself. The recombinant Vesicular Stomatitis Virus (rVSV) platform is a promising vector for vaccines against emerging viruses. However, limited studies have evaluated the genotoxicity and safety pharmacology of this viral vector vaccine, which is crucial to ensure the safety of vaccines developed using this platform. Hence, the present study aimed to assess the genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc COVID-19 vaccine using micronucleus and comet assays, as well as neurobehavioral, body temperature, respiratory, and cardiovascular assessments in Sprague-Dawley rats and beagle dogs. The intramuscular administration of rVSVInd(GML)-mspSGtc at doses up to 1.5 × 109 PFU/animal did not increase the number of bone marrow micronucleated polychromatic erythrocytes or cause liver DNA damage. Additionally, it had no significant impact on neurobehavioral functions in rats and showed marginal temporary changes in body temperature, respiratory rate, heart rate, and electrocardiogram parameters in rats and dogs, all of which resolved within 24 h. Overall, following genotoxicity and pharmacological safety assessments, rVSVInd(GML)-mspSGtc displayed no notable systemic adverse effects in rats and dogs, suggesting its potential as a vaccine candidate for human clinical trials.

Metabolomics captures the differential metabolites in the replication pathway of snakehead vesiculovirus regulated by glutamine.

Snakehead vesiculovirus (SHVV) is a negative-sense single-stranded RNA virus that infects snakehead fish. This virus leads to illness and mortality, causing significant economic losses in the snakehead aquaculture industry. The replication and spread of SHVV in cells, which requires glutamine as a nitrogen source, is accompanied by alterations in intracellular metabolites. However, the metabolic mechanisms underlying the inhibition of viral replication by glutamine deficiency are poorly understood. This study utilized liquid chromatography-mass spectrometry to measure the differential metabolites between the channel catfish Parasilurus asotus ovary cell line infected with SHVV under glutamine-containing and glutamine-deprived conditions. Results showed that the absence of glutamine regulated 4 distinct metabolic pathways and influenced 9 differential metabolites. The differential metabolites PS(16:0/16:0), 5,10-methylene-THF, and PS(18:0/18:1(9Z)) were involved in amino acid metabolism. In the nuclear metabolism functional pathway, differential metabolites of guanosine were observed. In the carbohydrate metabolism pathway, differential metabolites of UDP-d-galacturonate were detected. In the signal transduction pathway, differential metabolites of SM(d18:1/20:0), SM(d18:1/22:1(13Z)), SM(d18:1/24:1(15 Z)), and sphinganine were found. Among them, PS(18:0/18:1(9Z)), PS(16:0/16:0), and UDP-d-galacturonate were involved in the synthesis of phosphatidylserine and glycoprotein. The compound 5,10-methylene-THF provided raw materials for virus replication, and guanosine and sphingosine are related to virus virulence. The differential metabolites may collectively participate in the replication, packaging, and proliferation of SHVV under glutamine deficiency. This study provides new insights and potential metabolic targets for combating SHVV infection in aquaculture through metabolomics approaches.

Vaccine Platform Comparison: Protective Efficacy against Lethal Marburg Virus Challenge in the Hamster Model.

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.

Community composition of black flies during and after the 2020 vesicular stomatitis virus outbreak in Southern New Mexico, USA.

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.

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins.

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

Bivalent VSV Vectors Mediate Rapid and Potent Protection from Andes Virus Challenge in Hamsters.

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human-human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination.

Mechanisms of active diffusion of vesicular stomatitis virus inclusion bodies and cellular early endosomes in the cytoplasm of mammalian cells.

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.

UV-Inactivated rVSV-M2e-Based Influenza Vaccine Protected against the H1N1 Influenza Challenge.

BACKGROUND: To investigate the immune responses and protection ability of ultraviolet light (UV)-inactivated recombinant vesicular stomatitis (rVSV)-based vectors that expressed a fusion protein consisting of four copies of the influenza matrix 2 protein ectodomain (tM2e) and the Dendritic Cell (DC)-targeting domain of the Ebola Glycoprotein (EΔM), (rVSV-EΔM-tM2e). METHOD: In our previous study, we demonstrated the effectiveness of rVSV-EΔM-tM2e to induce robust immune responses against influenza M2e and protect against lethal challenges from H1N1 and H3N2 strains. Here, we used UV to inactivate rVSV-EΔM-tM2e and tested its immunogenicity and protection in BALB/c mice from a mouse-adapted H1N1 influenza challenge. Using Enzyme-Linked Immunosorbent Assay (ELISA) and Antibody-Dependent Cellular Cytotoxicity (ADCC), the influenza anti-M2e immune responses specific to human, avian and swine influenza strains induced were characterized. Likewise, the specificity of the anti-M2e immune responses induced in recognizing M2e antigen on the surface of the cell was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis. RESULTS: Like the live attenuated rVSV-EΔM-tM2e, the UV-inactivated rVSV-EΔM-tM2e was highly immunogenic against different influenza M2e from strains of different hosts, including human, swine, and avian, and protected against influenza H1N1 challenge in mice. The FACS analysis demonstrated that the induced immune responses can recognize influenza M2 antigens from human, swine and avian influenza strains. Moreover, the rVSV-EΔM-tM2e also induced ADCC activity against influenza M2e from different host strains. CONCLUSIONS: These findings suggest that UV-inactivated rVSV-EΔM-tM2e could be used as an inactivated vaccine against influenza viruses.

Histone deacetylase 8 promotes innate antiviral immunity through deacetylation of RIG-I.

Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.