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.

Application and development of a TaqMan-based real-time PCR assay for rapid detection of snakehead vesiculovirus.

Snakehead vesiculovirus (SHVV) is one of the primary pathogens responsible for viral diseases in the snakehead fish. A TaqMan-based real-time PCR assay was established for the rapid detection and quantification of SHVV in this study. Specific primers and fluorescent probes were designed for phosphoprotein (P) gene, and after optimizing the reaction conditions, the results indicated that the detection limit of this method could reach 37.1 copies, representing a 100-fold increase in detection sensitivity compared to RT-PCR. The specificity testing results revealed that this method exhibited no cross-reactivity with ISKNV, LMBV, RSIV, RGNNV, GCRV, and CyHV-2. Repetition experiments demonstrated that both intra-batch and inter-batch coefficients of variation were not higher than 1.66%. Through in vitro infection experiments monitoring the quantitative changes of SHVV in different tissues, the results indicated that the liver and spleen exhibited the highest viral load at 3 poi. The TaqMan-based real-time PCR method established in this study exhibits high sensitivity, excellent specificity, and strong reproducibility. It can be employed for rapid detection and viral load monitoring of SHVV, thus providing a robust tool for the clinical diagnosis and pathogen research of SHVV.

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.

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.

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.

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.

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.

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.

A single-point mutation in the rubella virus E1 glycoprotein promotes rescue of recombinant vesicular stomatitis virus.

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.

Snakehead vesiculovirus hijacks SH3RF1 for replication via mediating K63-linked ubiquitination at K264 of the phosphoprotein.

Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Host proteins Alpha-2-Macroglobulin and LRP1 associate with Chandipura virus.

Chandipura Virus is an emerging tropical pathogen with a high mortality rate among children. No mode of treatment or antivirals exists against CHPV infection, due to little information regarding its host interaction. Studying viral pathogen interaction with its host can not only provide valuable information regarding its propagation strategy, but also on which host proteins interact with the virus. Identifying these proteins and understanding their role in the infection process can provide more stable anti-viral targets. In this study, we focused on identifying host factors that interact with CHPV and may play a critical role in CHPV infection. We are the first to report the successful identification of Alpha-2-Macroglobulin (A2M), a secretory protein of the host that interacts with CHPV. We also established that LRP1 (Low-density lipoprotein receptor-related protein 1) and GRP78 (Glucose regulated protein 78), receptors of A2M, also interact with CHPV. Furthermore, we could also demonstrate that knocking out A2M has a severe effect on viral infection. We conclusively show the interaction of these host proteins with CHPV. Our findings also indicate that these host proteins could play a role in viral entry into the host cell.

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFU mL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

The Utilization and Development of Viral Vectors in Vaccines as a Prophylactic Treatment Against Ebola Virus as an Emerging and Zoonotic Infectious Disease.

Alongside the prescription of commonly used antivirals, such as acyclovir, remdesivir, oseltamivir, and ciprofloxacin, the most efficient way to prevent or treat communicable diseases is by vaccination. Vaccines have been the most efficient way to prevent or treat highly transmissible infectious agents, such as Ebola, Anthrax, and Dengue Fever. Most epidemics of these highly transmissible infectious agents occur in places, such as South America, Central America, Tropical Asia, and Africa, where the availability of resources and access to adequate healthcare are limited. However, recent events in history have proven that even with access to resources and proper healthcare, those in firstworld countries are not invincible when it comes to infectious diseases and epidemics. The Ebola virus outbreak in West Africa highlighted the gaps in therapeutic advancement and readiness and led to the rapid development of novel vaccine approaches. Viral vectors, in the case of the Ebola vaccine the Vesicular Stomatitis Virus (VSV), can be safely used to activate or initiate the innate adaptive immune response to protect against viral infection. When developed properly and with extensive study, novel vaccine approaches allow physicians and health experts to control the rate at which viruses spread or prevent transmission. This review will discuss the advantages of viral vector vaccines, their chemistry and development, and the pathophysiology of the Ebola virus to develop advantageous and efficacious treatments.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks.

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

Vesicular Stomatitis Virus Elicits Early Transcriptome Response in Culicoides sonorensis Cells.

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.

Single virus fingerprinting by widefield interferometric defocus-enhanced mid-infrared photothermal microscopy.

Clinical identification and fundamental study of viruses rely on the detection of viral proteins or viral nucleic acids. Yet, amplification-based and antigen-based methods are not able to provide precise compositional information of individual virions due to small particle size and low-abundance chemical contents (e.g., ~ 5000 proteins in a vesicular stomatitis virus). Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope for high-throughput fingerprinting of single viruses. With the identification of feature absorption peaks, WIDE-MIP reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses. Different nucleic acid signatures of thymine and uracil residue vibrations are obtained to differentiate DNA and RNA viruses. WIDE-MIP imaging further reveals an enriched ß sheet components in DNA varicella-zoster virus proteins. Together, these advances open a new avenue for compositional analysis of viral vectors and elucidating protein function in an assembled virion.

Rhesus macaques show increased resistance to repeated SHIV intrarectal exposure following a heterologous regimen of rVSV vector vaccine expressing HIV antigen.

Despite the human immunodeficiency virus (HIV) pandemic continuing worldwide for 40 years, no vaccine to combat the disease has been licenced for use in at risk populations. Here, we describe a novel recombinant vesicular stomatitis virus (rVSV) vector vaccine expressing modified HIV envelope glycoproteins and Ebola virus glycoprotein. Three heterologous immunizations successfully prevented infection by a different clade SHIV in 60% of non-human primates (NHPs). No trend was observed between resistance and antibody interactions. Resistance to infection was associated with high proportions of central memory T-cell CD69 and CD154 marker upregulation, increased IL-2 production, and a reduced IFN-γ response, offering insight into correlates of protection.

Discontinuous L-binding motifs in the transactivation domain of the vesicular stomatitis virus P protein are required for terminal de novo transcription initiation by the L protein.

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. IMPORTANCE RNA-dependent RNA polymerase L proteins of nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order require their cognate co-factor P proteins or their counterparts for genome transcription and replication. However, exact roles of these co-factor proteins in modulating functions of L proteins during transcription and replication remain unknown. In this study, we revealed that three discrete L-binding motifs within a transactivation domain of the P protein of vesicular stomatitis virus, a prototypic nonsegmented negative-strand RNA virus, are required for terminal de novo initiation mediated by the L protein, which is the first step of synthesis of the leader RNA as well as genome/anti-genome.