p38MAPK- and GSK3-Mediated Phosphorylation of Snakehead Vesiculovirus Phosphoprotein at Threonine 160 Facilitates Viral Replication.

Phosphoprotein (P), co-factor of the polymerase (large protein, L) of single-stranded negative-sense RNA viruses, is phosphorylated during viral infection and its phosphorylation has been reported to play important roles in viral replication. However, the function of P phosphorylation in viral replication is still far from clear. Snakehead vesiculovirus (SHVV) is a kind of fish rhabdovirus that has caused serious economic losses in snakehead fish culture in China without any effective preventive or therapeutical measures currently. In this study, 4D label-free phosphoproteomics sequencing of SHVV-infected cells identified five phosphorylated sites on SHVV P, among which threonine 160 (T160) was proved to be phosphorylated. Overexpression of wild-type P, but not P-T160A or P-T160E mutant, promoted SHVV replication, suggesting that the T160 phosphorylation on the P protein is critical for SHVV replication. Moreover, we found that T160A or T160E mutation on SHVV P had no effect on the interactions of P-nucleoprotein (N), P-P, or P-L. Further study revealed that p38 mitogen-activated protein kinase (p38MAPK) and glycogen synthase kinase 3 (GSK3) interacted with SHVV P and mediated the T160 phosphorylation. Besides, overexpression of p38MAPK or GSK3 facilitated, while knockdown or activity inhibition of p38MAPK or GSK3 suppressed, SHVV replication. Overall, p38MAPK- and GSK3-mediated phosphorylation of the P protein at T160 is required for SHVV replication, which provided targets for designing anti-SHVV drugs and developing live-attenuated SHVV vaccines. Our study helps understand the role of P phosphorylation in the replication of single-stranded negative-sense RNA viruses. IMPORTANCE Phosphorylation of viral proteins plays important roles in viral replication. Currently, the role of phosphorylation of phosphoprotein (P) in the replication of single-stranded negative-sense RNA viruses is far from clear. Identification of the phosphorylated sites on viral P protein and the related host kinases is helpful for developing live-attenuated vaccines and designing antiviral drugs. This study focused on identifying the phosphorylated sites on P protein of a fish rhabdovirus SHVV, determining the related host kinases, and revealing the effects of the phosphorylated sites and kinases on SHVV replication. We found that SHVV P was phosphorylated at T160, which was mediated by the kinases p38MAPK and GSK3 to promote SHVV replication. This study is the first time to study the role of P phosphorylation in fish rhabdovirus replication.

DDX3X Is Hijacked by Snakehead Vesiculovirus Phosphoprotein To Facilitate Virus Replication via Stabilization of the Phosphoprotein.

Asp-Glu-Ala-Asp (DEAD) box helicase 3 X-linked (DDX3X) plays important regulatory roles in the replication of many viruses. However, the role of DDX3X in rhabdovirus replication has seldomly been investigated. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was used to study the role of DDX3X in rhabdovirus replication. DDX3X was identified as an interacting partner of SHVV phosphoprotein (P). The expression level of DDX3X was increased at an early stage of SHVV infection and then decreased to a normal level at a later infection stage. Overexpression of DDX3X promoted, while knockdown of DDX3X using specific small interfering RNAs (siRNAs) suppressed, SHVV replication, indicating that DDX3X was a proviral factor for SHVV replication. The N-terminal and core domains of DDX3X (DDX3X-N and DDX3X-Core) were determined to be the regions responsible for its interaction with SHVV P. Overexpression of DDX3X-Core suppressed SHVV replication by competitively disrupting the interaction between full-length DDX3X and SHVV P, suggesting that full-length DDX3X-P interaction was required for SHVV replication. Mechanistically, DDX3X-mediated promotion of SHVV replication was due not to inhibition of interferon expression but to maintenance of the stability of SHVV P to avoid autophagy-lysosome-dependent degradation. Collectively, our data suggest that DDX3X is hijacked by SHVV P to ensure effective replication of SHVV, which suggests an important anti-SHVV target. This study will help elucidate the role of DDX3X in regulating the replication of rhabdoviruses. IMPORTANCE Growing evidence has suggested that DDX3X plays important roles in virus replication. In one respect, DDX3X inhibits the replication of viruses, including hepatitis B virus, influenza A virus, Newcastle disease virus, duck Tembusu virus, and red-spotted grouper nervous necrosis virus. In another respect, DDX3X is required for the replication of viruses, including hepatitis C virus, Japanese encephalitis virus, West Nile virus, murine norovirus, herpes simplex virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because DDX3X has rarely been investigated in rhabdovirus replication, this study aimed at investigating the role of DDX3X in rhabdovirus replication by using the fish rhabdovirus SHVV as a model. We found that DDX3X was required for SHVV replication, with the mechanism that DDX3X interacts with and maintains the stability of SHVV phosphoprotein. Our data provide novel insights into the role of DDX3X in virus replication and will facilitate the design of antiviral drugs against rhabdovirus 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.

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.

The role of sand flies as vectors of viruses other than phleboviruses.

Sand flies (Diptera: Phlebotominae) are proven vectors of various pathogens of medical and veterinary importance. Although mostly known for their pivotal role in the transmission of parasitic protists of the genus Leishmania that cause leishmaniases, they are also proven or suspected vectors of many arboviruses, some of which threaten human and animal health, causing disorders such as human encephalitis (Chandipura virus) or serious diseases of domestic animals (vesicular stomatitis viruses). We reviewed the literature to summarize the current published information on viruses detected in or isolated from phlebotomine sand flies, excluding the family Phenuiviridae with the genus Phlebovirus, as these have been well investigated and up-to-date reviews are available. Sand fly-borne viruses from four other families (Rhabdoviridae, Flaviviridae, Reoviridae and Peribunyaviridae) and one unclassified group (Negevirus) are reviewed for the first time regarding their distribution in nature, host and vector specificity, and potential natural transmission cycles.

Immunoinformatics based designing a multi-epitope vaccine against pathogenic Chandipura vesiculovirus.

Chandipura vesiculovirus (CHPV) is a rapidly emerging pathogen responsible for causing acute encephalitis. Due to its widespread occurrence in Asian and African countries, this has become a global threat, and there is an urgent need to design an effective and nonallergenic vaccine against this pathogen. The present study aimed to develop a multi-epitope vaccine using an immunoinformatics approach. The conventional method of vaccine design involves large proteins or whole organism which leads to unnecessary antigenic load with increased chances of allergenic reactions. In addition, the process is also very time-consuming and labor-intensive. These limitations can be overcome by peptide-based vaccines comprising short immunogenic peptide fragments that can elicit highly targeted immune responses, avoiding the chances of allergenic reactions, in a relatively shorter time span. The multi-epitope vaccine constructed using CTL, HTL, and IFN-γ epitopes was able to elicit specific immune responses when exposed to the pathogen, in silico. Not only that, molecular docking and molecular dynamics simulation studies confirmed a stable interaction of the vaccine with the immune receptors. Several physicochemical analyses of the designed vaccine candidate confirmed it to be highly immunogenic and nonallergic. The computer-aided analysis performed in this study suggests that the designed multi-epitope vaccine can elicit specific immune responses and can be a potential candidate against CHPV.

Hsp90 Is Required for Snakehead Vesiculovirus Replication via Stabilization of the Viral L Protein.

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.

Structural intermediates in the fusion-associated transition of vesiculovirus glycoprotein.

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

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Induction of apoptosis in SSN-1cells by Snakehead Fish Vesiculovirus (SHVV) via Matrix protein dependent intrinsic pathway.

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.

Leader RNA regulates snakehead vesiculovirus replication via interacting with viral nucleoprotein.

Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.

Characterization of Antibody Interactions with the G Protein of Vesicular Stomatitis Virus Indiana Strain and Other Vesiculovirus G Proteins.

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

MiR-214 inhibits snakehead vesiculovirus (SHVV) replication by targeting host GS.

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus).

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection.

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

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.

Distinct lineage of vesiculovirus from big brown bats, United States.

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

Multi-modal efficacy of a chimeric vesiculovirus expressing the Morreton glycoprotein in sarcoma.

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

Quantification of in vitro replication kinetics of Alagoas vesiculovirus isolates by digital droplet RT-PCR.

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

Evaluation of a novel severe combined immunodeficiency mouse model for antiviral drug evaluation against Chandipura virus infection.

Chandipura virus (CHPV) is a negative-sense single-stranded RNA virus known to cause fatal encephalitis outbreaks in the Indian subcontinent. The virus displays tropism towards the pediatric population and holds significant public health concerns. Currently, there is no specific, effective therapy for CHPV encephalitis. In this study, we evaluated a novel C.B-17 severe combined immunodeficiency (SCID) mouse model which can be used for pre-clinical antiviral evaluation. Inoculation of CHPV developed a lethal infection in our model. Plaque assay and immunohistochemistry detected increased viral loads and antigens in various organs, including the brain, spinal cord, adrenal glands, and whole blood. We further conducted a proof-of-concept evaluation of favipiravir in the SCID mouse model. Favipiravir treatment improved survival with pre-symptomatic (days 5-14) and post-symptomatic (days 9-18) treatment. Reduced viral loads were observed in whole blood, kidney/adrenal gland, and brain tissue with favipiravir treatment. The findings in this study demonstrate the utility of SCID mouse for in vivo drug efficacy evaluation and the potential efficacy of favipiravir against CHPV infection.