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Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

Background Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.

Lettuce necrotic yellows virus is a type of species in the Cytorhabdovirus genus and appears to be endemic to Australia and Aotearoa New Zealand (NZ). The population of lettuce necrotic yellows virus (LNYV) is made up of two subgroups, SI and SII. Previous studies demonstrated that SII appears to be outcompeting SI and suggested that SII may have greater vector transmission efficiency and/or higher replication rate in its host plant or insect vector. Rhabdovirus glycoproteins are important for virus–insect interactions. Here, we present an analysis of LNYV glycoprotein sequences to identify key features and variations that may cause SII to interact with its aphid vector with greater efficiency than SI. Phylogenetic analysis of glycoprotein sequences from NZ isolates confirmed the existence of two subgroups within the NZ LNYV population, while predicted 3D structures revealed the LNYV glycoproteins have domain architectures similar to Vesicular Stomatitis Virus (VSV). Importantly, changing amino acids at positions 244 and 247 of the post-fusion form of the LNYV glycoprotein altered the predicted structure of Domain III, glycosylation at N248 and the overall stability of the protein. These data support the glycoprotein as having a role in the population differences of LNYV observed between Australia and New Zealand.

In order to understand the immune response of largemouth bass against Micropterus salmoides Rhabdovirus (MSRV), assisting disease resistance breeding, three largemouth bass breeding varieties Micropterus salmoides “Youlu No 3” (U3), “Youlu No 1” (U1) and “Zhelu No 1” (P1) were challenged intraperitoneally with MSRV. Serum and tissues were sampled to study the changes in non-specific immune parameters, viral loads, and transcript levels of immune-related genes, and the cumulative mortality rate was recorded daily for 14 days. The results showed that the cumulative mortality rates in the U1, P1, and U3 groups were 6.66% ± 2.89%, 3.33% ± 2.89%, and 0, respectively. The higher mortality may attribute to the increased viral loads after infection in the liver (2.79 × 105 and 2.38 × 105 vs. 1.3 × 104 copies/mg), spleen (2.14 × 105 and 9.40 × 104 vs. 4.21 × 103 copies/mg), and kidney (3.59 × 104 and 8.40 × 103 vs. 2.42 × 103 copies/mg) in the U1 and P1 groups compared to the U3 group. The serum non-specific immune parameters (lysozyme, catalase, and acid phosphatase) were found to be increased significantly in the U3 group. In addition, the transcripts of interferon-related genes (IFN-γ, IRF3, and IRF7) and pro-inflammatory-related genes (TNF-α and IL-1β) exhibited up-regulation and peaked at 6 h post infection in the U3 group, which also exhibited up-regulation but peaked at 12–24 h post infection in the U1 and P1 groups. In conclusion, these findings indicate that earlier activation of interferon and pro-inflammatory response is beneficial to largemouth bass against MSRV infection. This experiment may provide an insight into understanding the immune mechanism of largemouth bass against MSRV infection and contributes to molecular-assisted selection.

Piscine novirhabdovirus = Viral Hemorrhagic Septicemia Virus (VHSV) first appeared in the Laurentian Great Lakes with large outbreaks from 2005 to 2006, as a new and novel RNA rhabdovirus subgenogroup (IVb) that killed >30 fish species. Interlude periods punctuated smaller more localized outbreaks in 2007, 2010, and 2017, although some fishes tested positive in the intervals. There have not been reports of outbreaks or positives from 2018, 2019, or 2020. Here, we employ a combined population genetics and phylogenetic approach to evaluate spatial and temporal evolutionary trajectory on its G‐gene sequence variation, in comparison with whole‐genome sequences (11,083 bp) from a subset of 44 individual isolates (including 40 newly sequenced ones). Our results show that IVb (N = 184 individual fish isolates) diversified into 36 G‐gene haplotypes from 2003 to 2017, stemming from two originals (“a” and “b”). G‐gene haplotypes “a” and “b” differed by just one synonymous single‐nucleotide polymorphism (SNP) substitution, remained the most abundant until 2011, then disappeared. Group “a” descendants (14 haplotypes) remained most prevalent in the Upper and Central Great Lakes, with eight (51%) having nonsynonymous substitutions. Group “b” descendants primarily have occurred in the Lower Great Lakes, including 22 haplotypes, of which 15 (68%) contained nonsynonymous changes. Evolutionary patterns of the whole‐genome sequences (which had 34 haplotypes among 44 isolates) appear congruent with those from the G‐gene. Virus populations significantly diverged among the Upper, Central, and Lower Great Lakes, diversifying over time. Spatial divergence was apparent in the overall patterns of nucleotide substitutions, while amino acid changes increased temporally. VHSV‐IVb thus significantly differentiated across its less than two decades in the Great Lakes, accompanied by declining outbreaks and virulence. Continuing diversification likely allowed the virus to persist at low levels in resident fish populations, and may facilitate its potential for further and future spread to new habitats and nonacclimated hosts., This study analyses the evolutionary trajectory of a novel virus over its 17‐year history, examining the dynamics of its host relationships and its patterns across geographic space and evolutionary time. We sequence its entire genome and discern that the virus has significantly changed, with its original genotypes disappearing and being replaced in a quasispecies trajectory. It also has become less virulent, appearing to adapt for long‐term persistence at low levels in the host populations.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales, of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales, which were strongly decreased in Czech bees compared to the Varroa- and DWV-naive Australian bees. ispartof: MSYSTEMS vol:7 issue:3 ispartof: location:United States status: published

European perch (Perca fluviatilis) are increasingly farmed as a human food source. Viral infections of European perch remain largely unexplored, thereby putting farm populations at incalculable risk for devastating fish epizootics and presenting a potential hazard to consumers. To address these concerns, we applied metatranscriptomics to identify disease-associated viruses in European perch farmed in Switzerland. Unexpectedly, in clinically diseased fish we detected novel freshwater fish filoviruses, a novel freshwater fish hantavirus, and a previously unknown rhabdovirus. Hantavirus titers were high, and we demonstrated virus in macrophages and gill endothelial cells by using in situ hybridization. Rhabdovirus titers in organ samples were low, but virus could be isolated on cell culture. Our data add to the hypothesis that filoviruses, hantaviruses, and rhabdoviruses are globally distributed common fish commensals, pathogens, or both. Our findings shed new light on negative-sense RNA virus diversity and evolution. ispartof: EMERGING INFECTIOUS DISEASES vol:27 issue:12 pages:3082-3091 ispartof: location:United States status: published

Rhabdoviridae is the most diverse family of the negative, single-stranded RNA viruses, which includes 40 ecologically different genera that infect plants, insects, reptiles, fishes, and mammals, including humans, and birds. To date, only a few bird-related rhabdoviruses among the genera Sunrhavirus, Hapavirus, and Tupavirus have been described and analyzed at the molecular level. In this study, we characterized seven additional and previously unclassified rhabdoviruses, which were isolated from various bird species collected in Africa during the 1960s and 1970s. Based on the analysis of their genome sequences obtained by next generation sequencing, we observed a classical genomic structure, with the presence of the five canonical rhabdovirus genes, i.e., nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). In addition, different additional open reading frames which code putative proteins of unknown function were identified, with the common presence of the C and the SH proteins, within the P gene and between the M and G genes, respectively. Genetic comparisons and phylogenetic analysis demonstrated that these seven bird-related rhabdoviruses could be considered as putative new species within the genus Sunrhavirus, where they clustered into a single group (named Clade III), a companion to two other groups that encompass mainly insect-related viruses. The results of this study shed light on the high diversity of the rhabdoviruses circulating in birds, mainly in Africa. Their close relationship with other insect-related sunrhaviruses raise questions about their potential role and impact as arboviruses that affect bird communities.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

Trans-neuronal viruses are frequently used as neuroanatomical tools for mapping neuronal circuits. Specifically, recombinant one-step rabies viruses (RABV) have been instrumental in the widespread application of viral circuit mapping, as these viruses have enabled labs to map the direct inputs onto defined cell populations. Within the neuroscience community, it is widely believed that RABV spreads directly between neurons via synaptic connections, a hypothesis based principally on two observations. First, the virus labels neurons in a pattern consistent with known anatomical connectivity. Second, few glial cells appear to be infected following RABV injections, despite the fact that glial cells are abundant in the brain. However, there is no direct evidence that RABV can actually be transmitted through synaptic connections. Here we review the immunosubversive mechanisms that are critical to RABV’s success for infiltration of the central nervous system (CNS). These include interfering with and ultimately killing migratory T cells while maintaining levels of interferon (IFN) signaling in the brain parenchyma. Finally, we critically evaluate studies that support or are against synaptically-restricted RABV transmission and the implications of viral-host immune responses for RABV transmission in the brain.

Vesicular stomatitis virus (VSV) emerges periodically from its focus of endemic transmission in southern Mexico to cause epizootics in livestock in the US. The ecology of VSV involves a diverse, but largely undefined, repertoire of potential reservoir hosts and invertebrate vectors. As part of a larger program to decipher VSV transmission, we conducted a study of the spatiotemporal dynamics of Simulium black flies, a known vector of VSV, along the Rio Grande in southern New Mexico, USA from March to December 2020. Serendipitously, the index case of VSV-Indiana (VSIV) in the USA in 2020 occurred at a central point of our study. Black flies appeared soon after the release of the Rio Grande’s water from an upstream dam in March 2020. Two-month and one-year lagged precipitation, maximum temperature, and vegetation greenness, measured as Normalized Difference Vegetation Index (NDVI), were associated with increased black fly abundance. We detected VSIV RNA in 11 pools comprising five black fly species using rRT-PCR, five pools yielded a VSIV sequence. To our knowledge, this is the first detection of VSV in the western US from vectors that were not collected on premises with infected domestic animals.

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Rice stripe mosaic disease (RSMD) is caused by the rice stripe mosaic virus (RSMV; genus Cytorhabdovirus, family Rhabdoviridae). In recent years, significant progress has been made in understanding several aspects of the disease, especially its geographical distribution, symptoms, vectors, gene functions, and control measures. Since RSMD was first detected in southern China in 2015, it has been found in more and more rice growing areas and has become one of the most important rice diseases in southern China. RSMV is transmitted by the leafhopper Recilia dorsalis in a persistent-propagative manner, inducing yellow stripes, a slight distortion of leaves, increased tillers, and empty grains in rice plants. The virus has a negative-sense single-strand RNA genome of about 12.7 kb that encodes seven proteins: N, P, P3, M, G, P6, and L. Several molecular and serological tests have been developed to detect RSMV in plants and insects. The disease cycle can be described as follows: RSMV and its vector overwinter in infected plants; viruliferous R. dorsalis adults transmit the virus to spring rice and lay eggs on the infected seedlings; the next generation of R. dorsalis propagate on infected seedlings, become viruliferous, disperse, and cause new disease outbreaks. Control measures include monitoring and accurate forecasting, selecting disease-resistant varieties, improving cultivation systems, covering rice seedling nurseries with insect-proof nets, and using pesticides rationally. Inappropriate cultivation systems, pesticide overuse, and climatic conditions contribute to epidemics by affecting the development of vector insects and their population dynamics.

Background Circularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses. Methods The role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing. Results We have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR. Conclusions The results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43–55) and the remaining region (aa 56–295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis., A cytorhabdovirus phosphoprotein forms mobile inclusion bodies that traffic along the actin/endoplasmic reticulum network in a process that is important for viral RNA synthesis.

The family Rhabdoviridae contain important human and mammalian pathogens that are vectored by different arthropod species. The ground supernatants of mosquitoes were used to inoculate in BHK-21 and C6/36 cells for virus isolation. Then, the viral complete genome sequence was obtained and used for phylogenetic analysis. In this study, we observed a cytopathic effect (CPE) in mosquito cells (C6/36) and rod-like virion after inoculating a pool of Armigeres subalbatus samples collected in Shanxi Province, China, in 2019 (SX1916). Meta-transcriptomics sequencing revealed the presence of two distinctive rhabdoviruses with similar abundance levels, namely, Shanxi Armigeres subalbatus rhabdovirus (SXARV) and Shanxi Arboretum virus (SXABTV). Despite the fact that the SXARV genome (9590 nt) was much shorter than that of SXABTV (11,480 nt), both belonged to the Almendravirus group within Rhabdoviridae whose genomes encoded five proteins (N, P, M, G, and L) and a small hydrophobin (U1) and the difference in lengths is mainly caused by a substantially shorter N protein encoded by SXARV. On the phylogenetic tree, SXABTV was closely related (90.7% amino acid identity at L protein) with the Arboretum virus isolated from Psorophora albigenu mosquitoes in Peru in 2014, whereas SXARV was distantly related to Rio Chico virus (63.3% amino acid identity), a genetic distance large enough to be defined as a new species within Rhabdoviridae. Collectively, we report a simultaneous isolation of two related rhabdoviruses from Armigeres subalbatus that marked the circulation of almendraviruses in Shanxi, China.

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV. Instituto de Patología Vegetal Fil: Kuhn, Jens H. National Institute of Allergy and Infectious Diseases. National Institutes of Health. Integrated Research Facility at Fort Detrick; Estados Unidos Fil: Adkins, Scott. United States Department of Agriculture. Agricultural Research Service. US Horticultural Research Laboratory; Estados Unidos Fil: Agwanda, Bernard R. National Museums of Kenya. Zoology Department; Kenia Fil: Agwanda, Bernard R. Jomo Kenyatta University of Agriculture & Technology; Kenia Fil: Kubrusli, Rim Al. Humboldt-Universität zu Berlin. Faculty of Life Sciences. Division Phytomedicine; Alemania Fil: Alkhovsky, Sergey V. D.I. Ivanovsky Institute of Virology of N.F. Gamaleya National Center on Epidemiology and Microbiology of Ministry of Health of Russian Federation; Rusia Fil: Amarasinghe, Gaya K. Washington University School of Medicine. Department of Pathology and Immunology; Estados Unidos Fil: Avšič-Županc, Tatjana. University of Ljubljana. Faculty of Medicine; Eslovenia Fil: Ayllón, María A. Universidad Politécnica de Madrid—Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Campus de Montegancedo. Centro de Biotecnología y Genómica de Plantas; España Fil: Ayllón, María A. Universidad Politécnica de Madrid. Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas. Departamento de Biotecnología-Biología Vegetal; España Fil: Bahl, Justin. University of Georgia. Center for Ecology of Infectious Diseases, Department of Infectious Diseases, Department of Epidemiology and Biostatistics, Insitute of Bioinformatics; Estados Unidos Fil: Balkema-Buschmann, Anne. Institute of Novel and Emerging Infectious Diseases. Friedrich-Loeffler-Institut. Federal Research Institute for Animal Health; Alemania Fil: Bejerman, Nicolas Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Bejerman, Nicolas Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Zhou, Xueping. Chinese Academy of Agricultural Sciences. Institute of Plant Protection. State Key Laboratory for Biology of Plant Diseases and Insect Pests; China

Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat (Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.

Glioblastoma multiforme is the most common and malignant form of brain cancer in humans. The current prognosis for glioblastoma patients undergoing standard treatment is extremely low, with a median patient survival of 1 to 2 years. Oncolytic viruses are currently being evaluated as promising novel therapies for the treatment of glioblastoma. Our laboratory’s preliminary data has demonstrated that the oncolytic rhabdovirus Farmington effectively eliminates brain tumours, but is also neurosafe. However, the biological mechanisms underlying Farmington’s neurosafety and oncotropism were unknown. Through studying Farmington infection in normal brain cells and brain cancer cells in vitro, we determined that Farmington’s distinct tropism is dependent on the Farmington glycoprotein and the type 1 interferon response.

VSV fusion machinery, like that of many other enveloped viruses, is triggered at low pH in endosomes after virion endocytosis. It was suggested that some histidines could play the role of pH-sensitive switches. By mutating histidine residues H22, H60, H132, H162, H389, H397, H407, and H409, we demonstrate that residues H389 and D280, facing each other in the six-helix bundle of the post-fusion state, and more prominently H407, located at the interface between the C-terminal part of the ectodomain and the fusion domain, are crucial for fusion. Passages of recombinant viruses bearing mutant G resulted in the selection of compensatory mutations. Thus, the H407A mutation in G resulted in two independent compensatory mutants, L396I and S422I. Together with a crystal structure of G, presented here, which extends our knowledge of G pre-fusion structure, this indicates that the conformational transition is initiated by refolding of the C-terminal part of the G ectodomain.

Prime-boost vaccination employing heterologous viral vectors encoding an antigen is an effective strategy to maximize the antigen-specific immune response. Replication-deficient adenovirus serotype 5 (Ad5) is currently being evaluated clinically in North America as a prime in conjunction with oncolytic rhabdovirus Maraba virus (MG1) as a boost. The use of an oncolytic rhabdovirus encoding a tumor antigen elicits a robust anti-cancer immune response and extends survival in murine models of cancer. Given the prevalence of pre-existing immunity to Ad5 globally, we explored the potential use of DEC205-targeted antibodies as an alternative agent to prime antigen-specific responses ahead of boosting with an oncolytic rhabdovirus expressing the same antigen. We found that a prime-boost vaccination strategy, consisting of an anti-DEC205 antibody fused to the model antigen ovalbumin (OVA) as a prime and oncolytic rhabdovirus-OVA as a boost, led to the formation of a robust antigen-specific immune response and improved survival in a B16-OVA tumor model. Overall, our study shows that anti-DEC205 antibodies fused to cancer antigens are effective to prime oncolytic rhabdovirus-boosted cancer antigen responses and may provide an alternative for patients with pre-existing immunity to Ad5 in humans., Graphical Abstract, Replication-deficient Ad5 vectors are used for gene therapy and vaccination. Tzelepis and colleagues show that seropositivity to Ad5, highly prevalent in the human population, abrogates an Ad5 vaccination response. Alternatively, they show the efficacy of αDEC205 antibody-antigen fusion in the context of an oncolytic rhabdovirus cancer vaccine targeting mouse melanoma.

Background: Rabies is a major zoonotic disease affecting humans, domestic and wildlife mammals. Cattle are the most important domestic animals impacted by rabies virus in the New World, leading to thousands of cattle deaths per year and eliciting large economic losses. In the New World, virus transmission in cattle is primarily associated with Desmodus rotundus, the common vampire bat. This study analyses the association of weather fluctuations and the El Niño Southern Oscillation (ENSO), with the occurrence and magnitude, in terms of associated mortality, of cattle rabies outbreaks. Data from the 100 cattle rabies outbreaks recorded between 1985 and 2016 in Costa Rica were analyzed. Periodograms for time series of rabies outbreaks and the El Niño 4 index were estimated. Seasonality was studied using a seasonal boxplot. The association between epidemiological and climatic time series was studied via cross wavelet coherence analysis. Retrospective space-time scan cluster analyses were also performed. Finally, seasonal autoregressive time series models were fitted to study linear associations between monthly number of outbreaks, monthly mortality rates and the El Niño 4 index, temperature, and rainfall. Results: Large rabies mortality occurred towards the Atlantic basin of the country. Outbreak occurrence and size were not directly associated with ENSO, but were sensitive to weather variables impacted by ENSO. Both, ENSO phases and rabies outbreaks, showed a similar 5 year period in their oscillations. Cattle rabies mortality and outbreak occurrence increased with temperature, whereas outbreak occurrence decreased with rainfall. These results suggest that special weather conditions might favor the occurrence of cattle rabies outbreaks. Conclusions: Further efforts are necessary to articulate the mechanisms underpinning the association between weather changes and cattle rabies outbreaks. One hypothesis is that exacerbation of cattle rabies outbreaks might be mediated by impacts of weather conditions on common vampire bat movement and access to food resources on its natural habitats. Further eco-epidemiological field studies could help to understand rabies virus transmission ecology, and to propose sound interventions to control this major veterinary public health problem. Antecedentes: La rabia es una importante enfermedad zoonótica que afecta a los seres humanos y a los mamíferos domésticos y salvajes. El ganado es el animal doméstico más importante afectado por el virus de la rabia en el Nuevo Mundo, lo que provoca la muerte de miles de reses al año y provocan grandes pérdidas económicas. En el Nuevo Mundo, la transmisión del virus en el ganado se asocia principalmente a con el Desmodus rotundus, el murciélago vampiro común. Este estudio analiza la asociación de las fluctuaciones meteorológicas y El Niño Oscilación del Sur (ENSO), con la aparición y la magnitud, en términos de mortalidad asociada, de brotes de rabia en el ganado. Se analizaron los datos de los 100 brotes de rabia bovina registrados entre 1985 y 2016 en Costa Rica se analizaron los datos de los 100 brotes de rabia bovina registrados entre 1985 y 2016 en Costa Rica. Se estimaron los periodogramas de las series temporales de los brotes de rabia y el índice de El Niño 4. Se estudió la estacionalidad mediante un boxplot estacional. La asociación entre las series temporales epidemiológicas y climáticas se estudió mediante un análisis de coherencia de ondículas cruzadas. También se realizaron análisis de conglomerados de barrido espacio-temporal retrospectivo de clusters espacio-temporales retrospectivos. Por último, se ajustaron modelos de series temporales autorregresivas estacionales para estudiar las asociaciones lineales entre número mensual de brotes, las tasas mensuales de mortalidad y el índice de El Niño 4, la temperatura y las precipitaciones. Resultados: La mayor mortalidad por rabia se produjo hacia la cuenca atlántica del país. La ocurrencia y el tamaño de los brotes no se asociaron directamente con el ENSO, pero fueron sensibles a las variables meteorológicas afectadas por el ENSO. Tanto las fases del ENSO y los brotes de rabia, mostraron un período de 5 años similar en sus oscilaciones. La mortalidad por rabia del ganado y la aparición de brotes aumentaron con la temperatura, mientras que la aparición de brotes disminuyó con las precipitaciones. Estos resultados sugieren que unas condiciones meteorológicas especiales podrían favorecer la aparición de brotes de rabia bovina. Conclusiones: Es necesario realizar más esfuerzos para articular los mecanismos que subyacen a la asociación entre los cambios meteorológicos y los brotes de rabia bovina. Una hipótesis es que la exacerbación de los brotes de rabia bovina podría estar mediada por el impacto de las condiciones meteorológicas en los movimientos del murciélago vampiro común y el acceso a los recursos alimenticios en sus hábitats naturales. Otros estudios eco-epidemiológicos de campo podrían ayudar a entender la transmisión del virus de la rabia y a proponer intervenciones adecuadas para controlar este importante problema de salud pública veterinaria. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Background Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease. Materials and methods In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention. Results Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group. Conclusion This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.

Aquatic vertebrates are very abundant in the world, and they are of tremendous importance in providing global food security and nutrition. However, emergent and resurgent viruses, such as ranavirus (e.g., Rana grylio virus, RGV and Andriasd avidianus ranavirus, ADRV), herpesvirus (e.g., Carassius carassius herpesvirus, CaHV), reovirus (e.g., grass carp reovirus 109, GCRV-109, Scophthal musmaximus reovirus, SMReV and Micropterus salmoides reovirus, MsReV), and rhabdovirus (e.g., Siniper cachuatsi rhabdovirus, SCRV and Scophthal musmaximus rhabdovirus, SMRV) can cause severe diseases in aquaculture animals and wild lower vertebrates, such as frogs, giant salamanders, fish, and so on. Here, we will briefly describe the symptoms produced by the aforementioned viruses and the molecular basis of the virus–host interactions. This manuscript aims to provide an overview of viral diseases in lower vertebrates with an emphasis on visible symptomatic manifestations and pathogenesis.

Simple Summary The generation of tumor-specific T cells remains a pillar of modern cancer immunotherapy. Exogenous vaccines often rely on targeting tumor-associated antigens. The anti-apoptotic protein survivin has been deemed a high priority target due to its overexpression in a wide variety of tumor types. To support the analysis of tumor-associated T cell responses, optimization of epitope mapping would be valuable. A heterologous prime-boost vaccination strategy was designed to target survivin to induce anti-tumor immune responses. However, survivin-specific T cell responses could not be detected in mice. Potential mechanisms to explain this failure were explored. To confirm the robustness of the vaccination platform, enhanced green fluorescent protein (eGFP) was targeted since it has been defined as a protein with relatively low immunogenicity. In this context the vaccination strategy uncovered novel T cell epitopes from eGFP in two strains of mice. This research highlighted the utility of the vaccine platform to triage potential target antigens based on their immunogenicity. Abstract Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

In 2010, a severe disease of lucerne (Medicago sativa L.) named alfalfa dwarf disease (ADD) was reported in Argentina. High throughput sequencing of diseased plants indicated the presence of five viruses, the (−) ssRNA virus, alfalfa dwarf virus (ADV), the (+) RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus (BLRV) and alfalfa enamovirus 1 (AEV-1) and the ssDNA virus, alfalfa leaf curl virus (ALCV). In this study, we determined which ADD-associated viruses are present in Australian lucerne that showed diverse virus-like symptoms. A duplex RT-PCR was developed for simultaneous detection of ADV and AMV using a cloned non-infectious ADV RNA fragment as positive control. Similarly, the presence of BLRV and AEV-1 was determined by duplex RT-PCR, and ALCV by PCR. Only AMV and BLRV that are endemic to Australia were detected. None of the novel exotic viruses ADV, AEV-1 and ALCV were detected in lucerne samples collected between 2015 and 2017. However, AMV and BLRV were detected in 78% and 70% of tested samples, respectively. Based on analysis of coat protein (CP) nucleotide sequences, Australian BLRV isolates are closely related to each other and to the Argentine Manfredi isolate. Phylogenetic analyses based on CP gene nucleotide sequences confirmed separation of AMV isolates into two subgroups. The majority of AMV isolates, including all those from Argentina and Australia, clustered in subgroup I with isolates from various hosts and geographic origins. All Australian AMV isolates were closely related to AMV isolated from ADD-affected lucerne in Argentina. Fil: Samarfard, S.. The University Of Queensland; Australia Fil: Bejerman, Nicolas. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet. Córdoba. Unidad de Fitopatología y Modernización Agrícola (UFYMA); Argentina Fil: Sharman, M.. The University Of Queensland; Australia Fil: Trucco, Verónica Milagros. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Giolitti, Fabián José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Dietzgen, R. G.. The University Of Queensland; Australia

The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material.

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12 792 nt long and organized into seven ORFs with the gene order 3′-N-X-P-Y-M-G-L-5′, which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. Cloned ORFs for each gene, except L, were used to construct a protein interaction and localization map (PILM) for this virus, which shares greater than 80 % amino acid similarity in all ORFs except X and P with the sanguinolenta strain of this species (SYDV). Protein localization patterns and interactions unique to each viral strain were identified, resulting in strain-specific PILMs. Localization of CYDV and SYDV proteins in virus-infected cells mapped subcellular loci likely to be sites of replication, morphogenesis and movement.

Background Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. Methods In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5′ termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Results Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Conclusion Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0776-7) contains supplementary material, which is available to authorized users.

In September and October 2015, suspected cases of bovine ephemeral fever (BEF) were reported in the mainland region of Kagoshima Prefecture and on Tanegashima Island. The genome of the BEF virus (BEFV) was detected in the diseased cows and the cows that had recovered. The serum obtained from the affected cows contained high titers of BEFV-neutralizing antibody. In total, 18 affected cows were demonstrated to be infected with BEFV during the outbreak. Our findings showed evidence that BEF occurred in mainland Japan after a 23-year absence. Phylogenetic analysis based on the surface glycoprotein (G) gene revealed that BEFVs detected in the affected cows were genetically distinct from previous Japanese BEFVs, but were close to BEFVs circulating in Taiwan and mainland China in recent years. Amino acid substitution in the neutralizing epitope domains of the G protein was limited between the detected viruses and the vaccine strain (YHL isolate), and high titers of the neutralizing antibody against the YHL isolate were induced in the infected cattle during the disease occurrences. Therefore, current BEF vaccines probably elicit protective immunity against the BEFVs detected in 2015, although their effectiveness should be assessed. Since the BEFV vaccination rates are estimated to be low, a BEF outbreak should be considered a possibility in mainland Japan.

Arboviruses circulate in the biota of northern Australia, sometimes causing disease in animals and humans. Five different arboviruses, isolated and stored for over 30 years, were re-cultured and sequenced using high-throughput sequencing technology (Ion Torrent PGM) for identification and genetic characterization. Phylogenetic analysis, primarily of sequence fragments of the large segment, indicated classification as members of the Family Bunyaviridae (Belmont, Little Sussex, Parker's Farm, and Thimiri viruses). Another was identified as a member of the Family Rhabdoviridae (Beatrice Hill virus) based on phylogenetic analysis of the RNA polymerase region.

Epizootic congenital abnormalities caused by Akabane, Aino, and Chuzan viruses have damaged the reproduction of domestic ruminants in East Asia for many years. In the past, large outbreaks of febrile illness related to bovine ephemeral fever and Ibaraki viruses severely affected the cattle industry in that region. In recent years, vaccines against these viruses have reduced the occurrence of diseases, although the viruses are still circulating and have occasionally caused sporadic and small-scaled epidemics. Over a long-term monitoring period, many arboviruses other than the above-mentioned viruses have been isolated from cattle and Culicoides biting midges in Japan. Several novel arboviruses that may infect ruminants (e.g., mosquito- and tick-borne arboviruses) were recently reported in mainland China based on extensive surveillance. It is noteworthy that some are suspected of being associated with cattle diseases. Malformed calves exposed to an intrauterine infection with orthobunyaviruses (e.g., Peaton and Shamonda viruses) have been observed. Epizootic hemorrhagic disease virus serotype 6 caused a sudden outbreak of hemorrhagic disease in cattle in Japan. Unfortunately, the pathogenicity of many other viruses in ruminants has been uncertain, although these viruses potentially affect livestock production. As global transportation grows, the risk of an accidental incursion of arboviruses is likely to increase in previously non-endemic areas. Global warming will also certainly affect the distribution and active period of vectors, and thus the range of virus spreads will expand to higher-latitude regions. To prevent anticipated damages to the livestock industry, the monitoring system for arboviral circulation and incursion should be strengthened; moreover, the sharing of information and preventive strategies will be essential in East Asia.

Tese de Doutoramento em Medicina, Lassa fever (LF), is a viral hemorrhagic fever caused by Lassa virus (LASV), for which neither an approved vaccine or effective treatment is available. LASV is an endemic virus in western Africa and a major health and economic burden, causing an estimate 100,000-300,000 infections yearly, with the number of reported infections increasing in the last years. This thesis describes the development of LASSARAB, a dual LF and rabies vaccine based on recombinant rabies vector. Rabies is another equally important disease in Africa that is estimated to cause thousands of deaths every year, despite vaccination being available. LASSARAB uses a codon optimized version of LASV’s glycoprotein (GPC) as its LASV immunogen. After confirming that LASSARAB expresses and incorporates GPC in the virions, LASSARAB’s potential as an LF vaccine was tested using several LASSARAB based vaccine candidates: inactivated LASSARAB formulated in PBS only; inactivated LASSARAB formulated with GLA-SE adjuvant (a TLR-4 agonist); live-LASSARAB; and live-LASSARAB-ΔG, a variant of LASSARAB that lacks the Rabies G gene. Inactivated LASSARAB formulations induced a significant GPC specific IgG response above background with LASSARAB+GLA-SE inducing higher IgG titers than LASSARAB alone as well as a lower IgG1/IgG2c ratio. Neither live- LASSARAB vector induced a significant LASV GPC immune response. In LASV challenge studies, in both guinea pig and mouse, inactivated LASSARAB+GLA-SE, significantly protected 80% of the animals against LF disease. Higher titers of anti-GPC IgGs were correlated with protection independently of LASV neutralizing titers. Instead, non-neutralizing LASV GPC-specific antibodies, through antibody-dependent cellular functions (ADCC and ADCP) appear to the main drivers of protection against LF as demonstrated by in vitro and in vivo studies. Overall, these findings demonstrate an effective inactivated LF and rabies vaccine and suggest a novel correlate of protection for LF. Currently, further LASSARAB immunogenicity studies using NHPs are underway determine its eligibility for clinical phase 1 trials., A febre de Lassa (LF), é uma febre hemorrágica viral causada pelo vírus Lassa (LASV), para a qual não existe nem vacina ou tratamento aprovado. O LASV é um vírus presente na África ocidental que incute um pesado encargo na saúde pública regional, e estima-se que causa 100,000 a 300,000 infeções por ano. Esta tese é sobre o desenvolvimento de LASSARAB, uma potencial vacina contra a LF e Raiva baseada num vetor do vírus da Raiva recombinante. A Raiva, é outra doença igualmente importante em África que causa milhares de mortes todos anos apesar de haver vacina disponível. LASSARAB usa uma versão codon-optimized da glicoproteína de LASV (GPC) como o imunogénio de base contra LASV. Após confirmar que LASSARAB expressa e incorpora o LASV GPC nas partículas virais, o potencial de LASSARAB foi testado através de vários candidatos incluindo: LASSARAB inativado formulado em PBS apenas; LASSARAB inativado formulado com o adjuvante GLA-SE (um agonista TLR4); LASSARAB vivo; e LASSARAB-ΔG vivo, uma variante de LASSARAB que não expressa o gene da glicoproteína de raiva. Ambos os candidatos LASSARAB inativados induziram uma elevada resposta imune contra GPC, com LASSARAB+GLA-SE induzindo níveis de IgGs contra GPC mais elevados assim como um ratio IgG1/IgG2c mais baixo. Em estudos de exposição com LASV em porquinhos da Índia e ratinhos, o LASSARAB+GLA-SE inativo foi capaz de proteger em 80% os animais contra LF. Níveis mais elevados de IgGs específicos para GPC, foram correlacionados com proteção independentemente dos níveis de anticorpos neutralizantes contra LASV. Pelo contrário, IgGs específicos contra GPC não neutralizantes, através de funções celulares dependentes de anticorpos (ADCC e ADCP) parecem ser os principais intervenientes na proteção contra LF. Em conjunto, estes resultados demonstram uma vacina inativada eficaz contra LF e Raiva e sugerem uma correlação de proteção contra LF, baseado na resposta de IgGs específicos contra GPC. Atualmente, estudos em primatas estão a decorrer para testar a imunogenicidade de LASSARAB neste modelo em preparação para clinical phase 1 trials., Financial support for this PhD was provided by FCT (Fundação para Ciência e Tecnologia), by the fellowship PD/BD/105847/2014, awarded through the MD/PhD program of University of Minho.

The aquaculture of salmonid fishes is a multi-billion dollar industry with production over 3 million tons annually. However, infectious hematopoietic necrosis virus (IHNV), which infects and kills salmon and trout, significantly reduces the revenue of the salmon farming industry. Currently, there is no effective treatment for IHNV infected fishes; therefore, early detection and depopulation of the infected fishes remain the most common practices to contain the spread of IHNV. Apart from hygiene practices in aquaculture and isolation of infected fishes, loss of fishes due to IHNV infection can also be significantly reduced through vaccination programs. In the current review, some of the diagnostic methods for IHNV, spanning from clinical diagnosis to cell culture, serological and molecular methods are discussed in detail. In addition, some of the most significant candidate vaccines for IHNV are also extensively discussed, particularly the DNA vaccines.

Background Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence). Methods Overlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection. Results Complete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0–10% mortality). Conclusions When chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.

International audience; Rhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus.

Rhabdoviruses are single stranded RNA enveloped viruses. They own a unique glycoprotein G anchored on the viral membrane. G is involved in the early stages of the viral cycle. At first G binds a cellular receptor, leading to the virus endocytosis. Then G orchestrates the membrane fusion between the viral and endosomal membranes. Membrane fusion allows the release of the viral genome into the cytoplasm of the infected cell.The low-density lipoprotein receptor (LDLR) is the main receptor of VSV. The ectodomain of the LDLR is composed of a large ligand binding domain constituted of cysteine-rich domains (CR1 to CR7).In order to understand the molecular basis of the interaction between G and its receptor, we performed binding test of G with all the CR domains of the LDLR. This revealed that only CR2 and CR3 domains could bind G. When CR2 and CR3 are present in the VSV inoculum, they protect the cells from VSV infection. We crystallized G in complex with each of these CR domains. The structures reveal that CR2 and CR3 binding sites on G are identical, and that the same residues on G are involved in the binding of the two CR domains. HAP-1 cells in which the gene encoding the LDLR has been invalidated are still susceptible to VSV infection. This confirms that VSV can use other receptors than the LDLR itself to enter the cells. However, mutation of G residues that are key in the interaction with the CR domains of the LDLR abolish the infectivity of VSV in mammalian and insect cells. This indicates that the only VSV receptors in these cells are members of the LDLR family and that VSV G has specifically evolved to interact with their CR domains. Moreover, we have shown that G mutated for key residues involved in the interaction with CR domains, are still able to induce fusion. This work shows that receptor recognition and G fusion activities can be decoupled. This paves the way to develop glycoproteins derived from G with modified tropism.In Rhabdoviruses, G is the only target of neutralizing antibodies. There is no structure of a Rhabdovirus G in complex with an antibody. The 8G5F11 antibody neutralizes several genotypes of the genus Vesiculovirus including VSV. We have shown that FAB 8G5F11 prevents infection of cells by VSV Indiana on the one hand and that they recognize the pre- and post-fusion form of VSV G with a 1: 1 G: FAB stoichiometry on the other hand. We have developed the observation conditions of the G-FAB complex in cryo-electron microscopy, which could be useful to obtain the structure of the G-FAB complex.We also started a study to characterize the glycoproteins of other Rhabdoviruses of the Lyssavirus genus. We produced and purified G ectodomains of rabies virus (RABV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV). We characterize these G by electron microscopy at different pHs. The measurements made on G at pH 8 are compatible with those expected for a pre-fusion monomer of G. At pH 6, these ectodomains could correspond to a monomeric late intermediate in the structural transition. Obtaining the crystallographic structure of the G ectodomain of MOKV could validate these results., Les Rhabdovirus sont des virus enveloppés à ARN simple brin de polarité négative. Ils possèdent une unique protéine transmembranaire à leur surface, la glycoprotéine G. G est impliquée dans les étapes précoces du cycle viral. G lie dans un premier temps un récepteur cellulaire, menant à l’endocytose du virus. Ensuite, G orchestre la fusion membranaire entre les membranes virale et endosomale. Cette fusion membranaire permet la libération du génome viral dans le cytoplasme de la cellule infectée.Le récepteur des lipoprotéines de basse densité (LDLR) est le récepteur principal du VSV. L’ectodomaine du LDLR est composé d’un grand site de liaison au ligand constitué de domaines riches en cystéines (CR1 à CR7).Afin de comprendre les bases moléculaires de l’interaction entre G et son récepteur, nous avons réalisé des tests de fixation de G aux différents domaines CR du LDLR. Cela a révélé que seuls les domaines CR2 et CR3 pouvaient lier G. Lorsque CR2 et CR3 sont incubés avec VSV dans l’inoculum, ils protègent les cellules de l’infection par VSV. Nous avons cristallisé G en complexe avec chacun de ces domaines CR. Les structures révèlent que le site de liaison de CR2 et de CR3 sur G sont identiques, et que les mêmes résidus sur G sont impliqués dans la liaison des deux domaines CR. Des cellules HAP-1 dans lesquelles le gène codant pour le LDLR a été invalidé, sont toujours sensibles à l’infection par VSV. Ceci confirme que VSV peut utiliser d’autres récepteurs que le LDLR pour entrer dans les cellules. Cependant la mutation des résidus de G qui sont clefs dans l’interaction avec les domaines CR du LDLR abolissent l’infectiosité de VSV aussi bien dans les cellules de mammifères que dans les cellules d’insectes. Ceci indique que les seuls récepteurs de VSV dans ces cellules sont des membres de la famille du LDLR et que VSV G a spécifiquement évolué pour interagir avec leur domaines CR. Par ailleurs nous avons montré que les G dont les résidus clef pour l’interaction avec les domaines CR sont mutés, ont conservé leur activité de fusion. Ces travaux montrent que les activités de reconnaissance du récepteur et de fusion de G peuvent être découplées. Ceci ouvre la possibilité de développer des glycoprotéines dérivées de G au tropisme modifié.Chez les Rhabdovirus, G est la seule cible des anticorps neutralisants. A ce jour il n’existe aucune structure de G de Rhabdovirus en complexe avec un anticorps. L’anticorps 8G5F11 neutralise plusieurs génotypes du genre Vésiculovirus dont VSV. Nous avons montré que les FAB 8G5F11 empêchent l’infection des cellules par VSV Indiana d’une part et qu’ils reconnaissent la forme pré- et post-fusion de VSV G avec une stœchiométrie G : FAB de 1 : 1 d’autre part. Nous avons mis au point les conditions d’observation du complexe G-FAB en cryo-microscopie électronique, ce qui permet d’envisager à terme une résolution de la structure du complexe G-FAB.Enfin, nous avons débuté une étude visant à caractériser les glycoprotéines d’autres Rhabdovirus du genre Lyssavirus. Pour cela, nous avons produit et purifié les ectodomaines de G du virus de la rage (RABV), du virus Mokola (MOKV) et du virus de la chauve-souris ouest-caucasienne (WCBV). Ces G ont été caractérisées par microscopie électronique à différents pH. Les mesures effectuées sur G à pH 8 sont compatibles avec celles attendues pour un monomère de G pré-fusion. A pH 6, les ectodomaines observés pourraient correspondre à un intermédiaire monomérique allongé apparaissant tardivement lors de la transition structurale. Ces résultats pourraient être prochainement validés par l’obtention de la structure cristallographique de l’ectodomaine de G MOKV.

Oncolytic viruses (OVs) are a class of cancer therapy that is currently undergoing clinical trials on its way to full regulatory approval. At present, the downstream processing of OVs relies on a combination of chromatography and membrane-based processes to remove process-related (e.g. host-cell proteins and nucleic acids) and product-related impurities (e.g. aggregated virus particles). This thesis explores various methods that can potentially be used to address the challenges associated with downstream processing during the production of OVs. To this end, the Rhabdoviral vector, which is currently undergoing clinical trials (phase I/II) for use in treating advanced or metastatic solid tumors, was selected as a promising oncolytic virus. One potential improvement in the downstream process that was investigated was the use of monolithic column chromatography for Rhabdovirus purification. Two monolithic anion-exchange columns (2 and 6 µm pore size) and one hydrophobic interaction column (6 µm pore size) were used to examine how column pore size affects virus recovery and contaminant removal. This investigation ultimately inspired the development of a purification process based on monolithic hydrophobic interaction column chromatography. Furthermore, this work is also the first to investigate how additives, namely glycerol, impact the hydrophobic interaction chromatography of virus particles. The developed process could be readily implemented for the scaled-up purification of the Rhabdoviral vector. Another challenge associated with the downstream processing of OVs is membrane fouling, which is characterized by a dramatic rise in transmembrane pressure (TMP) and low virus recovery. Indeed, membrane fouling poses a significant challenge, as some recent studies have reported that it can result in viral vector titer losses of over 80%. One critical use of membranes in downstream processing is for the sterile filtration of OVs, which is a required final step that is conducted right before vialing and involves passing the virus particles through a validated sterile filter. One of the main objectives of this thesis was to develop a fundamental understanding of the sterile filtration process and to optimize it in order to achieve higher throughput and lower losses, which are both essential to the large-scale production of OVs. To this end, a dead-end sterile filtration setup was designed, and various commercially available filters were evaluated to examine how membrane morphology affects fouling and product recovery. The results of these tests showed that double-layered composite filters enabled higher virus recovery and filtration capacity compared to single-layered sterile filters. Another cause of membrane fouling is the aggregation of virus particles, which is mediated by various interactions in the solution. To study this, the above-described setup was re-designed to create an effective procedure that utilizes minimal volumes of virus solution, while also enabling the rapid assessment of microscale filtration performance and a comprehensive understanding of virus-virus and virus-membrane interactions. This setup was used to study how different additives, including various proteins (bovine serum albumin and α-lactalbumin) and polymers (polyethylene glycol and polyvinylpyrrolidone), affect the microfiltration of the Rhabdoviral vector and, consequently, the TMP profile. Furthermore, the correlation between the membrane fouling rate (via TMP profiles) and virus recovery was also investigated. This investigation revealed that proteins significantly increase virus transmission and that polymers are incapable of mimicking the effects of the proteins. To explain this phenomenon, a theory based on the biophysical structure of proteins, mainly heterogenicity in charge distribution, was proposed. Moreover, membrane surface modification tests were conducted using bovine serum albumin, with the results indicating that this approach has considerable potential for enhancing virus transmission. Due to the similarities between the test setup and actual downstream processing unit operations, the results from this part of the thesis could be easily and accurately applied to process optimization. Thesis Candidate in Philosophy There is considerable interest in the development of oncolytic viruses for cancer immunotherapy. Indeed, at the time of this thesis’ writing, a Canadian team of researchers is conducting the world’s first clinical trial using a combination of two viruses to kill cancer cells and stimulate an immune response. The process of manufacturing oncolytic viruses is generally divided into two major steps: upstream processing and downstream processing. While upstream processing focuses on virus propagation, downstream processing aims at removing process-related and product-related impurities. However, research into downstream process design and optimization has largely been neglected in favour of a focus on upstream processing, aimed at increasing bioreactor yields and achieving high viral titers. Consequently, downstream processing has become the main bottleneck in virus manufacturing processes, accounting for as much as 70% production costs. This thesis aims to identify and develop a fundamental understanding of the main challenges associated with the downstream processing of oncolytic viruses and to investigate methods for addressing them. Specifically, the present work focuses on the purification and final sterile filtration steps in the manufacturing of oncolytic Rhabdoviral vectors.

Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15&ndash, 39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.

International audience; Mayotte is an island located in the Mozambique Channel, between Mozambique and Madagascar, in the South Western Indian Ocean region. A severe syndrome of unknown aetiology has been observed seasonally since 2009 in cattle (locally named "cattle flu"), associated with anorexia, nasal discharge, hyperthermia and lameness. We sampled blood from a panel of those severely affected animals at the onset of disease signs and analysed these samples by next-generation sequencing. We first identified the presence of ephemeral bovine fever viruses (BEFV), an arbovirus belonging to the genus Ephemerovirus within the family Rhabdoviridae, thus representing the first published sequences of BEFV viruses of African origin. In addition, we also discovered and genetically characterized a potential new species within the genus Ephemerovirus, called Mavingoni virus (MVGV) from one diseased animal. Finally, both MVGV and BEFV have been identified in cattle from the same herd, evidencing a co-circulation of different ephemeroviruses on the island. The clinical, epidemiological and virological information strongly suggests that these viruses represent the etiological agents of the observed "cattle flu" within this region. This study highlights the importance of the strengthening and harmonizing arboviral surveillance in Mayotte and its neighbouring areas, including Africa mainland, given the importance of the diffusion of infectious diseases (such as BEFV) mediated by animal and human movements in the South Western Indian Ocean area.

In 2012, the genome of a novel rhabdovirus, Bas-Congo virus, was discovered in the acute-phase serum of a Congolese patient with presumed viral hemorrhagic fever. In the absence of a replicating virus isolate, fulfilling Koch's postulates to determine whether Bas-Congo virus is indeed a human virus and/or pathogen has been impossible. However, experiments with vesiculoviral particles pseudotyped with Bas-Congo glycoprotein suggested that Bas-Congo virus particles can enter cells from multiple animals, including humans. In 2015, genomes of two related viruses, Ekpoma virus 1 and Ekpoma virus 2, were detected in human sera in Nigeria. Isolates could not be obtained. Phylogenetic analyses led to the classification of Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2 in the same genus, Tibrovirus, together with five biting midge-borne rhabdoviruses (i.e., Beatrice Hill virus, Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus) not known to infect humans. Using individual recombinant vesiculoviruses expressing the glycoproteins of all eight known tibroviruses and more than 75 cell lines representing different animal species, we demonstrate that the glycoproteins of all tibroviruses can mediate vesiculovirus particle entry into human, bat, nonhuman primate, cotton rat, boa constrictor, and Asian tiger mosquito cells. Using four of five isolated authentic tibroviruses (i.e., Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus), our experiments indicate that many cell types may be partially resistant to tibrovirus replication after virion cell entry. Consequently, experimental data solely obtained from experiments using tibrovirus surrogate systems (e.g., vesiculoviral pseudotypes, recombinant vesiculoviruses) cannot be used to predict whether Bas-Congo virus, or any other tibrovirus, infects humans.

A wide range of viruses from different virus families in different geographical areas, may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the central nervous system, frequently leaving the patient or affected animal with a poor or fatal prognosis. Mechanisms that govern neuropathogenesis and immunopathogenesis of viral infections are highlighted, using examples of well-studied virus infections that are associated with these alterations in different populations throughout the world. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of mechanisms that underlie their clinical manifestations may be expected to provide tools for the development of more effective intervention strategies and treatment regimens.

Rabies virus (RABV) causes a fatal neurological disease in both humans and animals. Understanding the mechanism of RABV infection is vital for prevention and therapy of virulent rabies infection. Our previous proteomics analysis based on isobaric tags for relative and absolute quantitation to identify factors revealed that RABV infection enhanced AP-2-associated protein kinase 1 (AAK1) in N2a cells. In this study, to further confirm the role of AAK1, we showed that RABV infection increased the transcription and expression of AAK1 in N2a cells. AAK1 knockdown significantly decreased RABV infection in both N2a and BHK-21 cells. AAK1 knockout inhibited RABV infection in N2a cells. Furthermore, inhibition of AAK1 kinase activity using sunitinib decreased RABV infection. However, AAK1 overexpression did not change RABV infection in vitro. Therapeutic administration of sunitinib did not significantly improve the survival rate of mice following lethal RABV challenge. In addition, AAK1 knockdown decreased infection in N2a cells by vesicular stomatitis virus, which is another rhabdovirus. These results indicate that rhabdovirus infection is dependent on AAK1 and inhibition of AAK1 is a potential strategy for the prevention and therapy of rabies.

Obligate hematophagous ectoparasitic flies of the superfamily Hippoboscoidea are distributed worldwide, but their role as vectors and reservoirs of viruses remains understudied. We examined hippoboscoid bat flies (family Nycteribiidae) parasitizing Angolan soft-furred fruit bats (Lissonycteris angolensis ruwenzorii) from Bundibugyo District, Uganda. Using metagenomic methods, we detected 21 variants of the rhabdovirid genus Ledantevirus, which contains medically important &ldquo, bat-associated&rdquo, viruses. These 21 viruses, representing at least two divergent viral lineages, infected 26 bat flies from 8 bats in a single roost. Cophylogenetic analyses of viruses and bat flies resulted in strong evidence of virus-host codivergence, indicating vertical transmission of bat fly ledanteviruses. Examination of oral swabs from bats revealed ledantevirus RNA in the saliva of 1 out of 11 bats, with no evidence of insect genetic material in the mouth of this bat. These data demonstrate that bat flies can harbor diverse ledanteviruses even in a single roost and that the predominant mode of transmission is likely vertical (among bat flies), but that bats can become infected and shed viruses orally. In conclusion, bat flies may serve as ectoparasitic reservoirs of &ldquo, viruses that only transiently or sporadically infect bats.

Rhabdoviruses are a large and ecologically diverse family of negative-sense RNA viruses (Mononegavirales: Rhabdoviridae). These viruses are capable of infecting an unexpectedly wide variety of plants, vertebrates, and invertebrates distributed over all human-inhabited continents. However, only a few rhabdoviruses are known to infect humans: a ledantevirus (Le Dantec virus), several lyssaviruses (in particular, rabies virus), and several vesiculoviruses (e.g., Chandipura virus, vesicular stomatitis Indiana virus). Recently, several novel rhabdoviruses have been discovered in the blood of both healthy and severely ill individuals living in Central and Western Africa. These viruses—Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2—are members of the little-understood rhabdoviral genus Tibrovirus. Other than the basic genomic architecture, tibroviruses bear little resemblance to well-studied rhabdoviruses such as rabies virus and vesicular stomatitis Indiana virus. These three human tibroviruses are quite divergent from each other, and each of them clusters closely with tibroviruses currently known only from biting midges or healthy cattle. Seroprevalence studies suggest that human tibrovirus infections may be common but are almost entirely unrecognized. The pathogenic potential of this diverse group of viruses remains unknown. Although certain tibroviruses may be benign and well-adapted to humans, others could be newly emerging and produce serious disease. Here, we review the current knowledge of tibroviruses and argue that assessing their impact on human health should be an urgent priority.

RNA viruses compartmentalize their replication machinery to evade detection by host pattern recognition receptors and concentrate the machinery of RNA synthesis. For positive-strand RNA viruses, RNA replication occurs in a virus-induced membrane-associated replication organelle. For NNS RNA viruses, the replication compartment is a cytoplasmic inclusion that is not circumscribed by a cellular membrane. Such structures were first observed in the cell bodies of neurons from humans infected with rabies virus and were termed Negri bodies. How the replication machinery that forms this inclusion remains associated in the absence of a membrane has been an enduring mystery. In this article, we present evidence that the VSV replication compartments form through phase separation. Phase separation is increasingly recognized as responsible for cellular structures as diverse as processing bodies (P-bodies) and nucleoli and was recently demonstrated for rabies virus. This article further links the fields of host-pathogen interaction with that of phase separation., RNA viruses that replicate in the cell cytoplasm typically concentrate their replication machinery within specialized compartments. This concentration favors enzymatic reactions and shields viral RNA from detection by cytosolic pattern recognition receptors. Nonsegmented negative-strand (NNS) RNA viruses, which include some of the most significant human, animal, and plant pathogens extant, form inclusions that are sites of RNA synthesis and are not circumscribed by a membrane. These inclusions share similarities with cellular protein/RNA structures such as P granules and nucleoli, which are phase-separated liquid compartments. Here we show that replication compartments of vesicular stomatitis virus (VSV) have the properties of liquid-like compartments that form by phase separation. Expression of the individual viral components of the replication machinery in cells demonstrates that the 3 viral proteins required for replication are sufficient to drive cytoplasmic phase separation. Therefore, liquid-liquid phase separation, previously linked to organization of P granules, nucleolus homeostasis, and cell signaling, plays a key role in host-pathogen interactions. This work suggests novel therapeutic approaches to the problem of combating NNS RNA viral infections.

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. This work was supported by the European Research Council (ERC starting grant 2014 GA639249).

Rhabdovirus infections are an emerging problem for both wild and farmed freshwater fish in Northern Europe. In October 2005, a clinical outbreak with an approximate mortality rate of 40% occurred in a single batch of juvenile perch on a farm in the Republic of Ireland. Clinical signs developed slowly and were consistent with a perch rhabdovirus infection: signs included haemorrhages at the base of the fins and apparent impairment of the central nervous system (manifested as loss of equilibrium and erratic swimming behaviour). Studies suggest that the infected fish originated from a hatchery within the country which relied on wild fish broodstock to supplement the production of perch juveniles. A related rhabdovirus was subsequently isolated from this hatchery. Virus isolation studies have shown that rhabdoviruses were often isolated from wild fish in the vicinity of the hatchery between 1993 and 2005. All isolates were analysed using a generic primer set specific for the L gene of fish vesiculotype viruses. Phylogenetic analysis revealed that all isolates recovered from perch clustered together with the European lake trout rhabdovirus (903/87) of the genus Perhabdovirus. In addition to this, anguillid rhabdovirus was isolated from eel, and the partial L-gene sequence of a previously reported isolate from tench clustered with the pike fry rhabdoviruses, in the genus Sprivivirus.

Highlights • Three new rhabdoviruses were detected in bats, China. • JHBV and BXBV found in Rhinolophus bats could be new members of the genus Vesiculovirus. • Four strains of YJBV detected in Hipposideros larvatus formed a new lineage., The Rhabdoviridae is among the most diverse families of RNA viruses and currently classified into 18 genera with some rhabdoviruses lethal to humans and other animals. Herein, we describe genetic characterization of three novel rhabdoviruses from bats in China. Of these, two viruses (Jinghong bat virus and Benxi bat virus) found in Rhinolophus bats showed a phylogenetic relationship with vesiculoviruses, and sequence analyses indicate that they represent two new species within the genus Vesiculovirus. The remaining Yangjiang bat virus found in Hipposideros larvatus bats were only distantly related to currently known rhabdoviruses.

The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.