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Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s).

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT-PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.

Background In Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and D’Aguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014. Results Blood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D’ Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle (ages 6–12 months: had only been alive for one summer) clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan. Conclusions We showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0747-z) contains supplementary material, which is available to authorized users.

International audience; More than two thirds of emerging viruses are of zoonotic origin, and among them RNA viruses represent the majority. Ceratopogonidae (genus Culicoides) are well-known vectors of several viruses responsible for epizooties (bluetongue, epizootic haemorrhagic disease, etc.). They are also vectors of the only known virus infecting humans: the Oropouche virus. Female midges usually feed on a variety of hosts, leading to possible transmission of emerging viruses from animals to humans. In this context, we report here the analysis of RNA viral communities of Senegalese biting midges using next-generation sequencing techniques as a preliminary step toward the identification of potential viral biohazards. Sequencing of the RNA virome of three pools of Culicoides revealed the presence of a significant diversity of viruses infecting plants, insects and mammals. Several novel viruses were detected, including a novel Thogotovirus species, related but genetically distant from previously described tick-borne thogotoviruses. Novel rhabdoviruses were also detected, possibly constituting a novel Rhabdoviridae genus, and putatively restricted to insects. Sequences related to the major viruses transmitted by Culicoides, i.e., African horse sickness, bluetongue and epizootic haemorrhagic disease viruses were also detected. This study highlights the interest in monitoring the emergence and circulation of zoonoses and epizooties using their arthropod vectors.

A major obstacle in the implementation of adoptive cell therapy (ACT) for solid tumors is CD8+ T cell quantity and functional quality. In order to address this issue, the ACT field has directed considerable effort toward the generation of less-differentiated memory T cells (Tm), which demonstrate superior effector function and engraftment over effector T cells. An obstacle in using Tm for ACT is their requirement for in vivo activation before full effector function can be acquired. We sought to determine if a rhabdovirus expressing a defined tumor antigen (i.e. a rhabdoviral oncolytic vaccine) could activate adoptively-transferred Tm in vivo and eliminate established tumors. We used ex vivo cultured DUC18 TCR-transgenic Tm combined with a rhabdoviral oncolytic vaccine to target established CMS5 fibrosarcomas in both balb/c and NRG mice, and we compared the efficacy of the combination treatment versus monotherapies. Our data demonstrate that the rhabdoviral oncolytic vaccine was capable of expanding adoptively-transferred Tm in order to eliminate established tumors. Furthermore, synergy between ACT and oncolytic vaccination was required for optimal therapeutic outcome. Interestingly, we observed a population of endogenous, tumor-primed lymphocytes which appeared to be required for complete tumor elimination and subsequent memory formation. This was in contrast to the current consensus in the ACT field which is that endogenous lymphocytes are detrimental to therapeutic outcome, thus necessitating lymphodepletion prior to the commencement of therapy. Our data suggest that endogenous lymphocytes may be a beneficial cell population which is overlooked by current approaches to ACT. Thesis Master of Science (MSc) Current approaches to the T cell therapy of cancer are hindered by poor cell quality. It is simple to grow higher quality T cells, but it is difficult to grow very large numbers of them. Furthermore, higher quality T cells need a signal in order to “switch on” before they can start killing cancer cells. Here, we use a cancer-targeting virus as a signal for these cells to activate, grow to very large numbers in the patient, and destroy their tumor. Our vaccine also switches on other immune cells in the patient, which help guarantee the destruction of the tumor. The significance of this work is that it will improve T cell therapy for cancer by opening the possibility of using higher-quality T cells which are much better at killing cancer than the currently used type of T cells.

Surface glycoproteins of enveloped virus are potent elicitors of both innate and adaptive host immune responses. Therefore, the identification of viral glycoprotein determinants directly implicated in the induction of these responses might be of special interest for designing new therapeutical/adjuvant molecules. In this work we review the contribution of the "pepscan" approach to the screening of viral functions in the sequence of glycoprotein G (gpG) of the fish rhabdovirus of viral hemorrhagic septicemia (VHSV). Among others, by scanning gpG peptides, it has been possible to identify and validate minimal determinants for gpG directly implicated in initiating the fish type I Interferon-associated immune responses as well as in the antiviral autophagy program. Further fine-tunning of the identified peptides in the gpG of VHSV has allowed designing novel adjuvants that decrease DNA vaccine requirements and identify possible innovative antiviral molecules. In addition, these results have also contributed to improve our knowledge on how to stimulate the fish immune system. Refereed/Peer-reviewed

Kotonkan virus (KOTV) and Obodhiang virus (OBOV) are rhabdoviruses that were isolated from arthropods in Africa and formerly classified as lyssaviruses. KOTV causes clinical bovine ephemeral fever in cattle; the ecology and pathogenicity of OBOV is poorly understood. In this paper, we report the complete genome sequences of KOTV and OBOV, their gene expression profiles, and their serological and phylogenetic relationships to other rhabdoviruses. The 15,870 nt KOTV genome (3′-l-N-P-M-G-GNS-α1-α2-β-γ-δ-L-t-5′) is similar to that of bovine ephemeral fever virus but encodes an additional protein (δ) that shares homology with the pleckstrin homology domain of coactivator-associated arginine methyltransferase. The 14,717 nt OBOV genome (3′-l-N-P-M-G-GNS-α1-α2-β-L-t-5′) is similar to that of Adelaide River virus from which it is distinguishable serologically. In each virus, all ORFs, except α1 and α2, are transcribed as monocistronic mRNA. Genetic and serological data indicate that KOTV and OBOV should be classified as new species in the genus Ephemerovirus.

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

The sperm of the heteropteran bug Raphigaster nebulosa (Poda) are of two types, differing in length and size of their flagella. The thicker sperm are shorter than the thinner ones and have large mitochondrial derivatives. The presence of virus particles associated with the plasma membrane of thinner sperm is described for the first time; thicker sperm are immune to virus infection. The fact that virus particles are present on thinner sperm only initiates considerations on the transmission of virus.

The Rhabdoviridae is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. The availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. The five canonical rhabdovirus structural protein genes (N, P, M, G and L) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. Although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. Others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. However, most encode proteins of unknown function that are unrelated to any other known proteins. Understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine.

Publisher Summary This chapter discusses the structure and working of viral fusion machinery. The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Structural studies have defined three classes of viral membrane fusion proteins. Despite their different structural organizations, all seem to have a common mechanism of action that generates the same lipid organizations during the fusion pathway. The entry of enveloped viruses into host cells requires binding of the virus to one or more receptors present at the cell surface, followed by fusion of the viral envelope with a cellular membrane. These steps are mediated by virally encoded glycoproteins that promote both receptor recognition and membrane fusion. The first crystal structure of a viral fusion protein ectodomain that has been determined is that of influenza virus hemagglutinin (HA) in its prefusion conformation. The structures of viral fusion glycoproteins, of which the conformational change is triggered at low pH, has allowed the identification of amino acid residues that play the role of pH-sensitive molecular switches.

A diverse range of endosymbionts are found within the cells of animals. As these endosymbionts are normally vertically transmitted, we might expect their evolutionary history to be dominated by host-fidelity and cospeciation with the host. However, studies of bacterial endosymbionts have shown that while this is true for some mutualists, parasites often move horizontally between host lineages over evolutionary timescales. For the first time, to our knowledge, we have investigated whether this is also the case for vertically transmitted viruses. Here, we describe four new sigma viruses, a group of vertically transmitted rhabdoviruses previously known in Drosophila . Using sequence data from these new viruses, and the previously described sigma viruses, we show that they have switched between hosts during their evolutionary history. Our results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts.

Viral hemorrhagic septicemia virus (VHSV) is one of the most serious pathogens of finfish worldwide in terms of its wide host-range, pathogenicity, disease course, and mortality rates. The disease was first documented in the 1930s in Europe in association with heavy losses in rainbow. Data collected over 50 years show that VHSV is a virus of marine origin: its ability to alternate between marine and freshwater environments remains an enigma which requires further investigation. In 2003, VHSV invaded the Laurentian Great Lakes basin causing devastating losses. VHSV is believed to have negative impacts on a number of important Atlantic, Pacific, and Great Lakes fish species. Phylogenetic analysis has offered clues into the geographic and host range of the virus, but sporadic outbreaks of the disease in uncommon locations have imparted unforeseen challenges in delineating the virus’ regional distribution. The virus’ ability to gain access to aquaculture farms has also positioned it as a disease of utmost priority once detected in these settings. Current diagnostic methods, while greatly improved, are hampered by the variability of disease course among susceptible species. In general, VHSV causes severe degeneration in the hematopoietic tissues of affected fish. Based on historical and current data, it is feared that VHSV will continue to mutate, expand to other geographic areas, and infect new host species. As a result, immediate international attention and coordination of efforts are needed. The objective of this review article is to provide an updated synopsis on the current status of VHSV epizootiology and pathogenicity.

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:Trout RBCs were obtained from peripheral blood, ficoll purified and exposed toViral 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 profilingResults:VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon (ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-induciblemxandpkrgenes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the 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 Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV.Conclusions:A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.

Like many animal viruses, those of the Rhabdoviridae family, are able to antagonize the type I interferon response and cause disease in mammalian hosts. Though these negative-stranded RNA viruses are very simple and code for as few as five proteins, they have been seen to completely abrogate the type I interferon response early in infection. In this review, we will discuss the viral organization and type I interferon evasion of rhabdoviruses, focusing on vesicular stomatitis virus (VSV) and rabies virus (RABV). Despite their structural similarities, VSV and RABV have completely different mechanisms by which they avert the host immune response. VSV relies on the matrix protein to interfere with host gene transcription and nuclear export of anti-viral mRNAs. Alternatively, RABV uses its phosphoprotein to interfere with IRF-3 phosphorylation and STAT1 signaling. Understanding the virus-cell interactions and viral proteins necessary to evade the immune response is important in developing effective vaccines and therapeutics for this viral family.

Antimicrobial peptides (AMPs) are one of the components of the non-specific immune system that operate first lines of protection in many animal species including fish. They exert broad-spectrum antimicrobial activity, apart from many other potential roles in innate immunity, and represent a promising class of antiviral agents. Recent advances in understanding the mechanisms of their antiviral action(s) indicate that they have a dual role in antiviral defence, acting not only directly on the virion but also on the host cell. Despite the acute problems of viral diseases and restrictions in using chemicals in aquaculture, few but successful attempts to assess the antiviral activities of fish AMPs have been reported. This review focuses on the antiviral activities and mechanisms of action of some AMPs, and their potential relevance in the aquaculture industry, one of the most important sources of fishery products in the near future. It is a matter of notable concern to understand whether the AMPs can be used as model molecules for designing antiviral drugs that might help to solve the problems with viruses in the fish farming industry worldwide. In addition, because fish rely more heavily on their innate immune defences than mammals, they might constitute a potential rich source of antiviral compounds for fighting against mammalian viral infections. © 2009 Bentham Science Publishers Ltd.

It is well known that human alpha-defensin-1, also designated as human neutrophil peptide 1 (HNP1), is a potent inhibitor towards several enveloped virus infecting mammals. In this report, we analyzed the mechanism of the antiviral action of this antimicrobial peptide (AMP) on viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Against VHSV, synthetic HNP1 possesses two antiviral activities. The inactivation of VHSV particles probably through interfering with VHSV-G protein-dependent fusion and the inhibition of VHSV replication in target cells by up-regulating genes related to the type I interferon (IFN) response, such as Mx. Neither induction of IFN-stimulated genes (ISGs) by HNP1 nor their antiviral activity against fish rhabovirus has been previously reported. Therefore, we can conclude that besides to acting as direct effector, HNP1 acts across species and can elicit one of the strongest antiviral responses mediated by innate immune system. Since the application of vaccine-based immunization strategies is very limited, the used of chemicals is restricted because of their potential harmful impact on the environment and no antimicrobial peptides from fish that exhibit both antiviral and immunoenhancing capabilities have been described so far, HNP1 could be a model molecule for the development of antiviral agents for fish. In addition, these results further confirm that molecules that mediate the innate resistance of animals to virus may prove useful as templates for new antivirals in both human and animal health. © 2007 Elsevier B.V. All rights reserved.

Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5′ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order.

Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies., This research was supported by Karadeniz Technical University, Trabzon, Turkey (KTU-BAP 9102 and 9585) and the Spanish Ministry of Science and Innovation (AGL2011-30352-C02-02). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

To the Editor: Rhabdoviruses (family Rhabdoviridae) are enveloped single-stranded negative-sense RNA viruses belonging to the Mononegavirales order. The International Committee on Taxonomy of Viruses recognizes 11 genera (Cytorhabdovirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Tibrovirus, Tupavirus, Vesiculovirus) (1). In addition, many recently described rhabdoviruses remain unassigned. Rhabdoviruses contain 5 major genes, encoding for nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). The Rhabdoviridae family includes pathogens of various animal species, humans, and plants. Viruses of the genus Lyssavirus are the most relevant to public health because they can cause rabies. Bats are the driving force within this genus; foxes and various other species of wild carnivores also can be infected with lyssaviruses and transmit them to humans and dogs (2). During a viral metagenomic survey, conducted as described previously (3), of fecal samples collected from 4 red foxes (Vulpes vulpes) that were found dead in Alava, Basque Country, Spain, we identified the complete coding sequence and the partial leader and trailer sequence of a novel rhabdovirus, tentatively called red fox fecal rhabdovirus (RFFRV; 15,541 nt, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KF823814","term_id":"645393534","term_text":"KF823814"}}KF823814; Technical Appendix) by mapping 8,287 of the 56,519 sequence reads in the sample of a red fox. A proportion of obtained reads contained sequences that were >99% identical to mitochondrial DNA of V. vulpes, which confirmed that the sample was collected from a red fox. The obtained sequence of RFFRV was partially confirmed by specific primers and Sanger sequencing of PCR amplicons. Five major and 3 minor open reading frames (ORFs) were identified that had a genome organization similar to that of other rhabdoviruses (Figure, panel A). No significant hits were obtained by BLAST analysis (http://blast.ncbi.nlm.gov/Blast.cgi) of N, P, M, and G nucleotide and amino acid sequences, which was reported previously for novel divergent rhabdoviruses (4). Figure Genome organization and phylogenetic analysis of RFFRV. A) Genome organization of RFFRV. Indicated are the locations of the major ORFs (including the positions of the first and last nucleotide) and 3 theoretical minor ORFs between the G and L genes. B) ... Predicted N, P, and M genes of RFFRV consist of 1,629, 2,490, and 813 nt, respectively, encoding for 543, 830, and 271 aa (Technical Appendix Table 1). In addition to the absence of significant hits observed by BLAST analysis, no significant sequence homology was observed with known rhabdovirus proteins in pairwise alignments. Furthermore, no conserved motifs were detected in N, P, and M genes of RFFRV that are commonly observed in rhabdoviruses. However, intergenic regions between all major ORFs contained relatively conserved motifs that could be transcription termination/polyadenylation sequences (A/U) CU7, similar to other rhabdoviruses (5). Adjacent to this termination signal was a stretch of conserved nucleotides that might function as a transcription initiation signal Technical Appendix Table 1). The amino acid sequence of the G protein consisted of 669 aa and contained an N terminal signal peptide (1-MYHLIVLLVMLGQRAVA-17), a noncytoplasmic domain (aa 18–646), a transmembrane domain (647-ITAILMPLLSLAVVVGIIMCC-667), and a cytoplasmic tail of 2 aa, similar to other rhabdovirus G proteins as predicted by using Phobius and TMHMM (http://www.cbs.dtu.dk/services/TMHMM) (6,7). We predicted 3 potential glycosylation sites in the ectodomain at positions 38–40 (NKT), 554–556 (NAS), and 592–594 (NIS) using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc). Between the G and L genes, a complex intergenic region was present that contained 3 ORFs of 246 nt (7,413–7,658 aa), 231 nt (7,716–7,946 aa), and 459 nt (7,893–8,355 aa), of which 2 were overlapping frames (U1–3). Additional ORFs between G and L genes were detected previously in other rhabdoviruses (8,9). We detected transmembrane domains in the amino acid sequences of all 3 additional ORFs, suggesting they might act as viroporin (8,9). The L gene of RFFRV contained 6,591 nt (2,197 aa). We detected several conserved domains and motifs, including RNA-dependent RNA polymerase, mRNA-capping region, mRNA capping enzyme, and virus-capping methyltransferase. Alignment of the deduced amino acid sequence of the L gene with the L gene of various other viruses belonging to the Mononegavirales order by using MAFFT version 7 (http://mafft.cbrc.jp/alignment/software/) and subsequent phylogenetic reconstruction by using a maximum-likelihood tree (WAG+F+I+G model with 100 bootstrap replicates in MEGA5 [http://www.megasoftware.net]) suggested that this virus belongs to a novel genus of the Rhabdoviridiae family. In addition, pairwise identities of the deduced amino acid sequence of the L gene of RFFRV with that of other rhabdoviruses of the Rhabdoviridae family were only

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

International audience; Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass À,fi'croptertts salmoides exhibiting clinical signs of disease, Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previotrsiy isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion.The relationship between these viruses and the previously characterised fish rhabdoviruses, pike fry rhabdovirus (PFRV), spring viraemia of carp virus (SVCV) and lake trout rhabdovirus, was investi- Eated. Viruses were propagated in bluegill fry (BF-2) cells and were characterised using electron microscopy, serum neutralisation tests, immunofluorescence tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nucleotide sequence analysis. The bullet-shaped viral particles appeared to be compact, with spikes visible at the surface, a morphology similar to that of the vesiculovirus group of rhabdoviruses. Serum neutralisation tests showed that the viruses were antigenically closely related to the previously characterised perch rhabdovirus, but were not significantly neutralised by antisera to PFRV, SVCV or viral haemorrhagic septicaemia virus (VHSV), In immunofluorescence tests with perch rhabdovirus antisera, strong specific fluorescence was observed in cell cultures infected with the new rhabdovirus isolates, but no fluorescence was observed with antisera to PFRV, SVCV or VHSV. SDS-PAGE analysis reveaied a polypeptide profile typical of vesiculoviruses, but the novel virus isolates had different relative mobilities of their P and M proteins compared to PFRV and SVCV. Nucleotide sequence analysis was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing of a 439 base-pair region of the viral L gene. The novel rhabdovirus isolates had 95o/" identity to perch rh.abdovirus. Phylogenetic analysis using both maximum parsimony and neighbour-joining methods assigned the perch rhaboviruses to a separate group to that of PFRV, SVCV and lake trout rhabdovirus, These data are the initial characterisation of a group of emerging fish vesiculo-type viruses that are biochemically and geneticaliy distinct from the PFRV, SVCV and lake trout rhabdoviruses.

A homologous fish cell line stably expressing the recombinant Japanese flounder Mx (JFMx) was infected with hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), both of which are negative single-stranded RNA viruses belonging to the Rhabdoviridae family. Analysis of primary transcription of the two rhabdoviruses showed that there was lower expression level and copy number of the viral nucleoprotein transcript in the JFMx-transfected cell line than the infected, control cells, although no significant difference was observed. This suggests that JFMx may not be a potent inhibitor of rhabdoviral primary transcription. Kinetics of rhabdovirus expression by RT-PCR and quantitative real-time RT-PCR showed reduced levels of the rhabdoviral glycoprotein and nucleoprotein transcripts over time, indicating the possible role of JFMx in blocking rhabdoviral replication by interfering with the transcription of the viral subgenomic mRNAs. Significant inhibition in rhabdovirus replication consequently resulted in the synthesis of fewer viral particles. This may explain why JFMx-expressing cells are less susceptible to virus-induced cell lysis, and thus, why they would have a significantly higher survival than the infected, control cells. These results provide direct evidence that JFMx has an antiviral effect in vitro.

The complete nucleotide sequence of RNA1 from Lettuce big-vein virus (LBVV), the type member of the genus Varicosavirus , was determined. LBVV RNA1 consists of 6797 nucleotides and contains one large ORF that encodes a large (L) protein of 2040 amino acids with a predicted M r of 232,092. Northern blot hybridization analysis indicated that the LBVV RNA1 is a negative-sense RNA. Database searches showed that the amino acid sequence of L protein is homologous to those of L polymerases of nonsegmented negative-strand RNA viruses. A cluster dendrogram derived from alignments of the LBVV L protein and the L polymerases indicated that the L protein is most closely related to the L polymerases of plant rhabdoviruses. Transcription termination/polyadenylation signal-like poly(U) tracts that resemble those in rhabdovirus and paramyxovirus RNAs were present upstream and downstream of the coding region. Although LBVV is related to rhabdoviruses, a key distinguishing feature is that the genome of LBVV is segmented. The results reemphasize the need to reconsider the taxonomic position of varicosaviruses.

A highly attenuated, recombinant rabies virus (RV) vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against hepatitis C (HCV) glycoprotein E2. We showed previously that RV-based vectors are able to induce strong immune responses against human immunodeficiency virus type I (HIV-1) antigens. Here we constructed and characterized three replication-competent RV-based vectors expressing either both HCV envelope proteins E1 and E2 or a modified version of E2 which lacks 85 amino acids of its carboxy terminus and contains the human CD4 transmembrane domain and the CD4 or RV glycoprotein cytoplasmic domain. All three constructs stably expressed the respective protein(s) as indicated by Western blotting and immunostaining. Moreover, surface expression of HCV E2 resulted in efficient incorporation of the HCV envelope protein regardless of the presence of the RV G cytoplasmic domain, which was described previously as a requirement for incorporation of foreign glycoproteins into RV particles. Killed and purified RV virions containing HCV E2 were highly immunogenic in mice and also proved useful as a diagnostic tool, as indicated by a specific reaction with sera from HCV-infected patients. In addition, RV vaccine vehicles were able to induce cellular responses against HCV E2. These results further suggest that recombinant RVs are potentially useful vaccine vectors against important human viral diseases.

Viruses of the family Rhabdoviridae infect a broad range of hosts from a variety of ecological and geographical niches, including vertebrates, arthropods, and plants. The arthropod-transmitted members of this family display considerable genetic diversity and remarkable genomic flexibility that enable coding for various accessory proteins in different locations of the genome. Here, we describe the genome of Holmes Jungle virus, isolated from Culex annulirostris mosquitoes collected in northern Australia, and make detailed comparisons with the closely related Ord River and Wongabel viruses, with a focus on identifying very small open reading frames (smORFs) in their genomes. This is the first systematic prediction of smORFs in rhabdoviruses, emphasising the intricacy of the rhabdovirus genome and the knowledge gaps. We speculate that these smORFs may be of importance to the life cycle of the virus in the arthropod vector.