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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

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

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) plants growing in 25 districts across Ethiopia, Kenya, Tanzania and Uganda were surveyed for four RNA viruses. Leaf samples from 392 plants were tested for cucumber mosaic virus (CMV), dasheen mosaic virus (DsMV), taro vein chlorosis virus (TaVCV) and Colocasia bobone disease-associated virus (CBDaV) by RT-PCR. No samples tested positive for TaVCV or CBDaV, while CMV was only detected in three tannia samples with mosaic symptoms from Uganda. DsMV was detected in 40 samples, including 36 out of 171 from Ethiopia, one out of 94 from Uganda and three out of 41 from Tanzania, while none of the 86 samples from Kenya tested positive for any of the four viruses. The complete genomes of nine DsMV isolates from East Africa were cloned and sequenced. Phylogenetic analyses based on the amino acid sequence of the DsMV CP-coding region revealed two distinct clades. Isolates from Ethiopia were distributed in both clades, while samples from Uganda and Tanzania belong to different clades. Seven possible recombination events were identified from the analysis carried out on the available 15 full-length DsMV isolates. Nucleotide substitution ratio analysis revealed that all the DsMV genes are under strong negative selection pressure.

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.

Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 μg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.

Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 μg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro-inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.

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 exampl

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. © 2016.

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.

A wide range of viruses from different virus families in different geographical areas, may cause immediate or delay

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.

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.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

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.

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.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year. © 2014 Landes Bioscience.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific. Copyright © 2013 by The American Association of Immunologists, Inc.

The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific. Copyright © 2013 by The American Association of Immunologists, Inc.

Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV 860) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G 860). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV 860 lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization. © 2012.

Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV860) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G860). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV860 lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization.

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV)-coding plasmid induces an anti-VHSV state, similar to that induced by poly IC. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of GVHSV protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9-CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding GVHSV gene are due to GVHSV rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants. © 2010 Elsevier Ltd.

One of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.

K13965, an uncharacterized virus, was isolated in 1993 from Anopheles annulipes mosquitoes collectedin the Kimberley region of northern Western Australia. Here, we report its genomic sequence, identifyit as a rhabdovirus, and characterize its phylogenetic relationships. The genome comprises a P (C) and SH protein similar to the recently characterized Tupaia and Durham viruses, and shows overlap between G and L genes. Comparison of K13965 genome sequence to other rhabdoviruses identified K13965 as a strain of the unclassified Australian Oak Vale rhabdovirus, whose complete genome sequence we also determined. Phylogenetic analysis of N and L sequences indicated genetic relationship to a recently proposed Sandjima virus clade, although the Oak Vale virus sequences form a branch separate from the African members of that group.

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.

A new tool for DNA transfer to fish cell lines such as epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG2), has been optimized by testing commercially available polyethylenimine (PEI) polymers as transfectant reagents. Deacylated 25 kDa PEI polymers were selected amongst the most active and then low toxicity deacylated PEIs fractions around 15 kDa were obtained by gel filtration chromatography to increase 3-4-fold their initial in vitro transfection efficiency. The EPC and plasmids coding for reporter genes were first used to optimize variable values for best expression by transfection with deacylated low toxicity PEI while both EPC/RTG2 and a plasmid coding for the glycoprotein G gene of the fish pathogen, viral haemorrhagic septicemia virus (VHSV) were then used to demonstrate some of their practical applications. Due to its relatively low price, defined chemical composition and availability, low toxicity deacylated PEI might be used for numerous applications for all those studying fish cell immunology in vitro as well as in vivo. © 2009 Elsevier Ltd. All rights reserved.

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.

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.

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.

In rainbow trout (Oncorhynchus mykiss), at least three CXC chemokines and eighteen CC chemokine sequences have been discovered in the past years, although no studies concerning their bioactivity have been performed yet. We have studied the expression of five different CC chemokines that cluster into the group of inducible chemokines (CK5A, CK5B, CK6, CK7A and CK7B) in trout injected with a plasmid coding for the glycoprotein G gene (pMCV1.4-G) of viral haemorrhagic septicemia virus (VHSV). In a previous work, we had demonstrated that a plasmid coding for the CXC chemokine interleukin 8 (IL-8, pIL8+) when co-administered with pMCV1.4-G was able to modulate the expression of induced pro-inflammatory cytokines, therefore, we have also studied the expression of these inducible CC chemokines in fish injected with pMCV1.4-G in the presence of pIL8+, study that will also help establish the relations among the different chemokine groups. All chemokines were induced in the head kidney of fish injected with the DNA vaccine, and the co-administration of pIL8+ together with the vaccine modulated the expression of all CC chemokines studied. In this sense, expression of the inducible CC chemokines was also studied in trout head kidney leucocytes treated with supernatants from EPC cells transfected with pIL8+. In this case, all CC chemokines studied except for CK5B were significantly induced by rainbow trout IL-8.

Symptoms of citrus leprosis were observed during 2004 - 2005 in the East plains of Colombia, Departments of Meta and Casanare. To identify the causal agent, CORPOICA collected leaves of Valencia sweet orange exhibiting typical lesions of leprosis from several locations in the Departments of Casanare (Yopal, Aguazul) and Meta (Guamal, Villavicencio and Cumaral). Virus identification was done by RT-PCR and transmission electron microscopy (TEM). Ten of the 21 samples examined by TEM resulted positive to the leprosis virus (Citrus Leprosis Virus, CiLV-C). Short bacilliform particles characteristic of CiLV-C were found in the endoplasmic reticulum, and irregularly shaped viroplasms were present in the cytoplasm. Fourteen of the 32 samples processed by RT-PCR were positive to CiLV-C. RT-PCR analyses amplified cDNA fragments of expected size for samples collected in Meta. One of the amplifications was sequenced (GenBank accession No. DQ272491) and found to have 98% nucleotide sequence identity to the Brazilian CiLV-C isolate (GenBank accession No. AY289190.1). Sequence and primers were provided to ICA for CiLV-C diagnosis in Colombia. Mites collected from affected plants were identified as Brevipalpus phoenicis (Geijskes), one of the most important vectors of CiLV-C., Durante los años 2004 y 2005 se observaron síntomas de la leprosis de los cítricos en los departamentos de Meta y Casanare. Para confirmar la causa de esta sintomatología, se colectaron hojas de naranja ‘Valencia’ con lesiones típicas en municipios de Casanare (Yopal y Aguazul) y Meta (Guamal, Villavicencio y Cumaral). Las muestras fueron procesadas identificar el virus por medio de microscopía electrónica de transmisión (MET) y transcripción reversa - reacción en cadena de la polimerasa (RT-PCR). Diez de las 21 muestras analizadas por MET resultaron positivas para el virus de la leprosis (Citrus Leprosis Virus, CiLV-C). En algunas muestras se observaron partículas baciliformes características del CiLV-C en el retículo endoplasmático y formas irregularles de viroplasmas en el citoplasma. Catorce de las 32 muestras procesadas por RT-PCR resultaron positivas al CiLV-C. Los análisis RT-PCR amplificaron fragmentos de cADN del tamaño esperado para las muestras colectadas en el Meta. Una de las ampliaciones fue secuenciada (GenBank accessión No. DQ272491) y la secuencia obtenida guarda identidad del 98% con la secuencia brasilera aislada de nucleótidos para CiLV-C (GenBank accessión No. AY289190.1). La secuencia obtenida y los primers fueron entregados al Instituto Colombiano Agropecuario —ICA— para el diagnóstico del virus CiLV-C en Colombia. Entre los ácaros recolectados en las plantas afectadas se identificó a Brevipalpus phoenicis (Geijskes) reconocido como principal vector de CiLV-C.

Symptoms of citrus leprosis were observed during 2004 - 2005 in the East plains of Colombia, Departments of Meta and Casanare. To identify the causal agent, CORPOICA collected leaves of Valencia sweet orange exhibiting typical lesions of leprosis from several locations in the Departments of Casanare (Yopal, Aguazul) and Meta (Guamal, Villavicencio and Cumaral). Virus identification was done by RT-PCR and transmission electron microscopy (TEM). Ten of the 21 samples examined by TEM resulted positive to the leprosis virus (Citrus Leprosis Virus, CiLV-C). Short bacilliform particles characteristic of CiLV-C were found in the endoplasmic reticulum, and irregularly shaped viroplasms were present in the cytoplasm. Fourteen of the 32 samples processed by RT-PCR were positive to CiLV-C. RT-PCR analyses amplified cDNA fragments of expected size for samples collected in Meta. One of the amplifications was sequenced (GenBank accession No. DQ272491) and found to have 98% nucleotide sequence identity to the Brazilian CiLV-C isolate (GenBank accession No. AY289190.1). Sequence and primers were provided to ICA for CiLV-C diagnosis in Colombia. Mites collected from affected plants were identified as Brevipalpus phoenicis (Geijskes), one of the most important vectors of CiLV-C., Durante los años 2004 y 2005 se observaron síntomas de la leprosis de los cítricos en los departamentos de Meta y Casanare. Para confirmar la causa de esta sintomatología, se colectaron hojas de naranja ‘Valencia’ con lesiones típicas en municipios de Casanare (Yopal y Aguazul) y Meta (Guamal, Villavicencio y Cumaral). Las muestras fueron procesadas identificar el virus por medio de microscopía electrónica de transmisión (MET) y transcripción reversa - reacción en cadena de la polimerasa (RT-PCR). Diez de las 21 muestras analizadas por MET resultaron positivas para el virus de la leprosis (Citrus Leprosis Virus, CiLV-C). En algunas muestras se observaron partículas baciliformes características del CiLV-C en el retículo endoplasmático y formas irregularles de viroplasmas en el citoplasma. Catorce de las 32 muestras procesadas por RT-PCR resultaron positivas al CiLV-C. Los análisis RT-PCR amplificaron fragmentos de cADN del tamaño esperado para las muestras colectadas en el Meta. Una de las ampliaciones fue secuenciada (GenBank accessión No. DQ272491) y la secuencia obtenida guarda identidad del 98% con la secuencia brasilera aislada de nucleótidos para CiLV-C (GenBank accessión No. AY289190.1). La secuencia obtenida y los primers fueron entregados al Instituto Colombiano Agropecuario —ICA— para el diagnóstico del virus CiLV-C en Colombia. Entre los ácaros recolectados en las plantas afectadas se identificó a Brevipalpus phoenicis (Geijskes) reconocido como principal vector de CiLV-C.

We have sequenced the monopartite RNA genome of a Fijian isolate of Taro vein chlorosis virus (TaVCV) and confirmed that it is a definitive rhabdovirus with most similarity to members of the genus Nucleorhabdovirus. The 12020 nt TaVCV negative-sense RNA genome contained six ORFs in the antigenomic sequence, equivalent to genes N, P, 3, M, G and L identified in other rhabdoviruses. The putative gene products had closest identity to those of the nucleorhabdovirus, Maize mosaic virus (MMV). A characteristic 3'AAUUCUUUUUGGGUUGU/A 5' sequence was identified in each of the intergenic regions, and the TaVCV leader and trailer sequences comprised 140 nt and 61 nt, respectively. Assignment of TaVCV into the genus Nucleorhabdovirus was supported by thin-section electron microscopy of TaVCV-infected taro leaves, which identified virions budding from nuclear membranes into the perinuclear space. Variability studies identified high levels of TaVCV sequence diversity. Within the L gene of 20 TaVCV isolates from Fiji, Federated States of Micronesia, New Caledonia, Papua New Guinea, Solomon Islands and Vanuatu, maximum variability at the nucleotide level was 27.4%. Within the N gene, maximum variability among 15 isolates at the nucleotide level was 19.3%. The high level of TaVCV variability observed suggested that the introduction of TaVCV to the Pacific Islands is not a recent occurrence.

Several recombinant infectious hematopoietic necrosis viruses (IHNV) were produced by reverse genetics and their pathogenicity in trout was evaluated and compared to that of the wild type (wt) viruses IHNV and viral haemorrhagic septicemia virus (VHSV). Recombinant IHNVs used in this study were: rIHNV, identical to the wtIHNV; rIHNV-Gvhsv, a recombinant virus expressing the VHSV G gene instead of the IHNV G gene; rIHNV-Gmut, which possesses 2 targeted mutations in the glycoprotein; and rIHNVmut-Gmut, which is similar to the rIHNV-Gmut, but exhibits additional mutations along the genome. Results obtained in experimental infections showed that the rIHNV and rIHNV-Gmut were the most virulent recombinant viruses. Severity of the lesions induced by the different recombinant viruses was in agreement with mortality data. The kidney and the liver were the organs most affected by the most pathogenic viruses, and the lesions observed resembled those produced by wtIHNV. The introduction of mutations did not alter the tissue tropism of the virus. The recombinant viruses were able to replicate in fish, as shown by immunoperoxidase assay and RT-PCR. Antibodies against IHNV were detected in the fish inoculated with IHNV, rIHNV, rIHNV-Gmut and rIHNVmut-Gmut, and antibodies against VHSV were also found in fish infected with rIHNV-Gvhsv. Finally, antibody production was highest in fish infected with the rIHNVmut-Gmut even though this virus was the least virulent.

In 2001, a potentially lethal pathogen was identified in sardines landed in Southern California. The pathogen was found to be a rhabdovirus with similarities to the rabies virus. The virus attacks the lining of a fish’s blood vessels causing hemorrhaging. Fish with these symptoms have what is known as viral hemorrhagic septicemia (VHS), a disease that has caused mass fish kills in the wild and at farms. Although California sardines were infected with the VHS virus, none of the fish sampled showed symptoms of disease.

In 2001, a potentially lethal pathogen was identified in sardines landed in Southern California. The pathogen was found to be a rhabdovirus with similarities to the rabies virus. The virus attacks the lining of a fish’s blood vessels causing hemorrhaging. Fish with these symptoms have what is known as viral hemorrhagic septicemia (VHS), a disease that has caused mass fish kills in the wild and at farms. Although California sardines were infected with the VHS virus, none of the fish sampled showed symptoms of disease.

Antibody linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout antibodies among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa and Coll 1996; Virology 21660-70) and leucocyte stimulating peptides (Lorenzo et al 1995; Virology 212348-355). Among 17 monoclonal antibodies (MAbs), only 2 non-neutralizing MAbs, 110 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None of the neutralizing MAbs tested reacted with any of the gpG peptides previously mapped MAb resistant mutants in the gpG did not coincide with any of the linear epitopes defined by the pepscan strategy, suggesting the complementarity of the 2 methods for the identification of antibody recognition sites.

The rhabdovirus causing viral haemorrhagic septicemia (VHS), an important disease of salmonids, induced formation of large syncytia in fish cell monolayers by cell fusion. For maximal percentage of fusion, not only was a pH of 5.6 needed but also a temperature of at least 14°C. The fusion was dependent on the G protein of VHSV as shown by inhibition of fusion with specific anti-G polyclonal and monoclonal antibodies. Part of the region of the G protein involved in fusion was mapped to amino acids 82 to 109 because anti-peptide antibodies binding to that region also inhibited fusion. The fusion assay described here can be useful for studying the functionality of the G protein of fish rhabdoviruses in the early steps of infection and this information can, in turn, help to designing effective vaccination and/or therapeutic strategies against fish rhabdoviruses. The described fusion assay could also he applied to other fish rhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) anti spring viraemia of carp virus (SVCV).