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Background Ticks are implicated as hosts to a wide range of animal and human pathogens. The full range of microbes harbored by ticks has not yet been fully explored. Methods As part of a viral surveillance and discovery project in arthropods, we used unbiased high-throughput sequencing to examine viromes of ticks collected on Long Island, New York in 2013. Results We detected and sequenced the complete genome of a novel rhabdovirus originating from a pool of Amblyomma americanum ticks. This virus, which we provisionally name Long Island tick rhabdovirus, is distantly related to Moussa virus from Africa. Conclusions The Long Island tick rhabdovirus may represent a novel species within family Rhabdoviridae.

15 páginas, 7 figuras, 1 tabla.-- This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited, 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., This work was supported by the Spanish Ministry of Economy and Competence’s grants AGL2011-28921-C03 and CONSOLIDER INGENIO 2010-CSD2007-00002 as well as by the Generalitat Valenciana’s grant ISIC-2012-003 ACOMP/2013/230.

We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpG(VHSV)), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpG(VHSV). In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31 + p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1 mu g of the construct pMCV1.4-p31 + p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31 + p33 construct was assayed for molecular adjuvant city's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-G(SVCV)). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-GSVCV without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system Refereed/Peer-reviewed

To the Editor: Rhabdoviruses are enveloped, single-stranded, negative-sense RNA viruses that comprise a large and diverse family in the order Mononegavirales and infect arthropods, plants, fish, and mammals. There are 9 genera of rhabdoviruses: Cytorhabdovirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Tibrovirus, and Vesiculovirus. In addition, a substantial number of plant, vertebrate, and invertebrate rhabdoviruses have not been classified (1). Three genera (Novirhabdovirus, Perhabdovirus, and Vesiculovirus) comprise members that infect fresh water and marine fish (2). Fish rhabdoviruses pose a serious problem for aquaculture because of worldwide outbreaks of disease caused by novirhabdoviruses, perhabdoviruses, and vesiculoviruses (3,4). In 1992, a rhabdovirus-like virus was isolated from lung and kidney of a white-beaked dolphin (Lagenorhynchus albirostris) that had beached along the coast of the Netherlands (5). Although no macroscopic or microscopic lesions were observed at necropsy, negative contrast electron microscopy showed typical rhabdovirus-like, bullet-shaped particles in Vero cell cultures that showed a focal cytopathic effect (5). After this rhabdovirus-like virus was injected intracerebrally into brains of 1-day-old suckling mice, they died within 5 days (5). We report genetic and phylogenetic characterization of a dolphin rhabdovirus (DRV) and evaluated the seroprevalence of DRV-neutralizing antibodies by using serum samples from various marine mammals collected during a 10-year period (2003–2012). To characterize DRV, we performed random sequence amplification and deep sequencing with the 454 GS Junior Instrument (Roche, Basel, Switzerland) with DRV-infected Vero cell supernatants as described (6). From this analysis, we determined the complete coding sequence of DRV covered by 42,080 of 49,292 reads (minimum coverage 4 reads, average coverage 872 reads). Genomic termini of DRV were determined by using a 3′ and 5′ rapid amplification of cDNA ends PCR and Sanger sequencing of obtained PCR amplicons. The complete genome of DRV (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KF958252","term_id":"657638886","term_text":"KF958252"}}KF958252) consists of 11,141 nt and has a typical rhabdovirus gene arrangement of 5 major open reading frames (ORFs) in the order 3′-nucleoprotein (N), phosphoprotein (P), matrix (M) protein, glycoprotein (G), and large (L) protein-5′ (Figure, panel A, Appendix). No additional ORFs ≥300 nt were detected. Between the major ORFs of DRV, intergenic sequences were present that ranged in size from 34 (P–M) to 83 (G–L) nucleotides. Putative transcription initiation and transcription termination polyadenylation sequences were AACA(G/U) and AUGA7, respectively. Figure Genome organization and phylogenetic analysis of dolphin rhabovirus (DRV). A) Genome organization of DRV showing locations of major open reading frames and start and stop codons at the nucleotide level. N, nucleoprotein; P, phosphoprotein; M, Matrix; ... The deduced amino acid sequence of genes of DRV and several other rhabdoviruses were aligned by using MUSCLE in MEGA5 version 5.2) (7). Ambiguous aligned regions were removed by using the Gblocks program (8). Phylogenetic analysis of the L and G genes was performed by using the neighbor-joining method in MEGA5 (7). This analysis showed that DRV is most closely related to fish rhabdoviruses of the genera Perhabdovirus and Vesiculovirus and unassigned fish rhabdoviruses with strong bootstrap support (Figure, panels B–D, Appendix). Deduced amino acid sequences of the 5 major genes had the highest, although weak, homology with those of various fish rhabdoviruses by pairwise identity analyses: N (48%) with hybrid snakehead virus (HSHV), Monopterus albus rhabdovirus (MARV), and Siniperca chautsi rhabdovirus (SCRV); P (18%–20%) with eel virus European X (EVEX), HSHV, MARV, and SCRV; M (27%–33%) with lake trout rhabdovirus, Swedish sea trout rhabdovirus, and EVEX; G (30%–32%) with perch rhabdovirus, lake trout rhabdovirus, Swedish sea trout rhabdovirus, HSHV, MARV, SCRV, and EVEX; and L (54%–56%) with perch rhabdovirus, HSHV, and EVEX. This close relationship with fish rhabdoviruses is surprising because DRV was isolated from tissues of a mammal and propagated in mammalian cell lines at 37°C, which does not occur with related viruses isolated from fish. To evaluate whether DRV or related viruses circulate among species of cetaceans, we performed serologic screening by using a virus neutralization assay as described (5). The specificity of this assay was tested by using a panel of rhabdovirus-specific antisera obtained from cetaceans of various species (5). The serum samples had been collected for diagnostic purposes from mainly juvenile cetaceans stranded along the coast of the Netherlands during 2003–2012. These species included 2 Atlantic white-sided dolphins (Lagenorhynchus acutus), 79 harbor porpoises (Phocoena phocoena), 9 striped dolphins (Stenella coeruleoabla), and 6 white-beaked dolphins (Lagenorhynchus aIbirostris). Serum samples from 145 bottlenose dolphins (Tursiops truncates) from the collection of the Dolphinarium Harderwijk (Harderwijk, the Netherlands) were also tested. DRV-neutralizing antibodies were detected in serum samples from 1 bottlenose dolphin (7%), 5 striped dolphins (55%), 1 white-beaked dolphin (17%), and 3 harbor porpoises (4%). These results suggested that DRV or closely related viruses continue to infect members of cetacean species (6). Although rhabdovirus evolutionary pathways are complicated (9), our analysis suggests that DRV is a possible derivative of fish rhabdoviruses. DRV might have originated from an unidentified fish rhabdovirus and might cycle between fish and marine mammals, similar to that suggested for cycling of vesicular stomatitis virus between arthropods and terrestrial mammals (10). Future analyses of sequences from other marine mammal rhabdovirus sequences might support the validity of our phylogenetic analysis and result in creation of a new group containing marine mammal rhabdoviruses.

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. This work was partially funded by the Virgo Consortium, funded by the Dutch government project number FES0908, by Netherlands Genomics Initiative (NGI) project number 050-060-452 and ZonMW TOP project 91213058. A. Ruiz-Gonzalez holds a Post doc fellowship awarded by the Department of Education, Universities and Research of the Basque Government (Ref. DKR-2012-64) and was partially supported by the Research group on "Systematics, Biogeography and Population Dynamics" (Basque Government; Ref. IT317-10; GIC10/76).

By means of bioassays performed in arenas of 3 cm in diameter, manufactured with coffee leaves floating on water, the phases of the coffee ringspot mite Brevipalpus phoenicis (Geijskes) were studies on the preference by several developmental stages of the predaceous mites Euseius alatus DeLeon and Iphiseiodes zuluagai Denmark & Muma. The experiment was conducted in laboratory at 25 ± 2ºC, 70 ± 10% RH and 14 h photophase. The preferred stage of coffee ringspot vector virus was the larva followed by nymph and egg. Adults were least preyed on. In general, the most aggressive phase of the predators was the adult female followed by the nymph; the larval phase was the least aggressive. The average predation of E. alatus and I. zuluagai for the different phases of B. phoenicis were, respectively: larva (79% and 90%) > egg (47% and 83%) > nymph (40% and 77%) > adult (1% and 18%). This result indicated that I. zuluagai is more aggressive than E. alatus in relation to its predation upon B. phoenicis. Através de bioensaios realizados em arenas com 3 cm de diâmetro, confeccionadas com folhas de cafeeiro flutuando em água, foram estudadas as fases do ácaro da mancha-anular do cafeeiro Brevipalpus phoenicis (Geijskes) quanto à preferência pelos diversos estádios do desenvolvimento dos ácaros predadores Euseius alatus DeLeon e Iphiseiodes zuluagai Denmark & Muma. Os experimentos foram conduzidos em laboratório a 25 ± 2ºC, 70 ± 10% de UR e 14 horas de fotofase. O estádio do ácaro vetor mais predado foi o de larva, seguido pelo de ninfa e ovo. A fase adulta teve muito pouca predação. De modo geral, a fase mais agressiva dos predadores foi a de fêmea adulta, seguida pela de ninfa. A fase de larva foi a menos eficiente na predação. As médias de predação de E. alatus e I. zuluagai para as diferentes fases do B. phoenicis foram respectivamente: larva (79% e 90%) > ovo (47% e 83%) > ninfa (40% e 77%) > adulto (1% e 18%), o que demonstra que I. zuluagai mostrou maior atividade predatória que E. alatus.

Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers.

This work describes the characterization of trout haematopoietic in vitro long-term cell cultures showing specific viral antigen-dependent cell (ADC) proliferation. The ADC cultures were developed from outbreed trout after surviving viral hemorrhagic septicaemia virus (VHSV) infections or after immunization with purified VHSV. For in vitro long-term proliferation of the ADC cultures, adherent (Ad) cells obtained from autologous trout were pulsed with VHSV recombinant glycoprotein G4 (G4-pulsed Ad cells) and added periodically to the cultures. ADC did not proliferate in cultures obtained from non-infected control trout treated in parallel with G4 or from VHSV survivor/VHSV immunized trout kidney donors treated with non-viral proteins. After months in culture, the ADC acquired an increasingly homogeneous morphology compatible with that of mature trout lymphocytes, secreted supernatant `factors', and were stained with rabbit antibodies to the ectodomain of recombinant trout T-cell receptor (TcR) β-chain. Together with all the above mentioned properties, the presence of TcR sequences in the ADC cultures confirmed by the expression of α- and β-chain TcR by nested PCR amplification and sequencing of the amplified bands, suggests that these ADC cultures contain trout T-like cells engaged in a VHSV response. These trout ADC cultures offer a first opportunity to further analyze fish anti-viral immunological responses.

Lyssavirus surveillance in bats was performed in Bangladesh during 2003 and 2004. No virus isolates were obtained. Three serum samples (all from Pteropus giganteus, n = 127) of 288 total serum samples, obtained from bats in 9 different taxa, neutralized lyssaviruses Aravan and Khujand. The infection occurs in bats in Bangladesh, but virus prevalence appears low.

A method based on reverse transcription (RT)-polymerase chain reaction (PCR) was established for detection and identification of infectious hematopoietic necrosis virus (IHNV). A set of primers was prepared for amplification of a 510 by nucleotide which encodes a region of IHNV nucleoprotein (N) gene. The PCR product was confirmed as the IHNV specific nucleotide by southern hybridization with an oligonucleotide probe synthesized from the N gene. The PCR was able to amplify the target se quence from five representative strains of IHNV from Japan and North America. There was no PCR product from five other fish rhabdoviruses. Viruses from ovarian fluid of masu salmon collected at Shibetsu River, Ichani River in Hokkaido and kidney tissue of rainbow trout in Aichi Prefecture were isolated and identified using the RT-PCR.

It is well known that β-defensins are key components of the host innate immune response against pathogens and potentially provide a link between innate and adaptive immunity. In zebrafish (Danio rerio), a vertebrate model species in numerous biomedical fields, three β-defensin isoforms were recently identified. To our knowledge, however, studies describing antimicrobial or immunomodulatory properties of any of the zebrafish β-defensins isoforms are absent today. Since it is indubitable that deepening the study of zebrafish β-defensins would be of interest in this work we investigated whether or not the zebrafish β-defensin 2 (zfBD2) has the antiviral properties described for their vertebrate counterparts. Our in vitro and in vivo studies showed that zfBD2 has antiviral activity, immunomodulatory properties and, most importantly, is a potent viral DNA vaccine molecular adjuvant. In addition, a potential relationship between zfBD2 activity and the NF-κB signaling pathway is suggested. Altogether these results show that the zebrafish could be a suitable in vivo animal model to study the roles played by β-defensin 2 in viral diseases, vaccinology and even in clinical dermatology. To note that psoriasis can be induced in zebrafish and the over-expression of β-defensin 2 is implicated in the inflammatory response associated with this human skin disorder. © 2013 Elsevier B.V. All rights reserved.

Chantier qualité GA; Health control is a major issue in animal breeding and a better knowledge of the genetic bases of resistance to diseases is needed in farm animals including fish. The detection of quantitative trait loci (QTL) will help uncovering the genetic architecture of important traits and understanding the mechanisms involved in resistance to pathogens. We report here the detection of QTL for resistance to Viral Haemorrhagic Septicaemia Virus (VHSV), a major threat for European aquaculture industry. Two induced mitogynogenetic doubled haploid F2 rainbow trout (Oncorhynchus mykiss) families were used. These families combined the genome of susceptible and resistant F0 breeders and contained only fully homozygous individuals. For phenotyping, fish survival after an immersion challenge with the virus was recorded, as well as in vitro virus replication on fin explants. A bidirectional selective genotyping strategy identified seven QTL associated to survival. One of those QTL was significant at the genome-wide level and largely explained both survival and viral replication in fin explants in the different families of the design (up to 65% and 49% of phenotypic variance explained respectively). These results evidence the key role of innate defence in resistance to the virus and pave the way for the identification of the gene(s) responsible for resistance. The identification of a major QTL also opens appealing perspectives for selective breeding of fish with improved resistance.

Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5′ and 3′ regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies. © 2012 Springer-Verlag.

Potato (Solanum tuberosum subsp. tuberosum) alterations characterized by severe necrosis of vascular rings and adjacent flesh were observed in potato cv. Vivaldi in Emilia Romagna (northen Italy). The presence of Eggplant mottled dwarf virus (EMDV) was demonstrated in diseased plants by bioassays, electron microscopy, serology and RT-PCR. Symptoms similar to those observed in cv. Vivaldi were observed in other potato cultivars and areas of central and southern Italy. This is the first report of natural occurrence of EMDV in potato in Italy.

DOI ne fonctionne pas. Chantier qualité GA; La truite arc-en-ciel ( Oncorhynchus mykiss) est une espèce d'intérêt aquacole majeur en Europe et notamment en France. Le système immunitaire de cette espèce est également l'un des mieux connus au sein des poissons téléostéens. Différentes maladies virales affectent la truite arc-en-ciel, en particulier deux rhabdoviroses, causées par le virus de la septicémie hémorragique virale (VSHV) et par le virus de la nécrose hématopoïétique infectieuse (VNHI), qui sont à l'origine de pertes importantes dans les élevages. Une variabilité significative de la résistance naturelle à ces infections a été décrite chez la truite arc-en-ciel, entre et au sein des populations. L'étude de cette variabilité génétique est à la fois pertinente pour améliorer les approches de sélection d'individus naturellement résistants à ces virus, et pour progresser dans la dissection des mécanismes sous-jacents. Les méthodes employées, et les premiers résultats de l'analyse des bases génétiques de la résistance de la truite arc-en-ciel à la septicémie hémorragique virale sont présentés ici, de l'identification d'un QTL majeur à l'étude du rôle des interférons.

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.

In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was adapted to antiviral purposes by means of monoclonal antibodies targeting the surface G glycoprotein of the fish viral haemorrhagic septicemia rhabdovirus (VHSV). We also studied the antiviral action of these immunoliposomes in vitro and the results showed that they are capable of inhibiting the VHSV infectivity by 95.2% via direct inactivation of the virus. Furthermore, the inhibition of the infectivity when treatments were added at different times post-infection and the analysis of the infection foci sizes suggested altogether that they also act by reducing the VHSV spread in cell culture and by killing the infected cells which express the G glycoprotein in their plasmatic membranes. © 2012 Elsevier B.V.

Two new rabies-related viruses were discovered in Russia during 2002. Viruses were isolated from bats in Eastern Siberia near Baikal Lake and in the western Caucasus Mountains. After preliminary antigenic and genetic characterization, we found that both viruses should be considered as new putative lyssavirus genotypes.

Background Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World non-human Primates (NHP). To investigate the suitability of the owl monkey Aotus nancymaae asissue sections examined were unremarkable for inflammation or other histologic signs of rabies a viable animal model for rabies virus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus. Methods Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. Results Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p = 0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies within the asymptomatic animal. Similarly none of the brain sections exhibited immunoreactivity for rabies virus antibody. Discussion This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. Conclusions Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.

Chantier qualité GA; Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis using fragments of PYDV-N identified a karyophilic region in the carboxy-terminal 122 amino acids. Alanine-scanning mutagenesis was performed across this region in the context of the full-length N protein. Mutants were assayed for their ability to nuclear localize using live-cell imaging and a yeast-based assay. Two amino acid motifs, (419)QKR(421) and (432)KR(433) were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or -M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. GFP fused to aa 419-434 established that the PYDV-N-NLS can function outside the context of this protein. Taken together, it was determined that PYDV-N contains the bipartite NLS (419)QKRANEEAPPAAQKR(433).

7 páginas, 5 figuras, 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., This work was supported by the projects Consolider Ingenio 2010, CSD2007-00002 and AGL2011-28921-C03 both from the Spanish Ministerio de Ciencia e Innovación (MICINN).

In the Spring of 1990, Croton vein yellowing nucleorhabdovirus (CVYV) was found for the first time in some dwarfed plants of croton (Codiaeum variegatum L.) “Fred Sander” showing malformation and yellow or pink veins. CVYV was mechanically transmitted to Nicotiana glutinosa and Chenopodium amaranticolor and, by leaf-graft, to healthy croton plants in which it induced the same original disease. After about twenty years from the first finding, CVYV has been serologically identified and molecularly characterized as an isolate of Eggplant mottled dwarf virus (EMDV). In electron microscopy of negatively stained crude extracts from croton, fixed in gluteraldeid solution, typical bacilliform virus particles were observed; by applying immunoelectron microscopy tests, using an antiserum to an EMDV Iranian potato isolate, all these particles were clearly decorated. EMDV was also detected in original croton by DAS-ELISA (Bioreba kit; Switzerland) and by RT-PCR using a virus-specific primer pair designed to amplify ca. 900 bp fragment of the P1 polymerase gene. The amplicon was cloned and sequenced at MWG (Ebersberg, Germany) on both strands. Sequence obtained showed 86.5% identity with EMDV eggplant isolate from Greece, confirming the identity of rhabdovirus infecting symptomatic croton. From this serological and molecular study, it appears evident that CVYV can be definitively considered an isolate of EMDV. In the last decade, EMDV has been frequently detected infecting two common ornamental species in Mediterranean area, Hibiscus rosa-sinensis and Pittosporum tobira. From this study croton, typical pot-houseplant appreciated for its large and glossy leaves, can be considered a new natural host of EMDV.

Two novel rhabdoviruses were isolated from birds during surveillance for arboviral encephalitis in the northeastern United States. The first, designated Farmington virus, is a tentative new member of the Vesiculovirus genus. The second, designated Rhode Island virus, is unclassified antigenically, but its ultrastructure and size are more similar to those of some of the plant rhabdoviruses. Both viruses infect birds and mice, as well as monkey kidney cells in culture, but their importance for human health is unknown.

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.

7 figuras, 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., This work was supported by the projects FAIRCT 98-4398 from the European Union and BIO 2000-0906 from the Spanish Ministerio de Ciencia y Tecnología. European structural funds were used for confocal microscopy. Fondo Europeo de Desarrollo Regional (FEDER) y Ministerio de Ciencia e Innovación. A. Romero acknowledges the CSIC for the I3P fellowship.

The sigma virus (DMelSV), which is a natural pathogen of Drosophila melanogaster , is the only Drosophila -specific rhabdovirus that has been described. We have discovered two new rhabdoviruses, D. obscura and D. affinis , which we have named DObsSV and DAffSV, respectively. We sequenced the complete genomes of DObsSV and DMelSV, and the L gene from DAffSV. Combining these data with sequences from a wide range of other rhabdoviruses, we found that the three sigma viruses form a distinct clade which is a sister group to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they deserve to be recognized as a new genus. Furthermore, our analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing us to reconstruct the major transitions that have occurred during the evolution of the family. Our data suggest that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects.

A fully intact immune system would be expected to hinder the efficacy of oncolytic virotherapy by inhibiting viral replication. Simultaneously, however, it may also enhance antitumor therapy through initiation of proinflammatory, antiviral cytokine responses at the tumor site. The aim of this study was to investigate the role of a fully intact immune system on the antitumor efficacy of an oncolytic virus. In this respect, injection of oncolytic vesicular stomatitis virus (VSV) into subcutaneous B16ova melanomas in C57Bl/6 mice leads to tumor regression, but it is not associated with viral replicative burst in the tumor. In contrast, intratumoral delivery of VSV induces an acute proinflammatory reaction, which quickly resolves concomitantly with virus clearance. Consistent with the hypothesis that therapy may not be dependent on the ability of VSV to undergo progressive rounds of replication, a single-cycle VSV is equally effective as a fully replication-competent VSV, whereas inactivated viruses do not generate therapy. Even though therapy is dependent on host CD8+ and natural killer cells, these effects are not associated with interferon-gamma-dependent responses against either the virus or tumor. There is, however, a strong correlation between viral gene expression, induction of proinflammatory reaction in the tumor and in vivo therapy. Overall, our results suggest that acute innate antiviral immune response, which rapidly clears VSV from B16ova tumors, is associated with the therapy observed in this model. Therefore, the antiviral immune response to an oncolytic virus mediates an intricate balance between safety, restriction of oncolysis and, potentially, significant immune-mediated antitumor therapy.