Transcriptome-wide analysis of wild Asari (=Manila) clams affected by the Brown Muscle Disease: Etiology and impacts of the disease.

Recently, we reported an emerging pathology named Brown Muscle Disease (BMD) affecting Asari clams inhabiting the most productive area for this species in France, the Arcachon Bay. The main macroscopic feature of the pathology relies on the atrophy of the posterior adductor muscle, affecting the ability of clams to burry. The research of the etiological agent of BMD privileged a viral infection. Contrary to healthy clams, infected animals are always found at the surface of the sediment and exhibit 30 nm virus-like particles in muscle, granulocytic and rectal cells. In order to get more insights on the etiology and impacts of the BMD on clams, we took advantage in the present study of next generation sequencing technologies. An RNA-Seq approach was used (i) to test whether viral RNA sequences can be specifically found in the transcriptome of diseased animals and (ii) to identify the genes that are differentially regulated between diseased and healthy clams. Contrary to healthy buried animals, in diseased clams one sequence showing extensive homologies with retroviridae-related genes was detected. Among the biological processes that were affected in diseased clams, the synaptic transmission process was the most represented. To deepen this result, a new sampling was carried out and the transcription level of genes involved in synaptic transmission was determined in healthy and diseased clams but also in clams with no visible sign of pathology but located at the surface of the sediment. Our findings suggest that muscle atrophy is a latter sign of the pathology and that nervous system could be instead a primary target of the BMD agent.

Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish.

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

[Alteration in quality of life in anorexia nervosa and improvement under treatment: validation of a new questionnaire, the QUAVIAM]. / Altération de la qualité de vie dans l'anorexie mentale et amélioration sous traitement : validation d'un nouveau questionnaire, le QUAVIAM.

Anorexia nervosa (AN) is a chronic and often severe eating disorder, which could have a serious impact on various life domains. AN may lead to physical, mental, behavioural and socioprofessional impairment. Thus, one could expect a poor quality of life (QoL) in AN patients. QoL is certainly a key factor to provide quantitative measurement of treatment efficacy that will facilitate clinical decision-making and treatment planning. Despite that QoL was rarely prospectively analyzed in AN patients, one could conclude that AN patients showed reduced QoL, as compared to normal controls and other psychiatric-disordered patients. It seems that mental health components of QoL are more impaired than the physical ones in AN patients, who showed a modest impact in the physical domain. Thus, our aim was to analyse the QoL using a new, French, questionnaire, the QUAVIAM (qualité de vie dans l'anorexie mentale). After a bibliography research (including EDE, EDI, SF-36, QOL.ED), the choice of 12 themes, regrouped in six scores, was made by three eating disorder specialists and two recovered patients. For each score, 10 to 15 questions were written by the experts, and then corrections and validation were made by the five experts and 21 patients. After this, we prospectively determined the reproducibility (3 days interval), the specificity, and the sensitivity for short-term change in patients exhibiting an "active" AN (n=54, mean age: 31 ± 9 yrs, mean BMI: 14.1 ± 2.8 kg/m(2), AN duration: 2.6 ± 1.9 yrs), and again after cognitive behavioral therapy (CBT). We also analyzed the QUAVIAM score and subscores in 48 recovering patients and in 56 subjects without eating disorder. The QUAVIAM final version (61 questions) was collected in 76 patients and the 56 healthy controls matched for sex and age. Its reproducibility was 91% (intra-questionnaire) and 94% (inter-questionnaire), its specificity 98% (versus controls; P<0.0001) and its sensitivity 99%. The QUAVIAM global score of the AN patients was more impaired (389 ± 87) than that of the recovering patients (157 ± 82) and the normal controls (89 ± 49; P<0.0001). Each of the six subscores was higher (more altered) in active AN than in recovering AN patients and in normal subjects: the somatic, the psychological, the hedonic, the socioprofessional, the affective and the TCA-related ones (P<0.001 for each comparison). The QUAVIAM global score and its subscores were significantly improved (decreased) by the 3-month CBT: 385 ± 25 before and 189 ± 30 after CBT (P<0.0001). The changes were observed for all the subscales (P<0.0001). The somatic subscore did not decrease less than the other subscores. Thus, the present study permits proposing the QUAVIAM for analysis of physical, mental, behavioural and socioprofessional impairment or improvements in AN patients.

[Triggers of bulimia and compulsion attacks: validation of the "Start" questionnaire]. / Les inducteurs des crises dans la boulimie et la compulsion : validation du questionnaire « Start ¼

AIM: There are few published studies on the triggers of binge eating in anorexia nervosa of binge/purging subtype (BPAN), bulimia nervosa (BN) and binge eating disorder (BED). PATIENTS AND METHODS: We validated in 29 patients (10 BPAN, 10 BN and 9 BED) the perspicuity, the clarity and the intra- (doubles) and inter- (test-retest) reproducibility of a 24-item Start questionnaire on the triggers of binge eating. Then the Start questionnaire was administered to 176 patients (65 BPAN, 62 BN and 59 BED patients) being 27.5+9.1 yr old, having 15+9 binge eating (BE) episodes/week, with a mean binge duration of 1 hr 36min (+ 38min)/day. RESULTS: BE episodes occurred mainly during the second part of the day: afternoon after work (67% of the patients), "tea" time (55%), evening after dinner (42%) and at night (22%). The principal place for BE episodes was at home (96%). The BED patients avoided binges at the parents' home (89%) more often than the BPAN (62%, P<0.02). The binges occurred mainly in the living room (44%), in the kitchen (43%), and less in the bedroom (31%). Hunger pangs seemed to be a trigger of binges in 31% of the patients, and a stronger trigger in BED (42%) than in the BPAN and BN patients (24%; P=0.04). Binge eating episodes could occur despite a high satiety level (just after lunch or dinner) in 29% of the BN and in 16% of the BED patients (P<0.02). Concerning food, the major triggers were high energy-density food (77%) and comfort food (60%), such as chocolate, cakes, bread and pasta. The food consumed for binge episodes (in-binge food) was more often a strong trigger than the other food (not used for binges): olfaction (19% versus 10%), sight (52% versus 25%) and placing in the mouth (71% versus 26%; P<0.02 for all, in the 3 groups). Being tired could be a strong trigger in 37% of the patients, but "being aroused" in the other 38 % of the patients. Stressful events (65%), anxiety (74%), "being under pressure" or irritated (51% and 55%) were of course major triggers in a majority of the patients, as well as sadness (61%), feeling of powerlessness (62%), inefficiency (73%) and depressive state (71%). Flashback from traumatism (sexual trauma in 17% of the patients) was a strong trigger of binges more often in BPAN and BED (44%) than in BN (23%; P<0.05). The binge eating was painful (and "not at all a pleasure") in 69% of the patients, but could also be a relaxing behavior in 31% of the patients, more often in the BED (43%) than in the BPAN patients (20%; P<0.05). The binge eating behavior was quoted as obsessive in 63% of BPAN, 92% of BN and only 34% of BED patients (P<0.001). The patients said that they were unable to avoid the binge (76% of the patients), more often in BPAN and BN than in BED patients (P<0.01). As a whole, 62% of BPAN, 89% of BN and only 4 % of BED patients (P<0.05) were unable to avoid purging (vomiting). In 12% of the cases, there was a pleasure felt when binging. For the other patients, shame, filth and incapacity were the feelings related to binges in 58% of the BPAN, 45% of BN and 43% of BED patients (P<0.04). The global score of addiction (zero=not addicted, 10=very addicted) was 8.56+1.2 in BPAN, 8.42+1.5 in BN and 6.74+1.1 in BED patients (NS between BPAN and BN; P<0.01 between BPAN and BN on the one hand and BED on the other). CONCLUSION: The present study has demonstrated the usefulness of the Start questionnaire. It also evidences the key role of intrinsic factors, both metabolic and emotional, as strong triggers for binge eating episodes in BPAN, BN and BED. It has also demonstrated the role of environmental determinants.

Zebrafish ISG15 exerts a strong antiviral activity against RNA and DNA viruses and regulates the interferon response.

ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Sequence analysis of the large (L) polymerase gene and trailer of the peste des petits ruminants virus vaccine strain Nigeria 75/1: expression and use of the L protein in reverse genetics.

The large (L) polymerase gene and the 5'-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.

The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin.

Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed "U-tube," to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

Reverse genetics on fish rhabdoviruses: tools to study the pathogenesis of fish rhabdoviruses.

Rhabdoviruses, mainly in rainbow trout, are among the most devastating viruses for worldwide aquaculture. To date no effective treatments to fight against these viruses are available. During the past years, several approaches to develop efficient vaccines have been undertaken such as the use of immunogenic recombinant viral proteins, naked DNA or inactivated viruses. However, although these vaccines have been proven to be very effective on a small scale, they have never been used in the field because the vaccines would have to be injected into thousands of yearling trouts. The only alternative to injection consists of the development of attenuated live vaccines that can be administrated to trouts by bath immersion. Reverse genetics on trout rhabdoviruses offer the possibility of recovering a series of live recombinant viruses in which the viral genome has been irreversibly modified to generate cost-effective live, safe vaccines.

Comparison of the eIF-2alpha homologous proteins of seven ranaviruses (Iridoviridae).

The alpha-subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is a key component of the translation machinery of the cell. In response to cellular stress such as viral infections, eIF-2alpha is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis. The importance of eIF-2alpha as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR. Thus, Vaccinia virus encodes K3L protein, which resembles eIF-2alpha and acts as a pseudo-substrate inhibitor of PKR. Nucleotide sequencing of the genome of epizootic haematopoietic necrosis virus (EHNV), a member of the genus ranavirus of Iridoviridae, has revealed an eIF-2alpha equivalent gene. We have cloned and sequenced eIF-2alpha genes of several iridoviruses of fishes and frogs. The eIF-2alpha open reading frames and deduced proteins of the iridoviruses investigated exhibit a high degree of homology of both nucleotide and amino acid sequences. At the N-terminus, the iridoviral eIF-2alpha shows significant homology to the N-termini of cellular initiation factor 2-alpha of various species, to full-length poxviral eIF-2alpha proteins, and to the S1 domain of ribosomal proteins. Comparison of amino acid sequences of corresponding iridoviral proteins with eIF-2alpha homologous proteins of poxviruses and eukaryotes has revealed a high conservation of motifs. A phylogenetic analysis of eukaryotic eIF-2alpha and poxvirus and iridovirus eIF-2alpha sequences has demonstrated the relationship of these iridoviruses. In order to investigate the role of the eIF-2alpha equivalent, respective genes have been expressed in prokaryotic and eukaryotic (insect, fish and chicken cell) systems. The iridoviral eIF-2alpha protein has a molecular weight of 31 kDa and is cytoplasmic. The cellular and viral protein synthesis of iridoviruses is probably regulated by a mechanism similar to that of Vaccinia virus. Frog-virus 3, the type species of the genus ranavirus of Iridoviridae, has a unique translational efficiency and, moreover, down-regulates the cellular protein synthesis of infected cells.

Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes.

Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14 degrees C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.

An RT-PCR-based method for the diagnosis of the sleeping disease virus in experimentally and naturally infected salmonids.

The sleeping disease (SD) of rainbow trout (Oncorhynchus mykiss) is a worldwide disease for which the causative agent, the sleeping disease virus (SDV), has been recently characterized as an atypical alphavirus (Villoing et al. 2000). Up to now, no diagnostic tools were available and thus no epidemiological studies have been undertaken to evaluate the occurrence of this disease on the field. We present in this paper a sensitive and highly specific 1 working day method, which allows the detection of SDV from experimentally and naturally infected fishes. This method, based on a reverse transcriptase/polymerase chain reaction (RT-PCR) assay on total RNA extracted from SDV-infected fish organs, enables the specific DNA amplification of part of the gene encoding the SDV glycoprotein E2, as early as 2 d post-infection (d.p.i.) and as late as 70 d.p.i., at which time clinical signs of infection are no longer apparent. Moreover, we show that this RT-PCR method can be successfully used for the diagnosis of fish infected by a closely related virus, namely salmon pancreas disease virus (SPDV). This report is the first description of a very powerful diagnostic assay which could provide a more accurate replacement for the classical virological, histological and immunochemistry methods.

Rainbow trout sleeping disease virus is an atypical alphavirus.

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented. To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV). Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing. Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length. Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined. Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins. Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases. But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined.

Fish rhabdovirus cell entry is mediated by fibronectin.

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.

Comparison of European systemic piscine and amphibian iridoviruses with epizootic haematopoietic necrosis virus and frog virus 3.

Iridovirus-like agents isolated from systemic infected fish (Silurus glanis, SFIR; Ictalurus melas, CFIR I, CFIR II, CFIR III) and from frogs (Rana esculenta, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Perca fluviatilis), and Frog Virus 3 (FV 3) isolated from frogs (Rana pipiens) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. All virus isolates proved to be similar in morphology and in size and reacted with EHNV polyclonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates cleaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3. Southern-blot analysis of viral BamH I digested DNA using an EHNV DNA probe revealed cross-hybridization with DNA of the investigated iridoviruses. Using a set of primers designed for an open reading frame of the EHNV genome, PCR products of about 250 bp were obtained with the DNA of systemic piscine and amphibian iridoviruses. The data suggest that the systemic piscine and amphibian iridoviruses should be regarded as members of the the genus Ranavirus within the family Iridoviridae.

An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein.

The gene encoding the nucleoprotein (N) and PCR-derived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in Escherichia coli BL21(DE3) transformed by recombinant expression vector pET-14b containing N and PCR-generated sub-fragment cDNAs under the control of the T7 RNA polymerase promoter. Following induction with IPTG, recombinant His-tagged proteins were expressed, purified by affinity metal chelation chromatography under denaturing conditions and renatured. Protein blots were hybridized with various radiolabelled nucleic acid probes. Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain. To confirm this observation, two more accurate approaches were undertaken: (i) a gel retardation assay of RNA and purified protein complexes was done; and (ii) RNA-protein complexes were cross-linked by UV light and analysed on a denaturing polyacrylamide gel. All these experiments led us to conclude that the middle part of N is the domain which interacts with RNA, despite the absence of homology with known consensus amino acid sequences of other RNA-binding proteins.