Monitoring for Anguillicoloides crassus, Anguillid herpesvirus 1, aquabirnavirus EVE and rhabdovirus EVEX in the European eel population of southern Spain.

European eel is critically endangered in Europe. Among other stressors, pathogens are well-known to harm eels' fitness. One hundred and eighty-two eels were captured in three Eel Management Units in Andalucía (SE Spain) and analysed for Anguillicoloides crassus, Anguillid herpesvirus 1 (AngHV1), the rhabdovirus Eel Virus European X (EVEX) and the aquabirnavirus Eel Virus European (EVE). A. crassus adults and preadults were isolated and morphometrically identified, and the eel swimbladders were artificially digested to count A. crassus larvae. Also, eel tissues were examined by PCRs for the presence of viruses. EVEX and EVE were not detected in any of the eels. The estimated prevalence (95% confidence limits) was 71 (64-78)% for A. crassus and 35 (28-42)% for AngHV-1, varying these prevalences significantly between and within EMUs. Moreover, A. crassus prevalence was highest in smaller eels, in sites closest to the sea and eels sampled in the autumn. By contrast, AngHV-1 prevalence was highest in biggest eels, in sites far from the sea and sampled in the summer or winter. However, in mixed effects logistic models including site as a random variable, the risk of infection was associated with distance to the sea in both A. crassus and AngHV-1 infections and also to winter sampling in the case of AngHV-1 and not to other variables. These results are evidence that both pathogens are highly endemic in eels from Andalusian habitats. Further studies are needed to better understand the risk factors associated with these pathogens on eel populations.

Establishment of a brain cell line (SaB-1) from gilthead seabream and its application to fish virology.

Gilthead seabream (Sparus aurata) is among the most important cultured fish species in the Mediterranean area and pathogen diseases one of the bottlenecks to the aquaculture sector. For this reason, generation of laboratory tools for diagnostic and research applications would be beneficial to improve the seabream aquaculture. In this sense, we aimed to generate a seabream cell line for biological studies. Thus, we have obtained a brain-derived cell line (SaB-1) that is continuously growing for more than 4 years. Cellular characterization of the SaB-1 cells shows that they express both neural and glial cell markers, suggesting they are neural-stem cells, have a neuron-like morphology and show a rapid growth in culture. We evaluated their susceptibility to the main fish viruses: nervous necrosis virus (NNV), spring viremia carp virus (SVCV), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). SaB-1 cells are susceptible to all the tested viruses. In addition, the transcription of genes related to the type I interferon (IFN) is greatly up-regulated by the NNV infection whilst the viral infection with SVCV, IPNV or VHSV failed to do so. These data demonstrate that the seabream SaB-1 cell line is continuous, stable and could be useful, at least, for fish virology and immunity applications.

First examination of the Lough Neagh European eel (Anguilla anguilla) population for eel virus European, eel virus European X and Anguillid Herpesvirus-1 infection by employing novel molecular techniques.

Lough Neagh is home to the largest wild-caught European eel (Anguilla anguilla) commercial fishery in the EU, producing 14% of the EU catch and worth £3.2 million to the local economy. Viral infections have been suggested to play a contributory role in the decline of the worldwide eel stock, but previous studies of the Lough Neagh European eel population had not observed either acute or chronic viral signs. Eel virus European (EVE), Eel virus European X (EVEX) and Anguillid herpesvirus-1 (HVA) have been detected throughout Europe and as the Lough Neagh eel fishery is supplemented by re-stocking of eels from France, Spain and the United Kingdom and these viral infections may be asymptomatic, it is vital that the viral pathogen prevalence in the Lough is accurately determined. This study aimed to ascertain the presence of these viruses in the Lough Neagh European eel population by employing novel molecular techniques testing specifically for the presence of EVE, EVEX and HVA. No evidence was found of HVA infection, whereas EVE and EVEX were found, albeit at a very low prevalence.

Complete genome sequence and phylogenetic analyses of an aquabirnavirus isolated from a diseased marbled eel culture in Taiwan.

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Molecular characterization of Tasmanian aquabirnaviruses from 1998 to 2013.

Tasmanian aquabirnaviruses (TABVs) have been isolated intermittently since 1998 from healthy Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss farmed in Macquarie Harbour, Tasmania, Australia. However, beginning in 2011, TABVs have been isolated from rainbow trout in association with mortality events. To determine if recent molecular changes in TABV were contributing to increased mortalities, next generation sequencing was undertaken on 14 TABVs isolated from 1998 to 2013. Sequencing of both genome segments and analysis of the 5 viral proteins they encode revealed that minimal changes had occurred in the past 15 yr. Of the amino acid changes detected only 1, alanine to aspartic acid at position 139 of the minor structural VP3 protein, was unique to the recent disease events. The most dramatic changes observed were in the length of the non-structural VP5 protein varying from 43 to 133 amino acids. However, the amino acid substitution in VP3 and variable VP5 length were unlikely to have resulted in increased TABV pathogenicity. The genome of a novel Australian aquabirnavirus, Victorian trout aquabirnavirus (VTAB) was also sequenced and compared to TABV isolates.

Infection Profiles of Selected Aquabirnavirus Isolates in CHSE Cells.

The wide host range and antigenic diversity of aquabirnaviruses are reflected by the presence of a collection of isolates with different sero- and genotypic properties that have previously been classified as such. Differences in cytopathogenic mechanisms and host responses induced by these isolates have not been previously examined. In the present study, we investigated infection profiles induced by genetically and serologically closely related as well as distant isolates in-vitro. CHSE-214 cells were infected with either E1S (serotype A3, genogroup 3), VR-299 (serotype A1, genogroup 1), highly virulent Sp (TA) or avirulent Sp (PT) (serotype A2, genogroup 5). The experiments were performed at temperatures most optimum for each of the isolates namely 15°C for VR-299, TA and PT strains and 20°C for E1S. Differences in virus loads and ability to induce cytopathic effect, inhibition of protein synthesis, apoptosis, and induction of IFNa, Mx1, PKR or TNFα gene expression at different times post infection were examined. The results showed on one hand, E1S with the highest ability to replicate, induce apoptosis and IFNa gene expression while VR-299 inhibited protein synthesis and induced Mx1 and PKR gene expression the most. The two Sp isolates induced the highest TNFα gene expression but differed in their ability to replicate, inhibit protein synthesis, and induce gene expression, with TA being more superior. Collectively, these findings point towards the adaptation by different virus isolates to suit environments and hosts that they patronize. Furthermore, the results also suggest that genetic identity is not prerequisite to functional similarities thus results of one aquabirnavirus isolate cannot necessarily be extrapolated to another.

Genetic positioning of aquabirnavirus isolates from cultured Japanese eel Anguilla japonica in Korea.

Aquabirnavirus is an epizootic virus in Japanese eel Anguilla japonica farms in Korea, although its origin is unclear. In the present study, nucleotide sequences of the VP2/NS junction region of 9 Korean aquabirnaviruses from cultured eel in various areas of Korea during 2000-2009 were analyzed to evaluate their genetic relatedness to worldwide isolates. The nucleotide sequences showed more than 94.2% identity among the 9 Korean eel isolates, 71.2% identity among 16 Korean isolates from freshwater and marine fish, and 71.1% identity among 25 worldwide isolates. All 9 isolates in this study were phylogenetically classified into genogroup II, including isolates from Denmark, Spain, Taiwan and Japan, and were discrete from salmonid and marine fish isolates (genogroup I and VII) in Korea. These results suggest that the Korean eel isolates have most likely been introduced from outside the country and not from coastal areas of Korea.

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

Crystal structure of a viral protease intramolecular acyl-enzyme complex: insights into cis-cleavage at the VP4/VP3 junction of Tellina birnavirus.

Viruses of the Birnaviridae family are characterized by their bisegmented double-stranded RNA genome that resides within a single-shelled non-enveloped icosahedral particle. They infect birds, aquatic organisms, and insects. Tellina virus 1 (TV-1) is an Aquabirnavirus isolated from the mollusk Tellina tenuis. It encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is cleaved by the self-encoded protease VP4 to yield capsid precursor protein pVP2, peptide X, and ribonucleoprotein VP3. Here we report the crystal structure of an intramolecular (cis) acyl-enzyme complex of TV-1 VP4 at 2.1-Šresolution. The structure reveals how the enzyme can recognize its own carboxyl terminus during the VP4/VP3 cleavage event. The methyl side chains of Ala830(P1) and Ala828(P3) at the VP4/VP3 junction point into complementary shallow and hydrophobic S1 and S3 binding pockets adjacent to the VP4 catalytic residues: nucleophile Ser738 and general base Lys777. The electron density clearly shows that the carbonyl carbon of Ala830 is covalently attached via an ester bond to the Oγ of Ser738. A highly ordered water molecule in the active site is coordinated in the proper position to act as the deacylating water. A comparative analysis of this intramolecular (cis) acyl-enzyme structure with the previously solved intermolecular (trans) acyl-enzyme structure of infectious pancreatic necrosis virus VP4 explains the narrower specificity observed in the cleavage sites of TV-1 VP4.

Expression, purification and crystallization of VP4 protease from Tellina virus 1.

Tellina virus 1 is an aquabirnavirus that was isolated from the sand-dwelling marine bivalve mollusc Tellina tenuis. The self-encoded protease viral protein 4 (VP4) processes its own polyprotein to yield the individual proteins VP2 and VP3 that are required for viral assembly. VP4 protease utilizes a serine-lysine catalytic dyad in its mechanism. A full-length VP4 construct was overexpressed in Escherichia coli and purified to homogeneity using nickel-affinity chromatography. Ion-exchange and size-exclusion chromatographic steps were utilized to isolate a monomeric fraction of the protein. The purified monomeric VP4 was subjected to limited proteolysis to yield crystallizable protein. Crystal growth was performed using the hanging-drop vapour-diffusion method and was carried out at room temperature (∼296 K). Hexagonal crystals grew in the presence of PEG 8000, ammonium sulfate and urea. These crystals diffracted to beyond 2.1 Šresolution and belonged to space group P6(4)22, with unit-cell parameters a=59.1, b=59.1, c=208.1 Å, one molecule in the asymmetric unit and a solvent content of 42%.

Infectious pancreatic necrosis virus (IPNV) enumeration through epifluorescence microscopy: technical aspects

A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.

Genetic analysis of aquabirnaviruses isolated from wild fish reveals occurrence of natural reassortment of infectious pancreatic necrosis virus.

In this study, we report the sequencing of the whole genome [including the 5' and 3' non-coding regions (NCR) of both segments A and B] of seven birnavirus strains isolated from wild fish from the Flemish Cap (FC) fishery at Newfoundland, Canada. From analysis and comparison of the sequences, most of the FC isolates clustered with the North American reference strains West Buxton (WB), Dry Mill and Jasper. One strain was included in the same genotype as the European strain Ab. In addition, at least in one case cohabitation of both type strains in an individual fish was demonstrated. These results clearly suggest the acquisition of the viruses from two different sources. The prevalence of the American type is easily explained by the close proximity of this fishing bank to the American coast whereas, although surprising, the presence of the European type strain could be because of migration of fish from European waters. In one strain, segment A and B sequences were typed differently (WB and Ab, respectively). These findings indicate natural reassortment between two strains of aquabirnaviruses in a host.

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg).

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.

Distribution of marine birnavirus (MABV) in marine organisms from Okinawa, Japan, and a unique sequence variation of the VP2/NS region.

Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25 degrees C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.

Phylogenetic evidence for homologous recombination within the family Birnaviridae.

Birnaviruses are bi-segmented double-stranded RNA (dsRNA) viruses infecting insects, avian species and a wide range of aquatic species. Although homologous recombination is a common phenomenon in positive-sense RNA viruses, recombination in dsRNA viruses is rarely reported. Here we performed a comprehensive survey on homologous recombination in all available sequences (>1800) of the family Birnaviridae based on phylogenetic incongruence. Although inter-species recombination was not evident, potential intra-species recombination events were detected in aquabirnaviruses and infectious bursal disease virus (IBDV). Eight potential recombination events were identified and the possibility that these events were non-naturally occurring was assessed case by case. Five of the eight events were identified in IBDVs and all of these five events involved live attenuated vaccine strains. This finding suggests that homologous recombination between vaccine and wild-type IBDV strains may have occurred; the potential risk of mass vaccination using live vaccines is discussed. This is the first report of evidence for homologous recombination within the family Birnaviridae.

Proposal for a fourth aquabirnavirus serogroup.

Four putative aquabirnaviruses, based on morphology, nucleic acid type and partial RNA-dependent RNA polymerase gene (VP1) sequence, isolated from three tropical freshwater fish species were not neutralised by antisera against type members of the Aquabirnavirus genus serogroups A, B or C. Antisera produced against two of the isolates neutralised the homologous and heterologous isolates, but not any type member of Aquabirnavirus serogroups A, B or C. The serological comparisons suggest that the four isolates should be regarded as members of a fourth Aquabirnavirus serogroup, D.

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea.

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.

Poly I:C induces Mx transcription and promotes an antiviral state against sole aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup).

Mx is an interferon-induced protein that protects against viral infections. In this study the absolute number of Mx transcripts after poly I:C injection (a synthetic dsRNA) or sole aquabirnavirus (solevirus) inoculation in Senegalese sole (Solea senegalensis Kaup) has been quantified. Mx expression profiles differed clearly in both experimental conditions; the induction response was faster and more intense after poly I:C injection than after solevirus inoculation. Moreover, pre-injection of soles with poly I:C prior to solevirus infection eliminated the induction of Mx expression associated with this virus. To evaluate the possible interference of poly I:C treatments on solevirus replication, the mRNA levels of the virus capsid protein (VP2) were determined by RT-PCR. VP2 transcripts were hardly detected in poly I:C pre-injected animals from 12 to 72 h after solevirus inoculation. All these data suggest that poly I:C is able to induce an antiviral state that interferes with solevirus replication, and support the suitability of Mx expression analysis as a marker to study the defensive response against solevirus.

In vitro inhibition of sole aquabirnavirus by Senegalese sole Mx.

Senegalese sole, Solea senegalensis, is a flat fish of growing interest in European aquaculture. In its culture viral infections are constant threats, thus understanding antiviral defences is a key factor for a successful industry. Mx proteins are IFN-induced proteins widespread in eukaryotes; however, their antiviral activity is unclear and the results variable among species. Therefore assessment of the putative Mx antiviral activity in each species is of interest. Our group has recently cloned the Senegalese sole Mx (SsMx) cDNA and in this study its antiviral activity was assessed by infecting CHSE-214 cells expressing recombinant SsMx, with sole aquabirnavirus. The antiviral activity against this pathogen was demonstrated by reduction in induced cytopathic effects, reduction in virus yield and decrease in viral transcripts. These findings contribute to our understanding of fish antiviral mechanisms and open the possibility of using this protein as a tool for fighting viral infections in aquaculture.

Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family.

We characterized tellina virus 1 (TV-1), a birnavirus isolated from the marine bivalve mollusk Tellina tenuis. Genome sequence analysis established that TV-1 is representative of a viral cluster distant from other birnaviruses. The maturation process of the polyprotein encoded by the genomic segment A was delineated with the identification of the N-termini of the viral protease VP4 and the ribonucleoprotein VP3, and the characterization of peptides deriving from the processing of pVP2, the VP2 capsid protein precursor. One of these peptides was shown to possess a membrane-disrupting domain. Like the blotched snakehead virus, the polyprotein exhibits a non-structural polypeptide (named [X]) located between pVP2 and VP4. Mutagenesis analysis allowed the identification in VP4 of a catalytic Ser-Lys dyad that does not possess the common Gly-X-Ser signature of the serine hydrolases. The genomic segment B encodes the viral RNA-dependent RNA-polymerase VP1 with the unique sequence motif arrangement identified in other birnavirus VP1s.