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.

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.

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.

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.

An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region.

Aquabirnaviruses, represented by Infectious pancreatic necrosis virus (IPNV), have been isolated from epizootics in salmonids and a variety of aquatic animals in the world; six genogroups of aquabirnaviruses have been identified. In comparisons of nucleotide sequences of the VP2/NS junction region, maximum nucleotide diversities of 30.8 % were observed among 93 worldwide aquabirnavirus isolates. A phylogenetic tree revealed the existence of a new genogroup, VII, for Japanese aquabirnavirus isolates from marine fish and molluscan shellfish. Nucleotide diversities between genogroups VII and I-VI were 18.7 % or greater. At the nucleotide level, Japanese IPNV isolates from epizootics in salmonids were nearly identical to a genogroup I strain from the USA or Canada. It is suggested that Japanese IPNV isolates belonging to genogroup I were originally introduced from North American sources, whereas Japanese aquabirnavirus isolates of genogroup VII were from marine aquatic animals indigenous to Japan.

Quantification of aquabirnaviruses isolated from different host species by real-time RT-PCR.

Real-time reverse transcription (RT)-PCR was developed to detect and quantify successfully aquatic birnaviruses (ABVs) from fish tissue and seawater samples. This method detected marine birnavirus (MABV) RNA in several samples, and the detection was specific for ABVs. Monitoring the MABV strain Y-6 RNA quantification by real-time RT-PCR showed replication kinetics of MABV after experimental infection in vitro. We found the quantity of ABVs isolated from different host species by using combined virus absorption in cell culture and real-time RT-PCR. Although all specimens showed no symptoms of viral infection, ABVs were detected regardless of host species. In conclusion, real-time RT-PCR was shown to be a sensitive and reliable tool to detect and quantify ABVs in cultured fish. This method is useful procedure to show details of horizontal or vertical infections by ABVs in the breeding water of aquatic animals.

Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain.

Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.

Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures.

An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10(3) TCID50/ml, whilst the labelling appears at 24h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent.

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA.

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.

Concentration of marine birnavirus from seawater with a glass fiber filter precoated with bovine serum albumin.

In the present study an efficient method for sampling the marine birnavirus (MABV) gene from seawater was developed. MABV gene was monitored by a specific polymerase chain reaction. When Millipore filters were used, MABV was efficiently collected on a filter with 0.05- micro m pore size. When both millipore and glass fiber filters were used, MABV was recovered from both filters. Use of plain glass fiber filters resulted in poor recovering efficiency. However, coating the glass fiber filters with 1% bovine serum albumin trapped MABV efficiently. Combining concentration on glass fiber filters with polymerase chain reaction is quantitative, economic and fast, suggesting that this method can be used to detect genetically identified fish disease viruses, algal viruses, and phages.

Early interactions of marine birnavirus infection in several fish cell lines.

Marine birnavirus (MABV), a member of the genus Aquabirnavirus, family Birnaviridae, is an unenveloped icosahedral virus with two genomes of double-stranded RNA. The mechanisms of MABV adsorption and penetration are still undetermined. This work examined MABV infection in susceptible and resistant fish cell lines. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. Thus, restriction of the MABV replication cycle is different between resistant FHM and EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. Thus, this 250 kDa protein may be a major MABV receptor that exists in the plasma membranes of all four cell lines examined. This result suggests further that another receptor for virus penetration may exist in CHSE-214, RSBK-2 and FHM cells but not in EPC cells.

Detection of marine birnavirus genome in zooplankton collected from the Uwa Sea, Japan.

Marine birnaviruses (MABVs) infect a wide range of fish and shellfish, yet their mode of transmission is still unclear. To determine whether marine plankton serve as a vector for MABVs, we examined plankton collected from the Uwa Sea, Japan. The phytoplankton and zooplankton were collected monthly, at depths of 0 and 40 m, from May to November 2001. Detection of the MABV genome was carried out using 2-step PCR and virus isolation. Viral genome was detected in zooplankton collected at 0 m depth in September and at 40 m depth in November. The virus could not be isolated in the PCR-positive samples. These results suggest that zooplankton may act as a vector of MABVs, although the infective and/or accumulated virus titer in zooplankton was low.

Detection of marine birnavirus in the Japanese pearl oyster Pinctada fucata and seawater from different depths.

This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 m). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 m depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6% were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 m depth. In contrast, the MABV genome in seawater was present through the year at 15 m depth, but was not detected in summer at 2 m. This suggests that the virus is destroyed by UV and/or other factors at 2 m in summer, but is stable in deeper waters.

Infectivity of aquabirnavirus strains to various marine fish species.

To determine the infectivity of marine birnavirus (MABV) in various marine fish species, experimental infection was performed in combination groups of 5 fish species with 7 strains of MABV and 1 strain of infectious pancreatic necrosis virus (IPNV). Mortality was observed in yellowtail Seriola quinqueradiata and amberjack S. dumerili infected with MABV strains Y-6, Y-10K and H-1, but not in other infected species. MABV was reisolated from most combination groups, but the virus isolation rate and virus infectivity titer were often significantly different among groups with the same fish species or with the same virus strain. All MABV strains replicated well in makogarei Limanda yokohamae, but only slightly in tiger puffer Takifugu rubiipes. IPNV also replicated in all fish species without causing death. The isolation rate and infectivity titer of IPNV were similar to or higher than those of non-virulent strains of MABV. In conclusion, the infectivity of MABV for different fish species is considered to change, which is an important factor in the development of the infection cycle of this virus among marine organisms.

Seasonal change of infective state of marine birnavirus in Japanese pearl oyster Pinctada fucata.

This study examines the seasonal occurrence and infective state of marine birnavirus (MABV) in cultured Japanese pearl oyster (Pinctada fucata). Planted oysters were sampled monthly in 1997 and 1998. To detect MABV in the oysters, PCR and virus isolation were carried out. Also, the indirect fluorescent antibody technique (IFAT) was performed to know the organs expressing viral antigens. The detection rate of the MABV genome by PCR was low during July to October, but increased after November. This virus was isolated only after October, with a 10-40% isolation rate. Results of the IFAT showed that the specific fluorescence was observed in hemocytes in September. Fluorescence in hemocytes decreased in January, but increased in liver parenchymal cells. These results suggest that MABV persistently infected hemocytes in summer with a small amount of genome and protein, and then the virus spread in winter into the parenchymal cells.

Surveillance for aquabirnavirus in fish hatcheries in Victoria.

OBJECTIVE: To determine whether or not aquabirnavirus is present in Victorian fish hatcheries. PROCEDURE: Milt and ovarian fluids were collected from brood stock at 12 hatcheries and cultured in two sensitive cell lines for the presence of viruses. RESULTS: No cytopathic effect was detected indicating the absence of virus. CONCLUSION: There is no evidence of infection with aquabirnavirus or Epizootic Haematopoietic Necrosis virus in Victorian fish hatcheries.

Viral coinfection in salmonids: infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus.

Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log10 units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48-72 h post infection, in contrast to the 50-80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections.