Virulence marker candidates in N-protein of viral haemorrhagic septicaemia virus (VHSV): virulence variability within VHSV Ib clones.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.

Virulence of viral haemorrhagic septicaemia virus (VHSV) genotype III in rainbow trout.

In general, viral haemorrhagic septicaemia virus (VHSV) isolates from marine fish species in European waters (genotypes GIb, GII and GIII) are non- to low virulent in rainbow trout. However, a VHSV isolation was made in 2007 from a disease outbreak in sea farmed rainbow trout in Norway. The isolate, named NO-2007-50-385, was demonstrated to belong to GIII. This isolate has attracted attention to assess which of the viral genome/proteins might be associated with the virulence in rainbow trout. In this study, we describe the difference of virulence in rainbow trout between the NO-2007-50-385 and 4p168 isolates as representatives of virulent and non-virulent GIII isolates, respectively. Rainbow trout were bath challenged with VHSV NO-2007-50-385 for 1 and 6 h, resulting in cumulative mortalities of 5 and 35%, respectively. No mortality was observed in the rainbow trout groups immersed with the genotype III VHSV isolate 4p168 for 1 and 6 h. The viral titre in organs from fish challenged with NO-2007-50-385 for 6 h increased more rapidly than those exposed for 1 h. By in vitro studies it was demonstrated that the final titres of VHSV DK-3592B (GI), NO-2007-50-385 and 4p168 inoculated on EPC cells were very similar, whereas when inoculated on the rainbow trout cell line RTG-2 the titre of the non-virulent 4p168 isolate was 3-4 logs below the two other VHSV isolates. Based on a comparative analysis of the entire genome of the genotype III isolates, we suggest that substitutions of amino acids in positions 118-123 of the nucleo-protein are candidates for being related to virulence of VHSV GIII in rainbow trout.

Isolation and characterization of hirame aquareovirus (HAqRV): A new Aquareovirus isolated from diseased hirame Paralichthys olivaceus.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

Identification of RNA regions that determine temperature sensitivities in betanodaviruses.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment, RNA1 (~3.1 kb), encodes an RNA-dependent RNA polymerase (protein A), and the smaller genomic segment RNA2 (~1.4 kb) codes for the coat protein. These viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in their partial RNA2 sequences. The optimal temperatures for the growth of these viruses are 20-25°C (SJNNV), 25-30°C (RGNNV), 20°C (TPNNV), and 15-20°C (BFNNV). However, little is known about the mechanisms underlying the temperature sensitivity of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at 25°C. However, the levels of all of the viruses but RGNNV were markedly reduced at 30°C. These results indicate that both RNA1 and RNA2 control the temperature sensitivity of betanodaviruses by modulating RNA replication or earlier viral growth processes. We then constructed ten mutated RGNNVs, the RNA1 segments of which were chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA1 region, encoding amino acid residues 1-445, grew similarly to the parental RGNNV at 30°C. This portion of protein A is known to serve as a mitochondrial-targeting signal rather than functioning as an enzymatic domain.

Genetic analysis and pathogenicity of betanodavirus isolated from wild redspotted grouper Epinephelus akaara with clinical signs.

Diseased wild redspotted grouper Epinephelus akaara were collected from Seto Inland Sea, Ehime Prefecture, in August 2002. Fish showed erratic swimming behavior and inflation of the swim bladder. The fish brains were positive for nodavirus in both RT-PCR and nested PCR. The sequence of the nested PCR product (177 nt) was closely related to that of a known betanodavirus, redspotted grouper nervous necrosis virus. When juvenile sevenband grouper E. septemfasciatus were challenged intravitreously with virus, abnormal swimming behavior and high mortality were observed. This is the first report on viral nervous necrosis in a wild population of redspotted grouper with clinical signs.

Variable region of betanodavirus RNA2 is sufficient to determine host specificity.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.

New validated rapid screening methods for identifying Kudoa septempunctata in olive flounder (Paralichthys olivaceus).

Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2-3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBA-NAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata.

Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2.

Striped jack nervous necrosis virus (SJNNV), which infects fish, is the type species of the genus Betanodavirus. This virus has a bipartite genome of positive-strand RNAs, designated RNAs 1 and 2. A small RNA (ca. 0.4 kb) has been detected from SJNNV-infected cells, which was newly synthesized and corresponded to the 3'-terminal region of RNA1. Rapid amplification of cDNA ends analysis showed that the 5' end of this small RNA (designated RNA3) initiated at nt 2730 of the corresponding RNA1 sequence and contained a 5' cap structure. Substitution of the first nucleotide of the subgenomic RNA sequence within RNA1 selectively inhibited production of the positive-strand RNA3 but not of the negative-strand RNA3, which suggests that RNA3 may be synthesized via a premature termination model. The single RNA3-encoded protein (designated protein B2) was expressed in Escherichia coli, purified and used to immunize a rabbit to obtain an anti-protein B2 polyclonal antibody. An immunological test showed that the antigen was specifically detected in the central nervous system and retina of infected striped jack larvae (Pseudocaranx dentex), and in the cytoplasm of infected cultured E-11 cells. These results indicate that SJNNV produces subgenomic RNA3 from RNA1 and synthesizes protein B2 during virus multiplication, as reported for alphanodaviruses. In addition, an Agrobacterium co-infiltration assay established in transgenic plants that express green fluorescent protein showed that SJNNV protein B2 has a potent RNA silencing-suppression activity, as discovered for the protein B2 of insect-infecting alphanodaviruses.

In vivo and in vitro analysis of the resistance against viral haemorrhagic septicaemia virus in Japanese flounder (Paralichthys olivaceus) precedingly infected with aquabirnavirus.

The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.

Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses.

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5' cap. Both RNAs were largely resistant to 3' polyadenylation and ligation, suggesting the presence of an interfering 3' structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5' and 3' non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT-PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3' terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0.4 kb) that was newly synthesized and corresponded to the 3'-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.

Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus.

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.