Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Japan
Correspondence
Toyohiko Nishizawa
jjnishi{at}fish.hokudai.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences determined in this study are AB179699AB179712.
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MAIN TEXT |
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Aquabirnaviruses have a non-enveloped, icosahedral capsid approximately 60 nm in diameter containing the bisegmented, double-stranded RNA genome (segments A and B; Leong et al., 2000). Segment B (2·8 kb) encodes a minor internal polypeptide VP1 (Mr 94 kDa), the putative RNA-dependent RNA polymerase, whilst segment A (3·1 kb) contains two partially overlapping open reading frames (ORFs), a large ORF for the polyprotein (Mr 106 kDa, NH2-pVP2-NS-VP3-COOH) and a small ORF for VP5 (Mr 17 kDa). The polyprotein is cleaved into three polypeptides: pVP2, the precursor of the major capsid protein VP2; NS, a non-structural protein with protease activity associated with the cleavage of the polyprotein; and VP3, a minor capsid protein (Duncan & Dobos, 1986
; Duncan et al., 1987
; Håvarstein et al., 1990
; Manning & Leong, 1990
; Manning et al., 1990
; Magyar & Dobos, 1994
; Leong et al., 2000
). Recently, molecular phylogenetic analysis, based on the VP2-coding region of IPNV and other aquabirnaviruses, has revealed the existence of six discrete genogroups that are correlated to the previously established serotypes, A1A9 and B1 (Blake et al., 2001
; Cutrín et al., 2004
). The previous studies unfortunately included only a limited number of Japanese isolates; thus, in this study, a total of 14 Japanese isolates of IPNV and other aquabirnaviruses, differing in the year of isolation and geographical and host origins, together with worldwide isolates, were subjected to molecular phylogenetic analysis based on the nucleotide sequence of the VP2/NS junction region in order to evaluate their genetic relatedness.
Six Japanese isolates of IPNV from affected salmonids (Eniwa, Tomakomai, Oippe, Towada, Gifu and Nagano) and eight Japanese aquabirnavirus isolates from apparently healthy marine fish (Obama10, Obama29, Izu6, Izu18, Akk02SS, Akk02RS, Akk02SC and Akk02JD) were used in this study. Eniwa and Tomakomai were isolated from rainbow trout (Oncorhynchus mykiss) at Hokkaido in the 1970s, whilst Oippe was also from masu salmon (Oncorhynchus masou) at Aomori Prefecture in the 1980s. Towada, Gifu and Nagano isolates were from rainbow trout on the mainland of Japan in 1987, the 1980s and 2000, respectively (Kimura et al., 1984; Yoshimizu et al., 1993
). Obama10, Obama29, Izu6 and Izu18 were isolated from free-living Japanese flounder at Obama and Minami-Izu, Japan, in 1999 (Takano et al., 2001
). Isolates Akk02SS, Akk02RS, Akk02SC and Akk02JD were isolated respectively from the wild marine fish snowy sculpin (Myoxocephalus blandti), saffron cod (Eleginus gracilis), rainbow smelt (Osmerus eperlanus mordax) and Japanese dace (Tribolodon hakonensi) captured in the Akkeshi coastal area of Hokkaido, Japan, in 2002 (Kobayashi et al., 2005
).
Viral genomic RNA, prepared with an RNA-extraction kit (Isogen-LS; Nippon Gene) according to the manufacturer's instructions, was used as the template for RT-PCR with an annealing temperature of 52 °C. PCR primers ABV-P1 (5'-AGAGATCACTGACTTCACAAGTGAC-3') and ABV-P2 (5'-TGTGCACCACAGGAAAGATGACTC-3') were used for amplification of the VP2/NS junction region between nucleotide positions 1403 and 1761 of segment A of the viral genome (Heppell et al., 1992). The PCR products were purified with a PCR-purification kit (Stratagene) and subjected to nucleotide sequence analysis. The resulting sequences were assembled with DNASIS (hitachi) and multiple alignments of the sequences were constructed by using CLUSTAL W (Thompson et al., 1994
) to search for an optimal phylogenetic tree with neighbour-joining criteria. The final phylogenetic tree was drawn with the NJplot program (Perrière & Gouy, 1996
). The determined nucleotide sequences were registered with GenBank/DDBJ under accession nos AB179699AB179712. Deposited nucleotide sequences of 79 isolates of IPNV and aquabirnavirus in GenBank/DDBJ were used for comparative purpose (Fig. 1
). Although 93 isolates were employed in total in the present study, the corresponding countries, host species and serotypes of 39 representative isolates are provided in Table 1
.
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The Japanese isolates of IPNV, Eniwa, Tomakomai, Oippe, Towada, Gifu and Nagano, were all classified into genogroup I, but were divided into two of the three minor clusters of this genogroup (Fig. 1). The Tomakomai isolate was nearly identical to the Jasper strain, whereas the other five isolates were related closely to VR299 or the Buhl strain (Fig. 1
; Table 1
). It is thus indicated that the Tomakomai isolate is serotyped as A9, the same as the Jasper strain, whereas the other five isolates are classified as serotype A1, the same as the VR299 and Buhl strains. Additionally, genogroup II contained a Japanese strain, EEV, which was isolated from an imported European eel (Sano et al., 1981
) and serotyped as A3 (Blake et al., 2001
). Therefore, it is shown that aquabirnaviruses belonging to at least three different genogroups (including four serotypes) have been present in Japan and that genogroups I (serotype A1) and VII of aquabirnaviruses are predominant in Japan.
IPNV is thought to be spread from continent to continent with the importation of salmonid fish or fish eggs, but it is likely that the virus had a global distribution prior to the widespread dissemination of salmonids in the 19th and the early 20th centuries (Reno, 1999). Aquabirnaviruses from a variety of aquatic animal species are generally infected with the same genogroup as particular IPNV isolate(s) in the same area. However, it is interesting that Japanese IPNV and other aquabirnaviruses are classified into two different genogroups, I and VII, which are segregated completely by the environment in which they are distributed, i.e. the former is isolated from salmonids in freshwater environments, whereas the latter is detected in marine aquatic animals. Against this unique situation in Japan, we consider the following. Aquabirnaviruses of genogroup VII may have existed as indigenous viruses in Japanese marine environments prior to the widespread dissemination of salmonids, because genogroup VII isolates have been found to be limited to Japan and Korea; moreover, these isolates are not currently associated with any disease in their host. This assumption is further supported by the nucleotide diversity within genogroup VII (3·5 %), the same degree as that within other discrete clades, genogroups II and V (Table 2
). In contrast, Japanese IPNV isolates belonging to genogroup I could have been introduced from North American sources by transportation of infected fish or fish eggs in the 1950s, because Japan was an important client for importation of salmonid fish and fish eggs from North America at that time (Kimura & Yoshimizu, 1991
; Yoshimizu, 1996
). As a consequence, the introduced viruses may have become established in Japanese freshwater environments. This is supported by the degree of nucleotide diversity (8·4 %) exhibited by Japanese IPNV isolates; however, the Tomakomai isolate was nearly identical to the Canadian Jasper strain (1·0 %), whereas the other remaining Japanese isolates were nearly identical to USA strains VR299 and Buhl (
1·9 %) (Tables 1 and 2
). Therefore, we speculate that the segregated distribution of IPNV and aquabirnaviruses in Japan is an interesting situation that suggests that IPNV may originate in marine environments, as well as a theory for a marine origin of Viral hemorrhagic septicemia virus, a fish-pathogenic rhabdovirus (Smail, 1999
; Nishizawa et al., 2002
; Einer-Jensen et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 13 July 2004;
accepted 23 March 2005.