Abstract
Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.
Highlights
Mass mortality from erythrocytic inclusion body syndrome (EIBS) occurs frequently and causes severe economic losses on coho salmon (Onchorhynchus kisutchi) farms in Japan [1]
No double-stranded RNA (dsRNA) corresponding in size to small segments (1.0–1.3 kbp) was detected (S2 Fig), but cDNAs corresponding to small, medium, and large segments were successfully amplified with the full-length amplification of cDNAs (FLAC) method (S3 Fig)
70.4%–73.5% (Fig 2), whereas the sequence identities between the PRV isolates were ! 79.0% [19], suggesting that our virus has a novel segment S1. These results indicate that the virus purified from EIBS-affected erythrocytes is a member of the piscine orthoreoviruses
Summary
Mass mortality from erythrocytic inclusion body syndrome (EIBS) occurs frequently and causes severe economic losses on coho salmon (Onchorhynchus kisutchi) farms in Japan [1]. Fish suffering EIBS are always characterized by erythrocytic inclusion bodies and anemia [7]. Hyperbilirubinemia and the accumulation of bilirubin in the liver [9] may be one of the causes of the jaundice and yellow-colored livers of diseased coho salmon. The causative agent of EIBS is an icosahedral virus, approximately 70– 80 nm in diameter, and the virions are often observed within membrane-bound structures that are frequently associated with the cellular membranes [7, 10]. The virions are occasionally free in the cytoplasm of erythrocytes or combined within large cytoplasmic vacuoles [10]. The preliminary characterization of this virus with electron microscopy revealed similar characteristics to those of a member of the family Togaviridae [11], but the isolation of the virus in a fish cell line has been unsuccessful, preventing its further characterization [12]
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