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The relationship of histopathological changes and the infection of Piscine orthoreovirus 2 (PRV‐2) was investigated in coho salmon that were suffering from the erythrocytic inclusion body syndrome (EIBS). Immunohistochemical observations revealed abundant σ1 protein of PRV‐2 in the spongy layer of the ventricle of the heart, where severe myocarditis was observed. In the spleen, the virus protein was detected in many erythrocytes, some of which were spherical‐shaped and apparently dead. The number of erythrocytes was decreased in the spleen compared to the apparently healthy fish. The virus protein was also detected in some erythrocytes in blood vessels. The viral protein was often detected in many macrophages ingesting erythrocytes or dead cell debris in the spleen or in the kidney sinusoids. Large amounts of the viral genomic segment L2 were also detected in these organs by RT‐qPCR. Many necrotic foci were found in the liver, although the virus protein was not detected in the hepatocytes. These results suggest that the primary targets of PRV‐2 are myocardial cells and erythrocytes and that clinical symptoms such as anaemia or jaundice and histopathological changes such as myocarditis in EIBS‐affected coho salmon are caused by PRV‐2 infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Coho salmon (Oncorhynchus kisutch) is a Pacific salmon species that is commercially farmed in Japan, and is susceptible to erythrocyte inclusion body syndrome (EIBS), which is characterized by anemia, erythrocyte inclusion bodies, and cardiovascular muscle necrosis. At present, no vaccine for this disease is available. Piscine orthoreovirus-2 (PRV-2) is the causative agent of EIBS in Japan, and is one of three genotypes of PRV that induce the formation of erythrocyte inclusion bodies. However, due to a deficiency in cell lines able to culture PRV-2, the development of vaccines for diseases caused by PRV-2 has been difficult. A recent strategy involved the combination of a DNA vaccine, a σ1, and a non-structural protein gene for moderate PRV-1 protection. In the present study, analysis of antiserum obtained from PRV-2 infection survivors identified σ1 as a PRV-2 antigenic protein. Since a DNA vaccine incorporated with only σ1 did not prove efficacious against PRV-2 infection in a preliminary study, the present study tested the enhanced protective effect of the DNA vaccine with various sequences linked to the σ1 gene. Antigenic PRV-2 gene products were first screened using recombinant PRV-2 proteins and serum from coho salmon with a history of EIBS. Six vaccine groups of DNA vaccines were then created by the linkage of several sequences to the PRV-2 gene. Two trials were conducted to identify effective vaccines and a third vaccination trial confirmed the reproducibility of an effective vaccine. Trial I showed insignificant differences in the mean antibody level after 42 days. However, an outlier test indicated significantly higher antibody levels in coho salmon vaccinated in signal-linked σ1 gene DNA vaccine group and chaperone-linked σ1 gene DNA vaccine group. Trial II evaluated the effects of individual DNA vaccines and showed that individuals in the Sec-σ1, TF-σ1, and mixed vaccine group had significantly higher antibody levels and significantly lower virus loads. Trial III confirmed the suppression of viral replication and increase in antibody levels of the TF-σ1 DNA vaccine. However, this DNA vaccine was only effective for increasing antibody levels and inhibiting viral growth in some individuals, indicating the necessity for further experimentation involving analyses of the effect of individual differences on the efficacy of the vaccine. • DNA vaccines having limited effect against Piscine orthoreovirus 2 was developed. • Plasmids for DNA vaccination were constructed with the PRV-2 σ1 gene alone or with various sequences linked to the σ1 gene. • TF gene and secretory signals linked toσ1 gene enhanced vaccine efficacy. [ABSTRACT FROM AUTHOR]