Antibodies to infectious salmon anaemia virus (ISAV) augment virus entry in macrophage-like fish cell lines

Infectious salmon anaemia virus (ISAV), a member of the family Orthomyxoviridae, is an important fish pathogen in marine aquaculture in the Northern Hemisphere. The pathogenesis of ISAV infection at the cellular level is not well studied. We investigated three permissive cell lines for ISAV, (SHK-1, TO and CHSE-214) as indicator systems for virus neutralization (VN) with anti-ISAV polyclonal antibodies of rabbit or fish (Atlantic salmon) origin. It was shown that the rabbit antiserum showed higher VN antibody titres in the CHSE-214 cell line than in macrophage-like fish cell lines SHK-1 and TO. With the fish antiserum, VN was observed only on CHSE-214 cells. The input virus used in the CHSE-214 cell system (i.e., 100TCID50) was found to be 1000 and 2000 times more than that used in the SHK-1 and TO cell systems, respectively, indicating that the true VN antibody titres of the antisera used were higher than the titres obtained on CHSE-214 cells. Neutralization of infectious pancreatic necrosis virus (IPNV) by rabbit anti-IPNV polyclonal serum in both CHSE-214 and TO cells demonstrated that effective virus neutralization is possible in TO cells. The VN antibody titre of rabbit anti-ISAV serum was increased 48-fold in TO cells when the assay was performed in presence of staphylococcal Protein A, which binds to the Fc moiety of immunoglobulins. Antibody-facilitated entry of ISAV in TO cells was confirmed using fluoresceine isothiocyanate (FITC)-labelled ISAV and rabbit anti-ISAV serum. These findings indicate that antibodies to ISAV augment the virus infection of macrophage-like fish cells. In conclusion, this study has demonstrated, for the first time, possible Fc receptor-mediated infection of macrophage-like fish cell lines by a fish virus, ISAV.

Piscine orthoreovirus sequences in escaped farmed Atlantic salmon in Washington and British Columbia

Abstract Background Piscine orthoreovirus (PRV) is an emergent virus in salmon aquaculture belonging to the family Reoviridae. PRV is associated with a growing list of pathological conditions including heart and skeletal inflammation (HSMI) of farmed Atlantic salmon. Despite widespread PRV infection in commercially farmed Atlantic salmon, information on PRV prevalence and on the genetic sequence variation of PRV in Atlantic salmon on the north Pacific Coast is limited. Methods Feral Atlantic salmon caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound were sampled. Fish tissues were tested for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. Results Following the escape of 253,000 Atlantic salmon from a salmon farm in Washington State, USA, 72/73 tissue samples from 27 Atlantic salmon captured shortly after the escape tested PRV-positive. We estimate PRV-prevalence in the source farm population at 95% or greater. The PRV found in the fish was identified as PRV sub-genotype Ia and very similar to PRV from farmed Atlantic salmon in Iceland. This correlates with the source of the fish in the farm. Eggs of infected fish were positive for PRV indicating the possibility of vertical transfer and spread with fish egg transports. Conclusions PRV prevalence was close to 100% in farmed Atlantic salmon that were caught in Washington State and British Columbia following a large containment failure at a farm in northern Puget Sound. The PRV strains present in the escaped Atlantic salmon were very similar to the PRV strain reported in farmed Atlantic salmon from the source hatchery in Iceland that was used to stock commercial aquaculture sites in Washington State. This study emphasizes the need to screen Atlantic salmon broodstock for PRV, particularly where used to supply eggs to the global Atlantic salmon farming industry thereby improving our understanding of PRV epidemiology

Formal comment on: Piscine reovirus: Genomic and molecular phylogenetic analysis from farmed and wild salmonids collected on the Canada/US Pacific Coast

<p>Formal comment on: Piscine reovirus: Genomic and molecular phylogenetic analysis from farmed and wild salmonids collected on the Canada/US Pacific Coast</p

First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV

Background: Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic salmon Salmo salar, first recognized in 1999 in Norway, and recently associated with piscine orthoreovirus (PRV) infection. To date, HSMI lesions with presence of PRV have only been described in marine-farmed Atlantic salmon in Norway. A new HSMI-like disease in rainbow trout Oncorhynchus mykiss associated with a PRV-related virus has also been reported in Norway. Methods: Sampling of Atlantic salmon and coho salmon was done during potential disease outbreaks, targeting lethargic/moribund fish. Fish were necropsied and tissues were taken for histopathologic analysis and testing for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. Results: The Atlantic salmon manifested the classical presentation of HSMI with high PRV virus loads (low Ct values) as described in Norway. The coho salmon with low Ct values had myocarditis but only in the spongy layer, the myositis of red muscle in general was mild, and the hepatic necrosis was severe. Upon phylogenetic analysis of PRV segment S1 sequences, all the Chilean PRV strains from Atlantic salmon grouped as sub-genotype Ib, whereas the Chilean PRV strains from coho salmon were more diversified, grouping in both sub-genotypes Ia and Ib and others forming a distinct new phylogenetic cluster, designated Genotype II that included the Norwegian PRV-related virus. Conclusions: To our knowledge the present work constitutes the first published report of HSMI lesions with presence of PRV in farmed Atlantic salmon outside of Europe, and the first report of HSMI-like lesions with presence of PRV in coho salmon in Chile. The Chilean PRV strains from coho salmon are more genetically diversified than those from Atlantic salmon, and some form a distinct new phylogenetic cluster, designated Genotype II.Centro de Investigaciones Biologicas Aplicadas (CIBA) Puerto Montt, Chile; Virology Research Laboratory at the Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada; University of San Sebastian Puerto Montt, Chile; Salmon from Chile

Phylogenetic tree for sequences of piscine orthoreovirus (PRV) segment S1 listed in S1 Table.

<p>All 127 available robust isolates are included in this tree. The phylogenetic tree was constructed using the neighbor-joining method and Tamura-Nei genetic distances (Saitou and Nei 1987 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188690#pone.0188690.ref018" target="_blank">18</a>]). Bootstrapping was performed 1,000 times. Bootstrap supports of topology of 70% or higher are shown at the nodes. The PRV grouping of Genotype I sub-genotypes Ia, Ib, and Genotype II are indicated. The mutation direction was determined by an outgroup sequence.</p

Aquaculture Association of Canada Special Publication 6 64 68 Aquaculture Association of Canada [St. Andrews, NB]

Infectious salmon anaemia virus (ISAV) is a member of the family Orthomyxoviridae. Hybridization using riboprobes of segments 6 and 8 and histology were combined in situ to demonstrate the tissues that harbour ISAV during the clinical phase of ISAV infection in Atlantic salmon (Salmo salar L.) The choice of fixative (paraformaldehyde or formalin) and conditions used to permeabilize tissues (proteinase K or Tween 20%) prior to in situ hybridization were first optimized with the segment 8 riboprobe of ISAV. Atlantic salmon parr (~620 g) were infected with ISAV in freshwater. Six tissues/organs were removed from seven fish 15-21 days post infection. Reverse transcription-polymerase chain reaction (RT-PCR) on tissue pools confirmed the presence of ISAV in each fish. Microscopic lesions were seen in 6 livers and consisted of sinusoidal congestion and coagulative necrosis. One fish had a mild vascular congestion and ingestion and coagulative necrosis. One fish had a mild vascular congestion and increased erythrophagia in the kidney. Small blood vessels and capillaries in the cardiac ventricles were markedly dilated and contained prominent endothelial and macrophage-like cells in 3 fish. The purplish-blue hybridization signal was seen in the endothelial cells lining small blood vessels of all tissues examined, with the heart consistently showing the most intense signal in the endothelial and macrophage-like cells. Signal intensity, in decreasing order, was in liver, spleen, pyloric caeca, gill, and kidney. There was no significant difference in the hybridization signal with the different fixatives. The riboprobe for segment 8 showed more hybridization signal in all tissues studied and therefore more mRNA presence in the cells than the segment 6 riboprobe

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (<i>Salmo salar</i>) during 2021–2022

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV