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

Abstract

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.

Keywords: Atlantic salmon; HSMI; HSMI-like; Heart and skeletal muscle inflammation; PRV; Piscine orthoreovirus; coho salmon.

Fig. 1

Main gross pathology findings in Chilean farmed Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch: Panels a and b: Atlantic salmon with characteristic gross lesions of heart and skeletal muscle inflammation (HSMI). Panels c and d: Coho salmon with gross lesions of HSMI-like disease

Fig. 2

Photomicrographs showing the main microscopic findings in farmed Chilean Atlantic salmon Salmo salar with HSMI-like disease. Panel a: Heart section stained with haematoxylin and eosin (H&E) showing diffuse infiltration of mononuclear cells in epicardium (arrow) and infiltration of mononuclear cells, degeneration and necrosis of muscle fibers in myocardium (asterisk) (100X). Panel b: Red muscle section stained with H&E showing infiltration of mononuclear cells, degeneration and necrosis of muscle fibers (arrow head) (scale bar = 100 μm). Panel c: Liver section showing a blood vessel surrounded by a mononuclear inflammatory cell infiltrate (perivasculitis) focal hepatocellular necrosis (arrow head) (scale bar = 100 μm)

Fig. 3

Photomicrographs showing the main microscopic findings in farmed Chilean coho salmon Oncorhynchus kisutch with HSMI-like disease. Panel a: Heart section stained with haematoxylin and eosin (H&E) showing infiltration of the spongious layer (arrow) by mononuclear cells (scale bar = 100 μm). Panel b: Red muscle section stained with H&E showing a focal infiltration of mononuclear cells among muscle fiber bundles (arrow head) (scale bar = 100 μm). Panel c Liver section showing focal necrosis of hepatocytes (asterisk) close to a blood vessel (scale bar = 100 μm)

Fig. 4

Frequency distribution of the cycle threshold (Ct) values for all farms positive for PRV. The PRV positive Atlantic salmon (n = 57) are shown in red, and the PRV positive coho salmon (n = 80) are shown in blue

Fig. 5

Phylogenetic tree of segment S1 sequences from PRV isolates from Norway, Chile, and Canada. The phylogenetic tree was constructed using the Maximum Likelihood method (software package: PhyML 3.0) [53]. An outgroup (Genbank accession number: AF059720) was used to determine its root, but the outgroup itself was not included in the tree. The bootstrapping procedure was applied for 1000 times and the branches with 60 % or higher bootstrapping support values were marked