Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska

Abstract

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

Keywords: emerging diseases; histopathology; in situ hybridization; next-generation sequencing; piscine orthoreovirus; salmon; virus discovery.

Figure 1.

Myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis in both the compact and trabecular myocardium of a coho salmon (Oncorhynchus kisutch) fingerling. H&E.

Figures 2–5.

Raw Oxford Nanopore Technologies (ONT) reads mapped to piscine orthoreovirus (PRV) segment S1 (upper panel) and M2 (lower) using minimap2, visualized on IGV (https://igv.org/app/), shows high concordance with PRV2 (Fig. 2) and low concordance with PRV1a (Fig. 3), PRV1b (Fig. 4), and PRV3 (Fig. 5). Colored positions highlight discordance of basecalls against reference, and gray indicates concordance to reference.

Figure 6.

Phylogeny of concatenated coding sequences supports clustering of piscine orthoreovirus 2–like (PRV2-like) with published PRV2. Where available, PRV1 isolates subtyped PRV1a or PRV1b are indicated. Branch labels indicate number of substitutions per site.

Figures 7–12.

Matching H&E histopathology and tissue distribution of piscine orthoreovirus 2–like (PRV2-like) virus by in situ hybridization (ISH) from multiple infected fish. Figure 7. Whole heart from infected fingerling with myocardial degeneration and myocarditis. Asterisk indicates luminal blood. H&E. Figure 8. Diffuse abundant positive reactivity for PRV2-like viral genome in the ventricular trabecular myocardium, mild reactivity to the atrium, and abundant blood cell reactivity (asterisk). ISH. Figure 9. Mild renal histiocytosis. H&E. Figure 10. Moderate multifocal positive reactivity for PRV2-like viral genome in the hematopoietic tissue of the kidney. ISH. Figure 11. Moderate splenic histiocytosis. H&E. Figure 12. Abundant multifocal positive reactivity for PRV2-like viral genome in the hematopoietic tissue of the spleen. ISH.