Endangered wild salmon infected by newly discovered viruses

Mordecai, Gideon J; Miller, Kristina M; Di Cicco, Emiliano; Schulze, Angela D; Kaukinen, Karia H; Ming, Tobi J; Li, Shaorong; Tabata, Amy; Teffer, Amy; Patterson, David A; Ferguson, Hugh W; Suttle, Curtis A

RNA-ISH was performed using RNAscope 2.5 HD Duplex assay (Advanced Cell Diagnostics, Newark, California, USA, catalog# 322500) according to the manufacturer's instructions. Briefly, consecutive sections of Chinook and sockeye salmon samples utilised for the histopathological analysis were dewaxed by incubating for 60 min at 60ËšC and endogenous peroxidases were quenched with hydrogen peroxide for 10 min at room temperature. Slides were then boiled for 30 min in RNAscope target retrieval reagents (Advanced Cell Diagnostics, Newark, California, USA) and incubated for 30 min in RNAscope Protease Plus reagent prior to hybridization. The slides underwent hybridization with RNAscope probes against a portion of SPAV-1 and SPAV-2 genome (Advanced Cell Diagnostics, Newark, California, USA, catalog #513591-C2 and 538881-C2, respectively). A RNAscope probe against Coil-p84 housekeeping gene in Chinook salmon (Advanced Cell Diagnostics, Newark, California, USA, catalog #512391) was used as positive control probe to confirm the efficacy of the probes and the viability of the samples. Two samples which were negative for SPAV-1 and SPAV-2 were used as negative controls to confirm absence of background and (or) non-specific cross-reactivity of the assay. Signal amplification was performed according to the manufacturer's instructions, followed by counterstaining with Gill's hematoxylin and visualisation by bright field microscopy.