Localization of infectious salmon anaemia virus (ISAV) in experimentally infected fish by in situ hybridization using two riboprobes of the virus

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.