Detection of rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

Formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that succumbed to rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) during the 2019 outbreak in Washington, USA, were utilized for in situ hybridization via RNAscope (ACDBio). This detection method was both sensitive and specific, with no staining in tissues from RHDV- (Lagovirus GI.1) and RHDV2-negative rabbits, and only slight background staining of RHDV-positive rabbits; RHDV2-positive tissues had bright-red cytoplasmic staining. Although much of the viral mRNA detection was consistent with previously described antigen detection via immunohistochemistry of the liver, lungs, and spleen, there was also significant glomerular staining in the kidneys, and endothelial staining within blood vessels of almost all organs. We validated the RNAscope technique for detection of RHDV2 mRNA in formalin-fixed, paraffin-embedded tissues, with increased sensitivity from previous techniques, and identified additional affected cell types that may contribute to the understanding of pathogenesis.