Localization and tissue tropism of viral haemorrhagic septicemia virus (VHSV) in experimentally infected juvenile olive flounder, Paralichthys olivaceus: An in situ hybridization and immunohistochemical study

An insight on localization of viral pathogen is imperative to better understand the host-pathogen interaction. Previously, we developed an in-situ hybridization (RNA-ISH) assay to detect viral hemorrhagic septicemia virus (VHSV), an OIE reportable fish pathogen, in an in vitro model. Here, we utilized its potential and applicability in vivo to further our understanding about the localization and tissue tropism of VHSV in experimentally infected juvenile olive flounder (Paralichthys olivaceus), an economically important flatfish in Asian aquaculture. Two separate digoxigenin (DIG) labeled antisense RNA probes targeting a fragment of viral nucleoprotein (N) and glycoprotein (G) gene segments were employed to localize VHSV in five infected flounder tissues viz., kidney, spleen, heart, liver and brain. Further, immunohistochemistry (IHC) assay was also used to observe and substantiate the spatial localization of the viral particles using a monoclonal antibody (MAb) against the viral nucleoprotein (N). Following the RNA-ISH and IHC assays, VHSV localization was observed in different cells and areas of the tested tissues. The positive reaction in both the assays correlated with histopathological alterations confirming the presence of VHSV within the tested tissues. Information gained from the present study using both genome and protein based localization sheds light on viral tropism which can help to further elucidate VHSV pathogenesis in olive flounder.