Early detection and localization of viral haemorrhagic septicemia virus (VHSV) genomic mRNAs in external tissues of juvenile olive flounder (Paralichthys olivaceus) following an immersion challenge

The present study is a continuation of our previous work on in-situ detection and localization of viral haemorrhagic septicemia virus (VHSV) in olive flounder. As VHSV must cross and overcome the external barriers to reach the target organs, the role of external tissues becomes important in early viral uptake. However, investigations in this line are scarce and have not received much attention. Here, we investigated VHSV uptake in olive flounder to decipher the role of external tissues in early viral trafficking using a natural infection model. Juvenile olive flounder were bath (immersion) challenged with live VHSV for 1 h and tissue samples from gill, skin, fin and muscle were aseptically sampled at 15 min, 30 min, 3 h, 12 h, 24 h and 48 h post-challenge. Afterwards, samples were processed for in-situ hybridization (RNA-ISH) to observe spatio-temporal localization of VHSV using our previously designed DIG-labeled antisense RNA probes viz., nucleoprotein (N) and glycoprotein (G) riboprobes. In parallel, a time-dependent quantitation of the viral mRNA transcript (N & G) was done at similar time points using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the viral load. Whereas, VHSV uptake was confirmed in all the tested tissues using qRT-PCR, a weak signal was observed as early as 15 min in the gill using RNA-ISH. The signal intensity gradually increased at the corresponding time points and marked difference was evident at 12 hpc, 24 hpc and 48 hpc in all the tested tissues; particularly in the gill, skin and fin, followed by muscle. Our results suggest that VHSV uptake in flounder possibly occurs via gill immediately after exposure to the virus. Subsequently, virus gains entry through skin and fin base after it crosses the mucosal barriers of the host and then actively replicates in the external tissues. The current findings can be helpful in designing efficient immersion vaccination strategies for VHSV infection in olive flounder.