Development of a duplex fluorescent in situ hybridization (FISH) technique to detect and localize redspotted grouper nervous necrosis virus sense and antisense genomic RNA1 and its application to the tissues of aquatic vertebrates and invertebrates

The causative agent of viral encephalopathy and retinopathy (VER), the nervous necrosis virus (NNV), is considered one of the most impacting agents in the Mediterranean aquaculture and it has already been described in several aquatic vertebrates and invertebrates including bivalve molluscs.

In this study a novel duplex fluorescent in situ hybridization (FISH) assay was developed for the detection of sense and antisense RGNNV genomic RNA1 fragment, coding for the RNA-dependent RNA polymerase (RdRp), and applied to aquatic vertebrates and invertebrates' tissues. The detection of sense and antisense RNA provides useful information not only on viral localization at cellular level, but also on viral replication, improving the understanding of the host-pathogen interactions between NNV and its hosts.

The designed assay resulted in the specific detection of the RdRp of the two NNV strains most widespread in the Mediterranean basin: RGNNV and RGNNV/SJNNV.

FISH was applied to the brain of European sea bass (Dicentrarchus labrax) showing the localization at cellular level of both NNV genome and cRNA produced during viral replication.

The assay was also able to localize the NNV genome and its replicative intermediate form in clams (Ruditapes philippinarum). Clear positive signals were found in oocytes' cytoplasm confirming the intracellular localization of NNV in invertebrates. Altogether, these findings suggest NNV active replication status in clams' oocytes and its potential transmission through vertical route. In conclusion, the developed assay can find wide application in the deep understanding of host-pathogen interactions, contributing to pointing out the real role that invertebrates play in VER epidemiology.