Isolation and genetic characterization of Betanodavirus from wild marine fish from Mediterranean Sea

Betanodavirus infection is responsible of mortality and economic losses in European sea bass farm in the Mediterranean Sea. Symptoms generally are not observed in wild fish, despite infection was detected in several species. Interspecies transmission are possible and sub clinically infected carrier are important for horizontal transmission. The roles of wild fish as carrier and reservoir are suspected, but need to be further investigate. In this study several fish species collected from fish market of two regions of Italy: Sicily and Emilia-Romagna were tested for Betanodavirus infection on SSN-1 cell line. Fish brains were analysed as a pool of homogeneous fish. Seven out of 73 (9,5%) and 13 out of 109 (11.9%) of samples from Sicily and Emilia-Romagna respectively were positive for Betanodavirus. All samples positive to viral isolation were confirmed by RT-PCR. Eleven new fish species (black goby; bogue; pilchard; garpike; gurnard; hake; mackerel; Pagellus sp.; poor cod; red mullet; whiting) and one previously signalised specie (Mugil cephalus) were evidenced infected. Viruses isolated were genetically characterized by RT-PCR and sequencing. Alignment and phylogenetic analysis were conducted with sequences of reference strains and DlEVs available from genbank database. 99.8% to 100% and 93.6% to 100% identity was observed among viruses isolated from Sicilian and Emilia-Romagna samples respectively. 93.9% to 100% identity was observed among viruses isolated from the same species, while 93.6% to 100% identity was observed among viruses from different species showing that no specie-specificity was present for analysed strains. In phylogenetic analysis all our strains clustered with RGNNV. Sub cluster were not supported by bootstrap analysis. Viruses isolated from wild and farmed fish showed high identity values and were not separated in different groups by phylogenetic analysis. The spreading of the same strains in both wild and farmed fish could be potentially responsible for interspecies transmission and need to be monitored by virus detection and characterization.