SPREAD OF BETANODAVIRUS IN WILD MARINE FISH FROM ADRIATIC SEA: USE OF CELL CULTURE ISOLATION OR NESTED RT-PCR?

Betanodaviruses are the causal agents of viral nervous necrosis that is responsible of mortality and economic losses worldwide in aquaculture. Furthermore these viruses were detected in an increasing number of cultured and wild fish specie without report of symptoms. Presence of Betanodavirus infection in wild asymptomatic fish were reported in Mediterranean Sea, Japan, Northeastern Atlantic, Korea and East China Sea. For some species a specific \u201ccarrier\u201d role was established as in the case of Gilthead seabream (Sparus aurata) that is responsible of experimental virus transmission to European sea bass (Dicentrachus labrax). In the other cases interspecies transmission from asymptomatic fish are strongly suspected. Free-living fish, in fact, could be responsible for transmission between different farms through long distance migration and/or winter persistence of Betanodavirus. For this study we tested for Betanodavirus infection 263 fish, belonging to 22 species, collected from fish market of Emilia-Romagna between May and November 2005. Fish brains were analysed as a pool of homogeneous fish on SSN-1 cell line. Thirteen out of 109 (11.9%) samples were positive for Betanodavirus. Three blind passages were necessary to evidence the virus in most of the samples, due to low viral load. The virus was isolated from 9 new fish species (black goby; bogue; pilchard; garpike; gurnard; hake; mackerel; red mullet; whiting) and one previously signalised specie (Mugil cephalus). Betanodavirus were found in fish along all the analysed period apart from temperature condition. Twenty two negative samples were further tested by nested RT-PCR using primers S6-S7 for the first RT-PCR and F2-R3 for nested PCR. Seven samples gave positive results despite they were negative to cell culture analysis. On the base of this result one more specie resulted infected by Betanodavirus: Atlantic mackerel (Scomber scombrus). Viruses isolated in cell culture were genetically characterized by sequencing and phylogenetic analysis. On the base of this analysis all isolated viruses clustered with RGNNV. Further genetic analysis including viruses detected only by nested RT-PCR will be necessary to explain if the negative result obtained with cell culture isolation was to be ascribed to different sensitivity of methods or a different nature of viruses