Pathogenesis studies and detection of betanodavirus in marine fish species from the iberian peninsula

Resumo

The Viral Nervous Necrosis Virus (VNNV) is the causative agent of the Viral Encephalopathy and Retinopathy (VER), which causes high mortalities and economical losses in fish farms worldwide. One of the most susceptible fish species is European sea bass (Dicentrarchus labrax), with 2-50 % mortality, in the juvenile stage, and up to 100% in larvae. VNNV, Betanodavirus genus, Nodaviridae family, has a genome composed of two single-stranded positive-sense RNA segments: RNA1 (enconding the RNA dependent RNA polymerase, RdRp), and RNA2 (encoding the coat protein, CP). This genus has been classified into four genotypes: SJNNV, RGNNV, BFNNV and TPNNV. SJNNV and RGNNV are the only genotypes reported in the Iberian Peninsula to date. These studies, based on the RNA2 amplification, showed the predominance of the SJNNV genotype. However, the amplification of both segments showed a high percentage of reassortant viruses (RGNNV RNA 1/SJNNV RNA2). The results derived from this Thesis have demonstrated a high percentage of fish specimens (wild and cultured) carrying both genotypes simultaneously. Moreover, a VNNV carrier state has been reported for the first time in wild meager (Argyrosomus regius) and thicklip grey mullet (Chelon labrosus). Based on these results, an in vitro study was conducted to analyze the effect of this coexistence (coinfection and superinfecions) on the multiplication of the viruses involved. This study showed that SJNNV replication is negatively affected in coexistence, whereas RGNNV replication is favoured. In addition, several challenges have been conducted to evaluate the susceptible to RGNNV replication is favoured. In addition, several challenges have been conducted to evaluate the susceptibility of juvenile European seabass to different VNNV isolates, showing that this fish species is susceptible to RGNNV, SJNNV and a reassortant strain (RG1/SJ2, isolated from Senegalese sole, Solea senegalensis); however, mortality and clinical signs were not observed after the SJNNV challenge. One of the RGNVV isolates tested in this study did not cause mortality in European seabass after intramuscular injection, leading us to perform a comparative study between the virulent and avirulent RGNNV isolates. Thus, the predictive secondary structure of the CP displays differences that could be responsible for the lack of virulence. Finally, a comprenhensive study of RGNNV distribution in nervous and non-nervous tissues of juvenile European seabass was conducted in spleen, kidney and caudal fin of European seabass for the first time. In this study, a qPCR to specifically detect the RGNNV RNA1 segment has been developed. This PCR allows the viral detection at early stages of infection, before the mortality onset, and permits the exact characterización of the viral genotype.