Zebrafish Reveals Genotype-Specific Differences in Betanodavirus Infection and Host Responses

Fish viral encephalopathy and retinopathy (VER) is a significant infectious disease caused by the nervous necrosis virus (NNV), which affects cultured and wild species. Four NNV genotypes with different virulence in susceptible species have been identified: red-spotted grouper NNV (RGNNV), striped jack NNV (SJNNV), barfin flounder NNV (BFNNV), and tiger puffer NNV (TPNNV), in addition of reassortment between RGNNV and SJNNV. In light of this threat, zebrafish emerged as a promising model to further characterize these pathogens and identify the cellular and molecular mechanisms responsible for susceptibility/resistance phenotypes. Thus, the aim of the present study was to compare the susceptibility of zebrafish to RGNNV, SJNNV, and RGNNV/SJNNV infection and to characterize the pathogenesis of these isolates in terms of virulence and early innate host immune response. Disease symptoms were recorded in all groups of intracranially infected larvae, and quantification of viral genome evidenced that the three isolates replicate in zebrafish larvae. The highest mortality and proportion of larvae showing disease symptoms were registered in the RGNNV group, where the highest viral replication level was also recorded. Thus, RGNNV seems to be the most virulent isolate for zebrafish larvae. Interestingly, transcriptional patterns of IFN I response and apoptosis genes were similar for RGNNV- and RGNNV/SJNNV-inoculated larvae, being interferon regulatory factor 3, irf3, and receptor transporter protein 3, rtp3, the most induced genes, whereas a much lower gene upregulation was recorded in SJNNV-inoculated larvae. RGNNV infection also induced stronger inflammatory responses, including neutrophil recruitment. RGNNV infection of zebrafish larvae by immersion lead to a significant increase of viral genome; however, the infection was quickly controlled. Infection of juvenile zebrafish with RGNNV by intramuscular injection lead to virus migration to the central nervous system; however, no viral RNA replication was recorded. The potential of zebrafish to study NNV-host interplay prompts further studies into the underlying mechanisms of these observations, which could contribute to design effective strategies to protect farmed fish susceptible to NNV infections.