Genomic Differences Between Nervous Necrosis Virus (NNV) Reassortants Isolated From Wild and Farmed Fish: Implications for Viral Fitness, Temperature Adaptation and Virulence.

Nervous necrosis virus (NNV), one of the most widespread fish pathogens, is classified into four genotypes: Barfin flounder-, Redspotted grouper-, Striped Jack- and Tiger puffer NNV (BFNNV, RGNNV, SJNNV and TPNNV, respectively), which show different thermotolerance and geographical distribution. Reassortant RGNNV/SJNNV strains are detected in Southern Europe, associated to disease outbreaks in Senegalese sole and gilthead seabream larvae or early juveniles, with water temperatures around 22-23°C. These strains contain amino acid changes in the capsid and polymerase protein when compared with the reference strains of each genotype. We have assessed the effect of temperature on the replicative fitness of four reassortants obtained from wild pilchard and mackerel and their pathogenic potential against sole and turbot. In vitro replication assays showed an improved replication of the mackerel isolate at 15°C while it was delayed at 20 and 25°C. Substitutions in the viral polymerase, particularly Arg237, and differences in the non-coding regions (NCR) might account for this adaptation to replicate at a suboptimal temperature for reassortants. In addition, in the in vivo assays at different temperatures, the mackerel isolate caused the lowest mortality and showed limited replication in sole brain tissue. However, in the experimental infection in turbot at 15°C, it displayed an exponential replication, although it did not cause mortality. The analysis of the capsid protein (Cp) of this isolate points to position 237 as a putative host specificity determinant that might favour the interaction with turbot cell receptors. In conclusion, substitutions observed in the mackerel strain suggest an adaptation to replicate at low temperature, which would enable it to spread to the cold waters of the North Atlantic Ocean. In addition, it also highlights the potential risks associated with the introduction of NNV strains from the wild into fish farms or new areas.