Construction of red-spotted grouper nervous necrosis virus (RGNNV) nonstructural protein B1/B2 mutant strains and dynamics during infection

Nervous necrosis virus (NNV) is a neurotropic pathogen that causes serious losses in aquaculture worldwide. B1 and B2 proteins, nonstructural proteins of NNV, play vital roles in viral function. In this study, a plasmid-based reverse genetics system was established for red-spotted grouper nervous necrosis virus (RGNNV), and codon mutations on B1 and B2 proteins were introduced. One recombinant virus (recomb) and five mutant viruses (B1QM, B2QM, BoQM, B1JM, and B2JM) were successfully rescued. Mature virions of approximately 30 nm in diameter within intracellular vacuoles were observed under transmission electron microscopy. The rescue results were further confirmed by PCR and western blotting. Compared with wild-type virus, all recombinant viruses displayed similar infectivity in vitro and in vivo. B2QM induced enhanced vacuolation-associated cytopathic effects at 5 days post-infection (dpi). Viral genome copy number peaked at 5 dpi in B1QM, whereas other recombinant viruses peaked at 3 dpi. RNA-dependent RNA polymerase expression showed significant differences only at 5 dpi, with 0.29-fold change (recomb) and 0.19-fold change (BoQM). Capsid protein expression level decreased in the B1QM group by 0.35-fold and increased by 2.29-fold in the B2QM group at 2 dpi. In vivo challenge showed that all groups except B2QM group reached 100% mortality within 30-hour post infection (hpi), with peak mortality at 24 hpi. In the B2QM group, viral genome copy number remained relatively stable in brain and heart tissues but declined over time in eye tissue. Sequencing analyzes and immunofluorescence assay revealed that all mutated codons had been reverted to the original sequence, and B1 and B2 protein expression occurred in all mutant strains. These may suggest that the comparable infectivity between mutant and wild-type viruses is related to the reversion of codon mutations in nonstructural proteins, and revertant sequences that emerged during passaging probably account for this similarity. These findings provide new insights into NNV pathogenesis, but whether these mutations remain stable during passaging requires further investigation.