Characterization of betanodavirus quasispecies influences on the subcellular localization and expression of tumor necrosis factor (TNF)

The aim of this study was to investigate the influence of variant coat proteins (CPs) from different quasispecies of betanodavirus on diverse aspects of nodavirus-induced pathogenesis. It is known that variant CPs can acquire either nuclear or cytoplasmic localization, depending on the nodavirus CP genotype, and this variation may arise during viral replication and influence the regulation of host and viral gene transcription. To investigate the role of these variant CPs in pathogenesis, six variant CP expression plasmids were constructed, each containing different quasispecies CP variants from nodavirus genotype red spotted grouper nervous necrosis virus (RGNNV). The CP expression plasmids were transiently transfected into grouper GF-1 cells. At different times, the cell cycle and cell proliferation were assayed using flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. The proportion of G₂/M-phase GF-1 cells transfected with CP expression plasmids was higher than that of cells transfected with the blank plasmid, especially in regards to quasispecies 2 (QS2). The proliferation ratio of cells transfected with the CP expression plasmids was significantly higher than that of cells transfected with the blank plasmid, with the exception of QS6. We also found that the different quasispecies CPs downregulated the promoter activity of the tumor necrosis factor (TNF) gene to different degrees. In addition, this is the first report showing the betanodavirus CP derived from different quasispecies of RGNNV provide evidence of a chronically nodavirus-infected grouper. Overall, this study represents the first comprehensive analysis of variant CPs from grouper with persistent nodavirus infections and their effects on different aspects of pathogenesis.