Infectivity titers and aggregation states of intracellular and extracellular nervous necrosis virus in cell lines with cytolytic and persistent infections

Nervous necrosis virus (NNV) in the genus Betanodavirus (Nodaviridae) is highly lethal to a wide range of fish species. Although striped snakehead (SSN-1) cell lines have been widely used for culturing NNV, cell lines persistently infected (PI) with NNV have only recently been established. This study investigated the infectivity titers of intracellular and extracellular NNV and the associated aggregation states. The intracellular NNV infectious doses were higher than those of extracellular NNV, irrespective of the cell lines. In SSN-1 cells, the intracellular-to-extracellular-NNV ratio was approximately 50–60-fold on days 1 and 2 after NNV infection, although it decreased following the onset of the cytopathic effect (CPE), reaching 3.5-fold on day 4. In the PI-cell lines, both the intracellular and extracellular NNV were in a nearly monomeric state. While the extracellular NNV were in a monomeric state in the SSN-1 cells, more than 92 % of the intracellular virus were in an aggregated state. When the NNV accumulated intracellularly at a median tissue culture infectious dose (TCID₅₀)/cell of 10⁴ to 10^4.5, SSN-1 cells appeared to exhibit CPE and eventually died. We believe that the aggregates of intracellularly accumulated NNV particles may be related to the cellular CPE onset and/or cell death.