In vitro infection efficiency of nervous necrosis virus alters depending on amount of viral particles adsorbed onto cells

Nervous necrosis virus (NNV) in the family Nodaviridae is one of the simplest spherical RNA viruses and is pathogenic to many fish species. We investigated the effect of purified NNV on striped snakehead cells (SSN-1) in terms of adsorption ratio and infection efficiency using the 96-well titration system. The proportion of cytopathic effect (CPE)-positive wells among total number of wells inoculated with the virus (CPE appearance ratio) reduced by 17% each time the NNV infectivity dose was halved (y = 55.7x + 50.6). Thus, subtle differences in NNV infectivity could be accurately detected using this system. Experiments performed to observe alteration of CPE appearance ratio with changing viral doses and adsorption times showed that NNV particles introduced into microplate wells as suspensions in ≤ 100 µl inoculum were adsorbed almost completely onto cells seeded on the wells within 4 days of incubation. Density profile analysis of NNV coat proteins revealed that the NNV suspension at 1 50% tissue culture infectious dose (TCID(50)) contained 60 particles. Infection efficiency/NNV peaked at 20 particles (1.20%/particle) and then declined gradually with increasing NNV doses. Therefore, in vitro infection efficiency of NNV may alter depending on the quantity of viral particles adsorbed onto cells.