SSN-1

Correlation between NNV Infectivity and Cytopathic Effect (CPE) Appearance Ratio

Nervous necrosis virus (NNV), a simple spherical RNA virus in the family Nodaviridae, is pathogenic to many fish species. Lee et al. studied the adsorption ratio and infection efficiency of purified NNV on striped snakehead cells (SSN-1) using a 96-well titration system.

The purified NNV suspension (4.0 TCID₅₀/100 µl) was serially diluted twice, and infectivity titration was performed using the 96-well system (Fig. 1). The infectivity titer of the NNV suspension, calculated using a modified Kärber's formula based on the CPE appearance ratio at each dilution, was 4.56 TCID₅₀/100 µl (average of two replicates: 4.46 and 4.66 TCID₅₀/100 µl) (Fig. 1A). The CPE appearance ratio, which is the proportion of CPE-positive wells among all inoculated wells at each dilution, indicates the probability of NNV infection. Plotting these data on a semilogarithmic graph with CPE appearance ratio (%) on the Y axis and NNV infectivity (log10 TCID₅₀/100 µl) on the X axis (Fig. 1B) yielded a regression line (y = 55.7x + 50.6, R2 = 0.9930), showing a close relationship between CPE appearance ratio and NNV infectivity titer. This line indicated that the CPE appearance ratio decreased by 17% each time the NNV infectivity dose was halved. Subsequent NNV infectivity titers were calculated using this regression line with the 96-well system.

Fig. 1. Correlation of NNV infectivity with CPE appearance ratio demonstrated by titration using the 96-well system (Lee H S, Gye H J, et al., 2023).

Cytopathic Effects in SSN-1 Cells Inoculated with the Filtrate of Naturally Infected Murry Cod Fry Tissue Samples

In December 2019, a severe mass mortality event occurred among cultured Murray cod fry in Foshan city, China, with a cumulative mortality rate reaching 45% within 15 days. The affected fish exhibited abnormal swimming behavior, loss of appetite, and dark body coloration. To explore the cytopathic effect of betanodavirus, SSN-1 cells was inoculated with the tissue filtrate of diseased Murry cod fry. Typical CPE characterized by vacuole formation in cytoplasmic was observed in SSN-1 cells after 3 days of inoculation with the filtrate of tissue samples prepared from moribund fish and the monolayer degenerated utterly. Cells floated in the medium eventually 6 to 7 days post-inoculation (Fig. 2). Following 2 to 4 passages of supernatants to new cultures, the same pattern of CPE was also observed. The virus titre of the filtered tissue homogenates of moribund fish was 10^7.8 TCID₅₀/ml. In the cytoplasm of SSN-1 cells showing CPE, intracellular viruses occurred as free single particles or aggregates (Fig. 3) within cytoplasmic inclusions. The viruses were non-enveloped, spherical and measured 22 to 28 nm in diameter.

Fig. 2. Cytopathic effects in SSN-1 cells inoculated with the filtrate of naturally infected Murry cod fry tissue samples (Wang Y, Xu L, et al., 2021).

Fig. 3. Electron micrograph showing viral aggregates (arrows) within inclusion body of SSN-1 cells infected by the filtrate of naturally infected Murry cod fry tissue samples (Wang Y, Xu L, et al., 2021).