Establishment and application of a rapid assay for inactivated infectious spleen and kidney necrosis virus based on viral mRNA

In order to establish a rapid assay for inactivated infectious spleen and kidney necrosis virus (ISKNV), ISKNV ORF099 was selected as a promising target gene based on the results of transcriptomic profiles of Mandarin fish brain cells (CPB) infected with ISKNV and qRT-PCR. Standard curve of C_T value and plasmid copy number was drawn with standard plasmid pMD099, and the linear equation was: C_T= –3.42lgx+39.455, and the minimum detection limit was 3 copies/μL. Results of repeated trials showed variation coefficients of intra-and-inter groups were less than 2%, indicating the method has high sensitivity and repeatability. The ISKNV suspension was diluted into 10⁰–10³ copies/mL, and each T25 cell culture flask was inoculated with 1 mL virus diluted solution. Thereafter, total RNAs were extracted at 7, 9 and 11 d post inoculation. The mRNA of ORF099 gene was detected using routine qRT-PCR method. The results showed that the mRNAs of ISKNV could be detected from cells inoculated with one copy ISKNV virus at 7 days post inoculation. CPB cells have been inoculated with three batches of ISKNV inactivated by different concentrations (0.05%, 0.1%, 0.2%) of formaldehyde for 9 days. Results showed that ISKNV ORF099 mRNA was only detected from cells inoculated with 0.05% and 0.1% formaldehyde inactivated ISKNV at 7 days post inoculation by qRT-PCR. However, after blind passaging three generations, cells inoculated with ISKNV inactivated by 0.1% final concentration of formaldehyde did not exhibit CPE. The inactivated ISKNV did not cause clinical symptoms and death of fish, indicating this rapid virus inactivation test has higher sensitivity than cell blind passage trial and the fish safety test. The established method will be important for improving the ISKNV inactivated vaccine production.