乙型脑炎疫苗株SA14-14-2包膜蛋白279位氨基酸回复突变对其毒力的影响

Objective: To investigate the effects on virulence of M279K reversion in envelope protein of Japanese encephalitis virus (JEV) vaccine SA14-14-2. Methods: The full-length cDNA harbouring M279K mutation of JEV SA14-14-2 was cloned into the low- copy plasmid pACNR with overlapping PCR and gene recombination technologies, the resulting plasmids were then identified with restriction enzyme digestion and sequence analysis. RNA was transcribed from the plasmid template in vitro and electroporated into BHK21 cells, and the culture supernatant was collected and recovery virus rJEV (M279K) was detected with plaque assay and sequence analysis. The form and size of the plaques and virulence of the recovery viruses were compared with that of the parental strain SA14-14-2. Results: Restriction enzyme digestion and sequence analysis showed that the plasmid pACNR-JEV (M279K) containing M279K reversion was constructed successfully and sequence analysis showed that there was no mutant detected except an engineered mutation at nucleotide 1813 (T-,A) in the envelope protein of rJEV (M279K). The plaque size of rJEV (M279K) in BHK21 was smaller than that of vaccine strain, but the neurovirulence phenotype in Kun-miug mouse has no difference in rJEV (M279K) and vaccine virus. Conclusion: M279K reversion affects the size of plaque of SA14-14-2 but not virulence.