The Effect Of Envelope Protein 279 On The Neurovirulence Of Japanese Encephalitis Virus

Objective:The infectious cloning of envelope protein K279M mutant from JEV wild strain SA14 was constructed to save the virus.The effect of279 amino acid mutation of JEV envelope protein on the neurovirulence of the virus was investigated by animal experiments.Methods:Using JEV envelope protein cDNA as template,a full-length cDNA plasmid pACNR-JEV SA14?K279M?containing 279 amino acids of rJEV SA14 envelope protein mutated from lysine?K?to methionine?M?was constructed by overlapping extended PCR technology and molecular cloning technology.The full-length cDNA plasmid pACNR-JEV SA14?K279M?was transcribed by using itself as a template in vitro to obtain RNA,which was transfected into BHK21 cells for packaging to recover the virus JEV SA14?K279M?.The rescued virus was identified by plaque assay,indirect immunofluorescence assay and sequencing.The plaque size and growth characteristics of the recovered virus JEV SA14?K279M?,rJEV SA14,the mutant JEV SA14-14-2?M279K?and JEV vaccine strain?rJEV SA14-14-2?were compared,as well as the differences in neurovirulence between Kunming mice and 7-day-old lactating mice.Results:The infectious clone was successfully constructed by restriction enzyme digestion and sequencing.The results of virus sequencing explained that there were no other mutations in the restored virus JEV SA14?K279M?except for the mutation of A to T introduced artificially at the base of 1813?resulting in K279M mutation?.Indirect immunofluorescence assay confirmed that the recovered virus JEV SA14?K279M?infected BHK21 cells could be recognized by anti-JEV envelope protein antibodies.Viral plaque assay demonstrated that JEV SA14?K279M?had the largest plaque diameter,followed by rJEV SA14,rJEV SA14-14-2 and JEV SA14-14-2?M279K?.The virus growth curve showed that the viral titre of JEV SA14?K279M?reached its peak(8.03 log₁₀PFU/ml)after 48 hours of infection when multipicity of infections was 0.01,while that of rJEV SA14,rJEV SA14-14-2,JEV SA14-14-2?M279K?reached its peak in 60 hours.Neurovirulence studies found that the neurovirulence?LD50?of rJEV SA14 and JEV SA14?K279M?to Kunming mice were 0.05 PFU/0.03ml and 0.21 PFU/0.03ml,respectively.The neurovirulence?LD50?of rJEV SA14-14-2 and JEV SA14-14-2?M279K?to 7-day-old suckling mice were 0.52 PFU/0.02ml,0.19PFU/0.02ml,respectively.Conlusion:The infectious clone containing K279M mutation virus was successfully constructed and the rescued virus JEV SA14?K279M?was obtained.Mutations at envelope protein 279 site can significantly affect the plaque size of the virus,and have certain effects on the growth and neurovirulence of the virus.It was also found that the size of plaque did not necessarily reflect the virulence of the virus,and there was a big deviation between in vitro and in vivo experiments.