The Effect On Neurovirulence In Mice Of Mutant Envelope Protein Y158H Or V169I Of Japanese Encephalitis/Zika Chimeric Virus

Objective:To explore the effect of the mutation of Amino Acids Y158H or V1691 of Zika virus Envelope protein(E)in Japanese Encephalitis/Zika chimeric virus(JE/ZIKV)on the neurovirulence in mice,and to explore the key amino acid sites controlling the neurovirulence of JE/ZIKV chimeric virus in mouse,and to provide the theoretical basis for the safety evaluation of Zika virus vaccine candidate strains.Methods:Regarding the full-length CDNA plasmid of chimeric virus JE/ZIKV(MR766)as a template,the full-length cDNA of the chimeric virus was obtained by overlap extension PCR and gene recombination.The chimeric virus RNA was obtained by transcribing in vitro and then electro-transfected into BHK21 cells to rescue the mutated virus JE/ZIKV(Y158H)and JE/ZIKV(V1691).Plaque assay,indirect immunofluorescence assay,gene sequencing and growth curve experiment were used to identify the virus,and 3-week-old Kunming mice were used to evaluate the neurovirulence of the mutant virus in mice.Results:Sequencing and enzyme digestion confirmed the successful construction of full-length cDNA of JE/ZIKV(Y158H)and JE/ZIKV(V1691)viruses.Plaque assay confirmed the presence of virus in the supernatant of cell culture medium transfected with RNA.Indirect immunofluorescence assay showed that the two rescued mutant viruses could be recognized by anti-JEV-NS1 antibody,but not by anti-JEV-E antibody.Virus sequencing results confirmed the successful rescue of chimeric viruses and the successful introduction of Y158H or V169I mutation sites.The plaque test showed that the diameter of JE/ZIKV plaque was 1.7±0.2mm and that of JE/ZIKV(Y158H)plaque was 1.6±0.1mm,and the difference between the two was not statistically significant(t=2.46,P=0.09>0.05).JE/ZIKV plaque diameter was 1.6±0.4mm,JE/ZIKV(V1691)plaque diameter was 1.4±0.4mm,the difference was not statistically significant(t=0.99,P=0.35>0.05).The growth curve showed that the proliferation rate of JE/ZIKV(Y158H)and JE/ZIKV(V169I)peaked at 60h,and the proliferation rate of JE/ZIKV(Y158H)was similar to that of JE/ZIKV(V169I),but the proliferation efficiency of JE/ZIKV(Y158H)was slightly lower than that of the The original virus JE/ZIKV(MR766)during the whole process of virus proliferation.JE/ZIKV(V169I)proliferated as fast as the parent virus in the first 60h,and then decreased more than that of the original virus.The results of animal experiments showed that the brain neurovirulence of JE/ZIKV(Y158H)mice was 1964.33 PFU/mL,the brain neurovirulence of JE/ZIKV(V169I)mice was 621.33 PFU/mL,and the brain neurovirulence of original virus JE/ZIKV(MR766)mice was 73.67 PFU/mL.Conclusion:Mutation of Zika virus E protein Y158H or V169I can reduce the neuro virulence in the brain of mice injected JE/ZIKV(MR766).E158 and E169 are two important amino acids that control the neurovirulence in the brain of mice injected JE/ZIKV(MR766),and E158 plays a more significant role.