Zika Virus Mutants Attenuated Strains Constructed And The Preliminary Screening Selected

[Objective]Firstly,to compare the differences of nucleotide sequence homology?capsid protein?pre-membrane protein coding sequence and amino acid sequence between Asian and African genomes.Taking the virulence site of dengue virus to the reference of zika virus,then explore the effect of replication of dengue virus type ? in C6/36 and Vero cells on its virulence to analyze the possible virulence sites.Based on the correlation between virulence and site mutation,the mutant strain of Zika virus was constructed by reverse genetics technology and screening of ZIKV attenuated strain preliminary.[Methods]The reference viral sequences of Asian and African genotypes of ZIKV were search and collected from the GenBank sequence database.The comparative analysis of assembled nucleotide sequences and translated amino acid sequences were performed by BioEdit software.Nucleotide and amino acid mutation were collected between different types.Two dengue virus type ? strains were inoculated and cultured on C6/36 cells.The virulence of the two strains was stable and then inoculated into Vero cells for continuous passage.The number of copies of different generations of dengue virus was detected by using dengue type ? standard plasmid.The full-length sequence was obtained by RCR amplification and compared with the original virus sequence;The full-length cloned plasmid of ZIKV was analyzed to determine the feasible virulence sites.The mutant plasmid was obtained by mutating the virulence sites on the full-length cloned plasmid of ZIKV by site-directed mutagenesis.The transformed skeleton was transcribed in vitro and transfected into Vero cell to rescue the virus.The rescued virus was identified by RT-PCR,immunofluorescence,analysis of virus proliferation characteristics and plaque-phagocytosis.The biological characteristics of suckling mice were identified by inoculation experiments.[Results]The Nucleotide similarity ranged from 88.00%to 89.00%between Asian and African ZIKV genotypes.Comparing to African genetype,a total of 19 amino acid mutation in protein C and 50 amino acid mutation in protein prM were discovered in Asian genetype,and all these 69 mutations were nonsynonymous mutation.A strain of dengue virus type ?(DV3-1)was found to be more virulent on C6/36(P17-C6/36)and less virulent on Vero cells(P10-Vero).Total 16 base mutations were found among them,leading to 12 amino acid nonsynonymous mutations.Among them,P0 and P10-Vero were the same at site 5826.6251 and 7907,while P17-C6/36 was different from them,and led to amino acid substitution at site 1942,2084 and 2636.Virulence site analysis showed that one attenuation site mutation and four mutagenesis sites could be carried out,and the mutant virus Pzika-FL-G53D,Pzika-FL-T47SS,Pzika-FL-S64T,Pzika-FL-V255A and Pzika-FL-S260T could be successfully constructed and rescued.Preliminary screening showed that Pzikv-FL-G53D had a certain attenuation effect at the cellular level compared with Pzikv-FL.The two-day-old suckling mice Zhaika card injection experiment showed that Pzikv-FL-G53D had a certain attenuation effect.It shows that Pzikv-FL-G53D has lower neurotoxicity than Pzikv-FL.[Conclusion]Mutations in unit sites may affect the pathogenicity and infectivity of the virus strains.The virulence of dengue virus is affected by the replacement of passaged cells,and the recovery mutation may occur after repeated passages and replacement of cells.This study provides a reference for the study of the biological and pathogenicity differences of ZIKV and provides a basis for the follow?up study of ZIKV attenuation strategy.