Isolation Of Japanese Encephalitis Virus And Full-length Cloning And Construction Of Attenuated Japanese Encephalitis Vaccine Strain

Objectives:Japanese encephalitis(JE)is a common mosquito-borne zoonotic disease caused by Japanese encephalitis virus(JEV).The case fatality rate is high and sequelae are serious.In recent years,the epidemic areas of JEV are spreading,posing a serious threat to human health.At present,the virulence determinants of JEV are not fully understood,so it is necessary to analyze the genome and protein of JEV.In this study,a strain of JEV was isolated from the serum and cerebrospinal fluid samples of a patient with severe JEV infection.The whole genome sequence of the virus was analyzed,and the protein structure and function,as well as the physical and chemical properties and molecular functions of the protein were predicted.In order to further identify whether the predicted virulence related sites will really affect the virulence of JE virus,we constructed a full-length infective clone of attenuated JE virus vaccine strain SA14-14-2,so as to facilitate constructing the site-specific mutation clone based on full-length JE vaccine strain,and observing the replication ability and virulence difference between the mutant and the vaccine strain,and then identify virulence sites.Methods:The serum and cerebrospinal fluid of a patient with severe JE were collected in Xishuangbanna,Yunnan province at the beginning of the study,the viral RNA were then extracted,followed by RT-PCR and sequenceing.The whole gene sequence of the JEV is obtained by splicing obtained its complete sequence.Through the DNAMAN and MEGA6.0 software for the whole genome nucleotide and amino acid sequence analysis,and through ProtParam,PSIPRED and Phyre2 and SWISS-MODEL software of NS3 and NS4A for the structure and function of the protein structure prediction.,two possible virulence related sites were observed.Based on JEV vaccine strain SA14-14-2.as the completed viral gene were amplification and connection to the pcr2.1 plasmid.obtained by the method of in vitro transcription virus RNA transcription,transcriptional body will save RNA transfection to C6/36 cells,make the virus in cell packing into a complete,has the activity of virus particles,and then the cytopathic effect was observed,and evaluate their protein in the cell by indirect fluorescent immune power of expression.This laid a foundation for the subsequent evaluation of virulence related sitesConclusions:18 PCR products were obtained using the extracted viral RNA as the template,and the full-length gene sequence of JEV was obtained by sequencing and splicing of the amplified fragments.By comparing the sequences with other 43 strains in NCBI database.the full-length viral genome and protein sequence were close to isolates of pig in 2008 and 2016,China,respectively,which belonged to JEV G?group.Through the comparison of similar strains,vaccine strains(SA14-14-2)and virulent strains,two nucleic acid site in NS3 and NS4A were worth noting.Further prediction analysis showed that these two sites did affect the function and structure of the protein,which may be potential virulence related sites.In addition,a full-length infectious clone of JEV vaccine strain SA14-14-2 was constructed by the method of homologous recombination.The attenuated strain was divided into eight sides in turn connected to pcr2.1 plasmid.Then the result showed that the transcriptional body RNA can be packaged into a complete virus particles in the cells,and cause cells to CPE effect,IFA showed rescue virus can express the protein in the cell cytoplasm,sequencing results showed rescue virus has good genetic stability,The above results preliminarily demonstrated the availability of full-length infectious clonal plasmids in attenuated je vaccine strains.