Construction And Characterization Of Japanese Encephalitis Viruses With Mutations In The VR Of 3’ Untranslated Region

Japanese encephalitis virus(JEV)is an arthropod-borne virus that can infect humans when bitten by infected mosquitoes,causing permanent neurological and/or psychiatric sequelae and even death.JEV is an enveloped virus belonging to the genus Flavivirus of the family Flaviviridae.Its genome consists of a single stranded plus RNA with a length of about 11KB.The two ends are 5’untranslated region(UTR)and 3’untranslated region(UTR),with an open reading frame in the middle.ORF encodes 3structural proteins(C,pr M and E)and 7 non-structural proteins(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5).The sequence of the starting region of the 3’UTR of the flavivirus genome is low conserved,which is called the variable region(VR).On the one hand,changes in the VR region may affect viral RNA folding,thus interfering with viral replication.On the other hand,the low conserved VR region can accommodate a certain length of exogenous sequences,thus endowing the virus with new biological functions.Therefore,exploring the relationship between the structure and function of the VR region of JEV will deepen our understanding of the replication mechanism of JEV and provide new ideas for the design and transformation of the virus vector based on JEV.In this study,we first modified the 5’end of the 3’UTR of JEV by molecular biological manipulation on the basis of the previously constructed infectious clone of JEV,and then obtained a new recombinant clone which could facilitate the integration of the exogenous sequence into the VR domain.On this basis,we attempted to insert the poly U/UC motives of the hepatitis C virus genome which can stimulate the innate immune response into the VR region of JEV,and obtained a series of recombinant viruses with VR deletion or insertion,and evaluated the basic biological properties of the recombinant viruses.1.Construction and identification of a full-length c DNA clone of recombinant Japanese encephalitis virus genomeUsing the reverse genetics technology in our laboratory,based on the previously constructed full-length infectious cloning plasmid p JE70 of Japanese encephalitis virus by our laboratory,the linear enzyme restriction site Xho I of p JE70 and the single enzyme restriction site Xba1,upstream of the termination codon were selected to design PCR primers to amplify the fragment.After restriction enzyme digestion,the fusion fragment was ligated and transformed into competent cells in vitro,and the plasmid was extracted to obtain p JE70＿Kpn1.The constructed plasmid p JE70＿Kpn1 was identified preliminarily.By reference to the above methods,plasmid p JE70＿Kpn1 was used as a template to introduce poly U/UC at 20nt,40nt,60nt,80nt and 100nt after the termination codon in the 3’untranslated region of the virus genome.A full-length clone of Japanese encephalitis virus carrying poly U/UC at different locations in the 3’untranslated region of the genome was constructed and obtained.We transfected the transcriptome RNA of the mutant virus plasmid into BHK-21 cells to save and obtain the recovered virus.After sequencing identification,it was found that the polymeric U/UC motif was completely lost,but the VR region was partially deleted or inserted,and the mutant virus carrying the mutation could also be passed stably.2.Identification of biological properties of Japanese encephalitis virus with VR mutation in 3’untranslated regionIn order to preliminarily explore the effect of insertion/deletion mutation of the 3’untranslated VR region sequence on the biological characteristics of the virus.Based on the numbers of bases mutated in VR region,the Japanese encephalitis mutant viruses were named as DEL12,DEL10 and INS8 respectively.Through in vitro cell infection experiment and mouse experiment,we identified the difference of biological characteristics between the mutant virus and its parent strain E70-Kpn1,such as the morphology of the lesions,the proliferation characteristics in different cells,and the analysis of neurovirulence and neuroinvasion in mice.The results showed that the size and diameter of plaque formed by the mutant virus on BHK-21 cells were similar to that of the parent strain E70-Kpn1,but there was no significant difference.In BHK-21 and SH-SY5Y cell lines,the replication level of the mutant virus was not significantly different from that of E70-Kpn1.In the mouse experiment,there was no significant difference in neurovirulence between the mutant virus and its parent strain at the doses of50PFU and 5 PFU,and no significant difference in neurovirulence between the mutant virus and its parent strain at the doses of 10~6PFU and 5×10~6PFU.The evaluation of neurovirulence and neuroinvasiveness needs further investigation.In conclusion,we successfully constructed a full-length infectious clone of Japanese encephalitis virus with a single enzyme cut-site of Kpn1,and rescued the corresponding recombinant Japanese encephalitis virus,which showed no significant difference in vitro proliferation characteristics and mouse neurovirulence from the parent strain.On this basis,through the insert U/UC polymer components into the method of VR area,save the VR series loss and insertion mutant viruses,a preliminary analysis of the biological properties of the results showed that the series of the mutant viruses plaques in morphology,cell replication capacity and parental strains had no significant difference,but the virulence and nerve invasion force.In this study,the constructed recombinant clone with single enzyme restriction site provides a research tool for the design and modification of VR domain.The specific mechanism of the neuropathogenicity induced by the deletion and insertion of VR domain is worthy of further study,which is expected to provide new scientific data for the understanding of the structure and function of VR.