The Study On The Construction Of Infectious Clone Of Zika Virus

Background and Objects Zika virus（ZIKV）is a member of the Flavivirus genus within the Flaviviradae family,which is an important new mosquito-borne virus.Zika virus infection is closely related to severe neurological diseases such as Guillain-Barre syndrome in adults and microcephaly in neonates.Since the large-scale epidemic in 2013-2014,it has caused a heavy burden on global public health.Unfortunately,the pathogenic mechanism of Zika virus is currently not fully understood,and the targeted vaccines and antiviral drugs have not appeared.The reverse genetic system is an important method for studying RNA viruses.It can modify RNA viruses at the gene level,Providing a powerful tool for studying the life behavior,pathogenic mechanism,and virus-host cell interaction mechanisms of viruses.Therefore,constructing infectious clone of Zika virus is an indispensable experimental technique to understand the biology of Zika virus and research related treatment methods.Methods1.Construction of the infectious clone vector of Zika virusThe infectious clone of Zika virus was constructed using vitro transcription method by T7 promoter.The entire genome of Zika virus ZKC2 strain（GenBank accession number KX253996）was divided into four PCR fragments for amplification,there were over 200bp overlapping sequences between the two adjacent fragments.The four fragments cover the entire Zika virus genome,in which the T7 promoter is introduced into the upstream primer of the first fragment,the fragment④is ligated to HDVr by overlapping PCR.Subsequently,the T7 promoter,the full length genome of Zika ZKC2 strain（GenBank accession number KX253996）,HDVr（Hepatitis D ribozyme）was ligated into the PWSK29 low copy vector by In-Fusion homologous recombination.Lastly,they were transformated into XL 10 super competent cells.2.Rescue of the recovery virus Q5 superfidelity enzyme was used to amplify the DNA templates（including T7 promoter,The entire genome of Zika virus ZKC2 strain,HDVr）for vitro transcription,transcription RNA is obtained in vitro transcription and transfected into vero cells by electroporation to observe the CPE of the susceptible cells.3.Testing the virulence of the recovery virus in vivo and vitro experiments.We used qPCR and virus plague formation assay to evaluate the supernatant infectivity titers and the replication kinetics.Immunofluorescence assay was used to verify the expression of the protein of recovery virus,The inoculum was administered to C57BL/6 and Kunming 1 day old neonatal mice by multipoint subcutaneous injection.All of the vivo and vitro experiments is to test the virulence and the stability of the recovery virus.Result1.After homologous recombination,T7 promoter,the entire genome of Zika virus ZKC2 strain,HDVr successfully ligated into PWSK29 low copy vector.Compared with ZKC2 strain,the insertion sequence has 6 mutation sites（3149,3730,4015,5633,5744,7007）,the first three site mutations result in amino acid changes,and the last three mutations are synonymous mutations.2.NS2A 55-74 extramembrane protein simulation results have showed that D62G mutation may lead to a-helix extension and β-turn formation,making the structure more stable.After 3730 site-directed mutagenesis experiments and in vitro transcription,the full-length RNA of the virus was successfully transcribed.After electroporated RNA to Vero cells,the cells produced significant CPE.No mutations occurred in the plasmid and the recovery virus for five consecutive generations.3.The titer of the recovery virus in the supernatant of Vero cells was 1010 copies/ml measured by qPCR,and the plaque assay showed that the titer of the recovery virus was 107 PFU/ml.Growth kinetics curves indicate that the virulence of the recovery virus is similar to that of the parental virus in vitro experiments4.After administered the recovery virus to C57BL/6 and Kunming 1 day old neonatal mice,both the neonatal mice showed lower limbs weakness,slow movements,hump,tremor,abnormal gait,weight loss and other symptoms,and then all died,with a mortality rate of 100%.The brain of the dying suckling rat had obvious histopathological changes,and the virus titer in the brain reached 1010 copies/g.The course of the recovery virus on the two neonatal mice is similar to that of the parental virus.Conclusion After homologous recombination,the infectious clone of Zika virus was successfully constructed and genetically stable.In vitro and in vivo experiments showed that the virulence of the recovery virus was similar to that of the parental virus.The research in this article provides an effective tool for exploring the life behavior and drug research of Zika virus.