Construction Of ZIKV GZ01 Infectious Clone And Preliminary Study On The Biological Function Of Cyclization Sequence

Objective: Zika virus(ZIKV)belongs to the Flavivirus genus within the Family of Flaviviridae,together with many other pathogens with global impact,such as dengue virus(DENV),West Nile virus(WNV),Japanese encephalitis virus(JEV),and yellow fever virus(YFV).Since 2013,the Zika virus continued in the global raging and outbreak,to bring great harm to human health.Despite the timely and cooperative efforts of the scientific and public health community in the past years,the mechanisms of ZIKV emergence and pathogenicity remains largely unknown and no vaccine or specific antiviral is commercially available.The development of infectious clones of Zika virus has played a crucial role in resolving the virus crisis.The ZIKV reverse genetics system provides a reliable tool for studying the function of ZIKV protein and molecular pathogenesis.Method: The reverse genetics system represents one of the most powerful tools to study viral replication and pathogenicity,and have now widely utilized in vaccine design and antiviral screening.In this work,we successfully constructed a full-length infectious clone of Zika virus Guangzhou 01 strain,and established the reverse genetics system of Zika card virus,cloned the cDNA fragments of a contemporary ZIKV strain isolated in 2016 and successfully assembled the full-length cDNA using the self-splicing group II Intron-based strategy.Result: Using the full-length infectious clone of Zika virus Guangzhou 01 strain,the transcribed RNA was subjected to self-splicing in vitro,and transfection of the spliced RNA into BHK-21 cells resulted in the production of infectious progeny viruses.The parental strain and the recovered ZIKV has similar growth profiles,plaque morphologies and neurovirulences.Conclusion: The results suggest that the recovered ZIKV retains the biological properties of the parental strain well.Moreover,using this newly established reverse genetics system,we have revealed the critical role of a panel of RNA elements,including the cyclization sequences(CS)during ZIKV viral RNA replication,highlighting the reliability and potential of this new self-splicing Intron-based strategy in the generation of flavivirus infectious clones.