Rapid generation and characterization of recombinant HCoV-OC43-VR1558 infectious clones expressing reporter Renilla luciferase  

作　　者：Fei Ye Na Wang Qiongge Guan Mengwei Wang Jiewei Sun Desheng Zhai Baoying Huang Ying Zhao Wenjie Tan 

机构地区：[1]National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,NHC Key Laboratory of Biosafety,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China [2]School of Pharmacy,Xinxiang Medical University,Xinxiang 453003,China [3]School of Public Health,Xinxiang Medical University,Xinxiang 453003,China

出　　处：《Biosafety and Health》2024年第6期350-360,共11页生物安全与健康(英文)

基　　金：supported by the National Key Research and Development Program of China(2022YFC2304100 and 2021YFA1201003).

摘　　要：Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.

关 键 词：Infectious clones Human coronaviruses(HCoV)-OC43 Transformation-associated recombination(TAR) Reporter Renilla luciferase(Rluc) 

分 类 号：R511[医药卫生—内科学]