tetano
Editor, Senior Moderator
J Med Virol
. 2025 Jan;97(1):e70171.
doi: 10.1002/jmv.70171. Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly
Xinrong Zhou[SUP] 1 2 [/SUP], Hongchao Liu[SUP] 1 2 [/SUP], Sidi Yang[SUP] 1 2 [/SUP], Xiaoran Dong[SUP] 2 [/SUP], Cailing Xie[SUP] 2 [/SUP], Weixin Ou[SUP] 2 [/SUP], Jinwei Chen[SUP] 2 [/SUP], Zixiao Yang[SUP] 3 [/SUP], Yuzhen Ye[SUP] 1 2 [/SUP], Konstantin I Ivanov[SUP] 2 [/SUP], Lihong Liu[SUP] 1 2 [/SUP], Jie Zou[SUP] 3 [/SUP], Chunmei Li[SUP] 3 [/SUP], Deyin Guo[SUP] 1 2 4 [/SUP]
Affiliations
Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.
Keywords: BAC; Gibson assembly; antiviral drug screening; coronavirus; reverse genetic manipulation.
. 2025 Jan;97(1):e70171.
doi: 10.1002/jmv.70171. Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly
Xinrong Zhou[SUP] 1 2 [/SUP], Hongchao Liu[SUP] 1 2 [/SUP], Sidi Yang[SUP] 1 2 [/SUP], Xiaoran Dong[SUP] 2 [/SUP], Cailing Xie[SUP] 2 [/SUP], Weixin Ou[SUP] 2 [/SUP], Jinwei Chen[SUP] 2 [/SUP], Zixiao Yang[SUP] 3 [/SUP], Yuzhen Ye[SUP] 1 2 [/SUP], Konstantin I Ivanov[SUP] 2 [/SUP], Lihong Liu[SUP] 1 2 [/SUP], Jie Zou[SUP] 3 [/SUP], Chunmei Li[SUP] 3 [/SUP], Deyin Guo[SUP] 1 2 4 [/SUP]
Affiliations
- PMID: 39821287
- DOI: 10.1002/jmv.70171
Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.
Keywords: BAC; Gibson assembly; antiviral drug screening; coronavirus; reverse genetic manipulation.