ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Veterinary and Zoonotic Infection
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1614369
This article is part of the Research TopicUnveiling Host-Pathogen Interactions: Insights into Animal Cellular Immunity and Novel Diagnostics - Volume IIView all 14 articles
Rapid generation of HCoV-229E and HCoV-OC43 reporter viruses and replicons for antiviral research
Provisionally accepted
Yuyuan Zhang1
Jiannan Chen2
Hui Feng1Lulu Yang1Liyuan Hu1Yang Gao1Ziqiao Wang1Fei Feng1
Jincun Zhao3
Ping Zhang4
Rong Zhang1,4*- 1School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
- 2School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong Province, China
- 3State Key Laboratory of Respiratory Diseases (SKLRD), Guangzhou, Guangdong Province, China
- 4Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
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IntroductionThe large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration.MethodsUsing transformation-associated recombination (TAR) cloning in yeast and the in vitro CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established. Antiviral assays, high-content imaging, and NanoLuc luciferase assays were used to characterize reporter viruses and replicon systems.ResultsWe rapidly assembled infectious clones for two mild coronaviruses, HCoV-OC43 and HCoV-229E, using transformation-associated recombination (TAR) cloning in yeast. The infected clones could stably express the mGreenLantern reporter gene. We further generated T7 promoter-driven RNA replicon of HCoV-229E and CMV promoter-driven DNA replicon of HCoV-OC43, with the readout of NanoLuc luciferase activity. The effectiveness of these tools for antiviral study was evaluated using the broad-spectrum RNA-dependent RNA polymerase inhibitor remdesivir, exhibiting high sensitivity, efficiency, and convenience.DiscussionThe application of yeast-based TAR cloning significantly facilitates the rapid assembly of large viral genome, and the establishment of HCoV-OC43 and HCoV-229E reverse genetics systems provides valuable platforms for studying the biology and developing antivirals against coronaviruses.
Keywords: Human coronavirus, Reverse genetics system, TAR cloning, Reporter virus, Replicon, antiviral
Received: 18 Apr 2025; Accepted: 04 Jul 2025.
Copyright: © 2025 Zhang, Chen, Feng, Yang, Hu, Gao, Wang, Feng, Zhao, Zhang and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Rong Zhang, Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China
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