- Efficiency of reverse genetics methods for rescuing severe acute respiratory syndrome coronavirus 2
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Chang-Joo Park, Taehun Kim, Seung-Min Yoo, Myung-Shin Lee, Nam-Hyuk Cho, Changhoon Park
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J. Microbiol. 2025;63(2):e2411023. Published online February 27, 2025
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DOI: https://doi.org/10.71150/jm.2411023
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Bacteria-free reverse genetics techniques are crucial for the efficient generation of recombinant viruses, bypassing the need for labor-intensive bacterial cloning. These methods are particularly relevant for studying the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This study compared the efficiency of three bacteria-free approaches—circular polymerase extension reaction (CPER) with and without nick sealing and infectious sub-genomic amplicons (ISA)—to bacterial artificial chromosome (BAC)-based technology for rescuing SARS-CoV-2. Significant differences in viral titers following transfection were observed between methods. CPER with nick sealing generated virus titers comparable to those of the BAC-based method and 10 times higher than those of the standard CPER. In contrast, ISA demonstrated extremely low efficiency, as cytopathic effects were detected only after two passages. All rescued viruses exhibited replication kinetics consistent with those of the original strain, with no significant deviation in replication capacity. Furthermore, the utility of CPER and ISA in genetically modifying SARS-CoV-2 was demonstrated by successfully inserting the gene encoding green fluorescent protein into the genome. Overall, this study underscores the potential of bacteria-free methods, such as CPER and ISA, in advancing SARS-CoV-2 research while highlighting their significant differences in efficiency.
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Citations
Citations to this article as recorded by  - Research Progress of Coronavirus Reverse Genetics Technology
Ziqi Han, Jiaxu Han, Yan Zhao, Chao Xu, Xue Leng, Boyin Jia, Naichao Diao, Fei Liu, Chunmei Cui, Jian Liang, Yuhang Jiang, Rui Du
Journal of Medical Virology.2026;[Epub] CrossRef
- Infection Dynamics of Dengue Virus in Caco-2 Cells Depending on Its Differentiation Status
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Jayoung Nam, Jisu Lee, Geon A Kim, Seung-Min Yoo, Changhoon Park, Myung-Shin Lee
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J. Microbiol. 2024;62(9):799-809. Published online August 30, 2024
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DOI: https://doi.org/10.1007/s12275-024-00161-w
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Abstract
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Dengue virus (DENV), from the Flaviviridae family, is the causative agent of dengue fever and poses a significant global health challenge. The virus primarily affects the vascular system and liver; however, a growing body of evidence suggests its involvement in the gastrointestinal (GI) tract, contributing to clinical symptoms such as abdominal pain, vomiting, and diarrhea. However, the mechanisms underlying DENV infection in the digestive system remain largely unexplored. Prior research has detected viral RNA in the GI tissue of infected animals; however, whether the dengue virus can directly infect human enterocytes remains unclear. In this study, we examine the infectivity of human intestinal cell lines to the dengue virus and their subsequent response. We report that the Caco-2 cell line, a model of human enterocytes, is susceptible to infection and capable of producing viruses. Notably, differentiated Caco-2 cells exhibited a lower infection rate yet a higher level of virus production than their undifferentiated counterparts. These findings suggest that human intestinal cells are a viable target for the dengue virus, potentially elucidating the GI symptoms observed in dengue fever and offering a new perspective on the pathogenetic mechanisms of the virus.
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Citations
Citations to this article as recorded by - Efficient and modular reverse genetics system for rapid generation of recombinant severe acute respiratory syndrome coronavirus 2
Sojung Bae, Jinjong Myoung
Journal of Microbiology.2025; 63(7): e2504015. CrossRef - Domain-Specific Impacts of Spike Protein Mutations on Infectivity and Antibody Escape in SARS-CoV-2 Omicron BA.1
Tae-Hun Kim, Sojung Bae, Jinjong Myoung
Journal of Microbiology and Biotechnology.2025;[Epub] CrossRef
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