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- Efficiency of reverse genetics methods for rescuing severe acute respiratory syndrome coronavirus 2
- Chang-Joo Park, Taehun Kim, Seung-Min Yoo, Myung-Shin Lee, Nam-Hyuk Cho, Changhoon Park
- J. Microbiol. 2025;63(2):e2411023. Published online February 27, 2025
- DOI: https://doi.org/10.71150/jm.2411023
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Abstract
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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.
Research Support, Non-U.S. Gov't
- Development of a Chimeric Strain of Porcine Reproductive and Respiratory Syndrome Virus with an Infectious Clone and a Korean Dominant Field Strain
- Jung-Ah Lee , Nak-Hyung Lee , Sang-Won Lee , Seung-Yong Park , Chang-Seon Song , In-Soo Choi , Joong-Bok Lee
- J. Microbiol. 2014;52(4):345-349. Published online March 29, 2014
- DOI: https://doi.org/10.1007/s12275-014-4074-4
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- 16 Crossref
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Abstract
- The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 106 TCID50/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.
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Citations
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