Journal of Microbiology

Research Article

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 PDF: 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.

Review

Protective and pathogenic role of humoral responses in COVID-19: Uni Park , Nam-Hyuk Cho; J. Microbiol. 2022;60(3):268-275. Published online March 2, 2022; DOI: https://doi.org/10.1007/s12275-022-2037-8

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Since the advent of SARS-CoV-2 in Dec. 2019, the global endeavor to identify the pathogenic mechanism of COVID-19 has been ongoing. Although humoral immunity including neutralizing activity play an important role in protection from the viral pathogen, dysregulated antibody responses may be associated with the pathogenic progression of COVID-19, especially in high-risk individuals. In addition, SARS-CoV-2 spike-specific antibodies acquired by prior infection or vaccination act as immune pressure, driving continuous population turnover by selecting for antibody-escaping mutations. Here, we review accumulating knowledge on the potential role of humoral immune responses in COVID-19, primarily focusing on their beneficial and pathogenic properties. Understanding the multifaceted regulatory mechanisms of humoral responses during SARS-CoV-2 infection can help us to develop more effective therapeutics, as well as protective measures against the ongoing pandemic.

Citations

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Research Support, Non-U.S. Gov't

Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi: Sunyoung Bang , Chan-Ki Min , Na-Young Ha , Myung-Sik Choi , Ik-Sang Kim , Yeon-Sook Kim , Nam-Hyuk Cho; J. Microbiol. 2016;54(2):136-144. Published online February 2, 2016; DOI: https://doi.org/10.1007/s12275-016-5599-5

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Abstract

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide- repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNAmodifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukarytotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

Citations

Citations to this article as recorded by

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium
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Published Erratum

Erratum to: Protective and Pathogenic Role of Humoral Responses in COVID‑19: Uni Park , Nam-Hyuk Cho; J. Microbiol. 2023;61(7):713-713.; DOI: https://doi.org/10.1007/s12275-023-00058-0

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