A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation

Abstract The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicati...

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Autores principales:	Tomohiro Kotaki, Xuping Xie, Pei-Yong Shi, Masanori Kameoka
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/14dcdbd4693243798f8fae9ed7bc5980
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spelling	oai:doaj.org-article:14dcdbd4693243798f8fae9ed7bc59802021-12-02T14:16:26ZA PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation10.1038/s41598-021-82055-02045-2322https://doaj.org/article/14dcdbd4693243798f8fae9ed7bc59802021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-82055-0https://doaj.org/toc/2045-2322Abstract The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.Tomohiro KotakiXuping XiePei-Yong ShiMasanori KameokaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-7 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tomohiro Kotaki Xuping Xie Pei-Yong Shi Masanori Kameoka A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
description	Abstract The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.
format	article
author	Tomohiro Kotaki Xuping Xie Pei-Yong Shi Masanori Kameoka
author_facet	Tomohiro Kotaki Xuping Xie Pei-Yong Shi Masanori Kameoka
author_sort	Tomohiro Kotaki
title	A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
title_short	A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
title_full	A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
title_fullStr	A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
title_full_unstemmed	A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
title_sort	pcr amplicon-based sars-cov-2 replicon for antiviral evaluation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/14dcdbd4693243798f8fae9ed7bc5980
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A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation

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