Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination.
Int J Mol Sci
; 22(19)2021 Sep 22.
Article
in English
| MEDLINE | ID: covidwho-1438627
ABSTRACT
The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Genome, Viral
/
Cloning, Molecular
/
SARS-CoV-2
/
COVID-19
Type of study:
Prognostic study
Limits:
Animals
/
Humans
Language:
English
Year:
2021
Document Type:
Article
Affiliation country:
Ijms221910188
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