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Review
. 2020:107:383-416.
doi: 10.1016/bs.aivir.2020.06.003. Epub 2020 Jun 30.

Reverse genetic systems: Rational design of coronavirus live attenuated vaccines with immune sequelae

Zhiqian Ma  1 , Zhiwei Li  1 , Linfang Dong  1 , Ting Yang  1 , Shuqi Xiao  2
Affiliations
Review

Reverse genetic systems: Rational design of coronavirus live attenuated vaccines with immune sequelae

Zhiqian Ma et al. Adv Virus Res. 2020.

Abstract

Since the end of 2019, the global COVID-19 outbreak has once again made coronaviruses a hot topic. Vaccines are hoped to be an effective way to stop the spread of the virus. However, there are no clinically approved vaccines available for coronavirus infections. Reverse genetics technology can realize the operation of RNA virus genomes at the DNA level and provide new ideas and strategies for the development of new vaccines. In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. An efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine.

Keywords: Coronavirus; Live attenuated vaccine; Reverse genetics.

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Figures

Fig. 1
Fig. 1
The genomic structure of coronaviruses. (A) Classification of coronavirus, with the new coronavirus SARS-CoV-2 highlighted in red. (B) The genome structure of four genera of coronaviruses. Pp1a and pp1b represent the 2 long polypeptides that are processed into 16 nonstructural proteins. S, E, M, and N indicate the four structural proteins spike, envelope, membrane, and nucleocapsid proteins.
Fig. 2
Fig. 2
Timeline of emerging representative coronavirus events and their infectious clones generated using reverse genetics systems. The timeline spans from the first coronavirus in history to the emergence of 2019-nCoV in 2019. Red arrows indicate human coronavirus outbreaks and identification events. Blue arrows indicate animal coronavirus outbreaks and identification events. Green arrows indicate the publication of infectious clones using different reverse genetics methods (Almazan et al., 2000; Li et al., 2013; Thao et al., 2020; Yount et al., 2002).
Fig. 3
Fig. 3
Flowchart of three methods for constructing coronavirus infectious cloning. (A) Targeted RNA recombination scheme used to make interspecies chimeric viruses: mIBV (Stage 1) and recombinant IBV (Stage 2). (B) In vitro ligation. The SARS-CoV full-length genome is divided into seven segments, named A-F, with type II restriction endonucleases Bgl1 at both ends. (C) Bacterial artificial chromosome system. Restriction enzyme sites in the MERS-CoV genome were employed to ligate the full-length MERS-CoV genome into pBeloBAC11.
Fig. 4
Fig. 4
Flowchart of two methods for constructing coronavirus infectious clones. (A) Transformation-associated recombination (TAR) cloning. A schematic representation of the general workflow of TAR cloning for SARS-CoV-2 infectious clones. (B) Vaccinia virus vectors. The general workflow of vaccinia virus vectors for constructing HCoV-229E infectious clones.
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