Development of the coronavirus reverse genetic system: Core technology for pathogenesis mechanisms research and vaccine/drug development

Abstract

Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses that cause respiratory, gastrointestinal, hepatic, and neurological diseases in humans and other animals. In recent years, frequent outbreaks of emerging and re-emerging CoVs have threatened animal and human health. However, an insufficient understanding of the mechanisms underlying CoV pathogenicity and cross-species transmission limits the development of drugs and vaccines against CoVs. Reverse genetic technology is a powerful tool for manipulating the genomes of CoVs and acquiring recombinant viruses, which allows researchers to better understand viral pathogenesis and develop genetically attenuated and marked vaccines and antiviral drugs. However, the large genomes of CoVs and the instability and toxicity of viral sequences in bacteria represent serious obstacles to the development of reverse genetic systems of CoVs. With the development of molecular biological methods, various new construction strategies have emerged. Accordingly, this review summarizes the construction strategies of CoV reverse genetics systems and their applications in studying pathogenesis, cross-species transmission, vaccine development, and drug screening, with the aim of providing an important reference for the prevention and control of CoVs.

Keywords: Coronavirus; antiviral drug-screening; coronavirus tropism; infectious clone; pathogenesis; reverse genetic system.

Figure 1.

Genomic organization of representative α, β, γ and δ-CoVs. The open reading frame 1a (ORF1a) and ORF1b are posited in the two-thirds of the 5’ end of genome. The structural genes spike (S), envelope (E), membrane (M), nucleoprotein (N), and accessory proteins are placed in the one-third of the 3’ end of genome. The genes encoding accessory proteins are shown as gray boxes. The schematic diagram below shows the structural and accessory proteins in the regions of α-CoVs (TGEV), β-CoVs (SARS-CoV-2), γ-CoVs (IBV), and δ-CoVs (PDCoV). The sizes and positions of accessory genes are drawn, relative to the basic genes S, E, M, and N. The size of the genome and individual genes is not drawn to scale. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 2.

Reverse genetic construction strategy of MHV based on targeted RNA recombination [60]. The synthetic subgenomic RNA7 contains the MHV leader sequence, MHV N gene and 3’ UTR, and is flanked by the T7 promoter and Poly(A) tail at its termini. Repairing the deletion in Alb4 could be achieved through targeted RNA recombination with the Alb4 genome in 17CL–1 cells. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 3.

Reverse genetic construction strategy of PEDV deleting ORF3 gene based on targeted RNA recombination [61]. The transcripts containing the upstream and downstream homologous arms of the PEDV S gene and MHV S gene were electroporated into PEDV-infected Vero cells. The infected Vero cells were overlaid onto L cells to generate recombinant mPEDV virus. Then, the transcripts containing upstream and downstream homologous arms of the PEDV S-ΔORF3 gene were electroporated into mPEDV-infected L cells. The infected L cells were overlaid onto Vero cells to obtain recombinant PEDV-ΔORF3 virus. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 4.

Reverse genetic construction strategy for TGEV based on BAC vector [63]. The TGEV genome was amplified as five DNA fragments by RT-PCR. And the TGEV cDNA was flanked by the CMV promoter at 5’ end, and flanked by the poly(A) tail, the HdvRz and the BGH terminator at 3' end. Then, the DNA fragments was ligated to pBelobac11 to obtain the infectious TGEV clone plasmid. Then the plasmid was transfected into susceptible cells to rescue virus. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 5.

Reverse genetic technology for TGEV based on in vitro ligation [71]. The TGEV genome was divided into five DNA fragments (A-E). The T7 promoter was inserted at the 5' end of the fragment A, and a poly (A) tail at the 3' end of the fragment E. The unique BglI restriction sites were flanked at the junctions of each cDNA fragment by the primer-mediated PCR mutagenesis. in vitro ligation was performed to obtain the full-length cDNA, which was further used as a template for in vitro transcription. The obtained full-length RNA transcripts were electroporated into cells to rescue TGEV. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 6.

Reverse genetic technology for IBV based on poxvirus vectors [83]. The IBV genome was divided into three fragments (F1–F3), which were then used to construct three plasmids. The T7 promoter was added to the 5' end of F1, and a poly (A) tail was added to the 3' end of F3. These plasmids were digested by the enzymes shown in the figure, and then were ligated to obtain the full-length IBV cDNA using the T4 ligase. The acquired ligation products were ligated into the linearized genome of vaccinia virus vector vNoti/tk digested by NotI to acquire the recombinant vaccinia virus. After linearization of the vaccinia virus genome by NotI digestion, the IBV cDNA was obtained. Finally, the infectious mRNA that was transcribed through in vitro transcription was electroporated into cells to rescue IBV. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 7.

Reverse genetic technology for PDCoV based on yeast TAR cloning [90]. The PDCoV genome was amplified as six overlapping fragments by RT-PCR (A-F). The T7 promoter was added at the5' end of fragment A, and a poly (A) tail and ASCI restriction sites were followed by the fragment F. Then, these fragments and the linearized TAR cloning vector were assembled into the correct assembly of YAC through TAR cloning in yeast. The YAC plasmids were linearized for in vitro transcription. Finally, the infectious mRNA was electroporated into cells to rescue PDCoV. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 8.

Reverse genetic technology for SARS-CoV-2 based on CPER [95,96]. The SARS-CoV-2 genome was divided into six overlapping fragments (F1-F6). A linker fragment contains an overlapping sequence with the 3’ UTR, a poly(A) tail, HdvRz, a BGH terminator, the CMV promoter and an overlapping sequence with the 5’ UTR of SARS-CoV-2. The linker sequence and overlapping DNA fragments were assembled through CPER to generate circular full-length viral cDNA. Then, the CPER assembly was transfected into susceptible cells to rescue SARS-CoV-2. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 9.

Reverse genetic technology for SARS-CoV-2 based on ISA [98]. The SARS-CoV-2 genome covered eight overlapping fragments (F1-F8). The CMV promoter was inserted to the 5’end of F1, and the HdvRz and SV40pA were added to the 3’ end of F8. Then, these cDNA fragments were transfected into BHK-21 cells, and the infected BHK-21 cells were passaged on Vero E6 cells to acquire infectious viruses.

Figure 10.

Reverse genetic technology for SARS-CoV-2 based on ISA using digested plasmids [99]. Schematic representation of digested plasmids were shown in the figure. The fragments were cloned into the plasmids. After digestion with specific enzymes at unique restriction sites avr ii and NotI, the digested fragments were obtained, and then they were transfected into susceptible cells to rescue infectious viruses. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).

Figure 11.

Reverse genetic technology for SARS-CoV-2 based on ISA using the linker fragment [100]. Schematic representation of the linker fragment was shown in the figure. The linker fragment contains, from left to right, poly(A) tail, HdvRz, SV40pA downstream of the 3’ UTR, and CMV promoter upstream of the 5’ UTR. SARS-CoV-2 DNA fragments and the linker fragment were transfected into susceptible cells to rescue infectious viruses. This figure was created by BioRender.com accessed on 17 May 2025 (BioRender, Toronto, on, Canada).