Reverse Genetics Systems for Emerging and Re-Emerging Swine Coronaviruses and Applications

Figures

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  Figure 1. Targeted RNA recombination scheme to construct recombinant PEDV with ORF3 gene deletion [23]. MHV S transcripts with homologous arms of PEDV S gene are electroporated into PEDV-infected Vero cells. Then, the recombinant virus is obtained by plaque purification in murine L cells. Transcripts with expected mutations are electroporated into the recombinant virus-infected L cells; after 4 h post-infection, the recombinant virus with expected mutations can be obtained by plating the infected L cells onto monolayers of Vero cells.

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  Figure 2. Engineering the genome of PEDV by in vitro transcription [33]. The genome structures of PEDV and the in vitro ligation approach are shown. Cohesive overhangs are shown; the length is not to scale. The full-length cDNA of PEDV is directionally assembled in vitro and then transcribed into infectious genomic mRNA with a T7 transcription kit. The genomic mRNA is electroporated into Vero cells to rescue the recombinant PEDV.

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  Figure 3. Flowchart for construction of the PEDV infectious clone using BAC system [39]. The genome structure of PEDV followed by DNA fragments amplified by RT-PCR is shown. The F1 and F2 DNA fragments are ligated to an intermediate plasmid pBAC-M-PEDV which cloned the CMV promoter, PEDV genome segments [nucleotide (nt) 1–1,092, PacI cleavage site, followed by nt 22,199 to 3′ UTR] a poly(A) tail, HDVr, and BGH polyadenylation sequence by homologous recombination; similarly, F3, F4, and F5 are also ligated to pBAC-M-PEDV by homologous recombination. Finally, DNA fragments (F3–F5) were cut by restriction enzymes PacI and SacII and then ligated to the recombinant BAC plasmid cloned F1 and F2 with T4 DNA ligase to obtain an infectious PEDV clone plasmid. The virus could be rescued through transfection of the recombinant BAC plasmid into Vero cells.

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  Figure 4. Engineering the genome of PEDV through yeast-based vector. The complete genome of PEDV was divided into seven fragments with at least 30 nt overlaps with neighbor fragments. All the seven cDNA fragments were transformed together with a linearized vector (pYES1L) into yeast competent cells for assembly through transformation-associated recombination in yeast. After identification and extraction of the positive clones, the full-length cDNA clones were transfected into Vero cells for virus recovery.

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  Figure 5. Vaccinia virus vector-based reverse genetics system for PEDV [43]. The PEDV genome was divided into eight fragments (F1–F8), which were then used to construct four plasmids: pA, containing fragment 1 upstream and fragment 8 downstream of the GPT gene; pB, containing fragment 2 and 7; pC, containing fragment 3 upstream and fragment 6 downstream of the GPT gene; pD, containing fragment 4 and 5. The genome was introduced into vaccinia virus genome through four rounds of vaccinia virus-mediated homologous recombination with GPT as a positive or negative selection marker. After linearization of the vaccinia virus genome by NotI digestion, the infectious mRNA was transcribed and electroporated into Vero cells to rescue recombinant PEDV.

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  Figure 6. General workflow of TAR cloning for SARS-CoV-2 [44]. The genome of SARS-CoV-2 was divided into nine fragments with 45–500 base pair (bp) overlaps which were amplified by RT-PCR or overlapping PCR. The DNA fragments were co-transformed with TAR cloning vector into yeast for TAR cloning. The yeast plasmids were extracted from yeast culture and linearized. After being transcribed through in vitro transcription, the genomic mRNA was electroporated into susceptible cells for recovery.

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  Figure 7. Scheme of CPER method to engineer the coronavirus genome. The complete CoV genome was covered by eight overlapping DNA fragments. A linker DNA fragment contains homologous arms, CoV 3′ UTR, a poly(A) tail, HDVr, a BGH polyadenylation sequence, a spacer sequence, a CMV promoter sequence, and CoV 5′ UTR. The linker sequence and overlapping DNA fragments were assembled through CPER, and, then, the CPER mixture was transfected into susceptible cells for virus recovery.