The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus

Figures

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  Figure 1. Construction of recombinant plasmids with FMDV cDNA clones. Plasmid pKLS3 comprises FMDV O189 5′UTR downstream of T7 promoter followed by StuI restriction enzyme recognition site, FMDV 3′UTR, 48 adenine residues, and a ribozyme sequence of hepatitis delta virus. Three DNA fragments were designed for the GA reaction, in which FMDV 5′UTR at 3′ end of the linearized pKLS3 overlaps with 53 (O189) or 20 (NP05) bp at 5′ end of F1 fragment (~3.8 kb); 74 bp at 3′ end of F1 overlaps with 5′ end of F2 fragment (~3.2 kb); and 57 (O189) or 20 (NP05) bp at 3′ end of fragment F2 overlaps with 5′ end of FMDV 3′UTR within the linearized pKLS3. (A) Plasmid pKLS3 with FMDV type O, O189, coding sequence. (B) Plasmid pKLS3 with FMDV type A, NP05, coding sequence.

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  Figure 2. Electrophoretic photographs of two GA reactions. Input DNA fragments in each GA reaction include 4.3 kb of the linearized pKLS3 vector (V), 5′coding sequence of FMDV fragment 1 (AF1 or OF1), and 3′coding sequence of FMDV fragment 2 (AF2 or OF2). (A) FMDV type A, NP05, composes of 3.8 kb AF1 and 3.3 kb AF2. (B) FMDV type O, O198, comprises 3.8 kb OF1 and 3.3 kb OF2. The DNA fragments in each GA reaction (lane GA) compose of ~11.3 kb of pKLS3 plus the full-length FMDV coding region (GA), ~7 kb of assembled F1 and F2 or pKLS3 and F1/F2, ~3.8 kb of F1 and ~3.2 kb of F2, respectively.

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  Figure 3. Cytopathic effect (CPE) and RT-PCR products of the rescued and parental FMDVs at 24 h post-infection in BHK-21 cells. (A) The rescued FMDVs of both types A (NP05) and O (O198) produced CPE similarly to the corresponding parental viruses while mock-infected cells (control) did not develop CPE. (B) Electrophoretic photographs demonstrated the RT-PCR products amplified with common FMDV (FMDV 2B) and type-specific (VP1) primers. The FMDV 2B primers produced an approximately 420 bp DNA fragment, while the FMDV type-specific primers amplified the VP1 gene region and generated 630 and 714 bp DNA fragments for rNP05 and rO189, respectively. Lanes 1–4 are RT-PCR products amplified with FMDV 2B primers; control [1], rescued O189 [2], rescued NP05 [3], and wild-type FMDV [4]. Lanes 5 and 6 are PCR products amplified using cDNA from the rescued and parental O189 viruses, respectively, with OVP1 primers. Lanes 7 and 8 are PCR products amplified with AVP1 primers using cDNA from the rescued and parental NP05 viruses, respectively.

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  Figure 4. A chart of the plot between logarithmic based 10 of cDNA copy numbers of the rescued FMDVs and passage numbers. Passage 0 (p0) was supernatant harvested from transfected cells and media. p1-13 were the supernatants that were passed in BHK-21 cells 1–13 times, respectively. The rescued FMDV cDNAs were determined by RT-qPCR using the absolute quantification method. The cDNA copy numbers were calculated based on the mean Cq values.

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  Figure 5. The chart demonstrated the growth kinetics of the rescued and parental FMDVs. The rescued and their parental FMDV types O (O189) and A (NP05) were inoculated onto BHK-21 cells at 0.01 MOI and the supernatants were collected at four-hour intervals (0–24 hpi). Viral titers were transformed to logarithmic-based 10, which were plotted against collecting times in hours post inoculation (hpi). The growth characteristics of the rescued (rNP05 and rO189) and their parental viruses (NP05: A/TAI/NP05/2017 and O189: O/TAI/189/1987) were not significantly different.

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  Figure 6. Immunoperoxidase monolayer assay (IPMA) demonstrates specific reactivity between wild-type (WT) or rescued FMDVs and a monoclonal antibody (mAb) raised against FMDV (3E11). FMDV types O (O189) and A (NP05), and rescued virus types O (rO198) and A (rNP05) infected BHK-21 cells reacted strongly with the mAb 3E11 as shown by the brown color. mAb 3E11 did not bind to non-infected BHK-21 cells and porcine circovirus type 2 (PCV2) infected PK-15 cells, which served as 3E11 background and isotype controls, respectively. Both WT and rescued FMDVs did not react to mAb 12G3, a mAb specific to PCV2 [26]. PCV2-infected PK-15 cells show a positive signal with mAb 12G3, while non-infected PK-15 cells remained negative. (Magnification of 100×).

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  Figure 7. Plaque Morphologies of wild-type O189 (A), rescued O189 (B), wild-type NP05 (C), and rescued NP05 (D). The plaques were observed at 24 h post-infection. Non-infected BHK-21 cells were also included as a negative control (E).