Reverse genetics approach for arteriviruses using circular polymerase extension reaction

Figures

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  Fig. 1.. Designing of the three overlapped cDNA fragments spanning the full-length genome of EAV KY84 strain (GenBank accession number: MG137429) (F1 : 1 to 4,325; F2 : 4,276 to 8,625; and F3 : 8,576 to 12,729) (a). Designing of the five overlapping cDNA fragments spanning the full-length genome of PRRSV VR2332 strain (GenBank accession number: AY150564) (F1 : 1 to 2,228; F2 : 2,174 to 7,152; F3 : 7,096 to 12,052; F4: 11,995 to 14,354; and F5: 14,300 to 15,451) (b) to be used in the CPER assay. (c) Schematic representation of the reverse genetics systems based on CPER used for the generation of arterivirus infectious cDNA clones.

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  Fig. 2.. Designing of the overlapped cDNA fragments spanning the full-length genome incorporating reporter genes of EAV KY84-RFP mCherry, in which the RFP mCherry was inserted into ORF1a at a position located five codons downstream of the nsp1/nsp2 cleavage junction (a) and PRRSV VR2332-GFP, in which the GFP was inserted between the stop codon of ORF1b and the start codon of ORF2a (b) for use in the CPER assay.

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  Fig. 3.. Recovery of rEAV KY84 (0.05 (a) and 0.1 (b) pmol of CPER DNA) P1 in BHK-21 cells at 48 hpi compared to the negative controls (mock-infected, non-transfected BHK-21 cells (c) and CPER reaction product lacking F1/F3 (d). Recovery of rPRRSV VR2332 (0.05 (e) and 0.1 (f) pmol of CPER DNA) P1 in MARC-145 cells at 96 hpi compared to the negative controls (mock-infected, non-transfected MARC-145 cells (g) and CPER reaction product lacking F2/3 (h)). Magnification: 20×; scale=100 µm.

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  Fig. 4.. Confirmation of rEAV KY84 recovery following CPER. BHK-21 cells were infected with rEAV KY84 P0 (recovered from 0.05 and 0.1 pmol of CPER DNA) and immunolabelled with anti-N (a) and anti-nsp1 (b) antibodies at 24 hpi. Mock-infected and minus F1/F3 were included as negative controls. Magnification: 20×; scale: 100 µm.

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  Fig. 5.. Confirmation of rPRRSV VR2332 recovery following CPER. MARC-145 cells were infected with rPRRSV VR2332 P0 (recovered from 0.05 and 0.1 pmol of CPER DNA) and immunolabelled with anti-N (a) and anti-GP5 (b) antibodies at 48 hpi. Mock-infected and minus F2/F3 were included as negative controls. Magnification: 20×; scale: 100 µm.

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  Fig. 6.. Live fluorescence imaging monitoring expression of RFP mCherry reporter gene in rEAV KY84-RFP mCherry P0 in transfected BHK-21 from 24 to 72 hpt (a); magnification: 4×; scale: 400 µm. Recovery of the reporter virus was confirmed by immunofluorescence using anti-N (b) and anti-nsp1 antibodies (c). Magnification: 20×; scale: 100 µm.

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  Fig. 7.. Live fluorescence imaging monitoring expression of GFP reporter gene in rPRRSV VR2332-GFP P0 in transfected BHK-21 from 24 to 72 hpt (a); magnification: 4×; scale: 400 µm. Recovery of the reporter virus in MARC-145 cells was confirmed by immunofluorescence using anti-N (b) and anti-GP5 antibodies (c). Magnification: 20×; scale: 100 µm.

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  Fig. 8.. (a) Growth kinetics of rEAV KY84 (cyan) and rEAV KY84-RFP mCherry (red) compared to the parental EAV KY84 strain (magenta) in EEC cells (MOI=5). Each datapoint corresponds to triplicate wells and represents mean titre values±sd. (b) Growth kinetics of rPRRSV VR2332 (orange) and rPRRSV VR2332-GFP (green) compared to the parental PRRSV VR2332 strain (blue) in MARC-145 cells (MOI=5). Each datapoint corresponds to triplicate wells and represents mean titre values±sd. Light blue asterisks indicate statistically significant differences between rPRRSV VR2332 and parental PRRS VR2332, purple asterisks indicate statistically significant differences between rPRRSV VR2332-GFP and parental PRRS VR2332 and the red asterisk indicates statistically significant differences between rPRRSV VR2332 and rPRRSV VR2332-GFP (*P<0.05, **P≤0.01, ***P≤0.001). (c) Stability of RFP mCherry reporter gene following 15 serial passages of rEAV KY84-RFP mCherry in EEC cells, the expression of the RFP mCherry was stable until P6 and then, it decreased significantly to reach the lowest expression at P15. Magnification: 4×; scale: 400 µm. (d) Stability of GFP reporter gene following 15 serial passages of rPRRSV…