The 5′-end motif of Senecavirus A cDNA clone is genetically modified in 36 different ways for uncovering profiles of virus recovery

Figures

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  Figure 1. Schematic representations of putative HPH structure and wild-type cDNA clone. The HPH formation (A) is predicted through two online servers. The first hairpin, hairpin-1, is made up of one 17-nt-paired stem and one 6-nt-unpaired loop (Green stem-Yellow loop); the middle structure is an H-type pseudoknot, containing two stems and two loops (Blue stem-Blue loop); the second hairpin, hairpin-2 (Purple stem-Yellow loop), is structurally simpler than the hairpin-1. The N0 plasmid contains the wild-type SVA cDNA clone with a fusion sequence of eGFP-Thosea asigna virus 2A (B). The red star indicates the 5′ terminus of cDNA clone.

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  Figure 2. Schematic representations of 36 putative RNA structures, corresponding to N1 to N36 genotypes. The first ten nucleotides, “TTTGAAAGGG,” are deleted one by one from the N0 to construct N1 to N10 (A). The HPH-forming motif (nt 4 to 85) in the N0 is genetically modified to construct N11 to N36, including single-stranded deletion (B), single-stranded mutation [(C,E,H,I), Red circle-marked], double-stranded deletion (D), double-stranded substitution [(G,J), Brown circle-marked] and single-stranded insertion [(F), Gray circle-marked].

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  Figure 3. Rescue and passaging of rSVA-N0 to -N5. Green fluorescence is visible on cell monolayers during serial blind passaging. BF, bright field. P0: passage-0 at 72 hpt. P1, P6 and P9: passage-1, −6, and −9 at 48 hpi. Bar = 50 μm.

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  Figure 4. Rescue and passaging of rSVA-N26 to -N29, and -N0. Green fluorescence is visible on cell monolayers during serial blind passaging. BF, bright field. P0: passage-0 at 72 hpt. P1 and P3: passage-1 and −3 at 48 hpi. Bar = 50 μm.

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  Figure 5. RT-PCR detection, 5′-RACE analysis and Sanger sequencing. RT-PCR detection of rSVA-N0 to -N5 at P6 (A). PCR controls are assigned to demonstrate no interference of cDNA residues. RT-PCR detection of rSVA-N0 to -N5 at P9 (B). Nested PCR amplification for 5′-RACE products of rSVA-N0 to -N5 at P9 (C). Sanger sequencing chromatograms of 5′-end sequences of rSVA-N0 to -N5 at P9 (D–L). The “TTGA” and “TTTGA” genotypes are covered with red and green shadows, respectively. The partial 3′-end sequences of adapters are covered with gray shadows. The yellow shadows indicate one or two extra nucleotides inserted between the 3′ end of adapter and the 5′ end of viral cDNA.

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  Figure 6. Detection and characterization of rSVA-N26 to -N29. RT-PCR analysis of rSVA-N26 to -N29 at P6 (A). PCR controls are assigned to demonstrate no interference of plasmid residues. Sanger sequencing for hairpin-1-forming motifs of rSVA-N26 to -N29 at P6 (B–E). Since the reverse primer (RP4) is used for Sanger sequencing, four chromatogram-exhibited fragments are reverse-complement sequences of hairpin-1 motifs. Green, yellow, red and gray shadow-covered chromatograms (rSVA-N26 to -N29) match to green, yellow, red and gray circle-marked fragments in the hairpin-1 (N26 to N29), respectively. LFM, loop-forming motif; SFM, stem-forming motif.

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  Figure 7. Multistep growth curves of rSVAs at P4. Data at 0, 24, 48 and 72 hpi are representative of three independent experiments. Error bar indicates standard deviation.