AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome

Figures

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  Fig. 1. Construction of rSVA cDNA clone with SAc. Schematic representation of full-length genome of foreign sequence-free SVA (A). The viral Nc is located in the VP2 sequence. The wild-type AAACA residues are marked with red circles. Map of cDNA-wt plasmid (B). Schematic representation of cDNA-wt genome (C). The cDNA-wt genome contains a SNc, and a fused sequence “eGFP–Ac–T2A”. Two SDM-modified sites in the SNc are marked with blue crosses and circles in the normal and enlarged rectangles, respectively. Profile of SDM-modified sites in A1A2A3C4A5 motif of Ac (D). A total of thirteen single-site mutations (A1T, A1G, A2T, A2C, A2G, A3T, A3C, A3G, C4A, C4G, A5T, A5C and A5G) are designed for independently constructing thirteen SDM-modified cDNA clones. Mutated sites are shown with blue letters.

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  Fig. 2. Recovery, detection and characterization of replication-competent rSVAs. Cell monolayers transfected with cDNA clones and serial blind passaging (A). BF: bright field; Bar = 20 μm. Green fluorescence is observable always in cDNA-A5U and -A5C groups. RT-PCR detection of virus recovery at P5 (B). Two pairs of primers are separately used for amplifying SNc- and SAc-containing fragments. Only cDNA-A5U and -A5C groups show RT-PCR-positive results. Sanger sequencing chromatograms of SDM-modified motifs at P5 (C). Multi-step growth curves of rSVA-wt, -A5U and -A5C (D). Data at 0, 24, 48 and 72 hpi are representative of three independent experiments. Error bar indicates standard deviation.

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  Fig. 3. Genetic stabilities of SNc and SAc with serial viral passaging. Cell monolayers infected with P10, P15 and P20 viral progenies (A). BF: bright field; Bar = 20 μm. RT-PCR analyses of SNc- and SAc-containing fragments at P10 (B) and P20 (C). Sanger sequencing chromatograms of SDM-modified motifs at P10 (D) and P20 (E). Two SNP sites in SNc of rSVA-A5C at P10 (F). The mut-1 and -2 sites exhibit A–U and A–G coexistences, respectively (as shown in Supplementary 1).