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

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem–loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.