Serial passaging in vitro generates a Vero cell-adapted coxsackievirus A6 strain with distinct phenotypic characteristics

Background Coxsackievirus A6 (CVA6) has emerged as a major etiological agent of hand, foot, and mouth disease (HFMD), yet no commercial vaccine is available against CVA6. CVA6 exhibits poor adaptability to Vero cells, a WHO-approved substrate for human vaccine production, and the mechanisms underlying its infectivity and pathogenesis remain incompletely understood. Methods A Vero cell-adapted CVA6 strain was generated through serial passaging in vitro . Two recombinant viruses derived from the earlier and later generations, corresponding to passage 10 and passage 45, were generated and designated rV10 and rV45, respectively. Their phenotypic characteristics, including viral growth kinetics, cytopathic effects, receptor interaction, entry and release efficiency, in vivo pathogenicity, immunogenicity, and host transcriptomic responses, were systematically compared. Results Compared with rV10, rV45 exhibited a pronounced cytolytic phenotype, accompanied by higher viral titers, increased efficiency of viral entry and egress, and enhanced interaction with the receptor KRM1. Notably, rV45 was markedly attenuated in mice while retaining immunogenicity comparable to that of rV10. Comparative sequence analysis revealed ten amino acid substitutions in the structural protein VP1 and one in the non-structural protein 3A between rV10 and rV45. Transcriptome profiling revealed that rV45, but not rV10, preferentially activated several signaling pathways including MAPK, TNF, IL-17, and apoptosis. Further validation demonstrated that rV45 infection triggered caspase-3 cleavage and apoptosis, which facilitated viral proliferation. Conclusions Serial passaging of CVA6 in Vero cells drives coordinated molecular and phenotypic adaptation, predominantly mediated by VP1 mutations that enhance receptor KRM1 utilization and apoptosis-associated viral replication. This study provides novel insights into infectivity and pathogenesis of CVA6 and establish a potential foundation for development of attenuated vaccines against CVA6.