Intertypic Recombination Between Coxsackievirus A16 and Enterovirus A71 Structural and Non-Structural Genes Modulates Virulence and Protection Efficacy

<b>Background/Objectives</b>: Enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot and mouth disease (HFMD), often co-circulating and occasionally undergoing genetic recombination. While natural recombinants often involve genomic regions encoding non-structural proteins, their effects on replication and pathogenesis remain unclear. <b>Methods</b>: To address this, four chimera viruses (Chi-CCE, Chi-ECE, Chi-EEC, and Chi-CEC) were constructed with 5'UTR, capsid P1, and non-structural P2 and P3 genes, from CVA16 (denoted as C) or EV-A71 (denoted as E). These chimeras were tested for replication kinetics and cytopathic effects in rhabdomyosarcoma cells while <i>in vivo</i> virulence and protection efficacy were evaluated using a newborn BALB/c mouse model. <b>Results</b>: All chimeric viruses remained viable and exhibited higher replication than CVA16. <i>In vivo</i>, all chimeric viruses were avirulent except Chi-CCE and CVA16, which showed high virulence and viral titres in the brains and limbs of infected newborn mice. This suggests that 5'UTR and capsid P1 genes of CVA16 are critical genetic determinants of virulence. Notably, only the anti-inflammatory cytokine IL-10 was elevated, suggesting potential immune modulation during infection. Inactivated Chi-CCE immunisation conferred 100% protection against lethal CVA16 or mouse-adapted EV-A71 challenge revealing its potential as a bivalent vaccine candidate. <b>Conclusions</b>: Our study demonstrates that recombination between CVA16 and EV-A71 influences viral virulence and protection efficacy with implications for future development of multivalent vaccines.