EV-B 3D polymerase remodels viral populations through 5'UTR recombination to subvert cardiac antiviral innate immunity.
Viral myocarditis, a leading cause of morbidity in young populations, is strongly linked to Coxsackievirus B (CV-B) infections harboring dominant 5'-terminally deleted (5'TD) and minor full-length (FL) CV-B RNA populations in cardiac tissues. Here, we demonstrate how viral RNA-dependent RNA polymerase (3Dpol)-driven recombination in the 5'UTR orchestrates viral RNA populations dynamics and subverts type I interferon responses. In primary human cardiomyocytes (HCMs), 3Dpol-mediated copy-choice recombination enhances 5'TD RNA replication while suppressing FL populations. Infection of immunocompetent mice with recombination-deficient CV-B3 (3Dpol Y276H) shifted 5'TD populations ratios toward immune-sensing viral RNAs, elevating cardiac IFN-β/ISG15 and accelerating viral clearance. Transfection experiments confirmed that 50-nt 5'TD RNAs (TD50) evade innate immunity, whereas shorter deletions (9-36-nt, TD15) restore type I interferon responses in HCMs. Our findings establish 3Dpol-driven recombination as a critical mechanism sustaining pathogenic 5'TD RNAs that subvert antiviral innate immunity, highlighting recombination inhibition as a promising therapeutic strategy against CV-B myocarditis.