Genetic characterization of human enterovirus A71 genotypes C4 and B5 Circulating in Qingdao City, Shandong province, China, from 2023 to 2024.

Enterovirus A71 (EV-A71) is one of the main pathogens causing hand, foot, and mouth disease (HFMD), and this virus exhibits substantial genetic diversity. To clarify its genomic evolutionary features, this study conducted a phylodynamic analysis on EV-A71 viruses collected in Qingdao (a northern Chinese city) between 2023 and 2024. EV-A71 was identified using a commercial real-time quantitative PCR (qPCR) assay; EV-A71-positive samples were then inoculated into rhabdomyosarcoma (RD) cells for virus isolation. The complete genome sequences and VP1 gene sequences of EV-A71 strains were amplified and analyzed, with IQ-TREE, SimPlot, and RDP4 software used to evaluate their evolutionary characteristics. Among 2,083 clinical samples, 27 were EV-A71-positive, with 19 isolates successfully cultured. Whole-genome analysis confirmed the co-circulation of EV-A71 genotypes C4 (5 strains) and B5 (14 strains). The C4 strains showed high homology to a strain isolated in China in 2019 and carried six lineage-specific mutations. In contrast, the B5 strains clustered into two distinct lineages, including recombinants that had undergone genetic recombination with coxsackievirus A4 (CV-A4) and coxsackievirus A2 (CV-A2). Notably, all EV-A71 strains collected from Qingdao maintained a serine (S) at the 17th amino acid residue of the VP1 region. This work enhances our understanding of the geographical distribution of EV-A71 by confirming the sustained circulation of genotype B5 in northern China and identifying a novel C4/B5 co-circulation pattern in Qingdao-a pattern reflecting complex local evolutionary dynamics. It emphasizes expanding genomic sequencing coverage to monitor B5 and recombinant strains, refining surveillance, and guiding HFMD prevention and control.