Coxsackievirus A6 was the predominant pathogen of hand, foot, and mouth disease with frequent recombination and mutations in Shandong Province, 2023.

The pathogens of hand, foot, and mouth disease (HFMD) are changing, with CV-A6 becoming the predominant one and evolving rapidly. We collected data on monthly HFMD infections per county and related demographic variables in Shandong Province, China, during 2023. Viruses were isolated and sequenced for further bioinformatics analysis. An epidemiological follow-up study in Jinan city was conducted to explore the relationship between onychomadesis and CV-A6. The risk factors of atypical skin lesions were analysed using binary logistic regression models. Totally, 47 396 cases were clinically diagnosed as HFMD, among which 9543 samples were collected and 7133 (74.75%) were laboratory-confirmed Enterovirus (EV)-positive cases. CV-A6 became the predominant pathogen (5993, 84.02%). Forty-six representative CV-A6 strains were successfully isolated; by successfully constructing a novel amplicon-based next-generation sequencing method, the full genomes of the 46 strains were sequenced. All strains belonged to the D3c subgenotype. A new recombination lineage emerged with three specific amino acid mutations in the P3 region. Fourteen amino acid sites in the P3 coding region and three in the VP1 coding region were identified as highly variable amino acid sites. On the whole, viral evolution was predominantly driven by negative selection. Three amino acid sites in the VP1 coding region and one in the P3 coding region were under positive selection, among which P3-551 was both a highly variable amino acid site and underwent positive selection pressure. CV-A6 strains in Shandong, 2023, may have originated from Southwest China lineages, which emerged from Vietnam. CV-A6 was the primary pathogen of onychomadesis and atypical skin lesions. The increase in age, male individuals, living in suburban areas, with fever and respiratory symptoms were significant risk factors for atypical skin lesions. In 2023, CV-A6 was the predominant pathogen of HFMD in Shandong and underwent rapid evolution with frequent recombination and mutations. Therefore, surveillance for disease prevention and control should be strengthened.