Environmental surveillance reveals enterovirus diversity in Jinan, China: detection of types D68, A71, A76, B88, A90, and C99

Enterovirus (EV) exhibits high evolutionary activity and is associated with a wide spectrum of human diseases. Environmental surveillance employing next-generation sequencing (NGS) is an effective approach for elucidating EV type diversity and genetic variation; however, similar studies in China are limited. Sewage samples were collected monthly in Jinan, China, from January to December 2024 and subsequently concentrated. EV isolation was conducted using cell culture, followed by VP1 amplification and Sanger sequencing. Concurrently, VP1 amplicons directly obtained from sewage concentrates were subjected to NGS. Similarity and phylogenetic analysis were performed. A total of 104 EV strains representing nine serotypes were isolated via cell culture, with echovirus 11 (E11) and E3 being the most prevalent, accounting for 34.62% (36/104) and 27.88% (29/104) of the total isolates, respectively. NGS technology identified 31 EV types, with CVB4 and CVA16 exhibiting the highest read counts. Additionally, uncommon types such as D68, A71, A76, B88, A90, and C99 and 11 rhinovirus types were detected in the sewage samples. Phylogenetic analysis revealed that CVA16 and D68 sequences from sewage had close genetic relationship with recent Chinese strains, and multiple branches of B88 and C99 were observed. This study represents the first detection of D68 and B88 in sewage in China, demonstrating co-circulation of diverse enteroviruses with significant genetic variability in the region. These findings underscore the utility of environmental surveillance for tracking EV epidemiology and evolution.IMPORTANCEEnteroviruses are significant human pathogens associated with frequent epidemics and outbreaks. Although numerous types exist, some exhibit low prevalence and are rarely detected. By implementing next-generation sequencing (NGS) in wastewater surveillance, this study comprehensively characterized enterovirus diversity, revealing multiple uncommon types of interest. Notably, we detected EV-D68-an emerging agent linked to acute flaccid myelitis-in sewage, underscoring its public health relevance. Additionally, we identified multiple rhinovirus types. These data demonstrate the local circulation of diverse viruses with substantial public health implications and provide a critical foundation for disease monitoring and early warning systems.