A 3-Year Environmental Surveillance Reveals Multiple Enteroviruses Including Types A76, A89, A90 and C113 in Eastern China.

Since molecular typing method of enterovirus was introduced, many new types have been discovered. However, due to the low epidemic potential, the information on newer enteroviruses remains limited globally. This study aims to investigate the diversity and phylogeny of newer enterovirus types through environmental surveillance utilizing next-generation sequencing (NGS) technology. Sixty-six wastewater samples were collected from two cities in eastern China between 2019 and 2021 and concentrated 100-fold using a negatively charged membrane method. After cell culture, 388 enterovirus isolates representing 14 serotypes were recovered, including Sabin-like poliovirus type 1 (n = 22) and type 3 (n = 57). Concurrently, RNA extraction was performed on all 66 sewage concentrates, and VP1 semi-nested RT-PCR yielded 56 amplicons, which were subsequently subjected to NGS. The NGS analysis identified a total of 33 serotypes, with echovirus 11, coxsackievirus A10, echovirus 18, coxsackievirus B4, and coxsackievirus B5 being the most prevalent, accounting for 29.11%, 9.87%, 8.27%, 8.14%, and 6.10% of the total reads, respectively. Newer identified enterovirus types A76, A89, A90, and C113 were detected in 1 (1.52%), 17 (25.76%), 5 (7.58%), and 6 (9.09%) sewage samples, respectively. Phylogenetic analysis based on the partial VP1 coding region indicated that these local enteroviruses formed distinct lineages separate from previously identified strains. These findings demonstrate that sewage contains enteroviruses with considerable diversity. NGS-based sewage surveillance offers a significant advantage in data output compared to the cell culture method and can be effectively utilized for monitoring newer enterovirus strains.