To analyze the contamination status of human enteroviruses (HEVs) in untreated municipal sewage and identify viral serotypes based on the municipal sewage network in Guizhou province.

Influent sewage samples were collected from monitoring sites in Guiyang city (Nanming district), Zunyi city (Xinpu new district), Liupanshui city (Zhongshan district), and Tongren city (Bijiang district) in Guizhou province from November 2024 to January 2025. Ten virus-enriched sewage samples were processed by a combined anion-exchange membrane adsorption-elution and ultrasound-assisted elution method. The concentrated viral particles were then inoculated into human rhabdomyosarcoma (RD) cells and mouse L20B cells expressing the human poliovirus receptor. RNA was extracted from virus-positive isolates obtained through cell culture, followed by reverse-transcription polymerase chain reaction (RT-PCR) to amplify the VP1 gene of enteroviruses. Genetic sequencing and phylogenetic analysis were performed to characterize the obtained viral strains.

A total of 26 poliovirus (PV) vaccine-related strains and 11 non-polio enterovirus (NPEV) strains were isolated from the 10 sewage samples. Of the PV strains, 10 were of serotype I and 16 were of serotype Ⅲ. All the 26 PV vaccine-related strains exhibited fewer than 10 nucleotide variations in the VP1 region compared to the Sabin reference strains. The NPEV strains included seven echovirus 11 (E11) strains, one echovirus 6 (E6) strain, and one strain each of coxsackieviruses B2 (CVB2), B3 (CVB3), and B5 (CVB5). Phylogenetic analysis revealed that the seven E11 strains belonged to the genetic subcluster D5.

HEVs were detected in sewage samples from the municipal sewage system of Guizhou province, while no wild poliovirus or vaccine-derived poliovirus was identified. Viral surveillance data confirm the potential environmental contamination by HEVs and could inform the development of wastewater management policy standards.