Characterization of non-polio enteroviruses (Picornaviridae, Enterovirus) isolated from clinical specimens and wastewater samples collected in Saint Petersburg and the Leningrad Region

Introduction. Enteroviruses (genus Enterovirus, family Picornaviridae) are one of the common causes of infectious diseases with neurological and dermatological manifestations in children. Surveillance of these viruses is carried out as part of the program Epidemiological surveillance and prevention of enterovirus (non-polio) infection for 2023–2027. Objective. To study the type diversity of non-polio enteroviruses (NPEVs) isolated from clinical specimens and wastewater samples collected in Saint Petersburg and the Leningrad Region, and to perform molecular epidemiological characterization of Coxsackievirus B5 isolates. Materials and methods. Stool samples were collected from resident children with clinical symptoms, healthy migrants under 5 years of age; wastewater samples were also included in the study. Enterovirus isolation was performed by inoculation of specimens onto RD, HEp-2, and L20B cell cultures. Molecular genetic methods included PCR, Sanger sequencing, nucleotide sequence analysis and phylogenetic analysis using the R programming language. Estimation of tMRCA and construction of nucleotide identity heatmaps were performed using TreeTime and SDTv 1.3 software, respectively. Results. A total of 25 NPEV types were identified. At the species level, Enterovirus betacoxsackie predominated – 53/71 (74.6%; 95% CI: 62.9–84.2%), followed by Enterovirus alphacoxsackie – 17/71 (23.9%; 95% CI: 14.6–35.5%), and Enterovirus coxsackiepol – 1/71 (1.4%; 95% CI: 0.04–7.6%). In Saint Petersburg and the Leningrad Region, viruses belonging to Enterovirus betacoxsackie were most frequently detected: Coxsackievirus B5 (12.7%), ECHO 11 (11.3%), ECHO 13 (8.5%), ECHO 14 (8.5%), and ECHO 6 (8.5%). A notable prevalence of ECHO 25 (5.6%) and ECHO 29 (5.6%) was also observed. A comprehensive analysis of the phylogenetic tree, nucleotide identity heatmap, and tMRCA estimates showed that the CVB5 strains isolated from resident children in 2018 and from migrant children in 2021 belonged to two distinct genetic lineages, indicating multiple independent introductions of the virus into the Russian Federation. Conclusion. The frequent detection of Enterovirus betacoxsackie in the studied collection of isolates and the identification of two genetic lineages of Coxsackievirus B5 confirm the complexity of its epidemiological dynamics and highlight the necessity of continuous surveillance for the timely detection of new genetic variants.