Detection and Genotyping of Human Adenoviruses in Wastewater from Bulgaria by Targeted Surveillance, In Vitro Cultivation, and Whole-Genome Sequencing

Adenoviruses are detected in wastewater at significantly higher frequencies than most other enteric viruses. The aim of this study was to determine the abundance of human adenoviruses in samples collected from wastewater treatment plants in Sofia, Plovdiv, Burgas, and Varna using quantitative PCR (qPCR). During the study period in 2024, one or two peaks in adenovirus levels were detected in each city, with viral loads ranging from 10 to 30,000 genome copies/mL. Selected wastewater samples from Sofia were used to inoculate three different cell lines. Productive adenovirus replication was observed only in the Vero E6 cell line. Shotgun sequencing of DNA extracted from infected cells enabled the assembly of six complete adenovirus genomes from three qPCR-positive samples. Taxonomic analysis identified viruses belonging to the Mastadenovirus adami, Mastadenovirus blackbeardi, Mastadenovirus caesari, and Mastadenovirus dominans species, with the predominant genotype (three of six genomes) showing the highest similarity to HAdV-A31. Comparative genomic analysis provided evidence of molecular evolution driven by multiple recombination events. The recovered genomes contained hypervariable regions composed of genetic elements closely related to genotypes previously reported from different geographical regions. Overall, the integrated approach employed in this study demonstrates the value of combining wastewater surveillance, virus isolation, and whole-genome sequencing to monitor adenovirus circulation, characterize dominant variants, and assess their potential clinical significance.