Detection and Phylogenetic Characterization of Human Aichi Virus in Sewage by Next Generation Sequencing in Eastern China, 2019–2022

Aichi virus (AiV) infection is associated with gastroenteritis symptoms, and sewage surveillance has emerged as an effective approach for assessing AiV prevalence. However, a comprehensive understanding of its epidemiology and genetic diversity remains limited, particularly in China. In this study, monthly sewage samples were collected from Jinan, China, between 2019 and 2022. The AiV genome was quantitatively analyzed, and the VP1 region was amplified from sewage concentrates for subsequent next‐generation sequencing (NGS). The results revealed a remarkably high detection rate of 95.83% (46/48) over the 4‐year study period. Quantitative analysis showed AiV genome concentrations ranging from 1.4 × 10 3 to 2.7 × 10 5 genomic copies per liter in sewage, with distinct seasonal patterns and peak occurrences during summer and autumn. Phylogenetic analysis of the VP1 region revealed that Jinan sequences clustered into three groups within genotypes A and B, with an average p ‐distance of 0.091. Genotype A was detected in all sewage samples (100.00%), while genotype B was found in 52.50% of samples. In terms of relative abundance, they constituted 86.06% and 13.94% of the total clean reads, respectively. This study represents the first application of amplicon NGS for AiV detection in sewage samples in China, revealing the continuous co‐circulation of multiple transmission lineages and significant genetic diversity of AiV in wastewater systems. The findings demonstrate that NGS technology significantly enhances detection sensitivity and provides substantially more comprehensive data for the molecular epidemiology of AiV.