Detection of multiple human astroviruses in sewage by next generation sequencing

Human astrovirus (HAstV) composes of classic HAstV serotypes 1-8 and recently discovered novel HAstV-MLB and HAstV-VA strains. A number of studies have demonstrated that wastewater analysis is an effective approach to understand the prevalence and diversity of enteric viruses in local population. However, a comprehensive analysis of classic and novel HAstVs in sewage is still lacking. In this study, sewage samples were collected monthly from Jinan, China during 2018-2019. Quantification of HAstV genomes was performed by real-time quantitative PCR. Different from previous studies which focused on partial ORF1b or ORF2 gene, complete ORF2 region of HAstV was amplified from sewage concentrates, and amplicons were subjected to next generation sequencing (NGS) and genetic analysis. This methodology allowed detection of 18 astroviruses, of which 7 (HAstV-1, -2, -4, -5, VA1, VA2, and VA3) were detected in all sewage samples. A new strain VA6 mapped to the HMO clade was identified in 20.8% of samples, with 82.4%-83.3% nucleotide identities to the closest strain VA5. The viral load of classic, MLB and VA clades in sewage samples ranged from 3.7 × 10⁴ to 4.6 × 10⁷, 3.4 × 10⁴ to 3.9 × 10⁶, and 3.3 × 10⁴ to 4.1 × 10⁶ copies per liter, respectively. Phylogenetic analysis based on complete ORF2 region reflected local HAstVs within each genotype constituted multiple co-circulating lineages. Existence of several new lineages composed exclusively or predominantly of Chinese sequences was observed as well. These results demonstrate sewage contains astroviruses with considerable high diversities. NGS based environmental surveillance greatly improves the understanding of HAstV circulation and should be encouraged.