Diversity of human astroviruses in untreated domestic sewage: a next generation sequencing analysis in Jining city, Shandong province

ObjectiveTo investigate the prevalence and genetic diversity of human astrovirus (HAstV) strains in untreated domestic sewage. MethodsWe collected six domestic sewage samples (one liter for each) at an inlet of an urban sewage treatment plant quarterly from January 2018 to June 2019 in Jining city, Shandong province. After concentrated via anion membrane adsorption elution method, the viral nucleic acid in the samples was extracted with MagMAX Pathogen RNA/DNA Kit and complete open reading frame 2 (ORF2) region of HAstV was amplified using real-time reverse transcription PCR (RT-PCR). Then the amplicons were subjected to next generation sequencing (NGS). Clean data of NGS were assembled de novo to form contigs using CLC Genomics Workbench 12.0 (QIAGEN, USA) with default parameters for homology and phylogenetic analysis. ResultsAll the six sewage samples were positive for HAstV. Totally 43 ORF2 sequences of HAstV were obtained and classified into 9 genotypes including classic HAstV-1 to HAstV-5 and novel MLB1 and VA1-3. HAstV-5 was the most common genotype (47.53%). Phylogenetic analysis revealed that local and foreign sequences clustered together in some branches, indicating the transmission of various genotypes of HAstV among different regions in the world. ConclusionThe analysis results demonstrates that NGS-based environmental surveillance could greatly improve the understanding on HAstV genetic diversity.