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Omono River virus (OMRV) is a newly reported, unclassified RNA virus in the family Totiviridae, which infects mosquitoes and bats. In this study, we report the isolation of an OMRV strain SD76 from Culex tritaeniorhynchus captured in Jinan city, China. The cytopathic effect was characterized by cell fusion on C6/36 cell line. Its complete genome was 7611 nucleotides in length, with 71.4–90.4% similarities with other OMRV strains. Phylogenetic analysis based on complete genomes showed all OMRV-like strains can be divided into 3 groups with between-group distances ranging from 0.254 to 0.293. These results revealed that the OMRV isolate had high genetic diversity with those identified previously, and enriched the genetic information of family Totiviridae. [ABSTRACT FROM AUTHOR]

• We developed a complete ORF2 amplification method for detecting HAstVs. • Up to 18 astroviruses were detected, including a new strain, VA6. • 7 astroviruses (HAstV-1, −2, −4, −5, VA1, VA2, and VA3) were present in all samples. • Sewage contains astroviruses with considerable high diversities. • NGS based environmental surveillance has a broad application prospect. 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. [Display omitted] [ABSTRACT FROM AUTHOR]