| BackgroundHuman astrovirus(HAstV)is nonenveloped single-stranded RNA virus,which is one of the common viruses that cause acute gastroenteritis in children worldwide.Astrovirus have been reported to be associated with central nervous system complications in immunocompromised patients,such as meningitis and encephalitis.HAstV compose of classic HAstV serotypes-1 to-8 and recently discovered novel HAstV-MLB and HAstV-VA strains.A large number of studies have shown that environmental sewage surveillance is an effective way to understand the prevalence and diversity of gastroenteritis virus in local population.However,there is a lack of comprehensive research on classic and novel HAstV in environmental sewage.In recent years,the next generation sequencing(NGS)has begun to be applied in the environmental sewage monitoring of gastrointestinal viruses.NGS for PCR amplicons has greatly enriched the understanding on the virus spectrum in sewage.In this study,sewage samples were collected monthly from Guangda Sewage Treatment Plant in Jinan,Shandong Province from 2018 to 2021.Different from previous studies which focused on partial ORF1b or ORF2 gene,the amplification area of this study was ORF2 full-length ORF2 coding region,a more comprehensive sequence information will be obtained.On this basis,sequence analysis was carried out to explore distribution and temporal dynamic variation characteristics of HAstV type in sewage,in order to provide a scientific basis for the establishment of prevention and early warning mechanism for gastroenteritis diseases.Objectives1.Based on full-length ORF2 region amplification combined with NGS technology,we explore the type distribution of classic HAstV and novel HAstV in environmental sewage in Jinan City,Shandong Province in 2018-2021.2.Through the collection and analysis of HAstV prevalence information in environmental sewage,clinical surveillance will be supplemented.We obtain the epidemic data of whole population,and explore the long-term cyclic dynamic changes.3.The understanding of HAstV transmission will improve by conducting phylogenetic analysis of HAstV in Jinan,Shandong Province and other types in China and abroad.Methods1.Collection and concentration of environmental sewage:In 2018-2021,a total of 48 samples of sewage from the inlet of Jinan Guangda Sewage Treatment Plant were collected.Anion membrane adsorption ultrasonic oscillation method was applied to the concentration of sewage samples.2.Viral nucleic acid extraction:Viral nucleic acid was manually extracted utilizing the MagMAX pathogen RNA/DNA extraction kit.3.Amplification and sequencing of viral nucleic acids:The HAstV full-length ORF2 region was amplied and forwarded to NGS.NGS data was assembled and analyzed using CLC Genomics Workbench 12.0 software.4.Similarity analysis and phylogenetic analysis:BioEdit 7.0.9.0 software was used for similarity analysis;MEGA 11.0 software was used to construct phylogenetic trees.Results1.The detection of HAstV:A total of 48 domestic sewage samples were collected during the study,of which 40 were HAstV-positive,with a positive rate of 83.3%.In this study,94 amplicons were obtained,a total of 16 astrovirus were detected from all 40 sewage samples,including classic HAstV(genotypes 1,2,3,4,5,6 and 8),HastV-MLB(strains 1-3),HastVVA(strains 1-6).Three classic HAstVs(HAstV-1,HAstV-3,and HAstV-4)were detected in HAstV-positive sewage samples.In 22.5%(9/20)of sewage samples,a new type VA6 was identified and proposed with nine full-length ORF2 sequences obtained.They had 82.3%-83.3%nucleotide similarity with the closest strain VA5.2.Similarity and phylogenetic analysis based on full-length ORF2 coding region:According to the similarity analysis of HAstV type detected in this study,the Shandong strains within a single type had the highest similarity.Phylogenetic analysis showed that HAstV-1,HAstV-2,HAstV-3,HAstV-5 and VA1 of Shandong strains were divided into more than two genetic lineages with multiple co-circulating transmission chains.In addition,we also found a new lineage composed mostly of Shandong strains.All types of Shandong plants were clustered in the same type in the phylogenetic tree.Conclusions1.From 2018 to 2021,various types of HAstV were detected in environmental sewage of Jinan city,Shandong Province,including new types VA6 and relatively rare types such as MLB2 and MLB3.The type spectrum was significantly rich.2.Environmental monitoring based on NGS has greatly improved the understanding of the transmission of HAstV.Therefore,a special environmental monitoring system for HAstV should be established to fully understand and grasp the actual circulation of the transmission in the population combined with clinical case monitoring data.3.HAstV-1,HAstV-2,HAstV-3,HAstV-5 and VA1 were the most common types with different evolutionary branches in the monitored population in Shandong.Obvious spatial and temporal aggregation were observed for Shandong strains. |
