Molecular Epidemiology Of Norovirus And Rotavirus Based On Sewage Surveillance In Shandong Province

[Background]Gastroenteritis is one of the most widespread and most common diseases in the world.Its main clinical manifestations include diarrhea,vomiting and stomach pain.It maintains heavy social and economic burden in both developed and developing countries.Since the first large-scale survey in China,the incidence of acute gastroenteritis(AGE)has dropped from 0.76/person year to 0.56/person year.Despite the decline in morbidity,AGE still imposes a huge disease burden on society.Pathogens that cause the onset of AGE include bacteria,viruses,and parasites.With the spread of safe water and increased awareness of personal hygiene and the application of anti-infection,viral pathogens have become the main cause of AGE.Viral gastroenteritis(VGE)can be caused by a variety of different viruses,including primarily norovirus(NoV),rotavirus(RoV),astrovirus(AstV),sapovirus(SaV)and adenovirus(AdV).Among them,NoV and RV are the main causes of VGE.Strengthening the prevention and control of NoV and RV can effectively reduce the disease burden caused by AGE.Study on the molecular epidemiology of NoV and RV can effectively monitor the prevalence of in the local populations,provide a scientific basis for the prevention and control of gastroenteritis,and provide a theoretical basis for the development of diagnostic reagents and vaccines.At present,the surveillance of gastroenteritis virus is generally based on the monitoring of clinical specimens.Environmental surveillance is a simple,sensitive,effective and fast approach for enteric viruses.It can provide valuable information in the area with no or weak case-based surveillance.In view of the fact that NoV and RVA can be discharged from the infected person through the domestic sewage collection pipe into the domestic sewage treatment plant,the domestic sewage is collected from the water inlet of the urban domestic sewage treatment plant and virus detection and analysis is carried out,and the urban sewage pipe network coverage area can be obtained.Molecular epidemiological characteristics of NoV and RVA in the population can be obtained.[Objectives]To describe the temporal dynamics of Nov and RVA in sewage,understand the genotype distribution and changes,and to explore the genetic characterization,in order to lay scientific foundation for the establishment of integrated population and environment surveillance system.[Methods:]1.According to the geographical characteristics of plain,mountainous area and coastal area,domestic sewage sentinel sites were set up in the cities of Jinan,Linyi and Yantai of Shandong Province.Sewage samples were collected monthly from January 2017 to June 2018,and membrane adsorption-elution method was performed to concentrate viruses from obtained sewage samples.2.After viral RNA extraction,RT-PCR and TA cloning,NoV and RVA target genes were sequenced for genotyping.3.BioEdit 7.2.5was used for sequence homology comparison,and Mega 7.0 software was used for phylogenetic analysis by using the neighbor-joining method.[Results]1.Detection rate of NoV and RVA in domestic sewages.We collected 54 raw sewage from January 2017 to June 2018 in Jinan,Yantai and Linyi.Among 54 samples,53 were positive for NoV GI genotype,with a detection rate of 98.1%.There were 52 positive samples of NoV G? genotypes,the detection rate was 96.3%;52 positive samples of RVA G genotypes,the detection rate was 96.3%;52 positive samples of RVA P genotypes,the detection rate was 96.3%.2.Distribution of NoV and RVA genotypes.We collected 382 sequences of NoV I belonging to 6 genotypes including G?.1,G?.2,G?.3,G?.4,G?.5 and GI.6.The two most frequently detected genotypes were G?.6 and GI.5,accounting for 40.3%(154/382)and 39.8%(152/382)of the total detection,respectively.A total of 282 NoV ? sequences belonged to 6 genotypes including G?.2,G?.3,G?.4,G?.8,G?.13 and G?.17.GIL 17(70.9%,200/282)was the most common genotype.There were 305 RVA G sequences obtained in this study including G1,G2,G3,G4,G6,G8 and G9.G9(86.9%,265/305)was the most common genotype.A total of 410 RVA P sequences were obtained belonging to 6 genotypes including P[1],P[3],P[4],P[8],P[9]and P[14].P[4](45.1%,185/410)and P[8](48.3%,198/410)were two most common genotypes.3.homologous comparison and phylogenetic analysis of NoV and RVA.In this study,the sequence similarities among NoV GI.5 sequences were low,and the phylogenetic tree showed that it was divided into three evolutionary branches.GI.6 sequences had high sequence homology among themselves and the phylogenetic tree showed that it was divided into 4 evolutionary branches.NoV GII.2 had high sequence homology,and the phylogenetic tree showed that it was divided into three evolutionary branches.The sequence homology was low for GII.17 genotype,and the obtained GII.17 was divided into 2 clusters in the phylogenetic tree,most of the sequences were closely related to Kawasaki 308 variant.The obtained RVA G9 sequence had low homology,and the phylogenetic tree showed that it was divided into two evolutionary branches;the RVA P[4]and P[8]sequences had high homology,and the phylogenetic tree showed the multiple transmission lineages of these genotypes co-circulated among local population.[Conclusions]1.NoV and RVA are highly detected in the local population,and there are multiple transmission lineages of NoV G?.5,G?.6 and G?.2,G?.3,G?.17 and RVA G9 and P[4],P[8]co-circulated.2.NoV GI.5 detection increased significantly,which may become the local dominant genotype in the near future,the dominant genotypes of RVA P appeared alternately during this monitoring,but phylogenetic analysis did not show evidence of newly emerging variant,suggesting that this phenomenon may be related to the changes of immune level in the same period.3.100%nucleotide identities can be observed for sequences from different time or region,indicating the continuous circulation and inter-city transmission.