Molecular Epidemiology Of Group A Rotavirus In Domestic Sewage In Jinan By Next Generation Sequencing

[Background]Rotavirus(RV)is the main pathogen causing the death of infants and young children from diarrhea.Its transmission route is the fecal-oral route,and the main clinical manifestations are fever,vomiting,diarrhea,dehydration,electrolyte imbalance and so on.Although there are already available vaccines for RV,there are hundreds of thousands of deaths caused by RV every year,which brings a heavy burden of disease to society.According to the antigenicity of VP6,RV can be divided into 10 groups A-J.Among them,group A rotavirus(RVA)is the main pathogen causing infant diarrhea,and 90%of infant RV diarrhea is caused by RVA.According to the antigenicity of VP7 and VP4,RV can be further divided into G genotype and P genotype.So far,36 G genotypes and 51 P genotypes have been found.Diarrhea caused by RVA is prevalent all year round without obvious regional characteristics.These genotypes,G1-G4,G9,P[8]and P[4],circulate together to form the common prevalent types in the world.However,the G and P genotypes and their combination types that are popular in various countries in the world mostly show complex and changeable characteristics in space and time.By analyzing the molecular epidemiological characteristics of RV,it is helpful to have a comprehensive understanding of the prevalence of RV in the population of the study area during a certain period,which is of great public health significance for taking targeted prevention and control measures and effectively reducing the burden of related diseases.The current RVA population surveillance data mainly comes from hospitalized and outpatient diarrhea children,but there are still a large number of patients who do not seek medical attention such as light,hidden infections and convalescent carriers in the population.Therefore,pathogen information derived from clinical specimens mainly reflects the prevailing dominant types in the population,and other non-mainstream types or even potential dominant types are usually unable to provide comprehensive information,which is prone to surveillance information bias and even surveillance gaps,thereby underestimating the true prevalence of RVA in a region.Environmental surveillance,as a sensitive means of virus surveillance,has been well used in the molecular epidemiology of enterovirus(EV).In view of the fact that the infection and transmission of RVA are mainly through the fecal-oral route,it is determined that the RVA surveillance information from domestic sewage is more comprehensive,can better reflect the prevalence of RVA in the whole population,and make up for the lack of clinical surveillance data,especially in some areas where RVA surveillance system is relatively weak,environmental surveillance of RVA is more important.The newly emerging next generation sequencing(NGS)technology is more advantageous in exploring the complex pathogen species distribution in the specimen,and its application in the RVA surveillance in environmental sewage can provide more comprehensive data to further clarify the prevalence diversity and genetic variation rule of RVA in the population.[Objectives]In this study,the RVA VP7 and VP4 genes in domestic sewage in some months of 2018 and the whole year of 2019 in Jinan area were amplified,and the amplicons were compared and analyzed by TA cloning and sequencing and NGS technology respectively,so as to achieve the following research objectives:1.Establish an amplicon-based NGS method to detect RVA in sewage,and evaluate its advantages compared with traditional TA cloning and sequencing methodology.2.Understand the distribution of RVA types and dynamic characteristics in domestic sewage in Jinan.3.Explore the phylogenetic characteristics of RVA epidemic types in Jinan.[Methods]1.Sewage collection and concentration:Domestic sewage surveillance sites were designated and sewage samples were collected monthly from January to December 2019 using the timed and quantitative collection method.Membrane adsorption-elution method was used to concentrate and enrich virus from sewage samples.2.RNA extraction,RT-PCR and sequencing:RNA was extracted from sewage in 2019 and September and November of 2018 for RVAG and P gene amplification.The RT-PCR positive products of sewage samples from two months in 2018 were sequenced by two different methods.Method 1 was TA cloning and Sanger sequencing,and method 2 was NGS sequencing of the positive amplicons.The latter was used for all the month samples in 2019.3.Sequence analysis:RVA Sanger sequencing results from two months in 2018 were assembled using the Bioedit 7.0.9.1 software.The CLC Genomics Workbench 12.0 software was used to analyze the obtained NGS results,and all the obtained sequences were subjected to BLAST comparison and finalization.Two methods were used to detect and analyze sewage samples from two months in 2018 for methodological evaluation.The RVA sequences obtained in 2019 were analyzed using BioEdit 7.0.9.1 software for nucleotide and amino acid homology,and Mega 7.0 software was used for phylogenetic analysis.[Result]1.Comparison of different methods for detecting RVA genotypesIn the water samples from September and November 2018,the RVA G and P genotype tests of both methods were all positive.Method 1 only obtained the G9 and P[8]genotype sequences in two months.Method 2 detected 5 G genotypes in the water samples in September,namely G2,G3,G9,G14,and G27;and detected 5 G genotypes in the water samples in November,namely G1,G2,G3,G9 and G14;2 P genotypes,P[4]and P[8]were detected in both months.G9 and P[8]were the genotypes with the highest proportion detected in method 2.The detection rates of RVA genotypes by the two methods were 25%and 87.5%,respectively.2.Detection,distribution and temporal dynamics of RVA genotypes in 2019A total of 12 sewage samples were collected in 12 months of 2019 and tested by RVA VP7 and VP4 RT-PCR,among which 11 samples were positive for RVA G genotype,with a detection rate of 91.7%(11/12).All 12 samples were positive for RVA P genotype,and the detection rate was 100%(12/12).A total of 10 RVA G genotypes were found in this study,which were G1,G2,G3,G4,G6,G9,G11,G12,G27 and G32,among which G9 genotype accounted for the highest reads proportion,followed by G3 genotype,which accounted for 75.60%and 19.24%,respectively.A total of 14 P genotypes were detected,and they were P[3],P[4],P[6],P[8],P[9],P[14],P[17],P[24],P[30],P[31],P[35],P[36],P[38]and P[45],respectively.Among them,P[8]has the highest detection rate(86.70%).G9 was the dominant genotype of each month in the first half of 2019,and the detection proportion of G3 genotype increased month by month from July,the G3 genotype in October and November became the dominant genotype of the month.P[8]has always been the P genotype with the highest detection rate,but the detection rates of P[3]and P[4]both showed a brief increase.3.RVA homology and phylogenetic analysisAfter NGS sequencing,assembly,and analysis of samples in 2019,there were many sequences obtained,most of which were inconsistent in length.In this study,sequences close to the full length of the PCR target fragment were selected for homology and phylogenetic analysis.A total of 7 G genotype and 8 P genotype sequences were obtained.The 7 G genotypes were G1(1),G2(5),G3(22),G4(1),G6(11),G9(11)and G12(2),respectively.Among them,G9 has high intratype homology,and the phylogenetic tree shows that it can be divided into two evolutionary branches.G3 has low intratype homology,and the phylogenetic tree shows that it can be divided into 4 evolutionary branches.The intratype homology between G2 and G12 types are high,and the phylogenetic tree shows that they are both in the same evolutionary branch.The intratype homology within the G6 type is low,and the phylogenetic tree shows that it can be divided into two evolutionary branches.The 8 P genotype sequences are P[3](13),P[6](3),P[8](11),P[9](2),P[14](1),P[17](2),P[30](1)and P[31](5).Among them,P[8]has high intratype homology,and the phylogenetic tree shows that it can be divided into two evolutionary branches.Except for the low intratype homology of the P[17]sequence,the genotypes of more than one sequence obtained have high intratype homology.The phylogenetic tree shows that P[6]can be divided into two evolutions branches.[Conclusions]1.There is abundant RVA genetic information in domestic sewage,and sewage surveillance is an effective means to grasp the regional prevalence of the human digestive tract viruses.2.The detection rate of RVA in domestic sewage in the surveillance area was very high,suggesting that RVA was persistently prevalent among the population in the surveillance area.3.Great genotype diversity of RVA was observed in the surveillance area.G9 and P[8]showed obvious dominant prevalence trend..4.Phylogenetic analysis showed that RVA G9 and P[8]genotypes in the surveillance area had multiple transmission chains.5.Compared with the traditional TA cloning and Sanger sequencing method of amplicons,NGS method can provide more information on RVA diversity.