| [Background]Gastroenteritis is one of the common diseases in humans,presenting with the clinical symptoms of nausea,vomiting,diarrhea and abdominal pain.It can also lead to the dehydration and electrolyte disorder in the infant and old people with low immunity,and is the main reason for hospitalization and death of the infant and old people.Currently,variable pathogens are responsible for gastroenteritis.Of these,rotavirus(RoV),norovirus(NoV)and human asrtrovirus(HAstV)are the significant viral agents.RoV can be divided into seven groups,and group A,B and C can infect humans.RoV group A(RVA)is the predominant virus associated with gastroenteritis worldwide.Based on the difference in VP4 and VP7 sequences,RVA can be divided into at least 35 P genotypes and 27 G genotypes.Of these,G1～G4,P[8]and P[4]are the most prevalent genotypes.The detection rate of NoV in the patients with gastroenteritis is second to RVA.More importantly,NoV is the most frequently detected virus in the gastroenteritis outbreaks.According to the difference in VP1 sequences,NoV can be divided into seven genogroups(GⅠ,GⅡ and GⅣ can infect humans)and more than 40 genotypes.Of these,GⅡ.4 is one of the most important genotypes,because novel GⅡ.4 variant emerges every two years and can spread globally.HAstV is detected at a lower detection rate than RVA and NoV in the clinical cases.HAstV consists of eight serotypes,and HAstV-1 is the most prevalent serotype globally.RVA,NoV and HAstV have a high genetic diversity and evolve frequently.The molecular characteristics of these viruses vary in the different populations,regions and periods,and epidemiology surveillance for these viruses is essential,which will contribute to take measures to reduce the disease burden of gastroenteritis.At present,case-based surveillance is the main approach to obtain epidemiology characteristics of these viruses.As a novel surveillance method,environmental sewage surveillance has been successfully used in enterovirus(EV)research.Fecal-oral route is the main transmission pathway of RVA,NoV and HAstV,and viral particles can also be shed from infected humans and finally transported to the wastewater treatment plants just like EV.In view of these,environmental sewage analysis can also be an important supplement to the surveillance for RVA,NoV and HAstV.The case-based surveillance for viral gastroenteritis is not well established in China.The limited information on molecular characteristics of RVA,NoV and HAstV can only be obtained from rare case reports.As a novel and independent surveillance system,environmental sewage surveillance is expected to be an important approach to evaluate the epidemiology of RVA,NoV and HAstV in China.[Objectives]1.To investigate the distribution and time fluctuation of RVA,NoV and HAstV genotypes in urban sewage in Shandong Province from 2013 to 2016.2.To analyze the phylogenetic relationships between RVA,NoV and HAstV sequences from raw sewage in this study and reference sequences downloaded from GenBank database.3.To evaluate the recombination event and evolutionary genetics characteristics of the predominant RVA,NoV and HAstV genotypes in Shandong Province.[Methods]1.From 2013 to 2016,raw sewage samples were monthly collected in the inlet collector canal of wastewater treatment plants in Jinan,Linyi and Yantai of Shandong Province,and an adsorption-elution method was used to concentrate viruses from sewage samples.2.RT-PCR,TA cloning and sequencing of genes of RVA,NoV and HAstV for genotyped were performed after viral RNA extraction.3.BioEdit 7.1.9 was used to carry out sequence alignment and homologous comparison.Mega 5.02 was used to compute mean distance and construct the phylogenetic tree by using the neighbor-joining method.4.Recombination event was analyzed by RDP 4.0 software package.5.Based on Bayesian Markov Chain Monte Carlo(MCMC)method,evolutionary genetics characteristics of RVA,NoV and HAstV were estimated by BEAST 1.7.5.[Results]1.Detection rates of RVA,NoV and HAstV in environmental sewages.From 2013 to 2016,a total of 132 samples were collected.RVA G-genotype,RVA P-genotype,NoV GⅠ,NoV GⅡ and HAstV were detected at the detection rates of90.9%,89.4%,100%,97.0%and 81.8%,respectively.2.Distribution of RVA,NoV and HAstV genotypes in environmental sewages.A total of 758 RVA G-genotype sequences were obtained,belonging to 7 genotypes(G2,G3,G4,G6,G8,G9 and GⅡ).Of these,G9 was the predominant one(91.4%,693/758).The number of RVA P-genotype sequences obtained in this study was 950,and P[8]and P[4]were two most common genotypes,with the proportions of 53.7%(510/950)and 39.1%(371/950)。There were 1138 NoV GⅠ sequences obtained,belonging to 8 genotypes(GⅠ.1,GⅠ.2,GⅠ.3,GⅠ.4,GⅠ.5,GⅠ.6,GⅠ.8 and GⅠ.9),and GⅠ.6 was the most frequently detected genotype(44.1%,502/1138).Since 2014,the dominant genotype of NoV GⅠshifted from GⅠ.2 to GⅠ.6.There were 886 NoV GⅡ sequences obtained,belonging to14 genotypes(GⅡ.1,GⅡ.2,GⅡ.3,GⅡ.4,GⅡ.5,GⅡ.6,GⅡ.8,GⅡ.9,GⅡ.10,GⅡ.13,GⅡ.14,GⅡ.15,GⅡ.17 and GⅡ.21).and GⅡ.17(39,0%,345/886),GⅡ.3(27.1%,240/886),GⅡ.4(12.2%,108/886)and GⅡ.6(8.7%,77/886)were four most common genotypes.A total of 526 HAstV sequences were obtained in this study,belonging to 5 genotypes(HAstV-1 to HAstV-5),and HAstV-5 was the most prevalent genotype(45.4%,239/526).Since 2015,the dominant genotype shifted from HAstV-1 to HAstV-1 in the study area.3.Homologous comparison and phylogenetic analysis of dominant genotypes of RVA,NoV and HAstV.The nucletide and amino acid identities of RVA G9,P[8]and P[4]sequenceswere low,and multiple transmission lineages of these genotypes co-circulated in the local population.NoV GI.2 sequences had high identities among themselves,and located at one branch in the phylogenetic tree.NoV GⅠ.6 sequences had low identities among themselves,and can be divided into two clusters.GⅡ.4 sequences in this study belonged to Den Haag 2006b and Sydney 2012 variant.However,Den Haag 2006b variant was only detected in Jinan in 2014.Since 2015,all GⅡ.17 sequences obtained from sewage samples shifted to Kawasaki 2014 variant,proving that this novel variant circulated in Shandong.Besides,GⅡ.17 Hong Kong 2015 variant which was rare reported was also detected in Yantai in 2014.HAstV-1 and HAstV-5 sequences showed low identities among themselves,and multiple transmission lineages co-circulated in Shandong.4.Recombination analysis of the dominant genotypes of RVA,NoV and HAstV.We identified 4 recombination sequences in RVA sequences.Of these,two were G9 and P[8]intragenetic recombination sequences,and another two were recombination sequences between P[4]and P[8].A total of 11 NoV GⅠ recombination sequences were recognized,presenting 6 recombination combinations,and GⅠ.2/GⅠ.5 was the most common one.There were 3 NoV GⅡ recombination sequences obtained,presenting one recombination combination(GⅡ.3/GⅡ.6).The number of HAstV recombination sequences was up to 36,and HAstV-1/HAstV-5 was the most common recombination combination.5.Evolutionary and genetics analysis of the dominant genotypes of RVA,NoV and HAstV.RVA G9 originated from the year of 1970.6,and evolves at the rate of 1.3×10-3 s/s/y(nucleotide substitutions per site per year).P[8]evolves faster than P[4],and originated at a earlier time.NoV GI.6 also evolves faster than GI.2,and originated at an earlier time.The time to the most recent common ancestor(TMRCA)of GⅡ.4 and GⅡ.17 were 1983.4 and 1956.5,and evolutionary rates were 3.6×10-3 s/s/y and 2.8×10-3 s/s/y,respectively.Besides,the evolutionary genetics characteristics of HAstV-1 and HAstV-5 were reported for the first time in this study,and TMRCA of these two genotypes were 1960.4 and 1993.The evolutionary rates of them were 1.3×10-3 s/s/y and 4×10-3 s/s/y,respectively.[Conclusions]1.RVA,NoV and HAstV infections are common in the local population based on the high detection rates of these viruses in sewages.2.G9,P[8]and P[4]were the most prevalent RVA G-and P-genotypes in Shandong.GⅠ.2,GⅠ.6 and GⅡ.17,GⅡ.3,GⅡ.4 were the dominant genotypes of NoV GⅠ and GⅡ.HAstV-1 and HAstV-5 were the most frequently detected genotypes of HAstV.In addition,multiple transmission lineages of aforementioned genotypes co-circulated in the study area.3.GⅡ.4 Sydney 2012 variant was the main circulating GⅡ.4 variant in Shandong.However,Den Haag 2006b variant has been detected in Jinan in 2014.Long-term surveillance should be conducted to verify whether Den Haag 2006b variant is still circulating in the local population.Moreover,since 2015,all GⅡ.17 sequences obtained from sewage samples belonged to GⅡ.17 Kawasaki 2014 variant,proving the circulation of this novel GⅡ.17 variant in Shandong.4.During the study period,the dominant genotype of NoV GⅠ shifted from GⅠ.2 to GⅠ.6,and the dominant genotype of HAstV shifted from HAstV-1 to HAstV-5.However,the results of phylogenetic analysis showed that no novel GⅠ.6 and HAstV-5 variant emerged.We speculate that the change of herd immunity level may lead to this phenomenon.5.Recombination events were detected in RVA,NoV and HAstV sequences,and co-infections may be common in the local population. |
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