| [Background]Infectious diarrhea also referred to gastroenteritis is the second leading cause of morbidity and mortality worldwide.Acute gastroenteritis remains consistently as one of the leading six causes of all deaths.The most common cause is an infection of the intestines mainly due to a virus.To date,five viruses definitely causing gastroenteritis have been discovered:rotavirus,norovirus,enteric adenoviruses,astrovirus,and sapovirus.Sapoviruses ranked second to fourth as the major viral pathogens among patients with sporadic gastroenteritis.Sapovirus and norovirus which mainly infect human species are known as human calicivirus.Although many animal calicivirus strains have been passaged in vitro,human calicivirus has not been grown successfully in cell cultures yet.The invention of PCR in 1980s facilitated the study about human calicivirus reaching to molecular level.Sapovirus is a nonenveloped virus containing a positive-sense,single-stranded RNA genome with highly genetical diversity.Current sapoviruses were classified into 15 genogroups and further multiple genotypes.Four genogroups(GⅠ,GⅡ,GⅣ,and GⅤ)were widely accepted to mainly infect human.Clinical manifestations in human include asymptomatic infection,diarrhea which may be milder,severe,complicated or dehydrated,and even death.Diarrhea is often associated with substantial medical and healthcare costs and thus has a high economic impact on society.Sporadic and outbreak gastroenteritis cases related to sapovirus have been reported in Asia,Europe,Africa,North America,South America and Oceania.Sapovirus showing Lower infective dose,diversified transmission routes,and resistance to external environment can cause acute gastroenteritis in all age groups in different settings throughout the year.Asymptomatic infections are often the potential source of outbreaks.In recent years,sapovirus as an emerging role of public health problems is recognized gradually.The outbreaks related to sapovirus have occurred frequently and the prevalence is increasing worldwide.New genogroups,genotypes,variants and recombination strains are emerging.However,people always lack immunity to these emerging viruses.To date,research about acute gastroenteritis,a common infectious disease,is not a high priority items appealing not enough attention from the public resulting less epidemiological data about circulation in specific population.Hence,more etiological data is demanded to access through extra ways such as monitoring of diarrhea syndrome,foodborne diseases surveillance,outbreak study,and environmental surveillance,beyond national notifiable disease reporting system.Among them,environmental surveillance based on the unique shedding mode of viruses is an indirect tool to acquire current circulating viral sequences in the population.Since the superiority of sensitivity,wide representative range and close correlation to the population,environmental surveillance can serve as the powerful complement to case-based surveillance system.[Objectives]Based on the accumulated sewage samples during longitudinal environmental surveillance,applying molecular biology technology like gene amplification,cloning and sequencing,this study on genotypes distribution,phylogeny,and intra-genogroup recombination aiming at human sapoviruses is expected to complete 3 following tasks:1.To monitor sapoviruses in environmental sewage,describe and predict their distribution of genogroups and genotypes within the monitoring area,analyze the genetic diversities and characteristics about the temporal variation,and meanwhile focus on the emergence of new strains and prevalence trend of the potential epidemic.2.To analyze the genetic characteristics of sapoviruses in environmental sewage,and then tentatively explore their variation and evolution during the studied period.3.To explore the temporal,geographical and phylogenetic relationships among major genotypes in sewage samples collected in this study and other environmental samples or clinical specimens from other countries or regions.[Methods]1.Sewage samples from Jinan and Yantai WWTPS were monthly collected by grab sampling method usually in the early part of one month from Jan 2009 and Sep 2013,respectively.while samples from Linyi WTPS were collected twice in the early and last part of each month.The samples were transported to our laboratory and then concentrated by an adsorption-elution method.2.Viral nucleic acid was extracted from the concentrated samples,and nested RT-PCR primer pairs targeting the partial capsid region were used for gene amplification.Positive PCR products gone through a series of assays from gel extraction purification,to TA cloning,and to blue-white selection,and finally sequencing by a company.3.BioEdit7.0.9.0 was used to align sapovirus sequences obtained from this study and those reference strains downloaded from GenBank further calculating and exporting their related nucleotide homology values.4.MEGA6.0 was used to generate phylogenetic trees with neighbor-joining method consisting both sapovirus sequences from our samples and other reference strains.5.SimPlot3.5.1 was used to recognize the intra-genogroup recombinant strains and determine their break points.[Results]1.General detection results during environmental surveillance in 3 citiesThe studied period of Jinan.Linyi and Yantai were from January 2009,April 2010,September 2013 respectively to December 2016.The number of months collecting sewage samples are 87,68,and 36 respectively amounting to 191 months,147 of which saw detection of sapoviruses.Among all sapovirus sequences obtained,1405 were successful s.equenced and effectively used for next analysis,of which non-recombinant strains accounted for 96.2%(1352/1405)covering 3 gene groups:GⅠ.2(79.5%,1075/1352)、GⅠ.6(9.9%,134/1352)、GⅠ.1(79.5%,112/1352)、GⅡ.1(1.8%,24/1352)、GV.1(0.5%,7/1352)in decreasing order.In addition,56 two-fragment and 1 three-fragment recombinant sequences were determined.Another 1 sequences failed to be genotyped.2.Regional distribution of sapovirus genotypes in sewage samplesIn sewage samples from Jinan,GⅠ.2(69/87),GⅠ.1(12/87),GⅠ.6(9/87),GⅡ.1(4/87),and recombinant genotypes(16/87)were identified;in those from Linyi,GⅠ.2(10/46).GⅠ.1(37/46),GⅠ.6(18/46),GⅤ.1(1/46),and recombinant genotypes(16/46)were identified;in those from Yantai,GⅠ.2(9/36),GⅠ.1(35/36),GⅠ.6(9/36),recombinant genotypes(10/36)were identified.3.The temporal distribution of sapovirus genotypes in sewage samplesDuring the studied period.although not being detected in several months,GⅠ.2 had been the single dominant genotype while other major genotypes showed short epidemic periods of 3 to 7 months successively.The gap of detection rates between GⅠ.2 and other genotypes were narrowing at the end of studied period.The overall detection rate of sapovirus was slightly higher in cold seasons(spring and winter)than that in warm seasons(summer and autumn)in samples both from Jinan and Linyi.The detection rates of GⅠ.1 in three regions were all much lower in warm seasons than that in cold seasons,yet other genotypes did not observe such pattern.Of note,the number of GⅠ.6 and recombinants in the 3 regions increased at the end of studied period.4.Homology analysis of non-recombinant sapovirus sequences in sewage samplesOverall,the majority of sequences within each genotype of GⅠ.1,GⅠ.2,and GⅠ.6 were closely homologous to each other showing high homology(>97%).while GⅡ.1 sequences showed lower homology(>95%).The yearly GⅠ.6 sequences indicated a drop trend at the end of the studied period(2015 and 2016).Sequences sharing 100%homology could be found both in the same and different samples.Intervals of nucleotide homologies in each genotype fluctuates over time.5.Phylogenetic analysis of major sapovirus genotypes in sewage samplesGⅠ.1,GⅠ.2 and GⅠ.6 sequences detected from 3 regions in different months or years were closely related and also showed close relationships with strains detected from other regions in the world,interestingly,a significant part of which were isolated in Japan.No relation between the virus variants and the sampling date or place was observed in phylogenetic trees of GⅠ.1 and GⅠ.2,while the GI.6 phylogenetic tree showed temporal clustering among part of sequences detected from 2015 to 2016.6.Homology comparison,phylogenetic analysis and SimPlot similarity curves of recombinant sequences in sewage samplesThe fragments of recombinants shared much higher homologies with the internal sequences in the same sample,if available,than with external reference strains,while both two sources contained the sequence sharing 100%identity with the recombination fragments.The phylogenetic trees showed that each fragment of recombinants clustered within the genotype it belonged to.SimPlot analysis clearly described the types of sapovirus recombinants and their breakpoints.These results above provided evidences for novel inter-genotype strains in genogroup Ⅰ and uncommon sites of breakpoints.[Conclusions]1.The study confrmed continuous existing of GI.2 during the studied period in 3 cities(Jinan,Linyi,Yantai),and GI.2 was predicted to remain predominant in the coming years with successive short-term epidemics of GⅠ.1 and GⅠ.6.At the end of studied period(2015～2016),the detection rate of GⅠ.6 showed a rising trend sequences,while the homology among GI.6 sequences each year demonstrated a drop trend and part of them displayed time clustering meanwhile,which probably indicated the emergence of potential new variants within GI.6 genotype.2.Among the 3 major genotypes.only GⅠ.1 showed a seasonal trend.That is,GⅠ.1 were more frequently detected in cold seasons than in warm seasons.When it came to whether such pattern was intrinsic to GⅠ.1,more accumulated data and statistical analysis was demanded in subsequent surveillance to confirm the seasonal pattern.3.GⅠ.1 and GⅠ.2 sequences with close phylogenetic relationships were circulating in multiple countries or regions.Sequences of GⅠ.1 and GI.2 in this study were highly similar to those from other places of the world suggesting they came from the same source of infection.The rare genotypes might be related to exchange of people among various geographical areas warning the potential risk that unusual genotypes might gradually adapt to the local population and even cause pandemic after being introduced into new territory.4.Inter-genotype recombinants of genogroup Ⅰ emerged since the first year(2009)of the studied period,and these recombinants were detected more frequently at the end of studied period(2015～2016).Sequence analysis showed that 6 types of recombinants as well as one three-fragments recombinant,and their recombination sites were all located in the conserved regions outside the RdRp-VP1 junction region with no previous reports before.5.Silent outbreaks through silent routes might exist in the population.Data on sapoviruses circulating in the human population is still quite limited.and environmental surveillance should be promoted to hold more comparable data among different laboratories around the country. |
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