| [BACKGROUND]Enterovirus(EV)is single-stranded positive-stranded RNA virus belonging to Picornavirales family Enterovirus genus.It was initially divided into polioviruses(PVs),Coxsackievirus A(CVA),Coxsackievirus B(CVB),Echovirus(E)and EV new types such as EV-D68,EV-A71 according to the pathogenicity.Based on the molecular biological typing method of VP1 coding region,Enterovirus genus is divided into 15 groups:Enterovirus A to L(EV-A-EV-L),Rhinovirus A to C group(RV-A-RV-C),among which EV-A-EV-D can infect humans.The poliomyelitis caused by PV has caused tens of thousands of children’s disability and death in China.Meanwhile,poliomyelitis is a typical kind of vaccine-preventable disease.The immunization program with 4 doses of trivalent Oral Polio Vaccine(tOPV)has been being used since the initial implementation of childhood immunization in China.In the final stage of the Global Polio Eradication Initiative(GPEI),in order to decrease the prevalence of vaccine-associated paralytic polio cases(VAPP)and the pandemic of vaccine-derived poliovirus(VDPV),as well as to keep polio-free status in China,the first adjustment of the polio vaccine immunization strategy was conducted in May 2016 according to the globally consistent arrangement of WHO.Polio vaccine sequential immunization program was adopted with the removement of Sabin II in tOPV.In this immune program,the first dose of basic immunization was Inactivated Polio Vaccine(IPV),the second,third dose and booster immunization were replaced by bivalent OPV(bOPV)composed of PV1 and PV3.At present,two kinds of nationwide surveillance systems are carried out to maintain polio-free status in China.Acute flaccid paralysis(AFP)surveillance system is focused on AFP symptoms in children under 15 years old for detecting the circulation of imported wild PV and VDPV timely.AFP surveillance has been operating continuously in our country for decades since its establishment in 1994.AFP surveillance can also detect a variety of non-polio enterovirus(NPEV)in addition to PV,which is an effective means to explore the NPEV circulation under the circumstance that a special EV-related disease surveillance system is absent in China.Environmental surveillance(ES)is recommended by WHO as a supplement to AFP surveillance system.It is only carried out in a few provinces like Shandong Province in China.Its sensitivity to the detection of locally circulating PVs and NPEVs has been improved in many regions around the world.The introduction of new strategies for polio vaccines in our country will cause changes in the prevalence of the vaccine strain PV in the population,leading to a series of theoretical and practical scientific problems.Exploring the dynamics of poliovirus transmission and prevalence in the population after the adjustment of polio immunization strategies through ES methods will help improve our understanding of poliovirus epidemics in the population and provide a basis for early warning of the occurrence of VDPV and even cVDPV.At the same time,the continuous environmental wastewater monitoring research of NPEV will help to clarify the dynamic changes of NPEVs and its association with clinical case isolates,and greatly enrich the molecular epidemiology data of NPEVs,which makes sense to prevent EV-related diseases outbreaks.[OBJECTIVES]1.To evaluate the dynamic distribution and the genetic characteristics of PV types in environmental surveillance around the immune strategy switch period of polio vaccine.2.To investigate the prevalence and circulation patterns of NPEVs in Shandong province from 2015 to 2018.3.To understand the genetic evolutionary characteristics of the dominant epidemic serotypes of NPEV.[METHODS]1.Environmental sewage samples were collected from Jinan,Linyi,and Yantai city and perform virus concentration;Fecal specimens of AFP cases were collected and conducted processing.2.Sewage concentration and speimens processing supernatant of AFP cases were inoculated to RD,Hep-2,and L20B cell lines for virus isolation,harvest the EV isolates.3.Viral RNA was extracted and the entire VP1 coding region of EV was amplified.4.Sanger sequencing was conducted and EV serotype was identified.5.Sequence analysis:Sequence screening and multiple sequence alignment were performed by BioEdit(v7.0.9.0)software;Phylogenetic trees based on the neighbor method were constructed by MEGA(v.7.0)software;Evolutionary analysis of the dominant NPEV strains were conducted by BEAST(v.1.10.4)software.6.Statistical analysis:SPSS 27.0 was used to conduct descriptive research for epidemiological data.Test the rate or composition ratio by χ2 test,the result was considered statistically significant when P<0.05.[RESULTS]1.PV type composition and dynamic circulation in sewageDuring the study period,430 PV isolates were isolated from ewage samples,with 215 isolates(50%)in Jinan City,186 isolates(43.3%)in Linyi City,and 29(6.7%)in Yantai City.Before the immunue strategy switch of polio vaccine in May 2016,all 3 PV types were detected,of which PV1 accountedfor 35.2%,PV2 accounted for 29.0%,and PV3 accounted for 35.8%.After the switch,PV2 vanished and PV3 came into the predominant type.The number of PV1 and PV3 isolates were 71(26.5%)and 197(73.5%),respectively.During the observation period,no WPV or VDPV isolates were detected in environmental sewage.Among 8 detected PV vaccine highly variable isolates(pre-VDPV),7 isolates were isolated before vaccine switch,and one isolated after that.2.EV Genotype distribution in AFP surveillance systemFrom 2015 to 2018,a sum of 105 NPEV isolates of 24 serotypes were isolated from AFP surveillance system.Six serotypes in EV-A group including CVA2,CVA4-6,CVA10 and EV-A71 as well as 19 serotypes in EV-B group including CVA9、CVA21、CVB1～B5、E1、E3、E6、E7、E11、E14、E18、E20、E25、E30、E39 and E33 were detected successfully.In terms of time distribution,the type composition of NPEV in AFP surveillance system from 2015 to 2018 was not always consistent.In 2015,37 NPEV isolates in 13 types were detected,accounting for 35.2%of all NPEV strains in AFP surveillance system,including 4 EV-A types and 9 EV-B types.In 2016,a total of 31 NPEV(29.5%)strains were isolated,in which E25,E1 and CVB1 were only detected in that year.In 2017,although less quatity of NPEVs were isolated,three E30 isolates,the important pathogen of encephalitis/meningitis,and one EV-A71 isolate,the main cause of HFMD,were detected.The number of NPEV detected in 2018 increased significantly comparing to 2017.A total of 28 NPEV strains were isolated,in which E11,CVB5 and CVA4 are the dominant types.Besides,CVB5 was only frequently detected in 2018.3.Enterovirus detection in environmental surveillance systemA total of 180 environmental sewage samples were collected including 48 in Jinan,86 in Linyi,and 46 in Yantai.Generally,the positive rate of PV and NPEV was 46.3%and 70.7%,respectively.The positive rates of PV and NPEV in Jinan were higher than those of the other two cities.The positive rates of NPEV in Jinan,Linyi,and Yantai were all higher than those of PV in 2015～2018.The annual positive rate of PV was above 50%,and that of NPEV was always above 70%except for in 2018.4.Distribution and dynamic circulation of NPEV in urban sewageDuring the observation period,a total of 977 NPEV isolates in 21 serotypes were detected in urban sewage,including 14 NPEV types detected in AFP surveillance system during the same period.The predominant types circulating in environmental surveillance are E-11(32.9%),CV-B5(12.1%),E-3(10.8%),CV-B3(8.9%),and E-6(8.7%).The type composition of NPEV in ES showed difference before and after the switch of polio vaccine immunization strategy.Before the switch,a total of 311 NPEV strains of 17 types were isolated in sewage,among which the most popular types were E6(57/311,18.3%),E3(56/311,18.0%),and CVB3.(55/311,17.7%),CVB5(38/311,12.2%)and Ell(20/311,6.4%),with a relaticely small proportion difference in the five types.The dominant serotypes changed after the vaccine switch:the number of E11 isolates increased significantly while the proportion of CVB5 did not change distinctly;the isolated number of previous prevalent serotypes E3,CVB3,and E6 were dropped.Generally,NPEV presented a popular pattern in summer and autumn,and less prevalent in winter and spring.It is worth noting that Ell has become the predominant type of NPEV since 2017,especially in Jinan city,and continued to be popular in 2018.5.Molecular epidemiology and evolutionary genetics of NPEV dominant typesThe genetic analysis of E11,CVB5,E3,CVB3,E6 and E30 revealed that there are multiple transmission chains of each serotype circulating concurrently worldwide.Local isolates showed a temporal and spatial aggregation.Sewage isolates in Shandong province have close genetic relationships with the clinical isolates,also sharing a common ancestral source.The most recent common ancestor(tMRCA)of the six main types is from 1870(E30)to 1922(E3),and the mean evolutionary rate is from 3.79×10-3(CVB3)to 5.42×10-3(E11)nucleotide substitutions per site per year.[CONCLUSIONS]1.The prevalence of PV in Shandong Province from 2015 to 2018 changed in keep with the implementation of immune switch strategy of polio vaccine.PV2 disappeared rapidly after the vaccine switch.The detection ratio of PV1 and PV3 types also changed,PV3 type has increased significantly since May 2016,becoming the dominant type in the following period.2.The predominant NPEVserptypes were E11,CVB5,CVB3,E3 and E6 in 2015-2018.Several transmission chains of each serotype are circulating in local areas.These serotypes have a higher frequency of detection in sewage and AFP cases.Respective phylogenetic analysis showed that environmental isolates were closely related to case isolates.Various predominant types were circulating during different years3.Environmental surveillance is an effective measure to grasp the regional epidemic of poliovirus and other enteroviruses. |
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