Recombination,Evolutionary Origin And Spatiotemporal Dynamic Transmission Of Enterovirus A Species In Shandong Province,China

[Background]Enteroviruses(EVs),including EV-A to EV-H and EV-J,are among the most common human pathogens within the Enterovirus genus of the family Picornaviridae in conjunction with three rhinovirus species A,B and C.EVs are ubiquitous,a range of different serotypes are circulating in human populations at any one time point.Although most EV infections are usually asymptomatic,they also lead to a wide spectrum of clinical diseases ranging from mild symptoms to fatal diseases like acute flaccid paralysis(AFP),hand,foot,and mouth disease(HFMD),aseptic encephalitis and meningitis(AM),acute hemorrhagic conjunctivitis(AHC),herpangina,myocarditis,type 1 diabetes and epidemic myalgia.Among the diseases caused by EV-A,HFMD is most common,which mostly occurs in infants under 5 years old with fever and rash in hands,feet and mouth.EV-A71 and coxsackievirus A16(CV-A16)are the two major pathogens leading to the ourbreak and epidemic of HFMD.The EV genome contains a non-enveloped positive single-stranded RNA of approximately 7.5 kb,with a single long open reading frame flanked by 5' and 3'non-translated regions.The open reading frame encodes a polyprotein comprising four structural proteins,VP1-VP4,and seven non-structural proteins,2A-2C and 3A-3D.The VP1 protein is highly exposed and is thought to play an important role in viral pathogenesis and virulence.Appropriately,molecular typing methods based on the complete or partial VP1 sequence are widely used to identify EV serotypes by sequencing of amplified VP1 products and pairwise alignments with those of prototype and variant EV strains.In recent years,phylogenetic analysis based on the VP1 sequence is explored to be a reliable method for epidemiological purposes and evolution rules in combination with phylogenetic analysis is able to shed light on important topics in EV epidemiology and to identify emerging new EV variants or serotypes.In the study of EV recombination and evolution,the BEAST program based on the Bayesian theory has been used to calculate and analyze the origin and evolution rate of EV.Using the markov chain monte carlo(MCMC)method,we can analyze the evolutionary genetic characteristics(such as gene origin,evolution rate,epidemic history and population dynamics)of different EV serotypes.Additionally,evolutionary reconstructions that integrate various sources of information alongside sequence data are becoming increasingly widespread in statistical phylogenetics.At present,statistical phylogenetics are used to simultaneously infer sequence and trait evolutionary processes and have become particularly populare in infectious desease study in inferring spatiotemporal history.Therefore,it will be important to carry out the statistical phylogenetic analysis of EV to infer the temporal and spatial evolution of different EV types with the Bayesian Stochastic Search Variable Selection(BSSVS)model in BEAST software.EV-A comprises 25 serotypes,and many serotypes including EV-A71 and CV-A16 have caused worldwide ourbreaks.Due to the limited EV-A sequences in the world,the molecular epidemiological study of most EV-A serotypes is insufficient.Shandong center for disease control and prevention has accumulated a number of EV-A isolates and all viruses have accurate information.Hence,to explore the molecular epidemiology of EV-A will contribute theoretical and practical significance forthe establishment of mechanism and prevention and control of related diseases.[Objectives]1.To clarify the cociruculation pattern of EV-A serotypes and their relationship with dis.ease in Shandong province.2.To establish the databases of VP1 and 3D sequences of 251 Shandong strains which belonging to 12 EV-A serotypes(CV-A2,CV-A4,CV-A5,CV-A6,CV-A8,CV-A10,CV-A12,CV-A14,CV-A16,EV-A71,EV-A76 and EV-A90)?and to analyze the genetic characterization.3.To analyze the genetic recombination of EV-A Shandong strains,and to investigate the effect of virus genetic variation on the pathogenicity and epidemic pattern.4.To calculate and analyze the evolutionary genetics characteristics(such as gene origin,evolution rate,epidemic history and population dynamics)of EV-A Shandong strains based on the Markov Chain Monte Carlo method.5.To explore the statistical phylogenetics of EV-A with the Bayesian Stochastic Search Variable Selection model,and to infer the temporal and spatial dynamics of different EV-A types.[Methods]1.Virus isolation.EV-A strains included in our study were isolated in Shandong province in 1989-2016.All EV-A isolates were stored at-80?,RD cell and Hep-2cell were used for virus proliferation before further processing.2.RNA extraction.A total of 50?l viral RNA was extracted from 140?l cell culture supernatant using QIAamp viral RNA mini kit,and all RNA was stored at-80 ? as 1 ?l(40U)of Rnasin(QIAGEN)was added.3.RT-PCR and nucleotide sequencing.RT-PCR was performed using Access RT-PCR System(Promega,USA)to amplify the VP1 and 3D sequences.The PCR amplicons were visualized in agarose gels(1%),and positive products were purified and sequenced.4.Serotype identification.Molecular typing based on the complete VP1 sequences was performed to identify the serotypes of EV strains using a BLAST search,obtained online from the National Center for Biotechnology Information(NCBI).5.Bioinformation analysis.The phylogenetic analysis was conducted using MEGA 6.0 software and recombinant analysis was carried out by comparing the VP1 and 3D phylogenetic trees.Evolutionary genetics of EV-A Shandong strains were caculated via BEAST 1.8.3,and the population dynamics of EV-A were inferred with bayesian skyline plot analysis.The spatiotemporal dynamics of EV-A were inferred by the Bayesian Stochastic Search Variable Selection model,and SPREAD v1.0.6 was used to reconstructed the routes of migration and the most significant rates of spread.[Results]1.Serotype distribution of EV-A Shandong strains.In this study,molecular serotyping method was used to identify the serotypes in Shandong province between 1989 and 2016,and 251 EV-A strains were detected.They belonged to 12 EV-A serotypes:CV-A2(12),CV-A4(43),CV-A5(3),CV-A6(13),CV-A8(1),CV-A10(40),CV-A12(4),CV-A14(1),CV-A16(27),EV-A71(102),EV-A76(2)and EV-A90(3).There were 237 strains belonging to EV-A71,CV-A4,CV-A10,CV-A16,CV-A6 and CV-A2,and were the predominant serotypes in Shandong province.Since 2008,the epidemic of EV-A in Shandong province became active,and 223 strains were isolated,accounting for 88.8%of all identified EV-A Shandong strains.2.Phylogenetic and recombinant analysis of EV-A Shandong strains.Totally,251 EV-A Shandong strains were selected for the phylogenetic and recombination analysis based on their VP1 and 3D sequences.The phylogenetic tree based on VP1 region revealed that all 12 EV-A serotypes isolated in Shandong province were clustered into different branches according to the genetic distance,and no recombination evidence was found in the VP1 sequences.In addition,all EV-A strains exhibited temporal dynamics in Shandong province,as was reflected via the different clusters of strains with different collection years in the phylogenetic tree.The phylogenetic tree of 3D region showed frequent recombinant was occurred in the non-structural gene among EV-A Shandong strains.There were 10 groups were divided in accordance with the phylogenetic relationship of EV-A Shandong strains in the 3D phylogenetic tree.(1)In the 3D phylogenetic tree,serotypes of Shandong isolates contained in each group were different,and the same serotype of Shandong strains wassegarated into different groups.Also,the collection date of EV-A Shandong strains in each group was different.The longest time span of the group was about 25 years,and the predominant collection dateineach group was about 10 years(10.4±7.6 years).(2)EV-A Shandong strains in group 1 were isolated between 2003 and 2015,and all strains were EV-A71 except for one strain of CV-A12(F424).The strains in group 2 were collected from 1996 to 2011 and they belonged to five different EV-A serotypes:CV-A4,CV-A6,CV-A12,CV-A14 and CV-A16.Group 3 was also the dominant group in the 3D phylogenetic tree of Shandong strains,and this group contained strains belonging to CV-A2,CV-A4,CV-A6,CV-A10 and CV-A14 with the time span of 24 years(1992-2015).Group 10 consisted one EV-A76 stain and four EV-A90 strains.The EV-A76 strain was isolated in 2005,and EV-A90 Shandong strains were collected between 1999 and 2003.3.Evolutionary origin of predominant EV-A serotypes.The most recent common ancestors of CV-A2,CV-A4,CV-A6,CV-A10,CV-A16 and EV-A71 can be traced back to 1925,1944,1946,1904,1905 and 1936,respectively.In the past ten years,the relative genetic diversity of VP1 region for the 6 predominant EV-A serotypes has been increased.The mean evolution rate of VP1 sequences for different EV-A serotypes was 2.615×10-3?6.431×10-3 substitutions per site per year,of which EV-A71 showed the lowest mean evolution rate and CV-A4 had the highest VP1 evolution rate.Moreover,different genegroups of the same EV-A serotype in the phylogenetic tree exhibited different mean evolution rate.EV-A Shandong strains showed the same ancestor with strains isolated in other provinces of China.4.Spatiotemporal dynamics of predominant EV-A serotypes.In recent years,the transimission pattern of EV-A had become more active in the world.The most probable ancestral geographical location for CV-A16 was predicted to be the Republic of South Africa,while phylogeographic reconstruction identified the same location for the origin of the global CV-A2,CV-A4,CV-A6,CV-A10 and EV-A71 in the USA regions.However,the spread of the predominant EV-A types in their first origin location had become silent over the past few decades and the prevalence of CV-A2,CV-A4,CV-A6,CV-A10,CV-A16 and EV-A71 is now mostly contcentrated in Asia-Pacific region and European countries.The spatiotemporal dynasmics of EV-A showed geographical characteristics,and the dispersion of virus within the defined location would be stronger than between them.[Conclusions]1.A variety of E'V-A serotypes was identified in Shandong EV surveillance,of which EV-A71,CV-A4,CV-A10,CV-A16,CV-A6 and CV-A2 were the predominant serotypes.Altogether 251 Shandong strains of 12 EV-A serotypes were identified,as were CV-A2,CV-A4,CV-A5,CV-A6,CV-A8,CV-A10,CV-A12,CV-A14,CV-A16,EV-A71,EV-A76 and EV-A90.The strains isolated from AFP surveillance covered the above 12 serotypes;the strains from HFMD cases were predominantly EV-A71 and CV-A16;the strains from AM cases were mainly EV-A71;CV-A10 and CV-A6 were the main pathogens that most likely cause herpangina.2.EV-A Shandong strains showed great nucleotide divergence with foreign strains,and recombination was a significant evolutionary mode of non-structural coding region among EVs.EV-A Shandong strains showed distanct phylogenetic relationship with the foreign strains,and.a certain degree of time aggregation and spatial aggregation was observed in the phylogenetic analysis.The phylogenetic trees based on 3D region showed that frequent recombinant was occurred in the non-structural gene among EV-A Shandong strains.EV-A Shandong strains were divided into 10 groups in the 3D phylogenetic tree.The number of serotypes located in each group was different,and each serotype can be divided into several groups.3.Variation in the VP1 gene of predominant EV-A Shandong strains was extensive,and multiple transmission chains were co-circulating in Shandong.Statistical phylogenetic analysis estimated the origin of predominant EV-A serotypes was to have emerged between 1904 and 1946.The estimated rate of EV-A molecular evolution was inferred as 2.6×10-3?6.4×10-3substitutions/site/year,and substitution rates varied greatly among different branches.4.The spatiotemporal dynamics of EV-A showed regional distribution characteristics,and the spread of EV-A had obviously increased in recent years.Most EV-A serotypes originated in the United States,while the epidemic area of EV-A were now concentrated in the Asia-Pacific region and European countries.5.It is suggested that we should strengthen the surveillance and isolation of different EV-A serotypes,and systematic molecular epidemiology study on EV-A would be conducted in the future.Considering the increased prevalence of EV-A in the world and the diseases caused by EV in recent years are more serious in China,it is necessary to strengthen the research on EV etiology,epidemiology systematically.This will provide scientific basis in the establishment of EV laboratory testing methods,forecasting and early warning of epidemics and development of vaccines.