| Backgroud:According to Brown's research on enterovirus A71(EV-A71)genotyping and large amount of surveillance and research data of EV-A71 since 1999,EV-A71 genotypes has expanded from 3 genotypes(A-C)in 1999 to seven genotypes(A-G)as well as several subgenotypes.These genotypes and subgenotypes EV-A71 spread worldwide with epidemic and outbreaks of infectious diseases such as encephalitis,aseptic meningitis,hand foot and mouth disease(HFMD)and other infectious disease.The first EV-A71 strain in mainland of China was isolated in 1987 and the nucleotide sequence of VP1 region was determined after 10 years of isolation.In 1998,strains belong to C4 genotype were isolated for the first time,which provides important basic data for the study of the epidemic regularity of EV-A71 in China.A large outbreak of hand foot and mouth disease occurred in mainland of China in 2008,it is considered that the appearance and prevalence of C4a evolutionary branch of EV-A71 is significantly correlated with a large number of severe cases and deaths in this outbreak.Thus,EV-A71 has attracted more attention of researchers,becoming the most important enterovirus after poliovirus.At present,the C4a evolutionary branch of EV-A71 is the major EV-A71 genotype in mainland of China.However,a few other genotypes and subgenotypes of EV-A71 have been detected in individual years.There are only a few scattered reports about non-C4 subgenotype in mainland of China,including the first B5 subgenotype strain reported in Xiamen in 2009,and it was reported again in Chongqing in 2014.Due to lack of systematic investigation on the sequence of non-C4 subgenotypes in mainland of China,the effect of imported genotype EV-A71 on the prevalence of native EV-A71 genotypes is unclear.Therefore,monitoring and tracing the imported genotype and studying the evolutionary dynamics after their importation are very necessary.Objective:Systematic investigation of non-C4 EV-A71 in mainland of China over the past 1998-2017 years,based on the local epidemic regularity and the evolutionary topological relationship of genotypes,we make a preliminary qualitative judgement of the imported genotypes and native epidemic genotypes,and we also try to estimate the traceability and possible transmission pathway of the imported EV-A71 genotypes.Methods:The nucleotide sequences of entire VP1 region of EV-A71 strain sent by provincial laboratory during 2015-2017 were examined and analyzed,and at the same time,all the entire VP1 sequences of EV-A71 in Genbank were obtained.A local EV-A71 sequence database was set up after sequence sorting and data cleaning according to EV-A71 genotype.Non-C4 EV-A71 sequences in mainland of China were analyzed,and evolutionary dynamics of imported EV-A71 genotypes and traceability analysis based on viral genome were performed by using evolutionary analysis and other bioinformatics methods.Results1.Eight strains of non-C4 subgenotype EV-A71 were detected among HFMD surveillance samples during in 2015-2017.Among them,7 strains were identified as B5 subgenotype which were isolated from Yunnan Province in 2016,1 strain was identified as C1 subgenotype which were isolated from Liaoning Province in 2017;2.As of March 2018,a total of 5912 entire VP1 sequences of EV-A71 were obtained from Genbank,and after data cleaning,22 non-C4 subgenotypes of EV-A71 sequences were selcected for further analyzing,including A genotype(15 sequences),B5(3 sequences),C1(1 sequence)and C2(3 sequences)subgenotypes;3.In 2008-2011,A genotype EV-A71 appeared in 5 provinces and cities in mainland of China(Beijing,Wuhan,Anhui,Shanghai,Yunnan),but there were no direct epidemiological links.Population state analysis shows that these A genotype EV-A71 do not exhibit population expansion and do not possess the characteristics of time evolution.The nucleotide similarity of these viruses to the sequence of Japanese laboratory temperature sensitive mutant was 99.1-100%,while that of the BrCr strain was 98.2-98.7%;4.The C1 subgenotype of EV-A71 presents a global distribution pattern,and the imported C1 subgenotype of EV-A71 in China in 2017 belongs to the European EV-A71 epidemic cluster;5.There were at least four C2 subgenotypes of EV-A71 importatons occurred in China's mainland in 1996,2008,2012 and 2015.However,the nucleotide similarity between 4 imported C2 subgenotypes sequences is relatively small,which is far apart from phylogenetic tree and belongs to separate events;6.Multiple B5 subgenotype of EV-A71 importations were found in mainland of China during 2009-2016.There may be two different sources of transmission on spatial links,from Chinese Taiwan and Vietnam,and there are two clusters in the maximum likelihood tree;7.The full-length genome sequence analysis of B5 subgenotype of EV-A71 shows that there are two recombination patterns,the recombination site appears in the 2C region,and the recombinant donors may be EV-A120 and CV-A5,respectively;8.The tMRCA of B5 subgenotype of EV-A71 is about 1996.The posteriori probability statistics of nodes used to analyze the origin of geographic transmission indicated that Malaysia,Singapore,Vietnam,Thailand and Chinese Taiwan,occupy a greater posterior probability,whicih suggested that Asia plays an important role in the transmission of B5 subgenotype of EV-A71;9.A posteriori probability statistics indicates that the B5 subgenotype of EV-A71 in Yunnan and Chongqing provinces may originate from Vietnamese epidemic strains,and the B5 subgenotype of EV-A71 in Xiamen city and Jiangsu province may originate from Chinese Taiwan epidemic strains.Conclusions:1.After amino acid comparison among sequences from A genotype of EV-A71 strains found in mainland of China(Beijing,Wuhan,Anhui,Shanghai,Yunnan)during 2008 to 2011 and other A genotype of EV-A71 sequences,it suggested that Chinese 'A' genotype EV-A71 strains were posibly related to the leak and importation of laboratory temperature sensitive strains in Japan in 2005.2.Evolutionary analysis indicated that imported C1 subgenotype of EV-A71 appeared in mainland of China originated from European epidemic strains,and may be imported into China through Russia or Japan.3.Imported C2 subgenotype of EV-A71 found in Beijing city and Dalian city may originate from Japan,while the C2 subgenotype of EV-A71 found in Chinese HongKong may originate from France;4.Imported B5 subgenotype of EV-A71 found in Xiamen city and Jiangsu province may originate from Chinese Taiwan or Japan,and that of in Yunnan province and Chongqing city may probably originate from Thailand and Vietnam.Yunnan imported B5 subgenotype of EV-A71 found in 2016 has potential transmission possibility within the province,however,the association among the multiple importation events in mainland of China is very weak.5.There are probably at least two recombinatant patterns in B5 subgenotype of EV-A71,the recombination site appears in the 2C 'region region,and the recombinant donors may be CV-A5 and EV-A120,respectively. |
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