中國(guó)大陸引起手足口病柯薩奇病毒A組4型基因特征分析
發(fā)布時(shí)間：2018-08-31 20:43
【摘要】：[背景]人類腸道病毒(Human Enterovirus, HEV)屬于小核糖核酸病毒科(Picornavirdae)腸道病毒(Enterovirus)屬,目前已經(jīng)報(bào)道了119個(gè)血清型,被分為EV-A、EV-B、EV-C和EV-D共4組。多數(shù)情況下,HEV感染的病人是無(wú)癥狀的,或者只引起輕微癥狀,如發(fā)熱、皮疹、或輕微上呼吸道癥狀；但有時(shí)HEV感染也可以引起廣泛多樣的臨床疾病,如急性出血性結(jié)膜炎、無(wú)菌性腦膜炎、急性弛緩性麻痹(Acute Flaccid Paralysis, AFP)、手足口病(Hand, Foot and Mouth Disease、心肌炎、類感冒病癥等。近年來(lái),由HEV引起的各類疾病一直旱.現(xiàn)上升趨勢(shì)。HEV病毒的基因組為單股正鏈RNA,長(zhǎng)約7.5kb,整個(gè)基因組只含有一個(gè)開(kāi)放閱讀框架(Open Reading Frame, ORF),編碼一個(gè)多聚蛋白(Polyprotein),最后水解產(chǎn)生VP1-VP4共4個(gè)結(jié)構(gòu)蛋白和7個(gè)非結(jié)構(gòu)蛋白。其中VP1蛋白是最主要的衣殼蛋白,是HEV與宿主細(xì)胞上受體進(jìn)行識(shí)別、結(jié)合的主要結(jié)構(gòu),包含重要的血清型特異性中和位點(diǎn)；VP1蛋白位于病毒顆粒的最外表而,在機(jī)體免疫壓力下,會(huì)發(fā)生變異而形成不同的流行株。因此,對(duì)VPl蛋白核苷酸序列的研究,不僅可以確定HEV的血清型別,而且可以說(shuō)明病毒核酸之間同源性的大小,從而明確病毒進(jìn)化變異的方向及其傳播鏈。手足口病是多種腸道病毒(EV)引起的常見(jiàn)傳染病。自中國(guó)大陸地區(qū)在2007年出現(xiàn)HFMD的爆發(fā)流行后,手足口病漸漸受到關(guān)注,據(jù)近年來(lái)的相關(guān)文獻(xiàn)報(bào)道看,中國(guó)大陸手足口病的疫情有愈演愈烈的傾向,HFMD已經(jīng)成為嚴(yán)重威脅廣大人群特別是兒童身體健康與生命安全的重大公共衛(wèi)生問(wèn)題之一。EV71和CA16是引起HFMD的主要病原體,對(duì)手足口病病原學(xué)的研究多也集中在EV71和CA16,對(duì)其他腸道病毒的研究相對(duì)較少,在但近年來(lái),一些地區(qū)的HFMD監(jiān)測(cè)數(shù)據(jù)表明,其他型腸道病毒的病原構(gòu)成比例有所升高,CVA4就是其中之一。本研究針對(duì)2008-2012年中國(guó)大陸地區(qū)HF-MI)病例中分離的CVA4毒株VPI編碼區(qū)和3D區(qū)基因特征,開(kāi)展分子流行分析。[目的]通過(guò)對(duì)2008-2012年中國(guó)大陸地區(qū)HFMD病例中的CVA4毒株VP1和3D基因特征進(jìn)行系統(tǒng)性的分析,闡明其在我國(guó)的基因變異變遷規(guī)律。豐富我國(guó)和世界上CVA4的毒株庫(kù)和基因信息庫(kù),為我國(guó)CVA4引起的HFMD的診斷、預(yù)防和控制提供科學(xué)支持。[研究方法]1.毒株來(lái)源：2012年江蘇省贛榆縣的1246份HFMD、病毒性腦炎、皰疹性咽峽炎臨床標(biāo)本分離的CVA4病毒57株；2008-2012年中國(guó)大陸HFMD監(jiān)測(cè)網(wǎng)絡(luò)實(shí)驗(yàn)室在其他地區(qū)HFMD病例中分離到19株CVA4。2.基因測(cè)序：提取病毒RNA, RT-PCR擴(kuò)增VP1和3D區(qū)基因全長(zhǎng),并對(duì)PCR產(chǎn)物進(jìn)行序列測(cè)定。3.生物信息學(xué)分析：采用BioEdit Sequence Alignment Editor software 7.0軟件對(duì)本研究CVA4的VP1和3D基因與GenBank中檢索到的中國(guó)及其它國(guó)家和地區(qū)的CVA4的VP1和3D基因進(jìn)行核苷酸序列進(jìn)行同源性比較,并對(duì)VP1基因核苷酸的基因變異所提示的氨基酸序列變異進(jìn)行對(duì)比分析。使用Mega5.0軟件,以相鄰連接方法(Neighbor-joining Method)構(gòu)建VP1區(qū)和3D區(qū)系統(tǒng)進(jìn)化樹(shù),進(jìn)行親緣性和進(jìn)化分析。[結(jié)果]1.江蘇省贛榆縣HFMD、病毒性腦炎、皰疹性咽峽炎等臨床標(biāo)本共分離到350株毒株,其中CVB3(142株)、CVA4(57株),CVA10(31株)、CVA16(29株)、EV71(24株)等。CVA4排第二位,所占比例為16%。贛榆縣分離到的57株CVA4毒株VP1 5’端564個(gè)核苷酸序列同源性為94.0%-100%,氨基酸序列同源性為97.8%-100%,選取2株做VPl編碼區(qū)基因915個(gè)核苷酸的序列測(cè)定。2.中國(guó)大陸分離到的21株CVA4代表株的VP1區(qū)序列相互間核苷酸序列的同源性為94.1%-100%,氨基酸序列同源性為97.3%-100%,同源性較高,進(jìn)化關(guān)系密切。3.本次研究獲得的2008-2012年21株CVA4代表株VP1序列和在GenBank中獲得分離于中國(guó)大陸的10株CVA4的VP1序列做同源性分析：中國(guó)大陸分離到的31株CVA4的VP1區(qū)編碼區(qū)基因的同源性在85.5%-100%之間,氨基酸序列同源性為97.5%-100%,同源性較高,屬于同一個(gè)基因型,核苷酸的突變多為同義突變；1998年分離于山東省AFP病例的兩株CVA4與其他年份分離的毒株核苷酸序列同源性在88.5%-89.9%,序列差異較大。排除1998年分離株,其余HFMD與AFP分離株之間的核苷酸序列同源性為94.8%,氨基酸序列同源性為98.4%,核苷酸和氨基酸在2種病例類型中未見(jiàn)特異差異何點(diǎn)；HFMD輕癥和重癥病例分離株之間的核苷酸序列同源性為95.5%,氨基酸序列同源性為98.8%,核苷酸和氨基酸在HFMD的2種病例類型中也未見(jiàn)特異差異位點(diǎn)。4.本次研究獲得的21株CVA4代表株VP1序列和在GenBank中獲得全球21株CVA4的VP1序列做親緣進(jìn)化樹(shù)：基于國(guó)際腸道病毒基因型分型標(biāo)準(zhǔn),依據(jù)VP1區(qū)編碼基因全長(zhǎng)可以將CVA4劃分為5個(gè)基因型,分別為A、B、C、D、E基因型；這是首次基于VP1基因全長(zhǎng)對(duì)CVA4進(jìn)行基因型的劃分。各基因型毒株核苷酸序列組間遺傳距離為14.5%-26.7%,組內(nèi)遺傳距離為2.0%-3.5%。分離于中國(guó)大陸的31株CVA4病毒都屬于E基因型,E基因型又被劃分為E1和E2基因亞型,其中E1基因亞型包括1998年分離于山東AFP病例的2株毒株和2006年分離于吉林省AFP病例的1株毒株：E2基因亞型包括了2008-2012年分離于HFMD病例的22株CVA4毒株,以及2006-2008年分離于山東省和吉林省AFP病例的6株毒株。E1和E2基因亞型毒株組間的核苷酸序列平均遺傳距離為12.6%,可見(jiàn)在中國(guó)大陸至少存在兩條大的傳播鏈,2006年及以前以E1基因亞型流行為主,2006以后以E2基因亞型流行為主。GenBank中CVA4的VP1區(qū)3’端的序列信息較VP1區(qū)全長(zhǎng)更為豐富,將本次研究獲得的21株CVA4毒株VP1區(qū)3’端420個(gè)核苷酸序列和在GenBank中獲得44株CVA4的VP1區(qū)3’端序列代表株做親緣進(jìn)化樹(shù)：基于VP1區(qū)3’端420個(gè)核苷酸序列,CVA4毒株可以劃分為5個(gè)進(jìn)化分支,分別為L(zhǎng)ineage A～E。各Lineage毒株核苷酸序列組間遺傳距離為13.4%-26.1%,組內(nèi)遺傳距離為5.8%-9.6%。其進(jìn)化分支與以VP1區(qū)基因全長(zhǎng)所做進(jìn)化樹(shù)結(jié)果相符,基因型A～E所包括毒株與對(duì)應(yīng)各Lineage A～E毒株相一致。分離于中國(guó)大陸的31株CVA4病毒都屬于Lineage E, Lineage E同樣可以分為L(zhǎng)ineage E1和Lineage E2,并且和VP1全長(zhǎng)劃分的結(jié)果也一致,Lineage E除包括中國(guó)大陸的毒株外,還包括分離于臺(tái)灣的毒株。57株分離于江蘇贛榆縣的CVA4毒株,基于5’端的序列測(cè)定提示全部為E2基因亞型。5.選取5株CVA4代表毒株完成3D區(qū)序列的測(cè)定,并與其他可以檢索到的CVA4、HEV-A組所有腸道病毒原型株、BLAST后同源性較高的毒株和EV71 C4a基因亞型代表株的3D區(qū)序列進(jìn)行基因同源性比較并構(gòu)建親緣進(jìn)化樹(shù)：5株CVA4毒株和2009年分離于中國(guó)深圳的CVA4毒株(HQ728260)和2009年分離于廣東省的EV71毒株(JF 799986)在一個(gè)進(jìn)化分支上,序列同源性高達(dá)94.6%-96%；這株EV71與EV71的原型株BrCr株或其他EV71 C4a基因亞型的代表株遺傳距離較遠(yuǎn),序列同源性只有73.3%-81.7%,推測(cè)這一株EV71病毒的3D區(qū)可能來(lái)源于與中國(guó)大陸流行的CVA4的3D區(qū)重組。[結(jié)論]1.CVA4是2012年引起江蘇贛榆縣手足口病、病毒性腦炎以及皰疹性咽峽炎等疾病的主要病原體之一,在當(dāng)?shù)匕l(fā)生了較大規(guī)模的循環(huán),并有多個(gè)病毒傳播鏈。2.本研究首次基于CVA4毒株VP1區(qū)編碼基因全長(zhǎng),將CVA4劃分為5個(gè)基因型,分別為A、B、C、D、E基因型,基于VP1區(qū)3’端序列親緣進(jìn)化分析也進(jìn)一步驗(yàn)證了基因型的劃分。分離于中國(guó)大陸的所有CVA4病毒都屬于E基因型中的E1和E2基因亞型。3.2009年1株廣東省的EV71分離株在3D區(qū)可能與中國(guó)大陸現(xiàn)流行的CVA4發(fā)生了重組,應(yīng)關(guān)注CVA4的3D區(qū)作為供體在腸道病毒重組的作用,同時(shí)關(guān)注EV71重組CVA4對(duì)EV71生物學(xué)性狀和傳播力和致病力的影響。4.本研結(jié)果為CVA4引起的HDMD等傳染病的診斷、防控工作提供了重要的病毒學(xué)基礎(chǔ)資料。
[Abstract]:[BACKGROUND] Human Enterovirus (HEV) belongs to the genus Enterovirus of the Picornavirdae family. At present, 119 serotypes have been reported and divided into four groups: EV-A, EV-B, EV-C and EV-D. In most cases, patients infected with HEV are asymptomatic or cause only mild symptoms, such as fever, rash, or EV-D. Mild upper respiratory symptoms; but sometimes HEV infection can also cause a wide range of clinical diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, acute flaccid paralysis (AFP), hand, foot and mouth disease (Hand, Foot and Mouth Disease, myocarditis, cold-like diseases, etc. In recent years, caused by HEV a variety of diseases. The genome of the HEV virus is a single strand of positive strand RNA, about 7.5 KB long. The whole genome contains only one Open Reading Frame (ORF), encodes a polyprotein, and finally hydrolyzes to produce VP1-VP4 with four structural proteins and seven non-structural proteins. VP1 protein is the main structure that recognizes and binds to receptors on the host cells and contains important serotype-specific neutralizing sites; VP1 protein is located in the outermost appearance of viral particles and mutates under the immune pressure to form different epidemic strains. Hand-foot-mouth disease (HFMD) is a common infectious disease caused by a variety of enteroviruses (EV). Since the outbreak of HFMD in the mainland of China in 2007, hand-foot-mouth disease (HFMD) has gradually attracted attention, according to the relevant literature in recent years. It is reported that HFMD has become one of the major public health problems that seriously threaten the health and life safety of the broad population, especially children. EV71 and CA16 are the main pathogens causing HFMD. EV71 and CA16 are the main pathogens of HFMD. The etiological studies of HFMD are mostly focused on EV71 and CA16, and other intestines. However, in recent years, HFMD surveillance data in some areas show that the pathogenic composition of other enteroviruses has increased, and CVA4 is one of them. [Objective] Through systematic analysis of VP1 and 3D gene characteristics of CVA4 strains from HFMD cases in mainland China from 2008 to 2012, to elucidate their genetic variation in China and enrich the CVA4 strain library and gene information database in China and the world, so as to provide scientific support for the diagnosis, prevention and control of HFMD caused by CVA4 in China. Source of the virus: In 2012, 1246 HFMD, viral encephalitis and herpes angina were isolated from clinical specimens of Ganyu County, Jiangsu Province, 57 strains of CVA4 virus were isolated; in 2008-2012, 19 strains of CVA4.2 were isolated from HFMD cases in other areas by HFMD Surveillance Network Laboratory of mainland China. Gene sequencing: virus RNA was extracted, VP 1 and 3D region genes were amplified by RT-PCR. Bioinformatics analysis: The VP1 and 3D genes of CVA4 in this study were compared with the VP1 and 3D genes of CVA4 retrieved from GenBank in China and other countries and regions by using BioEdit Sequence Alignment Editor software 7.0. The phylogenetic tree of VP1 and 3D regions was constructed by Neighbor-joining method, and the phylogenetic relationship and evolution were analyzed. [Results] 1. HFMD, viral encephalitis, herpes angina and other clinical specimens in Ganyu County, Jiangsu Province were separated. Among 350 strains, CVB3 (142 strains), CVA4 (57 strains), CVA10 (31 strains), CVA16 (29 strains), EV71 (24 strains), etc. CVA4 ranked second, accounting for 16%. The 564 nucleotide sequence homology of 57 CVA4 strains isolated from Ganyu County was 94.0%-100%, amino acid sequence homology was 97.8%-100%. Two strains were selected as 915 nucleotides of VPl coding region gene. The nucleotide sequence homology and amino acid sequence homology were 94.1% - 100% and 97.3% - 100% respectively. The homology was high and the evolutionary relationship was close. 3. The VP1 sequences of 21 CVA4 representative strains from 2008 to 2012 obtained from China and isolated from GenBank were analyzed. The VP1 sequences of 10 CVA4 isolates from mainland China were analyzed for homology: 31 CVA4 isolates from mainland China had 85.5% - 100% homology in VP1 region, 97.5% - 100% homology in amino acid sequence and high homology, belonging to the same genotype. The nucleotide mutations were mostly synonymous mutations; two AFP isolates from Shandong Province in 1998. The nucleotide sequence homology of CVA4 strain was 88.5% - 89.9% compared with other isolates. Excluding the isolates from 1998, the homology of nucleotide sequence and amino acid sequence between other HFMD and AFP isolates was 94.8% and 98.4%, respectively. There was no specific difference between the two types of cases. The nucleotide sequence homology and amino acid sequence homology were 95.5%, 98.8% and 95.5%, respectively. There were no specific differences in nucleotides and amino acids between the two types of HFMD cases. 4. The VP1 sequences of 21 representative CVA4 strains obtained in this study and the VP1 sequences of 21 global CVA4 strains obtained in GenBank were used as phylogenetic trees. Based on the international enterovirus genotyping standard, CVA4 can be divided into five genotypes according to the full length of VP1 coding gene, namely A, B, C, D and E genotypes. This is the first genotyping of CVA4 based on the full length of VP1 gene. 31 strains of CVA4 virus isolated from mainland China belong to E genotype, and E genotype is divided into E1 and E2 genotype. E1 genotype includes 2 strains isolated from AFP cases in Shandong Province in 1998 and 1 strain isolated from AFP cases in Jilin Province in 2006. E2 genotype includes 22 strains of CVA4 isolated from HFMD cases in 2008-2012. The average genetic distance of nucleotide sequences between E1 and E2 genotypes was 12.6%. It was found that there were at least two large transmission chains in mainland China. E1 genotype was predominant in 2006 and before, and E2 genotype was predominant after 2006. The sequence information of VP1 region 3'end of CVA4 in ank was more abundant than that of VP1 region. Based on the VP1 region 3'end of 420 nucleotide sequences of 21 CVA4 strains in this study, 420 nucleotide sequences of VP1 region and 44 CVA4 VP1 region 3'end of representative strains in GenBank were used as phylogenetic trees. The genetic distances were 13.4%-26.1% and 5.8%-9.6% respectively among the nucleotide sequences of Lineage A-E. The evolutionary branches were consistent with the results of the evolutionary tree of VP1 gene length. The genotypes A-E contained 31 strains of CVA4 isolated from mainland China. Viruses belong to Lineage E. Lineage E can also be divided into Lineage E1 and Lineage E2, and the results are consistent with the VP1 full-length division. Lineage E includes not only the Chinese mainland strains, but also the Taiwan strains. 57 strains of CVA 4 isolated from Ganyu County, Jiangsu Province. Sequence analysis based on the 5'end indicates that all of them are E2 genotypes. 5 CVA4 representative strains were selected to complete the 3D region sequencing and compared with other retrievable CVA4, HEV-A group enterovirus prototypes, BLAST high homology strains and EV71 C4a genotype representative strains to construct phylogenetic tree: 5 CVA4 strains and 2009 isolation from China. The CVA 4 strain from Shenzhen (HQ728260) and EV71 strain (JF 799986) isolated from Guangdong Province in 2009 had a high homology of 94.6% - 96% in one evolutionary branch; the genetic distance between the prototype strain of EV71 and BrCr strain of EV71 or other representative strains of EV71 C4a genotype was relatively long, and the sequence homology was only 73.3% - 81.7%. CVA4 is one of the main pathogens causing hand-foot-mouth disease, viral encephalitis, herpes angina and other diseases in Ganyu County, Jiangsu Province in 2012. It has a large-scale circulation and multiple transmission chains. 2. This study is the first time to compile the VP1 region of CVA4 strain. All CVA4 viruses isolated from mainland China belong to E1 and E2 subtypes of E genotype. 3. A Guangdong EV71 strain isolated in 2009 may be associated with a 3 D region EV71 strain. The present epidemic CVA4 in China has been recombined. We should pay attention to the role of the 3D region of CVA4 as a donor in the recombination of enteroviruses. At the same time, we should pay attention to the effects of the recombinant CVA4 on the biological characteristics, transmission and pathogenicity of EV71. 4. The results of this study provide important virological basic data for the diagnosis and prevention of infectious diseases such as HDMD caused by CVA4.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.5
本文編號(hào)：2216104
[Abstract]:[BACKGROUND] Human Enterovirus (HEV) belongs to the genus Enterovirus of the Picornavirdae family. At present, 119 serotypes have been reported and divided into four groups: EV-A, EV-B, EV-C and EV-D. In most cases, patients infected with HEV are asymptomatic or cause only mild symptoms, such as fever, rash, or EV-D. Mild upper respiratory symptoms; but sometimes HEV infection can also cause a wide range of clinical diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, acute flaccid paralysis (AFP), hand, foot and mouth disease (Hand, Foot and Mouth Disease, myocarditis, cold-like diseases, etc. In recent years, caused by HEV a variety of diseases. The genome of the HEV virus is a single strand of positive strand RNA, about 7.5 KB long. The whole genome contains only one Open Reading Frame (ORF), encodes a polyprotein, and finally hydrolyzes to produce VP1-VP4 with four structural proteins and seven non-structural proteins. VP1 protein is the main structure that recognizes and binds to receptors on the host cells and contains important serotype-specific neutralizing sites; VP1 protein is located in the outermost appearance of viral particles and mutates under the immune pressure to form different epidemic strains. Hand-foot-mouth disease (HFMD) is a common infectious disease caused by a variety of enteroviruses (EV). Since the outbreak of HFMD in the mainland of China in 2007, hand-foot-mouth disease (HFMD) has gradually attracted attention, according to the relevant literature in recent years. It is reported that HFMD has become one of the major public health problems that seriously threaten the health and life safety of the broad population, especially children. EV71 and CA16 are the main pathogens causing HFMD. EV71 and CA16 are the main pathogens of HFMD. The etiological studies of HFMD are mostly focused on EV71 and CA16, and other intestines. However, in recent years, HFMD surveillance data in some areas show that the pathogenic composition of other enteroviruses has increased, and CVA4 is one of them. [Objective] Through systematic analysis of VP1 and 3D gene characteristics of CVA4 strains from HFMD cases in mainland China from 2008 to 2012, to elucidate their genetic variation in China and enrich the CVA4 strain library and gene information database in China and the world, so as to provide scientific support for the diagnosis, prevention and control of HFMD caused by CVA4 in China. Source of the virus: In 2012, 1246 HFMD, viral encephalitis and herpes angina were isolated from clinical specimens of Ganyu County, Jiangsu Province, 57 strains of CVA4 virus were isolated; in 2008-2012, 19 strains of CVA4.2 were isolated from HFMD cases in other areas by HFMD Surveillance Network Laboratory of mainland China. Gene sequencing: virus RNA was extracted, VP 1 and 3D region genes were amplified by RT-PCR. Bioinformatics analysis: The VP1 and 3D genes of CVA4 in this study were compared with the VP1 and 3D genes of CVA4 retrieved from GenBank in China and other countries and regions by using BioEdit Sequence Alignment Editor software 7.0. The phylogenetic tree of VP1 and 3D regions was constructed by Neighbor-joining method, and the phylogenetic relationship and evolution were analyzed. [Results] 1. HFMD, viral encephalitis, herpes angina and other clinical specimens in Ganyu County, Jiangsu Province were separated. Among 350 strains, CVB3 (142 strains), CVA4 (57 strains), CVA10 (31 strains), CVA16 (29 strains), EV71 (24 strains), etc. CVA4 ranked second, accounting for 16%. The 564 nucleotide sequence homology of 57 CVA4 strains isolated from Ganyu County was 94.0%-100%, amino acid sequence homology was 97.8%-100%. Two strains were selected as 915 nucleotides of VPl coding region gene. The nucleotide sequence homology and amino acid sequence homology were 94.1% - 100% and 97.3% - 100% respectively. The homology was high and the evolutionary relationship was close. 3. The VP1 sequences of 21 CVA4 representative strains from 2008 to 2012 obtained from China and isolated from GenBank were analyzed. The VP1 sequences of 10 CVA4 isolates from mainland China were analyzed for homology: 31 CVA4 isolates from mainland China had 85.5% - 100% homology in VP1 region, 97.5% - 100% homology in amino acid sequence and high homology, belonging to the same genotype. The nucleotide mutations were mostly synonymous mutations; two AFP isolates from Shandong Province in 1998. The nucleotide sequence homology of CVA4 strain was 88.5% - 89.9% compared with other isolates. Excluding the isolates from 1998, the homology of nucleotide sequence and amino acid sequence between other HFMD and AFP isolates was 94.8% and 98.4%, respectively. There was no specific difference between the two types of cases. The nucleotide sequence homology and amino acid sequence homology were 95.5%, 98.8% and 95.5%, respectively. There were no specific differences in nucleotides and amino acids between the two types of HFMD cases. 4. The VP1 sequences of 21 representative CVA4 strains obtained in this study and the VP1 sequences of 21 global CVA4 strains obtained in GenBank were used as phylogenetic trees. Based on the international enterovirus genotyping standard, CVA4 can be divided into five genotypes according to the full length of VP1 coding gene, namely A, B, C, D and E genotypes. This is the first genotyping of CVA4 based on the full length of VP1 gene. 31 strains of CVA4 virus isolated from mainland China belong to E genotype, and E genotype is divided into E1 and E2 genotype. E1 genotype includes 2 strains isolated from AFP cases in Shandong Province in 1998 and 1 strain isolated from AFP cases in Jilin Province in 2006. E2 genotype includes 22 strains of CVA4 isolated from HFMD cases in 2008-2012. The average genetic distance of nucleotide sequences between E1 and E2 genotypes was 12.6%. It was found that there were at least two large transmission chains in mainland China. E1 genotype was predominant in 2006 and before, and E2 genotype was predominant after 2006. The sequence information of VP1 region 3'end of CVA4 in ank was more abundant than that of VP1 region. Based on the VP1 region 3'end of 420 nucleotide sequences of 21 CVA4 strains in this study, 420 nucleotide sequences of VP1 region and 44 CVA4 VP1 region 3'end of representative strains in GenBank were used as phylogenetic trees. The genetic distances were 13.4%-26.1% and 5.8%-9.6% respectively among the nucleotide sequences of Lineage A-E. The evolutionary branches were consistent with the results of the evolutionary tree of VP1 gene length. The genotypes A-E contained 31 strains of CVA4 isolated from mainland China. Viruses belong to Lineage E. Lineage E can also be divided into Lineage E1 and Lineage E2, and the results are consistent with the VP1 full-length division. Lineage E includes not only the Chinese mainland strains, but also the Taiwan strains. 57 strains of CVA 4 isolated from Ganyu County, Jiangsu Province. Sequence analysis based on the 5'end indicates that all of them are E2 genotypes. 5 CVA4 representative strains were selected to complete the 3D region sequencing and compared with other retrievable CVA4, HEV-A group enterovirus prototypes, BLAST high homology strains and EV71 C4a genotype representative strains to construct phylogenetic tree: 5 CVA4 strains and 2009 isolation from China. The CVA 4 strain from Shenzhen (HQ728260) and EV71 strain (JF 799986) isolated from Guangdong Province in 2009 had a high homology of 94.6% - 96% in one evolutionary branch; the genetic distance between the prototype strain of EV71 and BrCr strain of EV71 or other representative strains of EV71 C4a genotype was relatively long, and the sequence homology was only 73.3% - 81.7%. CVA4 is one of the main pathogens causing hand-foot-mouth disease, viral encephalitis, herpes angina and other diseases in Ganyu County, Jiangsu Province in 2012. It has a large-scale circulation and multiple transmission chains. 2. This study is the first time to compile the VP1 region of CVA4 strain. All CVA4 viruses isolated from mainland China belong to E1 and E2 subtypes of E genotype. 3. A Guangdong EV71 strain isolated in 2009 may be associated with a 3 D region EV71 strain. The present epidemic CVA4 in China has been recombined. We should pay attention to the role of the 3D region of CVA4 as a donor in the recombination of enteroviruses. At the same time, we should pay attention to the effects of the recombinant CVA4 on the biological characteristics, transmission and pathogenicity of EV71. 4. The results of this study provide important virological basic data for the diagnosis and prevention of infectious diseases such as HDMD caused by CVA4.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 吉彥莉;王永全;;手足口病患兒柯薩奇病毒A4的鑒定及其5′UTR-VP4-VP2區(qū)序列分析[J];中國(guó)預(yù)防醫(yī)學(xué)雜志;2012年09期
2 劉麗艷;朱啟昒;;腸道病毒71型分子生物學(xué)研究進(jìn)展[J];微生物與感染;2010年01期
3 劉建鋒;張勇;李慧;趙曉虹;甄曉榮;嚴(yán)冬梅;付鴻;陳瑛;王東絕;祝雙利;安洪秋;郭學(xué)斌;許文波;;從手足口病病例分離的兩株柯薩奇病毒A組4型毒株的VP_4～VP_2區(qū)基因特征分析[J];中國(guó)疫苗和免疫;2009年04期
4 毛乃穎;王海巖;朱貞;李巖;紀(jì)峰;陶澤新;崔愛(ài)利;許文波;;山東省人類腸道致細(xì)胞病變孤兒病毒13型分離株的基因特征分析[J];中國(guó)疫苗和免疫;2009年03期
5 朱理業(yè);丁振濤;萬(wàn)俊峰;劉奇泉;將_";徐海洋;李曉鵬;張勇;薛彬;王維;田亞貞;見(jiàn)海暉;;阜陽(yáng)市手足口病(EV71感染)重癥病例流行病學(xué)調(diào)查分析[J];安徽醫(yī)學(xué);2008年05期
6 陸一涵;姜慶五;;人腸道病毒71型與手足口病[J];疾病控制雜志;2008年03期
7 ;Expression,purification and characterization of enterovirus-71 virus-like particles[J];World Journal of Gastroenterology;2006年06期
8 李錫太;王全意;黃芳;李潔;曲梅;李爽;劉白薇;吳曉娜;張莉;黎新宇;;北京市2007-2010年手足口病流行特征分析[J];國(guó)際病毒學(xué)雜志;2011年01期
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