本文選題：人類腸道病毒　切入點(diǎn)：B組　出處：《山東大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：[背景] 人類腸道病毒(HEV)是一類在人類腸道中繁殖的常見病毒,根據(jù)生物學(xué)及遺傳特性HEV被劃分為A、B、C、D共4個基因組。HEV感染可引起多種疾病。無菌性腦膜炎(AM)主要是由各種病毒引起的一組以精神和意識障礙為突出表現(xiàn)的中樞神經(jīng)系統(tǒng)感染性疾病,輕者能自行緩解,危重者可導(dǎo)致后遺癥及死亡。文獻(xiàn)報道AM全球每年發(fā)病率為11～27/10萬。在所明確引起AM的病原體中,HEV約占85%～95%。 20世紀(jì)80年代以來,隨著全球成功開展消滅脊髓灰質(zhì)炎活動,許多國家已相繼實(shí)現(xiàn)無脊髓灰質(zhì)炎目標(biāo),與此同時,由非脊髓灰質(zhì)炎腸道病毒(NPEV)引起的各類疾病如AM和手足口病(HFMD)的發(fā)病近年一直呈上升趨勢。 本研究從1994～2008年山東省急性弛緩性麻痹病例(AFP)的常規(guī)監(jiān)測中分離到大量的HEV～B,同時對部分引起疾病暴發(fā)的優(yōu)勢毒株進(jìn)行了序列的測定和同源性分析,初步建立起HEV-B山東地方株的VPl區(qū)全基因數(shù)據(jù)庫。本研究對于明確山東省HEV～B基因型的分布,以及通過分子流行病學(xué)方法闡明疾病的發(fā)生與病毒的遺傳進(jìn)化之間的內(nèi)在聯(lián)系均具有重大的理論和現(xiàn)實(shí)意義。 [研究的目的] 1.描述山東省1994～2008年HEV-B的分離情況,研究HEV-B山東地方株的基因型分布； 2.建立HEV-B山東地方株的VP1完整編碼區(qū)基因數(shù)據(jù)庫； 3.分析HEV-B部分優(yōu)勢毒株的VPl完整編碼區(qū)基因序列的特征,并與國外和國內(nèi)其他省份的分離株進(jìn)行比較,分析其進(jìn)化來源； 4.探討HEV-B主要型別與其所致疾病之間的聯(lián)系,分析遺傳變異對其致病性和流行規(guī)律的影響,為相關(guān)疾病的預(yù)防控制提供理論依據(jù)。 [研究方法] 1.對1994-2008年山東省AFP監(jiān)測系統(tǒng)中的病例標(biāo)本以及2003～2008年3次AM暴發(fā)中的部分病例標(biāo)本,采用細(xì)胞培養(yǎng)的方法進(jìn)行病毒分離。對分離到的陽性分離物采用組合血清進(jìn)行中和試驗(yàn),確定其血清型。 2.按照毒株的分離年份及分離地點(diǎn),選取具有代表性的部分毒株進(jìn)行核酸的提取和VPl區(qū)序列的測定。 3.將測得的基因序列進(jìn)行剪裁、拼接,利用NCBI提供的BLAST服務(wù)器進(jìn)行序列比對,確定HEV-B山東地方株的基因型別。 4.將HEV-B山東地方株與GenBank中檢索到的其它國家和地區(qū)分離株,采用BioEdit Sequence Alignment Editor software 5.0.9軟件,對其VP1區(qū)核苷酸序列及其推導(dǎo)氨基酸序列進(jìn)行同源性比較。 5.采用Mega4.1軟件,以相鄰連接方法(Neighbor-joining Method)構(gòu)建VP1區(qū)系統(tǒng)發(fā)生樹,對HEV-B山東地方株進(jìn)行親緣進(jìn)化分析。 [主要結(jié)果] 1.山東省HEV-B的分離情況：1994～2008年山東省AFP病例的糞便標(biāo)本共分離出NPEV 1021株,各年度的分離率在7.7%-17.2%之間；2003～2008年山東省3次AM暴發(fā),其病原體均屬于HEV-B,分別為CVB3、CVB5和ECH030。 2.HEV-B山東地方株基因型分布及其VPl區(qū)全基因進(jìn)化樹：HEV-B山東地方株分屬于29個基因型,其中ECHO11、CVB3、ECHO6、ECHO14、ECHO25是分布最為廣泛的HEV-B；各基因型HEV-B無明顯的地區(qū)分布。 山東省HEV-B可以分為3個簇(Cluster), Cluster1包括ECH030在內(nèi)的12種ECHO病毒,Cluster2包括CVB 1-5, Cluster3包括ECHO 12在內(nèi)的另外11種ECHO病毒及CVA 9。每種血清型病毒單獨(dú)成簇。 3.主要的HEV-B山東地方株遺傳進(jìn)化分析 (1)HEV-B山東地方株VPl區(qū)全基因數(shù)據(jù)庫的建立及其同源性分析：根據(jù)VP1區(qū)全基因數(shù)據(jù)庫毒株備選原則,從山東省AFP病例中分離到的HEV-B中選取171株,與HEV-B所致疾病暴發(fā)中的33株分離株,一起構(gòu)建了山東地方株VP1區(qū)全基因數(shù)據(jù)庫。 VP1區(qū)全基因序列同源性分析顯示：山東地方株型內(nèi)核苷酸差異最大的為ECHO1(6.7%-24.6%),差異最小的為CVB4(2.9%-5.6%)；CVB2與其原型株相比核苷酸差異最小(4.8%-7.7%),EV73型與其原型株相比核苷酸差異最大(24.6%)。 (2)CVB3山東地方株遺傳進(jìn)化分析：CVB3山東地方株與原型株之間的核苷酸同源性為77-8%-80.0%,氨基酸同源性為96.1%-97.5%。分離株177/2008TC/SD/CHN的核苷酸變異位點(diǎn)及變異結(jié)果與其余4株都有較大的差異；5株山東地方株氨基酸變異的數(shù)目為9或10。CVB3毒株可劃分為A、B、C共3個基因型,山東地方株分布在A基因型內(nèi)。A1-A3 3個基因亞型內(nèi)的山東地方株分離年份依次為2004～2008年、1994～2002年、2004年和2008年。 (3)CVB5山東地方株遺傳進(jìn)化分析：CVB5山東地方株與原型株之間的核苷酸同源性為81.2%-81.6%,氨基酸同源性均為96.8%；山東地方株之間的核苷酸同源性為83.0%-94.6%,氨基酸同源性為96.4%-100.0%。在VP1完整編碼區(qū)中核苷酸的變異位點(diǎn)分布較為均勻,也沒有顯示時間上的聚集性。相對于原型株,山東地方株發(fā)生了9個氨基酸變異。CVB5毒株劃分為A、B、C、D 4個基因型,山東地方株分布在C和D兩個基因型內(nèi)。進(jìn)化樹中與山東地方株同源性最近的是浙江分離株Cox B5/Zejiang/12/02 (CSF)。 (4)ECH06山東地方株遺傳進(jìn)化分析：ECH06山東地方株之間的核苷酸同源性為77.6%～98.6%,氨基酸同源性為93.7%-99.6%,與原型株之間的核苷酸同源性為75.2%-78.8%,氨基酸同源性為92.7%-95.5%。VP1完整編碼區(qū)核苷酸的變異顯示出時間上的聚集性；氨基酸的變異存在三處變異較為密集的區(qū)域,變異數(shù)目為13-21。ECH06毒株劃分為A、B、C共3個基因型,山東地方株分布在B和C兩個基因型內(nèi)。山東地方株來源于3個不同的進(jìn)化分支。 (5)ECHO11山東地方株遺傳進(jìn)化分析：ECHO11山東地方株之間的核苷酸同源性為76.4%～100.0%,氨基酸同源性為91.4%-100.0%；ECHO11山東地方株與原型株Gregory之間的核苷酸同源性為77.7%-80.7%,氨基酸同源性為90.7%-94.8%。ECHO11山東地方株氨基酸變異數(shù)目大于20,存在時間上的聚集性以及變異較為密集的區(qū)域。ECHO11毒株劃分為A、B、C、D 4個基因型,山東地方株分布在A和C兩個基因型內(nèi)。山東地方株構(gòu)成了A1、A2和C1共3個新的基因亞型,A1基因亞型內(nèi)的毒株均分離自1998年之后,A2和C1兩個基因亞型內(nèi)的毒株均分離自1994-1997年。 (6)ECHO19山東地方株遺傳進(jìn)化分析：ECHO19山東地方株之間的核苷酸同源性為83.3%～98.6%,氨基酸同源性為98.6%～99.3%。山東地方株與原型株之間的核苷酸同源性為78.6%～79.2%,氨基酸同源性為97.6%～99.3%。核苷酸與氨基酸的比對結(jié)果顯示：氨基酸變異數(shù)目大于20,分離年份相近的毒株之間基因序列變異情況相似,毒株97206/SD/CHN/1997/E19基因序列變異與其他分離株相比差異較大。進(jìn)化樹顯示9株國內(nèi)毒株存在集聚現(xiàn)象,形成一個基因簇。 (7)ECHO30山東地方株遺傳進(jìn)化分析：ECHO30山東地方株與原型株之間的核苷酸同源性為81.9%～83.5%,氨基酸同源性為92.4%～93.1%；山東地方株之間的核苷酸同源性為86.0%～98.5%,氨基酸同源性為96.9%～98.9%。毒株06350/SD/CHN/2006/E30與其他山東地方株之間的同源性較小。核苷酸的變異情況顯示：06530/SD/CHN/2006/E30和017/2008TC/SD/CHN 2株毒株與其他山東地方株的變異數(shù)目和變異位點(diǎn)均存在較大差異。山東地方株氨基酸變異數(shù)目大于20。ECHO30毒株劃分為A～E共5個基因型,國內(nèi)株全部分布在E基因型內(nèi),山東地方株分布在E1、E3兩個基因亞型內(nèi)。 4.部分HEV-B山東地方株引起的AM暴發(fā)：2003年章丘市AM暴發(fā)中各年齡別罹患率在7歲前隨年齡增加而有所升高,7歲后隨年齡的增加而減少�；颊呔哂休^為明確的接觸史,神經(jīng)系統(tǒng)癥狀較為明顯。引起此次疾病暴發(fā)病原體為單一的ECHO30病毒,與同時期山東省泰安市、浙江省和江蘇省AM暴發(fā)中的分離株之間存在較為密切的流行病學(xué)聯(lián)系。 2005年山東省兗州市AM暴發(fā)以6歲以下兒童為主,2歲以下嬰兒罹患率最高。病例及其看護(hù)人均無明確的接觸史,患者的臨床癥狀較輕。引起此次疾病暴發(fā)的病原體為CVB5毒株,主要分離自腦脊液標(biāo)本,與2002的浙江分離株CoxB5/Zejiang/12/02 (CSF)流行病學(xué)聯(lián)系密切。 2008年山東省郯城縣AM暴發(fā)中1歲以下兒童發(fā)病數(shù)最多,各年齡別罹患率在隨年齡增加而降低。大部分患者有AM病例接觸史,臨床癥狀相對較輕。引起此次疾病的病原體為CVB3,此次暴發(fā)由同一傳播鏈的CVB3毒株所致,毒株與進(jìn)化樹中其他2008年的分離株同源性較近,為同一進(jìn)化來源。 [主要結(jié)論] 1.HEV-B在山東省分布廣泛,對人群健康存在著重要的影響,AFP病例監(jiān)測系統(tǒng)每年可以分離到大量的NPEV；不同基因型毒株有不同的時間循環(huán)模式；某些優(yōu)勢基因型如CVB5、CVB3、ECHO30在山東省呈中度流行狀態(tài),優(yōu)勢毒株的變遷往往伴隨著疾病的暴發(fā)。 2.VP1區(qū)全基因序列同源性分析顯示,各基因型HEV-B山東地方株與其原型株相比均發(fā)生了較大的變異,相對于ECHO病毒,CV病毒核苷酸的變異大多為同義突變,并沒有引起氨基酸的變異；ECHO病毒在進(jìn)化中較為活躍。 3.相同基因型的HEV-B山東地方株內(nèi)部,根據(jù)其遺傳距離的遠(yuǎn)近可以劃分為不同的基因亞型,進(jìn)化樹分析顯示相同基因亞型毒株之間在地理和時間分布上存在著一定的聚集性。 4.在山東省,HEV-B引起的疾病暴發(fā)仍以AM為主,在HFMD的流行中偶見HEV-B組病毒,多數(shù)疾病暴發(fā)中分離到的HEV-B為單一的血清型,這與HEV-A病毒之間常見的協(xié)同流行略有不同。 5.對于部分HEV-B優(yōu)勢毒株的分子流行病學(xué)研究顯示了其各自的流行特征：①CVB3山東地方株為原型株近似株而非變異株近似株,病毒在國內(nèi)傳播迅速,廣泛流行,相同的年份有多個傳播鏈的CVB3病毒在山東省流行；②CVB5毒株的變異進(jìn)化速度相對較慢,進(jìn)化樹中D1基因亞簇內(nèi)毒株2002-2005年在亞洲和歐洲的流行,導(dǎo)致了2002年浙江省和2005年山東省充州市AM的暴發(fā)；③ECH06毒株變異進(jìn)化較為迅速,山東省近年流行株與往年流行株之間的核苷酸序列存在較大差異,可能與往年毒株為不同進(jìn)化來源；④ECHO11病毒在進(jìn)化上較為活躍,1994-1997年期間山東省存在兩個差異較大的ECHO11基因亞型共循環(huán),而A1亞型內(nèi)的毒株為近年流行的基因亞型；⑤ECH019山東地方株的進(jìn)化變異速度較慢,引起2003年泰安市疾病暴發(fā)的毒株在山東省流行廣泛；⑥2003年章丘市AM暴發(fā)的ECH030分離株存在較大的抗原變異,感染性強(qiáng),傳播迅速,為山東省近幾年的主要流行株,在2003-2008年山東省存在兩個不同基因亞型的毒株流行。
[Abstract]:[background]
Human enterovirus (HEV) is a kind of breeding in the human gut the common virus, is divided into A, according to the biological and genetic characteristics of HEV B, C D, a total of 4 genomic.HEV infection can cause many diseases. Aseptic meningitis (AM) is the main disease caused by various viruses in a group of fine God and consciousness for the outstanding performance of the central nervous system infection, it can relieve itself, can lead to severe sequelae and death. The reported AM global annual incidence rate of 11 ~ 27/10 million. In the clear the causative agent of AM, HEV was about 85% ~ 95%.
Since 1980s, with the successful development of global polio eradication activities, polio free target, has been achieved in many countries at the same time, the non polio enterovirus (NPEV) caused by various diseases such as AM and hand foot mouth disease (HFMD) incidence in recent years has been on the rise.
In this study, 1994~2008 years from the Shandong province of acute flaccid paralysis (AFP) isolated from a large number of HEV ~ B in routine monitoring, at the same time on the part of the cause of outbreaks of dominant strains were determined and sequence homology analysis, initially established a whole genome database of HEV-B isolated in Shandong area VPl. This study to clarify Shandong province HEV ~ B genotype, the intrinsic link between genetic and molecular epidemiological methods to elucidate and by the viral diseases are of great theoretical and practical significance.
[the purpose of the study]
1. to describe the isolation of HEV-B in 1994~2008 years in Shandong Province, and to study the genotype distribution of HEV-B local strains in Shandong.
2. the VP1 complete coding region gene database of HEV-B Shandong local strain was established.
3., we analyzed the characteristics of VPl complete coding region gene sequence of some HEV-B dominant strains, and compared with the isolates from other provinces and other provinces in China, and analyzed their evolutionary sources.
4., we discussed the relationship between the main types of HEV-B and its diseases, and analyzed the effects of genetic variation on their pathogenicity and epidemic rule, so as to provide a theoretical basis for the prevention and control of related diseases.
[research methods]
1. in 1994-2008 years, the samples from AFP monitoring system in Shandong province and 2003~2008 cases of AM outbreak in 2003~2008 years were separated by cell culture. The positive isolates were neutralized by combinatorial serum, and their serotypes were determined.
2. the extraction of nucleic acid and the determination of VPl region sequence were selected according to the separation year and the separation site of the strains.
3. the gene sequence was tailored and spliced, and the sequence alignment of the BLAST server provided by NCBI was used to determine the genotypes of the local strains of HEV-B in Shandong.
4., we isolate the isolates from HEV-B Shandong and other countries and regions retrieved in GenBank. We used BioEdit Sequence Alignment Editor software 5.0.9 software to compare their nucleotide sequences and deduced amino acid sequences in VP1 region.
5. Mega4.1 software was used to construct a VP1 region phylogenetic tree with adjacent connection method (Neighbor-joining Method), and the phylogenetic evolution of local strains of HEV-B in Shandong was analyzed.
[main results]
1., the separation of HEV-B in Shandong Province: 1994~2008 years, 1994~2008 cases of NPEV were isolated from fecal specimens of AFP cases in Shandong province. The isolation rate in each year was between 7.7%-17.2%. In 2003~2008 years, 3 times AM outbreak in Shandong Province, all the pathogens were HEV-B, CVB3, CVB5 and ECH030. respectively.
2.HEV-B genotype distribution and VPl region gene evolution tree of Shandong local strain: HEV-B, Shandong local strain belongs to 29 genotypes, ECHO11, CVB3, ECHO6, ECHO14, ECHO25 are the most widely distributed HEV-B, and genotype HEV-B has no obvious regional distribution.
Shandong HEV-B can be divided into 3 clusters (Cluster), Cluster1 includes ECH030, including 12 ECHO viruses, Cluster2 includes CVB 1-5, Cluster3 includes ECHO 12, including 11 other ECHO viruses and CVA 9., each serotype virus is clustered separately.
Analysis of genetic evolution of 3. main HEV-B Shandong local strains
(1) establishment and homology analysis of whole genome database VPl HEV-B isolates from Shandong: according to the complete gene database VP1 strains isolated from the alternative principle, Shandong Province in the case of AFP HEV-B in the selected 171 strains, and HEV-B caused by disease outbreaks in 33 isolates, together to construct the whole gene database VP1 Shandong local strains.
VP1 gene sequence homology analysis showed that: Shandong local plant nucleotide difference maximum is ECHO1 (6.7%-24.6%), the smallest difference is CVB4 (2.9%-5.6%); CVB2 and its prototype strains compared to nucleotide differences (4.8%-7.7%), minimum EV73 and its prototype strain by nucleotide difference (24.6%).
(2) CVB3 analysis of Shandong local strains of genetic evolution: CVB3 isolates from Shandong and nucleotide homology between prototype strains of 77-8%-80.0%, amino acid homology of nucleotide variation and variation of the loci for 96.1%-97.5%. isolates of 177/2008TC/SD/CHN and other 4 strains are different; 5 strains of amino acid variation in the number of isolates from Shandong was 9 or 10.CVB3 strains can be divided into A, B, C a total of 3 genotypes, Shandong local strains of the year Shandong local strain distribution of.A1-A3 in the A genotype in 3 genotypes were within 2004~2008 years, 1994~2002 years, 2004 and 2008.
(3) CVB5 analysis of Shandong local strains of genetic evolution: CVB5 isolates from Shandong and nucleotide homology between the prototype strain 81.2%-81.6%, the homology of amino acid was 96.8%; Shandong local strains of the homology between 83.0%-94.6% and amino acid homology was 96.4%-100.0%. in VP1 complete nucleotide mutation sites in the encoding region is more evenly distributed, also show no aggregation time. Compared with the prototype strain, Shandong strain had 9 amino acid variant.CVB5 strains are divided into A, B, C, D 4 genotypes, Shandong local strain distribution in C and D two genotypes. And Shandong local strains recently isolated in Zhejiang is Cox B5/Zejiang/12/02 phylogenetic tree (CSF).
(4) ECH06 analysis of Shandong local strains of genetic evolution: Shandong ECH06 strains and the homology between 77.6% ~ 98.6%, the homology of amino acid was 93.7%-99.6%, and the nucleotide homology between the prototype strain 75.2%-78.8%, the homology of amino acid mutations for nucleotide 92.7%-95.5%.VP1 complete encoding region shows the time aggregation of the amino acid variation; there are three variants in more populated areas, the number of variant 13-21.ECH06 strains are divided into A, B, C a total of 3 genotypes, Shandong local strain distribution in B and C two genotypes. Shandong local strains derived from 3 different evolutionary branches.
(5) ECHO11 analysis of Shandong local strains of genetic evolution: Shandong ECHO11 strains and the homology between 76.4% ~ 100% amino acid homology to 91.4%-100.0%; ECHO11 between Shandong local strains and prototype strain Gregory nucleotide homology was 77.7%-80.7%, amino acid homology is the number of amino acid variation of 90.7%-94.8%.ECHO11 isolates from Shandong more than 20, the presence of time the aggregation and variation of relatively dense region of the.ECHO11 strain is divided into A, B, C, D 4 genotypes, Shandong local strain distribution in A and C two genotypes. Shandong local strains constitute the A1, A2 and C1 were 3 new genotypes, A1 gene subtype strains the separation since 1998, A2 and C1 strains of two genotypes were isolated from within 1994-1997 years.
(6) ECHO19 analysis of Shandong local strains of genetic evolution: Shandong ECHO19 strains and the homology between 83.3% ~ 98.6% amino acid homology is 98.6% ~ 99.3%. Shandong local strains and prototype strains nucleotide homology is 78.6% ~ 79.2%, the homology of amino acid was 97.6% ~ 99.3%. compared the nucleotide and amino acid showed that the amino acid the variation is greater than the number of 20 gene sequences similar to the separation between year similar strains, gene sequences of 97206/SD/CHN/1997/E19 strain compared with other strains. The phylogenetic tree showed large differences in strains of 9 strains of endotoxin agglomeration, forming a gene cluster.
(7) ECHO30 analysis of Shandong local strains of genetic evolution: ECHO30 isolates from Shandong and nucleotide homology between prototype strains ranged from 81.9% to 83.5%, the amino acid homology is 92.4% ~ 93.1%; Shandong local strains of the homology between 86% ~ 98.5% amino acid homology is 96.9% ~ 98.9%. 06350/SD/CHN/2006/E30 strain and other places of Shandong strains the homology of nucleotide variation is small. Display: 06530/SD/CHN/2006/E30 and 017/2008TC/SD/CHN 2 isolates and other isolates from Shandong the number variation and variation sites are quite different. The number of amino acid variations in Shandong local strains greater than 20.ECHO30 strains divided into A to E a total of 5 genotypes, the E genotype distribution in all strains in Shandong the local strain distribution in E1, E3 two subtypes.
The 4. part of the HEV-B Shandong strain caused by the outbreak of AM in Zhangqiu city in 2003: the outbreak of AM in each age incidence at the age of 7 with the increase of age increased, after 7 years of age decreased with the increasing age. Patients with definite contact history, nervous system symptoms are obvious. That caused the outbreak as a single pathogen the ECHO30 virus, with the same period in Shandong city of Tai'an Province, there is an epidemiological link more closely in Zhejiang province and Jiangsu Province in the outbreak of AM isolates.
Yanzhou City, Shandong province in 2005 outbreak of AM in children under 6 years old, infants under the age of 2 with the highest rate of patients and their carers. No clear history of exposure, the clinical symptoms of the patients were mild. Caused the disease outbreaks is the pathogen of CVB5 strains, mainly isolated from cerebrospinal fluid specimens, and 2002 strains of CoxB5/Zejiang/12/02 isolated in Zhejiang (CSF epidemiology) closely.
In 2008, Shandong County of Tancheng province AM in children under 1 years of age the largest incidence, prevalence rate in each age and decreased with the increasing of age. Most of the patients with AM cases of contact history, clinical symptoms are relatively mild. Caused by the pathogen of this disease is CVB3, by the outbreak of CVB3 strain caused by the same transmission chain, the other in 2008 strain and phylogenetic tree of isolates homology is near, for the same evolutionary origin.
[main conclusions]
1.HEV-B is widely distributed in Shandong Province, has an important impact on the health of the population, AFP surveillance system every year can be separated to a large number of NPEV; different strains have different circulation patterns; some dominant genes such as CVB5, CVB3, ECHO30 showed a moderate epidemic situation in Shandong Province, the dominant strains are often accompanied by changes an outbreak of disease.
2.VP1鍖哄叏鍩哄洜搴忓垪鍚屾簮鎬у垎鏋愭樉紺，

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