【作者】 翟友刚；

【导师】 梁国栋；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2008， 博士

【摘要】 甘肃省和陕西省位于我国西北部,是通往大西北内陆的门户,也是我国南北气候分界线所在。这两省长期以来是我国虫媒传染病——乙型脑炎的流行疫区,特别是陕西省近年来的乙型脑炎发病率一直较高。甘肃省天水、陇南和平凉市位于甘肃东南部,紧邻乙脑高发区陕西省和四川省,存在着多种虫媒病毒的传播媒介。对陕西省和甘肃省的病毒性脑炎流行病学监测结果显示,在蚊虫活动高峰的夏秋季节,当地存在病因不明的发热和病毒性脑炎患者,提示当地存在其它虫媒病毒性疾病的可能性。目前我国正进行西部大开发,将有大量人员和物资的来往和流动,可能会造成虫媒病毒病的传入、甚至爆发流行。因此,对甘肃省和陕西省进行虫媒病毒病原学的调查,全面了解当地存在和循环的虫媒病毒种类与分布情况,对于研究我国西北的虫媒病毒生物学及分子生物学特征、预防和控制当地虫媒病毒病等均具有重要意义。本课题对甘肃和陕西省部分地区进行虫媒病毒调查,以明确当地存在的虫媒病毒种类与分布,并对近年在虫媒病毒调查中分离到的部分病毒进行详细鉴定和序列分析,以了解我国新分离株的系统分类地位、分子生物学特征及其与国外分离株之间的分子生物学差异。1.甘肃省虫媒病毒调查2006年和2007年7～8月在甘肃省天水、陇南和平凉市的11个县区采集蚊虫标本13872只,分206批进行研磨和病毒分离。结果分离到30个阳性分离物,来自5个蚊种——三带喙库蚊、中华按蚊、骚扰阿蚊、淡色库蚊和伊蚊。经过系统鉴定,共得到4株乙脑病毒(Japanese encephalitis virus,JEV)、2株盖塔病毒(Getah virus,GETV)、10株版纳病毒(Banna vires,BAV)和17株骚扰阿蚊单组分RNA病毒(Armigeres subalbatus Totivirus,AsTV),共计33株。从三带喙库蚊和淡色库蚊中分离到的病毒最多,分别是22株和4株;4种病毒均能从三带喙库蚊中均分离到。对新分离病毒分子生物学特征分析显示,4株JEV均为基因Ⅰ型病毒,分离株之间E基因核苷酸和氨基酸序列同源性为98.3%～100%和99.8%～100%。新分离JEV与疫苗株SA14-14-2在E基因区段存在11处共同的氨基酸差异,但差异位点均不处于决定抗原性的关键氨基酸位点3’非编码区中均有典型的基因Ⅰ型序列缺失特征。2株新分离的GETV与我国其它省份和其它国家分离株的遗传关系很相近,Capsid基因之间的核苷酸和氨基酸序列同源性在97%以上。发现分离株GS07-KD22在Capsid基因中紧邻第76位后出现1个氨基酸的插入。2株GETV在3’UTR区第45nt～54nt间都存在与我国其它省分离株及蒙古和俄罗斯分离株相同的10个核苷酸缺失。对10株新分离BAV的核酸带型分析发现新分离株与北京分离株(BJ95-75)在第6和第9条带稍有差异,第6条带稍小,而第9条带稍大。甘肃省分离株第12片段核苷酸和VP12蛋白序列中存在多个不同于其它地域分离株的共同差异位点,10株病毒在第689位核苷酸发生缺失,并且VP12基因有两个连续的起始密码子,终止密码子与印度尼西亚分离株相同(TGA),而不同于云南和北京分离株(TAA)。10株BAV与基因A型分离株的核苷酸和氨基酸同源性分别为91.4%～93.3%和91.8%～93.7%,与基因B型分离株的同源性分别为88.6%～89.9%和85.5%～87.9%。系统发生分析结果显示甘肃省分布的BAV具有明显的地域特征,属于基因A型内的一个新亚型。2.陕西省虫媒病毒调查2006年9月在陕西省安康市和宝鸡巿共4县区采集蚊虫标本2765只,分离到1株昆虫浓核病毒(Densovirus,DNV)和1株AsTV。对AsTV分离株SaX06-AK20进行了全基因组特征研究,AsTV全长7510 bp,有两个开放读码框(ORF),ORF1长4443nt,编码病毒衣壳蛋白CP,ORF2长2286 nt,编码聚合酶RdRp。AsTV与单分体病毒科成员的核苷酸同源性为17.5%～41.4%,氨基酸同源性为5.3%～43.1%,系统发生分析表明AsTV是该科内的一种新病毒,或者可能是一种新的感染节肢动物的dsRNA病毒科的代表。3.病毒分子生物学特征研究对我国分离到的10株GETV进行序列测定和分子特征分析,测定了其中3株(M1、HB0234、YN0540)的全基因组序列,和另7株的E2基因和3’UTR。3株病毒与其它国家分离株的全基因组序列同源性分别是97.4%～98.8%(核苷酸)和98.7%～99.6%(氨基酸)。10株病毒之间E2基因核苷酸序列同源性在97.7%～99.9%,发现所有分离株在3’UTR第45-54位均出现10个核苷酸的缺失(YN0540有9个缺失),这同我国台湾、蒙古和俄罗斯分离株的一致。系统发生分析显示我国分离的GETV之间均有很近的亲缘关系。对16株分离自辽宁、云南、新疆和贵州省的淡色库蚊浓核病毒(CppDNV)进行鉴定和分子特征分析,结果显示CppDNV的完整编码区长3,335nt,有3个开放读码框,ORF1,2,3分别为2,376 nt、1,092 nt和1,071 nt,分别编码非结构蛋白1(NS1)、非结构蛋白2(NS2)和衣壳蛋白(VP)。CppDNV(JZ-16)全编码区序列同AalDNV-1、AalDNV-2、AaeDNV、AalDNV-3和HeDNV的同源性在81.6%～93.0%(核苷酸)之间,而且16株CppDNV分离株之间的同源性在98%以上,序列差异和系统发生分析显示CppDNV是一类新的DNV变异株。4.其它工作此外,对7株河南省2006年病毒分离物、1株新疆病毒分离物,1株蝙蝠病毒分离物和6株贵州省2006年病毒分离物进行了鉴定和/或分子生物学特征分析。河南省7株病毒均为基因Ⅰ型JEV,贵州省鉴定出3株基因Ⅰ型JEV,而新疆的病毒分离物M0507JS9可能为芜箐黄花叶病毒科(Tymoviridae)内的新成员。本课题的意义本课题对甘肃省和陕西省部分地区进行了虫媒病毒调查,在甘肃省分离到4种病毒,在陕西省分离到2种病毒。在甘肃省首次分离到基因Ⅰ型JEV,同时发现甘肃省分布的BAV具有明显的地域特征,并且还分离到一种新病毒(AsTV)。这些数据不但丰富了我国和甘肃省、陕西省虫媒病毒的种类,而且为了解当地传染病病因以及积极预防和控制相关虫媒病毒疾病提供了重要的信息。对我国分离到的GETV、CppDNV以及河南、新疆、贵州等省的病毒分离物的鉴定和/或基因分子生物学特征分析,明确了这些病毒的分类地位及与国内外分离株之间的分子生物学差异,丰富了我国虫媒病毒分子生物学研究内容,为深入研究我国存在和流行的虫媒病毒积累了知识。更多还原

【Abstract】 Gansu and Shaanxi provinces are located in the northwest of China’s mainland,which is the gateway through northwestern inland and is also the boundary of distinguishing climates.The two provinces are still the epidemic area of Japanese encephalitis（JE） for many years.In addition,unknown viral encephalitis cases were also reported during the JE epidemic time every year.Several investigations showed that many different insect vectors distribute in these two provinces,which suggests that it could exist many arboviruses besides JE virus.This study conducted arboviruses investigation in Gansu and Shaanxi provinces from July to August in 2006 and 2007.The aim of this work is to know the arboviruses species and their distributions in these two provinces.This study had also identified and studied the molecular characterizations of arbovirus isolates isolated during arboviruses investigations in China in recent years.1.Arbovirus investigation in Gansu provinceMosquito samples were collected in the villages of 11 counties belonged to Tianshui, Longnan and Pingliang cities during July to August in 2006 and 2007.13872 individuals covering 4 genus and 6 species had been collected.The mosquitoes were sorted into 206 pools for virus isolation.Total 30 virus isolates were obtained by inoculating the grinded supernatants onto C6/36 and BHK-21 cells and followed by observing cytopathic effect（CPE）.The virus isolates were from five species including Culex tritaeniorhynchus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens pallens and Aedes sp.24 isolates were observed to cause CPE in C6/36 cells only, while the other 6 isolates caused CPE both in C6/36 cells and BHK-21 cells.33 virus strains from these 30 isolates were identified by using morphological,serological and molecular biological methods.They are 4 Japanese encephalitis viruses（JEV）,2 Getah viruses（GETV）,10 Banna viruses（BAV） and 17 Armigeres subalbatus Totiviruses（AsTV）.It was found that 20 isolates covering all these 4 different virus species could be isolated from Culex tritaeniorhynchus,which demonstrated that this species is the dominant vector in the local area.Molecular analysis of the new isolates showed that 4 JEVs are all clustered into genotypeⅠ.The new strains showed very high identities in nucleotides and amino acids of E gene,which are 98.3%-100%and 99.8%-100%respectively.11 sites of differences were found between the new strains and the vaccine strains SA 14-14-2 in E protein.These sites were not the key sites that affect the antigenic and virulence of JEV.It is the first time to report that genotypeⅠJEV has spread into Gansu province. Analysis of the 2 GETVs found that there are very close genetic relationship between Gansu strains and other GETV isolates.The identities of the Capsid gene between all strains were more than 97%both in nucleotide and amino acid levels.One amino acid insertion was found in the site aa 76 of Capsid protein.In 3’UTR,a ten nucleic acid deletion occurs in position 45-54.The genome electrophoresis profiles of the ten BAV isolates showed small differences from the Beijing strain（BJ95-75）.The 6^th fragment showed shorter and the 9^th fragment showed larger than the Beijing strains’ in the PAGE profile.Sequence analysis about the 12^th fragment found that one nucleic acid deletion occurred in all of the ten BAVs.The coding region of VP12 was 627 nt, which was different from the other strains including China and Indonesia isolates which had 624 nt long.Two continued initiation codon（ATG） were found only in Gansu strains,while the stop codon（TGA） was the same to Indonesia isolates but different from the others.The nucleotides and amino acids identities of VP12 were 91.4%-93.3%and 91.8%-93.7%between Gansu isolates and genotype A strains,and were 88.6%-89.9%and 85.5%-87.9%between Gansu isolates and genotype B strains, respectively.Sequence and phylogenetic analysis showed that the BAV Gansu isolates possess distinct regionally differentia and should be identified as a novel subtype of this species. 2.Arbovirus investigation in Shaanxi province2765 mosquito samples were collected in the villages of Ankang and Baoji cities during the arbovirus investigation in Shaanxi province in September of 2006.Virus isolation and identification results showed that two viruses were isolated from Armigeres sp.,one is insect densovirus CppDNV and one is AsTV.3.Molecular analysis of arboviruses isolated in China.10 strains of GETV isolated from five provinces during arboviruses investigations in China since 1964 were analyzed.The full-length sequences of three Chinese isolates （M1,HB0234,YN0540） were determined.The full-length sequences of these isolates were 11696nt,11686nt and 11690nt respectively,and showed the identities of 97.4%-98.8%and 98.7%-99.6%in nucleotide and amino acid level,respectively.The E2 gene of the 10 Chinese GETV isolates showed identity values of 98 to 100%for nucleic acids,when compared with other GETV isolates.The 3’UTRs of the Chinese isolates showed a deletion between positions 45 and 54,and nucleotide transition at positions 43,64 and 148.This deletion was the same to Taiwan district,Russia and Mongolia isolates.Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates,with sequence particularities specific to the Chinese isolates.16 Culex pipiens pallens densovirus isolated from Liaoning,Yunnan,Xinjiang and Guizhou provinces were identified and analyzed.Sequencing results showed that the complete coding region of CppDNV is 3335nt,and that it contains 3 open reading frames（ORFs）.These were ORF1（2376 nt） encoding non-structural protein 1（NS1）, ORF2（1092 nt） encoding non-structural protein 2（NS2） and ORF3（1071 nt） encoding capsid protein（VP）.CppDNV shares 82 to 93%identical nucleotides with isolates of the Aedes albopictus densovirus（isolates AalDNV-1,AalDNV-2 and AalDNV-3）,Aedes aegypti densovirus,and Haemagogus equines densovirus.The nucleotide sequence identity among CppDNV isolates exceeds 98%.Phylogenetic trees based on VP,NS1 and NS2 gene show that CppDNV clustered with the species Aedes aegypti densovirus and should be identified as a new variant of this species within genus Brevidensovirus. The complete sequence of AsTV strain SaX06-AK20 was determined and genomic characterization was analyzed.The result showed that AsTV has a genome with 7510 nt long,in which two ORFs were found.ORF1（4443 nt） encodes a structural protein Cote protein,ORF2（2286 nt） encodes a nonstructural protein RNA-dependent-RNA polymerase.Nucleotides and amino acids identities between AsTV and members of family Totiviridae were 17.5%-41.4%and 5.3%-43.1%respectively.Phylogenetic analysis showed that AsTV should be identified as a novel member of the family Totiviridae or the species representation of a novel dsRNA family that infects invertebrates.4.other workIn addition,this study have also identified and / or analyzed 7 strains of virus isolates isolated from Henan province in 2006,one isolate from Xinjiang in 2005 and 6 strains of isolates isolated from Guizhou province in 2006.The result showed that 7 Henan isolates and 3 Guizhou isolates were genotypeⅠJEV,while the Xinjiang isolate M0507JS9 should be identified a new member of the family Tymoviridae.In summary,arbovirus investigation in Gansu and Shaanxi provinces were carried out in this study during 2006 and 2007.Results showed genotypeⅠJEV was the first time to be isolated from Gansu and the BAV isolates was found to possess distinct regionally differentia.The new virus AsTV could be isolated from both Gansu and Shaanxi province.These findings give us a clew to strengthen arbovirus surveillance in order to prevent arbovirus diseases.Identification and / or molecular analysis of GETV,CppDNV,JEV and unknown virus isolates isolated from China can help to know more about the virus characterization,especially the molecular difference from foreign isolates,and can provide basic information for further study of the arboviruses distributed in Chinese territory.更多还原