【作者】 王静林；

【导师】 梁国栋； 张海林；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2010， 博士

【摘要】 本研究在我国云南省(中缅佬)边境地区开展连续两年的虫媒病毒调查,在采集到的蚊虫标本中分离到多株虫媒病毒,并利用血清学,分子生物学及生物信息学等理论与方法对本次调查中分离的病毒和在云南省分离的其他虫媒病毒进行了鉴定及分子特征研究,结果报告如下。1云南省(中缅佬)边境地区蚊虫种类及蚊传病毒调查本研究于2005年和2006年连续两年的夏季在中缅佬边境地区三个自然村开展蚊虫媒介及虫媒病毒调查。在该地区共采集到4属(库蚊属、Lufzia蚊属、按蚊属和阿蚊属)17种蚊虫。各采集点优势蚊种分析显示,三带喙库蚊是曼夕村(62.71%／1838／2931)和曼恩村(90.98%／5924／6511)的优势蚊种,中华按蚊是曼国村(67.11%／3544／5281)的优势蚊种。对采集到的14723只蚊虫标本分为152批常规处理,接种组织培养细胞(C6／36和BHK-21细胞)分离病毒。在当地采集的三带喙库蚊标本中共分离到14株病毒(其他蚊虫未分离到病毒),分别隶属于4病毒科5病毒属6种病毒。病毒包括,披膜病毒科(Togaviridae)甲病毒属(Alphavirus)辛德毕斯病毒(Sindbis virus)(1株),黄病毒科(flaviviridae)黄病毒属(Flavivirus)乙脑病毒(Japanese Encephalitis virus)(2株),呼肠孤病毒科(Reoviridae)东南亚12节段双链RNA病毒属(Seadornavirus)新东南亚12节段双链RNA病毒(Novel Seadornavirus)(1株)、呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)云南环状病毒(Yunnan orbivirus)(6株)和新环状病毒(Novel orbivirus)(1株)及细小病毒科(Parvoviridae)浓核病毒属(Densovirus)病毒淡色库蚊浓核病毒(Culex pipiens pallens Densovirus)(3株)。在当地采集的不明原因发热和病毒性脑炎患者急性期血清标本中检测到乙脑病毒,辛德毕斯病毒,云南环状病毒,新环状病毒和新东南亚12节段双链RNA病毒IgM抗体,抗体总阳性率为36.1%(178／493),提示当地存在以上病毒的急性期感染。其中辛德毕斯病毒,云南环状病毒,新环状病毒和新东南亚12节段双链RNA病毒IgM抗体阳性均是在当地首次报道。提示在云南省(中缅佬)边境地区地区存在多种虫媒病毒,并可能是当地传染病致病病原体或潜在病原体。2.云南省新分离病毒分子生物学鉴定及分子特征研究利用分子生物学方法对20世纪80年代以来从云南省多种蚊虫、蝙蝠和病人标本中分离到的26株病毒分离物进行分子生物学鉴定。结果显示19株分离物为乙脑病毒,其中17株属基因Ⅲ型乙脑病毒,2株属基因Ⅰ型乙脑病毒；3株分离物为4型登革病毒,进一步分析表明这3株病毒均为基因Ⅰ型登革病毒；1株分离物为科萨努尔森林病病毒；其余3株病毒为泰勒病毒。3.我国蝙蝠分离乙脑病毒全基因组分子特征研究对分离自云南省蝙蝠的两株乙脑病毒进行了全基因组序列测定与分析,结果显示两株病毒全基因组全长均为10977nt,编码3432氨基酸。两株病毒之间核苷酸和氨基酸序列同源性均高于99.9%。与国际基因库(GenBank)55株乙脑病毒全基因序列进行比较发现蝙蝠分离株与其他宿主来源分离株核苷酸序列同源性在88.6%(KV1899) -99.3%(Nakayama)之间,氨基酸同源性在97%(K94P05)-99.7%(JaGAr01)之间。分析还显示蝙蝠分离株与云南当地乙脑病例标本分离株Liyujie (1979)和蚊虫分离株BN19株(1982)处在同一进化簇,提示蝙蝠分离的乙脑病毒可能是当地乙脑病毒的保存宿主并参与当地流行性乙型脑炎的流行有关。这是国际上首次进行的蝙蝠分离乙脑病毒全基因组序列分析。4.云南省辛德毕斯病毒(MX10)株全基因组分子特征研究MX10病毒是在云南省中缅佬边境地区开展虫媒病毒调查时分离的病毒,经鉴定为辛德毕斯病毒。此后在实验室经病毒噬斑纯化得到噬斑形态具有明显区别的两个病毒株,分别命名为MX10-LP和MX10-SP。两病毒株在生物学及分子生学存在较大差异。MX10-SP病毒对BHK-21细胞感染能力比MX10-LP病毒低4倍；MX10-SP病毒在C6／36细胞上出现CPE时间比MX10-LP快48h。两株病毒接种1日龄乳鼠后,MX10-LP和MX10-SP病毒致死时间分别为4d和6d。病毒全基因组序列分析显示两株病毒全基因组(不包括5‘帽子结构和3’多聚A)均为11700nt,核苷酸同源性为99.9%。大小斑病毒基因组间存在7个核苷酸差异(5个氨基酸差异)。MX10-LP和MX10-SP与马来西亚原型株MRE16病毒全基因核苷酸同源性仅为91.3%,氨基酸同源性为98.1%。NS3基因分析结果显示,与Paleoarctic/Ethiopian和western/Australian genotype辛德毕斯病毒相比,Oriental/Australian genotype辛德毕斯病毒MRE16株在NS3基因插入了57个核苷酸,而MX10-LP和MX10-SP病毒则63个核苷酸。以上研究结果提示中国分离的辛德毕斯病毒(MX10)为澳大利亚东方型辛德毕斯病毒(Oriental/Australian genotype)与该基因型原型株(MRE16)相比在病毒生物学表型和分子生物特征均存在较大差异,表明Oriental/Australian genotype基因型辛德毕斯病毒可能存在多种分子差异的变异株。这是在我国首次发现的该基因型辛德毕斯病毒。5.云南省蝙蝠分离病毒的鉴定及其与疾病关系的研究对三株分离自蝙蝠脑组织的病毒分离物(B90-121-125、B90-126-130和B8613)采用非序列依赖扩增(SISPA)和RT-PCR的方法进行分子生物学研究。初步鉴定结果显示三株分离自蝙蝠的病毒与泰勒病毒DA株核苷酸同源性最高为94.7%,提示为泰勒病毒。B90-126-130病毒株全基因组须列分析,结果显示B90-126-130病毒基因组长8094 nt,编码2301 aa。与GenBank中46株脑心肌炎属病毒比较,B90-126-130病毒与泰勒病毒DA分离株全基因组序列同源性最高,核苷酸同源性均为90.3%,氨基酸同源性为96.4%,与其他8株泰勒病毒核苷酸同源性在86.9%-88%之间,氨基酸同源性在94.3%-96.3%之间。病毒脑内接种1日龄乳鼠,5天乳鼠发病死亡,B90-126-130病毒对乳鼠LD50为7.38。B90-126-130病毒不引起BHK-21、C6/36、Vero、MDCK和Tubli细等胞产生细胞病变,但在Tubli细胞第3代培养上清中病毒核酸检测阳性,其余4种细胞核酸检测阴性。此外,使用重组原核表达B90-126-130病毒VP1、3D和3ABC三个基因蛋白,建立ELISA检测方法检测病人和蝙蝠血清中病毒抗体。结果显示在云南省西双版纳和德宏州地区采集的461份发热脑炎病人血清中,17.14%标本(79／461)为B90-126-130病毒IgM抗体阳性,43.82%(202／461)标本为IgG抗体阳性。同时在云南省德宏州采集的32份蝙蝠血清中有7份标本B90-126-130抗体检测阳性。本研究结果明确了在云南省蝙蝠中分离的三株病毒均为泰勒病毒,该病毒仅能在蝙蝠来源的Tubli细胞复制和对乳鼠具有较强的神经毒力。此外还提示当地人群和蝙蝠中存在泰勒病毒感染,该病毒可能是引起当地不明原因发热和脑炎的病原体或潜在的病原体。本研究创新点及意义本研究首次在云南省边境(中缅佬)地区进行蚊虫媒介、虫媒病毒分布以及人群感染情况的综合性调查,在当地分离到大量虫媒病毒(其中多株为我国首次分离)并证明当地存在病毒感染,研究结果对于解释当地发热甚至病毒性脑炎的病因提供了重要的基础数据,具有重要的公共卫生意义。对当地多年来分离自多种蚊虫、蝙蝠和病人标本的26余株病毒分离物进行了分子生物学研究,明确了这些病毒在病毒学的分类地位及其与疾病的关系。以上结果极大地丰富了我国虫媒病毒的背景信息,为云南省及我国虫媒病毒病预防和控制以至于对虫媒病毒病的国际联防等均具有重要意义。更多还原

【Abstract】 An investigation was conducted to identify mosquito species and mosquito-borne arboviruses near the border of China-Myanmar-Laos in consecutive year. Several arboviruses have been isolated from the mosquito sample. Identification and molecular characteristics of virus isolated from Yunnan province were preformed using molecular biological methods. The results were reported as follows.1 Distribution of mosquitoes and mosquito-borne arboviruses in Yunnan Province near the China-Myanmar-Laos borderIn July 2005 and July 2006, an investigation was conducted to identify mosquito species and mosquito-borne arboviruses at three villages in Yunnan Province (Manxi, Manguo and Manen) on the border of China-Myanmar-Laos border. A total of 14,723 mosquitoes representing four genera (Culex, Anopheles, Armigeres, Lufzia) and seventeen species were collected. Culex tritaeniorhynchus was the dominant mosquito species in Manxi village (62.71%/1838/2931)and Manen village(90.98%/5924/6511), while Anopheles sinensis was the most abundant species in Manguo village (67. 11%/3544/5281). A total of 14,723 mosquitoes were divided into 152 mosquitoe pools. Fourteen strains of mosquito-borne viruses were isolated from Culex tritaeniorhynchus, representing six different viruses in five genera 4 familys:1 strain Sindbis virus (SINV) of the genus Alphavirus, family Togaviridae; 2 strains Japanese Encephalitis virus (JEV) of the genus Flavivirus, family flaviviridae; 1 strain Novel Seadornavirus (NSEAV) of the genus Seadornavirus, family Reoviridae; 6 strains Yunnan orbivirus (YUOV) and 1 strain Novel orbivirus (NOBV) of the genus Orbivirus, family Reoviridae; 3 strains Culex pipiens pallens Densovirus (CPPDenV) of the genus Densovirus, family Parvoviridae. In addition, IgM antibodies against Japanese Encephalitis virus, Sindbis virus, Yunan orbivirus and novel seadornavirus irus were detected in acute serum samples collected from hospitalized patients with fever and encephalitis near the areas where the viruses were isolated, and the total positive rate was 36.1%. This investigation suggests that JEV and SINV as well as lesser-known arboviruses circulate and are causing human disease in the China-Myanmar-Laos border region. Detection and monitoring of multiple arboviruses besides JEV should be strengthened in local areas.2. Molecular Identification of Arboviruses isolated from Yunnan ProvinceA total of 26 stains arboviruses isolated from mosquitoes, bats and patients in Yunnan Province since 1980s were identified using the method of molecular biology, including 19 strains of JEV (of which 17 strains are geneⅢ, two belong to genotypeⅠ),3 strains of Dengue Virus 4 (DENV-4) (belong to genotypeⅠ),1 strain of Kyasanur Forest disease virus (KFDV) and 3 strains of Theilovirus.3. Molecular Characteriistics of Full-genome of two JEVs isolated from bats collected from Yunnan ProvinceTo understand Molecular Characterization of Full-genome of two JEVs isolated from bats collected from Yunnan Province, the full-genome sequence of them was determined and analyzed. The results indicated that the two virus genomes have the same length at 10,977 nt each with a 95-nt 5’non-translated region (NTR) and a 583-nt 3’NTR. The two bat JEV isolates have an overall sequence identity of 99.9% both at nucleotide and protein levels. When compared to 55 known JEV isolates of known full-genome sequences, the nucleotide sequence identity varies from 88.6 to 99.3%, and aa sequence identity from 97.0 to 99.3%. Phylogenetic trees derived from full-length genome or the most variable E gene of selected JEV strains indicated that both JEV isolates are placed within the genotypeⅢ. A more detailed analysis indicated that the bat JEVs are most closely related to human isolate LiYujie and mosquito isolate BN19 within cluster. These data indicate that bats may play an important role as a reservoir and/or transmitting host of JEV in certain parts of the world. Sequence analysis of the full-genome of two JEVs isolated from bats was firstly performed in this study.4. Molecular Characteristics of Full-genome of sindbis virus (MX10) isolated from Yunnan ProvincePrevious investigation shows that MX10 virus recently isolated from Yunnan of China belongs to Oriental-Australian (O/A) genotype of Sindbis virus (SINV). Similar to MRE16 isolate, the prototype O/A genotype of SINV, two virus strains with obviously different plaque morphology were derived from MX10 virus by using plaque purification, and the diameters of larger plaque and small plaque were (1.92±0.08) mm (n=10) and (0.29±0.07) mm (n=10) respectively, which were accordingly denominated as MX10-LP and MX10-SP. The reproduction capacity of MX10-SP virus in BHK-21 cells was only 4 folds lower than that of MX10-LP virus. In contrast, MRE16SP (the small-plaque variant of MRE16 virus) released 100 folds less particles than MRE16 did. Moreover, the neurovirulence of MX10-LP and MX10-SP in neonatal mice showed significantly different. Analysis on whole viral genome suggested that the length of whole genome of MX10-LP and MX10-SP [not including 5’cap and 3’poly (A)] were both 11700nt, and 7 seven nucleotide differences were found between the whole genome of both viruses, whose nucleotide homology was 99.9%. The nucleotide homology between the whole genome of MX10-LP and MX10-SP as well as MRE16 was only 91.3%, and the amino acid (aa) homology was 98.1%. Compared with MRE16 virus, MRE16SP deleted 30 aa (aa 200-229) in E2 gene, which was proved to be the molecular basis for different plaque morphology. However, MX10-SP virus had no deletion of 30 amino acids in E2 gene. Alignment of NS3 gene showed that, compared with Paleoarctic-Ethiopian (P/E) and Western-Australian (W/A) genotype of SINV, O/A genotype of SINV inserts multiple nucleotide in NS3 gene (nt 1027-1107). For example,57 nucleotides of insertion was found in NS3 gene of O/A genotype prototype MRE16 virus, and both of MX10-LP and MX10-SP had 63 nucleotides insertions in corresponding position. These results demonstrate that the O/A genotype of Sindbis virus isolated from China exhibited itself phenotypic and molecular characteristics distinctly different from the prototype MRE16 of this genotype, indicating O/A genotype of SINV might have multiple variants with different molecular basis. It was the first isolate of O/A genotype of SINV from China.5 Identification of three virus isolates from bat of Yunnan Province and its infection in humanIdentification of three virus isolates (B90-121-125,B90-126-130 and B8613) from Rousettus leschenaulti were performed by using the method of molecular biology, such as sequence independent single primer amplification (SISPA) and RT-PCR. The results showed that partial nucleotide sequences of three virus isolates from bat were 94.7% identical to that of Theilovirus DA strain, and phylogenetic analysis indicate that three isolates from bat and Theilovirus were in the same genetic cluster. Analysis on full genome suggested that the length of whole genome of B90-126-130 virus was 8094nt, and encoding 2301 aa. Compared to the full-genome sequences of 46 known Cardiovirus genus virus isolates of, the nucleotide homology between the full genomes of B90-126-130 and Theilovirus DA strain was 90.3%, and aa sequence homology was 99.9%. The nucleotide homology between the full genomes of B90-126-130 and the other 8 Theilovirus isolates varied from 86.9% to 88%, and aa sequence from 94.3% to 96.3% respectively.3 isolates from bat could kill one-day-old neonatal mice on the 5th day post inoculation and could not caused CPE on C6/36, BHK-21, Vero, MDCK or Tubli cells. Nucleotide of B90-126-130 virus was detected in the 3rd passage supernarnt of Tubli cells culture by using RT-PCR by Theilovirus specific primers. A serosurvey focused on B90-126-130 viruse was carried out by ELISA in Xishuangbannan and Dehong Prefecture of Yunnan province. The results showed that the positive rate of IgM or IgG antibody against B90-126-130 virus was 17.14% or 43.82% in 461 cases with fever or encephalitis patients respectively. IgG antibody against B90-126-130 virus (7/32) was also detected from the serum of Bat collected form Dehong Prefecture of Yunnan province. These results suggest that 3 isolates from bat was identified to be Theilovirus, and can replicated in Tubi cell and have high neuroviluence. In addition, the results of serosurvey suggest that B90-126-130 virus was an emerging or potential pathogen causing unknown fever and encephalitis in the location.Conclusion and creationIntegrated entomological-viral-serological surveys were firstly conducted on the border area of China-Myanmar-Laos. Several arboviruses, including known and potential pathogens, were isolated from the border area, some of which were firtly isolated in China. Moreover, the human infection caused by these viruses has been proved in this investigation. These results lay basis for elucidating the pathogen of the local patients with unknown fever or encephalitis, and play a curial role in public health. Identification and classification of 26 stains arboviruses isolated from mosquitoes, bats and patients in Yunnan Province since 1980s were performed by using the method of molecular biology, and the relationship between these viruses and the local infectious disease was also clarified. Our findings contribute to growing information about arboviruses as well as to controlling and preventing the infectious disease caused by arboviruses in Yunan province, or in China, even internationally.更多还原