云南省狂犬病流行特征及病毒分子流行病学研究

发布时间：2018-05-17 00:46

本文选题：狂犬病 + 狂犬病病毒　；参考：《大理学院》2010年硕士论文

【摘要】：目的狂犬病病毒(Rabies virus)属弹状病毒科(Rhabdoviridaes)狂犬病病毒属(Lyssavirus),是有包膜的单股负链RNA病毒。该病毒具有较强的神经组织嗜性,是致死性传染病—狂犬病的病原体。狂犬病病死率几乎为100%,目前尚无有效治疗药物。云南省地域辽阔,自然条件较为复杂,垂直气候十分明显,许多地区属于热带、亚热带气候,也有高寒山区的广泛存在,适于各类宿主动物的繁殖和狂犬病病毒的存在及传播,是国内狂犬病发病率较高的省份之一。本研究对1976—2009年云南省狂犬病流行病学资料进行整理、统计和分析,以期阐明云南省狂犬病的流行病学特征和流行趋势等,并应用分子生物学技术对2008年和2009年云南的狂犬病病人及犬脑标本进行狂犬病病毒抗原检测、基因分型及序列测定和分析,以掌握云南狂犬病病毒的基因型分布和分子遗传特征,为进一步搞好狂犬病防治提供科学依据。方法收集云南省1976～2009年狂犬病疫情及流行病学调查资料,用Excel建立数据库,用SPSS13.0统计软件进行统计分析,运用描述流行病学方法分析全省狂犬病的疫情信息。从高发的地区、时间和人群分布进行分析,并进行发病率、死亡率和病死率的统计分析。应用分子生物学技术对云南省近2年来的狂犬病病毒流行株进行分子特征研究,通过病毒抗原检测,阳性标本经提取核酸,用逆转录聚合酶链反应(RT-PCR)进行扩增,测序公司返回的测序结果用ATGC软件拼接后,ClustalX软件进行完全比对；BioEdit软件进行序列的分析和剪切后再次用ClustalX软件对N和M基因核苷酸序列进行完全比对,用Mega3软件打开比对结果,选择Neighbor-Joining(NJ)法构建系统发生树,因为该建树方法适合分析种(生物分类中的级别)水平以下的基因关系,Bootstrap值选择1000。DNAStar(5.01)软件中MegAlign对分析并剪切后的序列分别进行核苷酸和氨基酸序列间同源性比较,生成相应的数据表；GeneDoc软件制成核苷酸及氨基酸差异显示图。结果 1976～2009年全省狂犬病发病死亡1497人,全省发病率波动范围在0.00～0.69/10万之间,年均发病率为0.115/10万,最高为0.69/10万(1989年)。20世纪70年代发病数较少,80年代流行最为严重,90年代逐渐下降,2006年以来发病数明显上升。13个州、市72个县(市、区)发生过病例,主要流行地区为昭通、曲靖、文山、红河州(市),其次为玉溪、楚雄和昆明市(州),近几年滇西德宏州和保山市也发生流行。全年均有病例发生,主要流行季节为5～10月。所有年龄组均有发病,最小年龄不到1岁,最大年龄为85岁。其中5岁以下年龄占全部发病人数的4.16%,5～9岁年龄组占16.89%,10～14岁年龄组占14.97%,15～19岁年龄组占8.83%,20～49岁年龄组占33.21%,50岁以上占2.15%,主要发病年龄在20～40岁之间。男性发病占73%,女性发病占27%,男女发病性别比为1：0.38。绝大多数患者为农民,占发病人数的64%，，其次为学生(其中小学生占学生发病总数的75%)、儿童和工人等。 2008年～2009年在云南省文山、红河、德宏、西双版纳、曲靖、昭通、保山市(州)共采集标本616份,其中犬脑607份,唾液8份,脑脊液1份。免疫荧光法筛查犬脑607份,阳性为10份；试剂盒提取唾液、脑脊液9份,阳性6份。对16株云南狂犬病病毒N和M基因进行了RT-PCR扩增、克隆和测序(10份犬脑、5份唾液和1份脑脊液)。结果显示16株均含有完整的N和M基因序列。同源性分析表明,16株云南标本N和M基因核苷酸序列同源性范围分别为88.4%～100%和88.5%～100%,推导氨基酸序列的同源性分别为95.8%～99.8%和82.5%～99.5%。与狂犬病病毒基因3型(Mlkola)进行了比较,N基因核苷酸同源性和推导的氨基酸的同源性分别为65.8%和77.3%,M基因氨基酸和推导氨基酸序列同源性分别在71.4%～71.8%和77.8%～79.8%之间,同源性较低。结果显示,各毒株推导的氨基酸序列的同源性高于核苷酸序列的同源性。结论狂犬病在云南省广泛分布,全省16个州、市中有13个州、市72个县(市、区)发生过狂犬病流行,但各地发病率有明显差异,主要流行地区为滇东和滇南地区,病例主要集中在文山、红河、曲靖、昭通和西双版纳州(市)。云南省全年均有狂犬病流行,主要流行季节为5～10月。所有年龄组均有发病,主要发病年龄20-49岁,随着年龄组的增大发病数逐渐减少。病例男性多于女性,大多患者为农民。云南省2008年和2009年16株狂犬病病毒流行株的N和M基因同源性和系统进化树分析表明,均属于基因Ⅰ型狂犬病病毒；核苷酸和氨基酸序列分析显示云南狂犬病病毒呈现地域特征；云南狂犬病病毒基本按来源地区的不同显示出较高的亲缘特性；种系发生树分析提示,云南狂犬病病毒与周边省份和邻近国家狂犬病流行有关,狂犬病病毒株除来自其他省份疫源地的病毒株外,极可能还有来自东南亚的病毒株；云南省存在狂犬病有进一步流行和扩散的可能。研究结果为进一步做好云南省狂犬病防治工作提供了科学依据。
[Abstract]:objective
Rabies virus (Rabies virus) belongs to the rabies virus (Lyssavirus) family of the family of Rhabdoviridaes (Rhabdoviridaes). It is a coated single strand negative chain RNA virus. The virus has strong nerve tissue eosinophilia. It is a fatal infectious disease - the pathogen of rabies. The mortality rate of rabies is almost 100%. There is no effective therapeutic drug at present. The region of Yunnan province is not yet effective. The natural conditions are relatively complex and the vertical climate is very obvious. Many regions belong to the tropical, subtropical and high cold mountains. It is suitable for the propagation of various host animals and the existence and transmission of rabies virus, which is one of the provinces of higher incidence of rabies in China. The rabies flow in Yunnan province from 1976 to 2009 was studied in this study. In order to clarify the epidemiological characteristics and epidemic trends of rabies in Yunnan Province, the epidemiological characteristics and epidemiological trends of rabies in Yunnan province were clarified, and the rabies virus antigen, genotyping and sequence determination and analysis of rabies virus in the rabies and canine brain specimens of Yunnan in 2008 and 2009 were used in order to master the rabies in Yunnan. The genotype distribution and molecular genetic characteristics of the virus provide a scientific basis for further prevention and control of rabies.
Method
The data of the 1976~2009 year rabies epidemic situation and epidemiological survey in Yunnan province were collected, the database was established by Excel, and the statistical analysis was carried out by SPSS13.0 statistical software. The epidemic information of rabies in the province was analyzed by descriptive epidemiological methods. The analysis was made from the high incidence area, time and population distribution, and the incidence, mortality and fatality rate were carried out. The molecular characteristics of the rabies virus epidemic strains in Yunnan Province in recent 2 years were studied by molecular biology technology. Through the detection of virus antigen, the positive specimens were extracted from the nucleic acid and amplified by the reverse transcriptase polymerase chain reaction (RT-PCR). The sequencing results of the sequenced company were spliced with ATGC software, and the ClustalX software was carried out. Complete comparison; BioEdit software carries out sequence analysis and after shearing, the nucleotide sequence of N and M is compared completely with ClustalX software, Mega3 software is used to open comparison results, and Neighbor-Joining (NJ) method is selected to construct phylogenetic tree, because the construction method is suitable for the analysis of genes below the level of the species (biological classification level). The Bootstrap value selected 1000.DNAStar (5.01) software to compare the homology of nucleotide and amino acid sequences between the sequence of the analysis and the sequence after the shear. The corresponding data sheet was generated by the MegAlign. The GeneDoc software made the nucleotide and amino acid differential display.
Result
In the past 1976~2009 years, the incidence of rabies in the province was 1497. The incidence of morbidity in the province was between 0 and 0.69/10 million, the average annual incidence was 0.115/10 million, the highest was 0.69/10 million (1989) in 70s, the number of disease was less, the epidemic was most serious in 80s, the 90s gradually declined, and the number of diseases in 2006 was obviously increased in.13 States and 72 counties in the city. The main epidemic areas are Zhaotong, Qujing, Wenshan and Honghe (city), followed by Yuxi, Chuxiong and Kunming (state). In recent years, the central Yunnan and Baoshan cities are also popular. All the cases occur throughout the year, the main epidemic season is 5~10 months. The age group has the onset, the minimum age is less than 1 years and the maximum age is 85. Age group under 5 years of age accounted for 4.16% of the total number of patients, 5~9 years old age group accounted for 16.89%, 10~14 years old age group accounted for 14.97%, 15~19 age group accounted for 8.83%, 20~49 year old age group accounted for 33.21%, 50 years of age accounted for 2.15% age between 20~40 years. Most of the patients were farmers, accounting for 64% of the total number of cases, followed by students (including 75% of the total number of pupils), children and workers.
From 2008 to 2009, 616 specimens were collected in Wenshan, Yunnan Province, Honghe, Dehong, Xishuangbanna, Qujing, Zhaotong, and Baoshan city (state). Among them, there were 607 parts of the canine brain, 8 saliva and 1 cerebrospinal fluid. The immunofluorescence method was used to screen 607 copies of the canine brain and 10 copies. The kit was extracted from the saliva, 9 and 6, and 16 N and M genes of the Yunnan rabies virus were introduced into RT-PCR amplification, cloning and sequencing (10 copies of canine brain, 5 saliva and 1 cerebrospinal fluid). The results showed that 16 strains all contained a complete sequence of N and M genes. Homology analysis showed that the nucleotide sequence homology of 16 Yunnan specimens was 88.4% to 100% and 88.5% to 100% respectively, and the homology of the deduced amino acid sequence was 95.8% to 99.8, respectively. % and 82.5% ~ 99.5%. were compared with the rabies virus gene 3 (Mlkola). The nucleotide homology of the N gene and the derived amino acids were 65.8% and 77.3% respectively. The homology of the M gene amino acid and the deduced amino acid sequence was from 71.4% to 71.8% and 77.8% to 79.8%, respectively. The results showed that the amino acids derived from the strains were amino. The homology of acid sequences is higher than that of nucleotide sequences.
conclusion
Rabies is widely distributed in Yunnan province. There are 13 states in 16 prefectures and cities in the province, 72 counties (cities and districts) have been rabies epidemic. However, there are obvious differences in incidence rates. The main epidemic areas are East Yunnan and southern Yunnan. Cases mainly concentrated in Wenshan, Honghe, Qujing, Zhaotong and Xishuangbanna (city). Yunnan province has rabies epidemic all the year round. The main epidemic season is 5~10 months. All age groups have the onset of disease, the main onset age is 20-49 years old, with the increase of age group, the incidence of the disease gradually decreases. The cases of male more than women, most of the patients are farmers.
The homology of N and M genes and phylogenetic tree analysis of 16 strains of rabies virus strains in 2008 and 2009 in Yunnan province showed that all of them belong to the type I rabies virus. The nucleotide and amino acid sequence analysis showed that the Yunnan rabies virus presented regional characteristics, and the Yunnan rabies virus base was higher in the source region. Phylogenetic characteristics; phylogenetic tree analysis suggests that the rabies virus of Yunnan is related to the rabies epidemic in neighboring provinces and neighbouring countries. The rabies virus, except from the virus strains from other provinces, is likely to have the virus strains from Southeast Asia, and the possibility of further epidemic and diffusion of rabies in Yunnan province. It provides a scientific basis for further prevention and control of rabies in Yunnan province.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R512.99;R181.3

【参考文献】