本文選題：狂犬病 + 狂犬病病毒�。� 參考：《大理學(xué)院》2010年碩士論文

【摘要】：目的 狂犬病病毒(Rabies virus)屬彈狀病毒科(Rhabdoviridaes)狂犬病病毒屬(Lyssavirus),是有包膜的單股負鏈RNA病毒。該病毒具有較強的神經(jīng)組織嗜性,是致死性傳染病—狂犬病的病原體。狂犬病病死率幾乎為100%,目前尚無有效治療藥物。云南省地域遼闊,自然條件較為復(fù)雜,垂直氣候十分明顯,許多地區(qū)屬于熱帶、亞熱帶氣候,也有高寒山區(qū)的廣泛存在,適于各類宿主動物的繁殖和狂犬病病毒的存在及傳播,是國內(nèi)狂犬病發(fā)病率較高的省份之一。本研究對1976—2009年云南省狂犬病流行病學(xué)資料進行整理、統(tǒng)計和分析,以期闡明云南省狂犬病的流行病學(xué)特征和流行趨勢等,并應(yīng)用分子生物學(xué)技術(shù)對2008年和2009年云南的狂犬病病人及犬腦標本進行狂犬病病毒抗原檢測、基因分型及序列測定和分析,以掌握云南狂犬病病毒的基因型分布和分子遺傳特征,為進一步搞好狂犬病防治提供科學(xué)依據(jù)。 方法 收集云南省1976～2009年狂犬病疫情及流行病學(xué)調(diào)查資料,用Excel建立數(shù)據(jù)庫,用SPSS13.0統(tǒng)計軟件進行統(tǒng)計分析,運用描述流行病學(xué)方法分析全省狂犬病的疫情信息。從高發(fā)的地區(qū)、時間和人群分布進行分析,并進行發(fā)病率、死亡率和病死率的統(tǒng)計分析。應(yīng)用分子生物學(xué)技術(shù)對云南省近2年來的狂犬病病毒流行株進行分子特征研究,通過病毒抗原檢測,陽性標本經(jīng)提取核酸,用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)進行擴增,測序公司返回的測序結(jié)果用ATGC軟件拼接后,ClustalX軟件進行完全比對；BioEdit軟件進行序列的分析和剪切后再次用ClustalX軟件對N和M基因核苷酸序列進行完全比對,用Mega3軟件打開比對結(jié)果,選擇Neighbor-Joining(NJ)法構(gòu)建系統(tǒng)發(fā)生樹,因為該建樹方法適合分析種(生物分類中的級別)水平以下的基因關(guān)系,Bootstrap值選擇1000。DNAStar(5.01)軟件中MegAlign對分析并剪切后的序列分別進行核苷酸和氨基酸序列間同源性比較,生成相應(yīng)的數(shù)據(jù)表；GeneDoc軟件制成核苷酸及氨基酸差異顯示圖。 結(jié)果 1976～2009年全省狂犬病發(fā)病死亡1497人,全省發(fā)病率波動范圍在0.00～0.69/10萬之間,年均發(fā)病率為0.115/10萬,最高為0.69/10萬(1989年)。20世紀70年代發(fā)病數(shù)較少,80年代流行最為嚴重,90年代逐漸下降,2006年以來發(fā)病數(shù)明顯上升。13個州、市72個縣(市、區(qū))發(fā)生過病例,主要流行地區(qū)為昭通、曲靖、文山、紅河州(市),其次為玉溪、楚雄和昆明市(州),近幾年滇西德宏州和保山市也發(fā)生流行。全年均有病例發(fā)生,主要流行季節(jié)為5～10月。所有年齡組均有發(fā)病,最小年齡不到1歲,最大年齡為85歲。其中5歲以下年齡占全部發(fā)病人數(shù)的4.16%,5～9歲年齡組占16.89%,10～14歲年齡組占14.97%,15～19歲年齡組占8.83%,20～49歲年齡組占33.21%,50歲以上占2.15%,主要發(fā)病年齡在20～40歲之間。男性發(fā)病占73%,女性發(fā)病占27%,男女發(fā)病性別比為1：0.38。絕大多數(shù)患者為農(nóng)民,占發(fā)病人數(shù)的64%，，其次為學(xué)生(其中小學(xué)生占學(xué)生發(fā)病總數(shù)的75%)、兒童和工人等。 2008年～2009年在云南省文山、紅河、德宏、西雙版納、曲靖、昭通、保山市(州)共采集標本616份,其中犬腦607份,唾液8份,腦脊液1份。免疫熒光法篩查犬腦607份,陽性為10份；試劑盒提取唾液、腦脊液9份,陽性6份。對16株云南狂犬病病毒N和M基因進行了RT-PCR擴增、克隆和測序(10份犬腦、5份唾液和1份腦脊液)。結(jié)果顯示16株均含有完整的N和M基因序列。同源性分析表明,16株云南標本N和M基因核苷酸序列同源性范圍分別為88.4%～100%和88.5%～100%,推導(dǎo)氨基酸序列的同源性分別為95.8%～99.8%和82.5%～99.5%。與狂犬病病毒基因3型(Mlkola)進行了比較,N基因核苷酸同源性和推導(dǎo)的氨基酸的同源性分別為65.8%和77.3%,M基因氨基酸和推導(dǎo)氨基酸序列同源性分別在71.4%～71.8%和77.8%～79.8%之間,同源性較低。結(jié)果顯示,各毒株推導(dǎo)的氨基酸序列的同源性高于核苷酸序列的同源性。 結(jié)論 狂犬病在云南省廣泛分布,全省16個州、市中有13個州、市72個縣(市、區(qū))發(fā)生過狂犬病流行,但各地發(fā)病率有明顯差異,主要流行地區(qū)為滇東和滇南地區(qū),病例主要集中在文山、紅河、曲靖、昭通和西雙版納州(市)。云南省全年均有狂犬病流行,主要流行季節(jié)為5～10月。所有年齡組均有發(fā)病,主要發(fā)病年齡20-49歲,隨著年齡組的增大發(fā)病數(shù)逐漸減少。病例男性多于女性,大多患者為農(nóng)民。 云南省2008年和2009年16株狂犬病病毒流行株的N和M基因同源性和系統(tǒng)進化樹分析表明,均屬于基因Ⅰ型狂犬病病毒；核苷酸和氨基酸序列分析顯示云南狂犬病病毒呈現(xiàn)地域特征；云南狂犬病病毒基本按來源地區(qū)的不同顯示出較高的親緣特性；種系發(fā)生樹分析提示,云南狂犬病病毒與周邊省份和鄰近國家狂犬病流行有關(guān),狂犬病病毒株除來自其他省份疫源地的病毒株外,極可能還有來自東南亞的病毒株；云南省存在狂犬病有進一步流行和擴散的可能。研究結(jié)果為進一步做好云南省狂犬病防治工作提供了科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R512.99;R181.3

【參考文獻】

相關(guān)期刊論文 前10條

1 羅廷榮;李開鵬;劉芳;馮勵;潘艷;莫全記;李松;羅永莉;魏勇;余克倫;;應(yīng)用RT-PCR從蝙蝠和野鼠分離出狂犬病病毒[J];廣西農(nóng)業(yè)生物科學(xué);2005年04期

2 林燕鋒;黃金英;孫小康;;清遠市2001～2005年狂犬病流行病學(xué)分析[J];熱帶醫(yī)學(xué)雜志;2006年12期

3 李振平,王玉琳,劉燕,常婭莉,程夷;人狂犬病免疫球蛋白使用效果觀察[J];微生物學(xué)免疫學(xué)進展;2004年02期

4 張永振,肖東樓,孫玉輝,楊小冉,焉燕貞;中國1984～2002年狂犬病流行情況及防制對策[J];中華流行病學(xué)雜志;2003年10期

5 宋淼;唐青;許真;李浩;王定明;莫兆軍;高立東;郭綬衡;朱鳳才;胡岱霖;王顯軍;;中國2005年狂犬病流行相關(guān)因素分析[J];中華流行病學(xué)雜志;2006年11期

6 唐青;;狂犬病病毒致病機制研究概況[J];中華流行病學(xué)雜志;2007年08期

7 宋群鋒;王昭孝;余春;周敬祝;胡靜;吳昊;蔣維佳;;2004年貴州省人間狂犬病流行病學(xué)分析及控制對策[J];中國媒介生物學(xué)及控制雜志;2006年01期

8 張海林,何建華;云南省狂犬病流行及防制策略[J];中國人獸共患病雜志;2003年01期

9 張建明;嚴延生;;狂犬病病毒的基因分型及其分子流行病學(xué)研究進展[J];中國人獸共患病學(xué)報;2006年03期

10 宋淼;唐青;馮子健;李浩;杜加亮;梁國棟;;1996-2006年中國狂犬病流行特征分析[J];中國人獸共患病學(xué)報;2008年06期

本文編號：1899170