本文選題：狂犬病毒 + 流行病學(xué)　； 參考：《福建醫(yī)科大學(xué)》2009年博士論文

【摘要】： 目的 近些年,我國人間狂犬病報(bào)告病例數(shù)呈遞增態(tài)勢,狂犬病已成為當(dāng)前重大的公共衛(wèi)生問題。我國關(guān)于狂犬病流行病學(xué)、病原學(xué)的深入研究較少,整體資料不全,福建省這方面的研究更是薄弱,尚不清楚我省狂犬病毒主要宿主的病毒攜帶情況和免疫情況,不清楚居民對狂犬病的認(rèn)知程度及養(yǎng)犬情況等;國內(nèi)狂犬病毒街毒株的分離報(bào)道較少,尚未見在福建省境內(nèi)分離出狂犬病毒的報(bào)道,對福建省境內(nèi)狂犬病毒的流行毒株及其特征尚不清楚。因此,為了更好地預(yù)防和控制狂犬病,非常有必要開展系統(tǒng)的狂犬病流行病學(xué)研究,獲得狂犬病流行現(xiàn)狀的基礎(chǔ)資料,了解影響狂犬病發(fā)病率的主要因素;也很有必要進(jìn)行狂犬病毒流行毒株的分離鑒定、病原生物學(xué)和遺傳學(xué)特征的研究。因養(yǎng)犬?dāng)?shù)的增加導(dǎo)致犬傷患者也不斷增加,推進(jìn)和提高犬傷患者免疫后血清抗體及犬唾液帶毒的檢測方法也迫在眉捷。 方法 1、針對狂犬病高發(fā)的實(shí)際情況,通過系統(tǒng)的流行病學(xué)調(diào)查研究,以獲得狂犬病流行現(xiàn)狀的基礎(chǔ)資料;從生態(tài)學(xué)角度收集影響狂犬病發(fā)病率有關(guān)因素的資料,通過負(fù)二項(xiàng)回歸模型篩查出影響狂犬病發(fā)病率的主要因素。 2、運(yùn)用MIT和CIT法從疑似狂犬的腦組織中進(jìn)行狂犬病毒街毒株的分離與鑒定,采取分段RT-PCR的方法,對所分離的街毒株進(jìn)行序列擴(kuò)增、克隆、測序,拼接獲得全基因組序列,并運(yùn)用生物學(xué)軟件對基因組結(jié)構(gòu)進(jìn)行分析與比較; 3、通過收集福建省不同時(shí)間、不同地區(qū)的犬腦組織,進(jìn)行實(shí)驗(yàn)室檢測診斷,對陽性標(biāo)本進(jìn)行N基因全長的擴(kuò)增、克隆和測序,結(jié)合疫情資料進(jìn)行狂犬病的分子流行病學(xué)研究。 4、選取CTN株狂犬病毒糖蛋白富集表位基因片段進(jìn)行克隆、表達(dá)和純化,人工獲得狂犬病毒糖蛋白抗原,為探索犬傷患者免疫后血清抗體及犬唾液帶毒檢測方法的提高奠定基礎(chǔ)。 結(jié)果 1、從2000～2006年福建省每年均有人間狂犬病疫情發(fā)生,平均發(fā)病率為0.07/10萬。2002~2006年福建省人狂犬病報(bào)告病例,各地分布不一,夏秋季節(jié)發(fā)病相對較多,病例主要集中在30~60歲之間,男性多于女性,農(nóng)民發(fā)病人數(shù)最多。 2、福建省3個(gè)調(diào)查地區(qū)的平均家庭養(yǎng)犬率約為46.67%,人均養(yǎng)犬?dāng)?shù)約為0.13只,每家養(yǎng)犬?dāng)?shù)約為0.63只。家犬平均免疫率為5.54%。 3、福建省普通群眾對狂犬病的認(rèn)知情況較差,狂犬病專業(yè)技術(shù)人員,總體對狂犬病犬傷情況處理的知識水平較高。 4、研究認(rèn)為狂犬病的發(fā)病率與犬的免疫率及普通群眾狂犬病知識認(rèn)知能力有關(guān)。犬免疫率每增加1%,5年累積發(fā)病率為原來的79%即下降了21%。普通群眾狂犬病知識認(rèn)知能力平均每增加0.1分,5年累積發(fā)病率為原來的80%即下降了20%。 5、本研究通過RT-nested-PCR法從表觀健康犬腦組織中檢測出狂犬病毒,并經(jīng)基因測序證實(shí)表觀健康犬?dāng)y帶狂犬病毒,為狂犬病的預(yù)防和控制提供了有力的實(shí)驗(yàn)證據(jù)。 6、首次在福建省成功分離出狂犬病毒街毒株(7株),并完成其中兩株(FJ008、FJ009)的生物學(xué)特性和全基因組序列測定。 7、從福建省各地收集的犬腦組織標(biāo)本共89份,通過RT-nested-PCR擴(kuò)增、純化、克隆,獲得19條包含N基因完整讀碼框的序列。根據(jù)核苷酸和推導(dǎo)的氨基酸的同源性高低,把19份含有RABV RNA的標(biāo)本分為三個(gè)群組,a群組包括:FJ001、FJ002、FJ003、FJ012、FJ013、FJ014;b群組包括: FJ008、FJ009、FJ010、FJ011、FJ015、FJ016、FJ017、FJ018、FJ019;c群組包括: FJ004、FJ005、FJ005、FJ007。各群組內(nèi)部RABV N基因的核苷酸同源性在99.70~100%之間,群組間RABV N基因的核苷酸同源性在86.43~89.28%,各群組內(nèi)部RABV N基因的氨基酸同源性在98.86~100%之間,群組間RABV N基因的氨基酸同源性在95.33~98.44%。結(jié)合標(biāo)本來源地,證實(shí)福建省狂犬病具有顯著的地域分布特征。 8、福建省狂犬病的流行毒株與目前常用的各種疫苗株N基因序列比對同源性在86.47~98.89%之間,均屬于基因I型,提示目前使用的疫苗能較好地保護(hù)福建省RABV流行毒株的感染。 9、完成了重組質(zhì)粒pHTb-624的高效表達(dá)和純化,獲得一批高純度的狂犬病毒糖蛋白重組抗原,應(yīng)用于犬傷患者免疫后血清抗體的檢測,與市場上主流試劑盒檢測結(jié)果沒有明顯差異,提示該重組抗原具有較好的應(yīng)用價(jià)值。 結(jié)論 1、福建省近些年狂犬病高發(fā),居民養(yǎng)犬眾多,犬免疫率低下,普通群眾狂犬病相關(guān)知識薄弱。犬免疫率低下及普通群眾狂犬病相關(guān)知識薄弱是福建省狂犬病高發(fā)的主要影響因素。 2、首次在福建省成功分離出7株狂犬病毒街毒株,并完成其中兩株街毒株(FJ008、FJ009)的生物學(xué)特性和全基因組序列測定。 3、完成福建省狂犬病毒流行毒株的基因分型和分子流行病學(xué)研究。證實(shí)福建省存在表觀健康犬?dāng)y帶狂犬病毒的現(xiàn)象,為狂犬病的預(yù)防和控制提供了有力證據(jù)。雖然福建省狂犬病具有顯著的地域分布特征,本研究發(fā)現(xiàn)福建省狂犬病的流行毒株與目前使用的各種疫苗株N基因序列比對同源性在86.47~98.89%之間,均屬于基因I型,目前使用的疫苗還是可以較好地保護(hù)福建省流行毒株的感染。 4、完成了重組質(zhì)粒pHTb-624的高效表達(dá)和純化,獲得一批高純度的狂犬病毒糖蛋白重組抗原,應(yīng)用于犬傷患者免疫后血清抗體的檢測與市場上主流試劑盒檢測結(jié)果沒有明顯差異,提示該重組抗原具有較好的應(yīng)用價(jià)值。
[Abstract]:objective
In recent years, the number of human rabies reported in China has been increasing, and rabies has become a major public health problem. In our country, the epidemiology of rabies, the in-depth study of etiology, the incomplete data, the research in Fujian province are weak, and the virus carrying situation of the main host of rabies virus is not clear in our province. The situation and immunity are not clear about the residents' cognition of rabies and the situation of dog breeding. There are few reports on the isolation of rabies virus strains in domestic rabies virus street. Reports of rabies virus isolation in Fujian province have not yet been reported, and the epidemic strains and characteristics of rabies virus in Fujian Province are not yet clear. Canine disease, it is very necessary to carry out a systematic epidemiological study of rabies, obtain the basic information of the current situation of rabies epidemic, understand the main factors affecting the incidence of rabies, and also be necessary to separate and identify the rabies virus strains, the study of the pathogenic biology and the genetic characteristics of the pathogens. Also increasing, advancing and improving the detection of serum antibodies and dog saliva toxicity after immunization in dogs is also pressing on eyebrow.
Method
1, in view of the actual situation of high incidence of rabies, a systematic epidemiological investigation was conducted to obtain the basic data of the prevalence of rabies, and the data on the factors affecting the incidence of rabies were collected from the ecological perspective, and the main factors affecting the incidence of rabies disease were screened by negative two regression models.
2, the isolation and identification of rabies virus Street strains were carried out from the brain tissue of suspected rabies by MIT and CIT. The isolated RT-PCR was amplified, cloned, sequenced and spliced to obtain the whole genome sequence, and the genome structure was analyzed and compared with the biological software.
3, the whole length of N gene was amplified, cloned and sequenced, and the molecular epidemiological study of rabies was carried out in combination with the epidemic data by collecting the brain tissues of different regions of Fujian province.
4, the gene fragment of the glycoprotein concentration epitope of CTN strain of rabies virus was cloned, expressed and purified, and the rabies virus glycoprotein antigen was obtained artificially. It laid the foundation for the exploration of serum antibody after immunization and the improvement of the detection method of dog saliva virus.
Result
1, from 2000~2006 years, there were human rabies epidemic in Fujian province every year. The average incidence rate was 0.07/10 million.2002~2006 year report cases of rabies in Fujian province. The distribution of rabies in different places was different, the onset of summer and autumn season was relatively more. The cases were mainly concentrated in the age of 30~60, more men than women, and the number of farmers had the most incidence.
2, the average family dog rate in 3 survey areas in Fujian province is about 46.67%, and the per capita dog number is about 0.13, and the number of dogs in each family is about 0.63. The average immune rate of domestic dogs is 5.54%..
3, the cognition of rabies among the general public in Fujian is poor. The knowledge level of rabies professionals is generally higher than that of rabies dogs.
4, the study suggested that the incidence of rabies is related to the dog immune rate and the knowledge cognitive ability of common rabies. The immune rate of dogs increases by 1%, and the cumulative incidence of 5 years is 79%, which is reduced by 0.1 points per average of 21%. common people's knowledge of rabies knowledge, and the cumulative incidence of 5 years is reduced by 80% of the original 80%, 20%.
5, in this study, the rabies virus was detected in the brain tissue of the healthy dogs by RT-nested-PCR, and it was confirmed by gene sequencing that the epigenetic dogs carried rabies virus, which provided powerful experimental evidence for the prevention and control of rabies.
6, the rabies virus Street strain (7 strains) was successfully isolated in Fujian province for the first time, and the biological characteristics and complete genome sequencing of two strains (FJ008, FJ009) were completed.
7, 89 specimens of canine brain tissue collected from all over Fujian province were amplified, purified, and cloned by RT-nested-PCR. 19 sequences containing the complete reading code frame of the N gene were obtained. According to the nucleotide and deduced amino acid homology, 19 samples containing RABV RNA were divided into three groups, and a groups included FJ001, FJ002, FJ003, FJ012, FJ013, FJ014. B groups include: FJ008, FJ009, FJ010, FJ011, FJ015, FJ016, FJ017, FJ018, FJ019, and C groups including the nucleotide homology of the genes. The amino acid homology of the RABV N gene among the groups showed that the rabies in Fujian province had a significant geographical distribution in the origin of 95.33~98.44%. binding samples.
8, the epidemic strains of rabies in Fujian province are compared with the common N gene sequences of various vaccine strains which are commonly used in 86.47~98.89%, all of which belong to the gene I type, suggesting that the current vaccine can better protect the infection of the RABV epidemic strains in the RABV.
9, the recombinant plasmid pHTb-624 was highly expressed and purified, and a group of high purity rabies virus glycoprotein recombinant antigen was obtained. It was applied to the detection of serum antibody after immunization in dog injured patients. It was not significantly different from the main reagent box test results on the market, suggesting that the recombinant antigen has good application value.
conclusion
1, in recent years, Fujian province has a high incidence of rabies, there are many dogs in the population, the immune rate of dogs is low, and the knowledge of ordinary rabies is weak. The poor immune rate of dogs and the weak knowledge related to common rabies are the main factors affecting the high incidence of rabies in Fujian province.
2, 7 rabies virus Street isolates were successfully isolated in Fujian province for the first time, and the biological characteristics and complete genome sequencing of two street virus strains (FJ008, FJ009) were completed.
3, the genotyping and molecular epidemiology of the rabies virus strains in Fujian province were completed. It was confirmed that the presence of rabies virus in Fujian province has provided strong evidence for the prevention and control of rabies. Although the rabies in Fujian province have significant regional distribution characteristics, this study found the flow of rabies in Fujian Province. The line between the strain and the N gene sequence of various vaccine strains currently used is a genotype I between 86.47~98.89% and I. The current vaccine can better protect the infection of the epidemic strains in Fujian province.
4, the recombinant plasmid pHTb-624 was highly expressed and purified, and a group of high purity rabies virus glycoprotein recombinant antigen was obtained. The detection of serum antibody after immunization in dog injured patients was not significantly different from that in the market, suggesting that the recombinant antigen has good application value.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R512.99;R373

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 肖方震;鄧艷琴;陳亮;王靈嵐;;福建省2006-2010年人狂犬病流行特征分析[J];中華疾病控制雜志;2012年10期

相關(guān)碩士學(xué)位論文 前1條

1 羅世民;狂犬病弱毒疫苗Flury-lep株生物學(xué)特性檢測與分析[D];湖南農(nóng)業(yè)大學(xué);2010年

，

本文編號：1945102