福建省狂犬病流行病学及病毒学特征研究

发布时间：2018-05-28 03:34

本文选题：狂犬病毒 + 流行病学　；参考：《福建医科大学》2009年博士论文

【摘要】： 目的近些年,我国人间狂犬病报告病例数呈递增态势,狂犬病已成为当前重大的公共卫生问题。我国关于狂犬病流行病学、病原学的深入研究较少,整体资料不全,福建省这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携带情况和免疫情况,不清楚居民对狂犬病的认知程度及养犬情况等;国内狂犬病毒街毒株的分离报道较少,尚未见在福建省境内分离出狂犬病毒的报道,对福建省境内狂犬病毒的流行毒株及其特征尚不清楚。因此,为了更好地预防和控制狂犬病,非常有必要开展系统的狂犬病流行病学研究,获得狂犬病流行现状的基础资料,了解影响狂犬病发病率的主要因素;也很有必要进行狂犬病毒流行毒株的分离鉴定、病原生物学和遗传学特征的研究。因养犬数的增加导致犬伤患者也不断增加,推进和提高犬伤患者免疫后血清抗体及犬唾液带毒的检测方法也迫在眉捷。方法 1、针对狂犬病高发的实际情况,通过系统的流行病学调查研究,以获得狂犬病流行现状的基础资料;从生态学角度收集影响狂犬病发病率有关因素的资料,通过负二项回归模型筛查出影响狂犬病发病率的主要因素。 2、运用MIT和CIT法从疑似狂犬的脑组织中进行狂犬病毒街毒株的分离与鉴定,采取分段RT-PCR的方法,对所分离的街毒株进行序列扩增、克隆、测序,拼接获得全基因组序列,并运用生物学软件对基因组结构进行分析与比较; 3、通过收集福建省不同时间、不同地区的犬脑组织,进行实验室检测诊断,对阳性标本进行N基因全长的扩增、克隆和测序,结合疫情资料进行狂犬病的分子流行病学研究。 4、选取CTN株狂犬病毒糖蛋白富集表位基因片段进行克隆、表达和纯化,人工获得狂犬病毒糖蛋白抗原,为探索犬伤患者免疫后血清抗体及犬唾液带毒检测方法的提高奠定基础。结果 1、从2000～2006年福建省每年均有人间狂犬病疫情发生,平均发病率为0.07/10万。2002~2006年福建省人狂犬病报告病例,各地分布不一,夏秋季节发病相对较多,病例主要集中在30~60岁之间,男性多于女性,农民发病人数最多。 2、福建省3个调查地区的平均家庭养犬率约为46.67%,人均养犬数约为0.13只,每家养犬数约为0.63只。家犬平均免疫率为5.54%。 3、福建省普通群众对狂犬病的认知情况较差,狂犬病专业技术人员,总体对狂犬病犬伤情况处理的知识水平较高。 4、研究认为狂犬病的发病率与犬的免疫率及普通群众狂犬病知识认知能力有关。犬免疫率每增加1%,5年累积发病率为原来的79%即下降了21%。普通群众狂犬病知识认知能力平均每增加0.1分,5年累积发病率为原来的80%即下降了20%。 5、本研究通过RT-nested-PCR法从表观健康犬脑组织中检测出狂犬病毒,并经基因测序证实表观健康犬携带狂犬病毒,为狂犬病的预防和控制提供了有力的实验证据。 6、首次在福建省成功分离出狂犬病毒街毒株(7株),并完成其中两株(FJ008、FJ009)的生物学特性和全基因组序列测定。 7、从福建省各地收集的犬脑组织标本共89份,通过RT-nested-PCR扩增、纯化、克隆,获得19条包含N基因完整读码框的序列。根据核苷酸和推导的氨基酸的同源性高低,把19份含有RABV RNA的标本分为三个群组,a群组包括:FJ001、FJ002、FJ003、FJ012、FJ013、FJ014;b群组包括: FJ008、FJ009、FJ010、FJ011、FJ015、FJ016、FJ017、FJ018、FJ019;c群组包括: FJ004、FJ005、FJ005、FJ007。各群组内部RABV N基因的核苷酸同源性在99.70~100%之间,群组间RABV N基因的核苷酸同源性在86.43~89.28%,各群组内部RABV N基因的氨基酸同源性在98.86~100%之间,群组间RABV N基因的氨基酸同源性在95.33~98.44%。结合标本来源地,证实福建省狂犬病具有显著的地域分布特征。 8、福建省狂犬病的流行毒株与目前常用的各种疫苗株N基因序列比对同源性在86.47~98.89%之间,均属于基因I型,提示目前使用的疫苗能较好地保护福建省RABV流行毒株的感染。 9、完成了重组质粒pHTb-624的高效表达和纯化,获得一批高纯度的狂犬病毒糖蛋白重组抗原,应用于犬伤患者免疫后血清抗体的检测,与市场上主流试剂盒检测结果没有明显差异,提示该重组抗原具有较好的应用价值。结论 1、福建省近些年狂犬病高发,居民养犬众多,犬免疫率低下,普通群众狂犬病相关知识薄弱。犬免疫率低下及普通群众狂犬病相关知识薄弱是福建省狂犬病高发的主要影响因素。 2、首次在福建省成功分离出7株狂犬病毒街毒株,并完成其中两株街毒株(FJ008、FJ009)的生物学特性和全基因组序列测定。 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学研究。证实福建省存在表观健康犬携带狂犬病毒的现象,为狂犬病的预防和控制提供了有力证据。虽然福建省狂犬病具有显著的地域分布特征,本研究发现福建省狂犬病的流行毒株与目前使用的各种疫苗株N基因序列比对同源性在86.47~98.89%之间,均属于基因I型,目前使用的疫苗还是可以较好地保护福建省流行毒株的感染。 4、完成了重组质粒pHTb-624的高效表达和纯化,获得一批高纯度的狂犬病毒糖蛋白重组抗原,应用于犬伤患者免疫后血清抗体的检测与市场上主流试剂盒检测结果没有明显差异,提示该重组抗原具有较好的应用价值。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R512.99;R373

【引证文献】