发热伴血小板减少综合征病毒（SFTSV）宿主与媒介的调查研究

发布时间：2018-06-28 12:01

本文选题：发热伴血小板减少综合征病毒 + 抗体检测　；参考：《中国疾病预防控制中心》2013年博士论文

【摘要】：2009-2010年,在湖北、河南和山东等地相继出现发热、乏力、消化道症状、血小板减少、白细胞减少及多脏器功能损伤等临床症状的病例,该病被命名为发热伴血小板减少综合征(SFTS),其病死率高达10-30%,部分患者有明确的蜱叮咬史,俗称“蜱叮咬病”。中国疾病预防控制中心专家对患者血清检测后发现,SFTS病例是由一种新型布尼亚病毒感染所致,并将其命名为发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)。经鉴定该病毒属于布尼亚病毒科(Bunyaviridae),白蛉病毒属(Phlebo virus)。布尼亚病毒科成员在自然界中的传播方式主要有三种：1、以蚊、蜱虫、白蛉等节肢动物为传播媒介,如裂谷热病毒、新疆出血热病毒等,其主要扩增宿主为小型啮齿动物(松鼠、花栗鼠等),感染病毒的节肢动物通过叮咬小型啮齿动物,在动物体内引起病毒血症,其他节肢动物叮咬被感染的动物引起该病毒进一步扩散,感染的节肢动物叮咬人,便可在人群中引起疾病。2、以啮齿动物为传播媒介,以汉坦病毒为例,主要有水平传播(撕咬、粪、尿和气溶胶)和垂直传播(感染的雌鼠将病毒传播给子代),人接触具有感染性的粪、尿、气溶胶或被咬伤也可导致病毒感染。3、病毒可以经卵传播,雌蚊通过卵将病毒传播给下一代,这也是该类病毒过冬的主要机制；此外,雌蚊雄蚊交配时也可以将病毒传给对方,引起病毒在蚊种群中的扩散[2]。除流行病学调查发现部分病例有明确的蜱叮咬史之外,发热伴血小板减少综合征病毒的宿主及在自然界中的传播-循环模式等尚不清楚。目前国内已有15个省份发现SFTS病例,包括湖北、山东、河南、辽宁、安徽、江苏、浙江、湖南、四川、广西、江西、北京、上海、陕西、云南。2008以来,全国累计报告SFTS病例1531例,其中死亡病例总计101例,作为主要疫区之一的山东省,累计病例370例,占总数的24.2%；死亡病例42例,占总数的41.6%。烟台市2010年和2011年SFTS病例数分别为44和81例,分别占当年山东省SFTS病例总数的39.3%和48.2%,是山东省SFTSV流行较为严重的地区。SFTS病例高度散发,多分布于丘陵和山区地带的农村,在丘陵、山地、森林地区生活的居民、从事农业生产和野外工作的劳动者以及赴该类地区户外活动的旅游者感染风险较高,病例多为中老年患者,没有明显的性别差异。本病有明显的季节分布,多发于春夏两季,与蜱虫等节肢动物的繁殖高峰季节呈正相关。目前,该病毒的传播途径尚不明确,高度怀疑为蜱虫叮咬传播。文献报道,密切接触危重患者体液也可以导致病毒在人与人之间传播,因此患者家属及医护人员应引起足够重视,做好个人防护。为了解SFTSV的宿主、传播媒介及病毒的进化等特征,从2011年4月开始到2011年11月,我们选择山东省烟台市莱州和蓬莱地区有过SFTS确诊病例的农村开展调查研究,具体方案设计如附录2所示。我们对该地区的羊、牛、狗、猪、鸡等与人类接触密切的家畜家禽进行现场调查。每月采集一次动物血液,分离血清,同时采集动物体表蜱虫,并用布旗法在目标村庄周围草地上收集游离蜱虫。采用双抗原夹心法ELISA检测所采集动物血清中针对SFTSV的总抗体,了解SFTSV在疫区家禽家畜中的流行情况,初步分析家养动物在SFTSV复制、传播中的作用。同时采用Realtime RT-PCR的方法对所采集动物血清和蜱虫进行核酸检测,从核酸阳性的标本中分离病毒,并进行全基因组序列测定、同源性分析和系统发生分析,分析比较该病毒的病原学特征和分子生物学特征,初步了解和掌握SFTSV的宿主、媒介及其感染状况。阐述家养动物、蜱虫作为发热伴血小板减少综合征病毒宿主和传播媒介的可能性,为发热伴血小板减少综合征的防控提供依据,同时为进一步开展相关工作打下基础。本研究主要分为以下三个部分：一、分别在莱州、蓬莱地区所选择村庄采集家养动物(羊、牛、狗、猪、鸡)血液、分离血清,通过双抗原夹心法ELISA对所采集血液进行针对SFTSV的总抗体检测,通过Realtime RT-PCR进行SFTSV的核酸检测。调查时间从2011年4月开始到2011年11月底,选择莱州和蓬莱地区2010年有SFTS确诊病例的村庄及周围的村庄作为采样地点,每种动物每月在两地各采集50份标本,分离血清,统一编号并记录相关信息(附录3),-40℃保存。用双抗原夹心法ELISA对所采集血液进行针对SFTSV总抗体的检测,提取核酸利用Realtime RT-PCR方法进行SFTSV的核酸检测,并将标本运送至中国疾病预防控制中心进行复检。根据上月的血清结果,剔除抗体阳性动物,重新寻找该类动物,补充队列,满足数量要求；重复采集核酸阳性动物,进行病毒分离,观察核酸和抗体的变化情况。分析SFTSV在家养动物中的流行情况,并对每种动物在不同月份之间SFTSV感染率进行比较。本次研究共采集家养动物血清3580份,抗体阳性率为40.4%,核酸阳性率为3.6%。其中,羊1013只,抗体阳性率为64.9%,核酸阳性率为5%；牛842头,抗体阳性率为52.5%,核酸阳性率为3.7%；狗359只,抗体阳性率为28.7%,核酸阳性率为2.8%；猪839只,抗体阳性率为1.3%,核酸阳性率为1.2%；鸡527只,抗体阳性率为44.6%,核酸阳性率为2.1%。分别从羊、牛、狗中各分离到一株病毒,进行了同源性分析,并对分离到病毒的狗进行了隔日采血,观察病毒核酸及其抗体在狗体内的变化情况。为了解SFTSV在家养动物体内的增殖及变化规律,我们于2011年6月份,在山东省烟台地区购买了38只经过血清抗体检测为阴性的羊,其中,莱州17只,蓬莱21只,统一进行耳钉标号,分别由2名当地牧羊人常规放养,每隔10天对38只羊进行血液采集,直至流行季节结束。对所收集的血清进行针对SFTSV核蛋白的IgG抗体检测和核酸检测。结果显示,从6月份到11月份,莱州的羊血清抗体阳转率为76.5%,蓬莱达到100%,有17只羊的血清检测到了SFTSV核酸,并都产生了中和抗体。二、对山东省烟台地区所采集的健康人血清进行了针对SFTSV核蛋白的IgG抗体检测。为了调查SFTSV在健康人群中的流行情况,2011年我们通过随机抽样的方法对山东省烟台地区的30个村进行了横断面人群血清采集,记录采集者基本信息(如附录4所示)利用间接ELISA的方法对所采集血清进行了SFTSV的IgG抗体的检测,并采用空斑减少中和试验对阳性血清进行了验证。本次调查共收集横断面人群血清2590份,其中按性别分类,男性1144人,女性1446人；按职业分类,学生576人,从事农业的劳动者2014人；按年龄分为7个年龄组：0-10,11-20,21-30,31-40,41-50,51-60,61岁年龄组,各年龄组人数分别为337、253、258、261、448、461、572人。经检测,140份血清SFTSV核蛋白的IgG抗体阳性,抗体阳性率为5.4%,统计学分析发现,抗体阳性率在不同性别和年龄间没有差异,但在不同职业间有明显差异。三、用Realtime RT-PCR方法对所采集蜱虫(动物体表蜱和草地游离蜱)进行核酸检测,用套式PCR的方法对阳性标本进行S片段扩增,测序,并尝试分离病毒。采集动物血液的同时,分别对莱州、蓬莱地区所采集的各类家畜家禽体表的蜱虫进行采集,对应血清编号,并用布旗法对所采集动物所在村庄周围的草地上的游离蜱进行收集。将所采集的蜱虫根据来源分类保存于收集管中,收集管内放置蘸水的滤纸条,室温放置5-7天,冻存于-80℃。由中国检验检疫科学院和军事医学科学院蜱虫专家进行蜱虫种类鉴定。根据不同来源、种类、发育周期分管研磨、提取核酸、进行Realtime RT-PCR检测。对SFTSV阳性标本进行套式PCR,扩增S片段,进行序列分析。本研究共收集蜱虫3145只,其中,长角血蜱是绝对优势蜱虫,共3003只,占96.5%,另外还有73只血红扇头蜱,10只微小牛蜱,5只中华革蜱和9只铃头血蜱。其中,SFTSV核酸阳性标本一共126份,阳性率为4%,其中,有11份标本扩增到S片段全序列。
[Abstract]:2009-2010 years, in Hubei, Henan and Shandong, such as fever, fatigue, digestive tract symptoms, thrombocytopenia, leukopening and multiple organ dysfunction, the disease was named fever accompanied by thrombocytopenia syndrome (SFTS) with a mortality of up to 10-30%, and some patients have a history of tick biting, commonly known as "ticks." The Chinese Center for Disease Control and Prevention found that the SFTS case was caused by a new type of Bunyan virus and named the fever with Severe fever with thrombocytopenia syndrome virus, SFTSV. The virus belonged to the Department of bunyavirus (Buny). Aviridae), the genus Phlebo (virus).
There are three main modes of propagation of the members of the family of Buyan virus in nature: 1, arthropods such as mosquitoes, ticks, and flies, such as Rift Valley fever virus, Xinjiang hemorrhagic fever virus, etc., whose main amplification hosts are small rodents (squirrels, chipmunks, etc.), and the arthropods infected with the virus by biting small rodents. The virus causes the virus in the body. Other arthropods bite the infected animals to cause the virus to spread further. The infected arthropods bite and bite people. They can cause the disease.2, the rodent is the transmission medium, and hantavirus is used as an example. It mainly has the horizontal transmission (bite, feces, urine and aerosol) and vertical transmission (infected female mice). To spread the virus to Zi Dai), human contact with infected feces, urine, aerosols, or bites can also cause the virus to infect.3, the virus can be transmitted through the egg and the female mosquito passes the virus to the next generation through the egg. This is also the main mechanism of the virus for the winter. In addition, the male mosquitoes can also pass the virus to each other and cause the virus to be in the mosquito. The spread of [2]. in the population, in addition to epidemiological investigation, found that some cases have a clear history of tick bites, and the host of fever with thrombocytopenia syndrome virus and the transmission and circulation pattern in nature are not clear.
At present, SFTS cases have been found in 15 provinces, including Hubei, Shandong, Henan, Liaoning, Anhui, Jiangsu, Zhejiang, Hunan, Sichuan, Guangxi, Jiangxi, Beijing, Shanghai, and.2008, all of which have reported a total of 1531 cases of SFTS cases, including 101 cases of death, which are one of the main epidemic areas, accounting for 370 cases, accounting for the total number of cases. The number of 24.2% cases, 42 cases of death cases, the total number of 41.6%. Yantai cities in 2010 and 2011 were 44 and 81 cases respectively, accounting for 39.3% and 48.2% of the total number of SFTS cases in Shandong Province, respectively, and the high emission of.SFTS cases in the more severe areas of Shandong Province, which were distributed in the hilly and mountainous areas, and in the hilly and mountainous areas. The residents living in the woodland area, the workers engaged in agricultural production and field work as well as the tourists who went to the area outdoors were more likely to have a higher risk of infection. The cases were mostly middle-aged and elderly, and there was no obvious gender difference. The disease had a distinct seasonal distribution, more frequently in the two seasons of spring and summer, and the peak season of arthropods such as ticks, Cheng Zhengxiang. At present, the transmission route of the virus is not clear and highly suspected to be transmitted by tick bite. It is reported that close contact with the body fluids of critically ill patients can also lead to the spread of the virus between people and people, so the family and medical staff of the patients should pay enough attention to personal protection.
In order to solve the SFTSV host, the transmission medium and the evolution of the virus, from April 2011 to November 2011, we selected the rural areas of Yantai Laizhou and Penglai, Shandong Province, which had SFTS confirmed cases in the rural areas, and the specific scheme was designed as Appendix 2. We were in contact with the human, sheep, cattle, dogs, pigs and chickens in this area. The animal blood was collected once a month, the serum was separated, the animal surface tick was collected, and the free tick was collected on the grassland around the target village by the cloth flag method. The total antibody against SFTSV in the serum of the animal was detected by the double antigen sandwich method ELISA, and the SFTSV was found in the poultry domestic animals. Epidemic situation, preliminary analysis of the role of domestic animals in SFTSV replication and transmission. At the same time, Realtime RT-PCR method was used to detect the nucleic acid of the collected animal serum and ticks, the virus was isolated from the positive specimens of nucleic acid, and the whole genome sequencing, homology analysis and phylogenetic analysis were carried out, and the disease of the virus was analyzed and compared. Primary characteristics and molecular biological characteristics, preliminary understanding and mastery of the host, media and infection status of SFTSV. The possibility of domestic animals and ticks as host and media of thrombocytopenia syndrome virus, providing the basis for the prevention and control of fever accompanied by thrombocytopenia syndrome, and further work on related work. Lay the foundation.
This study is divided into the following three parts:
First, the blood of domestic animals (sheep, cattle, dogs, pigs and chickens) was collected in the villages of Laizhou and Penglai, respectively, and the serum was separated. The total antibodies against SFTSV were detected by the double antigen sandwich method ELISA, and the nucleic acid test of SFTSV was carried out by Realtime RT-PCR.
The survey time began from April 2011 to the end of November 2011. The villages and surrounding villages in Laizhou and Penglai areas were selected as sampling sites in 2010, and each animal collected 50 samples each month in two places, separated serum, unified numbered and recorded related information (Appendix 3), and stored at -40 C, ELISA with double antigen sandwich method ELISA Collect blood for the detection of SFTSV total antibody, extract nucleic acid using Realtime RT-PCR method to carry out SFTSV nucleic acid detection, and carry the specimen to the Chinese disease prevention and control center for reexamination. Nucleic acid positive animals, virus isolation, observation of the changes of nucleic acid and antibody, analysis of the prevalence of SFTSV in domestic animals, and compare the rate of SFTSV infection among each animal in different months.
A total of 3580 domestic animal serum samples were collected, the positive rate of antibody was 40.4%, the positive rate of nucleic acid was 3.6%., 1013 sheep were sheep, the positive rate of antibody was 64.9%, the positive rate of nucleic acid was 5%, cattle 842, the positive rate of antibody was 52.5%, the positive rate of nucleic acid was 3.7%, and the dog 359 was 28.7%, the positive rate of nucleic acid was 2.8%, pig 839, antibody positive The rate was 1.3%, the positive rate of nucleic acid was 1.2%, 527 chickens of the chicken, the positive rate of antibody was 44.6%, the positive rate of nucleic acid was 2.1%. isolated from sheep, cattle and dogs to one virus respectively. The homology analysis was carried out, and the dog isolated from the virus was collected every other day to observe the changes of virus nucleic acid and its antibody in the dog.
In order to understand the proliferation and change rules of SFTSV in domestic animals, in June 2011, we bought 38 sheep in Yantai area of Shandong Province, which were tested negative by serum antibody. Among them, 17 in Laizhou and 21 in Penglai were unify for ear nail marking. 2 local sheep people were kept regularly, and 38 sheep were collected every 10 days for blood collection. At the end of the epidemic season, the IgG antibody test and nucleic acid test for SFTSV nucleoprotein were performed on the collected serum. The results showed that from June to November, the positive rate of serum antibody to sheep was 76.5% in Laizhou and 100% in Penglai. The serum of 17 sheep had detected SFTSV nucleic acid, and the neutralization antibody was produced.
Two, IgG antibodies to SFTSV nucleoprotein were detected in healthy human serum collected from Yantai District of Shandong province.
In order to investigate the prevalence of SFTSV in healthy people, in 2011, we sampled 30 villages in Yantai, Shandong province by random sampling, and recorded the basic information of the collectors (as shown in Appendix 4) using the indirect ELISA method to detect the IgG antibody of SFTSV in the collected serum, and used it. The positive serum was verified by the plaque reduction neutralization test. A total of 2590 sera in the cross section population were collected in this survey, which were classified by sex, 1144 men and 1446 women. According to the occupational classification, 576 students and 2014 workers engaged in agriculture; the age group was divided into 7 age groups: 0-10,11-20,21-30,31-40,41-50,51-60,61 years old age. Group, the number of all age groups was 337253258261448461572. After testing, 140 serum SFTSV nucleoprotein IgG antibodies were positive, and the positive rate of antibody was 5.4%. Statistical analysis showed that the positive rate of antibody was no difference between different sex and age, but there was significant difference between different occupations.
Three, the Realtime RT-PCR method was used to detect the nucleic acid of the ticks (the ticks of animal body surface ticks and the free ticks), and the positive specimens were amplified, sequenced and sequenced using the set of PCR method, and the virus was isolated.
Collect animal blood and collect ticks on the body surface of livestock and poultry in Laizhou and Penglai regions, corresponding serum numbers, and collect free ticks from the grassland around the village where the animals are located. The collected tick roots are classified in the collection tube and collected in the tube. The filter paper dipped in water was placed at room temperature for 5-7 days and stored at -80 C. The species identification of ticks was carried out by the Chinese Academy of inspection and Quarantine Science and the tick expert of Military Medical Science Academy of the PLA. According to different sources, species, development cycle, lapping, nucleic acid extraction and Realtime RT-PCR detection. SFTSV positive specimens were set with PCR, amplification of S fragments and sequence sequence. Column analysis.
A total of 3145 ticks were collected in this study, of which 3003 were absolute dominant ticks, 96.5%, and 73 red chlorpyrifos, 10 tiny bovine ticks, 5 Chinese gram ticks and 9 Blood ticks. The positive rate of SFTSV nucleic acid was 126, and 11 specimens were amplified to the full sequence of S fragments.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R510

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