本文選題：發(fā)熱伴血小板減少綜合征病毒 + 抗體檢測(cè)��； 參考：《中國(guó)疾病預(yù)防控制中心》2013年博士論文

【摘要】：2009-2010年,在湖北、河南和山東等地相繼出現(xiàn)發(fā)熱、乏力、消化道癥狀、血小板減少、白細(xì)胞減少及多臟器功能損傷等臨床癥狀的病例,該病被命名為發(fā)熱伴血小板減少綜合征(SFTS),其病死率高達(dá)10-30%,部分患者有明確的蜱叮咬史,俗稱“蜱叮咬病”。中國(guó)疾病預(yù)防控制中心專家對(duì)患者血清檢測(cè)后發(fā)現(xiàn),SFTS病例是由一種新型布尼亞病毒感染所致,并將其命名為發(fā)熱伴血小板減少綜合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)。經(jīng)鑒定該病毒屬于布尼亞病毒科(Bunyaviridae),白蛉病毒屬(Phlebo virus)。 布尼亞病毒科成員在自然界中的傳播方式主要有三種：1、以蚊、蜱蟲(chóng)、白蛉等節(jié)肢動(dòng)物為傳播媒介,如裂谷熱病毒、新疆出血熱病毒等,其主要擴(kuò)增宿主為小型嚙齒動(dòng)物(松鼠、花栗鼠等),感染病毒的節(jié)肢動(dòng)物通過(guò)叮咬小型嚙齒動(dòng)物,在動(dòng)物體內(nèi)引起病毒血癥,其他節(jié)肢動(dòng)物叮咬被感染的動(dòng)物引起該病毒進(jìn)一步擴(kuò)散,感染的節(jié)肢動(dòng)物叮咬人,便可在人群中引起疾病。2、以嚙齒動(dòng)物為傳播媒介,以漢坦病毒為例,主要有水平傳播(撕咬、糞、尿和氣溶膠)和垂直傳播(感染的雌鼠將病毒傳播給子代),人接觸具有感染性的糞、尿、氣溶膠或被咬傷也可導(dǎo)致病毒感染。3、病毒可以經(jīng)卵傳播,雌蚊通過(guò)卵將病毒傳播給下一代,這也是該類病毒過(guò)冬的主要機(jī)制；此外,雌蚊雄蚊交配時(shí)也可以將病毒傳給對(duì)方,引起病毒在蚊種群中的擴(kuò)散[2]。除流行病學(xué)調(diào)查發(fā)現(xiàn)部分病例有明確的蜱叮咬史之外,發(fā)熱伴血小板減少綜合征病毒的宿主及在自然界中的傳播-循環(huán)模式等尚不清楚。 目前國(guó)內(nèi)已有15個(gè)省份發(fā)現(xiàn)SFTS病例,包括湖北、山東、河南、遼寧、安徽、江蘇、浙江、湖南、四川、廣西、江西、北京、上海、陜西、云南。2008以來(lái),全國(guó)累計(jì)報(bào)告SFTS病例1531例,其中死亡病例總計(jì)101例,作為主要疫區(qū)之一的山東省,累計(jì)病例370例,占總數(shù)的24.2%；死亡病例42例,占總數(shù)的41.6%。煙臺(tái)市2010年和2011年SFTS病例數(shù)分別為44和81例,分別占當(dāng)年山東省SFTS病例總數(shù)的39.3%和48.2%,是山東省SFTSV流行較為嚴(yán)重的地區(qū)。SFTS病例高度散發(fā),多分布于丘陵和山區(qū)地帶的農(nóng)村,在丘陵、山地、森林地區(qū)生活的居民、從事農(nóng)業(yè)生產(chǎn)和野外工作的勞動(dòng)者以及赴該類地區(qū)戶外活動(dòng)的旅游者感染風(fēng)險(xiǎn)較高,病例多為中老年患者,沒(méi)有明顯的性別差異。本病有明顯的季節(jié)分布,多發(fā)于春夏兩季,與蜱蟲(chóng)等節(jié)肢動(dòng)物的繁殖高峰季節(jié)呈正相關(guān)。目前,該病毒的傳播途徑尚不明確,高度懷疑為蜱蟲(chóng)叮咬傳播。文獻(xiàn)報(bào)道,密切接觸危重患者體液也可以導(dǎo)致病毒在人與人之間傳播,因此患者家屬及醫(yī)護(hù)人員應(yīng)引起足夠重視,做好個(gè)人防護(hù)。 為了解SFTSV的宿主、傳播媒介及病毒的進(jìn)化等特征,從2011年4月開(kāi)始到2011年11月,我們選擇山東省煙臺(tái)市萊州和蓬萊地區(qū)有過(guò)SFTS確診病例的農(nóng)村開(kāi)展調(diào)查研究,具體方案設(shè)計(jì)如附錄2所示。我們對(duì)該地區(qū)的羊、牛、狗、豬、雞等與人類接觸密切的家畜家禽進(jìn)行現(xiàn)場(chǎng)調(diào)查。每月采集一次動(dòng)物血液,分離血清,同時(shí)采集動(dòng)物體表蜱蟲(chóng),并用布旗法在目標(biāo)村莊周圍草地上收集游離蜱蟲(chóng)。采用雙抗原夾心法ELISA檢測(cè)所采集動(dòng)物血清中針對(duì)SFTSV的總抗體,了解SFTSV在疫區(qū)家禽家畜中的流行情況,初步分析家養(yǎng)動(dòng)物在SFTSV復(fù)制、傳播中的作用。同時(shí)采用Realtime RT-PCR的方法對(duì)所采集動(dòng)物血清和蜱蟲(chóng)進(jìn)行核酸檢測(cè),從核酸陽(yáng)性的標(biāo)本中分離病毒,并進(jìn)行全基因組序列測(cè)定、同源性分析和系統(tǒng)發(fā)生分析,分析比較該病毒的病原學(xué)特征和分子生物學(xué)特征,初步了解和掌握SFTSV的宿主、媒介及其感染狀況。闡述家養(yǎng)動(dòng)物、蜱蟲(chóng)作為發(fā)熱伴血小板減少綜合征病毒宿主和傳播媒介的可能性,為發(fā)熱伴血小板減少綜合征的防控提供依據(jù),同時(shí)為進(jìn)一步開(kāi)展相關(guān)工作打下基礎(chǔ)。 本研究主要分為以下三個(gè)部分： 一、分別在萊州、蓬萊地區(qū)所選擇村莊采集家養(yǎng)動(dòng)物(羊、牛、狗、豬、雞)血液、分離血清,通過(guò)雙抗原夾心法ELISA對(duì)所采集血液進(jìn)行針對(duì)SFTSV的總抗體檢測(cè),通過(guò)Realtime RT-PCR進(jìn)行SFTSV的核酸檢測(cè)。 調(diào)查時(shí)間從2011年4月開(kāi)始到2011年11月底,選擇萊州和蓬萊地區(qū)2010年有SFTS確診病例的村莊及周圍的村莊作為采樣地點(diǎn),每種動(dòng)物每月在兩地各采集50份標(biāo)本,分離血清,統(tǒng)一編號(hào)并記錄相關(guān)信息(附錄3),-40℃保存。用雙抗原夾心法ELISA對(duì)所采集血液進(jìn)行針對(duì)SFTSV總抗體的檢測(cè),提取核酸利用Realtime RT-PCR方法進(jìn)行SFTSV的核酸檢測(cè),并將標(biāo)本運(yùn)送至中國(guó)疾病預(yù)防控制中心進(jìn)行復(fù)檢。根據(jù)上月的血清結(jié)果,剔除抗體陽(yáng)性動(dòng)物,重新尋找該類動(dòng)物,補(bǔ)充隊(duì)列,滿足數(shù)量要求；重復(fù)采集核酸陽(yáng)性動(dòng)物,進(jìn)行病毒分離,觀察核酸和抗體的變化情況。分析SFTSV在家養(yǎng)動(dòng)物中的流行情況,并對(duì)每種動(dòng)物在不同月份之間SFTSV感染率進(jìn)行比較。 本次研究共采集家養(yǎng)動(dòng)物血清3580份,抗體陽(yáng)性率為40.4%,核酸陽(yáng)性率為3.6%。其中,羊1013只,抗體陽(yáng)性率為64.9%,核酸陽(yáng)性率為5%；牛842頭,抗體陽(yáng)性率為52.5%,核酸陽(yáng)性率為3.7%；狗359只,抗體陽(yáng)性率為28.7%,核酸陽(yáng)性率為2.8%；豬839只,抗體陽(yáng)性率為1.3%,核酸陽(yáng)性率為1.2%；雞527只,抗體陽(yáng)性率為44.6%,核酸陽(yáng)性率為2.1%。分別從羊、牛、狗中各分離到一株病毒,進(jìn)行了同源性分析,并對(duì)分離到病毒的狗進(jìn)行了隔日采血,觀察病毒核酸及其抗體在狗體內(nèi)的變化情況。 為了解SFTSV在家養(yǎng)動(dòng)物體內(nèi)的增殖及變化規(guī)律,我們于2011年6月份,在山東省煙臺(tái)地區(qū)購(gòu)買(mǎi)了38只經(jīng)過(guò)血清抗體檢測(cè)為陰性的羊,其中,萊州17只,蓬萊21只,統(tǒng)一進(jìn)行耳釘標(biāo)號(hào),分別由2名當(dāng)?shù)啬裂蛉顺Ｒ?guī)放養(yǎng),每隔10天對(duì)38只羊進(jìn)行血液采集,直至流行季節(jié)結(jié)束。對(duì)所收集的血清進(jìn)行針對(duì)SFTSV核蛋白的IgG抗體檢測(cè)和核酸檢測(cè)。結(jié)果顯示,從6月份到11月份,萊州的羊血清抗體陽(yáng)轉(zhuǎn)率為76.5%,蓬萊達(dá)到100%,有17只羊的血清檢測(cè)到了SFTSV核酸,并都產(chǎn)生了中和抗體。 二、對(duì)山東省煙臺(tái)地區(qū)所采集的健康人血清進(jìn)行了針對(duì)SFTSV核蛋白的IgG抗體檢測(cè)。 為了調(diào)查SFTSV在健康人群中的流行情況,2011年我們通過(guò)隨機(jī)抽樣的方法對(duì)山東省煙臺(tái)地區(qū)的30個(gè)村進(jìn)行了橫斷面人群血清采集,記錄采集者基本信息(如附錄4所示)利用間接ELISA的方法對(duì)所采集血清進(jìn)行了SFTSV的IgG抗體的檢測(cè),并采用空斑減少中和試驗(yàn)對(duì)陽(yáng)性血清進(jìn)行了驗(yàn)證。本次調(diào)查共收集橫斷面人群血清2590份,其中按性別分類,男性1144人,女性1446人；按職業(yè)分類,學(xué)生576人,從事農(nóng)業(yè)的勞動(dòng)者2014人；按年齡分為7個(gè)年齡組：0-10,11-20,21-30,31-40,41-50,51-60,61歲年齡組,各年齡組人數(shù)分別為337、253、258、261、448、461、572人。經(jīng)檢測(cè),140份血清SFTSV核蛋白的IgG抗體陽(yáng)性,抗體陽(yáng)性率為5.4%,統(tǒng)計(jì)學(xué)分析發(fā)現(xiàn),抗體陽(yáng)性率在不同性別和年齡間沒(méi)有差異,但在不同職業(yè)間有明顯差異。 三、用Realtime RT-PCR方法對(duì)所采集蜱蟲(chóng)(動(dòng)物體表蜱和草地游離蜱)進(jìn)行核酸檢測(cè),用套式PCR的方法對(duì)陽(yáng)性標(biāo)本進(jìn)行S片段擴(kuò)增,測(cè)序,并嘗試分離病毒。 采集動(dòng)物血液的同時(shí),分別對(duì)萊州、蓬萊地區(qū)所采集的各類家畜家禽體表的蜱蟲(chóng)進(jìn)行采集,對(duì)應(yīng)血清編號(hào),并用布旗法對(duì)所采集動(dòng)物所在村莊周圍的草地上的游離蜱進(jìn)行收集。將所采集的蜱蟲(chóng)根據(jù)來(lái)源分類保存于收集管中,收集管內(nèi)放置蘸水的濾紙條,室溫放置5-7天,凍存于-80℃。由中國(guó)檢驗(yàn)檢疫科學(xué)院和軍事醫(yī)學(xué)科學(xué)院蜱蟲(chóng)專家進(jìn)行蜱蟲(chóng)種類鑒定。根據(jù)不同來(lái)源、種類、發(fā)育周期分管研磨、提取核酸、進(jìn)行Realtime RT-PCR檢測(cè)。對(duì)SFTSV陽(yáng)性標(biāo)本進(jìn)行套式PCR,擴(kuò)增S片段,進(jìn)行序列分析。 本研究共收集蜱蟲(chóng)3145只,其中,長(zhǎng)角血蜱是絕對(duì)優(yōu)勢(shì)蜱蟲(chóng),共3003只,占96.5%,另外還有73只血紅扇頭蜱,10只微小牛蜱,5只中華革蜱和9只鈴頭血蜱。其中,SFTSV核酸陽(yáng)性標(biāo)本一共126份,陽(yáng)性率為4%,其中,有11份標(biāo)本擴(kuò)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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R510

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7 張s，

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