昆明地区戊型肝炎流行病学调查及猪戊型肝炎病毒感染动物模型的建立

发布时间：2018-03-29 13:15

本文选题：昆明地区　切入点：戊型肝炎　出处：《扬州大学》2014年博士论文

【摘要】：戊型肝炎(Hepatitis E, HE)是由戊型肝炎病毒(Hepatitis E virus, HEV)感染引起的一种人畜共患病,主要暴发和流行于亚洲和非洲一些卫生条件相对较差的发展中国家。人对HEV普遍易感,易感人群以大龄儿童和青壮年为主。在孕妇中可导致流产或引起死亡,病死率高达到15%-30%。2002-2004年卫生部传染病疫情报告显示我国是戊型肝炎高发的国家,呈现持续增长的态势,HE已经成为一个重要的公共卫生问题。越来越多的研究证据表明HEV可感染猪、犬、牛、山羊、绵羊、鹿、灵长类、鸡、贝类、啮齿类等多种动物,在这些动物宿主中感染率最高是猪,而且从猪中分离到的病毒基因序列和人源毒株有较高的同源性。加之HEV与其它嗜肝性病毒相似,易降解、不稳定,很难从粪便、胆汁等中分离到较大量的纯HEV,缺乏合适的细胞培养系统,在很大程度上限制了HEV深入的研究。为了解昆明地区戊型肝炎流行情况和探索建立猪HEV感染动物模型,我们开展了昆明地区人、猪、犬HE的血清流行病学调查,并以长爪沙鼠和树嗣建立猪HEV感染模型。本研究为进一步研究猪HEV的感染机制、研制有效的防控手段奠定了基础。具体研究内容如下：1.昆明地区戊型肝炎血清流行病学调查研究。为了解昆明地区人、猪、犬戊型肝炎的血清流行病学特征,应用双夹心抗原ELISA对采集自昆明地区人血清样品456份、猪血清样品835份、犬血清样品285份进行HEV IgG抗体检测。结果表明昆明地区人、猪、犬血清样品中HEV IgG抗体阳性分别为43.20%、55.45%、20.35%。人群中男性和女性HEV IgG抗体阳性率分别为43.25%和43.11%,男性和女性二者之间无显著差异(尸0.05)；21-30岁(48.82%)、 31-40岁(50.86%)、41-50岁(42.86%)人群的HEV抗体阳性率显著高于10-20岁(23.08%)、51岁以上(32.89%)人群(P0.05)。猪群中仔猪(1月龄-2月龄)、育肥猪(2月龄-6月龄)、种猪(6月龄以上)HEV IgG抗体阳性率分别为46.85%、57.06%、65.42%。不同年龄组猪群抗HEV IgG阳性率存在差异且差异显著(P(0.05)。犬群中城市流浪犬、家庭散养犬和养殖场犬HEV IgG抗体阳性率分别为59.18%、13.16%和10.71%,家庭散养犬、养殖场犬HEV与流浪犬HEV IgG抗体阳性率差异显著(P0.05)。德国牧羊犬、昆明犬与雪橇犬、其他品种之间及雪橇犬与其他品种之间HEV IgG抗体阳性率显著差异(P0.05)。1岁以下与1-5岁、5-10岁、10岁以上犬HEV IgG抗体阳性率显著差异(P0.05)。研究表明昆明地区人、猪、犬中存在戊型肝炎的流行,人群-猪群和犬HEV IgG阳性率与年龄有关,21-50岁人群HEV IgG抗体阳性率相对10～20岁、51岁以上较高；猪群HEV IgG阳性率随年龄的增长而逐步升高；犬HEV IgG抗体阳性率随年龄的增长而逐步下降。2.昆明及周边县猪戊型肝炎病毒分子流行病学调查研究。为了解昆明地区规模化猪场中仔猪HEV的分子背景和流行特征,针对GenBank中HEV的ORF2保守区域序列,设计合成两对巢式PCR引物。分别对采集自昆明及周边县的187份仔猪粪便样品进行扩增,PCR扩增出348bp左右的片段为阳性,阳性率为6.95%。禄劝县、江川县、富民县阳性率分别为4.54%、8.00%、9.52%。对扩增片段进行核苷酸同源性比较和遗传进化分析,9株猪HEV 348bp扩增片段的核苷酸同源性为87.1%-99.4%,与HEVⅠ、Ⅱ、Ⅲ型同源性分别为77.0%-81.9%、52.2%～53.6%、77.0%～88.2%,与HEV Ⅳ型的同源性为77.9%～96.8%。通过遗传进化树分析得出所检测到的9株猪HEV毒株均为HEV基因Ⅳ型。研究表明昆明地区规模化猪场中仔猪群中存在HEV感染并且都为基因Ⅳ型。3.戊型肝炎病毒感染猪肝脏的组织病理学观察。为了解猪戊型肝炎病毒感染肝的组织病理学变化,应用免疫组织化学染色和苏木素一伊红染色对采集自昆明地区农贸市场上的待售猪肝样品176份进行了组织病理学观察。免疫组织化学染色观察得出,市售猪肝呈HEV抗原阳性率为65.34%。苏木素一伊红染色观察得出,51.30%的猪肝组织出现炎性细胞浸润,28.70%的猪肝组织出现纤维结缔组织增生,20.87%的猪肝出现肝细胞萎缩,19.13%的猪肝出现肝细胞变性、坏死。研究表明昆明地区市售猪肝中普遍存在HEV感染并出现相应的病理学变化。4.猪戊型肝炎病毒感染长爪沙鼠的实验研究。为了探讨建立猪戊型肝炎病毒感染长爪沙鼠模型的可能性,应用HEV阳性猪粪便上清液腹腔注射接种长爪沙鼠,定期检测长爪沙鼠血清中丙氨酸转氨酶(ALT)和天门冬氨酸转氨酶(AST)水平的变化及肝脏、粪便、血液、小肠中HEV RNA的产生,病理组织学变化及HEV在肝脏中的分布情况。结果表明长爪沙鼠接种猪HEV后7天血清中ALT和AST均同步上升,此后开始降低。但ALT值到接种后35天还是没有降低到对照组水平,攻毒组比阴性对照组数值高出2倍左右。AST到接种后35天基本回落到正常范围内(根据阴性对照)。粪便和肝脏中均能检测到HEV RNA,血液和小肠偶能检测HEV RNA。肝脏组织观察到肝小叶间淋巴细胞浸润、肝细胞颗粒变性,局灶性淋巴细胞浸润、肝细胞索排列紊乱、胆管增生等。后期表现为多发性淋巴细胞浸润、肝细胞坏死,枯否细胞增多,汇管区纤维结缔组织增生。小肠、肾、脑、脾脏等组织也有不同程度病理变化。免疫组化检测结果可见到肝细胞浆中有强阳性信号。说明成功建立了猪戊型肝炎病毒感染长爪沙鼠模型。5.猪戊型肝炎病毒感染树晌的实验研究。为了探讨建立猪戊型肝炎病毒感染树(?)模型的可能性,应用HEV RNA阳性猪粪便上清液腹腔注射接种树(?),定期检测树(?)血清中丙氨酸转氨酶(ALT)和天门冬氨酸转氨酶(AST)水平的变化及肝脏、粪便、血液、小肠、肾脏、脾脏中HEV RNA的产生,病理组织学变化。结果表明树(?)接种猪HEV后7天血清中ALT和AST均同步升高,此后开始降低。但ALT和AST到接种第28天还是没有降低到对照组水平,攻毒组比阴性对照组数值高。树(?)接种猪HEV 7天后肝脏、粪便、血液、小肠中能检测到HEVRNA,肾脏和脾脏中偶能检测HEV RNA。肝脏可观察到静脉窦淤血、肝细胞空泡变性、多发性淋巴细胞浸润、枯否细胞增多、汇管区纤维结缔组织增生等病变。小肠、肾等组织也有不同程度病理变化。说明建立猪戊型肝炎病毒感染树昀模型是可行的,可为进一步研究HEV跨物种传播和致病机制等奠定基础。
[Abstract]:Hepatitis E (Hepatitis E, HE) is caused by hepatitis E virus (Hepatitis E, virus, HEV) infection is a zoonosis originated from, major outbreaks and epidemics in Asia and Africa some health conditions are relatively poor in developing countries. People of HEV generally susceptible, susceptible to older children and young adults. In pregnant women can cause miscarriage or cause death, high mortality rate reached epidemic reporting of infectious diseases 15%-30%.2002-2004 Ministry of health show that China is a country with high incidence of hepatitis E, showing sustained growth, HE has become an important public health problem. More and more evidence indicates that HEV can infect pigs, dogs and cattle. Goat, sheep, deer, primates, chicken, shellfish, rodents and other animal, in these animal hosts the highest infection rate of pigs, and isolated from swine virus gene sequence and human strains had high homology. In addition to HEV and other similar hepavirus, easy degradation and instability, it is difficult to separate from fecal bile, to a large number of pure HEV, the lack of suitable cell culture system, to a large extent limited the further study of HEV. To investigate the prevalence of hepatitis E in Kunming and explore the establishment of animal model of swine HEV we carried out infection in Kunming area, pig serum epidemiological investigation of canine HE, and Meriones unguiculatus and tree shrew establishment of pig model of HEV infection. This study for further study of the mechanism of infection of porcine HEV, laid the foundation for the development of effective means of prevention and control. The specific contents are as follows: sero epidemiological study on hepatitis E 1. Kunming area. In order to understand the Kunming region, pig, sero epidemiology of canine hepatitis E, application of double sandwich antigen ELISA in human serum samples collected from Kunming area of 456 copies, 835 copies of blood proof products, blood of dogs 285 proofs were tested for HEV IgG antibody. The results show that the Kunming region, pig, HEV IgG antibody positive canine serum samples were 43.20%, 55.45%, 20.35%. crowd of men and women HEV IgG antibody positive rates were 43.25% and 43.11%, between male and female two have no significant difference (P 0.05); at the age of 21-30 (48.82%), 31-40 (50.86%), 41-50 (42.86%) HEV antibody positive rate was significantly higher than that of 10-20 people (23.08%), 51 years old (32.89%) group (P0.05). Pigs piglets (1 month old -2 months), pigs (2 month old -6 months), pigs (6 month old above) the positive rate of HEV IgG antibody were 46.85%, 57.06%, the positive rate of 65.42%. among different age groups of anti HEV IgG differences and the difference was significant (P (0.05). The dogs in the city of stray dogs, family backyard dogs and farm dogs HEV IgG antibody positive rates were 59.18%, 13.16% and 10.71%, dog family farms scattered The difference in dogs HEV and stray dogs HEV IgG antibody positive rate significantly (P0.05). The German shepherd dog, Kunming dog and sled dogs, among other varieties and between the sled dogs and other varieties of HEV IgG antibody positive rate was significant difference (P0.05).1 years of age and 1-5 years old, 5-10 years old, 10 years old of canine HEV IgG antibody positive the rate of significant differences (P0.05). The results show that the Kunming area people, pigs, dogs have hepatitis E epidemic, population - pigs and dogs HEV positive rate of IgG is related with age, people aged 21-50 HEV IgG antibody positive rate relative to 10~20 years old, 51 years old is higher; the positive rate of swine HEV IgG with age gradually the positive rate of HEV increased; canine IgG antibodies with age gradually decreased the investigation of molecular epidemiology of hepatitis E virus.2. in Kunming and the surrounding counties. In order to understand the molecular background of pig farms in Kunming area HEV piglets and the epidemic characteristic, according to the GenBank ORF2 conserved region sequence of HEV, designed and synthesized two pairs of nested PCR primers. 187 fecal samples of piglets were collected from Kunming and the surrounding counties were amplified, amplified PCR fragment about 348bp was positive. The positive rate of 6.95%. in Luquan County, Jiangchuan County, the positive rate of Fumin County were 4.54%, 8%, 9.52%. the amplified fragment of the nucleotide homology and phylogenetic analysis, 9 strains of porcine HEV 348bp amplified fragment nucleotide homology was 87.1%-99.4%, and HEV I, II, III type homology were 77.0%-81.9%, 52.2% ~ 53.6%, 77% ~ 88.2%, the homology with HEV type IV was 77.9% ~ 96.8%. by phylogenetic analysis the detected 9 strains of Porcine HEV strains were HEV genotype. The results show that the scale of pig farms in Kunming province were pigs are infected by HEV and.3. for the genotype of hepatitis E virus infection of porcine liver tissue pathology Observation. In order to understand the swine hepatitis E virus infection of liver tissue pathological changes, immunohistochemical staining and hematoxylin eosin staining of collected from Kunming area farmers market on the sale of 176 samples of liver histopathology were observed. Immunohistochemical staining was used to observe the liver showed a positive rate of HEV the 65.34%. antigen for hematoxylin eosin staining, 51.30% liver tissue inflammatory cell infiltration, 28.70% liver tissue fiber connective tissue hyperplasia, 20.87% liver liver cell atrophy, 19.13% liver the liver cell degeneration, necrosis of liver. The results show that sold in Kunming in the prevalence of HEV infection and experimental study on the corresponding pathological changes of.4. swine hepatitis E virus infection in Mongolian gerbils. In order to explore the possibility of swine hepatitis E virus infection in Mongolian gerbil model, Application of HEV positive swine fecal supernatant by intraperitoneal injection inoculation of gerbil, Meriones unguiculatus regular detection of alanine transaminase in serum (ALT) and aspartate aminotransferase (AST) levels and changes of liver, feces, blood, intestine HEV RNA, histopathological changes and distribution of HEV in the liver. The results showed that gerbil pigs inoculated with ALT and AST HEV after 7 days in serum were increased, then began to decrease. But the value of ALT to 35 days after inoculation was not reduced to the level of control group, infection group and control group values as high as 2 times.AST to 35 days after inoculation the basic back to normal range than negative (according to the negative control). Feces and liver were detected in HEV RNA, blood and intestine I can detect HEV RNA. in liver tissue was observed between hepatic lobules of lymphocytic infiltration, degeneration of liver cell granules, focal lymphocytic infiltration, liver cell cords Disorder of bile duct hyperplasia. The late show multiple lymphocytic infiltration, necrosis of liver cells and Kupffer cells increased, hyperplasia of connective tissue fibers. Hui Guan Qu small intestine, kidney, brain, spleen tissues have different degrees of pathological changes. The immunohistochemical results can be seen a strong positive signal in liver cytoplasm. Successfully established experimental Study on gerbil model of swine hepatitis E virus.5. infection of tree of swine hepatitis E virus infection. In order to explore the establishment of swine hepatitis E virus infection tree (?) the possibility of application of HEV model, RNA positive swine fecal supernatant intraperitoneally inoculated trees (?), regular testing tree (?) in serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and changes of liver, feces, blood, intestine, kidney, spleen HEV RNA, histopathological changes. The results showed that the tree (?) HEV 7 days after inoculation of porcine serum ALT and A ST increased, then began to decrease. But the ALT and AST to twenty-eighth days of inoculation or not reduced to the level of control group, infection group than negative control group. High value tree (?) HEV vaccinated pigs after 7 days of feces, blood, liver, small intestine HEVRNA could be detected in the kidney and spleen, I can detect HEV RNA. can be observed in the liver sinus congestion, liver cell degeneration, multiple lymphocytic infiltration, Kupffer cells increased, periportal fibrous connective tissue hyperplasia lesions. The small intestine, kidney and other tissues have different degrees of pathological changes. The establishment of a porcine model of hepatitis E virus infection tree Yun is feasible, laid the foundation for further study on HEV cross species transmission and pathogenesis.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.63;S855.3

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