【作者】 司红丽；

【导师】 王建华； 王健伟；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2006， 博士

【摘要】 杯状病毒(Calicivirus)是一种人畜共患的病原体,是世界范围内人类急性病毒性胃肠炎的主要病原之一,对公共卫生有着重要影响。杯状病毒目前已知能感染人的包括诺如病毒(Norovirus)和札如病毒(Saporovirus)两个属,常在医院、餐馆、学校、托儿所、养老院、军队、家庭及其他人群中引起暴发性非菌性腹泻。根据RNA聚合酶区或外壳蛋白区的核苷酸和氨基酸序列,可将诺如病毒和札如病毒分为不同的基因组,目前已知诺如病毒基因组Ⅰ(GGⅠ)和Ⅱ(GGⅡ),札如病毒基因组SGⅠ和SGⅡ均可感染人。尽管杯状病毒引起的急性胃肠炎症状(呕吐、腹泻和腹部痉挛等)呈自限性,但若治疗不及时仍会导致死亡,因此检测该病毒的感染情况对于疾病控制和临床诊断均具有重要意义。近年来我国杯状病毒性腹泻发生率呈上升趋势,已在多个地区发生暴发流行。但是由于杯状病毒型别众多,变异频繁,其流行具有一定的地域和时间特征。因此,调查我国不同地区的杯状病毒流行特征、建立可靠的检测技术并开展其疫苗研究,对于全面掌握我国杯状病毒病的流行状况,评估其造成的经济社会负担及制订相应的控制策略具有重要意义。有鉴于此,本实验研究了山东济南地区存在杯状病毒感染的流行状况及其毒株的分子遗传特点,建立了针对流行毒株检测的荧光定量PCR技术,并对诺如病毒GGⅡ4型衣壳蛋白基因重组腺病毒(rvAdGⅡ4)免疫效果进行了研究。取得主要结果如下:一、济南地区婴幼儿腹泻病杯状病毒的流行特征收集了山东济南市儿童医院婴幼儿病毒性腹泻粪便标本,使用文献报道的杯状病毒特异性引物进行了RT-PCR检测。发现在212例病毒性腹泻标本中,杯状病毒RT-PCR阳性62例,检出率为29.25%,2月份为发病高峰。将杯状病毒阳性PCR产物进行克隆和序列分析,结果表明,所有杯状病毒均为诺如病毒基因组GGⅡ型,未见GGⅠ型和札如病毒。对39个毒株的序列分析表明,GGⅡ-4基因型占46.15%,GGⅡ-1基因型占35.90%,GGⅡ-2基因型占17.95%。说明杯状病毒是山东地区婴幼儿腹泻病的主要病原之一,目前以诺如病毒基因组Ⅱ-4型毒株流行为主,与我国其它地区相似。对测序毒株进行核苷酸序列的同源性分析,结果表明其中18株GGⅡ-4型同源性大于95.99%,与Lordsdale株同源性为89.05%～90.15%;14株GGⅡ-1型同源性大于94.16%,与Hawaii株同源性为85.04%～87.59%;7株GGⅡ-2型同源性为大于98.91%,与Melksham株同源性为80.29%～81.02%。39株诺如病毒之间的核苷酸同源性为81.45%～100%。本次测定的基因型内部毒株之间的核苷酸同源性大于94.16%,远高于与参考毒株核苷酸之更多还原

【Abstract】 Calicivirus are one of zoonosis pathogen and the most common causes of gastroenteritis in all age groups and large outbreaks remain an important public health problem worldwide. Norovirus and saporovirus were comprised to human calicivirus, due to the non- bacterium diarrhea outbreak in hospital, restaurant, school, nursery, gerocomium, armed forces, family and other crowd. Different genogroup were divided according to nucleotide and amino acid sequence of RNA polymerase and capsid, GGⅠand GGⅡof norovirus and SGⅠand SGⅡof saporovirus could infected human. Acute gastroenteritis by norovirus could autotherapy with vomit, diarrhea, abdomen, could be death if not treat promptly.The importance of calicivirus was underestimated due to the limit of detection method and it was recognized gradually with the high sensitivity diagnostic assay of RT-PCR. The studies for norovirus in our country were relatively late, and showed the infection with norovirus was at large, and it was the important pathogen causing diarrhea among children. However, the studies in our country were mainly focused on the detection of pathogen and antibody, and the results were short of representative, and were also limited. There was no clinical information and other epidemiological data, also.Hence, we want to reveal the incidence, clinical characteristic, molecule biology characteristic of calicivirus infection in Shandong. We want to establish a Taqman based real-time RT-PCR assay for the genogroupⅡ(GGⅡ) norovirus RNA in stool. Study the immune effect of the four genogroupⅡof norovirus capsid with recombinant adenovirus (rvAdGⅡ4). It will be favor of the development of detection reagents and effective vaccine. The result of experiment as follow:1 Characterization of human calicivirus in infants with acute diarrhea in Jinan area, China. To characterize the prevalence and genetics of human calicivirus (HuCV) infection in infants with acute diarrhea in Jinan area. The fecal specimens from children with acute nonbacterial gastroenteritis were collected and HuCV in the samples were detected by reverse transcription (RT)-PCR. HuCV was detectable in 62 cases out of the 212 specimens (29.25%). And the peak of epidemic was February every year.The amplified cDNA fragments were cloned into pMD18-T vector, sequenced and更多还原