【作者】 石建党；

【导师】 李晓眠；

【作者基本信息】 天津医科大学 ， 病原生物学， 2002， 博士

【摘要】 普通棉耳狨猴与人类的亲缘关系相近，加上体小、易饲养、好管理等优点，已广泛用于医学领域的研究。1987年天津医科大学从国外引进普通棉耳狨猴，用于地方病的基础研究，取得了满意的成果。经过努力已成功地繁育了第一代和第二代，使年拥有量在100只以上，成为国内目前唯一较为庞大的群体，并已向国家八五、九五攻关课题提供动物，产生了较大的社会效益和经济效益。 1999年6月，该动物中心饲养的普通棉耳狨猴群体中暴发急性呼吸道传染病，发病率几乎100％，死亡率达33％。本次发病具有明显的呼吸道病毒感染特征：突然暴发，迅速蔓延，发病率高并伴有一定的死亡率。为发展和保护我国唯一的狨猴种群基地，并了解病原体与人类的关系，我们对狨猴死亡的病因进行了深入研究。 第一部分研究主要是进行病毒分离及病原学鉴定。 在排除细菌感染的基础上，通过死亡狨猴肺组织匀浆接种鸡胚和MDCK细胞的分离培养，分离出一株具有很高血凝效价的病毒株。通过双份血清试验及动物接种试验，发现：(1)发病狨猴恢复期血清对分离病毒抗体效价有4倍以上升高；(2)分离病毒接种狨猴，狨猴出现呼吸道症状，4周后血清抗体与接种前相比有60倍以上升高；(3)分离病毒接种SPF小鼠，从死亡小鼠肺组织可分离出与接种病毒一致的病毒。确认该病毒是本次疾病流行的病原体。 我们在初步分析本次疾病流行规律的同时，发现动物中心工作人员都有此病原体的抗体，并且在正常人群(献血员)中也有此病毒的抗体，阳性率高达 46．7％门 人提示此病原体与人类有密切的关系，可能是一株人和诫猴共患 的呼吸道病毒。 第二部分研究的目的是对分离到的病原体进行进一步鉴定。 采用6种能够获得的呼吸道病毒标准血清及标准毒株（甲型流感HINI、 H3NZ；乙型流感BIBeijingl243197、BIBJll84193！the；新城疫病毒；仙台病毒） 用交叉血凝抑制试验对分离毒株进行鉴定，发现该毒株与仙台病毒血清学特征 最为接近，但与乙型流感病毒等也有交叉反应。 将病毒液浓缩后用磷钨酸负染，电镜观察进行形态学鉴定，分离毒株核衣 壳结构明显，具有典型的副粘病毒特征。 采用分子生物学方法，设计出 Inf A、Inf B、hPIV、仙台病毒、hPIVZ、 SVS、hPIV3和hPIV4等8对可能病毒的特异性PCR引物，用RTPCR技术检 测病毒基因组，发现只有仙台病毒的引物能够扩增出相应分子量大小的产物。 扩增产物经过序列测定后，用生物信息学方法与各基因库中核酸序列进行比较， 确认该分离毒株是副流感1型病毒中的仙台病毒。 第三部分主要是进行分离病毒与人群关系的研究，克隆病毒的HN基因并 将其与人副流感1型病毒进行比较，为开发一种可用于人类也可用于诫猴疾病 预防的疫苗提供理论和物质基础。 我们检验了天津市53份 14周岁以下）L童血清、36份 15～50岁青、中年 人血清、43份 50岁以上中、老年人血清和山西省 18份血清，抗体阳性率分别 为45．28％、41．67％、60．47％和50％。经统计学检验，不同年龄、省份的人群 血清中对应于仙台病毒Tiwiin株的抗体存在情况无差别，总体的抗体阳性率为 49．3 3％。提示该病毒与人关系密切。 仙台病毒的HN蛋白是其主要的抗原物质，有可能作为基因疫苗的成分之 一。为此，我们设计了扩增仙台病毒HN基因全部序列的PCR引物，用高保真 耐热 DNA聚合酶扩增出特异性 PCR产物，然后用普通 Tq酶为产物加上突出 4的3人碱基，再用A／T连接的方式克隆到质粒载体上，并对其进行了序列测定。根据测定的核酸序列推测出它的蛋白质序列，然后与人副流感1 型病毒humMashingtonll957毒株的HN蛋白进行了同源性分析。发现它们整体同源性为 71％，包膜外区同源性高达 76％。提示我们所克隆的 HN基因有望作为一种疫苗成分同时增强人体和诫猴对这两种副流感1型病毒——仙台病毒和人副流感1型病毒的抵抗力。更多还原

【Abstract】 As a close consanguinity of human race, the common cotton-eared marmoset (Callithrix Jacchus) has been used in a wide range of research as a primate mode] of human pathology, physiology, biochemistry and nutriology, etc. Their small size and adaptability make it possible for relatively large population to be maintained for experimental purposes. The Experimental Animal Center of Tianjin Medical University has fetched in the common cotton-eared marmosets from Australia in 1987, and now holds more than 100 marmosets. This colony is the biggest one in China.Acute respiratory infection (ARI) broke out in the colony in June, 1999. Almost all the marmosets got ill, and about 1/3 died. The fact that the infection spread out quickly immediately after the outbreak with a high incidence of the disease and a relatively high mortality rate shows distinct characteristics of respiratory virus infection. In order to protect and develop the only marmosets-colony center in P.R.China and find the relationship between the pathogen and human being, a meticulous study on the lethal pathogenicity was carried out.Part one is mainly about isolation & determination of the pathogen-causing ARI.A virus strain with high hemagglutinin liter was isolated out from embryonated egg and MDCK cells after they were inoculated with the lung tissues of the died marmosets. The hemagglutination inhibition (HI) test of paried serum samples and the animal inoculation tests show that: (1). The antibody liter in recovery period have more than 4-fold rise. (2). Symptoms of ARI manifested in the marmoset inoculated with the isolated virus strain are quite similar to that in the epidemic. Theserum antibody liter four weeks after the inoculation has 60-fold rise compared with that before inoculation. (3). Same virus was isolated from SPF mice inoculated with the virus strain. All these confirm that the isolated virus strain is the pathogen of the ARIIt is also found that all the faculty in the animal center have antibody specific to this virus, and 14 of 40 people who had never contacted with the marmosets have antibody to the virus. The result suggests that the virus has a close relation to human, and may be a common respiratory virus in human and marmoset.Part two is about the identification of the isolated virus strain.The cross HI test has been carried out to identify the isolated strain using 6 different sera & virus. It is found that the isolated virus strain is similar to Sendai virus in serology, and with cross reaction to influenza B though.After concentration, the virus was negatively stained and then observed under electron microscope. The structure of nucleocapsid can be observed easily, with the typical character of paramyxoviridae virus.RT-PCR technique was used to detect the genome of the agent. Out of 8 pairs of PCR primers for the possible respiratory viruses, only the primers for sendai virus can amplify product. The product was sequenced and then compared with known sequence in DNA database. BLAST analyses shows the strain is sendai virus, which belongs to parainfluenza virus type 1.Part three is the study on the relationship between the isolated pathogen and human, the virus HN gene clone, which can provide theoretic framework for developing an effective vaccine.The antibody liter of 53 sera from subjects under 14 years, 36 sera from between 15 and 50 years, 43 sera from older than 50 years in Tianjin, and 18 sera from ones in Shanxi-province were examined with HI test. The positive rates have no significantly statistical difference in these groups. About 50% of them have the antibody to the isolated virus strain.The HN gene of the isolated virus was amplified by RT-PCR, and cloned into aplasmid vector, and then sequenced. The deduced protein sequence was compared with the HN protein of human parainfluenza virus type l(hPIVl). These two proteins are very similar in composition, so it is promising that the HN gene of the isolated virus can be candidate for a vaccine against both sendai virus and hPI更多还原