Antigenic Relationships of SARS and Animal Coronaviruses

描述(申请人提供)：严重急性呼吸综合征(SARS)是一种新出现的全球性疾病，具有重大的经济影响和潜在的生物恐怖主义。它是由一种新的冠状病毒株(SARS CoV)引起的，在基因上与CoV I-III组只有遥远的亲缘关系。然而，SARS冠状病毒的系统发育分析表明，它与第二组冠状病毒有较远的亲缘关系，或者是通过哺乳动物(第一组)和鸟类(第三组)冠状病毒的重组而进化而来的。最近的数据表明，SARS是人畜共患病，可能有广泛的宿主范围。从中国的果子狸和浣熊身上分离到了基因相似的SARS冠状病毒，从中国最近的一例人类SARS病例中分离到了SARS冠状病毒，这表明后者是从动物水库中重新出现的。SARS冠状病毒实验感染猕猴、雪貂和猫。在SARS冠状病毒抗体的酶联免疫吸附试验中观察到抗原交叉反应，可能与其他冠状病毒抗体有关。这种交叉反应影响了对中国最近的SARS病例和加拿大2003年夏季疑似病例(可能是OC43)的快速诊断，阻碍了防止SARS传播和新的流行病所需的早期发现。它进一步阻碍了对动物和人类群体中SARS抗体的血清学筛选，以及对当前或正在出现的(大鼠、猫？)的识别。SARS的动物蓄水池。根据我们的初步数据和其他人的发现(Ksiazek等人，2003年)，这种交叉反应在SARS冠状病毒和I组冠状病毒(N蛋白)之间最常见，但其生物学基础尚不清楚。我们的目标是研究SARS冠状病毒和动物冠状病毒双向交叉反应的抗原性基础，建立SARS冠状病毒抗体特异性的ELISA法，并将其应用于家畜和野生动物的血清，以确定SARS的潜在动物宿主。具体地说，我们将：1)在血清学检测中评估SARS冠状病毒与动物I、II和III组冠状病毒的双向抗原交叉反应，包括ELISA、免疫荧光(IF)和Western blotting。2)利用在杆状病毒系统中表达的动物冠状病毒和动物冠状病毒重组N和S蛋白，确定参与动物冠状病毒和SARS冠状病毒双向抗原交叉反应的冠状病毒蛋白。3)用N片段和S片段评估引起交叉反应的冠状病毒蛋白(S)区域，并在血清学试验中截短该区域以消除交叉反应，以进行SARS冠状病毒特异性检测。4)用完整和截短的SARS CoV S和N蛋白检测实验接种和野外暴露动物的血清与SARS CoV的抗体交叉反应：a)作为一种创新方法测试从TGEV中回收并用灭活SARS CoV加强免疫的生猪血清，以评估体内交叉反应；b)测试家畜、家禽和野生动物(野生反刍动物和食肉动物)的血清。我们过去30年从事动物冠状病毒工作的知识和经验，我们广泛收集的包括野生动物在内的不同物种的多种动物冠状病毒株和相应的高免疫性抗血清和单抗，我们的TGEV冠状病毒S和N基因克隆，以及我们的实验室被世界卫生组织指定为动物冠状病毒SARS网络实验室，将提供深入研究包括SARS冠状病毒在内的动物和人类冠状病毒之间的抗原相互关系所需的专业知识和试剂。

DESCRIPTION (provided by applicant): Severe acute respiratory syndrome (SARS) is a newly emerged global disease of significant economic impact and bioterrorism potential. It is caused by a new strain of coronavirus (SARS CoV) genetically only distantly related to CoV groups I-III. However phylogenetic analysis of SARS CoV suggests distant relationships to CoV group II or an evolutionary origin through recombination between mammalian (group I) and avian (group III) CoVs. Recent data suggests that SARS is zoonotic and may have a broad host range. Genetically similar SARS CoV were isolated from civet cats and raccoon dogs in China and from a recent human SARS case in China suggesting the latter SARS virus reemerged from an animal reservoir. SARS CoV experimentally infects macaques, ferrets and cats. Antigenic cross-reactivity, likely related to antibodies to other CoVs, is observed in ELISA tests for SARS CoV antibodies. This cross-reactivity compromised rapid diagnosis of the recent SARS cases in China and the suspect 2003 summer cases in Canada (likely OC43), hindering early detection needed to prevent SARS spread and new epidemics. It further hinders serologic screening of SARS antibodies in animal and human populations and identification of current or emerging (rats, cats?) animal reservoirs for SARS. Based on our preliminary data and the findings of others (Ksiazek et al 2003), such cross-reactivity is most commonly seen between SARS CoV and group I CoVs (N protein) but the biologic basis is unknown. Our goal is to investigate the antigenic basis for the two-way crossreactivity between SARS CoV and animal CoVs, to develop SARS CoV antibody specific ELISA tests and to apply these to sera from domestic and wild animals to identify potential animal reservoirs for SARS. Specifically we will: 1) Assess the two-way antigenic cross-reactivity of SARS CoV with animal group I, II and III CoVs in serologic assays including ELISA, immunofluorescence (IF) and Western blots. 2) Determine the CoV proteins involved in the two-way antigenic cross-reactivity between animal CoVs and SARS CoV using both SARS and animal CoV recombinant N and S proteins expressed in a baculovirus system. 3) Evaluate the region of the CoV protein(s) responsible for the cross-reactivity using N and S fragments and truncate this region to eliminate cross-reactivity in serologic tests to make SARS CoV specific assays. 4) Test sera from experimentally inoculated and field exposed animals for antibodies cross-reactive with SARS CoV using the intact and truncated SARS CoV S and N proteins: a) As an innovative approach test serum from gnotobiotic pigs recovered from TGEV and boosted with inactivated SARS CoV to assess in vivo crossreactivity; b) Test serum from domestic livestock, poultry and wildlife (wild ruminants and carnivores). Our past 30 years of knowledge and experience working with animal CoVs, our extensive collections of multiple strains of animal CoVs from diverse species including wildlife and the corresponding hyperimmune antisera and monoclonal antibodies, our TGEV CoV S and N gene clones and appointment of our lab by WHO as an animal CoV SARS network lab will provide the expertise and reagents needed to thoroughly investigate the antigenic interrelationships among animal and human CoVs including SARS CoV.