Antigenic Relationships of SARS and Animal Coronaviruses

描述（由申请人提供）：严重急性呼吸系统综合征（SARS）是一种新出现的全球性疾病，具有重大经济影响和生物恐怖主义潜力。它是由一种新的冠状病毒株（SARS CoV）引起的，在遗传上与CoV组I-III只有很远的关系。然而，SARS冠状病毒的系统发育分析表明，通过哺乳动物（组I）和鸟类（组III）冠状病毒之间的重组，冠状病毒组II或进化起源的遥远关系。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狗以及中国最近的一个人类SARS病例中分离出了基因相似的SARS CoV，这表明后一种SARS病毒再次出现在动物宿主中。SARS CoV实验感染猕猴，雪貂和猫。在SARS CoV抗体的ELISA检测中观察到抗原交叉反应性，可能与其他CoV的抗体有关。这种交叉反应影响了对中国最近SARS病例和加拿大2003年夏季疑似病例（可能为OC 43）的快速诊断，阻碍了预防SARS传播和新流行病所需的早期检测。它进一步阻碍了SARS抗体在动物和人群中的血清学筛查，以及当前或新出现的SARS病毒（大鼠，猫？）SARS的动物宿主根据我们的初步数据和其他人的发现（Ksiazek et al 2003），这种交叉反应最常见于SARS CoV和I组CoV（N蛋白）之间，但生物学基础尚不清楚。我们的目标是调查SARS冠状病毒和动物冠状病毒之间的双向交叉反应的抗原基础，发展SARS冠状病毒抗体特异性ELISA检测，并将其应用于从国内和野生动物的血清，以确定潜在的动物水库SARS。具体而言，我们将：1）在包括ELISA、免疫荧光（IF）和Western印迹的血清学测定中评估SARS CoV与动物组I、II和III CoV的双向抗原交叉反应性。2)使用杆状病毒系统中表达的SARS和动物CoV重组N和S蛋白，确定参与动物CoV和SARS CoV之间双向抗原交叉反应的CoV蛋白。3)使用N和S片段评价负责交叉反应性的CoV蛋白区域，并截短该区域以消除血清学试验中的交叉反应性，从而进行SARS CoV特异性检测。4)使用完整的和截短的SARS CoV S和N蛋白，测试来自实验接种和现场暴露的动物的血清中与SARS CoV交叉反应的抗体：a）作为一种创新方法，测试来自从TGEV中回收并用灭活的SARS CoV加强的无菌猪的血清，以评估体内交叉反应性; B）测试来自家畜、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年的知识和经验与动物冠状病毒的工作，我们广泛收集的多种菌株的动物冠状病毒从不同物种，包括野生动物和相应的超免疫抗血清和单克隆抗体，我们的TGEV CoV S和N基因克隆以及世卫组织指定我们的实验室为动物CoV SARS网络实验室，将提供彻底研究TGEV CoV S和N之间抗原相互关系所需的专业知识和试剂。动物和人类的CoV，包括SARS CoV。

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.