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症（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。它是由一种新型冠状病毒（SARS CoV）引起的，在遗传上与冠状病毒I-III组有远亲关系。然而，系统发育分析表明，SARS冠状病毒与冠状病毒II族有遥远的关系，或者通过哺乳动物（I族）和鸟类（III族）冠状病毒之间的重组有进化起源。最近的数据表明，SARS是人畜共患的，可能有广泛的宿主范围。基因相似的SARS冠状病毒从中国的果子狸和貉以及中国最近的一例人类SARS病例中分离出来，这表明后者SARS病毒是从动物宿主中重新出现的。SARS冠状病毒实验感染猕猴、雪貂和猫。在SARS冠状病毒抗体的ELISA试验中观察到抗原交叉反应性，可能与其他冠状病毒的抗体有关。这种交叉反应性妨碍了对中国最近的SARS病例和加拿大2003年夏季疑似病例（可能是OC43）的快速诊断，阻碍了预防SARS传播和新流行病所需的早期发现。这进一步阻碍了在动物和人类群体中进行SARS抗体的血清学筛查，以及确定当前或新出现的SARS动物宿主（大鼠、猫？）。根据我们的初步数据和其他人的发现（Ksiazek et al 2003），这种交叉反应性在SARS冠状病毒和I类冠状病毒（N蛋白）之间最常见，但生物学基础尚不清楚。我们的目标是研究SARS冠状病毒与动物冠状病毒双向交叉反应的抗原基础，开发SARS冠状病毒抗体特异性ELISA检测方法，并将其应用于家畜和野生动物的血清中，以确定SARS潜在的动物宿主。具体而言，我们将：1)通过ELISA、免疫荧光（IF）和Western blots等血清学方法评估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性。2)利用杆状病毒系统中表达的SARS和动物冠状病毒重组蛋白N和S，确定参与动物冠状病毒与SARS冠状病毒双向抗原交叉反应的冠状病毒蛋白。3)利用N和s片段评估冠状病毒蛋白中负责交叉反应的区域，并在血清学检测中截断该区域以消除交叉反应性，以便进行SARS冠状病毒特异性检测。4)使用完整的和截断的SARS冠状病毒S和N蛋白检测实验接种和现场暴露动物血清中与SARS冠状病毒交叉反应的抗体：a)作为一种创新方法，测试从TGEV中恢复的非生猪血清，并用灭活的SARS冠状病毒增强，以评估体内交叉反应性；b)检测家畜、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年在动物冠状病毒方面的知识和经验，我们广泛收集了来自不同物种（包括野生动物和相应的超免疫抗血清和单克隆抗体）的多种动物冠状病毒菌株，我们的TGEV冠状病毒S和N基因克隆，以及世卫组织任命我们的实验室为动物冠状病毒SARS网络实验室，将提供彻底调查动物和人类冠状病毒（包括SARS冠状病毒）之间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.