Identification of new drugs and the evaluation of vaccines for treating and preventing canine distemper in dogs and raccoons

治疗和预防犬、浣熊犬瘟热的新药鉴定及疫苗评价

• 批准号: RGPIN-2019-06612
• 负责人: Richardson, Christopher
• 金额: $ 2.33万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684295
• 关键词: Identification; new; drugs; evaluation; vaccines

Canine distemper virus (CDV) is a morbillivirus that is closely related to measles virus (MeV). These viruses are highly contagious and infect lymphocytes, airway epithelial cells, and cells of the neurological system. There have been recent outbreaks of CDV in urban populations of raccoons in Halifax and Toronto. In addition, there are global concerns originating in China, where CDV was observed to jump from dogs into macaques at various primate centres. Our laboratory discovered the cellular receptors for morbilliviruses (SLAMF1, Nectin-4, and CD46). This application represents a new venture into antiviral drugs and the evaluation of vaccines directed against CDV. We propose to use ferret and a SLAMF1 transgenic model to evaluate drugs and vaccines. Ferrets are extremely susceptible to CDV and die within 2 weeks of contracting disease. We also have found that a single mutation in the CDV-H receptor binding protein permits the virus to replicate in our human SLAMF1 transgenic mouse model. In Aim 1 we propose to test the same mouse with wild type CDV strains including neurotropic Snyder Hill virus and local raccoon isolates. Infected mice will be dissected and tissue will be cryo-sectioned and analyzed for virus using immune fluorescence microscopy and RT-PCR. We will use the ferret and mouse models to evaluate new antiviral drugs directed against CDV in Aim 2. There are no therapeutic drugs for treating morbillivirus infections, but I previously discovered a small peptide Z-D-Phe-L-Phe Gly which blocked MeV-mediated membrane fusion and entry into the host cell [Richardson et al. (1980) Virology 105: 205-222]. A small molecular analogue to this peptide (AS48) has been synthesized and both molecules target a similar hydrophobic cavity in the MeV fusion (F) protein [Ha et al. (2017) J. Virol. 91(23):e01026-17]. Similar molecules which inhibit the F protein of CDV will be identified using a chemical library at the Li Ka Shing Virology Institute, Edmonton. Another target for antiviral drugs directed against CDV is the viral RNA polymerase (L). One lead compound (ERDRP-0519) appears effective against MeV and we propose to identify compounds that target the CDV polymerase. Aim 3 addresses a perennial problem with CDV vaccines characterized by vaccine-related encephalitis. The Rockborn vaccine is particularly prone to this debility. The safer Onderstepoort vaccine is attenuated exclusively in eggs, while the problematic Rockborn strain is propagated in mammalian cell culture. We propose to assess receptor usage of various strains of CDV using CHO, CHO-CD46, CHO-dogSLAMF1, CHO-dogNectin-4, CHO-chickNectin-4, and CHO-PVRL2 cell lines. We will study the poorly understood process of virus attenuation by studying vaccine and wild type CDV infections in canine peripheral blood lymphocytes and primary canine tracheal/bronchial epithelial cells. Infectious particle formation, viral protein synthesis, and interferon production by these cells will be evaluated.

犬瘟热病毒（CDV）是一种与麻疹病毒（MeV）密切相关的麻疹病毒。这些病毒具有高度传染性，可感染淋巴细胞、气道上皮细胞和神经系统细胞。最近在哈利法克斯和多伦多的城市浣熊群中爆发了CDV。此外，还有来自中国的全球关注，在中国，在各种灵长类动物中心观察到CDV从狗跳到猕猴。我们的实验室发现了麻疹病毒的细胞受体（SLAMF1， Nectin-4和CD46）。这一应用代表了抗病毒药物和针对CDV的疫苗评价的新尝试。我们建议使用雪貂和SLAMF1转基因模型来评价药物和疫苗。雪貂极易感染CDV，感染后两周内死亡。我们还发现，CDV-H受体结合蛋白的一个突变允许病毒在我们的人类SLAMF1转基因小鼠模型中复制。在目的1中，我们建议用野生型CDV毒株（包括嗜神经性施耐德希尔病毒和当地浣熊分离株）对同一只小鼠进行测试。被感染的小鼠将被解剖，组织将被冷冻切片，并使用免疫荧光显微镜和RT-PCR分析病毒。我们将利用雪貂和小鼠模型在Aim 2中评估针对CDV的新抗病毒药物。目前还没有治疗麻疹病毒感染的药物，但我之前发现了一种小肽Z-D-Phe-L-Phe Gly，它可以阻止mev介导的膜融合并进入宿主细胞[Richardson等人（1980）病毒学105:205-222]。该肽的小分子类似物（AS48）已被合成，两个分子都靶向MeV融合(F)蛋白中相似的疏水腔[Ha et al.(2017) .中国生物医学工程学报，91(23):e01026-17]。将利用埃德蒙顿李嘉诚病毒学研究所的化学文库鉴定抑制CDV F蛋白的类似分子。针对CDV的抗病毒药物的另一个靶标是病毒RNA聚合酶(L)。一种先导化合物（ERDRP-0519）似乎对MeV有效，我们建议鉴定靶向CDV聚合酶的化合物。目的3解决了以疫苗相关性脑炎为特征的CDV疫苗的长期问题。Rockborn疫苗尤其容易产生这种缺陷。更安全的Onderstepoort疫苗只在鸡蛋中减毒，而有问题的Rockborn菌株则在哺乳动物细胞培养中繁殖。我们建议使用CHO、CHO- cd46、CHO- dogslamf1、CHO- dognectin -4、CHO- chicknectin -4和CHO- pvrl2细胞系来评估不同CDV菌株的受体使用情况。我们将通过研究疫苗和野生型CDV在犬外周血淋巴细胞和原代犬气管/支气管上皮细胞中的感染，来研究尚不清楚的病毒衰减过程。将评估这些细胞的感染性颗粒形成、病毒蛋白合成和干扰素产生。