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

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种群中爆发了CDV。此外，还有源自中国的全球关注，在中国的多个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（SLAMF 1，Nectin-4和CD 46）。该应用代表了抗病毒药物和针对CDV的疫苗评估的新冒险。我们建议使用雪貂和SLAMF 1转基因模型来评估药物和疫苗。雪貂对CDV非常敏感，在感染疾病后2周内死亡。我们还发现CDV-H受体结合蛋白中的单一突变允许病毒在我们的人SLAMF 1转基因小鼠模型中复制。在目标1中，我们建议用野生型CDV毒株（包括嗜神经性斯奈德山病毒和当地浣熊分离株）测试同一只小鼠。将解剖感染的小鼠，并将组织冷冻切片，并使用免疫荧光显微镜和RT-PCR分析病毒。我们将使用雪貂和小鼠模型来评估新的抗CDV的抗病毒药物。没有治疗麻疹病毒感染的治疗药物，但我以前发现了一种小肽Z-D-Phe-L-Phe Gly，它阻断MeV介导的膜融合和进入宿主细胞[Richardson等（1980）Virology 105：205-222]。已经合成了该肽的小分子类似物（AS 48），并且两种分子都靶向MeV融合（F）蛋白中的类似疏水腔[Ha等人（2017）J. Virol. 91（23）：e01026-17]。埃德蒙顿的李嘉诚病毒学研究所将利用化学文库鉴定抑制犬瘟热病毒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CDV疫苗的一个长期问题，其特征在于疫苗相关性脑炎。Rockborn疫苗特别容易出现这种缺陷。更安全的Onderstepoort疫苗仅在鸡蛋中减毒，而有问题的Rockborn菌株在哺乳动物细胞培养中繁殖。我们建议使用CHO、CHO-CD 46、CHO-dogSLAMF 1、CHO-dogNectin-4、CHO-chickNectin-4和CHO-PVRL 2细胞系评估各种CDV毒株的受体利用。我们将通过研究犬外周血淋巴细胞和原代犬气管/支气管上皮细胞中的疫苗和野生型CDV感染来研究病毒减毒的知之甚少的过程。将评价这些细胞的感染性颗粒形成、病毒蛋白合成和干扰素产生。

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.