H蛋白Y549H CDV变异株跨种感染大熊猫和致病性增强的分子机制研究

As the number one killer of giant pandas, Canine distemper virus (CDV) poses a serious threat to their health and population safety. Over the period of December, 2014 through April, 2015, five of six CDV infected giant pandas died in the Shaanxi Rare Wild Animal Rescue and Research Center, China. The occurrence of the Y549H substitution of the CDV isolate giant panda/SX/2014 has aroused our attention, as it is involved in hemagglutinin (H) protein binding to the signaling lymphocytic activation molecule (SLAM) receptor and has been implicated in the emergence of highly pathogenic CDV and host range expansion. In this project we intend to take the perspective of the virus-receptor interaction to evaluate the influence of the Y549H substitution on receptor affinity, interaction of H/F, cell proliferation characteristic and pathogenicity both in vitro and in vivo by employing site-directed mutation, reverse genetics technique and CDV infected mice model which was established by transgenic mice expressing giant panda SLAM receptor previously. The results of these studies will clarify the molecular determinants of cross species-transmission and pathogenicity enhancement of giant panda/SX/2014 and provide theoretical basis for effective prevention and control of canine distemper for successful conservation of this endangered species.

犬瘟热病毒（CDV）作为“头号杀手”严重威胁大熊猫的生命健康及种群安全。陕西省珍稀野生动物抢救饲养研究中心暴发的犬瘟热疫情共造成5只大熊猫感染死亡。我们发现大熊猫源犬瘟热病毒giant panda/SX/2014H蛋白与宿主SLAM受体结合的549位关键氨基酸位点由犬源分离株Tyr（Y）突变为（H），推测H蛋白Y549H替换导致giant panda/SX/2014获得了跨种传播能力，且毒力增强。本研究拟以病毒与受体相互作用为出发点，以氨基酸定点突变、反向遗传学操作为主要技术手段，表达大熊猫SLAM受体的转基因小鼠为感染模型，分别在蛋白水平、细胞感染水平、动物模型上明确H蛋白Y549H突变对受体亲和力、H/F相互作用力、细胞增殖特性以及转基因小鼠致病性的影响，阐明CDV大熊猫分离株跨宿主感染及致病性增强的分子基础，为有效防控大熊猫犬瘟热、维护大熊猫种群安全提供理论依据。

为阐明CDV跨宿主感染大熊猫及致病性增强的分子基础，本研究首先通过CO-IP实验证实了CDV H549Y与H549H均可与犬SLAM受体发生相互作用，通过慢病毒包装系统构建了7株稳定表达大熊猫SLAM受体（gpSLAM）Vero细胞株，其中5株接种大熊猫源CDV后表现明显细胞融合病变。分别共转染F和H549H或H549Y表达质粒，结果发现H549H对gpSLAM细胞系具有较强融合能力，表明Y549H的突变可以更高效利用大熊猫SLAM受体。而构建的大熊猫源CDV H549Y的回复突变株均对大熊猫和犬SLAM受体具有高度亲和力，使感染后细胞呈现细胞融合现象。此外，将分离到的大熊猫源CDV在Vero-dogSLAM细胞系连续传代后，H蛋白549位氨基酸可发生H549Y自然回复突变，回复突变株同样均可利用大熊猫和犬SLAM受体，使感染细胞呈现明显的细胞融合现象。为评价大熊猫源CDV的致病能力，构建了大熊猫SLAM受体转基因小鼠，通过腹腔注射和滴鼻方式接种回复突变株，结果显示，与滴鼻方式相比，腹腔注射攻毒的小鼠体重降低更为显著，病毒体内分布检测发现，感染7天后仅小鼠肺灌洗液CDV核酸呈阳性，而21天后小鼠全血、肺灌洗液、腹腔洗液CDV核酸均呈阳性，表明已成功建立了大熊猫源CDV的转基因小鼠感染模型。为评价转基因小鼠感染病毒后组织器官的病毒载量，分别建立了荧光定量RT-PCR检测方法和可视化重组酶聚合酶扩增（RT-RPA-VF）快速检测方法，两者符合率为100%。上述结果提示CDV H549Y与H549H蛋白与不同种属的SLAM受体均可发生相互作用，病毒适应不同种属SLAM受体后会导致关键氨基酸发生变异。待拯救获得的野生型大熊猫源CDV亲本株传代后继续进行受体细胞和转基因小鼠的感染特性和致病性验证。