Host influence on cell tropism and immune response to MERS-CoV

人畜共患病病毒，如中东呼吸综合征冠状病毒(MERS-CoV)，可在人类身上引起严重的病理，但在宿主中，可能几乎没有疾病的证据。在作为放大宿主的骆驼身上，尽管病毒大量脱落，但在实验中观察到的主要是上呼吸道感染，几乎没有病理变化，也不会产生更长期的后果。在人类、非人类灵长类动物和小鼠动物模型中，会出现轻微到严重的疾病，病毒主要针对下呼吸道进行复制。了解病毒如何与不同的宿主相互作用，在病毒复制、脱落和最终疾病方面存在显著差异，可以为有益和有害的病毒与宿主相互作用提供许多见解。我的实验室正在建立骆驼的代用动物模型，使用羊驼，使我们能够研究病毒的复制、脱落、传播、组织趋向性和致病机制，以比较和对比宿主和疾病模型。初步数据显示，羊驼和骆驼是可比的型号。作为对比，我们将使用表达MERS-COV受体人二肽基肽酶4(HDPP4)的转基因小鼠。这个模型概括了与在人类中观察到的疾病相当的严重疾病。在这两个截然不同的模型中研究病毒与宿主的相互作用将使我们能够剖析病毒如何在不同的模型系统中传播、细胞趋向性和宿主反应的不同机制。这将有助于我们理解病毒和宿主都用来影响病毒复制和趋向性的机制--这最终决定了感染是导致一种相对储备库的状态，适合将病毒传播到新的宿主，还是导致重大疾病和/或死亡的疾病样状态。 首先，我们将确定MERS-CoV在小鼠和羊驼模型中的细胞趋向性，确定受体分布并量化呼吸道组织原代细胞培养中的病毒产量。体外研究结果将在动物模型中得到证实。到目前为止，所有的MERS-CoV病毒都是在猴子细胞中传代的，我们将在骆驼细胞系中产生一种骆驼适应病毒，随后进行基因组和生长动力学表征，以确定对趋向性和致病机制的影响。接下来，我们将使用细胞类型特定的基因标记，确定炎症细胞因子的水平，并确定哪些细胞被招募到感染部位，以确定两种动物模型中免疫细胞在呼吸道组织中招募和活动的差异。最后，我们将确定病毒在呼吸道组织中的传播机制。在有利于释放和扩散或细胞间传播的不同条件下的感染动力学决定了病毒在肺内的传播是否主要是细胞间传播。

Zoonotic viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pathology in humans, but in reservoir hosts, little to no evidence of disease may be present. In camels, which serve as a amplifying host, a primarily upper respiratory tract infection has been experimentally observed with little pathology and no longer term consequences, despite high levels of virus shedding. In humans, and nonhuman primate and mouse animal models, mild to severe disease is served, with virus primarily targeting the lower respiratory tract for replication. Understanding how a virus interacts with different hosts, where there is significant differences in viral replication, shedding and ultimately disease can provide many insights into beneficial and detrimental virus-host interactions. My lab is in the process of establishing a surrogate animal model for camels, using alpacas, to allow us to study virus replication, shedding, transmission, tissue tropism and pathogenesis to compare and contrast reservoir and disease models. Preliminary data indicates that alpacas and camels are comparable models. As a comparator, we will use transgenic mice that express human dipeptidyl peptidase 4 (hDPP4), the receptor for MERS-COV. This model recapitulates severe disease comparable to that observed in humans. Studying the virus interplay with the host in both these two widely different models will allow us to dissect out mechanisms of how virus spread, cellular tropism and the host response varies in different model systems. This will help us to understand the mechanisms that both the virus and the host use to influence virus replication and tropism - which ultimately determines whether infection results in a relatively reservoir-like state suitable for transmitting the virus on to new hosts or disease-like state that results in significant illness and/or death. First we will define the cellular tropism of MERS-CoV in the mouse and alpaca models, determining receptor distribution and quantifying virus output in primary cell cultures of respiratory tract tissues. In vitro findings will be confirmed in the animal model. As all MERS-CoV viruses to date have been passaged in monkey cells, we will generate a camelid-adapted virus in a camel cells line, with subsequent genomic and growth kinetic characterization to determine effects on tropism and pathogenesis. Next, we will determine differences in immune cell recruitment and activity in respiratory tract tissues in both animal models using cell type specific gene markers, determining levels of inflammatory cytokines and identifying what cells are recruited to sites of infection. Last, we will determine the mechanism of virus spread in respiratory tissues. The kinetics of infection under different conditions that favour either release and diffusion or cell-to-cell spread with determine if virus spread in the lung is primarily cell-to-cell.