Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures
Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures
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DOI：
10.1128/jvi.00080-19
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发表时间：
2019-05-01
影响因子：
5.4
通讯作者：
Maines，Taronna R.
中科院分区：
文献类型：
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作者：
Zeng，Hui;Goldsmith，Cynthia S.;Maines，Taronna R.
雪貂为研究流感病毒的发病机制和传播提供了宝贵的动物模型。为了进一步表征该模型，我们建立了一种分化的原代雪貂鼻上皮细胞（FNEC）培养模型，用于研究甲型流感病毒感染和病毒与宿主的相互作用。这种分化良好的培养物由各种细胞类型、粘液纤毛清除系统和紧密连接组成，代表鼻纤毛假层状呼吸上皮。α2,6-链和α2,3-链唾液酸（SA）受体分别优先结合人流感病毒和禽流感病毒的血凝素（HA），在培养物的顶端表面检测到具有不同细胞趋向性的α2,6-链唾液酸受体。根据SA受体的分布，我们观察到2009年前的季节性a （H1N1）病毒感染纤毛细胞和非纤毛细胞，而高致病性禽流感（HPAI） a （H5N1）病毒主要感染非纤毛细胞。透射电镜显示，病毒粒子从纤毛、非纤毛和分泌黏液的杯状细胞的顶膜释放或与之相关。感染后，高致病性甲型H5N1病毒在37℃下复制到高于人甲型H1N1病毒的滴度；然而，在33°C时，甲型H5N1病毒的复制明显减弱。此外，我们发现，与甲型H1N1病毒相比，甲型H5N1病毒感染可诱导更高水平的免疫中介基因表达，并导致更多的细胞损伤/损失。这个初级分化的FNEC培养模型概括了鼻上皮的结构，为流感病毒感染初期的细胞趋向性、感染性和发病机制的体外桥联研究提供了一个有用的模型。尽管雪貂是流感病毒感染的重要模型，但在细胞水平上，关于该物种中病毒与宿主的相互作用仍有许多未知之处。雪貂鼻上皮细胞分化原代培养的发展是了解细胞趋向性和流感病毒在该模型气道环境中感染和复制机制的重要一步。利用凝集素染色和显微镜技术，我们表征了唾液酸受体的分布和培养模型的细胞组成。然后，我们评估了在相关生理温度下人类和禽流感病毒的复制和免疫反应。我们的研究结果为预防流感病毒感染的第一道防线提供了重要的见解，并为在良好控制的体外环境下评估新出现的流感病毒提供了一个模型。
Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridgein vivoandin vitrostudies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCEAlthough ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlledin vitroenvironmental setting.