SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium.
SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium.
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DOI：
10.1016/j.virusres.2007.03.013
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发表时间：
2008-04
期刊：
影响因子：
5
通讯作者：
Pickles, Raymond J.
中科院分区：
文献类型：
--
作者：
Sims, Amy C.;Burkett, Susan E.;Yount, Boyd;Pickles, Raymond J.
SARS冠状病毒（SARS-CoV）于2002年出现，是人类严重下呼吸道感染的重要原因，需要体外肺模型来阐明病毒感染的细胞靶点和后果。在严重急性呼吸道病毒病例中，严重和突然出现的症状导致非典型肺炎伴干咳和持续高烧，这揭示了冠状病毒作为潜在致命人类病原体的重要性，并确定了几个人畜共患病宿主，这使得新菌株和未来流行病的重新出现变得更加可能。在这一章中，我们描述了SARS冠状病毒感染人类的病理学，并探讨了使用两种模型的人进行气道，以更好地了解复制和致病的SARS冠状病毒在相关的体外系统。第一种培养模型是人支气管上皮细胞系Calu-3，其可以作为非极化单层细胞或具有紧密连接和微绒毛的极化细胞接种病毒。第二个模型系统，来自原代细胞分离自人气道上皮细胞和Transwell上生长，形成一个假复层粘膜纤毛上皮细胞，概括的形态和生理特征的人进行气道在体内。使用这些肺上皮细胞模型的实验结果表明，与晚期病例中报道的病理学相反，SARS-CoV在传导气道的上皮细胞中复制到高滴度。SARS-CoV受体，即人血管紧张素1转化酶2（hACE 2），仅在极化Calu-3细胞和人气道上皮培养物（HAE）的细胞顶端表面检测到，表明hACE 2在腔内气道递送后可被SARS-CoV接近。此外，在HAE中，hACE 2仅定位于纤毛气道上皮细胞。支持hACE 2定位数据，在HAE的极化Calu-3和纤毛细胞中，接种和子代病毒体排出的最有效途径是顶端表面，表明将大量病毒释放到人肺腔中的机制。用抗hACE 2的抗血清预孵育培养物的顶面，可使病毒滴度降低两个对数，而抗DC-SIGN/DC-SIGNR的抗血清并不降低病毒复制水平，这表明hACE 2是SARS冠状病毒进入HAE培养物纤毛细胞的主要受体。为了评估来自易感动物物种的纤毛气道培养物的感染性，我们通过缺失开放阅读框7a/7 b（ORF 7a/7 b）和插入绿色荧光蛋白（GFP）产生了重组SARS-CoV GFP。SARS-CoV GFP在Vero E6、MA 104和CaCo 2细胞中复制到与野生型病毒相似的滴度。此外，SARS-CoV在金黄色叙利亚仓鼠气管产生的气道上皮培养物中的复制在感染后72小时达到与人类培养物相似的滴度。HAE中纤毛细胞类型的SARS-CoV有效感染为研究SARS-CoV复制和致病特性提供了有用的人肺起源的体外模型。
SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.
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影响因子：
64.8
作者：
Li W;Moore MJ;Vasilieva N;Sui J;Wong SK;Berne MA;Somasundaran M;Sullivan JL;Luzuriaga K;Greenough TC;Choe H;Farzan M
通讯作者：
Farzan M
影响因子：
15.8
作者：
Lawler JV;Endy TP;Hensley LE;Garrison A;Fritz EA;Lesar M;Baric RS;Kulesh DA;Norwood DA;Wasieloski LP;Ulrich MP;Slezak TR;Vitalis E;Huggins JW;Jahrling PB;Paragas J
通讯作者：
Paragas J
DOI：
10.1016/s0140-6736(03)13967-0
发表时间：
2003-07-26
期刊：
Lancet (London, England)
影响因子：
--
作者：
Kuiken T;Fouchier RA;Schutten M;Rimmelzwaan GF;van Amerongen G;van Riel D;Laman JD;de Jong T;van Doornum G;Lim W;Ling AE;Chan PK;Tam JS;Zambon MC;Gopal R;Drosten C;van der Werf S;Escriou N;Manuguerra JC;Stöhr K;Peiris JS;Osterhaus AD
通讯作者：
Osterhaus AD
影响因子：
5.4
作者：
Curtis, KM;Yount, B;Baric, RS
通讯作者：
Baric, RS
影响因子：
3.3
作者：
Franks TJ;Chong PY;Chui P;Galvin JR;Lourens RM;Reid AH;Selbs E;McEvoy CP;Hayden CD;Fukuoka J;Taubenberger JK;Travis WD
通讯作者：
Travis WD