Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures.
Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures.
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在三维培养物中生长的微绒毛的肠上皮细胞中人类诺如病毒传播的失败。
DOI：
10.1007/s00705-013-1806-4
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发表时间：
2014-02
影响因子：
2.7
通讯作者：
Wang, Qiuhong
中科院分区：
文献类型：
--
作者：
Takanashi, Sayaka;Saif, Linda J.;Hughes, John H.;Meulia, Tea;Jung, Kwonil;Scheuer, Kelly A.;Wang, Qiuhong
人诺如病毒（HuNoV）是急性胃肠炎的主要原因。用于体外HuNoV生长的细胞培养系统的建立仍然具有挑战性。已经报道了HuNoV在人肠细胞系（INT-407和Caco-2）中的复制，所述细胞系在旋转壁血管（RWV）三维培养物中分化以产生微绒毛（Straub等人，Emerg Infect Dis 13：396-403 2007，J Water Health 9：225-240 2011，和Water Sci Technol 67：863-868 2013）。我们使用类似的RWV系统、肠细胞系和相同的（基因组[G] I.1）加上额外的（GII.4和GII.12）HuNoV菌株来测试系统的再现性并扩展早期的发现。光镜和电镜下观察到两种细胞株表面均可见顶端微绒毛。然而，所测试的细胞类型中没有一种导致任何HuNoV毒株的生产性病毒复制，如通过实时逆转录PCR测定的HuNoV感染的细胞的上清液和细胞裂解物中的病毒RNA滴度的平台或下降所证实的。当添加培养补充剂时，这些趋势是相同的，这些培养补充剂已被报道在体外对其他苛养肠道病毒的复制有效。此外，通过共聚焦显微镜和orthoslice分析，病毒衣壳蛋白主要观察到以上的肌动蛋白丝信号，这表明，大多数病毒抗原的细胞表面。我们的结论是，即使肠细胞显示微绒毛不足以支持HuNoV复制的条件下测试。
Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis. Establishment of a cell culture system for in vitro HuNoV growth remains challenging. Replication of HuNoVs in human intestinal cell lines (INT-407 and Caco-2) that differentiate to produce microvilli in rotation wall vessel (RWV) three-dimensional cultures has been reported (Straub et al., Emerg Infect Dis 13:396–403 2007, J Water Health 9:225–240 2011, and Water Sci Technol 67:863–868 2013). We used a similar RWV system, intestinal cell lines, and the same (Genogroup [G] I.1) plus additional (GII.4 and GII.12) HuNoV strains to test the system’s reproducibility and to expand the earlier findings. Apical microvilli were observed on the surface of both cell lines by light and electron microscopy. However, none of the cell types tested resulted in productive viral replication of any of the HuNoV strains, as confirmed by plateau or declining viral RNA titers in the supernatants and cell lysates of HuNoV-infected cells, determined by real-time reverse transcription PCR. These trends were the same when culture supplements were added that have been reported to be effective for replication of other fastidious enteric viruses in vitro. Additionally, by confocal microscopy and orthoslice analysis, viral capsid proteins were mainly observed above the actin filament signals, which suggested that the majority of viral antigens were on the cell surface. We conclude that even intestinal cells displaying microvilli were not sufficient to support HuNoV replication under the conditions tested.
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影响因子：
3.8
作者：
Duizer, E;Schwab, KJ;Estes, MK
通讯作者：
Estes, MK
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Klein, Eileen J.;Boster, Daniel R.;Tarr, Phillip I.
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Black, Robert E.
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11.8
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Parashar, Umesh D.
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6.4
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通讯作者：
Estes, MK