Laboratory efforts to cultivate noroviruses
Laboratory efforts to cultivate noroviruses
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DOI：
10.1099/vir.0.19478-0
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发表时间：
2004-01-01
影响因子：
3.8
通讯作者：
Estes, MK
中科院分区：
文献类型：
--
作者：
Duizer, E;Schwab, KJ;Estes, MK
诺如病毒（NoV）是全球胃肠炎的主要原因，被认为是食源性疾病的首要原因。尽管许多努力，常规的细胞培养未能产生复制NoV。本文描述了用于尝试在两个实验室中体外生长NoV的方法。将细胞（A549、AGS、Caco-2、CCD-18、CRFK、CR-PEC、Detroit 551、Detroit 562、FRhK-4、HCT-8、HeLa、HEC、HEp-2、Ht-29、HuTu-80、I-407、IEC-6、IEC-18、Kato-3、L20 B、MA 104、MDBK、MDCK、RD、TMK、Vero和293）培养在固体或可渗透表面上。使用细胞培养补充剂如胰岛素、DMSO和丁酸诱导分化。在一些情况下，用生物活性消化添加剂处理细胞和含有NoV的粪便样品。在培养实验中评价的变量包括病毒接种物的制备方法、病毒的基因型、细胞单层的维持条件、维持培养基中的添加剂和细胞接种方法。常规进行系列盲传研究。除CPE评价外，还使用免疫荧光试验检测新产生的病毒衣壳抗原和RT-PCR试验检测病毒基因组，以寻找病毒复制的证据。尽管一些感染的培养物通过RT-PCR保持NoV阳性达5代，并且单层中的偶尔细胞显示特异性免疫荧光的证据，但未观察到可重复的NoV诱导的CPE，并且所有最初为阳性的RT-PCR结果在继续传代后均为阴性。因此，开发用于培养NoV的方法的尝试是不成功的。
Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide and are recognized as the foremost cause of foodborne illness. Despite numerous efforts, routine cell cultures have failed to yield replicating NoV. This paper describes methods used to try to grow NoV in vitro in two laboratories. Cells (A549, AGS, Caco-2, CCD-18, CRFK, CR-PEC, Detroit 551, Detroit 562, FRhK-4, HCT-8, HeLa, HEC, HEp-2, Ht-29, HuTu-80, I-407, IEC-6, IEC-18, Kato-3, L20B, MA104, MDBK, MDCK, RD, TMK, Vero and 293) were cultured on solid or permeable surfaces. Differentiation was induced using cell culture supplements such as insulin, DMSO and butyric acid. In some cases, the cells and the NoV-containing stool samples were treated with bioactive digestive additives. Variables evaluated in cultivation experiments included the method of preparation of the virus inoculum, the genotype of the virus, conditions for maintenance of cell monolayers, additives in the maintenance medium and the method of inoculation of the cells. Serial blind passage studies were performed routinely. In addition to evaluation for CPE, evidence of virus replication was sought using immunofluorescent assays to detect newly produced viral capsid antigen and RT-PCR assays to detect the viral genome. Although some infected cultures remained NoV positive by RT-PCR for up to five passages and an occasional cell in a monolayer showed evidence of specific immunofluorescence, no reproducible NoV-induced CPE was observed and all RT-PCR results that were positive initially were negative following continued passaging. Thus, attempts to develop a method for the cultivation of NoV were unsuccessful.