Human norovirus culture in B cells.
Human norovirus culture in B cells.
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DOI：
10.1038/nprot.2015.121
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发表时间：
2015-12
期刊：
影响因子：
14.8
通讯作者：
Karst SM
中科院分区：
文献类型：
--
作者：
Jones MK;Grau KR;Costantini V;Kolawole AO;de Graaf M;Freiden P;Graves CL;Koopmans M;Wallet SM;Tibbetts SA;Schultz-Cherry S;Wobus CE;Vinjé J;Karst SM
人类诺如病毒（HunoV）是食源性疾病和严重儿童腹泻的主要原因，并且它们引起全球大多数胃肠炎爆发。然而，有效和持久的HunoV疫苗和治疗剂的开发由于其不可培养性而受到极大阻碍。我们最近证明了HunoV在人类B细胞中复制，并且肠道细菌作为这种感染的辅因子。在本方案中，我们提供了直接在人B细胞中以及在共培养系统中培养GII.4-sydney HunoV毒株的详细方法，其中病毒必须穿过汇合的上皮屏障才能进入底层的B细胞。我们还描述了用于细菌刺激HunoV B细胞感染和用于测量病毒附着于B细胞表面的方法。最后，我们强调的变量，有助于在这个系统中的病毒复制的效率。感染试验需要3d，附着试验需要3 h。包括RNA提取和RT-QPCR的感染或附着样品的分析需要约6小时。
Human noroviruses (HunoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HunoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HunoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-sydney HunoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HunoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. analysis of infection or attachment samples, including rna extraction and rt-qpcr, requires ~6 h.