Jones Melissa K, Grau Katrina R, Costantini Veronica, Kolawole Abimbola O, de Graaf Miranda, Freiden Pamela, Graves Christina L, Koopmans Marion, Wallet Shannon M, Tibbetts Scott A, Schultz-Cherry Stacey, Wobus Christiane E, Vinjé Jan, Karst Stephanie M
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Nat Protoc. 2015 Dec;10(12):1939-47. doi: 10.1038/nprot.2015.121. Epub 2015 Oct 29.
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.
人诺如病毒（HuNoVs）是食源性疾病和儿童严重腹泻的主要病因，在全球范围内引发了大多数肠胃炎暴发。然而，由于其不可培养性，有效且持久的HuNoV疫苗和治疗方法的研发受到了极大阻碍。我们最近证明，一种HuNoV可在人B细胞中复制，并且共生细菌是这种感染的辅助因子。在本方案中，我们提供了在人B细胞中直接培养GII.4-悉尼HuNoV毒株的详细方法，以及在共培养系统中培养该毒株的详细方法，在该系统中，病毒必须穿过汇合的上皮屏障才能接触到下层的B细胞。我们还描述了细菌刺激HuNoV感染B细胞的方法以及测量病毒附着于B细胞表面的方法。最后，我们强调了影响该系统中病毒复制效率的变量。感染试验需要3天，附着试验需要3小时。对感染或附着样本的分析，包括RNA提取和RT-qPCR，大约需要6小时。
