title: Human norovirus culture in B cells.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Jones
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
authors.forename: Katrina R
authors.initials: KR
authors.lastname: Grau
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Veronica
authors.initials: V
authors.lastname: Costantini
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Abimbola O
authors.initials: AO
authors.lastname: Kolawole
authors.affiliation: Department of Viroscience; Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Miranda
authors.initials: M
authors.lastname: de Graaf
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Pamela
authors.initials: P
authors.lastname: Freiden
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Graves
authors.affiliation: Department of Viroscience; Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Marion
authors.initials: M
authors.lastname: Koopmans
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Wallet
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tibbetts
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Schultz-Cherry
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Vinjé
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Karst
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology*; D017250:Caliciviridae Infections / Q000378:metabolism* / Q000821:virology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods; D006801:Humans; D029322:Norovirus / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D014776:Virus Cultivation / Q000379:methods*; D053586:Virus Internalization; D014779:Virus Replication