title: Molecular and immunological methods to detect rotavirus in formalin-fixed tissue.
abstract: In 1999, a tetravalent rhesus-based rotavirus vaccine was withdrawn from the market after reports of intussusception cases among vaccinated infants. Methods to detect rotavirus in formalin-fixed pathology specimens from such patients will be important in examining the possible associations between the vaccine and intussusception, in investigating fatalities caused by natural rotavirus infection, and in furthering our understanding of the pathogenesis of rotavirus disease. Three different methods, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), were developed to detect rotavirus in infected cell lines that were fixed in formalin and embedded in paraffin. Using specific primer pairs to identify the VP4 gene with a one-step RT-PCR method, we detected simian rotavirus strains RRV and YK-1 in the liver of an RRV-infected SCID mouse and in the small intestine of an YK-1 infected macaque, respectively. Using a two-step indirect immunoalkaline phosphatase technique, we found RRV antigens in the liver of an infected SCID mouse with a rabbit polyclonal anti-group A rotavirus antibody and a murine monoclonal anti-rotavirus VP2 antibody. Using riboprobes designed to detect RRV genes, VP4 and NSP4, we obtained a positive hybridization signal in the same area of the infected SCID mouse liver as the area in which rotavirus antigens were localized. These techniques should prove valuable to detect rotavirus antigens and nucleic acids in tissues from patients infected naturally with rotavirus or with intussususception associated with rotavirus vaccine.
authors.affiliation: Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Infectious Disease Pathology Activity, 1600 Clifton Road, NE, Mailstop G30, Atlanta, GA 30333, USA. ket2@cdc.gov
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Tatti
authors.forename: Jon
authors.initials: J
authors.lastname: Gentsch
authors.forename: Wun-Ju
authors.initials: WJ
authors.lastname: Shieh
authors.forename: Tara
authors.initials: T
authors.lastname: Ferebee-Harris
authors.forename: Maureen
authors.initials: M
authors.lastname: Lynch
authors.forename: Joseph
authors.initials: J
authors.lastname: Bresee
authors.forename: Baoming
authors.initials: B
authors.lastname: Jiang
authors.forename: Sherif R
authors.initials: SR
authors.lastname: Zaki
authors.forename: Roger
authors.initials: R
authors.lastname: Glass
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005557:Formaldehyde; D006801:Humans; D007841:Latex Fixation Tests / Q000379:methods; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000276:immunology / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000145:classification / Q000175:diagnosis / Q000473:pathology*
chemical_list: D017931:DNA Primers; D005557:Formaldehyde