title: Detection of porcine reproductive and respiratory syndrome virus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe.
abstract: A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.
authors.affiliation: Laboratoire d'Hygiène Vétérinaire et Alimentaire, Agriculture Canada, Saint-Hyacinthe, Québec, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Larochelle
authors.forename: H
authors.initials: H
authors.lastname: Mardassi
authors.forename: S
authors.initials: S
authors.lastname: Dea
authors.forename: R
authors.initials: R
authors.lastname: Magar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D018018:Arterivirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018174:Arterivirus Infections / Q000175:diagnosis / Q000473:pathology / Q000662:veterinary*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004076:Digoxigenin; D019084:Fluorescent Antibody Technique, Indirect; D005557:Formaldehyde; D017403:In Situ Hybridization; D008168:Lung / Q000473:pathology / Q000821:virology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D013552:Swine; D013553:Swine Diseases; D013577:Syndrome
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D005557:Formaldehyde; D004076:Digoxigenin