DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN CELL-CULTURES AND FORMALIN-FIXED TISSUES BY IN-SITU HYBRIDIZATION USINGA DIGOXIGENIN-LABELED PROBE

A nonradioactive in situ hybridization method is described for the det ection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sectio ns originating from experimentally infected pigs and from 1 held 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 digoxigen in-dUTP using a commercially available kit. The ability of the digoxig enin-labeled probe to specifically detect PRRSV RNA was tested on cult ured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PR RSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed para ffin-embedded tissue sections from pigs experimentally infected with s everal Canadian isolates and from a field case, a strong signal withou t background staining was obtained. Our results indicate that nonradio active in situ hybridization could represent a useful tool for the det ection of PRRSV in routinely fixed and processed tissues. In situ hybr idization could also be used to differentiate infection by North Ameri can and European Lelystad-like PRRSV isolates.