Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification.
Two sets of oligonucleotide primers (1008PS-1009PR and 1010PLS-1011PLR) were designed according to the sequence of the nucleocapsid protein (N) gene of Quebec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and PCR (RT-PCR) experiments for detection of viral genomic RNA either from infected porcine alveolar macrophages (PAM) or tissues from experimentally infected specific-pathogen-free pigs. Considering the high degree of variation detected between the nucleotide sequences of the N genes of IAF-exp91 and Lelystad virus (LV) strains of PRRSV, the primers 1008PS-1009PR were referred to as the specific primers, since they were chosen in such a manner that they could amplify only sequences from IAF-exp91 RNA and not from LV. On the other hand, the primer pair 1010PLS-1011PLR was common to both strains of PRRSV. When analyzed by agarose gel electrophoresis, the products of RT-PCR from each set of primers were resolved as single band of the predicted size, the specificity of amplified products being confirmed by Southern blotting with a specific IAF-exp91 N gene probe. No amplification was observed when RNA was extracted from uninfected PAM or from other porcine viruses. As expected, only the common primer pair was able to amplify RNA from the Quebec reference strain and two European strains (LV and Weybridge). The resulting bands displayed differences in electrophoretic mobilities due to the absence of 37 nucleotides in both European strains, thus allowing their differentiation from the IAF-exp91 strain. Most of the tissue culture-adapted Quebec isolates were detected with both primer pairs. The sensitivity of the enzymatic amplification method for detection of PRRSV from lung tissues was a 50% tissue culture infective dose of 5. RT-PCR was found to be more sensitive than indirect immunofluorescence assay for detection of PRRSV in tissues from experimentally infected pigs and as sensitive as virus isolation in PAM, especially when combined with Southern blotting with the digoxigenin-labeled N probe and chemiluminescence detection.
Mardassi
H
H
Centre de Recherche en Virologie, Institut Armand-Frappier, Université du Québec, Laval, Canada.
Wilson
L
L
Mounir
S
S
Dea
S
S
D000034:Abortion, Veterinary / Q000453:epidemiology / Q000382:microbiology*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002213:Capsid; D002478:Cells, Cultured; D017931:DNA Primers; D005060:Europe / Q000453:epidemiology; D005260:Female; D016676:Macrophages, Alveolar / Q000821:virology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011792:Quebec / Q000453:epidemiology; D012328:RNA Viruses / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics*; D012141:Respiratory Tract Infections / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D013045:Species Specificity; D013047:Specific Pathogen-Free Organisms; D013552:Swine; D013553:Swine Diseases / Q000453:epidemiology / Q000382:microbiology*
D017931:DNA Primers; D012367:RNA, Viral