Detection of bovine retrospumavirus by the polymerase chain reaction.
A polymerase chain reaction (PCR) assay was developed for detection of bovine retrospumavirus (bovine syncytial virus; BSV) provirus DNA. Two different sets of oligonucleotide primers complementary to sequences located in the gag and the pol/env gene regions were used and compared for their ability to amplify the targeted BSV sequences by PCR. The results obtained from this study have shown that it is possible to amplify the BSV provirus DNA sequences not only from total DNA of BSV-infected cell cultures, but also from total DNA of various tissues and peripheral blood mononuclear cells (PBMCs) that were collected from two rabbits experimentally infected with BSV. Sensitivities of the PCR for amplification of BSV gag and pol/env nucleic acid sequences from cell culture total DNA were 10 ng and 10 pg of DNA, respectively, as determined by the analysis of the amplified PCR products on ethidium bromide-stained agarose gels. The specificity of the PCR for both primer sets tested was confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in Southern blot chemiluminescent hybridization assays. No amplification was obtained when the BSV-specific primers were used in the PCR with DNA material specific to either bovine leukemia virus (BLV) or bovine immunodeficiency virus (BIV) provirus genomic DNA. No cross-hybridization was obtained when the BSV-specific cDNA probes were allowed to react with BLV or BIV provirus DNA. The PCR targeting the gag and pol/env gene regions of the BSV provirus genome may be an alternative to conventional methods for the confirmation of the presence of BSV in cell cultures used for virus isolation, and for the diagnosis of BSV infection from bovine peripheral blood leukocytes.
Pamba
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R
University of Québec at Montréal, Department of Biological Sciences, Canada.
Jeronimo
C
C
Archambault
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D
D000818:Animals; D015139:Blotting, Southern; D002417:Cattle; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D015752:Genes, env / Q000235:genetics; D015750:Genes, gag / Q000235:genetics; D015751:Genes, pol / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D011817:Rabbits; D012192:Retroviridae Infections / Q000821:virology*; D012680:Sensitivity and Specificity; D016092:Spumavirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*
D017931:DNA Primers; D004279:DNA, Viral