Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes.
Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992-1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.
Oleksiewicz
M B
MB
Danish Veterinary Institute for Virus Research, Lindholm, Kalvehave.
Bøtner
A
A
Madsen
K G
KG
Storgaard
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D017931:DNA Primers / Q000737:chemistry; D003718:Denmark; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005062:European Union; D005260:Female; D016676:Macrophages, Alveolar; D008297:Male; D016366:Open Reading Frames; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012661:Semen / Q000821:virology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013047:Specific Pathogen-Free Organisms; D013552:Swine; D014481:United States
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D012367:RNA, Viral