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 highlighte d a requirement for supplementary tests for precise virus-typing. As a resu lt, we developed a RT-PCR assay able to detect as well as type PRRSV. To pr ovide maximal sequence information, complete viral open reading frames (ORF s 5 and 7) were targeted for amplification. The RT-PCR test was able to amp lify complete PRRSV ORFs from complex materials such as boar semen containi ng as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplishe d 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 ty ping strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV f ield isolates covering the period 1992-1997. The ORF 7-based test had parti cularly desirable characteristics, namely, highly sensitive detection of PR RSV without apparent type bias, typing of the detected virus, discriminatio n between pure and mixed virus populations, and semi-quantitative assessmen t of type ratios in mixed populations, all in a single PCR reaction. In add ition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligo nucleotide hybridization) for confirmation of the specificity of ORF 7 RT-P CR reactions. As such, the RT PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-t ypes. (C) 1998 Elsevier Science B.V. All rights reserved.