Differentiation of a Porcine Reproductive and Respiratory Syndrome Virus Vaccine Strain from North American Field Strains by Restriction Fragment Length Polymorphism Analysis of ORF 5

Abstract

Restriction fragment length polymorphism (RFLP) analysis of open reading frame 5 was developed for typing of Czech strains of porcine reproductive and respiratory syndrome virus (PRRSV). e set of restriction enzymes Acc I, Hae II and SnaB I allowed the differentiation of heterogeneous Czech strains of PRRSV clustered separately in the phylogenetic tree. e high-passage strain V-502 (164) was also differentiated from its parent strain V-502. e same restriction enzymes could distinguish the European-type vaccine strains Porcilis PRRS and Pyrsvac-183, registered in Czech Republic, from the Czech field isolates. e published ORF5 nucleotide sequences allowed us to presume that it will also be possible to distinguish most of European field strains from vaccine strains. PCR-based RFLP analysis can become a valuable tool in epidemiological studies of PRRSV in Europe.

Restriction Fragment Length Polymorphism of Porcine Reproductive and Respiratory Syndrome Viruses Recovered from Ontario Farms, 1998–2000

“…4,5 This study identified all of these except type 1-8-2. As well, 17 additional RFLP types of the virus were identified in this study.…”

“…2 In addition to detection of the virus, RT-PCR RFLP typing methods have been developed that make it possible to differentiate different field strains and the commercial vaccine. 4 The current study detected and typed PRRSV using PCR-based methods from clinical samples ob-tained from Ontario swine herds from January 1998 to July 2000.…”

“…j The code representing a digested pattern was assigned according to or adapted from those described previously. 4 For example, a PRRSV strain was assigned as type 2-5-2 if its RT-PCR amplicon was digested by MluI and generated 279-and 437-bp fragments, by HincII and generated 327-and 389-bp fragments, and by SacII and generated 53-and 663-bp fragments. As defined previously, 5 a field strain has 2 or more restriction enzyme sites different from a vaccine strain (type 2-5-2) and an intermediate strain has only 1 site different within ORF 5.…”

“…This suggests that the 716-bp RT-PCR primer sites of these strains may be different from that of the 716-bp RT-PCR typeable strains. The codes previously reported 4 were adapted to describe these strain types (those types marked with an asterisk below).…”

“…A PCR System 9600 thermocycler e was used with (4) 153 (1) 161 (1) 162 (3) 163 (3) 164 (1) 171 (1) the following thermocycling programs: cDNA synthesis at 42 C for 20 min, inactivation of reverse transcriptase and denaturation at 95 C for 3 min, 35 cycles of denaturation at 95 C for 20 sec, primer annealing at 60 C for 15 sec; and primer extension at 72 C for 20 sec, final extension at 72 C for 7 min, holding at 4 C. The PCR product was detected by electrophoresis in a 1.5% agarose g gel stained with ethidium bromide. h A previously described RT-PCR method 4 was modified to amplify a 716-bp fragment of ORF 5 and part of ORF 4 and 6 (716-bp RT-PCR). The PCR mix was 50 l in volume and contained reagents in the same concentrations as above, with the exception that 1.5 mM MgCl 2 , 0.2 M of each primer, and 4 l of template were used.…”