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

Abstract

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.

Instability of the Restriction Fragment Length Polymorphism Pattern of Open Reading Frame 5 of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Pig-to-Pig Passages

“…Such a high degree of heterogeneity among PRRS viruses prompted the development of molecular tools such as restriction fragment length polymorphism (RFLP) analysis (25), sequencing (2,8,10,11,13,14,17,21), and heteroduplex mobility assay (12) for use in further characterizing the genotypes of PRRS viruses and tracing virus movements within or among herds. All of these methods target open reading frame 5 (ORF5) of PRRS virus, which encodes the major envelope (gp5) protein (15,16,19), since ORF5 has shown the highest genetic variability among PRRS viruses compared to the variabilities of other genes (2, 10).Among the molecular tools available, RFLP analysis is a rapid and relatively inexpensive nucleic acid-based differential test; hence, the technique has been used in numerous fieldbased molecular epidemiological surveys of PRRS viruses reported previously (3,6,9,24). RFLP analysis of PRRS virus starts with PCR amplification of ORF5, followed by digestion of the PCR product with the three restriction enzymes MluI, HincII, and SacII.…”

“…Among the molecular tools available, RFLP analysis is a rapid and relatively inexpensive nucleic acid-based differential test; hence, the technique has been used in numerous fieldbased molecular epidemiological surveys of PRRS viruses reported previously (3,6,9,24). RFLP analysis of PRRS virus starts with PCR amplification of ORF5, followed by digestion of the PCR product with the three restriction enzymes MluI, HincII, and SacII.…”

Restriction fragment length polymorphism of ORF6 and ORF7 genes of porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strains registered in the Czech Republic

“…Finding of new RFLP pattern during a certain period of time has been described by Cai et al (2002). Moreover, high degree of genetic variability was confirmed among field isolates of PRRSV with the same digestion RFLP patterns and instability of RFLP patterns PRRSV during in vitro replication (Cha et al, 2004).…”

“…Therefore the use of primers selected from this region allows the highest possible sensitivity of reaction. Several studies have been devoted to RFLP analysis from this gene region (Gagnon and Dea, 1998;Umthun and Mengeling, 1999;Cai et al, 2002). RFLP analyses from part of PRRSV genome of ORF5 gene which codes for envelope glycoprotein E and as an antigenic agent is connected with virus neutralization suggest a possible high number of deviations in RFLP profiles (Cheon and Chae, 2000;Itou et al, 2001;Indik and Valicek, 2002).…”

Short communication. Differentiation of Type-I Porcine Reproductive and Respiratory Syndrome Virus vaccines and field strains by restriction fragment length polymorphism analysis

“…In those lines, a test based on the patterns produced by digestion of PCR products with different restriction enzymes has been developed for the differentiation of type-II field strains and Ingelvac PRRS MLV/Repro™, a MLV type-II vaccine frequently used for the control of the disease (Wesley et al, 1998). This method has frequently been used to typify PRRSV type-II isolates (Cai et al, 2002;Brar et al, 2011). Nonetheless, the extensive genetic differences between type-I and type-II strains of PRRSV make the application of this technique useless in the European situation.…”