Detection of U.S., Lelystad, and European-Like Porcine Reproductive and Respiratory Syndrome Viruses and Relative Quantitation in Boar Semen and Serum Samples by Real-Time PCR

Abstract

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via boar semen has been documented. Since semen is widely disseminated for artificial insemination and the virus can cause significant health and economic consequences, it is essential to have well-validated, rapid diagnostic techniques to detect and quantitate the virus for diagnostic and research purposes. Previously, boar semen was tested by a nested PCR (nPCR) assay which was compared to the "gold standard" swine bioassay. A correlation of 94% was observed, indicating that, most of the time, PCR detected infectious virus. Subsequently, a real-time PCR targeting the 3 untranslated region of the PRRSV genome was compared with nPCR by testing 413 serum and semen samples from PRRSV-inoculated and control boars. There was 95% agreement between the results of the two tests, with the majority of samples with discordant results containing virus at the lower range of detection by the assays. The virus in all samples was quantitated by using a standard curve obtained by serial dilution of an in vitro transcript. By using the in vitro transcript, the lower limit of sensitivity was observed to be approximately 33 copies/ml. Reactivity with a panel of more than 100 PRRSV isolates from various geographical regions in the United States was also documented. No reactivity with nine nonrelated swine viruses was noted. A real-time PCR was also developed for the detection of the European Lelystad virus and the Europeanlike PRRSV now found in the United States. In six of six PRRSV-inoculated boars, peak levels of viremia occurred at 5 days postinoculation (DPI) and were most consistently detectable throughout 22 DPI. In five of six boars, PRRSV was shed in semen for 0 to 2 days during the first 10 DPI; however, one of six boars shed the virus in semen through 32 DPI. Therefore, in general, the concentration and duration of PRRSV shedding in semen did not correlate with the quantity or duration of virus in serum. These differences warrant further studies into the factors that prevent viral replication in the reproductive tract.Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded RNA virus belonging to the order Nidovirales, family Arteriviridae, along with lactate dehydrogenaseelevating virus of mice, equine arteritis virus, and simian hemorrhagic fever virus (4, 13). PRRSV causes significant respiratory disease in nursery and growing-finishing pigs and late-term abortions in sows (29,46). Since the etiologic agent was identified in 1991, multiple methods of virus dissemination have been discovered, including transmission via semen (2,12,18,30,47,50). Transmission of PRRSV through the semen of infected boars may not always occur, but due to the widespread use of artificial insemination in the modern swine industry, dissemination of PRRSV via semen constitutes a major risk for swine herds. Therefore, it is important to provide diagnostic testing to ensure a PRRSV-free semen supply (41). Interestingly, the duration of PRRSV shedd...

Vascular Lesions in Pigs Experimentally Infected With Porcine Circovirus Type 2 Serogroup B

“…14,27 Serum samples collected from the pigs were tested by quantitative real-time reverse transcriptase PCR (RT-PCR) for porcine reproductive and respiratory syndrome virus (PRRSV) using a commercially available one-step multiplex RT-PCR test (Tetracore Inc., Rockville, MD) that can detect both North American and European strains of the virus. 36 In addition, the serum was tested for PPV by gelbased PCR as previously described. 4 Each reaction included negative and positive controls with, respectively, no added template nucleic acid and one or more samples known to contain the virus that was to be detected.…”

Evaluation of the Sensitivity of Reverse-Transcription Polymerase Chain Reaction to Detect Porcine Reproductive and Respiratory Syndrome Virus on Individual and Pooled Samples from Boars

“…25 For semen samples, a commercial RNA isolation column d was used for RNA isolation and purification after a modified cell-lysate homogenization protocol was performed. 4 Specifically, after centrifugation of the raw or extended semen at 660 x g for 15 min, the seminal cell fraction was obtained by pouring off the supernatant and resuspending the pellet in an equal volume of seminal plasma (extended semen) or phosphate-buffered saline (raw semen).…”

Experimental co-infection of pigs withBovine viral diarrhea virus 1andPorcine circovirus-2

“…16,41 Serum collected from germ-free pigs at days 0, 10, and 21/22 of the first study and from CDCD pigs at days -31, -24, and 35 of the second study was tested by quantitative real-time RT-PCR for PRRSV using a commercially available one-step multiplex RT-PCR test n that can detect both North American and European strains of the virus. 51 Finally, serum of germ-free pigs collected at days 0, 10, and 21/22 of the first study, and serum of CDCD pigs collected at days -31, -24, and -14 of the second study was tested for PPV viremia by gel-based PCR as previously described. 3 Filtered inocula and serum collected from each pig at the beginning and end of each study was tested for the presence of both Torque teno virus 1 and 2 (TTV-1 and -2, respectively).…”