Abstract. Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus or viral RNA was detected in the serum of all boars within 1 DPI by VI and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.Porcine reproductive and respiratory syndrome (PRRS) is an important disease in swine and occurs throughout the world. 8,13 The syndrome was first recognized in 1987 and is clinically characterized by poor conception rates, late-term abortions and stillborn and weak pigs from sows of reproductive age. Respiratory distress, fever, lethargy, and high mortality can occur in suckling, weaned, and grow-finish pigs. 8,12,16,24 The etiologic agent of this disease was first described in 1991 and was identified as a single-stranded RNA virus, currently classified as an arterivirus. 2,24 Although the primary route of PRRS virus (PRRSV) transmission may be through airborne spread, 13 recent investigations in the United Kingdom 12 and the United States 18,26 have indicated that infected boars can transmit PRRSV in semen. This information has heightened concern in the world swine industry, which is becoming more reliant on artificial insemination as a means to introduce new genetic information into "high health" status herds. There are also trade restrictions on the importation of semen from countries with PRRSV. 18 The contamination rate of semen and boars with PRRSV in the US and Europe is unknown. A polymerase chain reaction (PCR) assay using primers to open reading frame (ORF) 7 of the VR-2332 isolate has recently been developed to detect the PRRSV RNA in semen, and it was found to be more sensitive than virus isolation...

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4-to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32 P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 ؋ g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen. RESULTSOptimization of PCR amplification conditions. To improve the specificity and sensitivity of the PCR, various MgCl 2 concentrations (1.6 to 5 mM), primer concentrations (0.2 to 0.4 M), annealing (50 to 58ЊC) and denaturing (94, 95, or 98ЊC) temperatures, and numbers of cycles (30, 40, or 45) were tested. The optimum product yield was achieved with 5 mM MgCl 2 , 0.4 M primer, annealing and denaturing temperatures of 58 and 95ЊC, respectively, and 30 cycles.