Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

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 32P-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 x 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.     

Effect of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on European and North American PRRSV shedding in semen from infected boars

The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 10(0.1) to 10(1.0) viral genome copies per ml and 3.63 to 10(1.1) 50% tissue culture infective doses (TCID(50))/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 10(0.2) to 10(4.7) viral genome copies per ml and 1.14 to 10(3.07) TCID(50)/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 10(0.1) to 10(4.57) viral genome copies per ml and 1.66 to 10(3.10) TCID(50)/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 10(0.3) to 10(5.14) viral genome copies per ml and 1.69 to 10(3.17) TCID(50)/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.     

Detection of economically important viruses in boar semen by quantitative RealTime PCR technology

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests.

All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage.

In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.     

Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen

To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.     

Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.     

PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen

An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.     

Development and Validation of an Assay To Detect Porcine Reproductive and Respiratory Syndrome Virus-Specific Neutralizing Antibody Titers in Pig Oral Fluid Samples

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific neutralizing antibodies (NA) are important for clearing the virus. Pen-based pig oral fluid samples for disease surveillance are gaining in importance due to the ease of collection and low cost. The aim of this study was to develop a PRRSV-specific NA assay to determine NA titers in pig oral fluid samples. At first, we standardized the PRRSV NA assay using pen-based pig oral fluid samples collected over a period of 3 months from a herd of swine that received a PRRSV modified live vaccine (PRRS-MLV), and we also used oral fluid and serum samples collected from individual boars that were vaccinated with PRRS-MLV or infected with a virulent PRRSV strain. Our results suggest that a PRRSV NA titer of >8 in oral fluid samples is virus specific and can be detected beginning at 28 days after vaccination or infection. To validate the assay, we used 104 pen-based pig oral fluid and five representative serum samples from each pen of unknown history, as well as 100 serum samples from repeatedly vaccinated sows and oral fluid samples of their respective litters belonging to four different swine-breeding farms. Our results demonstrated that PRRSV NA titers in oral fluid samples are correlated with serum sample titers, and maternally derived PRRSV-specific NA titers could be detected in litters at the time of weaning. In conclusion, we have standardized and validated the pig oral fluid-based PRRSV NA assay, which has 94.3% specificity and 90.5% repeatability. The assay can be used to monitor herd immunity against PRRSV in vaccinated and infected herds of swine.     

Porcine reproductive and respiratory syndrome virus infection increases CD14 expression and lipopolysaccharide-binding protein in the lungs of pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) is a respiratory virus of swine that plays an important role in multifactorial respiratory disease. European strains of PRRSV cause mild or no respiratory signs on their own, but can sensitize the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial lipopolysaccharides (LPS). The inflammatory effect of LPS depends on the binding to the LPS receptor complex. Therefore, we quantified the levels of CD14 expression and LPS-binding protein (LBP) in the lungs of pigs throughout a PRRSV infection. Twenty-four gnotobiotic pigs were inoculated intranasally with PRRSV (10(6) 50% tissue culture infectious doses per pig, Lelystad strain) or phosphate-buffered saline (PBS), and euthanized 1-52 days later. Lungs were examined for CD14 expression (immunofluorescence and image analysis), LBP (ELISA), and virus replication. PRRSV infection caused a clear increase of CD14 expression from 3 to 40 days post-inoculation (DPI) and LBP from 7 to 14 DPI. Both parameters peaked at 9-10 DPI (40 and 14 times higher than PBS control pigs, respectively) and were correlated tightly with virus replication in the lungs. Double immunofluorescence labelings demonstrated that resident macrophages expressed little CD14 and that the increase of CD14 expression in the PRRSV-infected lungs was probably due to infiltration of highly CD14-positive monocytes in the interstitium. As both CD14 and LBP potentiate the inflammatory effects of LPS, their increase in the lungs could explain why PRRSV sensitizes the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to LPS.     

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds

Summary.  Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2–99.5% to the vaccine virus RespPRRS and 99.0–99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus. Received December 24, 1997 Accepted April 15, 1998     

Simultaneous detection of Classical swine fever virus and North American genotype Porcine reproductive and respiratory syndrome virus using a duplex real-time RT-PCR

Classical swine fever and porcine reproductive and respiratory syndrome are both notifiable diseases of the World Organization for Animal Health (OIE). The two diseases exhibit indistinguishable clinical symptoms and sometimes co-exist in swine herds. In this study, a duplex real-time RT-PCR for simultaneous detection of Classical swine fever virus (CSFV) and North American (NA) genotype Porcine reproductive and respiratory syndrome virus (PRRSV) based on two differently labeled TaqMan probes was developed and evaluated. The detection limit of the assay was 3.2 TCID(50) or 13 RNA copies for CSFV and 1.8 TCID(50) or 10 RNA copies for PRRSV, about 50 times more sensitive than conventional RT-PCRs. The duplex real-time RT-PCR was capable of specifically detecting different subgroups of wild-type CSFV and different strains of NA-genotype PRRSV, whereas a number of non-CSFV/PRRSV porcine viruses and bovine pestivirus were tested negative. Out of 155 field samples, 16 were tested positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. Therefore, the assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.     

Effects of North American Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-Based Modified Live Vaccines on Preimmunized Sows Artificially Inseminated with European PRRSV-Spiked Semen

The objective of the present study was to determine if the European porcine reproductive and respiratory syndrome virus (PRRSV) can be transmitted via spiked semen to preimmunized sows and induce reproductive failure. Sows were immunized with the North American PRRSV-based modified live vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) and were artificially inseminated. The sows were randomly divided into three groups. The vaccinated (group 2) and nonvaccinated (group 3) sows developed a PRRSV viremia at 7 to 28 days postinsemination with the European PRRSV-spiked semen. The number of genomic copies of the European PRRSV in serum samples was not significantly different between vaccinated and nonvaccinated sows. All negative-control sows in group 1 farrowed at the expected date. The sows in groups 2 and 3 farrowed between 103 and 110 days after the first insemination. European PRRSV RNA was detected in the lungs of 8 out of 11 live-born piglets and 46 out of 54 stillborn fetuses. In addition, PRRSV RNA was detected using in situ hybridization in other tissues from vaccinated sows that had been inseminated with European PRRSV-spiked semen (group 2). The present study has demonstrated that vaccinating sows with the North American PRRSV-based modified live vaccine does not prevent reproductive failure after insemination with European PRRSV-spiked semen.     

A simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is rapidly gaining worldwide importance as one of the most economically significant diseases of swine. The antibody of Porcine reproductive and respiratory syndrome virus (PRRSV) is detected currently by the combined use of an enzyme-linked immunosorbent assay, serum neutralization test, immunoperoxidase monolayer assay, indirect immunofluorescent antibody test. These methods are time-consuming and require specialized equipment operated by trained technicians. The purpose of this study was to evaluate a simple strip assay (based on a chromatographic and immunogold system) for specific detection of PRRSV antibody in swine sera. This "immunochromatographic strip" test uses Escherichia coli-expressed viral recombinant membrane protein antigen in combination with recombinant nucleocapsid protein as capture protein for detecting antibodies against PRRSV. In this study, the performance of this assay was evaluated with sera from both clinical samples and experimentally infected piglets. Detection by immunochromatographic strip test was compared with detection by a standard, available commercially, indirect enzyme-linked immunosorbent assay and an immunoperoxidase monolayer assay. The immunochromatographic test strip detected antibodies in sera known to contain antibodies to PRRSV in 95.7% sensitivity of samples from pigs infected experimentally and 98.6% sensitivity of clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was 97.8% and 98.2% for clinical and experimental serum samples, respectively.     

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.     

A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451bp (PRRSV), 343bp (CSFV), or 163bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 x 102, 2.90 x 103, and 6.16 x 103 copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology.     

A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus,porcine pseudorabies virus,and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.