Detection Of Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Prevalence of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and porcine parvovirus from aborted fetuses and pigs with respiratory problems in Korea

Porcine reproductive and respiratory syndrome virus(PRRSV)0, porcine circovirus type 2(PCV-2) and porcine parvovirus (PPV)0 infections were investigated as possible causes of the postweaning multisystemic wasting syndrome(PMWS). Specific primers for RT-PCR and PCR were designed for the differential detection of PRRSV, PCV-2 and PPV. Using PCR, these viruses were detected in homogenized tissue samples from pigs that had respiratory of reproductive problems in the time period between 1998 and 2000; the overall prevalences were: PRRSV 31.4%, PCV-2 46.5%, and PPV 8.1%. PCV-2 was also detected in aborted fetal tissues.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes

Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID 50 ml −1 of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992–1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.

Homologous challenge of porcine reproductive and respiratory syndrome virus immunity in pregnant swine

The clinical consequences of single or multiple exposure of pregnant gilts to porcine reproductive and respiratory syndrome virus (PRRSV) at various stages of gestation were determined. Thirty-three pregnant gilts were allotted to 6 experimental groups (5 to 7 gilts/group). Gilts of groups 1 to 5 were exposed to strain NADC-8 of PRRSV at the following times: group 1, gestation day (GD) 1; group 2, GDs 1 and 90; group 3, GD 30; group 4, GDs 30 and 90; group 5, GD 90. Virus exposure was by either intrauterine (GD 1) or oronasal (GDs 30 and 90) inoculation. Gilts of group 6 were kept as nonexposed controls. Gilts were either necropsied on or about GD 111 (groups 1 to 5) or were allowed to farrow (group 6). The detection of PRRSV in serum of fetuses and piglets (within 12 hof birth) was considered evidence of transplacentral infection. Transplacental infection and virus-induced death were and were not confirmed for groups 3, 4, and 5 and for groups 1, 2, and 6, respectively. Collectively, the results indicated that intrauterine exposure to PRRSV at GD 1 was without clinical effect (groups 1 and 2) and provided protection against subsequent exposure to the same strain of virus at GD 90 (group 2). The highest incidence of transplacental infection and fetal death followed a single exposure to PRRSV at GD 90 (group 5).

Development of a RT-PCR test coupled with a microplate colorimetric assay for the detection of a swine Arterivirus (PRRSV) in boar semen

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was developed based on amplification of the PRRSV RNA by reverse transcription and polymerase chain reaction (RT-PCR) followed by detection of the amplification products by hybridization and colorimetric assay in microwell plates. A highly reproducible and efficient method of viral RNA isolation from semen samples was set up. A combined RT-PCR procedure was performed, incorporating the use of uracil- N-glycosylase (UNG) in combination with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The colorimetric detection in microwell plates of amplification products from either PRRSV or IC RNA. gave specific and objective results and was automated. A cut-off value of 1000 RNA copies or 10 TCID 50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples collected from experimentally-infected boars were tested with this assay and showed PRRSV excretion early after infection and for an extended period.

Effect of post-coital intrauterine inoculation of porcine reproductive and respiratory syndrome virus on conception in gilts.

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on early gestation was investigated by exposing susceptible gilts to the virus shortly after they had been bred naturally. Sixteen gilts were exposed intrauterinely to PRRSV and 23 gilts received a sham inoculum. One day after exposure, and on or about seven, 14, and 30 days after exposure, the gilts were bled and the serum was tested for PRRSV and homologous antibody. The pregnancy status of each gilt was determined on day 30 by ultrasound, and near or at term either by necropsy or by allowing the gilts to farrow naturally. All 16 gilts exposed to PRRSV became infected, as evidenced by the detection of PRRSV in seven of the gilts and homologous antibody in the serum of all of them, whereas all the 23 gilts exposed to a sham inoculum remained free of both virus and antibody. Ten of the 16 infected gilts conceived, and 19 of the 23 uninfected gilts conceived, but the difference in conception rate was not statistically significant. Moreover, the mean numbers of live fetuses or pigs per litter of the infected and uninfected gilts were similar (9.7 and 9.3). These results suggest that the intrauterine infection of susceptible pigs with PRRSV at or near the time of conception may have little or no effect on their reproductive performance.

Nested PCR for detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs.

A nested polymerase chain reaction (PCR) was developed to detect porcine reproductive and respiratory syndrome (PRRS) virus. A common primer set for European and North American type isolates of PRRS virus was designed for reverse transcription PCR, and a specific primer set for each of the 2 type isolates was designed for nested PCR. The PCR that used a specific primer set detected the corresponding type of the virus at a level equivalent to 1 TCID50/100 microliters, but not the other type of isolates. Therefore, the method clearly differentiated the 2 types of virus from each other. The detection of PRRS virus by the nested PCR was as sensitive as virus isolation in cultures of porcine alveolar macrophages from infected pigs at the acute stages, and was more sensitive from pigs at the convalescent stages. The infecting virus type was determined by use of 2 specific primer sets even when virus isolation was negative in naturally infected pigs. It was concluded that the nested PCR is useful for diagnosis and typing of PRRS virus and studies of persistent infection by the virus.

Detection of porcine reproductive and respiratory syndrome virus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe.

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.