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. 1995 Jul;33(7):1730–1734. doi: 10.1128/jcm.33.7.1730-1734.1995

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

J Christopher-Hennings 1, E A Nelson 1, J K Nelson 1, R J Hines 1, S L Swenson 1, H T Hill 1, J J Zimmerman 1, J B Katz 1, M J Yaeger 1, C C Chase 1
PMCID: PMC228258  PMID: 7665637

Abstract

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.

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Selected References

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  1. Benfield D. A., Nelson E., Collins J. E., Harris L., Goyal S. M., Robison D., Christianson W. T., Morrison R. B., Gorcyca D., Chladek D. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J Vet Diagn Invest. 1992 Apr;4(2):127–133. doi: 10.1177/104063879200400202. [DOI] [PubMed] [Google Scholar]
  2. Chirnside E. D., Spaan W. J. Reverse transcription and cDNA amplification by the polymerase chain reaction of equine arteritis virus (EAV). J Virol Methods. 1990 Nov;30(2):133–140. doi: 10.1016/0166-0934(90)90014-7. [DOI] [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Collins J. E., Benfield D. A., Christianson W. T., Harris L., Hennings J. C., Shaw D. P., Goyal S. M., McCullough S., Morrison R. B., Joo H. S. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J Vet Diagn Invest. 1992 Apr;4(2):117–126. doi: 10.1177/104063879200400201. [DOI] [PubMed] [Google Scholar]
  5. Conzelmann K. K., Visser N., Van Woensel P., Thiel H. J. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology. 1993 Mar;193(1):329–339. doi: 10.1006/viro.1993.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Evenson D. P., Darzynkiewicz Z., Melamed M. R. Comparison of human and mouse sperm chromatin structure by flow cytometry. Chromosoma. 1980;78(2):225–238. doi: 10.1007/BF00328394. [DOI] [PubMed] [Google Scholar]
  7. Gerfen R. W., White B. R., Cotta M. A., Wheeler M. B. Comparison of the semen characteristics of Fengjing, Meishan and Yorkshire boars. Theriogenology. 1994 Feb;41(2):461–469. doi: 10.1016/0093-691x(94)90082-t. [DOI] [PubMed] [Google Scholar]
  8. Kim H. S., Kwang J., Yoon I. J., Joo H. S., Frey M. L. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol. 1993;133(3-4):477–483. doi: 10.1007/BF01313785. [DOI] [PubMed] [Google Scholar]
  9. Mermin J. H., Holodniy M., Katzenstein D. A., Merigan T. C. Detection of human immunodeficiency virus DNA and RNA in semen by the polymerase chain reaction. J Infect Dis. 1991 Oct;164(4):769–772. doi: 10.1093/infdis/164.4.769. [DOI] [PubMed] [Google Scholar]
  10. Meulenberg J. J., Hulst M. M., de Meijer E. J., Moonen P. L., den Besten A., de Kluyver E. P., Wensvoort G., Moormann R. J. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology. 1993 Jan;192(1):62–72. doi: 10.1006/viro.1993.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nelson E. A., Christopher-Hennings J., Drew T., Wensvoort G., Collins J. E., Benfield D. A. Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol. 1993 Dec;31(12):3184–3189. doi: 10.1128/jcm.31.12.3184-3189.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Porcine reproductive and respiratory syndrome (PRRS or blue-eared pig disease). Vet Rec. 1992 Feb 1;130(5):87–89. doi: 10.1136/vr.130.5.87. [DOI] [PubMed] [Google Scholar]
  13. Schultz R. D., Adams L. S., Letchworth G., Sheffy B. E., Manning T., Bean B. A method to test large numbers of bovine semen samples for viral contamination and results of a study using this method. Theriogenology. 1982 Feb;17(2):115–123. doi: 10.1016/0093-691x(82)90071-1. [DOI] [PubMed] [Google Scholar]
  14. Swenson S. L., Hill H. T., Zimmerman J. J., Evans L. E., Landgraf J. G., Wills R. W., Sanderson T. P., McGinley M. J., Brevik A. K., Ciszewski D. K. Excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars. J Am Vet Med Assoc. 1994 Jun 15;204(12):1943–1948. [PubMed] [Google Scholar]
  15. Van Woensel P., Van der Wouw J., Visser N. Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction. J Virol Methods. 1994 May;47(3):273–278. doi: 10.1016/0166-0934(94)90024-8. [DOI] [PubMed] [Google Scholar]
  16. Wiedmann M., Brandon R., Wagner P., Dubovi E. J., Batt C. A. Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay. J Virol Methods. 1993 Sep;44(1):129–139. doi: 10.1016/0166-0934(93)90015-j. [DOI] [PubMed] [Google Scholar]
  17. van Engelenburg F. A., Maes R. K., van Oirschot J. T., Rijsewijk F. A. Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen. J Clin Microbiol. 1993 Dec;31(12):3129–3135. doi: 10.1128/jcm.31.12.3129-3135.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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