Abstract
Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.
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References
Gu W, Zeng N, Zhou L, Ge X, Yang H (2014) Genomic organization and molecular characterization of porcine cytomegalovirus. Virology 460:165–1722
Done JT (1955) An “inclusion-body” rhinitis of pig (preliminary report). Vet Rec 67:525–527
Edington N (1986) Porcine cytomegalovirus. Dis Swine 138:330–336
Edington N, Broad S, Wrathall AE, Done JT (1988) Superinfection with porcine cytomegalovirus initiate infection. Vet Microbiol 16:189–1935
Hamel AL, Lin L, Sachvie C, Gredeski E, Nayar GPS (1999) PCR assay for detecting cytomegalovirus. J Clin Microbiol 37:3767–3768
Goltz M, Widen F, Banks M, Belak S, Ehlers B (2000) Characterization of the DNA polymerase loci of porcine cytomegalovirus from diverse geographical origins. Virus Genes 21:249–255
Clark DA, Fryer JFL, Tucker AW, McArdl PD, Hughes AE, Emery VC, Griffiths PD (2003) Porcine cytomegalovirus in pigs being bred for xenotransplant organs: progress towards control. Xenotransplantation 10:142–148
Liu X, Liao S, Zhu L, Xu Z, Zhou Y (2013) Molecular epidemiology of porcine cytomegalovirus (PCMV) in Sichuan Province, China: 2010–2012. PLoS One 8:e64648
Mueller NJ, Barth RN, Yamamoto S, Kitamura H, Patience C et al (2002) Activation of cytomegalovirus in pig-to-primate organ xenotransplantation. J Virol 76:4734–4764
Guedes MIMC, Risdahl JM, Wiseman B, Molitor TW (2004) Reactivation of porcine cytomegalovirus through allogenic stimulation. J Clin Microbiol 42:1756–1758
Neumann EJ, Ramirez A, Schwartz KJ (2010) Swine disease manuals, 4edn. Iowa State University, AASV, pp 53–54
Ramanan P, Razonable RR (2013) Cytomegalovirus infection in solid organ transplantation: a review. Infect Chemother 45:260–271
Yamada K, Tasaki M, Sekijima M, Wilkinson RA, Villani V et al (2014) Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model. Transplantation 98:411–417
Sekijima M, Waki S, Sahara H, Tasaki M, Wilkinson RA et al (2014) Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout swine. Transplantation 98:419–426
Fishman JA, Scobie L, Takeuch Y (2012) Xenotransplantation-associated infectious risk: a WHO consultation. Xenotransplantation 19:72–81
Denner J (2015) Xenotransplantation and porcine cytomegalovirus. Xenotransplantation. doi:10.1111/xen.12180
Widen BF, Lowings JP, Belak S, Banks M (1999) Development of a PCR system for porcine cytomegalovirus detection and determination of the putative partial sequence of its DNA polymerase gene. Epidemiol Infect 123:177–180
Fryer JFL, Griffiths PD, Fishman JA, Emery VC, Clark DA (2001) Quantitation of porcine cytomegalovirus in pig tissues by PCR. J Clin Microbiol 39:1155–1156
Whitteker JL, Dudani AK, Tackaberry ES (2008) Human fibroblasts are permissive for porcine cytomegalovirus in vitro. Transplantation 86:155–162
Fryer JFL, Griffiths PD, Emery VC, Clark DA (2004) Susceptibility of porcine cytomegalovirus to antiviral drugs. J Antimicrobial Chemother 53:975–980
Abrahante JE, Martins K, Papas KK, Hering BJ, Schuurman H-J, Murtaugh MP (2011) Microbiological safety of porcine islets: comparison with source pig. Xenotransplantation 18:88–93
Lefever S, Pattyn F, Hellemans J, Vandesompele J (2013) Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays. Clin Chem 59(10):1470–1480
Ohlinger V (1989) Porcine cytomegalovirus (PCMV). In: Wittmann G (ed) Herpesvirus disease of cattle, horses, and pigs. Developments in veterinary virology. Kluwer Academic Publishers, Norwell, pp 326–333
Hidenfelder B, Topal MD (2003) Effects of sequence on repeat expansion during DNA replication. NAR 31:7159–7164
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This project was financed in the frame of the Transregio Collaborative Research Centre 127 of the German Research Society (DFG).
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Morozov, V.A., Morozov, A.V. & Denner, J. New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV). Arch Virol 161, 1159–1168 (2016). https://doi.org/10.1007/s00705-016-2765-3
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DOI: https://doi.org/10.1007/s00705-016-2765-3