Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR

Abstract

A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/g of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo-and xenotransplantation.Porcine cytomegalovirus (PCMV) is a betaherpesvirus which causes generalized infection in newborn piglets. It has a worldwide distribution and is present in 90% of herds in the United Kingdom, many of which are from well-managed, high-healthstatus farms (3). Epidemiological features of the virus include evidence of latency and crossplacental infection. Xenotransplantation has the potential to relieve the current donor organ shortage associated with human allotransplantation, and for both practical and ethical reasons, the pig has become the most likely source of such tissues. As clinical trials of xenotransplantation are beginning, concerns have been raised about the potential transmission of porcine organisms to immunocompromised human recipients of porcine tissues (1, 4, 5). As with allotransplantation, there are potential risks associated with the transmission of porcine viruses through xenograft tissues. Although transmission to humans has not been demonstrated in vivo, porcine endogenous retroviruses have been shown to replicate in human cells in vitro (11,15). Human cytomegalovirus (HCMV) has a variety of direct and indirect effects in the allograft recipient (6). Given the high frequency of seropositivity of swine for PCMV and the efficiency of transmission of HCMV from donor to transplant recipient (6), PCMV may represent another potential risk to humans.At present the laboratory detection of PCMV is carried out by serology or qualitative PCR (8,13,14). We now describe the development of a quantitative-competitive PCR (QC-PCR) assay and its application to the measurement of viral load in porcine tissues. The QC-PCR is based on the coamplification of the test sample with an internal competitor, which differs from the wild-type sequence by the presence of a restriction endonuclease site in the middle of the amplicon. To our knowledge this is the first quantitative PCR assay for the quantification of PCMV to be reported.The qualitative and QC-PCR assays amplify a region of the polymerase gene (14). Primers PCMVF1 (5Ј CCTATGTTGG CACTGATACTTGAC 3Ј) and PCMVR1 (5Ј CCCTGAAAA TCACCGTCTGAGAGA 3Ј) were initially used to amplify a 236-bp region of the gene from tissue culture supernatant of porcine alveolar macrophages infected with PCMV strain B6. The amplicon was then cloned into the pGEM-T Easy vector (Promega). PCR-mediated site-directed mutagenesis, similar to the method described by Fox et al. (7), was used to create a SmaI restriction endonuclease site in the middle of the PCMV amplicon sequence by altering two nucleotides. Two complementary mutagenic primers were used in two separate PCRs; primers PCMVF1...

Cardiac Xenotransplantation: Progress Toward the Clinic

“…The primers and conditions for semi-quantitative PCR for BCMV and porcine CMV (PCMV) were used as previously described (26,27). Gamma herpesvirus evaluation included primers against baboon Herpes papio (28) and pig lymphotropic viruses (PLV) 1 and 2 as described by Ehlers and colleagues (29).…”

Porcine Endogenous Retroviruses PERV-A and PERV-B Infect neither Mouse Cells in vitro nor SCID Mice in vivo

“…However, the use of pig cells, tissues and organs still holds the risk of transmission of unknown pathogens. Porcine cytomegalo-Irgang/Karlas/Laue/Specke/Tacke/Kurth/ Schrezenmeir/Denner virus [1,2], porcine lymphotropic herpes-virus type 1 and 2 [3,4], porcine torovirus [5], porcine hepatitis E virus [6][7][8], Nipah virus [9], swine influenza virus [10], porcine encephalomyocarditis virus [11], porcine rotavirus [12] and porcine circovirus type 1 and 2 [13,14] are recently identified porcine viruses with possible human pathogenic properties, all of which can be eliminated by breeding under specified pathogen-free conditions [15]. Porcine endogenous retroviruses (PERVs), however, cannot be eliminated by specified pathogen-free breeding since they are integrated into the genome of all pigs [16].…”

Monitoring for Presence of Potentially Xenotic Viruses in Recipients of Pig Islet Xenotransplantation

“…Recipient monitoring has included testing peripheral blood mononuclear cells (PBMC) and blood plasma for PCV 2 (19), PLHV (7), PCMV (2,11,13), and PERV by PCR or reverse transcription-PCR (25). This testing has been carried out on each recipient before transplantation, at nine intervals during the first year posttransplantation, and once each subsequent year.…”