Real-Time Quantitative Polymerase Chain Reaction With SYBR Green I Detection for Estimating Copy Numbers of Porcine Endogenous Retrovirus From Chinese Miniature Pigs

Laboratory for Viral Safety of National Centre of Biomedical Analysis, Institute of Transfusion Medicine, the Academy of Military Medical Sciences, Beijing, China.
Transplantation Proceedings (Impact Factor: 0.98). 06/2010; 42(5):1949-52. DOI: 10.1016/j.transproceed.2010.01.054
Source: PubMed


Porcine endogenous retrovirus (PERV) in the pig genome represents a potential infectious risk in xenotransplantation. Chinese miniature pigs have been considered to be potential organ donors in China. However, an adequate level of information on PERV from Chinese miniature pigs has not been available. We established an SYBR Green I-based real-time quantitative polymerase chain reaction (PCR) assay for estimating copy numbers of PERV integrated in the host genome. The assay was 100-fold more sensitive compared with conventional PCR. We also evaluated the specificity and reproducibility of the assay. We statistically analyzed the difference in PERV copy numbers integrated into the genomes of Wuzhishan pigs versus Bama minipigs. This approach will be useful to screen donor pigs as well as to examine clinical samples from human subjects treated with porcine xenotransplantation products for evidence of PERV transmission.

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    • "In view of their clear genetic background, strong disease resistance and tiny interindividual differences, Chinese miniature pigs have been developed as the potential organ donors for xenotransplantation in China [16]. Previously, we performed a large-scale survey on the presence and expression status of PERV in seven breeds of Chinese miniature pigs [17] and the result showed that PERV present in all 348 genomic DNA samples, and no expression of PERV-C was found in Wuzhishan pigs (WZSPs), which were further confirmed to harbor lower PERV copy numbers compared with other breeds [18] and considered to be more suitable for xenotransplantation. Thus it is essential to study the molecular characteristics of PERV from WZSPs. "
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    ABSTRACT: Xenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs. The proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptalphaSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis. The ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F. Altogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced.
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    ABSTRACT: Xenotransplantation may be associated with the transmission of pig microorganisms including viruses, bacteria, fungi, and parasites. As the recipient may be immunosuppressed, infection and pathologic consequences may be more pronounced compared to non-immunosuppressed individuals. Transmission of most microorganisms with exception of porcine endogenous retroviruses (PERV) may be prevented by screening the donor pig and qualified pathogen-free breeding. PERVs represent a special risk as they are present in the genome of all pigs and infect human cells in vitro. Until now, no PERV transmission was observed in experimental and clinical xenotransplantations as well as in numerous infection experiments. Nevertheless, several strategies have been developed to prevent PERV transmission.
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    ABSTRACT: Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.
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