Real-time quantitative polymerase chain reaction with SYBR green i detection for estimating copy numbers of porcine endogenous retrovirus from Chinese miniature pigs.
ABSTRACT 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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ABSTRACT: Porcine endogenous retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.Journal of Virology 05/2006; 80(7):3135-46. · 5.08 Impact Factor
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ABSTRACT: Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV-pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT-PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.Journal of Virological Methods 11/2002; 106(1):97-106. · 1.90 Impact Factor
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ABSTRACT: Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.Journal of Virology 06/1998; 72(5):4503-7. · 5.08 Impact Factor