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: To effectively supress PERVs, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multi-targets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 hours after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multi-targeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression vector, which allowed for persistent and multiple targeting. This was the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics-resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and FISH assay and it demonstrated the highest knockdown efficiency in multi-targeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multi-targeting strategy could effectively inhibit PERVs. This article is protected by copyright. All rights reserved.Transplant International 10/2013; · 3.16 Impact Factor
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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.Virology Journal 07/2013; 10(1):228. · 2.09 Impact Factor
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ABSTRACT: In the context of the shortage of organs and other tissues for use in human transplantation, xenotransplantation procedures with material taken from pigs have come under increased consideration. However, there are unclear consequences of the potential transmission of porcine pathogens to humans. Of particular concern are porcine endogenous retroviruses (PERVs). Three subtypes of PERV have been identified, of which PERV-A and PERV-B have the ability to infect human cells in vitro. The PERV-C subtype does not show this ability but recombinant PERV-A/C forms have demonstrated infectivity in human cells. In view of the risk presented by these observations, the International Xenotransplantation Association recently indicated the existence of four strategies to prevent transmission of PERVs. This article focuses on the molecular aspects of PERV infection in xenotransplantation and reviews the techniques available for the detection of PERV DNA, RNA, reverse transcriptase activity and proteins, and anti-PERV antibodies to enable carrying out these recommendations. These methods could be used to evaluate the risk of PERV transmission in human recipients, enhance the effectiveness and reliability of monitoring procedures, and stimulate discussion on the development of improved, more sensitive methods for the detection of PERVs in the future.Viruses 01/2014; 6(5):2062-83. · 2.51 Impact Factor