Switzer W M, Shanmugam V, Chapman L, Heneine W
Division of AIDS, Sexually Transmitted Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, USA.
Transplantation. 1999 Jul 27;68(2):183-8. doi: 10.1097/00007890-199907270-00003.
Pigs offer an unlimited source of xenografts for humans. However, recipients of pig xenografts are inevitably exposed to the porcine endogenous retrovirus (PERV), which is carried in the pig germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients.
In response to the need to establish laboratory tests for the surveillance of PERV infection, we have developed polymerase chain reaction (PCR) assays to detect PERV pol and gag sequences by using conserved primers and probes. In addition, we have developed a PCR assay to detect pig-specific mitochondrial DNA (mtDNA) sequences as a marker of pig cells.
Analysis of assay sensitivities using cloned target copies in a background of human DNA demonstrated a detection threshold of 1, 5, and 1 copy for the PERV gag, pol, and pig mtDNA PCR assays, respectively. All three PCR assays gave negative results on peripheral blood lymphocyte samples from 69 humans, as well as 6 baboons and 6 macaques, demonstrating 100% specificity. The PERV and pig mtDNA assays were integrated into a simple testing algorithm that allows the differentiation between pig cell microchimerism and true xenogeneic infection. To allow for monitoring of PERV expression, a reverse transcriptase-PCR assay was also developed to detect cell-free PERV RNA.
The use of the diagnostic tests described here will help define the risks of PERV transmission associated with the use of pig xenografts in humans and nonhuman primates.
猪为人类提供了无限的异种移植来源。然而，猪异种移植的接受者不可避免地会接触到猪种系中携带的猪内源性逆转录病毒（PERV）。PERV在体外感染人类细胞的能力加剧了人们对PERV传播给猪异种移植接受者的安全担忧。
为了响应建立用于监测PERV感染的实验室检测方法的需求，我们开发了聚合酶链反应（PCR）检测方法，使用保守引物和探针来检测PERV pol和gag序列。此外，我们还开发了一种PCR检测方法来检测猪特异性线粒体DNA（mtDNA）序列，作为猪细胞的标志物。
在人类DNA背景下使用克隆的目标拷贝分析检测灵敏度表明，PERV gag、pol和猪mtDNA PCR检测的检测阈值分别为1、5和1拷贝。这三种PCR检测方法对69名人类以及6只狒狒和6只猕猴的外周血淋巴细胞样本均给出阴性结果，显示出100%的特异性。PERV和猪mtDNA检测方法被整合到一个简单的检测算法中，该算法能够区分猪细胞微嵌合体和真正的异种感染。为了监测PERV的表达，还开发了一种逆转录酶-PCR检测方法来检测无细胞PERV RNA。
使用本文所述的诊断测试将有助于确定与在人类和非人灵长类动物中使用猪异种移植相关的PERV传播风险。
