Optimization of XMRV and other MLV-related virus detection for screening of blood

描述(申请人提供)：异嗜性小鼠白血病病毒相关病毒(XMRV)是一种新发现的与前列腺癌和慢性疲劳综合征(CFS)有关的伽玛逆转录病毒。此外，与多嗜性MLV关系更密切的其他小鼠白血病病毒(MLV)样序列也被报道与CFS有关。由于这些病毒既可以在白细胞和血浆中检测到，也可以从白细胞和血浆中直接培养的XMRV中检测到，它们极有可能通过血液或血液制品传播。因此，迫切需要灵敏、特异和可靠的检测XMRV的存在，最好是通过核酸测试(NAT)，以进一步调查这一对血液安全的潜在严重威胁。最初将需要进行检测，以充分确定这些病毒的致病潜力，以及捐赠者群体中的真实流行率和输血传播(TT)的可能性。然后，这些结果将决定是否需要对血液供应进行大规模筛查。具体目标1：检测XMRV和其他MLV相关病毒。使用经过验证的临床样本的初步结果表明，XMRV的NAT检测充其量是可变的。然而，由于样品在处理前保持在4摄氏度，延迟2至4天，允许多个实验室更可靠地检测血浆部分中的XMRV。我们假设，常规处理的血浆不是检测XMRV和其他MLV相关病毒的最佳介质。因此，在目标1中，我们将扩展和进一步调查这些发现。我们将对多个XMRV/MLV阳性个体的处理方法进行详细和广泛的比较。在处理延迟一段时间后转为阳性的血浆将在超速离心前后用核酸酶、蛋白酶和洗涤剂处理，以确定病毒核酸是与病毒粒子相关还是以游离RNA/DNA的形式存在。具体目标2：加强核酸检测的机制。尽管血浆延迟处理的优化将极大地增强临床和基础研究的潜力，但它不太可能用于高通量献血者筛查，因为理想情况下，筛查结果需要在静脉采血后24小时内完成。因此，我们将研究多种技术，以实现与延迟处理类似的结果，首先从那些在血液采集环境中最容易和最便宜地实现的技术开始。

DESCRIPTION (provided by applicant): Abstract Xenotropic murine leukemia virus-related virus (XMRV) is a newly identified gammaretrovirus linked to prostate cancer and chronic fatigue syndrome (CFS). Furthermore, other murine leukemia virus (MLV)-like sequences more closely related to polytropic MLV have also been reported to be associated with CFS. Since these viruses can be both detected in, and in the case of XMRV directly cultured from, leukocytes and plasma, they are highly likely to be transmissible by blood or blood products. Thus, sensitive, specific and reliable detection of the presence of XMRV, ideally by nucleic acid testing (NAT), is urgently required to further investigate this potentially serious threat to blood safety. Testing will be required initially to fully characterize the pathogenic potential of these viruses, together with the true prevalence in donor populations and the likelihood of transfusion-transmission (TT). These results will then determine whether large scale screening of the blood supply is warranted. Specific Aim 1: Detection of XMRV and other MLV-related viruses. Preliminary results, using validated clinical samples, have demonstrated that NAT detection of XMRV was, at best, variable. However, a 2 to 4 day delay, with samples held at 4oC prior to processing, allowed a more reliable detection of XMRV in the plasma fraction, by multiple laboratories. We hypothesize that regularly processed plasma is not the optimal medium for the detection of XMRV and other MLV-related viruses. Thus in aim 1 we will extend and further investigate these findings. We will perform detailed and extensive comparison of processing methods from multiple XMRV/MLV positive individuals. Plasma that becomes positive after a delay in processing will be treated with nucleases, proteases and detergents before and after ultracentrifugation, to determine whether viral nucleic acid is virion associated or present as free RNA/DNA. Specific Aim 2: Mechanisms to enhance nucleic acid testing. Although the optimization of the delayed processing of plasma will greatly enhance the potential of clinical and basic research studies, it is not likely to be practical for high-throughput screening of blood donors, where screening results are ideally required less than 24 hours after phlebotomy. Thus, we will investigate a multitude of techniques in order to achieve similar results to delayed processing, starting with those most likely to be easily and cheaply achieved in the blood collection setting.