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

描述（由申请人提供）：摘要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）相关的γ逆转录病毒。此外，其他与多变性MLV更密切相关的鼠白血病病毒（MLV）样序列也被报道与CFS相关。由于这些病毒可以在白细胞和血浆中检测到，并且在XMRV直接从白细胞和血浆培养的情况下，它们很可能通过血液或血液制品传播。因此，迫切需要通过核酸检测（NAT）敏感、特异和可靠地检测XMRV的存在，以进一步研究这种对血液安全的潜在严重威胁。最初需要进行检测，以充分表征这些病毒的致病潜力，以及供体人群中的真实流行率和输血传播（TT）的可能性。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，2至4天的延迟，样品在处理前保持在4 ℃，允许多个实验室更可靠地检测血浆组分中的XMRV。我们假设，定期处理的血浆不是检测XMRV和其他MLV相关病毒的最佳培养基。因此，在目标1中，我们将扩展并进一步研究这些发现。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。处理延迟后变为阳性的血浆将在超离心前后用核酸酶、蛋白酶和去污剂处理，以确定病毒核酸是否与病毒体相关或以游离RNA/DNA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，但对于献血者的高通量筛查来说不太可能实用，因为理想情况下需要在静脉切开后24小时内获得筛查结果。因此，我们将研究多种技术，以实现与延迟处理相似的结果，从那些在血液采集环境中最容易和廉价实现的技术开始。 公共卫生相关性：异嗜性鼠白血病病毒相关病毒（XMRV）已被认为与前列腺癌和慢性疲劳综合征有关，而且已被证明存在于血细胞和血浆中。因此，存在通过血液或血液制品传播XMRV的真实的可能性，因此迫切需要灵敏且特异的检测测定以保护血液供应。我们计划确定最适合XMRV检测的血液成分，并将开发处理方法，以最大限度地提高XMRV的存在，从而在这些成分中进行检测。

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. PUBLIC HEALTH RELEVANCE: Xenotropic Murine Leukemia virus-Related Virus (XMRV) has been suggested to be involved in both prostate cancer and chronic fatigue syndrome, and furthermore has been demonstrated to be present in blood cells and plasma. Thus, there is real potential for XMRV transmission through blood or blood products and hence an urgent need for sensitive and specific detection assays in order to protect the blood supply. We plan to identify the most appropriate blood component for XMRV detection and will develop processing methods in order to maximize XMRV presence and hence detection within these components.