核黄素光化学技术对低载量病毒的灭活效果及其机制研究

Prevention and control of blood-borne pathogens have always been the focus in the field of blood safety. Although the nucleic acid test (NAT) has greatly reduced the risk of transmitting HIV, HBV and HCV infection via blood transfusion, the risk of transmission could not be eliminated completely due to the window period. Riboflavin photochemical method is expected to become an effective means to eliminate the risk of transfusion transmission completely. However, the effectiveness of riboflavin photochemical method in inactivating the low-viral-load virus during the window period in the blood/blood component has not been reported yet. The mechanisms of the riboflavin photochemical method are not very clear. In this study, plasma samples tested ELISA negative/NAT positive for HIV, HBV, HCV were used to assess the effect of riboflavin photochemical method in inactivating low-viral-load viruses in plasma. Besides, real-time fluorescent quantitative PCR, large fragments step-by-step, second-generation sequencing and nucleic acid mass spectrometry will be used to study nucleic acid degradation rules and fracture/mutation sites of the different types of viruses (pseudorabies virus, Sindbis virus, encephalomyocarditis virus, porcine parvovirus) after the riboflavin photochemical treatment. Finally, the potential hot spots will be identified and mechanisms for inactivating viruses will be explored. These studies will provide experimental evidence for the promotion and application of riboflavin photochemical technology in clinical practice.

经血传播病原体的预防和控制一直是血液安全领域关注的重点和热点。血液筛查核酸检测（NAT）极大降低了经血传播HIV、HBV、HCV的风险，但由于病毒“窗口期”导致的传播风险仍不能完全避免。核黄素光化学病原体灭活技术有望成为彻底解决病毒“窗口期”问题的有效手段，但其对血液/血液成分中处于检测“窗口期”的低载量水平病毒的灭活效果未见报道，灭活机制亦非十分清楚。本课题拟以HIV、HBV、HCV筛查ELISA阴性而NAT阳性的血浆样本为研究对象，评估核黄素光化学技术对血浆中低载量病毒的灭活效果；并通过实时荧光定量PCR、大片段步移法、二代测序及核酸质谱等技术，研究核黄素光化学技术对不同类型病毒（伪狂犬病毒、Sindbis病毒、脑心肌炎病毒、猪细小病毒）的核酸降解规律及断裂/突变位点，分析其靶向病毒核酸序列的潜在“热点”和规律，探索其灭活病毒的机制。为核黄素光化学技术在临床的推广和应用提供实验依据。

经血传播病原体防控是血液安全领域关注的重点和热点。血液筛查核酸检测极大降低了经血传播HIV、HBV、HCV的风险，但由于病毒“窗口期”导致的传播风险仍不能完全避免。核黄素光化学病原体灭活技术有望成为彻底解决病毒“窗口期”问题的有效手段，但其对血液/血液成分中处于检测“窗口期”的低载量水平病毒的灭活效果未见报道，灭活机制亦非十分清楚。本项目中，对核黄素光化学技术关键参数进行了系统性优化，确定了核黄素光化学技术应用于血液成分病原体灭活的最佳条件；完成低载量病毒血浆样品的收集与检测，通过实时荧光定量PCR和巢式PCR等技术评估了核黄素光化学技术对血浆中低载量病毒的灭活效果，并完成了更适用于病毒灭活效果评估的HBV、HCV、HIV病毒大片段逐步步移法的建立与评价；基于毛细管电泳和激光诱导荧光技术建立了聚合酶终止/线性扩增检测技术，在此基础上完成了核黄素光化学技术光敏损伤DNA序列特异性研究，研究发现核黄素光化学技术损伤核酸具有一定序列特异性，并且损伤位点的邻近核苷酸对其损伤程度亦有一定影响。此外，开展了核黄素光化学技术用于血浆α2-巨球蛋白制品的病毒灭活研究，并对核黄素光化学技术损伤蛋白活性的机制进行了初步探索。本项目研究为核黄素光化学技术的推广应用提供了数据支持，为深入研究核黄素光化学技术的病毒灭活机制奠定了基础，同时对于保障我国输血安全具有重要意义。本项目培养博士后1名，发表SCI论文1篇，接收国内期刊论文1篇，待发表英文论文3篇。

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