Yang Yu, Garver Lindsey S, Bingham Karen M, Hang Jun, Jochim Ryan C, Davidson Silas A, Richardson Jason H, Jarman Richard G
Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland.
Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland
Am J Trop Med Hyg. 2015 Dec;93(6):1377-82. doi: 10.4269/ajtmh.15-0440. Epub 2015 Sep 28.
Mosquito blood meals taken from humans and animals potentially represent a useful source of blood for the detection of blood-borne pathogens. In this feasibility study, Anopheles stephensi mosquitoes were fed with blood meals spiked with dengue virus type 2 (DENV-2) and harvested at serial time points. These mosquitoes are not competent vectors, and the virus is not expected to replicate. Ingested blood was spotted on Whatman FTA cards and stored at room temperature. Mosquito abdomens were removed and stored at -80°C. Control blood meal aliquots were stored in vials or applied onto FTA cards. After 4 weeks of storage, the samples were extracted using beadbeating and QIAamp Viral RNA kit (Qiagen Sciences, Germantown, MD). Recovered viral RNA was analyzed by DENV-2 TaqMan RT-PCR assay and next-generation sequencing (NGS). Overall viral RNA recovery efficiency was 15% from the directly applied dried blood spots and approximately 20% or higher for dried blood spots made by blotting mosquito midgut on FTA cards. Viral RNA in mosquito-ingested blood decreases over time, but remains detectable 24 hours after blood feeding. The viral sequences in FTA-stored specimens can be maintained at room temperature. The strategy has the potential utility in expedited zoonotic virus discovery and blood-borne pathogen surveillance.
从人类和动物身上获取的蚊子血餐可能是检测血源性病原体的有用血液来源。在这项可行性研究中，用含有2型登革病毒（DENV-2）的血餐喂养斯氏按蚊，并在连续时间点采集样本。这些蚊子不是有效的传播媒介，预计病毒不会复制。将摄入的血液点样在Whatman FTA卡上并在室温下保存。去除蚊子腹部并保存在-80°C。对照血餐等分试样保存在小瓶中或应用于FTA卡上。储存4周后，使用珠磨法和QIAamp病毒RNA试剂盒（Qiagen Sciences，德国小镇，MD）提取样本。通过DENV-2 TaqMan RT-PCR检测和下一代测序（NGS）分析回收的病毒RNA。直接应用的干血斑的总体病毒RNA回收效率为15%，通过将蚊子中肠印迹在FTA卡上制成的干血斑的回收效率约为20%或更高。蚊子摄入血液中的病毒RNA会随着时间减少，但在吸血后24小时仍可检测到。FTA储存样本中的病毒序列可在室温下保存。该策略在加速人畜共患病毒发现和血源性病原体监测方面具有潜在用途。
