台湾蝙蝠种群感染病毒监测的标准操作程序
Summary
该协议引入了标准实验室操作程序，用于在台湾蝙蝠中诊断病毒抗原。
Abstract
莱萨病毒属内的病毒是人畜共患的病原体，至少有七种莱沙病毒与人类病例有关。由于蝙蝠是大多数利沙病毒的天然宿主，自2008年以来，台湾一直在对蝙蝠进行感染病毒监测，以了解这些病毒在蝙蝠中的生态。在这个计划中，非政府的蝙蝠保护组织和当地动物疾病控制中心合作收集死于虚弱或疾病的蝙蝠。蝙蝠的脑组织通过尸检获得，并直接进行荧光抗体测试（FAT）和逆转录聚合酶链反应（RT-PCR），以检测利沙病毒抗原和核酸。对于 FAT，建议至少两种不同的狂犬病诊断联结。对于RT-PCR，使用两组引源（JW12/N165-146，N113F/N304R）来扩增裂源病毒核蛋白基因的部分序列。这个监测程序监测蝙蝠中的莱沙病毒和其他人畜人病。2016-2017年，台湾蝙蝠赖萨病毒在两例日本皮司特鲁斯（Pipistrellus abramus）病例中被发现。这些发现应该让公众、卫生专业人员和科学家了解接触蝙蝠和其他野生动物的潜在风险。
Introduction
莱萨病毒属内的病毒是人畜的病原体。至少有7种与人类病例1有关的莱沙病毒。除了该属的1、2、3、台湾蝙蝠利沙病毒（TWBLV）4和科塔拉赫蒂蝙蝠利沙病毒5最近已在蝙蝠身上鉴定的16个物种外，其分类状况尚未确定。
蝙蝠是大多数溶血病毒的天然宿主，除了莫科拉利萨病毒和伊科马利萨病毒，这些尚未在任何蝙蝠1，2，3，6中被识别。亚洲蝙蝠中有关感染病毒的信息仍然有限。据报道，亚洲蝙蝠（一种在印度，另一种在泰国）有2种未定性的莱萨病毒。2002年，中国报告发生了一起与蝙蝠咬伤有关的人类狂犬病病例，但诊断结果仅通过临床观察9。在中亚，1991年在吉尔吉斯斯坦较小的鼠耳蝙蝠（Myotisblythi）中发现了阿拉万利萨病毒，2001年在塔吉克斯坦的胡须蝙蝠（Myotismystacinus）中发现了胡扬德利萨病毒。在南亚，甘诺鲁瓦蝙蝠利萨病毒在2015年3月在斯里兰卡的印度飞狐（Pteropusmedius）中被鉴定。在东南亚，对菲律宾、泰国、孟加拉国、柬埔寨和越南蝙蝠的几项血清学研究表明，11、12、13、14、 15.虽然2012年中国吉林省大管鼻蝙蝠（Murinaleucogaster）中发现了伊尔库特流沙病毒，但东亚蝙蝠种群中流沙病毒的确切种类和位置仍不得而知。
为了评估台湾蝙蝠种群中是否存在感染病毒，启动了一项同时采用直接FAT和RT-PCR的监测计划。2016-2017年，在两例日本皮皮斯特勒（Pipistrellus abramus）4例中发现了台湾蝙蝠赖萨病毒。本文介绍了台湾蝙蝠种群的感染病毒监测实验室标准操作程序。图1显示了我们实验室中蝙蝠流沙病毒诊断的流程图。
Protocol
1. 处理莱沙病毒时的安全注意事项 确保所有处理蝙蝠标本的实验室工作人员接受接触前狂犬病预防17。事先监测工人的狂犬病抗体水平，每6个月重新检查一次。抗体水平低于0.5 IU/mL17的，需进行后续狂犬病疫苗接种。 根据实验室所在国家/地区的生物安全法规，确保在适当的生物安全级别实验室（例如台湾的 BSL-2 实验室和澳大?…
Representative Results
从2014年至2017年5月，收集了来自13个物种的332只蝙蝠尸体进行监测。两个检测呈阳性。在第一个蝙蝠案例中，使用FAT与一种商用FITC结合的抗狂犬病抗体（图2）进行脑印象测试呈阴性，而使用两个引物集（JW12/N165-146，N113F/N304R）的RT-PCRs则产生了正结果 （图 3）。获得428 bp序列的扩增素（用N113F/N304R扩增，并含有部分核蛋白基因）。其序列由GenBank数据库?…
Discussion
该实验室标准操作程序 （SOP） 提供了一个串行程序，用于测试蝙蝠样本是否存在在台湾的利沙病毒抗原。关键步骤包括使用 FAT 和 RT-PCR。选择合适的样本和成功分离病毒也很重要。此外，在监测蝙蝠感染病毒期间进行了一些故障排除。主要区别是目标动物。最初（2008-2009年），蝙蝠利沙病毒监测的目标动物是活蝙蝠，它们被困在台湾金门岛，在安乐死后进行利沙病毒检测。大多数被困蝙蝠都健?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢李天成、林益腾、蔡嘉荣和李亚兰在研究期间给予的协助。这项研究得到了台湾行政院动植物卫生检验检疫局第107AS-8.7.1-BQ-B2（1）号赠款的支持。
Materials
| 2.5% Trypsin (10x) | Gibco | 15090046 | Trppsin |
| 25 cm2 flask | Greiner bio-one | 690160 | |
| Acetone | Honeywell | 32201-1L | |
| Agarose I | VWR Life Science | 97062-250 | |
| Alcohol | NIHON SHIYAKU REAGENT | NS-32294 | |
| AMV Reverse Transcriptase | Promega | M5101 | |
| Antibiotic-Antimycotic(100X) | Gibco | 15240-062 | MEM-10 |
| Blade | Braun | BA215 | |
| Centrifuge | eppendorf | 5424R | |
| Chemilumineance system | TOP BIO CO. | MGIS-21-C2-1M | |
| Collection tube | Qiagen | 990381 | |
| Collection tube | SSI | 2341-SO | |
| Cover slide | Muto Pure chemical Co., LTD. | 24505 | |
| DNA analyzer | Applied Biosystems | 3700XL | |
| Fetal bovine serum | Gibco | 10437028 | MEM-10 |
| FITC Anti-Rabies Monoclonal Globulin | Fujirebio Diagnostic Inc. | 800-092 | FITC-conjugated anti-rabies antibodies: reagent B |
| Four-well Teflon-coating glass slide | Thermo Fisher Scientific | 30-86H-WHITE | |
| Gel Electrophoresis System | Major Science | MJ-105-R | |
| HBSS (1x) | Gibco | 14175095 | Trppsin |
| Incubator | ASTEC | SCA-165DS | |
| Inverted Microscope | Olympus | IX71 | |
| L-Glutamine 200 mM (100x) | Gibco | A2916801 | MEM-10 |
| LIGHT DIAGNOSTICS Rabies FAT reagent | EMD Millipore Corporation | 5100 | FITC-conjugated anti-rabies antibodies: reagent A |
| MagNA Pure Compact Instrument | Roche | 03731146001 | |
| MagNA Pure Compact NA Isolation Kit 1 | Roche | 03730964001 | |
| MEM (10x) | Gibco | 11430030 | MEM-10 |
| MEM NEAA (100x) | Gibco | 11140050 | MEM-10 |
| MEM vitamin solution | Gibco | 11120052 | MEM-10 |
| NaHCO3 | Merck | 1.06329.0500 | MEM-10 |
| Needle | Terumo | NN*2332R9 | |
| PBS | Medicago | 09-8912-100 | |
| Primer synthesis | Mission Biotech | ||
| RNasin ribonuclease inhibitor | Promega | N2111 | |
| Sequencing service | Mission Biotech | ||
| Slide | Thermo Scientific | AA00008032E00MNT10 | |
| Sodium Pyruvate (100 mM) | Gibco | 11360070 | MEM-10 |
| Stainless Steel Beads | QIAGEN | 69989 | |
| Sterile absorbent pad | 3M | 1604T-2 | |
| Syringe filter | Nalgene | 171-0045 | |
| Taq polymerase | JMR Holdings | JMR-801 | |
| Thermal cycler | Applied Biosystems | 2720 | |
| TissueLyser II | QIAGEN | 85300 | |
| Tongue depressor | HONJER CO., LTD. | 122246 | |
| Tweezer | Tennyson medical Instrument developing CO., LTD. | A0601 | |
| Tylosin Tartrate | Sigma | T6271-10G | MEM-10 |
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Citazione di questo articolo
Hsu, W., Hsu, C., Tu, Y., Chang, J., Tsai, K., Lee, F., Hu, S. Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan. J. Vis. Exp. (150), e59421, doi:10.3791/59421 (2019).