Name: standard operating procedure for lyssavirus surveillance of the bat population in taiwan
Uploaded: 2019-08-27T13:30:00
Description: Watch this Scientific Journal Video about Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan at JoVE.com

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。

1. 处理莱沙病毒时的安全注意事项
<ol><li>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。</li>
	<li>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?...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+real-time+assay+for+the+detection+of+Lyssaviruses.">A universal real-time assay for the detection of Lyssaviruses.</a> Journal of Virological Methods. 177 (1), 87-93 (2011).</li><li>Franka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+phylogenetic+lineage+of+rabies+virus+associated+with+western+pipistrelle+bats+(Pipistrellus+hesperus).">A new phylogenetic lineage of rabies virus associated with western pipistrelle bats (Pipistrellus hesperus).</a> Journal of General Virology. 87 (8), 2309-2321 (2006).</li><li>Trimarchi, C. V., Smith, J. S., Press, A., Jackson, A. C., Wunner, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+evaluation.">Diagnostic evaluation.</a> Rabies, 1st ed. , 307-349 (2002).</li><li>Moldal, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+detection+of+European+bat+lyssavirus+type+2+(EBLV-2)+in+Norway.">First detection of European bat lyssavirus type 2 (EBLV-2) in Norway.</a> BMC Veterinary Research. 13, 216 (2017).</li><li>Robardet, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assay+of+fluorescent+antibody+test+results+among+twelve+European+National+Reference+Laboratories+using+various+anti-rabies+conjugates.">Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.</a> Journal of Virological Methods. 191 (1), 88-94 (2013).</li><li>Hanlon, C. A., Nadin-Davis, S. A., Jackson, A. 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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?...

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。

<table>
	<tbody>
		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td>Trppsin</td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Greiner bio-one</td>
			<td>690160</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Honeywell</td>
			<td>32201-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose I</td>
			<td>VWR Life Science</td>
			<td>97062-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>NIHON SHIYAKU REAGENT</td>
			<td>NS-32294</td>
			<td></td>
		</tr>
		<tr>
			<td>AMV Reverse Transcriptase</td>
			<td>Promega</td>
			<td>M5101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic(100X)&#160;</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Braun</td>
			<td>BA215</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemilumineance system</td>
			<td>TOP BIO CO.</td>
			<td>MGIS-21-C2-1M</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube</td>
			<td>Qiagen</td>
			<td>990381</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube</td>
			<td>SSI</td>
			<td>2341-SO</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Muto Pure chemical Co., LTD.</td>
			<td>24505</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA analyzer</td>
			<td>Applied Biosystems</td>
			<td>3700XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>FITC Anti-Rabies Monoclonal Globulin</td>
			<td>Fujirebio Diagnostic Inc.</td>
			<td>800-092</td>
			<td>FITC-conjugated anti-rabies antibodies: reagent B</td>
		</tr>
		<tr>
			<td>Four-well Teflon-coating glass slide</td>
			<td>Thermo Fisher Scientific</td>
			<td>30-86H-WHITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Electrophoresis System</td>
			<td>Major Science</td>
			<td>MJ-105-R</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (1x)</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>Trppsin</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ASTEC</td>
			<td>SCA-165DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>LIGHT DIAGNOSTICS Rabies FAT reagent</td>
			<td>EMD Millipore Corporation</td>
			<td>5100</td>
			<td>FITC-conjugated anti-rabies antibodies: reagent A</td>
		</tr>
		<tr>
			<td>MagNA Pure Compact Instrument</td>
			<td>Roche</td>
			<td>03731146001</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA Pure Compact NA Isolation Kit 1</td>
			<td>Roche</td>
			<td>03730964001</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (10x)</td>
			<td>Gibco</td>
			<td>11430030</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>MEM NEAA (100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>MEM vitamin solution</td>
			<td>Gibco</td>
			<td>11120052</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Merck</td>
			<td>1.06329.0500</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>NN*2332R9</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Medicago</td>
			<td>09-8912-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer synthesis</td>
			<td>Mission Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin ribonuclease inhibitor</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing service</td>
			<td>Mission Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Thermo Scientific</td>
			<td>AA00008032E00MNT10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads</td>
			<td>QIAGEN</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile absorbent pad</td>
			<td>3M</td>
			<td>1604T-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Nalgene</td>
			<td>171-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>JMR Holdings</td>
			<td>JMR-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>2720</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tongue depressor</td>
			<td>HONJER CO., LTD.</td>
			<td>122246</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Tennyson medical Instrument developing CO., LTD.</td>
			<td>A0601</td>
			<td></td>
		</tr>
		<tr>
			<td>Tylosin Tartrate</td>
			<td>Sigma</td>
			<td>T6271-10G</td>
			<td>MEM-10</td>
		</tr>
	</tbody>
</table>

standard operating procedure for lyssavirus surveillance of the bat population in taiwan

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。

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?...

Watch this Scientific Journal Video about Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan at JoVE.com

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

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。

台湾蝙蝠种群感染病毒监测的标准操作程序

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

莱萨病毒是一种动物园无病原体。为了评估台湾蝙蝠种群中是否存在莱萨病毒，我们启动了一个监测计划。2016年到2018年，它成功鉴定了四种莱莎病毒。该协议是台湾病毒监测的分步介绍。我们希望对研究对病毒病毒监测感兴趣的人有用。连接正确的 tac-keg 符号非常重要。死亡或垂死的蝙蝠比活蝙蝠更适合对莱萨病毒的监测。根据实验室所在地国家的生物安全法规;在合适的生物安全水平实验室中执行以下程序。并确保研究人员目前合格并穿戴适当的个人防护装备。在收集弱蝙蝠或病蝙蝠或尸体后，让蝙蝠生态学家根据标本的形态特征识别蝙蝠物种。向实验室提交信息表，包括每个蝙蝠标本的采集地点、物种、临床体征和其他相关数据。在开始坏死镜检查之前，检查所有外部孔并拍摄蝙蝠的外部特征，特别是头部、耳朵和翅膀，以进行物种分化。收集口服拭子样本，将蝙蝠放在腹间，放在无菌解剖板上，用钳子固定头部。使用手术刀沿着小腿区域的中线切割皮肤;将皮肤拉到侧边，沿着小腿区域的中线在头骨上切口。用钳子打开头骨，露出脑组织，用钳子轻轻地将脑组织与连接的神经组织分离。切割大脑的横截面，包括脑干和小脑，用玻璃滑梯轻轻触摸脑组织的切面，留下涂片印象。按镜头组织上的幻灯片去除多余的组织，并在零下 20 摄氏度丙酮中固定涂片压片幻灯片 30 分钟。然后修复一小块新鲜脑组织，在形式，组织病理学检查。将其余组织放在emi-em 10中，用于核酸提取。接下来，沿着身体的中线将皮肤从歧管切到 Anus，并使用钳子抬起和分离皮肤并下划肌肉组织;允许收集位于曼迪贝骨附近的唾液腺。用钳子稍微抬起胸骨，用手术刀沿着中线切割胸骨和腹壁，并切割夹骨。使用针头将左右肋骨笼固定到解剖板上，打开胸腔。并记录整个军团和验尸后的变化程度。然后使用钳子和手术刀去除内脏组织，以适合计划下游分析。要在固定结束时进行直接荧光抗体测试，干燥涂片印象幻灯片，并选择至少两个荧光结合抗狂犬病抗体进行分析。通过0.45微米滤株将一个抗体过滤到每张幻灯片上。在潮湿的室内，在37摄氏度的温度下孵育滑梯30分钟。在孵育结束时，排出多余的结合，用PBS清洗幻灯片。然后使用 10% 甘油的小体积将盖滑到幻灯片上，然后通过荧光显微镜对幻灯片进行图像。当荧光抗体测试或RT-PCR分析显示正性时，在emi-en 10的10%悬浮液中使大脑标本均质化，通过离心沉淀组织。接种200微升的超级nat-en与三倍10至第6小鼠神经母细胞瘤细胞和1毫升emi-en10在37摄氏度和1%的二氧化碳1小时一小时。将大脑同质细胞悬浮液转移到一个25厘米的方形烧瓶中，里面装着6毫升的新鲜emi-en10。在一毫升脑均质细胞悬浮液中放入四孔特氟隆涂层的玻璃滑梯中，直径为六毫米，将滑梯在37摄氏度和1%的二氧化碳下放置3至4天。用100%丙酮固定后，用荧光抗体测试中使用的两种荧光结合抗狂犬病抗体染色幻灯片。并且通过荧光显微镜对幻灯片进行可视化，以确定内特拉西达-普拉斯-米内含物的数量。在根据标准协议对接种的细胞培养进行试青素和亚培养后，将冷培养物返回细胞培养箱三到四天，重复计算受感染的细胞数量，直到达到100%的感染率。从2014年1月到2017年5月，收集了13种332只蝙蝠尸体进行监测。在两个阳性蝙蝠病例的这种代表性分析中，使用荧光抗体测试对阴性测试，使用其中一种商业的、拟合的抗狂犬病抗体，而采用两种不同底型集的RT-PCR则均呈阳性结果。在Gen银行数据库中对获得的放大器序列进行爆炸查询的主观显示，序列与身份小于79%的Lyssa病毒相似，支持检测到的Lyssa病毒的身份。随后，荧光抗体测试和测序确认，两种Lyssa病毒随后从两个大脑中成功分离。确定莱萨病毒阳性率的关键步骤是荧光抗体测试和RT-CPR分析。强烈建议在底光组中使用两种或两种或两种（多）抗狂犬病抗体。除了莱萨病毒外，还可以使用封闭式诊断方法监测其他动物非病毒蝙蝠病原体。然后，这些数据可以用来告知公众避免与蝙蝠接触。我们越能够研究蝙蝠莱萨病毒，我们越能够了解其演变和对公共卫生的影响。

Research

JoVE Journal

Environment

A Small Volume Procedure for Viral Concentration from Water

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

科研

环境科学

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

清洁取样和痕量金属研究河和河口水域分析

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

海洋浮游生物和悬浮颗粒的超洁净多层采集装置

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

A Video Surveillance System to Monitor Breeding Colonies of Common Terns Sterna Hirundo

监测常见燕鸥繁殖菌落的视频监控系统 (燕鸥家燕)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread ...

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Autometallography 在鲸目组织中的定位和半定量银的应用

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of PlantÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Environment Interactions

植物与环境相互作用实时生理表型的遥测重力平台

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

通过元条形码分析监测智利有害藻华的标准化程序

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used ...

An Application for Pairing with Wearable Devices to Monitor Personal Health Status

The current protocol aims to showcase the technology integration, providing a detailed description of adopting the HealthCloud app, developed by the ...