The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. J.K.B., K.J., M.R.H., D.P., L.B., and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: J.H.K., an employee of Tunnell Government Services, Inc.; and M.G.L., an employee of Lovelace Respiratory Research Institute.

该协议符合下列动物护理指南。动物被安置在由该协会的评估和实验动物国际评审认可的设施。所有的实验过程由国家过敏和传染病研究所，临床研究部，动物护理和使用委员会批准，均符合动物福利法的规定，公共卫生服务政策和指南的保养和使用实验动物的建议。 1.大气生物学:动物生物安全4级（ABSL-4）套装实验室<ol><li> 实验室制 <ol><li>完成ABSL-4实验室西装入境手续（8详细介绍）。 </li><li>测试所有设备的功能（ 例如 ，体积描记法设备，笔记本电脑，生物危险垃圾桶，生物危害锐器容器，受监测装置多个）参与根据制造商的协议的ABSL -4-西装实验室内发生空气生物学过程。 </li><li>确保传输车为测试快速输送口（RTP），其通过壁连接运输车的III类BSC）的功能之前，生物清洁。 </li><li>手柄和仅在生物安全柜认证稀释病原。包含适当的消毒剂II级BSC内准备在适当的配方中的病原体。输送病原体在标记上的运输车湿冰上生物危害符号气密次级容器。通过RTP病原体进入III级BSC在ABSL-4箱体实验室， 如图1所示 。 </li></ol></li></ol> 2.体积描记法:动物生物安全4级（ABSL-4）套装实验室<ol><li> 体积描记法设置和校准 <ol><li>确定哪些体积描记采集方法（头出体积描记法或呼吸电感体积描记法[RIP]）将用于设备组件连接在一起。 </li><li>校准之前使用制造商的协议的实验的体积描记器。 </li></ol></li><li> 体积描记术采集 <ol><li>当处理NHPS，不要一对外乳胶或丁腈手套在西装手套顶部，以防止交叉污染，促进安全的做法。当处理完NHPS，去除这些多余的手套，并在房间内的生物危害垃圾桶丢弃。 </li><li>如果使用的头出体积描记法，附加一个新的橡胶/牙科坝到气缸的前面。切在坝的NHP的头部，以适应通过气缸的顶部的小孔。坐着时，大坝周围产生NHP的脖子密封。 </li><li>如果使用RIP，检查RIP带被正确安装围绕NHP的胸部和腹部和电子连接抢购tighTLY。 </li><li>发送从体积描记法处理中获得在ABSL-4箱体实验室研究人员的所有数据。通过兼容的程序导出每个动物的潮气量和每分钟通气量数据在使用过程中气溶胶的过程。 </li></ol></li></ol> 3.非人灵长类运输和处理:动物生物安全4级实验室套装<ol><li> NHP处理 <ol><li>监测并记录海图每个NHP任何物理操作，管理部门，或例行程序。 </li><li>当完成气溶胶挑战，放置NHP的运输集装箱内，并返回NHP到位于动物保持室家笼。 </li><li>当处理一个活的动物，按照需要2名工作人员在场的强制性规则。 </li></ol></li><li> NHP运输 <ol><li>确定麻醉的类型，麻醉持续时间（包括运输，体积描记法acquisit离子，和气溶胶攻击），并在给药前麻醉的相应剂量。基于由比较医学人员所选择的过程中充分麻醉NHP。如果需要额外的麻醉，确保所有的针，尖锐件，注射器和帽在位于任意的动物程序室的尖锐件容器被丢弃。使用后不要重新盖上任何针。 </li><li>交通运输在被闩锁固定在运输箱的盖子明确容器麻醉NHPS。 </li><li>负载运输容器上的移动车，以允许充分适合研究人员自由地使用呼吸空气线并通过空气压力抗性（APR）的门， 图 1中移动。 </li><li>至于NHP没有额外的呼吸空气供给到运输容器，最小化传输时间。 </li></ol></li></ol> 4.大气生物学:ABSL-4内阁实验室<ol><li> III级BSC设置 <ol><li>同时与比较医学人员进行的动物准备，准备III类BSC。直观地验证负压在III类BSC保持规定的范围内（以水柱（WG）最低125帕或-0.5;推荐250帕或-1.0在WG）。检查III类BSC为任何潜在的泄漏或裂缝（ 见图1）。 </li><li>身体和目视检查III类BSC合成橡胶手套和附着于III类平衡计分卡弱点，O型圈，眼泪，撕裂，或干腐病。更换损坏的III类合成橡胶手套和/或O形圈在使用前。在这一点上，III类BSC不受污染。 </li><li>如果在III类BSC被污染发生泄漏，找准突破口和警报设施管理和生物安全人员的位置。如果III类BSC集成手套被撕裂或破坏，遵循适当的培训技术和跨立即更换损坏的手套最终III类BSC标准作业程序。 </li><li>更改包含一个小撕裂或违背集成手套的曝光期间，先用双季铵盐的适当浓度喷洒撕裂或违反过度（正烷基二甲基苄基氯化铵，正 - 烷基二甲基乙基苄基氯化铵）消毒剂。在此期间，创造气流上升不要让过多的动作。 </li><li>小心地取下外O型圈（其中2个）仍留附着于III类BSC受损集成手套。同时确保综合手套密封完好无损略有端口移动受损集成手套袖口的路程。如果密封被破坏，报警器就会响起，指示不正确完成的步骤。第二O形环从第三类BSC除去后集成手套箍应保持连接到该端口。 </li><li>将新Ⅲ级BSC合成橡胶手套在旧手套我的ñ相同的方向。充分类似把这个新的手套上的端口到另一个III类BSC手套端口。 </li><li>在新的综合手套最接近更换O形圈的III类BSC。使用相邻的集成手套口，小心地将受损III级BSC合成橡胶手套III类BSC内。新的III类合成橡胶手套将作为屏障，以保持密封。一旦其他损坏的III类合成橡胶手套被删除（向内拉），取代其他外O形圈和继续工作。 </li><li>记录关于具体的III类BSC记录簿手套撕裂/破坏所有细节。如果损坏的集成手套被去除或在收容违反发生时，在受感染的综合手套/端口仍然保持0.47米3 /秒的向内气流。这内的气流与II类BSC使用，从而保持II类和III类的BSC之间的一致性同样的气流。 </li><li>检查扣篮坦克和验证扣篮罐填充有消毒剂扣篮罐， 图1内的显着水平。验证在扣篮罐消毒剂浓度是最小的使用电导率计3500μs。这个电导率相当于消毒剂的5％浓度。 </li><li>确保III类BSC高压釜是功能和操作，因此所有污染废物和设备可以被高压灭菌， 图1。高压灭菌唯一已知维持灭菌过程的苛刻条件的设备。 </li><li>测试的其他大气生物学设备（ 例如 ，AAMP部件，笔记本电脑），并参与了实验空气和真空线， 图2中的功能。 </li><li>广场上的第三类BSC迹象显示本机的当前污染状况。 </li></ol></li><li> NHP头只暴露商会大会和系统设置 <ol><li>组装一个16-1 NHP头只茶曝光通过将不锈钢输送和排气线， 图2 MBER。在一个推配置室/通过连接相应的空气，真空和压力线的AAMP拉，动态配置。所述AAMP通过位于第III类的BSC（ 图1）的顶部上的密封端口连接到III类BSC内一个电源和一台笔记本电脑。 </li><li>检查任何泄漏或裂缝的组装NHP头仅曝光室，并确保该室被正确组装。 </li><li>附加一个气溶胶发生器和空气动力学粒径读数仪到NHP头只曝光室。 </li><li>打开空气和真空源到AAMP。 </li><li>在笔记本电脑上启动气溶胶协议软件。输入适当的NHP头仅曝光室，气溶胶发生器和biosampler流量，和管理信息到软件的菜单。 </li><li>计算气溶胶挑战从体积描记过程中获得的数据的时间，步骤2.2.4。如果使用的是头出体积描记法，计算前的气溶胶曝光剂量。如果使用RIP，气溶胶曝光期间同时计算剂量。 </li><li>填与病原体的气溶胶发生器。 </li><li>通过气溶胶软件，转"上"的气溶胶​​发生器和喷NHP头仅曝光与挑战材料室的内部10分钟。 </li><li>关闭气溶胶发生器，清空挑战材料，并丢弃挑战材料到所在的III类BSC内部的生物危害垃圾袋。 </li></ol></li><li> NHP头只曝光 <ol><li>附加biosampler到NHP头仅曝光室，填充集媒体biosampler，及相应的真空管线连接到biosampler。 </li><li>检查NHP的麻醉深度。如果麻醉深度是足够（ <em&gt;例如，反应迟钝对外界刺激，肌张力，稳定的呼吸和心脏速率），通过快速输送口（RTP）传递麻醉NHP成III类BSC。如果麻醉深度不足，施用经由静脉附加麻醉，直接注射，或通过麻醉剂泵传递NHP通过RTP（比较医学人员在西服实验室）之前。通过RTP传递其他麻醉用品。 </li><li>放置NHP在仰卧位到NHP曝光坡道。 </li><li>轻轻通过连接于NHP头仅曝光室的头部门户在橡胶/牙科坝传递NHP的头部。橡胶/牙科坝确保了密封气溶胶曝光时围绕NHP的脖子上创建的。 </li><li>验证NHP的生命体征平稳视觉上和用便携式监视器主题。 </li><li>输入步骤4.2.6计算的气溶胶攻击的时间。每次和必要的设备标识符tinent每个气溶胶碰上气溶胶软件，并开始气溶胶挑战。 </li><li>与气溶胶粒度分析仪各气溶胶运行期间验证粒径数据，以确保所期望的粒度分布的实现。连续或间歇执行此验证整个曝光。 </li><li>一旦气溶胶攻击完成后，从头部仅曝光室除去NHP并用适当的消毒剂擦拭NHP的脸/头关闭以潜在的污染减少到实验室工作人员。 </li><li>吹扫气雾室或空气通过使空气和真空通过室洗5分钟，其余和滞后颗粒。此过程将"清理"，并从气溶胶曝光室的残留颗粒随后NHP气溶胶曝光。 </li><li>传回NHP通过RTP到位于ABSL-4空气生物学实验室的西装里面的研究人员。 </li><li>抛弃一切锐器ū在保留在BSC指定尖锐件容器中的III类BSC内的sed。当锐器容器是¾满，放入生物危害垃圾袋。 </li><li>空气溶胶发生器和任何剩余的挑战材料成含有垃圾，一次性设备的生物危害的垃圾袋的，和/或如果适用¾满尖锐件容器中。 </li><li>清空从烟雾biosampler收集介质插入适当标记收集管和放置在湿冰。 </li><li>直到所有预定的考试科目受到质疑重复步骤4.3.1到4.3.13。 </li><li>通过RTP所有气溶胶样品biosampler的研究人员气雾剂剂量量化和背部滴定。 </li><li>放置从气溶胶攻击成附连到三级BSC直通高压釜的垃圾和设备，并选择一个适用的消毒循环（ 图 3）。 </li><li>拆卸NHP头只有chamber和净化头仅室和III类BSC与生物学指标验证的多聚甲醛气体循环。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们概述在IRF-弗雷德里克用于高危险（风险组4）病原体的工作程序，大气生物学。可视化生物气溶胶程序的目的之一就是与这些病原体在实验过程中使用的是III类BSC时，为了避免实验室感染强调工作人员的安全。第三类的BSC保持一个向内定向的气流即排出到双HEPA过滤器，以确保病原体包含在实验室（ 图1）内。 作为Ⅲ类BSC是在生物气溶胶研究防止潜在的病原体暴露主要障碍，研究人员要求检查III类BSC的完整性，并在每次气溶胶实验后附综合手套泄漏。虽然尽一切努力尽快排除危险实验室的研究人员，违反III类BSC的整合可能会出现合成橡胶手套。人员必须提供无论教学和实践上的正确III类BSC应急响应程序的培训。这些程序包括从ABSL-4箱体实验室疏散，确保在遏制违反到III级BSC和个人防护装备穿戴在必要的时候。我们已经使用了过去依赖于该过程所需的精细运动技能不同厚度的其它手套。无论厚度，在执行这些步骤时所选择的所有手套都同样的保护。强大的培训，严格遵守安全协议和工程控制有助于在IRF-弗雷德里克使用III级生物安全柜时要确保员工的安全。上述过程如有变动，由于基于改善工作流程的新方法或安全重新评估。 而这里提出的aerobiological程序一般遵循BMBL建议7，这些程序是特定到IRF-弗雷德里克。 ËACH ABSL-4 / BSL-4设备有不同的建筑设计规范，影响实验室操作的具体方法。使用III类BSC实验室替代的方法和技术在很大程度上取决于这些实验室的设计和操作的部分。此外，在不同的国家不同的政府法规也可能对气溶胶研究程序的效果。尽管如此，ABSL-4气溶胶程序的一般理解和楼宇监控，支持实验室的研究人员的安全将帮助卫生管理员，谁正在考虑类似的建筑，以及参与的后果严重病原体的研究合作者以外的设计系统。 设计与外部合作者生物气溶胶协议时，有足够的时间应分配给执行甚至连基本的生物气溶胶的操作。交付成果时限的预期已被接受固有的工作中的困难进行调整在ABSL-4 III级BSC实验室。广义的假设是，在ABSL-2中执行的任何生物气溶胶实验（ 例如 ，2小时）将需要的时间量的两倍于ABSL-4（ 例如 ，4小时）来执行。

病毒的传播一般通过直接或身体接触，但许多重要的病毒性疾病（ 如麻疹，水痘，流感）通过由气雾剂或呼吸道飞沫传播的病原体引起的有发生。这样的病原体有可能导致其后果从与工作相关的损失普遍轻微的疾病大流行的可能性（ 如普通感冒），以罕见的高致命性（ 如天花）严重的疾病。后果严重的病原体，通过气溶胶或故意释放烟雾自然传播（生物武器）特别感兴趣大气生物学1。人类可以通过大的呼吸道飞沫或小颗粒核迅速成为感染了一些这些病原体，容易通过唾液分泌物传播这些病原体对他人，咳嗽，打喷嚏2。在美国生物防御社区，后果严重的病原体（ 如丝状病毒或其他NIAID的Category AC优先病原体和CDC生物恐怖代理）是由于相关感染3,4的高致命性的气溶胶研究项目的重点。在空气生物学领域内的科学显著进步已经在过去的十年内提出由于气溶胶设备和高封闭设施5,6的技术进步。研究在美国国立卫生研究院国家过敏和传染病研究所（NIH / NIAID），在位于弗雷德里克，MD，USA（IRF-弗雷德里克）德特里克堡综合研究机构的重点需要动物生物安全后果严重的新兴病原体4级（ABSL-4）遏制。在IRF-弗雷德里克的总体任务是评估和推动候选疫苗和疗法（医疗措施）的发展。 研究与在IRF-弗雷德里克后果严重的病原体是通过严格的生物安全和动物护理和使用要求的约束。这些requir对此语句在微生物和生物医学实验室 （BMBL）手册7和联邦动物福利法规的生物安全概述。这些必要的要求可能会限制可以进行的研究类型，并影响整体的研究设计。正如我们在本杂志如前所述，在ABSL-4环境中进行的所有研究需要特别谨慎，高度专业化的培训，以及强大的和冗余的设备基础设施8,9。 进入IRF-弗雷德里克ABSL-4实验室的西装穿上需要正压密封服8。不需要用于输入ABSL -4-柜实验室正压包封西装。穿上擦洗服，橡胶或丁腈手套，并靠近趾鞋在ABSL-4箱体实验室7认证的III级生物安全柜（BSC）内操作风险组4传染性物质时是合适的。 在IRF-弗雷德里克，气溶胶设备被设计，组装，并保持两个密封，不锈钢，气密，负压III类的BSC， 图1. IRF-弗雷德里克大气生物学核心采用一种自动化气雾剂管理平台（ AAMP）来控制和监视这些生物安全柜中气溶胶的实验， 如图2所示 。先前公布所述的IRF-腓特烈III级生物安全柜，并通过直通端口5连接到西装实验室的特定功能。之前的实验制备III类BSC的方法是特定于在IRF中。在其他机构使用的其他III类生物安全柜的功能类似于III类BSC在IRF的使用，但可能有交通工具，访问或对接不同的机制。 为了进一步了解高后果的病原体如何保持传染性，通过空气传播蔓延，安全AErobiological实验必须在这些III类的BSC根据一个特定的工作流程程序来进行。研究人员已经过仔细而彻底培训，确保此工作流之后在安全和一致的方式。到非人灵长类（NHP）气溶胶攻击，一些气溶胶表征或假气雾剂运行之前被执行，以测试的试剂的气雾剂形式的稳定性和生存能力时。气溶胶表征过程模拟实际的气溶胶挑战，研究人员评估与气溶胶研究相关的变量。 工作流的另一部分是记录于图表为每个NHP物理操作，管理或麻醉剂或其它试剂，或例行程序。这些主题图表深入分析，以确保程序的一致性和标准化。科目气溶胶曝光前麻醉。例如麻醉剂包括替来他明/唑拉西泮，氯胺酮/乙酰丙嗪，和ketami东北。麻醉药是基于减少呼吸抑制和促进控制，稳态呼吸选择。附加麻醉用品保持在动物操作室和输送在与NHP转印车到大气生物学ABSL -4-柜实验室。 内ABSL-4实验室的西装，NHPS通过两种方法（ 即头出体积描记法，呼吸感应体积描记[RIP]）来确定吸气潮气卷一接受体积描记法和呼吸率变化10-12。这些衍生的参数到或气溶胶曝光期间用于估计吸入剂量的病原体的精确计算之前立即。头出体积描记法使用长，圆柱形腔，里面的NHP 13。当动物是在气缸内产生的压力降由一个呼吸速度捕获，中继到放大器，由交流电流/直接柯伦处理T转换器，以及集成到软件来导出上述肺参数。 RIP使用由嵌入在围着人的胸部和腹部11,12松紧带感性盘绕铜线传感器。的感应电容的传感器产生的磁场。呼吸改变磁场，将所得的电压变化是由一个发射机通过短波长的超高频无线电波中继弹性带旁向接收机在计算机中。专用软件决定呼吸频率和总胸排量潮气量。 通过体积描记法得到的分钟量（MV）中的估计吸入剂量（D）的计算被使用。在产生和采样气雾剂，气溶胶浓度（AC）是由媒体（V）的该卷的biosampler浓度（BC）相乘并通过由所述biosampler（佛罗里达州）的流速乘以结果除以计算曝光时间（T）。简化式表示为AC = BC x垂直÷FL点¯xT.反过来，对于在NHPS实际气溶胶攻击时，D由MV和曝光时间（时间= T），乘以交流计算。该简化公式表示为D = AC点¯xMV点¯xT. 本文的目的是为了在视觉上演示使用NHPS从两个观点出发，ABSL -4-西装实验室侧和ABSL -4-柜实验室侧的整个气溶胶攻击过程。虽然这些程序可以针对提到的若干做法是一般性的，它们是专用于在IRF-弗雷德里克大气生物学Core和表示在该机构中使用的实际做法。本文着重讨论安全执行气溶胶的挑战，而不是实际的气溶胶攻击本身所需要的生物安全程序。在这些过程中，我们使用一个虚拟的主题展现生物安全的做法，由于与麻醉的NHP相关的风险。然而，PERFO的过程中最差气溶胶的挑战是写在一般的方式，因为程序是相同的，不管使用的高后果的病原体。我们的目标是提高有关最高封闭条件下进行严重后果的病原体气溶胶研究的严酷科学家的认识和了解。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micro-Chem Plus</td>
			<td style="width:239px;">National Chemical Laboratories</td>
			<td style="width:139px;">255</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol&#160;</td>
			<td>Fisher&#160;</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Class III BSC</td>
			<td>Germfree</td>
			<td>DGB-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Integrated BSC gloves</td>
			<td>Piercan</td>
			<td>10UY2032-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aerosol Management Platform (AeroMP)</td>
			<td>Biaera Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Head-out plethysmography</td>
			<td>Buxco/Data Sciences International</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Respriatory inductive plethysmography</td>
			<td>Data Sciences International</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centered flow tangential aerosol generator (CenTAG)</td>
			<td>CH Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collison nebulizer</td>
			<td>BGI Inc.&#160;</td>
			<td>CN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoclave</td>
			<td>Getinge</td>
			<td>GEB 2404 AMB-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sperian positive-pressure suit</td>
			<td>Honeywell Safety Products</td>
			<td>BSL 4-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (latex, Ansell Canners and Handlers)</td>
			<td>Fisher</td>
			<td style="width:139px;">19-019-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (nitrile/rubber, MAPA)</td>
			<td>Fisher</td>
			<td>2MYU1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scrubs</td>
			<td>Cintas</td>
			<td>60975/60976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Socks</td>
			<td>Cintas</td>
			<td>944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Duct tape</td>
			<td>Pack-N-Tape</td>
			<td>51131069695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Towels</td>
			<td>Cintas</td>
			<td>2720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O-rings</td>
			<td>O-ring warehouse</td>
			<td>AS568-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overshoes</td>
			<td>Amazon</td>
			<td>B0034KZE22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zip lube</td>
			<td>Amazon</td>
			<td>B000GKBEJA</td>
			<td></td>
		</tr>
	</tbody>
</table>

safety precautions and operating procedures in an (a)bsl-4 laboratory: 3. aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

所述III类生物安全柜（BSC）为含有ABSL -4-柜实验室内负压的ABSL-4环境密封的不锈钢箱体（ 图1）。材料可以被引入到由工作人员通过一个橱柜下式不锈钢罐中的ABSL -4-柜实验室工作在BSC（通常被称为在ABSL-4或BSL-4设置的"扣篮罐"）含有5 ％的双季铵盐（正烷基二甲基苄基氯化铵，正 - 烷基二甲基乙基苄基氯化铵）消毒剂溶液。因为在BSC内置于壁分离从ABSL -4-西装实验室柜实验室，材料，动物，和病毒病原体也可以被移动到使用传输车和快速传递从ABSL -4-西装实验室侧的BSC端口（RTP）。在BSC内的内容可以从外部由研究人员穿着来操纵<html>各类合成橡胶手套，氯丁橡胶专门/氯磺化聚乙烯。内容，不含传染性采样，被从BSC灭菌后经由扣篮罐双门高压釜或消毒除去。通过检查/检验的III类BSC和生物气溶胶设备（ 图2）是否正常，我们维持一个安全和正常运作的环境。正确维护和使用III类平衡计分卡是不可或缺的研究人员个人防护。继进行消毒气雾剂挑战气溶胶曝光，垃圾和设备放入连接到III级BSC直通高压灭菌器， 如图3所示 。通过严格遵守这些程序和做法，没有实验室获得性感染已经在记录在IRF-弗雷德里克生物气溶胶的研究。 2fig1.jpg"/&gt; 图1.在静止状态（从5转载） 机柜的IRF-弗雷德里克介绍了三级生物安全柜安装示意图演示 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53602/53602fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53602/53602fig2.jpg" /> 图2. 气溶胶管理平台。从5调整。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53602/53602fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53602/53602fig3.jpg" /> 图3.联锁双门高压灭菌器连接到III类BSC。一位研究员是选择一个预编程的高压釜周期，以确保高压釜室中的内容非感染性当外门最终打开。最靠近研究者门不能被打开，直到一个完整的消毒循环已经完成。高压釜室内生物指标进行分析，以确定灭菌过程（从5转载）之后剂灭活。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53602/53602fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

安全注意事项和操作步骤在（A）BSL-4实验室:3，大气生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

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Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

14.0K 次观看.  National Institutes of Health (NIH). As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

视频: Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测:非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

生产的非复制型犬2型腺病毒衍生载体与纯化

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

安全注意事项和操作步骤在（A）BSL-4实验室:1.生物安全4级实验室西服套房出入境手续

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

安全注意事项和操作步骤在（A）BSL-4实验室:2.常规做法

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

安全注意事项和操作步骤在（A）BSL-4实验室:4.医疗成像过程

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...