The authors wish to thank Mark Nilan for zebrafish care and maintenance and Meghan Breitbach and Deborah Bouchard for propagating NS1-GFP and determining IAV titers. This research was supported by NIGMS grant NIH P20GM103534 and the Maine Agricultural and Forest Experiment Station (Publication Number 3493).

所有工作都应该使用生物安全2级（BSL2或）由美国疾病控制中心（CDC），并按照机构动物护理和使用委员会（IACUC）制定的指令所描述的标准来执行。请有关官员商议，以确保安全和法规遵从。 1.斑马鱼保养与维护<ol><li>斑马鱼产卵，并收集所需数量的胚胎实验。必要时，大规模繁殖罐，像那些由Adatto 等人描述。 32，可用于收集大量的发育上演胚胎。 </li><li>允许胚胎发展到在低密度深培养皿中所需的发育阶段（48小时受精后的全身IAV感染[第3或5天受精后的本地化，鱼鳔感染[第4]）（&lt;含6无菌鸡蛋水100胚胎/皿）在28°C0微克/毫升海盐（ 例如 ，即时海洋）在蒸馏水。 <ol><li>用塑料吸管转移去除死胚胎和卵子变化，每天的水，以保证最佳的健康和发展。 </li></ol></li></ol> 2.材料和试剂的制备<ol><li>制备4毫克/毫升的库存三卡因-S的蒸馏水和高压釜溶液（调节pH值至7.0-7.4）。保存在4℃。 </li><li>用一个微量拉马长丝拉硼硅玻璃毛细管（ 例如微管普勒使用以下设置:压力设定= 100，热量= 550，拉= [无值]，速度= 130，时间= 110）。 注意:在此之前修整所述针，从那里的针开始逐渐变细，以在针的末端的长度应约为12-15毫米。然而，这可能会有所不同，根据自己的偏好和应用。更长，更渐进的锥度可能因为刺破胚胎增加的能力和降低RI更佳针弯曲或断裂的SK。 </li><li>消融2 g用微波在100毫升水蛋琼脂糖。使在其上排队并通过倾琼脂糖熔化成深培养皿中并使其固化注入胚胎板。 </li><li>使扎那米韦的10毫克/毫升的库存在无核酸酶的水。分装，冷冻在-20℃。 </li><li>利用微波使胚胎安装琼脂糖融化在50毫升水蛋0.5克琼脂糖。维持水浴在50℃下，直到准备成像期间使用。 </li></ol> 3.全身感染IAV（48小时受精后） 注意:所有的研究人员应与上级和医生就在开始工作之前与IAV疫苗接种咨询。 <ol><li>手动dechorionate使用＃5钳实验当天胚胎。小心取出，安乐死已经在200微克/毫升三卡因溶液的过程中受伤的胚胎。 </li><li> Ø准备˚FIAV显微注射 注:株已被证明感染斑马鱼胚胎是流感A / PR / 8/34（H1N1）（APR8），甲型流感的X 31 A /爱知县/ 68（H3N2）（X-31），以及荧光记者流感病毒NS1-GFP 33。有关APR8和X-31和其他菌株的详细信息，可以在流感研究数据库中找到<ol><li>传播IAV的NS1-GFP株鸡胚如前所述34,35。收集，分装和滴定含有IAV尿囊液，并储存在-80℃。预滴定APR8和X-31的病毒可以商业购买。 <ol><li>只是在感染前，迅速解冻的IAV等分在戴手套的手，然后迅速置于冰上减少滴度的损失。 </li></ol></li><li>病毒稀释<ol><li>如果使用的是APR8或X-31病毒，稀释至3.3×10 6个蛋感染剂量50％（EID 50）/ ml，在无菌的，冰冷的PBS supplemented用0.25％酚红在层流罩（在可视化助剂）。同时建立包含无菌PBS，0.25％酚红控制。 </li><li>如果使用NS1-GFP应变33，稀释至〜1.5×10 2噬菌斑形成单位（PFU）每NL在无菌的，冰冷的PBS含有0.25％酚红（在可视化助剂）在层流罩。同时建立含稀释就像在无菌PBS病毒与0.25％酚红未被感染的鸡蛋尿囊液控制。 </li><li>维持IAV冰上待用。 </li></ol></li><li>吸取病毒解决方案为使用微加载提示，使用前显微注射针。插入注射针头插入注射装置（ 例如 ，移动电话倡议-3-压喷射系统）的适当支架。 </li><li>在观看立体显微镜下，轻轻一夹有棱，消毒＃5镊子显微注射针头。 注:这是recommenDED的显微注射针尖逐渐裁剪。 </li><li>压低的压力喷射系统的脚踏板和注入显微镜浸油上的校准千分尺滑下降。测量液滴的直径。计算下降使用公式的体积，V = 4 /3πr3，其中V是体积在n1和r是在微米的半径。 注意:如果放置直径太小，附加玻璃可以被裁剪或压力和时序设置可调节。如果下降直径太大，压力和时序设置可调节。 </li><li>上的压力喷射器调节压力和时序设置和/或直至所需的注射体积达到（一般为1-3 NL）reclip针尖。 40和80毫秒之间的20和30 PSI和脉冲宽度设置之间的压力设置是典型的。调整回压，使其专柜毛细管压力，但不漏IAV（净零压力）。 </li></ol></li><li>对齐胚胎注射<ol><li>在200微克/毫升三卡因麻醉dechorionated胚胎。 </li><li>一旦运动停止，转移10-20胚胎至2％的琼脂糖平板（第2.3节中的方法制备）用塑料移液管。用塑料吸管以除去过量的液体。 </li><li>轻轻对准胚胎与火抛光和密封的硼硅玻璃毛细管注射。使用立体显微镜，轻轻东方胚胎以便居维叶的管道或后部主静脉与所述注射针（ 图1A）线。 </li></ol></li><li> IAV显微注射<ol><li>轻轻地插入注射针头插入居维叶管道或后主静脉。踩下脚踏注入IAV的所需体积进入胚胎（ 图1A）的循环系统。胚胎如有必要，可注入的一个以上的时间。 注意:注射小丸应该被扫成流通和DISTRIB整个身体的布式。如果注射体积收集在注射部位，从实验和安乐死移除胚胎。 </li><li>重复注射在不同的胚胎具有特定病毒所选择的应变控制解决方案。对于APR8和X-31，使用3.2.2.1节描述的控制;为NS1-GFP，使用3.2.2.2节描述的控制。 </li></ol></li><li>胚胎转移到含有无菌恒温箱鸡蛋水和地方在33°C标记的培养皿。 注:感染胚胎应在33℃下生长，以支持增强的病毒复制。另外，培养在33℃下更紧密地模仿人上呼吸道，这是在外部环境中的具有较低温度的紧密接触，并且在那里IAV感染发生的温度。胚胎可以适应于广泛的温度范围内34。 </li></ol> 4.本地化，鱼鳔IAV感染的Tg（MPX:mCherry） </EM&gt;转基因斑马鱼（5天受精后） <ol><li> IAV的制备显微注射<ol><li>如第3.2节所述稀释NS1-GFP应变和控制注射液。 </li><li>如在2.2节所述准备针。 </li><li>对齐的Tg（MPX:mCherry）含有与以下异常3.3节所述膨胀鱼鳔35幼虫。以这样的方式使针可穿透鱼鳔和病毒可以存入鱼鳔的后部，如前所述36（ 图2A）对齐幼虫。 </li></ol></li><li> IAV显微注射<ol><li>轻轻插入注射针头插入鱼鳔并朝向结构（ 图2A）的后注入所需量（ 例如 5 NL）。 注:注射小丸应收集朝着后和鱼鳔的气泡应该bË向前移位。 GFP表达应该开始在3小时后注射待观察。 </li><li>具体到病毒的选择，如3.4.2节中描述的应变控制解决方案，重复注射。 </li></ol></li><li>幼虫转移到含有无菌恒温箱鸡蛋和水发生在33°C的培养皿。 </li></ol> 5.抗病毒治疗药物注意:以下协议描述以前的Gabor 等所示的扎那米韦治疗。 19。该协议可以被修改以筛选其它抗病毒药物，并具有进行修改，以筛选在96孔板，高通量格式多种化合物的潜力。 <ol><li>在3小时后感染，用含0，16.7，33.3纳克/毫升无菌扎那米韦鸡蛋水代替NS1-GFP-的鸡蛋水和控制感染的鱼。 注意:这些浓度才能被选为试图复制FO达到的生理水平在人类患者中（100，200，或600毫克，四，每日两次或10mg吸入，每天两次）扎那米韦的llowing施用。在人类患者血清浓度范围为9.83至45.3纳克/毫升36。 </li><li>在5天的过程中，改变蛋水每12小时，并用含有扎那米韦适量无菌蛋水替换。 </li><li>追踪感染的课程。 <ol><li>在200微克/毫升三卡因麻醉斑马鱼。监测立体荧光显微镜，目视观察IAV感染和控制之间的鱼，而鱼浸泡在不同浓度的药物中的感染模式的差异GFP表达。 </li><li>跟踪发病率和死亡率在5天的过程中。 <ol><li>有关与疾病的病理感染的动态，包括嗜睡的症状和水肿的证据，色素沉着的差异，眼和颅面畸形，以及前凸记录观察结果。疾病的病理通常在24-48小时后喷射成为控制感染鱼显而易见，这取决于病毒的注入量。采集图像后24小时注射。 </li><li> 5天感染后每隔24小时，记录死亡，病态和健康幼虫的数目。死亡是由缺乏明显的心跳的定义。根据需要绘制的数据。例如，绘图数据作为堆积条形图，用健康，病态，或死鱼百分比在y轴和治疗组在x轴上。 19 注:死亡可能由Kaplan-Meier生存估计值进行评分。根据应用，也可适当开发用于评分鱼发病的各种途径，包括可能量化基于所观察到的上述病理特征的程度的发病率的评分矩阵。 </li><li>与统计学家咨询，以申请相应的统计分析。 </li></ol></li></ol></li></ol> 6。中性粒细胞迁移注:下面的协议描述用于跟踪中性粒细胞迁移到鱼鳔以下本地化，上皮IAV感染的方法。描述的方法可以被修改，以测试遗传和化学处理的效果。使用这种技术，将有可能在IAV感染期间表征底层嗜中性粒细胞的行为的机制。 <ol><li>安装斑马鱼成像<ol><li>麻醉幼虫在200微克/毫升三卡因进行成像。 </li><li>鸡蛋中的水（〜50-100微升）的一小滴已经感染了NS1-GFP到24的好啦幼虫，玻璃底板:传送单个的Tg（MPX mCherry）。与其他IAV感染和控制鱼重复。 </li><li>慢慢的1％琼脂糖胚胎的安装介质添加到每口井（2.5节），注意不要引入大泡。 <ol><li>这样它被安装在其一侧轻轻调整幼虫的位置。古迪和亨利37描述如何使使用被安装成与长丝硼硅玻璃毛细管和代替强力胶水粘上昆虫针在本申请中运作良好探针。 </li></ol></li><li>一旦琼脂糖变硬，轻轻地用含有200微克/毫升三卡因鸡蛋水填满个别井。 </li><li>用共聚焦显微镜，拍摄AZ系列堆栈使用20倍的目标明和荧光图像的集中在鱼鳔。 <ol><li>使得它们捕获所有明显可观察到的绿色和红色荧光设置上限和下限。 </li><li>在2.0微秒/像素的拍摄图像与那些≤4微米的步骤。增加时间每像素和还原步骤之间的距离（ 例如 ，0.5 - 1.0微米）增加图像的分辨率和质量。 </li><li>产生通过合并的层的二维图像。 </li><li>比较MPX-mCherry阳性嗜中性粒细胞目前我的数ñIAV感染和控制注入​​斑马鱼的鱼鳔。 </li></ol></li></ol></li></ol>

The authors have nothing to disclose.

为了最大限度地使用小动物到人类宿主 - 病原体相互作用的模型获得的利益，框架研究问题，并在测试假说，关于模型系统的固有优势利用是很重要的。作为人类IAV感染模型，斑马鱼有几个优势，包括高繁殖力，光学清晰度，顺从于药物筛选，以及该标签的免疫细胞嗜中性粒细胞一样的转基因株系的可用性。斑马鱼已经发展成为日益强大的替代鼠标模型系统的炎症和先天免疫方面的研究。因为他?...

据世界卫生组织（WHO），流感病毒感染的成年人的5％-10％，每年儿童的20-30％，并导致300万-500万严重病例和全世界1到500,000人死亡。对流感疫苗接种每年仍然以防止疾病的最佳选择。像世卫组织全球行动计划的努力，以降低发病率和季节性流感暴发2相关的死亡率增加了季节性流感疫苗的使用，疫苗生产能力，以及研究和开发更有效的疫苗策略。抗病毒药物像神经氨酸酶抑制剂（ 例如 ，扎那米韦和奥司他韦）是在一些国家可用以及缓解症状已被证明有效，当发病3，4，5的第一48小时内给药。尽管全球努力，季节性流感遏制欧tbreaks保持此时一个严峻的挑战，因为流感病毒抗原漂移往往超过适应病毒6的改变基因组中的电流的能力。疫苗策略靶向病毒的新菌株必须提前开发，有时由于在类型菌株，在一个流感季节最终占优势不可预见的变化呈现小于最佳效果。由于这些原因，有一个明确需要制定包含感染和死亡率降低替代治疗策略。通过实现更好的理解在主机病毒相互作用，有可能开发新的抗流感药物和辅助疗法7,8。 人宿主A型流感病毒（IAV）相互作用是复杂的。人类IAV感染的几种动物模型为了深入了解宿主病毒相互作用，大型的源码已经开发ING小鼠，豚鼠，棉鼠，仓鼠，雪貂，猕猴和9。同时提供具有增强宿主IAV动力学的理解的重要数据，每个模式生物拥有必须尝试将结果转化为人类医学时，应考虑显著的缺点。例如，小鼠，这是最广泛使用的模型，不容易与人流感分离株9感染时开发IAV诱导感染症状。这是因为小鼠缺乏对人流感的天然趋向性分离自小鼠上皮细胞表达α-2,3-唾液酸连接，而不是对人上皮细胞10表示的α-2,6唾液酸连接。存在于人类IAV的血凝素蛋白的分离从优结合并进入宿主细胞到通过受体介导的内吞作用9，11α-2,6唾液酸连接， </s达&gt; 12，13。因此，现在已被接受，在发育的小鼠模型的人流感，必须小心，以与流感的适当应变配对鼠标的适当应变，以达到疾病表型概括的人类疾病的各个方面。与此相反，在雪貂的上呼吸道上皮细胞具有类似于人类细胞14α-2,6唾液酸连接。感染雪貂共享许多在人类疾病中观察到的病理和临床特征，包括人类和禽流感病毒14的致病性和透射性，15。它们也是高度适合于疫苗功效试验。然而，对于人类流感鼬模型有几个缺点，主要与它们的大小和饲养成本，使统计上显的收购具有挑战性着数据。此外，雪貂以前已经显示在药代动力学，生物利用度和毒性，使测试功效很难区别。例如，雪貂表现出毒性M2离子通道抑制剂金刚烷胺16。因此，很显然，在选择的动物模型来研究有关人类IAV感染的问题，必须考虑其固有的优点和局限性，并在主机病毒相互作用是受调查的方面是非常重要的。 斑马鱼， 斑马鱼是动物模型，提供了独特的机会，研究微生物感染，宿主免疫反应，和潜在的药物治疗17，18，19，20，21，22，23，&lt;SUP类="外部参照"&gt; 24，25，26，27，28。 α-2,6-连接的唾液酸在斑马鱼细胞的表面上存在表明其易感性IAV，其在感染的研究证明了，用IAV 19的荧光报道菌株在体内成像。在IAV感染斑马鱼，抗病毒ifnphi1和MXA转录物的表达增加表明先天免疫应答已经刺激，并通过IAV感染斑马鱼，包括水肿和组织破坏显示的病理，是类似于在人类流感感染观察。此外，IAV抗病毒药神经氨酸酶抑制剂扎那米韦有限死亡率和在斑马鱼19减少病毒复制。 在这份报告中，对于引发体系协议在斑马鱼胚胎IC IAV感染描述。使用扎那米韦临床相应剂量的作为证明的原则，对于抗病毒活性的化合物筛选斑马鱼这种IAV感染模型的效用是证明。此外，用于产生局部的，上皮IAV感染在斑马鱼的协议鱼鳔，被认为是在解剖学和功能上类似于哺乳动物的肺21，29，30，31之间的一个器官，进行说明。使用这种局部IAV感染模型，中性粒细胞募集至感染部位可以跟踪，使调查中性粒细胞生物学在IAV感染和炎症中的作用。这些斑马鱼模型补充人类IAV感染的现有的动物模型，并且用于测试的小分子和由于增强了S的可能性免疫细胞应答特别有用tatistical功率，容量为中到高通量测定法，和能力来跟踪免疫细胞的行为和功能的光显微镜。

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			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller&#160;</td>
			<td>Sutter Instrument</td>
			<td>&#160;P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zanamivir</td>
			<td>AK Scientific</td>
			<td>G939</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope immersion oil</td>
			<td>Olympus</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope stage calibration slide&#160;</td>
			<td>AmScope</td>
			<td>MR095</td>
			<td></td>
		</tr>
		<tr>
			<td>MPPI-3 pressure injector&#160;</td>
			<td>Applied Scientific Instrumentation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Back pressure unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P-4758</td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature incubator</td>
			<td>VWR</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V12</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Zeiss</td>
			<td>HXP 200C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>Zeiss&#160;</td>
			<td>CL6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom multiwell plate, 24 well</td>
			<td>Mattek</td>
			<td>P24G-0-13-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr>
			<td>Fluoview software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism v6</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A/PR/8/34 (H1N1) virus&#160;</td>
			<td>Charles River&#160;</td>
			<td>490710</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A X-31, A/Aichi/68 (H3N2)&#160;</td>
			<td>Charles River&#160;</td>
			<td>490715</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza NS1-GFP</td>
			<td></td>
			<td></td>
			<td>Referenced in Manicassamy et al. 2010</td>
		</tr>
		<tr>
			<td>Tg(mpx:mCherry)</td>
			<td></td>
			<td></td>
			<td>Referenced in Lam et al. 2013</td>
		</tr>
	</tbody>
</table>

using zebrafish models of human influenza a virus infections to screen antiviral drugs and characterize host immune cell responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

这里，提供了示出如何全身IAV感染在斑马鱼可用于测试药物功效（ 图1A）的数据。在48小时后的受精胚胎经由居维叶的导管与APR8（ 图1C，1F）中，X-31（ 图1D，1G），或NS1-GFP（ 图1 H-1I）注入以引发病毒感染。在48小时后的受精胚胎另一个队列注射，作为病毒感染（ 图1B，1E）控制。通过48小时后感染，斑马鱼注?...

Watch this Scientific Journal Video about Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses at JoVE.com

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

using-zebrafish-models-human-influenza-virus-infections-to-screen

Research

JoVE Journal

Immunology and Infection

9.8K 次观看.  University of Maine. Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses. 

视频: Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...