Name: vector competence analyses on aedes aegypti mosquitoes using zika virus
Uploaded: 2020-05-31T14:00:00.000+00:00
Duration: 10 min 35 s
Description: Watch this Scientific Journal Video about Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus at JoVE.com

我们感谢世界新兴病毒和虫媒病毒参考中心（WRCEVA）的工作人员:Robert Tesh博士，Hilda Guzman博士，Kenneth Plante博士，Jessica Plante博士，Dionna Scharton和Divya Mirchandani，他们在策划和提供许多用于我们和其他团体的载体能力实验的病毒株方面所做的不懈努力。所介绍的工作由麦克劳克林奖学金基金（SRA）资助，NIH资助AI120942和AI121452。

这些协议中执行的所有程序均完全符合德克萨斯大学加尔维斯顿医学分校机构生物安全委员会和机构动物护理和使用委员会批准的协议。
1. 扩增Vero细胞中的ZIKV
<ol><li>在Dulbecco的Eagle最小必需培养基（DMEM）的修饰中培养Vero细胞（CCL-81或VeroE6），在150cm 2组织培养瓶中补充10%V / v热灭活胎儿牛血清（FBS）和1%（v / v）青霉素- 链霉素（分别为100 U / mL和100μg/ mL）的5%CO2至80-90%汇合度。</li>
	<li>在生物安全柜（BSC）中，取出培养基并处理在10%漂白剂或双季铵的工作稀释液中（材料表）。立即用1mL病毒储备接种单层，目标是每个细胞0.1-1个感染性病毒颗粒。立即搅拌烧瓶，使接种物与整个单层接触。</li>
	<li>使用补充有2%v / v热灭活FBS和1%（v / v）青霉素 - 链霉素的DMEM将培养基充注至5mL的体积。然后将烧瓶移入加湿的37°C培养箱中，用5%CO2 培养60分钟。</li>
	<li>从培养箱中取出烧瓶，然后将其放入BSC中。使用补充有 2% v/v 热灭活 FBS 和 1% （v/v） 青霉素-链霉素的 DMEM，将额外的培养基加入 15 mL 的总体积。将烧瓶移入加湿的37°C培养箱中，其中含有5%CO2。</li>
	<li>每天在相差显微镜下检查烧瓶，以寻找细胞病变效应（CPE）的证据。当只有大约40-50%的细胞留在单层中时，继续进行病毒收获（步骤1.7），通常在感染后3-5天，具体取决于所使用的ZIKV菌株。</li>
	<li>吸出上清液并放入50 mL锥形小瓶中。通过离心（3，500×g 20分钟）澄清细胞碎片的上清液。 注意:如果多个烧瓶的感染相同，则来自多个烧瓶的上清液可以组合成50 mL锥形管。</li>
	<li>将上清液从50mL锥形管中取出至新鲜管中，注意不要破坏沉淀。用热灭活FBS补充上清液至终浓度为30%（v / v）。将这种混合物等分到单独的螺旋盖管中，并在-80°C下冷冻直至使用。</li>
</ol>2. 人造血液制品的制备
<ol><li>在蚊子暴露于传染性血粉的那一天，打开节肢动物收容设施中的电源（材料表），以便在蚊子准备暴露时预热摄食装置（材料表）。</li>
	<li>使用下面描述的方法之一制备人造血液。<ol>
<li>方法1:将新鲜收获的（一周内）柠檬酸盐或肝素化的人血与病毒储备（如第1节所述制备）混合。 注意:这种方法取决于血液中是否存在任何针对病毒/病毒家族的抗体。</li>
		<li>如果无法确认抗体缺失，或者已知血源既往有黄病毒暴露史，请清洗并手动包装红细胞。<ol>
<li>在平衡计分卡中，将 30 mL 全人血加入 50 mL 锥形管中，并用磷酸盐缓冲盐水 （PBS） 补充至 50 mL。</li>
			<li>以3，500×g离心20分钟。</li>
			<li>通过轻轻倒入含有10%漂白剂的托盘锅或工作稀释双季铵来吸出上清液，注意不要丢弃红细胞沉淀。</li>
			<li>加入10 mL PBS，并将锥形管的底部轻轻拍打BSC的底部，使红细胞沉淀已经重建。使用PBS将悬浮液的体积调高至50 mL。通过轻柔的反转混合。</li>
			<li>重复步骤2.2.2.1−2.2.2.4，总共4−6倍。确认上清液是透明的或仅微粉红色，不再不透明。使用血清学移液器除去所有上清液。将红细胞沉淀重悬于1-2 mL的PBS中。</li>
			<li>组装血粉:使用补充2%v / v热灭活FBS和1%（v / v）青霉素的DMEM组装血粉:350μL包装的红细胞，100μL的10%蔗糖，200μL热灭活FBS，900μM重组ATP和2mL适当稀释的病毒储备。</li>
		</ol>
</li>
	</ol>
</li>
	<li>将标准的3 mL储液器单元（材料表）与未感染小鼠的皮肤覆盖（其他选项包括石蜡膜，胶原膜或香肠肠衣）。</li>
	<li>将有盖的水箱放在白纸巾上。一次向储液槽中加入~2 mL感染性血粉。检查喂食器下方的毛巾是否有任何泄漏迹象。如果存在泄漏，请从喂食器中回收血粉并丢弃盖子。用插头密封进纸器。再次确认不存在泄漏。</li>
</ol>3. 血粉背滴/斑块测定
<ol><li>使用制备的血粉的剩余体积，使用补充有2%v / v热灭活FBS和1%（v / v）青霉素 - 链霉素的DMEM进行10x连续稀释（即6次稀释，范围从稀释10x到1，000，000x）。</li>
	<li>将100μL稀释液等分到24或12孔板的孔中，从最稀释到最浓缩。</li>
	<li>在37°C，5%CO2 培养箱中孵育1小时。</li>
	<li>在1小时孵育期结束时，将板带回BSC中，并相应地向24孔或12孔板中加入1mL或2mL甲基纤维素覆盖层。</li>
	<li>将覆盖的板放回37°C，5%CO2 培养箱中，孵育3-7天（病毒株依赖性）。</li>
	<li>孵育后，从培养箱中取出板并带入BSC。将甲基纤维素覆盖物丢弃到装有10%漂白剂或双季铵消毒剂的托盘锅中。</li>
	<li>用PBS洗涤每个孔2x，将洗涤物丢弃到含有10%漂白剂或双季铵的托盘锅中。</li>
	<li>加入约1mL甲醇:丙酮（1:1 v:v），让细胞在室温（RT）下在BSC中固定在板上至少30分钟。根据机构对有机废物的政策丢弃甲醇:丙酮。</li>
	<li>使用下面描述的两种方法之一可视化ZIKV。<ol>
<li>除去甲醇:丙酮后，立即用结晶紫溶液（0.25%w / v在30%甲醇中）染色5分钟。在自来水中冲洗2次并晾干，然后直接用眼睛观察斑块或单层破坏的证据。</li>
		<li>或者，进行聚焦形成测定。<ol>
<li>让板风干，直到没有有机固定剂残留。 注意:这应该在平衡计分卡外需要约2-3小时，但可以通过在平衡计分卡或化学通风橱中风干来加速。</li>
			<li>在轨道板摇臂上，在非无菌PBS（Mg2 + 和Ca2 + 游离）中洗涤每个孔3x15分钟。取出PBS并向每个孔中加入1 mL封闭溶液（PBS + 3%FBS），并在室温下摇动15分钟。</li>
			<li>在封闭溶液中以1:2，000稀释度每孔加入100μL α-ZIKV或α-黄病毒一抗（例如，黄病毒群杂交瘤D1-4G2-4-15 [4G2]）。在室温下摇孵育至少4小时（最好过夜，不超过18小时）。</li>
			<li>除去一抗，在轨道板摇臂上用PBS（不含Mg2 + 和Ca2 +） 洗涤3x15分钟。</li>
			<li>在封闭缓冲液中每孔加入100μL二抗（山羊α小鼠HRP标记）稀释1:2，000。在室温下摇孵育1小时。</li>
			<li>用PBS（不含镁2+和钙2+）在轨道板摇臂上洗涤3x，每次15分钟。</li>
			<li>每孔等分100μL底物显影试剂（材料表）。在室温下岩石板15分钟。</li>
			<li>通过除去底物并用自来水冲洗板2x来停止病灶/斑块的发展反应。在定量之前，倒掉自来水，让板风干。</li>
			<li>计数病毒病灶以确定给定样本中存在的FFU数量。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 血液测量的管理
<ol><li>在关闭后2-4天使用 埃及伊蚊 。将雌性蚊子分分类到0.5升的纸板纸箱中，并带有屏蔽盖，并剥夺它们的糖（通常在传染性血粉前36-48小时）。通过水饱和的棉球 随意 提供水。</li>
	<li>在蚊子将暴露于传染性血粉的早晨去除水饱和的棉球。</li>
	<li>将含有人造血粉的储液槽连接到透明塑料手套箱内的喂养装置引线上。</li>
	<li>在手套箱内，将一个0.5升的纸板纸箱放在与水库相连的喂养装置下方，上面有一个带屏蔽盖的纸箱，里面装有50-100只饥饿 的埃及伊蚊 。 注意:适当饥饿的蚊子通常会在20分钟内进食。可以根据需要延长喂养时间以增加较慢群体中的样本量，尽管这不应超过60分钟，因为（ZIKV）病毒滴度可以在约60分钟后在饲养器内降低。</li>
	<li>喂料完成后，取出储液槽并浸入新鲜制作的10%漂白剂中。</li>
	<li>通过在-20°C下孵育30秒或在冰箱中孵育5分钟来冷麻醉蚊子。</li>
	<li>在手套箱内，将蚊子倒入冰上的培养皿中。从未参与的蚊子中计数并分类充血的雌性。将未吸水的蚊子浸入装有70%乙醇的50 mL管锥形管中处理。当蚊子仍然被麻醉时，将它们倒回0.5L纸板箱中，并迅速用屏幕和盖子盖住。修剪纸箱上多余的筛网，并用胶带固定网状物。</li>
	<li>将一个浸有无菌过滤的10%蔗糖水溶液饱和的棉球加入到每个纸箱的筛子中。将所有蚊盒放入装有湿海绵的大型塑料二级容器中，以保持湿度。</li>
	<li>将装有蚊子纸箱的二级容器放入温度为27±1°C的培养箱中（或酌情模拟感兴趣区域的条件），其80%±10%相对湿度和16:8的光:暗循环）。保持蚊子 随意 获得10%的蔗糖，直到实验完成。</li>
</ol>5. 样品采集和处理
<ol><li>在喂食后的指定日期，使用手套箱内的机械吸气器从适当的纸箱中吸出预定数量的蚊子。在获得必要数量的蚊子后，用棉轮盖住收集管。</li>
	<li>通过在-20°C下孵育30秒或在4°C下孵育5分钟来冷麻醉蚊子。</li>
	<li>在手套箱内，将蚊子倒入冰上的培养皿中。使用两对镊子，取出每只蚊子的所有六条腿，放入预先标记的2 mL圆底微量离心管中，该管中含有无菌不锈钢滚珠轴承（7/32"）和500μL蚊子收集培养基（MCM），由DMEM，2%FBS，1%青霉素 - 链霉素和2.5μg/ mL两性霉素组成。</li>
	<li>轻轻地将蚊子放在一滴矿物油上以约束它，注意不要让油与蚊子的头部和长鼻之间有任何接触。</li>
	<li>将蚊子长鼻插入装有10μL热灭活FBS的10μL移液器吸头中。 注意:或者，移液器可以填充蔗糖，血液或矿物油。该油允许通过光学显微镜直接可视化唾液气泡。</li>
	<li>让蚊子流口水30分钟。将含有FBS +唾液的微量移液管喷射到含有100μLMCM的微量离心管中，然后将胴体放入单独的2mL圆底微量离心管中，该离心管含有灭菌钢球轴承和500μLMCM。确保用于身体，腿部和唾液的管子被标记，以便清楚地表明所有三个样本都来自同一只蚊子。</li>
	<li>当蚊子垂涎时，对剩余的蚊子执行步骤5.3-5.5。</li>
	<li>将包含主体和支腿的管子输送到BSC中包含的珠磨组织均质化装置中。以26 Hz研磨所有身体和腿部样品5分钟，以将病毒颗粒释放到上清液中。通过以200×g离心5分钟以沉淀细胞碎片来澄清所有样品。 注意:此时，样品可以在-80°C下冷冻，或立即测定。</li>
</ol>6. 通过感染性检测检测ZIKV
<ol><li>在BSC中，在感染性测定开始前24小时用Vero细胞（每孔10个5 个细胞）制备24个孔组织培养板。用单个蚊子/样品的身份标记每个孔。</li>
	<li>如果样品在-80°C下冷冻，则允许解冻。</li>
	<li>在用样品一次一个板接种之前，从Vero细胞板中取出培养基。</li>
	<li>对于含有本体或腿的样品，小心地将100μL澄清的上清液等分到每个孔中，注意不要干扰沉淀中的蚊子细胞碎片。 注意:在接种到细胞上之前，唾液样品可以用MCM以1:1（v / v）稀释，以保存样品以供以后滴定（如有必要）。</li>
	<li>将板移入37°C，5%CO2 培养箱中并孵育1小时。</li>
	<li>将板返回BSC，并向每个孔中加入约1mL甲基纤维素覆盖层。将覆盖的板放回37°C，5%CO2 培养箱中，孵育3-7天（病毒/菌株依赖性）。</li>
	<li>按照步骤 3.6−3.9 中所述执行固定和可视化。</li>
	<li>关于通过焦点形成测定的评分，通过在光学显微镜下检查来量化阳性孔。检测孔中细胞的胞浆内染色表明存在病毒。样本仅按焦点正数或负数进行评分。</li>
</ol>

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作者没有什么可透露的。

此处描述的方法提供了执行矢量能力分析的通用工作流。作为一个一般框架，其中许多方法在整个文献中都是保守的。但是，有很大的修改空间（在Azar和Weaver1中进行了审查）。已知病毒（例如，病毒谱系、激发病毒的储存、病毒传代史）、昆虫学（例如，蚊子种群的实验室定植、先天免疫、蚊子微生物组/病毒组）和实验变量（例如，血粉组成、顺序喂养和孵育温度）都会影响?...

载体能力被定义为在物种，种群甚至个体水平上，给定节肢动物（如蚊子，蜱或放血沙蝇）在生物上获取和传播病原体的能力，并在节肢动物1，2中复制或发育。对于蚊子和节肢动物传播的病毒（即虫媒病毒），该病原体由雌性蚊子从病毒血症宿主体内吸收。摄入后，病毒必须有效地感染一小群中肠上皮细胞3，克服各种生理障碍，如消化酶的蛋白水解降解，微生物群的存在（中肠感染屏障，或MIB）和分泌的营养周基质。中肠上皮感染后必须复制病毒并最终从中肠逃逸到蚊子的开放循环系统或血淋巴中，这代表了克服中肠逃逸屏障（MEB）的播散性感染的发作。此时，病毒可以建立次级组织（例如，神经，肌肉和脂肪体）的感染并继续复制，尽管这种二次复制对于病毒感染唾液腺的腺泡细胞（克服唾液腺感染屏障）可能不是严格必要的。从唾液腺腺泡细胞流出到它们的顶端腔，然后移动到唾液管中，使病毒在叮咬时接种到随后的宿主中，并完成传播周期1，2，4，5，6，7。
鉴于这种表征良好且通常保守的蚊子媒介传播机制，实验室媒介能力评估通常在方法上相似，尽管方案确实存在差异1，2。通常，在口腔病毒暴露后，对蚊子进行解剖，以便可以分别测定中肠，腿部，卵巢或唾液腺等单个组织是否存在病毒感染，播散性感染，播散性感染/潜在经卵巢传播和播散性感染/潜在传播能力8。然而，仅仅唾液腺中存在病毒并不是传播能力的明确证据，因为在某些载体/病毒组合1，2，4，5，7，9中存在唾液腺逃逸/出口屏障（SGEB）。证明传播能力的标准方法仍然是蚊子传播给易感动物10，11，12。然而，鉴于对于许多虫媒病毒，这需要使用免疫功能低下的小鼠模型13，14，15，16，这种方法通常成本过高。一种常用的替代方案是收集蚊子唾液，可以通过逆转录聚合酶链反应（RT-PCR）或感染性测定进行分析，以分别证明病毒基因组或感染性颗粒的存在。值得注意的是，这种体外唾液收集方法可能高估了体内进食过程中沉积的12种或17种病毒的量，表明必须谨慎解释此类数据。尽管如此，当从唾液中仅存在病毒的角度进行分析时，体外方法非常有价值，这表明了传播潜力。
确定蚊媒在虫媒病毒病暴发中的作用有两种主要方法。第一种方法涉及现场监测，其中在主动传播的背景下收集蚊子18，19，20，21，22，23，24。然而，鉴于感染率通常相当低（例如，在美国寨卡病毒（ZIKV）传播活跃地区，蚊子的感染率估计为0.061%21），诱捕方法25、26和随机机会（例如，从1，600名未感染者中抽样一名受感染者中抽样一名）可能严重偏向于潜在病媒物种的犯罪21.考虑到这一点，一项特定的研究可能无法获得足够的蚊子的原始数量或物种多样性，以准确检测参与传播的蚊子。相比之下，载体能力分析在实验室环境中进行，允许严格控制口服剂量等参数。虽然不能完全代表现场环境中蚊子感染和传播能力的真正复杂性，但这些实验室评估仍然是arbovirology领域的有力工具。
基于ZIKV在几种蚊子物种，种群和方法27，28，29，30，31，32中的各种载体能力分析，以及最近对载体能力评估1的回顾，我们在这里描述了与典型载体能力工作流程相关的几种方案。在这些实验中，来自美洲（巴西萨尔瓦多市;多米尼加共和国;和美国德克萨斯州里奥格兰德河谷下游）的三个埃及伊蚊种群通过人工血粉以4，5或6对数10聚焦单位（FFU）/ mL剂量暴露于单一的ZIKV菌株（Mex 1-7，GenBank加入:KX247632.1）。随后，通过解剖和基于细胞培养的感染测定，在不同时间外在孵育（2，4，7，10和14天）后，对他们进行感染，播散性感染和传播能力的证据。尽管目前的工作流程/方案针对ZIKV进行了优化，但许多元素可直接转化为节肢动物收容和生物安全等级2和3（ACL / BSL2或ACL / BSL3）中的其他蚊子传播的虫媒病毒。

<table><tbody><tr><td>3mL Standard Reservoir</td><td>R37P30</td><td>Hemotek Ltd</td><td>Insectary Equipment</td></tr><tr><td>7/32&quot; Stainless Steel 440 Grade C Balls</td><td>4RJH9</td><td>Grainger</td><td>Grinding Media</td></tr><tr><td>Acetone, Histological Grade, Fisher Chemicals, Poly Bottle, 4L, 4/Case</td><td>A16-P4</td><td>FisherScientific</td><td>Fixative</td></tr><tr><td>Adenosine 5&#039;-triphospate disodium salt hydrat, microbial, BioReagent, suitable for cell culture</td><td>A6419-1G</td><td>MilliporeSigma</td><td>Reagent</td></tr><tr><td>Anti-Flavivirus Group Antigen Antibody, clone D1-4G2-4-15</td><td>MAB10216</td><td>MilliporeSigma</td><td>Primary Antibody for focus forming assay</td></tr><tr><td>Anti-Mouse IgG (H+L) Antibody, Human Serum Adsorbed and Peroxidase-Labeled, 1.0mL/Bottle</td><td>5450-0011</td><td>KPL/Seracare</td><td>Secondary Antibody for focus forming assay</td></tr><tr><td>Bleach</td><td>NC0427256</td><td>FisherScientific</td><td>Decontamination</td></tr><tr><td>Corning, Cell Culture Treated Flasks, 150cm2, Vented Cap, Case of 50</td><td>10-126-34</td><td>FisherScientific</td><td>Cell culture consumable</td></tr><tr><td>Costar Cell Culture Plates, 24-well, 5/bag, 100/case, Corning</td><td>07-200-740</td><td>FisherScientific</td><td>Cell culture consumable</td></tr><tr><td>Costar Cell Culture Plates, 96-well, 5/bag, 100/case, Corning</td><td>07-200-91</td><td>FisherScientific</td><td>Cell culture consumable</td></tr><tr><td>Crystal Violet</td><td>C0775-100G</td><td>MilliporeSigma</td><td>Stain</td></tr><tr><td>Eppendorf Snap Cap Microcentrifuge Safe-Lock 2mL Tubes, 500/Case</td><td>05-402-7</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon 15mL Conical Centrigue Tubes</td><td>14-959-70C</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon 50mL Conical Centrigue Tubes</td><td>14-959-49A</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon Disposable Polystyrene Serological 10mL Pipets, 200/Case</td><td>13-675-20</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon Disposable Polystyrene Serological 1mL Pipets, 1000/Case</td><td>13-675-15B</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon Disposable Polystyrene Serological 25mL Pipets, 200/Case</td><td>13-675-30</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon Disposable Polystyrene Serological 5mL Pipets, 200/Case</td><td>13-675-22</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Falcon Standard Tissue Culture Dishes</td><td>08-772B</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Fetal Bovine Serum-Premium, 500mL</td><td>S11150</td><td>Atlanta Biologicals</td><td>Cell culture reagent</td></tr><tr><td>Fisherbrand Economy Plain Glass Microscope Slides</td><td>12-550-A3</td><td>FisherScientific</td><td>Immobilization of Mosquitos</td></tr><tr><td>FU1 Feeder</td><td>FU1-0</td><td>Hemotek Ltd</td><td>Insectary Equipment; feeding units</td></tr><tr><td>Gibco DPBS with Calcium and Magnesium, 10 x 500mL Bottles</td><td>140-040-182</td><td>FisherScientific</td><td>Cell culture reagent</td></tr><tr><td>Gibco Fungizone, Amphotericin B, 250&amp;mu;g/mL, 50mL/Bottle</td><td>15-290-026</td><td>Fisher Scientific</td><td>Cell culture reagent</td></tr><tr><td>Gibco Penicillin-Streptomycin (10,000 U/mL), 100mL/Bottle, 20 Bottles/Case</td><td>15-140-163</td><td>FisherScientific</td><td>Cell culture reagent</td></tr><tr><td>Gibco, Tryptsin-EDTA (.25%), Phenol red, 20 x 100mL Bottles</td><td>25-200-114</td><td>FisherScientific</td><td>Cell culture reagent</td></tr><tr><td>Gibcom DMEM, High Glucose, 10 x 500mL Bottles</td><td>11-965-118</td><td>FisherScientific</td><td>Cell culture reagent</td></tr><tr><td>Human Blood, Unspecified Gender, Na-Citrate, 1 Unit</td><td>7203706</td><td>Lampire</td><td>Bloodmeal preparation</td></tr><tr><td>InsectaVac Aspirator</td><td>2809B</td><td>Bioquip</td><td>Insectary Equipment</td></tr><tr><td>Methanol, Certified ACS, Fisher Chemicals, Amber Glass Bottle, 4L, 4/Case</td><td>A412-4</td><td>FisherScientific</td><td>Fixative</td></tr><tr><td>Methyl cellulose, viscosity: 3,500-5,600 cP, 2 % in water(20 &amp;deg;C), 250g/Bottle</td><td>M0512-250G</td><td>MilliporeSigma</td><td>Cell culture reagent</td></tr><tr><td>Micro-chem Plus Disinfectant Detergent</td><td>C849T34</td><td>Thomas Scientific</td><td>Decontamination; working dilution of dual quaternary ammonium</td></tr><tr><td>Mineral Oil, BioReagent, for molecular biology</td><td>M5904-5X5ML</td><td>MilliporeSigma</td><td>Immobilization of Mosquitos</td></tr><tr><td>O-rings</td><td>OR37-25</td><td>Hemotek Ltd</td><td>Insectary Equipment</td></tr><tr><td>Plastic Plugs</td><td>PP5-250</td><td>Hemotek Ltd</td><td>Insectary Equipment</td></tr><tr><td>PS6 Power Unit (110-120V)</td><td>PS6120</td><td>Hemotek Ltd</td><td>Insectary Equipment; power source</td></tr><tr><td>Rubis Forceps, Offset blades, superfine points</td><td>4525</td><td>Bioquip</td><td>Insectary Equipment</td></tr><tr><td>Sarstedt Inc, 2mL Screw Cap Microtube, Conical Bottom, O-ring Cap, Sterile, 1000/Case</td><td>50-809-242</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>Sucrose, BioUltra, for molecular biology</td><td>84097-250G</td><td>MilliporeSigma</td><td>Reagent</td></tr><tr><td>ThermoScientific, ART Barrier Low Retention 1000&amp;mu;L Pipette Tips, 100 tips/Rack, 8 Racks/Pack, 4 Packs/Case</td><td>21-402-487</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>ThermoScientific, ART Barrier Low Retention 200&amp;mu;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td><td>21-402-486</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>ThermoScientific, ART Barrier Low Retention 20&amp;mu;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td><td>21-402-484</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>ThermoScientific, ART Barrier Low Retention, Extended Reach 10&amp;mu;L Pipette Tips, 96 tips/Rack, 10 Racks/Pack, 5 Packs/Case</td><td>21-402-482</td><td>FisherScientific</td><td>Plastic consumable</td></tr><tr><td>TissueLyser II</td><td>85300</td><td>QIAGEN</td><td>Homogenization</td></tr><tr><td>TrueBlue Peroxidase Substrate Kit, 200mL</td><td>5510-0030</td><td>Seracare</td><td>Developing solution for focus forming assay</td></tr><tr><td>Vero</td><td>CCL-81</td><td>American Type Culture Collection</td><td>Mammalian cell line to amplify virus and conduct infectious assay</td></tr><tr><td>Vero C1008 [Vero 76, clone E6, Vero E6]</td><td>CRL-1586</td><td>American Type Culture Collection</td><td>Mammalian cell line to amplify virus and conduct infectious assay</td></tr></tbody></table>

vector competence analyses on aedes aegypti mosquitoes using zika virus

所介绍的程序描述了在实验室条件下用寨卡病毒感染 埃及伊蚊 的通用方法，以确定病毒在相关蚊子种群中的感染率、播散性感染率和潜在传播率。这些程序在全球矢量能力评估中经过各种修改后被广泛使用。它们对于确定特定蚊子（即物种，种群，个体）在给定病原体的传播中可能发挥的潜在作用非常重要。

来自美洲（巴西萨尔瓦多;多米尼加共和国;和美国德克萨斯州里奥格兰德河谷）的三个 埃及伊 蚊种群暴露于来自美洲的ZIKV爆发菌株（ZIKV Mex 1-7，恰帕斯州，墨西哥，2015年），超过一系列血粉滴度（4，5和6 log10 FFU / mL），这些滴度在洗涤后基于红细胞的人造血粉中呈递。在感染后第2天、第4天、第7天、第10天和第14天，对蚊子亚群进行处理，以确定感染、传播和潜在传播率。
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Watch this Scientific Journal Video about Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus at JoVE.com

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

所提出的方案可以确定 埃及伊蚊 种群在收容环境中对特定病毒（如寨卡病毒）的载体能力。

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

vector-competence-analyses-on-aedes-aegypti-mosquitoes-using-zika

Research

JoVE Journal

Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

2.9K 次观看.  University of Texas Medical Branch. 所提出的方案可以确定 埃及伊蚊 种群在收容环境中对特定病毒（如寨卡病毒）的载体能力。

视频: Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

科研

遗传学

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

免疫与感染

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

生物学

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

强迫流涎法分析蚊虫的载体能力

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are &#34;mosquito-borne viruses (MBVs)&#34; and &#34;mosquito-specific viruses (MSVs)&#34;. These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 &#176;C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

从田间收集的蚊子中分离出蚊相关病毒

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

使用寨卡病毒对埃及伊蚊的载体能力分析

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胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

兹卡病毒感染细胞培养系统和

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

经口喂养和显微注射对成年蚊子进行实验性病毒感染

维护伊蚊蚊蚊子感染沃尔巴克

强迫流涎法分析蚊虫的载体能力

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力

从田间收集的蚊子中分离出蚊相关病毒

使用寨卡病毒对 埃及伊 蚊的载体能力分析

摘要

探索更多视频

此视频中的章节

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

兹卡病毒感染细胞培养系统和

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

经口喂养和显微注射对成年蚊子进行实验性病毒感染

维护伊蚊蚊蚊子感染沃尔巴克

强迫流涎法分析蚊虫的载体能力

使用荧光染料，罗达明B，研究雄性 伊蚊 的交配竞争力

从田间收集的蚊子中分离出蚊相关病毒

使用寨卡病毒对埃及伊蚊的载体能力分析

使用荧光染料，罗达明B，研究雄性伊蚊的交配竞争力