我们要感谢S.沃纳提供在这项研究中，并使用美国农业部的乌鸦，美国动植物检疫局，WS，NWRC照顾动物的工作人员对动物的照顾和监控。提及或使用产品并不意味着美国农业部认可。资助这项研究是由美国农业部动植物检疫局，兽医服务提供。

我们的协议是改编自一个大家此前公布的7。所有涉及动物的程序已获农业部美国农业部（USDA），动植物健康检验局（APHIS），野生动物服务（WS），国家野生动物研究中心（NWRC）的机构动物护理和使用委员会。 1。乌鸦Gavaging <ol><li>通过美国乌鸦的消化道估计的"伪大脑物质"通过时间。 <ol><li>将5毫升煮和炒鸡蛋全用蓝色染料和灌胃1乌鸦用灌胃针2（ 图1）。 </li><li>检查乌鸦每隔30分钟，直至蓝色/绿色染色的粪便不再被排出体外。 </li></ol></li><li>获得感染和患绝症的RML（钱德勒株）感染的C57BL / 6小鼠的大脑。 </li><li>产生20％的重量/体积正常和受感染的小鼠脑匀浆中的子弹搅拌器匀浆用1X磷酸盐缓冲盐水（PBS）和玻璃珠，然后离心以3,000×g离心1分钟，以除去较大的颗粒物质。除去上清，用1×PBS等体积的稀释以产生10％重量/体积的无菌PBS中。直到需要冻结在-80°C。 </li><li> 17前灌胃小时取出食物，但不能积水，从乌鸦笔。 </li><li>随机分配乌鸦到治疗组和在管饲针以2口服灌胃各乌鸦用5ml正常或感染的小鼠脑匀浆。 </li><li>乌鸦转移到单独的笼子，并收集和集中每个笼子内的所有粪便4小时后灌服（ 图2）。 </li><li>均质粪便汇集每个乌鸦在子弹搅拌器匀浆与玻璃珠直至均匀质地的实现。 *乌鸦粪便大多是液体，并且可以很容易被混淆。 </li><li>对于每个乌鸦，稀500μl的粪便匀浆成9.5毫升的1×PBS的10毫升的总体积。 </li><li>离心粪便homogenaTE 15分钟，1400×g离心并提取上清液。 </li><li>为了尽量减少二次微生物感染的威胁，治疗上清液用1微升100单位/毫升青霉素和100微克/毫升链霉素（Invitrogen公司，NY）中，每100微升匀浆，然后在UV光下放置在室温下20分钟，以减少病毒和细菌污染的风险。紫外线照射后，在3000万像素超声波仪在设置70下30秒以破坏任何剩余微生物膜的声处理样品。 </li></ol> 2。小鼠接种<ol><li>随机分配小鼠到以下治疗组（ 表1）: <ol><li>第1组 - 积极的治疗的小鼠腹腔接种（IP）用1毫升粪便匀浆从乌鸦喂饲用5ml感染小鼠的大脑。 </li><li>第2组 - 负治疗小鼠知识产权接种1毫升粪便匀浆从乌鸦喂饲用5ml正常小鼠的大脑。 </li><li>稀infecteD与正常小鼠脑匀浆组3和4至1:100 w / v的在1X PBS。 </li><li>组3 - 正对照小鼠接种IP用1ml的感染的小鼠脑匀浆。 </li><li>第4组 - 阴性对照小鼠接种IP用1毫升的正常小鼠脑匀浆。 </li></ol></li></ol><table border="1"><tbody><tr><td></td><td>治疗组</td><td>动物的数目</td></tr><tr><td>第1组</td><td>痒病+乌鸦屎</td><td> 100 </td></tr><tr><td>第2组</td><td>痒病 - 乌鸦屎</td><td> 25 </td></tr><tr><td>第3组</td><td>痒病+小鼠脑</td><td> 10 </td></tr><tr><td>第4组</td><td>痒病 - 小鼠脑</td><td> 5 </td></tr></tbody></table> 。 接种表1痒病状态（阳性+，负- ）和动物的使用数量7。 <ol start="2"><li>腹腔接种小鼠: <ol><li>颈背通过鼠标背部颈部的皮毛用拇指和食指轻轻转动，露出腹侧。 </li><li>提升鼠标后端所以头略低。 </li><li>将25号针头1厘米通过皮肤，中线为1厘米的横向，和1-2厘米用针锁式注射器前到骶髂关节。 </li><li>注入1毫升慢慢接种到小鼠体腔。 </li></ol></li></ol> 3。鼠标监测<ol><li>每天监测小鼠，直到他们表达小鼠瘙痒病的​​临床症状。临床症状包括:驼背，走路不稳，僵硬的尾巴，缺乏疏导，形体消瘦，嗜睡。 </li><li>得分老鼠各6临床体征时明显迹象是明显的，其中0 =没有可见的，1 =温和，2 =严重。 </li><li>安乐死在小鼠每日总成绩为每个符号1天达到≥8≥6连续3天，或在365天后接种（DPI）。 </li><li>收获的大脑紧随euthan亚洲并储存于-70℃。 </li><li>以确认诊断痒病，消化脑样品用3微升的蛋白酶K溶液（PK）50微克/毫升稀释如下:3.1。微升PK，12.5微升的500mM EDTA，pH为8，109.39微升1×PBS中的30分钟，在45℃，然后通过添加8微升加样缓冲液，并温育的样品在95℃下5分钟灭活的PK。负载样品到12％SDS-PAGE凝胶，electrophorese和转移到Immobilon PVDF膜，并用5％脱脂牛奶块中的0.2％吐温20的PBS溶液1小时，在室温下进行。然后探测与Bar224抗PrP的单克隆抗体缀合到辣根过氧化物酶，稀释于Superblock中，1小时在室温下。漂洗膜1小时，用PBS-0.2％吐温20。为了形象化，培养印迹5分钟，化学发光底物和图像上的G-box凝胶成像系统。 </li></ol>

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没有利益冲突的声明。

我们展示了一个过程，通过乌鸦的消化系统记录的PrP的住宅的材料通过。我们所用的常规方法，以确定是否有乌鸦易位的PrP 住宅材料到无病的地理区域的能力。他人已经评估的PrP 分辨率的电阻给反刍19-21和啮齿类22,23消化液，两者不能消除它。这些技术未来的应用应该可以应用到其他食肉动物24，因为他们也有可能遇到的PrP 住宅材料腐肉和运输这种材料在景观促进朊病毒疾病的传播。 我们添加了蓝色的食用色素给我们炒鸡蛋"伪大脑的物质，提供了一个简单的方式，通过乌鸦的消化道评估匀浆小鼠大脑近似通行时间。我们奉劝别人考虑这种技术首先插件他们期望研究动物的粪便中存在的颜色，然后用食用色素是最鲜明的对比。这将允许以易于识别的，通过研究动物已通过食色染色材料。我们选择使用食用色素来估计通过时间，但其他已使用荧光颜料25，氧化铁26，有色塑料标记物27，并涂上金属片状粉末（ 即闪耀）28。 尽量减少乌鸦粪便二次微生物感染的威胁，正确接种小鼠是主要的出发点，需要很长的潜伏期小鼠朊病毒研究。如果这些因素受到侵犯时，小鼠早期死亡率可能会导致。 调查的另一个可能的工具是串行的蛋白质错误折叠循环扩增或sPMCA评估乌鸦粪便样品随着时间的推移，以确定如何经过长期口服灌胃乌鸦传递传染性物质，具有出需要进行小鼠生物。通过Pulford酒店中PMCA粪便分析的最新进展， 等。表明，朊病毒分钟级别的扩增可以在哺乳动物粪便29被检测到，这表明PMCA也可能用于评估禽粪便中的有用工具。小鼠生物检测和sPMCA方法可用于评估残余感染的乌鸦喂饲CWD感染材料的粪便。一个cervidized转基因小鼠线须与脑内与腹腔接种将给予更快速的结果。 禽流清除剂，如乌鸦，秃鹫，和鹰，能够在谢疾病的传播起到一定的作用，即中华白海豚在北美。这些物种可能与患病屠体或内脏（在猎人杀死的鹿类的情况下）消耗CWD阳性的组织，并在其粪便转运感染性物质CWD自由区或鹿类种群。至于喂食谷物鹿类，在实践野生或圈养设置，吸引乌鸦可以排便的食物来源，并无意中鹿类消耗掉，因此是一个高风险的做法（VerCauteren个人观察）。此外，虽然CWD无动物通过随机粪便沉积遇到的PrP 住宅材料的几率可能较低，乌鸦在那里，因而其粪便集中的地区，如下面公用夜栖地点，可能成为疾病传播的高风险领域，因为朊病毒如此执着于环境30,31。

传染性海绵状脑病（TSE）是致命的感染性神经退行性疾病，影响野生动物，家养动物和人类。谢疾病的传染性病原体似乎是朊蛋白1的错误折叠或致病亚型（PRP 住宅 ）。动物TSE疾病包括慢性消耗性疾病（CWD）的黑尾鹿（ 白尾野驴 ），白尾鹿（ 白尾virginianus）来说 ，麋鹿（ 马鹿 ），和驼鹿（ 驼鹿 ）;痒病的绵羊和山羊，牛海绵状脑病（ BSE）在国内的牛，在养殖水貂传染性水貂脑病;猫猫海绵状脑病，在异国情调的异国情调的动物园有蹄类动物海绵状脑病反刍的牛科家族;及海绵状脑病在非人灵长类动物8。单人谢氏病，变异型克雅氏病，是罕见的，认为通过消耗PrP的住宅 -康泰明收购ATED食物9。同样，疯牛病可以感染人类，如果被污染的牛肉消耗10。所有TSE疾病，痒病和CWD是仅有的两个具有自我维持的流行病和源感染被假定为从动物到动物2,3,11以及从环境源4-6。研究表明，大多数TSE疾病需要从PrP的住宅的材料自然暴露事件显着延长潜伏期的临床症状表现2-4,6,8和表观物种界限减少，但不排除对，种间传播12-14的潜力。 识别机制传染性朊病毒（PrP的住宅 ）物质的扩散是回答有关TSE疾病在景观是如何移动的问题极为重要。实验研究表明，昆虫15,16，家禽和猪17日 ，美国和乌鸦（ 乌鸦座曼布拉克hyrhynchos）7,18是PrP的住宅材料的被动载体或传播者。通过乌鸦的消化系统通道的PrP的住宅的材料最近被记录在案，这表明他们在谢病7分散可能发挥的作用。这些结果使得合理的，乌鸦，一个清道夫，可能会遇到，消费，并通过粪便沉积运输传染性物质，对无病的地区。 我们在这里展示的程序是用来通过乌鸦的消解系统记录的PrP的住宅的材料通道，将极大地促进这些方法应用到其他净化剂和食肉动物物种特异性的车型在今后的相关研究。在这项研究中传统的方法被用来研究人口贩运的PrP 住宅材料，这可能有助于PrP的住宅材料的扩散和总体负担的非常规手段。

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

传染性朊病毒（PrP的住宅 ）的材料很可能是致命的，神经退行性传染性海绵状脑病（TSE）的疾病病因1。谢性疾病，如慢性消耗性疾病（CWD）的传输，被假定为从动物到动物2,3以及从环境来源4-6。清除剂和食肉动物有潜力通过消费和CWD污染腐肉的排泄，转运的PrP 住宅材料。最近的工作已经通过美国乌鸦的消化系统（ 乌鸦座brachyrhynchos），一个共同的北美清道夫7记载的PrP的住宅的材料通过。 我们描述的程序用于通过美国乌鸦记录的PrP的住宅的材料通过。乌鸦被灌服RML株小鼠适应痒病和他们的粪便收集4小时后灌服。乌鸦粪便，然后汇集和腹腔注射进C57BL / 6小鼠。小鼠每天监测，直到他们表示鼠标瘙痒病的临床症状，并随后实施安乐死。无症状的小鼠进行了监测，直到接种365天岗位。 Western blot分析进行确认疾病状态。结果表明，朊病毒通过乌鸦消化系统行驶后保持感染性和存在于粪便中，引起疾病中测试小鼠。

所使用的程序表明，乌鸦的消化系统并不之后的痒病脑匀浆7口灌胃消除朊病毒住宅感染4小时。这是灌服PrP的住宅的材料全部20只乌鸦随后通过粪便传播的PrP 住宅材料老鼠。病鼠均经临床鼠标痒病体征和疾病的确认通过Western blot分析完成的体现。 通过消化道的灌胃乌鸦为4小时，根据染料的粪便中存在（ 图1）的材料由乌鸦摄入的保留时间的调查显示流逝的时间。所有收集粪便用一次性移液管和汇集每个乌鸦（ 图2）。当原粪便上清液注入的IP为先导试验小鼠发生了未经处理的粪便乌鸦急性毒性。通过将粪便接种用青霉素和STREptomycin，紫外线，和超声处理我们缓解了这个问题。 接种或瘙痒病阳性的小鼠脑（9/9）或从痒病接种乌鸦屎所有小鼠（66 * / 84）开发的临床体征和测试通过Western印迹分析阳性（ 图3），说明痒病通过易通过乌鸦和引起疾病的消化系统。 * 18只在接种后3天死亡，可能是由于毒性。所有，但一（1/23）痒病阴性接种小鼠为阴性的Western blot分析。我们猜测，这款鼠标是无意中接种痒病阳性乌鸦粪便，而不是从痒病阴性乌鸦屎。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig1.jpg" width="500px" /> 图1。乌鸦手动内敛而用5毫升全蛋的蓝色染料（a）和b混合喂饲吕/绿粪染后4小时灌胃（B） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig1highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig2.jpg" width="500px" /> 图2。乌鸦粪便收集与吸管之前的同质化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig3.jpg" width="500px" /> 图3。 。小鼠大脑的生物测定代表性的SDS-PAGE电泳免疫印迹 NBH正常小鼠脑匀浆，痒病阴性，TX笼4 -大脑从小鼠接种从痒病接种乌鸦屎。用（+）和withou吨（ - ），PK（蛋白酶K）消化<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig3highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

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

清除剂具有潜在的易位感染传染性海绵状脑病朊病毒在其粪便到无病的区域。我们用来确定是否通路虽然美国乌鸦的消化道（ 乌鸦brachyrhynchos），死亡动物的共同消费后小鼠适应痒病朊病毒保持传染性细节的方法。

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 ...

procedures-for-identifying-infectious-prions-after-passage-through

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.0K 次观看.  USDA. 清除剂具有潜在的易位感染传染性海绵状脑病朊病毒在其粪便到无病的区域。我们用来确定是否通路虽然美国乌鸦的消化道（ 乌鸦brachyrhynchos），死亡动物的共同消费后小鼠适应痒病朊病毒保持传染性细节的方法。 

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

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

病毒浓度的测定通过空斑试验:用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

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 ...

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.

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 ...

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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...