We would like to thank the Texas Pierce&#8217;s Disease Research and Education Program and USDA-APHIS for their funding support for this project. We would also like to thank Hema Kothari at the University of Texas Health Science Center at Tyler for her assistance with confocal microscopy.

1，细胞培养注：由韦恩·亨特博士的实验室在美国农业部农业研究服务局（英尺皮尔斯，美国佛罗里达州）建立Homalodisca vitripennis细胞系用于启动一个实验室股混合细胞阶段，包括最初的成纤维细胞和单分子层组成。 <ol><li>在保持在20-24℃的温度范围为25 平方厘米培养瓶无菌的实验室环境，请执行以下步骤。 </li><li>培育和利用H2G +叶蝉介质中，修饰WH2蜜蜂媒体10（ 表1）中为25cm 2组织培养瓶中维持培养物。 </li><li>孵育培养瓶在24℃下以〜53％的湿度。 </li><li>采用倒置显微镜，总放大倍数100倍（TM）监测的文化发展， 例如，检查是否有异常细胞的生长，监视任何可能的污染物，并检查整个CUL成长进步TURE表面。 </li><li>执行完整的媒介变化（〜每瓶4毫升培养基）每7 - 11天无干扰的培养表面。 </li><li>通过培养物在培养表面是覆盖通过使用0.25％胰蛋白酶含有乙二胺四乙酸（EDTA）以解离细胞的细胞生长（汇合）约为80％。添加〜2 - 3 ml胰蛋白酶的每个培养瓶中，并露出培养（次），以酶为很短的时间（5 - 10分钟），以实现完全的细胞解离。 </li><li>加入新鲜培养基等量的培养瓶（次）（〜2 - 3毫升）以停止酶的活性和整个溶液转移到离心管中离心。 </li><li>离心沉淀细胞，在4℃下进行6分钟，在350×g下。 </li><li>除去上清液，而不会干扰细胞沉淀并加入〜加入8毫升的新鲜培养基到每个管中。轻轻均匀用吸管细胞。 </li><li>分离培养以1：2的比例25 平方厘米瓶（〜4毫升细胞soluti的每个烧瓶），并留下新鲜通过培养静置48小时，以允许细胞在悬浮液中牢固地附着在烧瓶的表面上。 注：培养的细胞在48孔无菌组织培养板中以1 cm 2的生长表面也是可能的，并且它们可以被保持在相同的方法培养烧瓶中，在〜250μl的培养基中，以体积减少。 </li></ol> 2，全病毒提取<ol><li>均质病毒阳性H.整个身体通过涡旋〜10秒的间隔vitripennis在磷酸盐缓冲液，pH〜7.2，用0.02％甲醇钠二乙三水合物（DETCA），直到没有组织存在的更大的团块。通过提取超高速离心病毒在124,500 XG 4小时，在4°C或22,000 XG 16小时在4℃。如果沉淀形成顶部，用消毒棉签11将其删除。 </li><li>弃上清。 </li><li>收集沉淀并溶于5毫升的10mM磷酸盐缓冲液（无DETCA），pH约7.2，含0.4％的Na-脱氧胆酸和4％聚乙二醇十六烷基醚（的Brij 52）。 <ol><li>以混合粒料良好，从该管的一侧除去颗粒和粉碎，直至在溶液中，如果有必要的。 </li><li>加在〜5个2ml增量更多的10mM磷酸缓冲液，以帮助颗粒进入溶液中，如果需要，结合成2个管11。 </li></ol></li><li>离心溶液以300×g离心15分钟11。 </li><li>除去上清液，并通过0.45μm的过滤器的溶液中，并收集在大量收集管11中的滤液。 </li><li>转移滤液至透析膜具有截留分子量（MWCO）为3.5 kD的，用10mM的少量的，如果需要11不含有DETCA磷酸盐缓冲液（pH 7）。 </li><li>将透析膜在一个大烧杯中，在4℃下充满DDH 2 O。改变DDH 2 O的每一个小时˚F或5 - 6小时，直至形成白色沉淀在膜11。 </li><li>从膜中收集纯化的病毒，并通过添加10微升的溶液中，以90微升的DDH 2 O中经受了100％的病毒溶液，以10倍稀释系列随后加入10微升稀释到另一个90微升DDH 2 O，直到达到稀释1：100,000。 </li><li>商店病毒解决方案在-80℃。 </li></ol> 3，病毒复制<ol><li>种子细胞在48孔培养板和成长中的所有行，直至细胞生长为80％汇合（约72小时后通，如果电池正在以平均速率）。 </li><li>接种细胞中的每一行与系列稀释的病毒一旦细胞生长达到汇合， 即，1行中的1:10稀释的，为1之一行：100稀释， 等等 ，除了最上面一行。使用顶行作为对照，并添加10微升DDH 2 O的每个以及音量控制。 <ol><li>为了建立一个起始基线细胞浓度与试验细胞计数相比，解离和计数每一行的第一个孔之前的初始接种在与病毒的培养物的任何孔。 </li></ol></li><li>监控培养皿病毒接种于任何颜色的变化在中显示的pH值变化和细胞形态的任何改变后，每24小时。 </li><li>试验板在每个时间点，使用在100X TM倒置显微镜的图象的一列。 </li><li>从被成像在每24小时的时间点的列中删除所有介质和存储它的短期在-20℃下提取RNA和病毒定量或长期在-80℃下。 </li><li> 1.9，只用〜250微升的0.25％胰蛋白酶EDTA和新鲜培养基以停止酶的活性和〜250微升新鲜培养基重新悬浮细胞PELLE -除去介质如先前在步骤1.6中所述后解离从孔中细胞吨。 </li><li>加入10微升的0.4％台盼蓝染色，以含有细胞的细胞解离为每24小时期间超过一周后进行细胞计数每个管。让污渍进行细胞计数前坐了10分钟。 <ol><li>在1小时内暴露于染色或存活的细胞进行细胞计数将开始摄取染色以及非活细胞。 </li></ol></li><li>慢慢加入10微升的染色细胞溶液以使用微量标准血细胞计数器的每一侧上。使溶液就由毛细管作用，避免气泡。 </li><li>计数存活的（非染色）细胞在血细胞计数器的两侧的数目（4计数的血细胞计数仪的16个正方形的等价物）。进行细胞计数为被删除的列的每个孔中。 </li><li>从每一列平均细胞数，并使用下面的公式来获得总细胞计数数：×稀释因子（细胞/ 4的平均数目）=细胞×10的数<suP&gt;4毫升。这是细胞密度或活细胞数在培养。 </li></ol>从细胞培养4。病毒提取<ol><li>删除处理H. vitripennis细胞从培养烧瓶中步骤1.6之前描述- 1.8，只用〜250微升的0.25％胰蛋白酶EDTA和新鲜的培养基中，以停止酶的活性。 </li><li>弃上清。 </li><li> 2.8 -从培养细胞按照先前在步骤2.3中所述的协议提取病毒。 </li></ol> 5，RNA提取<ol><li>从每一周病毒试验用硫氰酸胍 - 酚 - 氯仿提取设计用于根据制造商的协议液体样品中收集的培养基样品中提取RNA。 </li><li>商店提取的样品在-80℃。 </li></ol> 6，RT-PCR法<ol><li>通过运行传统的PCR使用建立的病毒标准的RT-PCR引物对HoCV的RT-PCR引物1（正向5&#39;-GCTCCCCGGCTTTGCTGGTT-3&#39;，反向5&#39;-ACGACGGATCTGCGTGCCAA-3&#39;）与病毒从全身H.隔离vitripennis。 </li><li>受试者的PCR产物在含有0.1％溴化乙锭的2％琼脂糖凝胶电泳60分钟，在120伏特。 </li><li>消费税从凝胶条带，纯化使用凝胶提取试剂盒按照制造商的协议。 </li><li>池中的所有切出，并凝胶纯化的产物和由基本乙醇沉淀进一步纯化。洗脱沉淀产物在约30微升的Tris EDTA（TE），以增加整体的样品浓度。 </li><li>通过采用核酸检测260/230波长分光光度法设置量化的cDNA水平混合样品中。 </li><li>进行10倍连续稀释系列的纯化的样品为57纳克/微升至57 AG /微升（10 -18）。执行一个类似的连续稀释描述íÑ2.8与浓度要求的不同进行调整。 </li><li>通过使用定量RT-PCR试剂盒用的低丰度转录物可靠的量化进行的qRT-PCR测定的稀释系列的检测限。 注意：超过5×10 -3份低病毒浓度是不可检测的。 </li><li>从实验样本中提取的RNA按照步骤5.1之前描述和量化使用分光光度法。 </li><li>所有标准化提取样本，以5纳克/微升用无核酸酶的水。 </li><li>对所有样品进行的qRT-PCR中，在重复的，因为使用单步定量RT-PCR试剂盒和用于感测低拷贝数的能力，将25μl的反应如下：50℃保持10分钟; 95℃，保持5分钟; 95℃的30个循环持续10秒，60℃30秒;熔体从50 - 99℃下进行5秒的每个步骤。 <ol><li>使主结构，使每个反应混合物中含有12.5微升1个maste的ř组合，正向引物1.0μL（0.3微米），1.0微升反向引物（0.3微米），0.25μL逆转录酶和模板的基础上的标准化值的变量金额。 </li><li>使总反应体积为25μl用无RNA酶的水。 </li><li>包括五个标准的浓度在每个PCR用下面的拷贝数运行：5×10 -10，5×10 -8，5×10 -6 5×10 -4，5×10 -2份。设置阈值的每个运行到刚好低于10倍-2.5荧光早期采集在每次运行的开始，以降低噪声。 </li></ol></li></ol> 7，激光共聚焦显微镜<ol><li>生长在12孔含有板玻璃盖玻片上测量直径18毫米，每孔Homalodisca vitripennis细胞。 </li><li>在平板上接种一列，每24小时为一个周期的四天，一旦单层实现。确保每个Column包含一个控制良好，低病毒稀释（1:10）以及和高病毒稀释液（1:10万）好。使用步骤2.8获得病毒解决方案。 </li><li>删除所有媒体的第五天和洗涤细胞用1×PBS（pH7.4）中的两倍。 </li><li>固定细胞用冷的4％多聚甲醛在4℃下进行30分钟。 </li><li>添加500μl的1X磷酸盐缓冲盐水（PBS）中的细胞，并在室温冲洗10分钟，在一个摇杆在低速。清洗细胞三次。 </li><li>加入500微升0.1％的Triton X-100的细胞通透它们。让我们坐在室温10分钟。 </li><li>洗涤细胞一次，在步骤7.5，如前所述。 </li><li>加入500微升的5％牛血清白蛋白（BSA）溶液以阻止细胞在RT。让我们坐了2小时，然后取出。 </li><li>在含5％BSA的1×PBS 250，并添加250μl的稀释液到每个孔中，以染色与F-肌动蛋白的稀释：股票罗丹明红缀合的鬼笔环肽（RCP）1。 </li><li>盖板铝˚F油，以防止染料的漂白和培养细胞在4°CO / N。 </li><li> 12小时温育后，除去RCP和其用250μl的4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI）（250微克/毫升）稀释在1×PBS中含有5％BSA，以染色细胞的核取代。 </li><li>孵育含有DAPI在RT 1小时的细胞。 </li><li>如先前在步骤7.5中所述用1×PBS洗涤细胞三次。 </li><li>轻轻地取出从井盖玻片并安装使用的安装介质具有防褪色试剂显微镜幻灯片。 </li><li>使载玻片干燥在遮光框，直到共聚焦显微镜下观察。查看幻灯片准备尽快染料能迅速褪色。 </li><li>使用装有含63X（油）计划，apochromate镜头显微镜共焦系统映像染色的细胞。 <ol><li>设置激光的波长为543±10 nm激发和575±10 nm发射的Rhoadmine红conjucated鬼笔环肽和369±10 nm激发和450±30 nm发射的DAPI染色。 </li><li>使用相同的增益和抵销设置探测器获得的所有图像。 </li><li>利用图像处理相应的软件和排序，进口所需的图像转换成图像管理软件过程的图像。 </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Exp. Cell. Res. 25, 585-621 (1961).</li><li>Goetz, I. E., Moklebust, R., Warren, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+some+antibiotics+on+the+growth+of+human+diploid+skin+fibroblasts+in+cell+culture.">Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture.</a> In Vitro. 15 (2), 114-119 (1979).</li><li>Coriell, L. L., Fogh, J. .. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+prevention+of+bacterial,+fungal,+and+other+contaminations.">Methods of prevention of bacterial, fungal, and other contaminations.</a> Contamination in Tissue Culture. , 29-49 (1973).</li><li>Hambor, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+Design+and+Bioprocess+Controls+for+Industrialized+Cell+Processing.">Bioreactor Design and Bioprocess Controls for Industrialized Cell Processing.</a> Bioprocess Tech. Bull. 10 (6), 22-33 (2012).</li><li>Abbot, A., Cyranoski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology&#8217;s+new+dimension.">Biology&#8217;s new dimension.</a> Nature. 424, 870-872 (2003).</li></ol>

The authors have nothing to disclose.

关于农业侵入物种的大量涌入上升的担忧引发了新的方法来对出现的害虫和病原体防御的需求增加。疾病预防和管理的重点包括病原载体的管理，是本研究的主要目标。经济学中生产这种类型的生物农药来管理病原载体的农业，因为在实际应用程序需要大量的广大地区，但以较低的成本12的决定起着至关重要的作用。利用细胞培养物的研究和开发的实践中已经变得越来越普遍，因此，本研...

玻翅神枪手（Homalodisca vitripennis Germar 1821）已被确定为Xylella难养 （ 十难养 ）的主要载体，小道消息（PD）的皮尔斯病的病原在北美1。昆虫种群的管理已经迅速成为研究的重点，以打击在加州和整个美国南部这个毁灭性的问题的葡萄种植业。属于家人正意义上说，单链RNA病毒Dicistroviridae，Homalodisca coagulata病毒01（HoCV-01）已经在野生H.确定vitripennis人口和证实会增加死亡率在这些人群2-4，同时降低昆虫的抗杀虫剂。 方法的发展，有效地后方感染H。 vitripennis在实验室环境到成年一直很困难，因为<eM&gt;小时。 vitripennis有需要多种寄主植物5-8不同阶段的特定营养需求。特定设备是必需的，以后部活H. vitripennis在美国;因此，细胞培养物是更经济，可行的替代方案，以及用于HoCV-01检测和复制2,9日益重要。而基本的方法用于建立H的细胞培养vitripennis进行了描述，这些方法还没有被用于商业化生产的生物控制剂，如病毒2。 以下程序的总体目标是生产出高浓度HoCV-01适合于利用作为生物控制剂。病毒复制需要一个活细胞，这就是为什么成功的培育和优化H. vitripennis文化是产生病毒的盈利水平的进步至关重要。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:93px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Corning&#160;cell culture flasks</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>CLS430168</td>
			<td>Surface area 25&#160;cm2, canted neck, cap (plug seal)</td>
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			<td height="21" style="height:21px;">Olympus DP30BW, IX2-SP, IX71</td>
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			<td style="width:119px;"></td>
			<td>Inverted microscope and camera</td>
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			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>T4049</td>
			<td>0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2&#160;g EDTA &#8226; 4Na per liter of Hanks&#8242; Balanced Salt Solution with phenol red</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Greiner CELLSTAR&#160;multiwell culture plates</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M8937</td>
			<td>48 wells (TC treated with lid)</td>
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		<tr height="63">
			<td height="63" style="height:63px;">DETCA&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>228680</td>
			<td style="width:167px;">Sodium diethyldithiocarbamate trihydrate</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning&#160;bottle-top vacuum filter system</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>CLS431206</td>
			<td>Cellulose acetate membrane, pore size 0.45&#160;&#956;m, membrane area 54.5&#160;cm2, filter capacity 500&#160;mL</td>
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			<td height="42" style="height:42px;">Brij 52</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>388831</td>
			<td>Polyethylene glycol hexadecyl ether</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffer solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P5244</td>
			<td>Recieved as 100mM diluted to 10mM with sterile water</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">TRIzol LS&#160;</td>
			<td style="width:93px;">Life Technologies</td>
			<td>10296-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agarose</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>A5304</td>
			<td>For electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ethidium bromide</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>E7637</td>
			<td>BioReagent, for molecular biology, powder</td>
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		<tr height="21">
			<td height="21" style="height:21px;">QIAquick</td>
			<td style="width:93px;">Qiagen</td>
			<td>28704</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">QuantiTect qRT-PCR kit&#160;</td>
			<td style="width:93px;">Qiagen</td>
			<td>204243</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">4% paraformaldehyde&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P6148&#160;</td>
			<td>Reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PBS&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>P5368</td>
			<td style="width:167px;">Phosphate buffered saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bovine serum albumin (BSA)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Rhodamine red-conjugated phalloidin&#160;</td>
			<td style="width:93px;">Life Technologies</td>
			<td>R415</td>
			<td>Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DAPI&#160;</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">ProLong Gold Antifade Reagent</td>
			<td style="width:93px;">Life Technologies</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM510 Meta Confocal System&#160;</td>
			<td style="width:93px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM Zen 2007 Software</td>
			<td style="width:93px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Grace&#8217;s Insect medium (supplemented, 1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G8142</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-histidine monohydrate</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>H8125</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium 199 (10X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M4530</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium 1066 (1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">C0422</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#8217;s Balanced Salts (1X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>51322C</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-Glutamine (100X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G3126</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM, amino acid mix (50X)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>56419C</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1 M MgCl solution</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>M8266</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pen-Strep (w/ Glutamine)</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>G6784</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nystatin</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>N6261</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gentamycin</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>46305</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextrose</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>D9434</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td>F2442</td>
			<td>H2G+ leafhopper medium component</td>
		</tr>
	</tbody>
</table>

propagation of homalodisca coagulata virus-01 via homalodisca vitripennis cell culture

玻翅神枪手（Homalodisca vitripennis）是整个美国西南部发现了一个非常vagile和杂食性昆虫。这些昆虫是Xylella难养（十难养）的主要载体，木质部限细菌是葡萄皮尔斯病（PD）的致病因子。皮尔斯病是造成经济损失;因此，H vitripennis已成为病原体管理战略目标。认定为Homalodisca coagulata病毒01（HoCV-01）一个dicistrovirus已在阁下的死亡率增加有关vitripennis人群。由于宿主细胞需要HoCV-01的复制，细胞培养为目标的复制是后勤和生物农药生产经济价值的统一环境。在本研究中，对于H的大规模繁殖系统通过组织培养vitripennis细胞发达，Providing病毒复制的机制。 HoCV-01是从整个身体的昆虫中提取并用于接种培养的幽门vitripennis细胞在不同的水平。将培养基除去，每24小时为168小时，萃取，用定量RT-PCR分析的RNA。细胞用台盼蓝染色并计数用光学显微镜来量化细胞生存能力。全病毒颗粒中提取多达96小时感染后，为确定为总细胞培养崩溃发生之前的时间点。细胞也进行荧光染色，并用共聚焦显微镜研究上的F-肌动蛋白的附接和细胞核完整性病毒活性观看。本研究的结论是H。 vitripennis细胞能够被培养，并用于批量生产HoCV-01在一个合适的水平，使生产的生物农药。

细胞的附着和生长在小型和大型培养瓶在48小时通行看出，从原代培养和传代延续。成纤维细胞生长和发展也在此时间范围内观察到。当新种子培养瓶48小时前不安，有细胞附着可见的下降，导致生长缓慢的文化和时有时无的附件或增长的。细胞在约80％汇合的传球的一个星期内，并在10-14天（ 图1）形成的单层。分别培养在本研究的开始即原代培养物，已存活30 +细胞的通道无任何形态...

Watch this Scientific Journal Video about Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture at JoVE.com

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

在这里，我们提出了一个协议来传播Homalodisca vitripennis细胞和HoCV-1 体外 。培养基从HoCV-1阳性培养去除和RNA的提取，每24小时为168小时。细胞生存力进行定量通过台盼蓝染色法。整个病毒粒子中提取的后感染。提取的RNA进行定量通过qRT-PCR。

传播<em&gt; Homalodisca coagulata病毒01</em&gt;通过<em&gt; Homalodisca vitripennis</em&gt;细胞培养

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

propagation-homalodisca-coagulata-virus-01-via-homalodisca

Research

JoVE Journal

Immunology and Infection

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

11.6K 次观看.  University of Texas at Tyler. 在这里，我们提出了一个协议来传播Homalodisca vitripennis细胞和HoCV-1 体外 。培养基从HoCV-1阳性培养去除和RNA的提取，每24小时为168小时。细胞生存力进行定量通过台盼蓝染色法。整个病毒粒子中提取的后感染。提取的RNA进行定量通过qRT-PCR。 

视频: Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

科研

免疫与感染

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

巨型囊泡内单细胞层的细菌细胞培养

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部主动脉瘤小鼠模型中脉冲传播速度、失散性和应变的测量

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...