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>

<ol>
	<li>Takiya, D. M., McKamey, S. H., Cavichioli, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+Homalodisca+and+of+H.+vitripennis+as+the+Name+for+Glassy-winged+Sharpshooter+(Hemiptera:+Cicadellidae:+Cicadellinae).">Validity of Homalodisca and of H. vitripennis as the Name for Glassy-winged Sharpshooter (Hemiptera: Cicadellidae: Cicadellinae).</a> Ann. Entomol. Soc. Am. 99 (4), 648-655 (2006).</li><li>Hunter, W. B., Katsar, C. S., Chaparro, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+capsid+protein+of+Homalodisca+coagulata+Virus-1,+a+new+leafhopper-infecting+virus+from+the+glassy-winged+sharpshooter,+Homalodisca+coagulata.">Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata.</a> J. Insect Sci. 6 (28), 1-10 (2006).</li><li>Hunnicutt, L. E., Hunter, W. B., Cave, R. D., Powell, C. A., Mozoruk, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+and+molecular+characterization+of+Homalodisca+coagulata+virus-1,+a+novel+virus+discovered+in+the+glassy-winged+sharpshooter+(Hemiptera:+Cicadellidae).">Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae).</a> Virology. 350 (1), 67-78 (2006).</li><li>Hunnicutt, L. E., Mozoruk, J., Hunter, W. B., Crosslin, J. M., Cave, R. D., Powell, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+natural+host+range+of+Homalodisca+coagulata+virus-1+(HoCV-1).">Prevalence and natural host range of Homalodisca coagulata virus-1 (HoCV-1).</a> Virology. 153, 61-67 (2008).</li><li>Jones, W. A., Setamou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+Biometry+of+Sharpshooter+Homalodisca+coagulata+(Homoptera)+Cicadellidae)+Reared+on+Cowpea.">Biology and Biometry of Sharpshooter Homalodisca coagulata (Homoptera) Cicadellidae) Reared on Cowpea.</a> Ann. Entomol. Soc. Am. 98 (3), 322-328 (2005).</li><li>Turner, W. F., Pollard, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+histories+and+behavior+of+five+insect+vectors+of+phony+peach+disease.">Life histories and behavior of five insect vectors of phony peach disease.</a> US Dep. Agric. Tech. Bull. 1188, 1-32 (1959).</li><li>Brodbeck, B. V., Andersen, P. C., Mizell, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+primary+nutrients+by+the+polyphagous+xylophage,+Homalodisca+coagulata,+reared+on+single+host+species.">Utilization of primary nutrients by the polyphagous xylophage, Homalodisca coagulata, reared on single host species.</a> Arch. Insect Biochem. Physiol. 32, 65-83 (1996).</li><li>Brodbeck, B. V., Andersen, P. C., Mizell, R. F. <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+total+dietary+nitrogen+and+nitrogen+form+on+the+development+of+xylophagous+leafhoppers.">Effects of total dietary nitrogen and nitrogen form on the development of xylophagous leafhoppers.</a> Arch. Insect Biochem. Physiol. 42, 37-50 (1999).</li><li>Kamita, S. G., Do, Z. N., Samra, A. I., Halger, J. R., Hammock, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Cell+Lines+Developed+from+the+Glassy-winged+Sharpshooter,+Homalodisca+coagulata+(Hemiptera:+Cicadellidae).">Characterization of Cell Lines Developed from the Glassy-winged Sharpshooter, Homalodisca coagulata (Hemiptera: Cicadellidae).</a> In vitro Cell Dev.-An. 41, 149-153 (2005).</li><li>Hunter, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+for+development+of+bee+cell+cultures+(Apis+mellifera:+Hymenoptera:+Apidae).">Medium for development of bee cell cultures (Apis mellifera: Hymenoptera: Apidae).</a> In vitro Cell Dev.-An. 46 (2), 83-86 (2010).</li><li>Bextine, B., Hunter, W., Marshall, P., Hail, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+whole+extraction+of+Homalodisca+coagulata-virus01.+(HoCV-01)+from+Texas+glassy-winged+sharpshooter+populations.">Identification and whole extraction of Homalodisca coagulata-virus01. (HoCV-01) from Texas glassy-winged sharpshooter populations.</a> , 9-12 (2009).</li><li>Rhoades, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economics+of+baculovirus+&#8211;+insect+cell+production.">Economics of baculovirus &#8211; insect cell production.</a> Cytotechnology. 20, 291-297 (1996).</li><li>Briske-Anderson, M. J., Finley, J. W., Newman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+culture+time+and+passage+number+on+the+morphological+and+physiological+development+of+Caco-2+cells.">The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells.</a> P. Soc. Exp. Biol. Med. 214 (3), 248-257 (1997).</li><li>Chin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+Rescues+the+Adverse+Effects+of+Telomere+Loss+and+Cooperates+with+Telomere+Dysfunction+to+Accelerate+Carcinogenesis.">Deficiency Rescues the Adverse Effects of Telomere Loss and Cooperates with Telomere Dysfunction to Accelerate Carcinogenesis.</a> Cell. 97 (4), 527-538 (1999).</li><li>Counter, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+shortening+associated+with+chromosome+instability+is+arrested+in+immortal+cells+which+express+telomerase+activity.">Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.</a> Embo. J. 11, 1921-1929 (1992).</li><li>Hayflick, L., Moorhead, P. 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。

<table><tbody><tr><td>Corning cell culture flasks</td><td>Sigma Aldrich</td><td>CLS430168</td><td>Surface area 25 cm&lt;sup&gt;2&lt;/sup&gt;, canted neck, cap (plug seal)</td></tr><tr><td>Olympus DP30BW, IX2-SP, IX71</td><td>Olympus</td><td></td><td>Inverted microscope and camera</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma Aldrich</td><td>T4049</td><td>0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA &amp;bull; 4Na per liter of Hanks&amp;prime; Balanced Salt Solution with phenol red</td></tr><tr><td>Greiner CELLSTAR multiwell culture plates</td><td>Sigma Aldrich</td><td>M8937</td><td>48 wells (TC treated with lid)</td></tr><tr><td>DETCA</td><td>Sigma Aldrich</td><td>228680</td><td>Sodium diethyldithiocarbamate trihydrate</td></tr><tr><td>Corning bottle-top vacuum filter system</td><td>Sigma Aldrich</td><td>CLS431206</td><td>Cellulose acetate membrane, pore size 0.45 &amp;mu;m, membrane area 54.5 cm&lt;sup&gt;2&lt;/sup&gt;, filter capacity 500 ml</td></tr><tr><td>Brij 52</td><td>Sigma Aldrich</td><td>388831</td><td>Polyethylene glycol hexadecyl ether</td></tr><tr><td>Phosphate buffer solution</td><td>Sigma Aldrich</td><td>P5244</td><td>Received as 100 mM diluted to 10 mM with sterile water</td></tr><tr><td>TRIzol LS</td><td>Life Technologies</td><td>10296-028</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A5304</td><td>For electrophoresis</td></tr><tr><td>Ethidium bromide</td><td>Sigma Aldrich</td><td>E7637</td><td>BioReagent, for molecular biology, powder</td></tr><tr><td>QIAquick</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QuantiTect qRT-PCR kit</td><td>Qiagen</td><td>204243</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>Reagent grade, crystalline</td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5368</td><td>Phosphate buffered saline</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>Rhodamine red-conjugated phalloidin</td><td>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><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>ProLong Gold Antifade Reagent</td><td>Life Technologies</td><td>P36934</td><td></td></tr><tr><td>LSM510 Meta Confocal System</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>LSM Zen 2007 Software</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect medium (supplemented, 1x)</td><td>Sigma Aldrich</td><td>G8142</td><td>H2G+ leafhopper medium component</td></tr><tr><td>L-histidine monohydrate</td><td>Sigma Aldrich</td><td>H8125</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Medium 199 (10x)</td><td>Sigma Aldrich</td><td>M4530</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Medium 1066 (1x)</td><td>Sigma Aldrich</td><td>C0422</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salts (1x)</td><td>Sigma Aldrich</td><td>51322C</td><td>H2G+ leafhopper medium component</td></tr><tr><td>L-Glutamine (100x)</td><td>Sigma Aldrich</td><td>G3126</td><td>H2G+ leafhopper medium component</td></tr><tr><td>MEM, amino acid mix (50x)</td><td>Sigma Aldrich</td><td>56419C</td><td>H2G+ leafhopper medium component</td></tr><tr><td>1 M MgCl solution</td><td>Sigma Aldrich</td><td>M8266</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Pen-Strep (w/ glutamine)</td><td>Sigma Aldrich</td><td>G6784</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Nystatin</td><td>Sigma Aldrich</td><td>N6261</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Gentamycin</td><td>Sigma Aldrich</td><td>46305</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>D9434</td><td>H2G+ leafhopper medium component</td></tr><tr><td>Fetal Bovine Serum</td><td>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

11.7K 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

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

生物学

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

生产高滴度性乙型肝炎病毒库的细胞培养模型

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

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 ...

Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

Source: Labadie, T. et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021).
This video describes a cellular impedance-based technique to study the survival kinetics of viruses that induce cytopathic effects. The method measures the electrical impedance of virus-infected adherent cells pre-treated with saline.

基于细胞阻抗的存活动力学测定，用于评估病毒存活

Source: Labadie, T. et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021).
This video describes ...

传播

摘要

探索更多视频

此视频中的章节