The authors have no acknowledgements to make.

1.样品采集<ol><li>收集来自不同的环境和区域70的样品。在这种情况下，使用以下命令:5脏水样本圣徒皮埃尔·德·Meyzoargues（法国），从马赛（法国）的博雷利公园湖中15个样品的村; 15海水样品从Samena在马赛（法国），从阿尔卑斯山（法国）25河水样品的岩石口泥沙，最后，从污水中10个样品中拉西约塔（法国）。 </li><li>涡旋样品均质接种，在室温下一分钟无任何治疗之前。 </li></ol> 2.分离过程<ol><li>主机准备的盲文化程序及抽样接种<ol><li> 五，使用vermiformis（株CDC19）作为共培养细胞的支持。 </li><li>保持在28℃的阿米巴，在用30毫升的蛋白酶蛋白胨-酵母提取物-葡萄糖培养基的75cm 2的细胞培养烧瓶（PYG）。请检查PYG成分见表1。 注:48小时后，变形虫是由正常的使用次数计数的幻灯片（ 例如 ，Kovaslides）量化。 </li><li>收获的细胞以在720×g离心10分钟的5×10 5〜10 6格/ ml，并将粒料变形虫的浓度通过离心。 </li><li>通过抽吸除去上清液，并重新悬浮在30毫升无菌页的amoebal盐水（PAS） 表1的阿米巴沉淀。重复此漂洗过程。 </li><li>以大约10 6变形虫/ ml的浓度重悬在30毫升饥饿培养基具有抗生素和抗真菌混合物变形虫。在饥饿培养基为变形虫生存介质，使其能够没有包囊或乘法活着。检查组合物在表1中。 注意:抗微生物剂混合物含有10微克/毫升万古霉素，10微克/毫升亚胺培南，20微克/毫升环丙沙星，20μ微克/毫升的强力霉素和20μg/ ml的伏立康唑。这种混合有助于减少细菌和真菌污染，可以减少或改变病毒的繁殖。 </li><li>直接接种25微升各样品到200微升变形虫单层在96孔板平底利于变形虫粘附和在潮湿环境下在30℃孵育（潮湿环境由放置湿敷包含内袋的板以避免蒸发）。这是初次 - 文化。离开没有任何样品接种变形虫的两口井;这充当阴性对照。孵育此初次 - 培养三天。 </li><li>三天后的第一个接种，亚文化，没有任何显微镜观察上述新鲜变形虫的初次 - 培养板。孵育相同的条件作为初次 - 培养下三天的新板。 </li><li>孵化三天后进行最后的文化方式与日同Ëprimo-和亚文化。孵育的最后培养两天。然后进行检测。 </li></ol></li><li>裂解检测自动通过流式细胞术 注:加上流动盲文化从术隔离以前的标准体系不同。它是更灵敏，准确和自动的。为了检测目的，我们应用开发以最高速度和无显微观察来检测裂解的新技术。这种技术是用于筛选的样品有利的，并且具有比旧的技术更好的灵敏度。7-9 <ol><li>使用最终共培养的96孔板来检测裂解。 </li><li>打开仪和以根据制造商的协议，以获得较低的背景噪声运行一个清洁和漂洗循环。 </li><li>启动高通量采样（HTS）与流动相结合仪自动加载从96孔板的样品和在40分钟内执行完全自动采集according至制造商的协议。 </li><li>成立了HTS如下:样品体积150微升，混合容积50微升，搅拌速度180，混合5号，每个样本400微升的洗涤容量，流速2.5微升/秒，记录的事件10000号。 </li><li>评估的最终共培养微板的阴性对照孔首先以栅极变形虫人口，并根据它们的物理特性，以确定这些细胞宿主的百分比。一定要有均匀的变形虫人口和相应的碎片和噪音更低事件的第二个群体。 注:此门程序由单细胞的大小，前向散射估计区分亚细胞碎片和团块。它准确地确定使用最佳选通过程的每个种群的百分比是重要的。这些百分比可以改变，尤其是在使用多种类型的原生动物，所以重要的是总是有一个是负面e控制，其可被用作样品的其余部分的参考群体。 </li><li>开始通过点击获取样品门。 </li><li>记录事件不大于10000的事件较少的数量。 </li><li>本地化使用箭头感兴趣的变形虫人口。这是如何定义的大门。 </li><li>门后，让现场HTS或通过单击"主页"定位板，然后自动运行整个板块。根据尺寸和结构参数执行数据采集和分析（分别为，FSC（前向散射）和SSC（侧向散射））。 注意:检测阿米巴 - 门控群体的损失信号负责阿米巴裂解致病剂的存在。与病毒感染相关的原生动物裂解与由分析器控和相比用作可靠阴性对照相同的非感染人群阿米巴人口的50％溶解的任意设计阈值检测。 </li></ol></li></ol><p类="jove_title"&gt; 3。裂解检测后的新分离的表征<ol><li>初步染色显示巨病毒的存在<ol><li>通过流式细胞仪检测溶解后，吸取裂解共培养暂停剩下的阿米巴。然后直接cytocentrifuge 50微升悬浮液在800×g离心10分钟。 </li><li>离心后，修复用纯甲醇溶液中的幻灯片。使用曙红/蓝色海岸和DAPI染色的商业快速染色染色的幻灯片，以检查病毒工厂的存在在1,000X倍的光学显微镜下观察。 <ol><li>为快速染色，沉浸所述滑入第一曙红溶液（4秒重复三次），然后直接传递到沉浸滑入含有蓝色海岸为6秒第二定影溶液，最后用磷酸盐缓冲溶液冲洗45秒的幻灯片pH值7.2。离开幻灯片在室温下干燥，然后进行observati上。 </li><li>对于DAPI染色，存款15微升的DAPI商业原液到固定干燥幻灯片。避光然后进行观察。 </li></ol></li></ol></li><li>电子显微镜<ol><li>在幻灯片上的第一个经过鉴定，评估的巨型病毒，通过培养上清电子显微镜观察的存在。 </li><li>存款5微升的阳性样品到辉光放电电网。等待在室温下大约20分钟。 </li><li>仔细擦干电网并沉积在其1％钼酸铵为10秒的小降。离开网格干燥5分钟。 </li><li>继续在200千电子伏电子显微镜。 </li><li>放置在保持器的网格;引进的持有人到显微镜。通过点击相应的图标检查真空概述。关闭阀，开始观察在光点大小5，并且x 25,000放大倍数。 </li><li>测量使用ImageJ或直接巨型病毒LY使用位于屏幕收购在显微镜测量工具显微镜。 注:分类的样品放入Faustovirus组具有大约200nm的衣壳大小。 </li></ol></li><li>分子生物学 注意:在此之后的识别，分子生物学试验是为确认，以便从Marseillevirus，其具有下式电子显微镜对相同衣壳大小和外观区分Faustovirus至关重要的，虽然它们不具有相同的宿主特异性。该Marseillevirus不能Vermamoeba增长，Faustovirus是特定于Vermamoeba和所有尝试生长它在其他阿米巴如棘阿米巴未能日期。在设计和Reteno 等人的作品中列出的特定的引物/探针系统的应用。启用的新分离出的病毒更精确的初步分类，通过病毒基因的扩增和测序。 <ol><li>前根据制造商的方案使用自动提取系统从培养阳性样品道的DNA。 </li><li>执行使用热循环仪的PCR作为Reteno 等人所述实时19 </li><li>序列采用了用于扩增的相同的引物。使用的引物靶向DNA指导的RNA聚合酶亚基1基因:Fstv_S2F 5&#39;-CCA GGA CAT GAT GGT CAC ATA G-3&#39;（正向）和Fstv_S2R 5&#39;-TTG CAC CTC CGC AGT TAA A-3&#39;（反向）与在Fstv_S2P FAM-TATGCTCCAATGGCCTTCAACGACA-TAMRA探针。 </li></ol></li></ol> 4.病毒生产，纯化和基因组测序<ol><li>继代培养的终点稀释克隆的单个病毒变形虫文化。 </li><li>为生产:制备装有30ml PYG，10毫升Vermamoeba vermiformis和5ml从井中已经转移到小瓶中分离的病毒的20烧瓶中。 </li><li>孵育在30℃，直到完全阿米巴溶解。倒置光学显微镜观察每一天，直到所有的阿米巴裂解。 </li><li>完成裂解后，全部集中到瓶一个收件人。用0.45微米的过滤器过滤，以消除碎屑。 </li><li>在89800 XG 75分钟开始通过超速离心提纯。确保在开始离心前校准管的重量。 </li><li>离心后，通过抽吸除去从每个管的上清液，用用于校准相同体积重悬浮在PAS沉淀，重复离心前。 </li><li>如上述，取出上清液并重新悬浮在1ml磷酸盐缓冲盐水（PBS）中的所有颗粒。 </li><li>纯化所产生，用25％的蔗糖（27.5在100ml PBS中的蔗糖，上过滤0.22微米过滤器）的病毒。 <ol><li>使用8毫升蔗糖和2ml病毒悬浮液。离心89800 XG为1小时15分钟。重新悬浮于1ml PBS中的沉淀。存储在-80℃。 </li&gt; </ol></li></ol>

<ol>
	<li>Raoult, D., 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=The+1.2-megabase+genome+sequence+of+Mimivirus.">The 1.2-megabase genome sequence of Mimivirus.</a> Science. 306 (5700), 1344-1350 (2004).</li><li>Claverie, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+viruses+in+the+oceans:+the+4th+Algal+Virus+Workshop.">Giant viruses in the oceans: the 4th Algal Virus Workshop.</a> Virol J. 2, 52-52 (2004).</li><li>Monier, A. A., 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=Marine+mimivirus+relatives+are+probably+large+algal+viruses.">Marine mimivirus relatives are probably large algal viruses.</a> Audio, Transactions of the IRE Professional Group on. 5, 12-12 (2007).</li><li>Monier, A., Claverie, J. M., Ogata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxonomic+distribution+of+large+DNA+viruses+in+the+sea.">Taxonomic distribution of large DNA viruses in the sea.</a> Genome Biol. , (2008).</li><li>Claverie, J. -. 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=Mimivirus+and+Mimiviridae:+Giant+viruses+with+an+increasing+number+of+potential+hosts,+including+corals+and+sponges.">Mimivirus and Mimiviridae: Giant viruses with an increasing number of potential hosts, including corals and sponges.</a> J Invertebr Pathol. 101 (3), 9-9 (2009).</li><li>Colson, P., 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=Evidence+of+the+megavirome+in+humans.">Evidence of the megavirome in humans.</a> J Clin Virol. 57 (3), 191-200 (2013).</li><li>La Scola, B., 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=Tentative+characterization+of+new+environmental+giant+viruses+by+MALDI-TOF+mass+spectrometry.">Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry.</a> Intervirology. 53 (5), 344-353 (2010).</li><li>Boughalmi, 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=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ Microbiol. , (2012).</li><li>Pagnier, I., 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=A+decade+of+improvements+in+mimiviridae+and+marseilleviridae+isolation+from+amoeba.">A decade of improvements in mimiviridae and marseilleviridae isolation from amoeba.</a> Intervirology. 56 (6), 354-363 (2012).</li><li>Philippe, N., 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=Pandoraviruses:+Amoeba+Viruses+with+Genomes+Up+to+2.5+Mb+Reaching+That+of+Parasitic+Eukaryotes.">Pandoraviruses: Amoeba Viruses with Genomes Up to 2.5 Mb Reaching That of Parasitic Eukaryotes.</a> Science. 341 (6143), 281-286 (2013).</li><li>Legendre, 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=Thirty-thousand-year-old+distant+relative+of+giant+icosahedral+DNA+viruses+with+a+pandoravirus+morphology.">Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.</a> PNAS. , (2014).</li><li>Saadi, H., 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=First+Isolation+of+Mimivirus+in+a+Patient+With+Pneumonia.">First Isolation of Mimivirus in a Patient With Pneumonia.</a> Clin Infect Dis. , (2013).</li><li>Saadi, H., 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=Shan+virus:+a+new+mimivirus+isolated+from+the+stool+of+a+Tunisian+patient+with+pneumonia.">Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia.</a> Intervirology. 56 (6), 424-429 (2013).</li><li>Claverie, J. -. M., Abergel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimivirus:+the+emerging+paradox+of+quasi-autonomous+viruses.">Mimivirus: the emerging paradox of quasi-autonomous viruses.</a> Trends Genet : TIG. 26 (10), 431-437 (2010).</li><li>Colson, P., 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=Viruses+with+more+than+1,000+genes:+Mamavirus,+a+new+Acanthamoeba+polyphaga+mimivirus+strain,+and+reannotation+of+Mimivirus+genes.">Viruses with more than 1,000 genes: Mamavirus, a new Acanthamoeba polyphaga mimivirus strain, and reannotation of Mimivirus genes.</a> Genome Biol Evol. 3, 737-742 (2010).</li><li>Claverie, J. -. M., Abergel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+questions+about+giant+viruses.">Open questions about giant viruses.</a> Adv Virus Res. 85, 25-56 (2013).</li><li>Fischer, M. G. M., Allen, M. J. M., Wilson, W. H. W., Suttle, C. A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+virus+with+a+remarkable+complement+of+genes+infects+marine+zooplankton.">Giant virus with a remarkable complement of genes infects marine zooplankton.</a> PNAS. 107 (45), 19508-19513 (2010).</li><li>Van Etten, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+really,+really+big+virus.">Another really, really big virus.</a> Viruses. 3 (1), 32-46 (2011).</li><li>Reteno, D. G., 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=Faustovirus,+an+asfarvirus-related+new+lineage+of+giant+viruses+infecting+amoebae.">Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.</a> J.Virol. , (2015).</li><li>Co&#351;kun, K. A., Oz&#231;elik, S., Tutar, L., Elald&#305;, N., Tutar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+free-living+amoebae+from+tap+water+in+Sivas,+Turkey.">Isolation and identification of free-living amoebae from tap water in Sivas, Turkey.</a> Biomed res Int. 2013, 675145 (2013).</li><li>Bradbury, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-living+amoebae+recovered+from+human+stool+samples+in+Strongyloides+agar+culture.">Free-living amoebae recovered from human stool samples in Strongyloides agar culture.</a> J Clin microbiol. 52 (2), 699-700 (2014).</li><li>Pagnier, I., Valles, C., Raoult, D., La Scola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vermamoeba+vermiformis+and+associated+bacteria+in+hospital+water.">Isolation of Vermamoeba vermiformis and associated bacteria in hospital water.</a> Microb Pathog. 80, 14-20 (2015).</li><li>Ogata, H., Toyoda, K., 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=Remarkable+sequence+similarity+between+the+dinoflagellate-infecting+marine+girus+and+the+terrestrial+pathogen+African+swine+fever+virus.">Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus.</a> Virol J. 6, 178-178 (2008).</li><li>Tarutani, K., Nagasaki, K., Itakura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+virus+infecting+the+novel+shellfish-killing+dinoflagellate+Heterocapsa+circularisquama.">Isolation of a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circularisquama.</a> Aquat Microb Ecol. 23, 103-111 (2001).</li></ol>

The authors declare that they have no competing financial interests.

该Faustovirus可能是一个新Megavirales家族接近非洲猪瘟病毒的第一个成员的可能性首先由Reteno。等，19建议，但有些差别仍可以区分。这似乎不清楚Faustovirus是否应该加入非洲猪瘟病毒科的家人或是否应该将形成一个新的公认的病毒家族。这个问题将需要进一步调查，特别是它的形态有更全面的描述，寄主范围，复制周期和基因剧目。更多Faustovirus基因型应以扩大泛基因组，并更好地理?...

巨病毒，特别是那些属于Megavirales顺序的发现，完全在粒度和基因组的复杂性的角度改变的病毒的世界。病毒以前被认为是小的实体，以及巨病毒的出现打破所有规则。1宏基因组数据表明巨病毒不仅在环境，2-5而且在人类中的普遍存在6因此，仍然需要来搜索这些病毒大规模。这些巨型病毒的多样性进行了筛选各种人体标本12,13和环境样品抽检不仅有各种水生环境及其相关的沉积物在世界范围内，7-11也评估7,9的棘阿米巴多食巨病毒是采用原生生物吞噬，这主要是棘阿米巴属共培养分离出14-16巨病毒的整个集合当时也从这个指定的原生生物的主机，这使得科学界对限制棘阿米巴属其研究和分离过程分离出来。显然，这依赖于一个单一的宿主物种已导致被忽视病毒的很大一部分。该巨病毒，CroV，是与高度能动海洋原生动物馆roenbergensis分离的事实，17,18表明，有必要使用范围更广原生动物以发现新的谱系或巨病毒家族。 Reteno 等人成功地选择其他原生动物细胞宿主以前从未被使用的，以及分离出的病毒巨头新Asfa酒店相关谱系（新命名的Faustovirus）。19 在试图为了扩大这个病毒家族的成员来隔离新Faustovirus基因型，我们修改隔离程序，并用它们来筛选能够收获新Faustoviruses的环境样本。然后，我们描述的整个协议来表征新菌株。我们评估Vermamoeba vermiformis，最常见的自由生活的原生生物在人类环境中发现的，20-22这是已经在第一Faustovirus原型E12 19此原生生物目前仍在主机的特异性Faustovirus的分离中使用。我们知道，没有任何已知病毒巨头病原是该变形虫，因为在我们的实验室中表明阿米巴裂解或病毒生长没有尝试种植其他巨型病毒。出于这个原因，我们认为五vermiformis是众所周知的隔离新Faustoviruses最好的和最独特的电池支持。

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">LSR FORTESSA cytometer&#160;</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:119px;">France&#160;</td>
			<td style="width:167px;">649225B4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TECNAI G2 F20</td>
			<td style="width:231px;">FEI</td>
			<td style="width:119px;">Germany&#160;</td>
			<td>5027/11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Optical inverted microscope</td>
			<td style="width:231px;">leica&#160;</td>
			<td style="width:119px;">France</td>
			<td>72643</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">DNA extraction</td>
			<td style="width:231px;">Qiagen EZ1 Advanced XL Extraction Robot</td>
			<td style="width:119px;">France&#160;</td>
			<td>L106A0452</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PCR Cycler CFX96</td>
			<td style="width:231px;">Bio rad</td>
			<td style="width:119px;">France</td>
			<td>785BR06298</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PYG medium , PAS, Starvation medium</td>
			<td style="width:231px;">In house laboratory production&#160;</td>
			<td style="width:119px;">Marseille URMITE</td>
			<td>x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Amoeba strain CDC-19</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:119px;">France</td>
			<td>50237</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Plates</td>
			<td style="width:231px;">Cellstar</td>
			<td style="width:119px;">France</td>
			<td>655180</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PCR materials, primers.&#160;</td>
			<td style="width:231px;">eurogentec</td>
			<td style="width:119px;">France</td>
			<td>Primers cited in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">glasstic slide 10 with grids</td>
			<td style="width:231px;">Kova&#160;</td>
			<td style="width:119px;">USA</td>
			<td style="width:167px;">H899871441F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Eosin/ blue Azur-Hemacolor stain</td>
			<td style="width:231px;">Merck milipore&#160;</td>
			<td style="width:119px;">France</td>
			<td>111955,6,57,109468</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum driven filters</td>
			<td style="width:231px;">Thermo scientific</td>
			<td style="width:119px;">France</td>
			<td>BPV4550 / 20170115</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Phosphate-Buffered Saline</td>
			<td style="width:231px;">Thermo Fisher scientific&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DAPI stain</td>
			<td style="width:231px;">Life Technologies&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>D1306</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">cytospin&#160; 4 cytocentrifuge</td>
			<td style="width:231px;">Thermo Fisher scientific&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>10522013JT184-31</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Single cytology tunnel</td>
			<td style="width:231px;">Biomedical polymers inc.</td>
			<td style="width:119px;">France</td>
			<td>BMP-cyto-S50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Carbon grids&#160;</td>
			<td style="width:231px;">Euromedex</td>
			<td style="width:119px;">France&#160;</td>
			<td>FCF400NI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Ammonium molibdate</td>
			<td style="width:231px;">VWR internationanl&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>21276185</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flasks&#160;</td>
			<td style="width:231px;">SARSTEDT</td>
			<td style="width:119px;">Germany</td>
			<td>833911</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">0.22&#956;m filters&#160;</td>
			<td style="width:231px;">Milex millipor&#160;</td>
			<td style="width:119px;">France</td>
			<td>SE2M229104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Ultracentrifuge Sorval WX 80</td>
			<td style="width:231px;">Thermo scientific</td>
			<td style="width:119px;">France</td>
			<td>9102448</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Rapid-flow filters</td>
			<td style="width:231px;">Nalgene</td>
			<td style="width:119px;">France</td>
			<td>450-0020</td>
		</tr>
	</tbody>
</table>

a rapid strategy for the isolation of new faustoviruses from environmental samples using vermamoeba vermiformis

巨病毒的隔离是病毒学的新时代的极大兴趣，特别是因为这些巨大的病毒都与原生生物。巨病毒可能是原生生物的许多种潜在的病原。它们属于Megavirales的最近描述的顺序。新血统Faustovirus已经从污水样品中分离是远亲哺乳动物病原体非洲猪瘟病毒。该病毒还具体到了amoebal主机，Vermamoeba vermiformis，保健水系统常见的原生生物。 这是继续以扩大其基因型收集并研究了其泛基因组中分离新Faustovirus基因型是至关重要的。我们通过改进使用抗生素和抗真菌组合以避免阿米巴共培养的细菌和真菌污染和有利于病毒增殖开发的附加菌株的分离的新策略。我们还实施了新的starvatioñ中保持V. vermiformis在病毒共培养的最佳条件。最后，我们使用流式细胞术而非显微镜观察，这是费时的，以检测细胞病变效应。我们获得了从污水样品2株，证明了这种方法的效率，从而扩大Faustoviruses的收集，以便更好地了解他们的环境，宿主特异性和遗传的内容。

在这个手稿研究该系统通过隔离两个新的验证Faustoviruses的概念证明。测试的70个样本中，有检测裂解的两集，而相比之下，我们的可靠阴性对照。用于裂解阴性对照含86％人口的阿米巴。相比之下，阳性标本（ST1为圣徒皮埃尔·德·Meyzoargues）和（LC9的拉西约塔样品9）显示门变形虫的大幅下降;变形虫的60％以上，用碎片的比例最高裂解。这些我们的方法的检测级的健壮的结?...

Watch this Scientific Journal Video about A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis at JoVE.com

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

a-rapid-strategy-for-isolation-new-faustoviruses-from-environmental

Research

JoVE Journal

Immunology and Infection

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

7.6K 次观看.  Aix Marseille University. We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba. 

视频: A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

科研

免疫与感染

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用"单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

耐高温溶菌酶的定向进化的一个新的筛选方法

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...