Name: a rapid strategy for the isolation of new faustoviruses from environmental samples using vermamoeba vermiformis
Uploaded: 2016-06-04T15:00:00.000+00:00
Duration: 9 min 27 s
Description: Watch this Scientific Journal Video about A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis at JoVE.com

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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>

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

免疫与感染

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 ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

发育生物学

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

使用脱脂牛奶絮凝和超滤法浓缩环境水和废水样品中的病毒颗粒

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

生物学

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

病毒测试,水样中检测病毒的一种使用点方法

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

一种快速战略新Faustoviruses从环境样品使用隔离

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