Name: a rapid strategy for the isolation of new faustoviruses from environmental samples using vermamoeba vermiformis
Uploaded: 2016-06-04T15:00:00
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

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><p class="jove_ti...

<ol>
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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。

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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 ...

The overall goal of this new isolation strategy is to widen the number of new virus isolates in order to study the pan-genome of this new viral family, and to better understand their ecosystems and potential pathogenicity to humans. This method can answer key questions in the virology field, to help virologists world wide interested in prospecting giant viruses and faustoviruses in particular. The main advantage of this technique is that it show new improvement to many stage like the antibiotic and antifungal mixture.The new starvation medium adapted for the amoeba and the use of floss-cytometry for faster and more sensitive lysis detection. After collecting dirty water samples according to the text protocol, prepare the host cell, V.vermiformis by adding the amoebae to a 75 square centimeter cell culture flask with 30 milliliters of protease peptone yeast extract glucose, or PYG medium and incubate at 28 degrees Celsius for 48 hours. Use counting slides to count the amoebae and when they reach a concentration of five times 10 to the 5th, to 10 to the 6th cells per milliliter, harvest the cells by centrifugation at 720 times g for 10 minutes.Aspirate the supernatant, and use 30 milliliters of sterile pages amoebal saline or PAS to resuspend the amoebae pellet. After rinsing the pellet a second time, use 30 milliliters of starvation medium, containing an antibiotic and an antifungal to resuspend the cells to approximately 10 to the 6th amoeba per milliliter. Starvation medium enables the amoebae to stay alive without encystment or multiplication.Next, seed 200 microliters of amoeba in the wells of a 96 well flat bottom plate and incubate at 30 degrees Celsius for 30 minutes. Then, after vortexing the water samples for one minute at room temperature, innoculate 25 microliters of each sample onto the amoebae cells, leaving two wells without sample as negative controls. To avoid evaporation, place the plate containing the primo culture into a bag with a wet compress, and place the humidified chamber with plate into an incubator at 30 degrees Celsius.Three days after the first innoculation, sub-culture the primo culture plate onto a fresh 96 well plate of amoebae as just demonstrated, without any microscopic observation. Incubate at 30 degrees Celsius for three days. Then, using the subculture, innoculate a third plate as just described, and incubate for two days.To detect lysis in the final cocultured plate, turn on the cytometer and run a clean and rinse according to the manufacturer's protocol to reduce the background noise. To carry out a 40 minute completely automated acquisition, launch the high throughput sampler or HTS to automatically load samples from the 96 well plate according to the manufacturer's protocol. Program the HTS settings as listed here.That include a sample volume of 150 microliters, a mixing volume of 50 microliters, a mixing speed of 180, the number of mixes as 5, a washing volume between samples of 400 microliters, a flow rate of 2.5 microliters per second, and the number of recorded events as 10, 000. Click on home to allow the HTS to spot or locate the plate. Then assess the negative control wells containing the host amoeba cells first.In order to gate the amoeba population, and to determine the cell percentage according to their physical characteristics. Be sure to have a homogeneous amoeba population and a second population of lower events corresponding to debris and noise. Next, start the gating by clicking acquire sample.Then, using the arrow, which defines the gates, localize the amoeba population of interest. After gating at least 10, 000 events, click on run to allow the HTS to automatically run the entire plate. Perform data acquisition and analysis according to the size parameter forward scatter and the structure parameter side scatter.Refer to the text for additional details. Following licence detection by flow-cytometry, pipette the lysed cocultures to suspend the remaining amoebae. After pipetting 50 microliters of each sample onto slides, directly cytocentrifuge the suspension at 800 times g for 10 minutes.Next, use pure methanol to fix the slides, then leave the slides to dry at room temperature. After fixation, plunge the slides into ESN solution three times for four seconds each before placing the slides into blue azur for six seconds. Finally, plunge the slides into PBS for 45 seconds.Leave the slides to dry at room temperature before proceeding to observation. To stain new fixed slides with DAPI, pipette 15 microliters of DAPI into the dry slides. Observe the slides under a light microscope at 1000x magnification in order to check for the presence of virus factories.To carry out electron microscopy, deposit five microliters of the positive sample onto a glow-discharged grid, and incubate at room temperature for approximately 20 minutes. Carefully dry the grid, then add a small amount of 1%ammonium molybdate and incubate for 10 seconds. After carefully removing the drop, allow the grid to dry for five minutes.Next, set the electron microscope to 200 kiloelectron volts. Then, place the grid on the holder and introduce the holder into the microscope. Click on the vacuum overview icon and check the vacuum.Then, close the valves, and set a spot size of five, and a magnification of 25, 000 before beginning observation. After locating a giant virus on the acquisition screen, use the measure tool to measure the virus. Carry out molecular biology and viral genome sequencing according to the text protocol.This figure shows results from the detection of two faustoviruses compared to the reliable negative controls. The negative control for lysis contained an 86%amoeba population. By contrast, more than 60%of the amoebae were lysed with the highest percentage of debris in the positive samples.As seen here, Eus and blue azur stains the nuclei of uninfected and infected amoebae cells, and the presence of virus factories inside the infected cells. DAPI staining also confirms the presence of virus factories inside amoebae. In these images, electron microscopy reveals the presence of faustovirus with the typical appearance of megavirals composed of an icosadedral capsid that lacks fibrils and a size of 200 nanometers.Once mastered and after the coculture, this technique of lysis detection and virus characterization can be done in an hour, if it's performed properly. While attempting this procedure, it is important to remember to control the amoebal viability and to check any probable contaminations. Following this procedure, other methods like molecular biology targeting giant viruses can be performed in order to answer additional questions like the virus family and or genotype.After its development, this technique paved the way for researchers in the field of virology to explore this viral family, their specific host, and their potential pathogenicity. After watching this video, you should have a good understanding of how to easily isolate giant viruses by widening the number and types of samples and by choosing the best host cell and the best control conditions. Don't forget that working with viruses potentially pathogenic for humans can be extremely hazardous, specially with huge samples.So precautions such as working in an SB2 labs with secure conditions should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

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

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

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 ...

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

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 ...

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

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

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

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

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

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 ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...