一种快速战略新Faustoviruses从环境样品使用隔离<em> Vermamoeba vermiformis</em
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements to make.
Materials
| LSR FORTESSA cytometer | BD Biosciences | France | 649225B4 |
| TECNAI G2 F20 | FEI | Germany | 5027/11 |
| Optical inverted microscope | leica | France | 72643 |
| DNA extraction | Qiagen EZ1 Advanced XL Extraction Robot | France | L106A0452 |
| PCR Cycler CFX96 | Bio rad | France | 785BR06298 |
| PYG medium , PAS, Starvation medium | In house laboratory production | Marseille URMITE | x |
| Amoeba strain CDC-19 | ATCC | France | 50237 |
| Plates | Cellstar | France | 655180 |
| PCR materials, primers. | eurogentec | France | Primers cited in manuscript |
| glasstic slide 10 with grids | Kova | USA | H899871441F |
| Eosin/ blue Azur-Hemacolor stain | Merck milipore | France | 111955,6,57,109468 |
| Vacuum driven filters | Thermo scientific | France | BPV4550 / 20170115 |
| Phosphate-Buffered Saline | Thermo Fisher scientific | France | 10010-023 |
| DAPI stain | Life Technologies | France | D1306 |
| cytospin 4 cytocentrifuge | Thermo Fisher scientific | France | 10522013JT184-31 |
| Single cytology tunnel | Biomedical polymers inc. | France | BMP-cyto-S50 |
| Carbon grids | Euromedex | France | FCF400NI |
| Ammonium molibdate | VWR internationanl | France | 21276185 |
| Flasks | SARSTEDT | Germany | 833911 |
| 0.22μm filters | Milex millipor | France | SE2M229104 |
| Ultracentrifuge Sorval WX 80 | Thermo scientific | France | 9102448 |
| Rapid-flow filters | Nalgene | France | 450-0020 |
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