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

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1. 샘플 컬렉션 <ol><li> 서로 다른 환경과 지역의 70 샘플을 수집합니다. 세인트 피에르 드 Meyzoargues (프랑스), 마르세유 (프랑스) 파크 Borély의 호수에서 15 샘플의 마을에서 5 더러운 물 샘플,이 경우, 다음을 사용 (15) 바다 물 25 강 물 샘플 알프스 (프랑스)에서 마르세유 Samena (프랑스)에서 바위 입구에서 퇴적물과 샘플, 그리고 마지막으로, 라 시오타의 하수에서 10 샘플 (프랑스). </li><li> 어떠한 처리...

<ol>
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Faustovirus이 ASFV에 가까운 새로운 Megavirales 가족의 첫 번째 구성원이 될 수 있다는 가능성을 먼저 Reteno 등., (19)에 의해 제안했지만 약간의 차이는 여전히 구별 할 수있다. Faustovirus이 Asfarviridae 가족에 가입해야할지 대신 새로운 추정 바이러스 제품군을 형성할지 여부를 명확 나타납니다. 이 문제는 추가 조사, 그 형태의 특히보다 포괄적 인 특성, 숙주 범위, 복제주기 및 유전자 레퍼?...

거대 바이러스의 Megavirales 순서에 속하는 특히 이들의 발견은 완전히 입경 게놈의 복잡성의 관점에서 바이러스의 세계를 바꾸었다. 바이러스는 이전 소기업 것으로 생각하고 Mimivirus 모든 규칙을 위반 등장 하였다. 1 군 유전체학 데이터 환경에서 2-5뿐만 아니라, 인간에서뿐만 아니라 거대 바이러스의 편재를 제안한다. (6) 따라서, 여전히 필요가있다 대규모 이러한 바이러스를 검색 할 수 있습니다. 이 거대한 바이러스의 다양성이 인간의 샘플 12, 13 및 환경 시료의 다양한 선별하여, 전 세계적으로도 7-11하지만를 수생 환경의 다양성과 관련 퇴적물뿐만 아니라 샘플링하여 평가 하였다. 7,9 가시 아메바의 풍뎅이 아목의 mimivirus이었다 식세포 원생 생물, 주로 가시 아메바 종을 사용하여 공동 문화입니다. 14-16 거대 바이러스의 전체 컬렉션은 또한 다음이었다과학계는 가시 아메바 종에 대한 연구 및 격리 절차를 제한 만든이 지정된 원생 호스트에서입니다. 분명히 하나의 호스트 종에이 신뢰는 바이러스의 많은 부분이 간과되고 가져왔다. 거대 바이러스 CroV가 높은 운동성 해양 원충 카페테리아 roenbergensis 절연 있다는 사실은 17,18 새로운 계통 또는 거대 바이러스 패밀리를 발견하기 위해 원생 동물의 넓은 범위를 사용할 필요성을 나타낸다. Reteno 등은. 이전에 사용 된 적이 셀 호스트와 같은 다른 원생 동물을 선택 관리, 거대한 바이러스의 새로운 아스파 관련 계통 (새로 명명 된 Faustovirus를)입니다. (19) 이 바이러스 계통의 구성원을 확장하기 위해 새로운 Faustovirus 유전자형을 분리하기위한 시도에서, 우리는 우리의 격리 절차를 수정하고 새로운 Faustoviruses을 수확 할 수있는 환경 시료를 선별를 사용했다. 우리 다음새로운 균주의 특성을하기 위해 전체 프로토콜을 설명했다. 우리는 Vermamoeba의 vermiformis, 이미 첫 번째 Faustovirus 프로토 타입 E12. 19 현재 여전히 호스트 특정되어 Faustovirus에 대한이 원생 생물의 분리에 사용되는 인간 환경에서 발견 된 가장 일반적인 자유 생활 원생 생물, 20-22을 평가 하였다. 우리는 우리의 실험실은 아메바 용해 또는 바이러스 성장을 보였다에 더 시도가 다른 거대한 바이러스를 성장하지 있기 때문에 알려진 거대한 바이러스의 것도,이 아메바에 대한 병원성 없다는 것을 알고 있었다. 이러한 이유로 생각 V. vermiformis 새로운 Faustoviruses을 분리하는 것으로 알려져 최고의 가장 독특한 세포 지원입니다.

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</table>

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

거대한 바이러스의 분리는이 거대한 바이러스가 원생 ​​생물에 관한 특히 때문에, 바이러스학의이 새로운 시대에 큰 관심이다. 자이언트 바이러스는 원생 생물의 많은 종에 대한 잠재적 병원성 수 있습니다. 그들은 Megavirales의 최근 기술 된 순서에 속한다. 하수 시료에서 분리 된 새로운 계보 Faustovirus는 포유 동물의 병원체 아프리카 돼지 열병 바이러스에 먼 관련이있다. 이 바이러스는 또한 amoebal 호스트, Vermamoeba의 vermiformis, 건강 관리 물 시스템에 공통 원생 생물에 고유합니다. 그것의 유전자형 수집을 확대하고 팬 게놈을 연구하기 위해 새로운 Faustovirus 유전자형을 분리 계속 중요합니다. 우리는 아메바 공동 배양 세균 및 진균 오염물을 방지하기 위해 항생제 및 항진균제의 조합의 사용을 개선하고 바이러스 증식을 선호하여 추가 균주의 분리를위한 새로운 전략을 개발했다. 우리는 또한 새로운 starvatio를 구현N 매체 V.을 유지할 바이러스 공동 배양을위한 최적의 조건에서 vermiformis. 마지막으로, 우리는 세포 병변 효과를 검출하기 위해, 시간 소모적 인 유동 세포 계측법보다는 현미경 관찰을 사용했다. 우리는 더 나은 자신의 환경, 호스트 특이성 유전자 내용을 이해하기 위해,이 방법의 효율성을 증명함으로써 Faustoviruses의 수집을 확대, 하수 시료에서 두 균주를 획득했습니다.

이 원고에서 공부 시스템은 두 개의 새로운 Faustoviruses을 분리하여 개념의 증거를 검증. 테스트 (70) 샘플, 용해의 두 에피소드는 우리의 신뢰할 수있는 음성 대조군 대조적으로 검출되었다. 용해에 대한 음성 대조군은 86 % 아메바 인구가 포함되어 있습니다. 대조적으로, 긍정적 인 샘플 (세인트 피에르 드 Meyzoargues에 대한 ST1)와 (라 시오타 샘플 9 LC9)는 게이트 아메바의 급격...

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.

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략<em&gt; Vermamoeba의 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

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