Name: a rapid strategy for the isolation of new faustoviruses from environmental samples using vermamoeba vermiformis
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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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Collection 1. Sample <ol><li> Recueillir 70 échantillons provenant de différents milieux et régions. Dans ce cas, utilisez les suivants: 5 échantillons d&#39;eau sale du village de Saint Pierre de Meyzoargues (France), 15 échantillons du lac dans le Parc Borély à Marseille (France); 15 échantillons d&#39;eau de mer avec les sédiments des calanques à Samena à Marseille (France), 25 échantillons d&#39;eau de la rivière des Alpes (France), et enfin, 10 échantillons provenant des eaux usées à La Ciotat (Fr...

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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. 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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>

La possibilité que Faustovirus pourrait être le premier membre d&#39;une nouvelle famille Megavirales près de ASFV a d&#39; abord été suggérée par Reteno et al., 19 mais certaines différences peuvent encore se distinguer. Il semble difficile de savoir si Faustovirus devrait se joindre à la famille Asfarviridae ou si elle devrait plutôt former une nouvelle famille virale putative. Cette question nécessitera une enquête plus approfondie, notamment une caractérisation plus complète de sa m...

La découverte des virus géants, en particulier ceux appartenant à l&#39;ordre Megavirales, a complètement changé le monde des virus en termes de taille des particules et de la complexité du génome. Les virus ont déjà été pensés pour être de petites entités, et l&#39;Mimivirus semblaient briser toutes les règles. 1 données métagénomique suggère l&#39;omniprésence des virus géants , non seulement dans l&#39;environnement, 2-5 , mais aussi chez l&#39; homme. 6 Par conséquent, il est encore nécessaire à la recherche de ces virus à grande échelle. La diversité de ces virus géants a été évaluée par échantillonnage non seulement une variété de milieux aquatiques et leurs sédiments associés dans le monde entier, 7-11 , mais aussi par criblage d&#39; une variété d&#39;échantillons humains 12,13 et les échantillons environnementaux. 7,9 Les mimivirus de polyphaga Acanthamoeba était isolé par co-culture en utilisant protistes phagocytaires, principalement Acanthamoeba spp. 14-16 toute une collection de virus géants étaient alors aussiisolé à partir de cet hôte de protistes spécifié, ce qui a rendu la communauté scientifique restreindre sa recherche et l&#39; isolement procédure de Acanthamoeba spp. Il est clair que cette dépendance sur une seule espèce d&#39;accueil a donné lieu à une grande partie des virus étant négligée. Le fait que le virus géant, CroV, a été isolé avec le très motiles marine protozoaires cafeteria roenbergensis, 17,18 démontre la nécessité d&#39;utiliser une plus large gamme de protozoaires afin de découvrir de nouvelles lignées ou familles de virus géants. Reteno et al. A réussi à choisir d&#39; autres protozoaires comme des cellules hôtes qui n&#39;a jamais été utilisé auparavant, et isolé la nouvelle lignée Asfar liée des virus géants (le Faustovirus nouvellement nommé). 19 Dans une tentative d&#39;isoler de nouveaux génotypes Faustovirus afin d&#39;élargir les membres de cette lignée virale, nous avons modifié nos procédures d&#39;isolement et les ont utilisés pour dépister les échantillons environnementaux capables de récolter de nouvelles Faustoviruses. Nous avons ensuitedécrit l&#39;ensemble du protocole pour caractériser les nouveaux isolats. Nous avons évalué vermiformis Vermamoeba, le protistes vivant en liberté le plus répandu dans les environnements humains, 20-22 , qui est déjà utilisé dans l&#39;isolement du premier prototype Faustovirus E12. 19 Cette protist actuellement encore l&#39; hôte spécifique pour Faustovirus. Nous savions que aucun des virus géants connus était pathogène pour cette amibe, car aucune tentative de se développer d&#39;autres virus géants dans notre laboratoire a montré une lyse amibe ou la croissance virale. Pour cette raison, nous croyons que V. vermiformis est le meilleur et le plus unique support cellulaire connue pour isoler de nouveaux Faustoviruses.

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

L&#39;isolement des virus géants est d&#39;un grand intérêt dans cette nouvelle ère de la virologie, d&#39;autant plus que ces virus géants sont liés à des protistes. virus géants peuvent être potentiellement pathogènes pour de nombreuses espèces de protistes. Ils appartiennent à l&#39;ordre des Megavirales récemment décrit. La nouvelle lignée Faustovirus qui a été isolé à partir d&#39;échantillons d&#39;eaux usées est lointainement apparenté au virus de la peste porcine africaine de l&#39;agent pathogène de mammifère. Ce virus est également spécifique à son hôte amibienne, vermiformis Vermamoeba, un protiste commun dans les systèmes d&#39;eau de soins de santé. Il est crucial de continuer à isoler de nouveaux génotypes Faustovirus afin d&#39;élargir sa collection de génotype et étudier son pan-génome. Nous avons développé de nouvelles stratégies pour l&#39;isolement de souches supplémentaires en améliorant l&#39;utilisation de combinaisons d&#39;antibiotiques et antifongiques, afin d&#39;éviter les contaminations bactériennes et fongiques de la co-culture de l&#39;amibe et de favoriser la multiplication du virus. Nous avons également mis en place un nouveau starvation moyen pour maintenir V. vermiformis dans des conditions optimales pour les virus co-culture. Enfin, nous avons utilisé la cytométrie en flux, plutôt que l&#39;observation au microscope, ce qui prend du temps, afin de détecter l&#39;effet cytopathogène. Nous avons obtenu deux isolats provenant d&#39;échantillons d&#39;eaux usées, ce qui prouve l&#39;efficacité de cette méthode et élargissant ainsi la collection de Faustoviruses, afin de mieux comprendre leur environnement, la spécificité d&#39;hôte et le contenu génétique.

Le système étudié dans ce manuscrit validé sa preuve de concept en isolant deux nouveaux Faustoviruses. Sur les 70 échantillons testés, deux épisodes de lyse ont été détectés, contrairement à nos contrôles négatifs fiables. Le contrôle négatif pour la lyse contenait une population amibe de 86%. En revanche, les échantillons positifs (ST1 pour Saint Pierre de Meyzoargues), et (LC9 pour l&#39;échantillon La Ciotat 9) ont montré une baisse spectaculaire des amibes fermée...

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.

Une stratégie rapide pour l&#39;isolement des nouveaux Faustoviruses d&#39;échantillons environnementaux Utilisation<em&gt; vermiformis Vermamoeba</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

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

Recherche

Immunologie et infection

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

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

L&#39;isolement et l&#39;analyse du génome des virions unique en utilisant la génomique du virus unique »

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

Mesures systolique ventriculaire droite pression en combinaison avec la récolte du poumon et des échantillons de tissus immunitaires chez des souris

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

Identification rapide des bactéries à Gram négatif de sang Bouillon de culture à l&#39;aide MALDI-TOF spectrométrie de masse

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

Analyse rapide des aberrations chromosomiques dans les lymphocytes B de souris par 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 ...

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

Isolement de leucocytes des tissus murins à l&#39;interface materno-foetale

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

Lingettes nasal pour la grippe détection d&#39;un virus et l&#39;isolement des porcs

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

Isolement et caractérisation des vésicules extracellulaires produites par des mycobactéries limitées en fer

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