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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Raccolta 1. Esempio <ol><li> Raccogliere 70 campioni provenienti da diversi ambienti e regioni. In questo caso, utilizzare i seguenti: 5 campioni di acqua sporca dal villaggio di Saint Pierre de Meyzoargues (Francia), 15 campioni dal lago nel Parc Borély a Marsiglia (Francia); 15 campioni di acqua di mare con sedimenti dalle insenature rocciose a Samena a Marsiglia (Francia), 25 campioni di acqua del fiume dalle Alpi (Francia), e, infine, 10 campioni di acque reflue a La Ciotat (Francia). </li><li> campioni Vortex pe...

<ol>
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A., 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=Marine+mimivirus+relatives+are+probably+large+algal+viruses.">Marine mimivirus relatives are probably large algal viruses.</a> Audio, Transactions of the IRE Professional Group on. 5, 12-12 (2007).</li><li>Monier, A., Claverie, J. M., Ogata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxonomic+distribution+of+large+DNA+viruses+in+the+sea.">Taxonomic distribution of large DNA viruses in the sea.</a> Genome Biol. , (2008).</li><li>Claverie, J. -. 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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. G., 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=Faustovirus,+an+asfarvirus-related+new+lineage+of+giant+viruses+infecting+amoebae.">Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.</a> J.Virol. , (2015).</li><li>Co&#351;kun, K. A., Oz&#231;elik, S., Tutar, L., Elald&#305;, N., Tutar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+free-living+amoebae+from+tap+water+in+Sivas,+Turkey.">Isolation and identification of free-living amoebae from tap water in Sivas, Turkey.</a> Biomed res Int. 2013, 675145 (2013).</li><li>Bradbury, R. 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 possibilità che Faustovirus potrebbe essere il primo membro di una nuova famiglia Megavirales vicino al ASFV è stato suggerito da Reteno et al., 19 ma alcune differenze possono ancora essere distinto. Appare chiaro se Faustovirus dovrebbe far parte della famiglia Asfarviridae o se debba invece formare una nuova famiglia virale putativo. Questo problema richiede ulteriori indagini, in particolare, una caratterizzazione più completa della sua morfologia, gamma degli ospiti, ciclo di replicazione ...

La scoperta del virus giganti, specialmente quelli appartenenti all&#39;ordine Megavirales, completamente cambiato il mondo dei virus in termini di dimensione delle particelle e del genoma complessità. I virus sono stati precedentemente pensato per essere piccole entità, e la mimivirus sembravano rompere tutte le regole. 1 metagenomiche dati suggeriscono l&#39;ubiquità di virus giganti, non solo per l&#39;ambiente, 2-5 ma anche negli esseri umani. 6 Pertanto, non vi è ancora la necessità per la ricerca di questi virus su larga scala. La diversità di questi virus giganti è stata valutata mediante il campionamento non solo una varietà di ambienti acquatici e delle loro sedimenti associati in tutto il mondo, 7-11 ma anche con la proiezione di una serie di campioni umani 12,13 e campioni ambientali. 7,9 I mimivirus Polyphaga Acanthamoeba è stato isolato dal co-coltura con protisti fagocitosi, soprattutto Acanthamoeba spp. 14-16 un&#39;intera collezione di virus giganti sono stati poi ancheisolata da questo host protista specificato, che ha reso la comunità scientifica limitare la sua procedura di ricerca e di isolamento per Acanthamoeba spp. Chiaramente questo affidamento su una singola specie ospite ha portato ad una grande frazione di virus essere trascurato. Il fatto che il virus gigante, CroV, è stato isolato con il altamente mobili marino protozoi Caffetteria roenbergensis, 17,18 dimostra la necessità di utilizzare una gamma più ampia di protozoi, al fine di scoprire nuove linee o famiglie di virus giganti. Reteno et al. Sono riusciti a selezionare altri protozoi come host di cellule che non era mai stato utilizzato in precedenza, e isolato la nuova stirpe Asfar correlati di virus giganti (il nuovo nome Faustovirus). 19 Nel tentativo di isolare nuovi genotipi Faustovirus al fine di espandere i membri di questo lignaggio virale, abbiamo modificato le nostre procedure di isolamento e li hanno usati per lo screening campioni ambientali capaci di raccolta nuovi Faustoviruses. abbiamo poidescritto l&#39;intero protocollo per caratterizzare i nuovi isolati. Abbiamo valutato vermiformis Vermamoeba, il protista a vita libera più comune in ambienti umani, 20-22 che è già utilizzato per l&#39;isolamento del primo prototipo Faustovirus E12. 19 Questo protista è attualmente ancora-host specifico per Faustovirus. Sapevamo che nessuno dei virus noti giganti era patogeni per questa ameba, perché nessun tentativo di crescere altri virus giganti nel nostro laboratorio ha mostrato lisi ameba o la crescita virale. Per questo motivo, riteniamo che V. vermiformis è il migliore e più unico supporto di cellule noto per isolare nuovi Faustoviruses.

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

L&#39;isolamento del virus giganti è di grande interesse in questa nuova era di virologia, soprattutto perché questi virus giganti sono legati alla protisti. i virus giganti possono essere potenzialmente patogeni per molte specie di protisti. Essi appartengono all&#39;ordine recentemente descritto di Megavirales. La nuova stirpe Faustovirus che è stato isolato da campioni di acque reflue è lontano parente del virus della peste suina africana mammiferi patogeno. Questo virus è anche specifico per il suo ospite amoebal, vermiformis Vermamoeba, un protista comune nei sistemi idrici sanitari. E &#39;fondamentale continuare a isolare nuovi genotipi Faustovirus al fine di ampliare la sua collezione genotipo e studiare la sua pan-genoma. Abbiamo sviluppato nuove strategie per l&#39;isolamento di ceppi supplementari migliorando l&#39;uso di combinazioni di antibiotici e antifungini per evitare contaminazioni batteriche e fungine della co-coltura amebe e favorendo la moltiplicazione del virus. Abbiamo anche implementato un nuovo starvation medio di mantenere V. vermiformis in condizioni ottimali per i virus co-coltura. Infine, abbiamo utilizzato la citometria a flusso anziché osservazione microscopica, che richiede tempo, per rilevare l&#39;effetto citopatico. Abbiamo ottenuto due isolati da campioni di depurazione, dimostrando l&#39;efficacia di questo metodo e ampliando così la raccolta di Faustoviruses, per capire meglio il loro ambiente, specificità dell&#39;ospite e contenuto genetico.

Il sistema studiato in questo manoscritto convalidato la sua prova di concetto, isolando due nuovi Faustoviruses. Dei 70 campioni esaminati, sono stati rilevati due episodi di lisi, contrariamente ai nostri controlli negativi affidabili. Il controllo negativo per lisi conteneva una popolazione un&#39;ameba 86%. Al contrario, i campioni positivi (ST1 per Saint Pierre de Meyzoargues), e (LC9 per il campione La Ciotat 9) hanno mostrato un drammatico calo delle amebe gated; più del 60% di a...

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.

Una strategia rapido per l&#39;isolamento di nuovi Faustoviruses da campioni ambientali Utilizzando<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

Separazione di DNA a singolo filamento, doppia elica del DNA e RNA da una Comunità Ambientale virale usando la cromatografia idrossiapatite

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

Ricerca

Immunologia e infezioni

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

Rilevazione multiplex di batteri in campioni clinici complessi ed Ambientali con oligonucleotidi accoppiati microsfere fluorescenti

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

Isolamento e analisi del genoma di virioni singole utilizzando &quot;Genomica singolo virus &#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

Ventricolo destro di pressione sistolica Misure in combinazione con raccolta di campioni di tessuto polmonare e immunitaria nei topi

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

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

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

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da 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

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

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

Panni nasale per l&#39;influenza A rilevazione antivirus e isolamento da Suina

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

Isolamento e caratterizzazione delle vescicoli extracellulari prodotti da micobatteri a limite di ferro

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