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. Sample Collection
<ol>
	<li>Collect 70 samples from different environments and regions. In this case, use the following: 5 dirty water samples from the village of Saint Pierre de Meyzoargues (France), 15 samples from the lake in Parc Bor&#233;ly in Marseille (France); 15 sea water samples with sediment from the rocky inlets at Samena in Marseille (France), 25 river water samples from the Alps (France), and finally, 10 samples from sewage in La Ciotat (France).</li>
	<li>Vortex samples for homogenization before inocu...

<ol>
	<li>Raoult, D., 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=The+1.2-megabase+genome+sequence+of+Mimivirus.">The 1.2-megabase genome sequence of Mimivirus.</a> Science. 306 (5700), 1344-1350 (2004).</li><li>Claverie, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+viruses+in+the+oceans:+the+4th+Algal+Virus+Workshop.">Giant viruses in the oceans: the 4th Algal Virus Workshop.</a> Virol J. 2, 52-52 (2004).</li><li>Monier, A. 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. -. M., 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=Mimivirus+and+Mimiviridae:+Giant+viruses+with+an+increasing+number+of+potential+hosts,+including+corals+and+sponges.">Mimivirus and Mimiviridae: Giant viruses with an increasing number of potential hosts, including corals and sponges.</a> J Invertebr Pathol. 101 (3), 9-9 (2009).</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=Evidence+of+the+megavirome+in+humans.">Evidence of the megavirome in humans.</a> J Clin Virol. 57 (3), 191-200 (2013).</li><li>La Scola, B., 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=Tentative+characterization+of+new+environmental+giant+viruses+by+MALDI-TOF+mass+spectrometry.">Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry.</a> Intervirology. 53 (5), 344-353 (2010).</li><li>Boughalmi, M., 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=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ Microbiol. , (2012).</li><li>Pagnier, I., 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=A+decade+of+improvements+in+mimiviridae+and+marseilleviridae+isolation+from+amoeba.">A decade of improvements in mimiviridae and marseilleviridae isolation from amoeba.</a> Intervirology. 56 (6), 354-363 (2012).</li><li>Philippe, N., 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=Pandoraviruses:+Amoeba+Viruses+with+Genomes+Up+to+2.5+Mb+Reaching+That+of+Parasitic+Eukaryotes.">Pandoraviruses: Amoeba Viruses with Genomes Up to 2.5 Mb Reaching That of Parasitic Eukaryotes.</a> Science. 341 (6143), 281-286 (2013).</li><li>Legendre, M., 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=Thirty-thousand-year-old+distant+relative+of+giant+icosahedral+DNA+viruses+with+a+pandoravirus+morphology.">Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.</a> PNAS. , (2014).</li><li>Saadi, H., 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=First+Isolation+of+Mimivirus+in+a+Patient+With+Pneumonia.">First Isolation of Mimivirus in a Patient With Pneumonia.</a> Clin Infect Dis. , (2013).</li><li>Saadi, H., 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=Shan+virus:+a+new+mimivirus+isolated+from+the+stool+of+a+Tunisian+patient+with+pneumonia.">Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia.</a> Intervirology. 56 (6), 424-429 (2013).</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=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>

The possibility that Faustovirus could be the first member of a new Megavirales family close to ASFV was first suggested by Reteno et al.,19 but some differences can still be distinguished. It appears unclear whether Faustovirus should join the Asfarviridae family or whether it should instead form a new putative viral family. This issue will require further investigation, in particular a more comprehensive characterization of its morphology, host range, replication cycle and gene repertoire. More Faus...

The discovery of giant viruses, especially those belonging to the Megavirales order, completely changed the world of viruses in terms of particle size and genome complexity. Viruses were previously thought to be small entities, and the Mimivirus appeared to break all the rules.1 Metagenomic data suggests the ubiquity of giant viruses not only in the environment, 2-5 but also in humans.6 Therefore, there is still a need to search for these viruses on a large scale. The diversity of these giant viruses was assessed by sampling not only a variety of aquatic environments and their associated sediments worldwide,7-11 but also by screening a variety of human samples12,13 and environmental samples.7,9 The Acanthamoeba polyphaga mimivirus was isolated by co-culture using phagocytic protists, primarily Acanthamoeba spp.14-16 An entire collection of giant viruses were then also isolated from this specified protist host, which made the scientific community restrict its research and isolation procedure for Acanthamoeba spp. Clearly this reliance on a single host species has resulted in a large fraction of viruses being overlooked. The fact that the giant virus, CroV, was isolated with the highly motile marine protozoa Cafeteria roenbergensis,17,18 demonstrates the need to use a wider range of protozoa in order to discover new lineages or families of giant viruses. Reteno et al. managed to select other protozoa as cell hosts which had never previously been used, and isolated the new Asfar-related lineage of giant viruses (the newly named Faustovirus).19
In an attempt to isolate new Faustovirus genotypes in order to expand the members of this viral lineage, we modified our isolation procedures and used them to screen environmental samples capable of harvesting new Faustoviruses. We then described the entire protocol to characterize the new isolates. We assessed Vermamoeba vermiformis, the most common free-living protist found in human environments,20-22 which is already used in the isolation of the first Faustovirus prototype E12.19 This protist is currently still host-specific for Faustovirus. We knew that none of the known giant viruses was pathogenic for this amoeba, because no attempts to grow other giant viruses in our lab showed amoeba lysis or viral growth. For this reason, we believe that V. vermiformis is the best and most unique cell support known to isolate new Faustoviruses.

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

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 viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

The system studied in this manuscript validated its proof of concept by isolating two new Faustoviruses. Of the 70 samples tested, two episodes of lysis were detected, in contrast to our reliable negative controls. The negative control for lysis contained an 86% amoeba population. By contrast, the positive samples (ST1 for Saint Pierre de Meyzoargues), and (LC9 for the La Ciotat Sample 9) showed a dramatic decline in gated amoebae; more than 60% of amoebae were lysed with the highest perc...

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.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

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 ...

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