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Immunology and Infection

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

Published: June 4th, 2016



1Faculty of Medicine and Pharmacy, Research Unit for Infectious and Tropical Emerging Diseases, Aix Marseille University, 2Pole of Infectious and Tropical Diseases, Clinical and Biological Sector, Federation of Bacteriology-Hygiene Virology, University Hospital Institute Mediterranean Infection

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.

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 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 ....

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1. Sample Collection

  1. 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 Borély 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).
  2. Vortex samples for homogenization before inocu.......

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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.......

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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.......

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The authors have no acknowledgements to make.


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Name Company Catalog Number Comments
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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