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

Jacques Yaacoub Bou Khalil; Julien Andreani; Didier Raoult; Bernard La Scola

doi:10.3791/54104

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Method Article

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

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DOI:

10.3791/54104

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June 4th, 2016

Jacques Yaacoub Bou Khalil¹ , Julien Andreani¹ , Didier Raoult¹^,² , Bernard La Scola¹^,²

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

Summary

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.

Abstract

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.

Introduction

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 ....