a-rapid-strategy-for-isolation-new-faustoviruses-from-environmental

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using <em>Vermamoeba vermiformis</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 ...

single-cell-micro-aspiration-as-an-alternative-strategy-to

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Aix Marseille University

Assistance Publique - Hôpitaux de Marseille (AP-HM)

Giant viruses are protist-associated viruses belonging to the proposed order Megavirales; almost all have been isolated from Acanthamoeba spp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses from their original protozoan hosts, we obtained eight isolates of a new giant viral lineage from Vermamoeba vermiformis, the most common free-living protist found in human environments. This new lineage was proposed to be the faustovirus lineage. The prototype member, faustovirus E12, forms icosahedral virions of ≈ 200 nm that are devoid of fibrils and that encapsidate a 466-kbp genome encoding 451 predicted proteins. Of these, 164 are found in the virion. Phylogenetic analysis of the core viral genes showed that faustovirus is distantly related to the mammalian pathogen African swine fever virus, but it encodes ≈ 3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to genes encoding any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy.

Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.

The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

Almost fifteen years ago, the discovery of Acanthamoeba polyphaga mimivirus, the first giant virus, changed how we define a virus. It was discovered incidentally in a process of isolating Legionella sp. from environmental samples in the context of pneumonia epidemics using a co-culture system with Acanthamoeba. Since then, much effort and improvement has been put into the original technique. In addition to the known families of Mimiviridae and Marseilleviridae, four new proposed families of giant viruses have been isolated: Pandoravirus, Pithovirus, Faustovirus and Mollivirus. Major improvements were based on enrichment systems, targeted use of antibiotics and high-throughput methods. The most recent development, using flow cytometry for isolation and presumptive identification systems, opens a path to large environmental surveys that may discover new giant virus families in new protozoa supports used for culture support.

Updating strategies for isolating and discovering giant viruses.

Most viruses are known for the ability to cause symptomatic diseases in humans and other animals. The discovery of  and other giant amoebal viruses revealed a considerable and previously unknown area of uncharacterized viral particles. Giant viruses have been isolated from various environmental samples collected from very distant geographic places, revealing a ubiquitous distribution. Their morphological and genomic features are fundamental elements for classifying them. Herein, we report the isolation and draft genome of Cedratvirus, a new amoebal giant virus isolated in , from an Algerian environmental sample. The viral particles are ovoid-shaped, resembling , but differing notably in the presence of two corks at each extremity of the virion. The draft genome of Cedratvirus-589,068 base pairs in length-is a close relative of the two previously described pithoviruses, sharing 104 and 113 genes with  and  genomes, respectively. Interestingly, analysis of these viruses' core genome reveals that only 21% of Cedratvirus genes are involved in best reciprocal hits with the two pithoviruses. Phylogeny reconstructions and comparative genomics indicate that Cedratvirus is most closely related to pithoviruses, and questions their membership in an enlarged putative  family.

Julien Andreani

Faculty of Medicine and Pharmacy,
Research Unit for Infectious and Tropical Emerging Diseases,
Faculty of Medicine and Pharmacy, Research Unit for Infectious and Tropical Emerging Diseases

Aix Marseille University

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

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

Julien Andreani

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Faculty of Medicine and Pharmacy,Research Unit for Infectious and Tropical Emerging Diseases,Faculty of Medicine and Pharmacy, Research Unit for Infectious and Tropical Emerging Diseases

Aix Marseille University

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

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

Faculty of Medicine and Pharmacy,
Research Unit for Infectious and Tropical Emerging Diseases,
Faculty of Medicine and Pharmacy, Research Unit for Infectious and Tropical Emerging Diseases