Julien Andreani

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

ABSTRACT 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. IMPORTANCE By using Vermamoeba, a protist living in humans and their environment, we isolated eight strains of a new giant virus that we named faustovirus. The genomes of these strains were sequenced, and their sequences showed that faustoviruses are related to but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae. Moreover, the faustovirus gene repertoire is ≈3 times larger than that of ASFV and comprises approximately two-thirds ORFans (open reading frames [ORFs] with no detectable homology to other ORFs in a database).

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

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.

Cedratvirus, a Double-Cork Structured Giant Virus, is a Distant Relative of Pithoviruses

Most viruses are known for the ability to cause symptomatic diseases in humans and other animals. The discovery of Acanthamoeba polyphaga mimivirus 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 Acanthamoeba castellanii, from an Algerian environmental sample. The viral particles are ovoid-shaped, resembling Pithovirus sibericum, 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 P. sibericum and Pithovirus massiliensis 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 Pithoviridae family.

Comparison of a Modern and Fossil Pithovirus Reveals Its Genetic Conservation and Evolution

Most theories on viral evolution are speculative and lack fossil comparison. Here, we isolated a modern Pithovirus-like virus from sewage samples. This giant virus, named Pithovirus massiliensis, was compared with its prehistoric counterpart, Pithovirus sibericum, found in Siberian permafrost. Our analysis revealed near-complete gene repertoire conservation, including horizontal gene transfer and ORFans. Furthermore, all orthologous genes evolved under strong purifying selection with a non-synonymous and synonymous ratio in the same range as the ratio found in the prokaryotic world. The comparison between fossil and modern Pithovirus species provided an estimation of the cadence of the molecular clock, reaching up to 3 × 10−6 mutations/site/year. In addition, the strict conservation of HGTs and ORFans in P. massiliensis revealed the stable genetic mosaicism in giant viruses and excludes the concept of a bag of genes. The genetic stability for 30,000 years of P. massiliensis demonstrates that giant viruses evolve similarly to prokaryotes by classical mechanisms of evolution, including selection and fixation of genes, followed by selective constraints.

Updating strategies for isolating and discovering giant viruses.

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.

Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses

ABSTRACT African swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii. Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus. IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.

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.

Orpheovirus IHUMI-LCC2: A New Virus among the Giant Viruses

Giant viruses continue to invade the world of virology, in gigantic genome sizes and various particles shapes. Strains discoveries and metagenomic studies make it possible to reveal the complexity of these microorganisms, their origins, ecosystems and putative roles. We isolated from a rat stool sample a new giant virus “Orpheovirus IHUMI-LCC2,” using Vermamoeba vermiformis as host cell. In this paper, we describe the main genomic features and replicative cycle of Orpheovirus IHUMI-LCC2. It possesses a circular genome exceeding 1.4 Megabases with 25% G+C content and ovoidal-shaped particles ranging from 900 to 1300 nm. Particles are closed by at least one thick membrane in a single ostiole-like shape in their apex. Phylogenetic analysis and the reciprocal best hit for Orpheovirus show a connection to the proposed Pithoviridae family. However, some genomic characteristics bear witness to a completely divergent evolution for Orpheovirus IHUMI-LCC2 when compared to Cedratviruses or Pithoviruses.

Genome Sequences of New Faustovirus Strains ST1 and LC9, Isolated from the South of France

ABSTRACT Faustoviruses are amoeba-infecting giant viruses closely related to the Asfarviridae family. Here, we report the isolation, genome sequencing, and annotation of ST1 and LC9, two new strains belonging to lineages L and E9 of faustoviruses, currently represented by only one representative each.

Deciphering viral presences: two novel partial giant viruses detected in marine metagenome and in a mine drainage metagenome

Nucleo-cytoplasmic large DNA viruses are doubled stranded DNA viruses capable of infecting eukaryotic cells. Since the discovery of Mimivirus and Pandoravirus, there has been no doubt about their extraordinary features compared to “classic” viruses. Recently, we reported the expansion of the proposed family Pithoviridae, with the description of Cedratvirus and Orpheovirus, two new viruses related to Pithoviruses. Studying the major capsid protein of Orpheovirus, we detected a homologous sequence in a mine drainage metagenome. The in-depth exploration of this metagenome, using the MG-Digger program, enabled us to retrieve up to 10 contigs with clear evidence of viral sequences. Moreover, phylogenetic analyses further extended our screening with the discovery in another marine metagenome of a second virus closely related to Orpheovirus IHUMI-LCC2. This virus is a misidentified virus confused with and annotated as a Rickettsiales bacterium. It presents a partial genome size of about 170 kbp.