Génomique comparative des virus géants du clade étendu des asfarviridae

Le premier virus géant, Acanthamoeba polyphaga mimivirus, a été découvert en 2003. Ce virus appartient au phylum des Nucleocytoviricota et se caractérisent par une grande taille, d’où leur dénomination de virus «géants». La technique de co-culture virus-amibe a permis la caractérisation de nouveaux Nucleocytoviricota. Le remplacement de Acanthamoeba comme cellule hôte par Vermamoeba vermiformis a aidé à la caractérisation de nouveaux virus. A ce jour, plusieurs dizaines de membres de cette famille ont été décrits, l’une d'entre elles est nommée et il comprend les virus du genre African Swine Fever Virus(ASFV) mais aussi Pacmanvirus, Abalone Asfavirus-like virus, Kaumoebavirus, et de genre Faustovirus ainsi que les Metagenome-Assembled Genome Asfarviruses.Dans cette thèse, je présente tout d'abord une synthèse sur les virus infectant Vermamoeba vermiformis puis la diversité génétique et l'évolution du clade des Asfarviridae, par des approches bioinformatiques. Pour cela, j’ai assemblé et annoté six nouveaux génomes de Faustovirus, un nouveau Kaumoebavirus, et uun nouveau Pacmanvirus. La comparaison génomique entre les souches virales a révélé plusieurs points marquants (i) L'organisation et l'évolution de la capside majeure au travers des Kaumoebavirus et des Faustovirus sont caractérisés par un grand nombre d'introns (ii) Les gènes de Kaumoebavirus présentent un biais de localisation sur les brins d’ADN du deux tiers génome contrairement aux autres Asfarviridae (iii) Des gènes non apparentés sont maintenus en ordre colinéaire dans tous les génomes de cette famille. Enfin j’ai également j'ai étudié les gènes de virulence du virus ASVF au sein de la famille Asfarviridae.The first giant virus was discovered in 2003 after Acanthamoeba polyphaga mimivirus was described. This virus belongs to the phylum Nucleocytoviricota, and due to its massive size has been categorized as "giant." The technique of virus-amoeba co-culture was prolific for Nucleocytoviricota characterization, while the substitution of the Acanthamoeba to Vermamoeba host cell led to the discovery of new viral species. Until nowadays, several tens of members of Nucleocytoviricota have been described, one of them is named extended Asfarviridae and is comprised by African Swine Fever Virus (ASFV), Pacmanviruses, Abalone Asfavirus-like virus, Metagenome-Assembled Genome Asfarviruses, Kaumoebaviruses and Faustoviruses.In this dissertation, I present a review study on the viruses infecting V. vermiformis. Then via bioinformatic approaches I explored the genetic diversity and evolution of the extended Asfarviridae clade. Six genomes of Faustoviruses, and one of Kaumoebavirus and Pacmanvirus were assembled and annotated by myself. Genomic comparison between the viral strains revealed two unique features. The organization and evolution of the Major Capsid Protein gene in Kaumoebavirus and Faustovirus which is characterized by a large number of introns. And the significant gene strand bias over two-thirds of Kaumoebavirus' genome length, a unique feature among the extended Asfarviridae viruses. Also, a comparative genomics study of all available extended Asfarviridae viruses was performed, revealing the groups of unrelated genes that maintain a collinear order in all family's genomes. Finally, I investigated the origin of ASVF virulence genes across the extended Asfarviridae family

Diversity of Giant Viruses Infecting Vermamoeba vermiformis

International audienceThe discovery of Acanthamoeba polyphaga mimivirus in 2003 using the free-living amoeba Acanthamoeba polyphaga caused a paradigm shift in the virology field. Twelve years later, using another amoeba as a host, i.e., Vermamoeba vermiformis , novel isolates of giant viruses have been discovered. This amoeba–virus relationship led scientists to study the evolution of giant viruses and explore the origins of eukaryotes. The purpose of this article is to review all the giant viruses that have been isolated from Vermamoeba vermiformis , compare their genomic features, and report the influence of these viruses on the cell cycle of their amoebal host. To date, viruses putatively belonging to eight different viral taxa have been described: 7 are lytic and 1 is non-lytic. The comparison of giant viruses infecting Vermamoeba vermiformis has suggested three homogenous groups according to their size, the replication time inside the host cell, and the number of encoding tRNAs. This approach is an attempt at determining the evolutionary origins and trajectories of the virus; therefore, more giant viruses infecting Vermamoeba must be discovered and studied to create a comprehensive knowledge on these intriguing biological entities

The Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among “Extended Asfarviridae”

International audienceThe Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among "Extended Asfarviridae"

Comparative Genomics Unveils Regionalized Evolution of the Faustovirus Genomes

International audienceFaustovirus is a recently discovered genus of large DNA virus infecting the amoeba Vermamoeba vermiformis, which is phylogenetically related to Asfarviridae. To better understand the diversity and evolution of this viral group, we sequenced six novel Faustovirus strains, mined published metagenomic datasets and performed a comparative genomic analysis. Genomic sequences revealed three consistent phylogenetic groups, within which genetic diversity was moderate. The comparison of the major capsid protein (MCP) genes unveiled between 13 and 18 type-I introns that likely evolved through a still-active birth and death process mediated by intron-encoded homing endonucleases that began before the Faustovirus radiation. Genome-wide alignments indicated that despite genomes retaining high levels of gene collinearity, the central region containing the MCP gene together with the extremities of the chromosomes evolved at a faster rate due to increased indel accumulation and local rearrangements. The fluctuation of the nucleotide composition along the Faustovirus (FV) genomes is mostly imprinted by the consistent nucleotide bias of coding sequences and provided no evidence for a single DNA replication origin like in circular bacterial genomes

Pacmanvirus S19, the Second Pacmanvirus Isolated from Sewage Waters in Oran, Algeria

International audienceAcanthamoeba castellanii is an amoeba host that was used to isolate a novel strain named pacmanvirus S19. This isolate is the second strain reported and belongs to the extended Asfarviridae family. Pacmanvirus S19 harbors a 418,588-bp genome, with a GC content of 33.20%, which encodes 444 predicted proteins and a single Ile-tRNA. I n 2017, the first pacmanvirus (strain A23) was isolated from Acanthamoeba castellanii (1) and nested phylogenetically within the extended Asfarviridae clade (2). This clade contains the African swine fever virus, a virus with an endemic background that causes swine disease and death (3-7). Faustoviruses (8), kaumoebaviruses (9), abalone asfarvirus-like virus (10), and asfarvirus metagenome-assembled genomes (MAGs) (11) complete the known diversity of this clade. Here, we report the genome sequence of pacmanvirus S19, which was isolated from a sewage sample collected in Cap Falcon, Oran, Algeria (35°46915.30N, 0°47947.20W), and stored at 4°C before analysis. This virus was isolated using a coculture technique on a 24-well plate, as described by Andreani et al. (1); viral DNA was extracted with an EZ1 Advanced XL automated system (Qiagen, France). A 2 Â 251-bp paired-end sequencing strategy was used, and limited-cycle PCR amplification (12 cycles) completed the tag adapters and introduced dual-index barcodes. After purification on beads, the library was normalized according to the Nextera XT protocol (Illumina) before sequencing on an Illumina MiSeq instrument (8) in a 39-hour single run. Sequencing yielded 2,586,744 raw reads, which were trimmed and quality controlled by AlienTrimmer (12) (with parameters p = 80, l = 100, and k = 10) before de novo assembly with SPAdes v 3.11.1 (13) with k-mer sizes of 21, 55, 77, 99, and 127. Remaining gaps were closed by subassembly of reads aligned with HISAT (14) onto orthologous A23 genomic regions corresponding to the gap and its surroundings (500 bp on both ends), as found by BLASTN (15). A linear contig of 418,588 bp (average coverage, 270Â), with a GC content of 33.20%, was generated. GeneMarkS (16) predicted 505 genes using the virus option; 61 of those genes were discarded from the final annotation because they were shorter than 300 bp and had no detectable hits in the nonredundant database (BLASTP E values of ,1E205; pacmanvirus A23 hits were excluded). The 444 predicted open reading frames (ORFs) were functionally annotated according to the best similarities against two protein databases and two motif databases, in the following order: Swiss-Prot and UniRef90 were searched using BLASTP (E values of ,1E205), excluding pacmanvirus A23 hits; Pfam-A and InterPro motif databases were searched using PfamScan (17) and InterProScan (18), respectively (E values of ,1E205). Proteins that did not yield detectable hits were annotated as hypothetical (Fig. 1A). Furthermore, one Ile-tRNA (Fig. 1B) was found by both ARAGON (19) and tRNAscan-SE (20). Pacmanvirus tRNAs were also found in some asfarvirus MAGs (11). For comparison, pacmanvirus A23 was reported to have a smaller genome (395,405 bp) containing 465 predicted protein genes and an Ile-tRNA gene, with a GC content of 33.62% (1). The average nucleotide identity (ANI) between the two strains, as calculated by OrthoANIu (21), was 84.97%