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%

Morphologic and Genomic Analyses of New Isolates Reveal a Second Lineage of Cedratviruses

International audienceGiant viruses have been isolated and characterized in different environments, expanding our knowledge about the biology of these unique microorganisms. In the last 2 years, a new group was discovered, the cedratviruses, currently composed of only two isolates and members of a putative new family, ``Pithoviridae,'' along with previously known pithoviruses. Here we report the isolation and biological and genomic characterization of two novel cedratviruses isolated from samples collected in France and Brazil. Both viruses were isolated using Acanthamoeba castellanii as a host cell and exhibit ovoid particles with corks at either extremity of the particle. Curiously, the Brazilian cedratvirus is similar to 20% smaller and presents a shorter genome of 460,038 bp, coding for fewer proteins than other cedratviruses. In addition, it has a completely asyntenic genome and presents a lower amino acid identity of orthologous genes (similar to 73%). Pangenome analysis comprising the four cedratviruses revealed an increase in the pangenome concomitant with a decrease in the core genome with the addition of the two novel viruses. Finally, phylogenetic analyses clustered the Brazilian virus in a separate branch within the group of cedratviruses, while the French isolate is closer to the previously reported Cedratvirus lausannensis. Taking all together, we propose the existence of a second lineage of this emerging viral genus and provide new insights into the biodiversity and ubiquity of these giant viruses. IMPORTANCE Various giant viruses have been described in recent years, revealing a unique part of the virosphere. A new group among the giant viruses has recently been described, the cedratviruses, which is currently composed of only two isolates. In this paper, we describe two novel cedratviruses isolated from French and Brazilian samples. Biological and genomic analyses showed viruses with different particle sizes, genome lengths, and architecture, revealing the existence of a second lineage of this new group of giant viruses. Our results provide new insights into the biodiversity of cedratviruses and highlight the importance of ongoing efforts to prospect for and characterize new giant viruses