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

Abstract : Acanthamoeba 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%.
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Submitted on : Friday, October 22, 2021 - 10:17:25 AM
Last modification on : Thursday, May 12, 2022 - 12:41:11 PM
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Guillaume Blanc, Khalil Geballa-Koukoulas, Souhila Abdi, Bernard La Scola, Julien Andreani. Pacmanvirus S19, the Second Pacmanvirus Isolated from Sewage Waters in Oran, Algeria. Microbiology Resource Announcements, American Society for Microbiology, 2021, 10 (42), ⟨10.1128/MRA.00693-21⟩. ⟨hal-03394563⟩



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