Draft Genome Sequence of Pandoravirus japonicus Isolated from the Sabaishi River, Niigata, Japan.

"Pandoraviridae" is a proposed family of the phylum Nucleocytoviricota Its features include an amphora-shaped capsid and the largest genome among all viruses. We report the isolation and genome sequencing of a new member of this family, named Pandoravirus japonicus, the third strain discovered in Japan.

ANNOUNCEMENT

Many members of the proposed family “Pandoraviridae” have previously been reported (1–7). Here, we report the isolation of a new member, Pandoravirus japonicus, from a freshwater sample (taken in a 50-ml tube) from the mouth of the Sabaishi River in Niigata, Japan (37°23′21.7″N, 138°33′59.4″E).

The sample (4.5 ml) was mixed with 4.5 ml of 2× peptone-yeast-glucose (PYG) medium, 50 μl of Acanthamoeba castellanii (amoeba) cells (1.5 × 105 cells), and 360 μl of antibiotic solution (8). This mixture was added to a 96-well plate (100 μl per well). After 3 days of culture at 26°C, 10 μl supernatant from each well showing microscopic evidence of cytopathic effects (CPE; delayed proliferation or cell rounding) was serially diluted to 1011-fold with PYG medium. Then, 10 μl of each dilution was mixed with 90 μl of PYG medium (16 ml PYG medium and 5.0 × 103 amoeba cells) in a 96-well plate. After several days, amoeba cells (1.5 × 106) were inoculated with the supernatants from wells showing CPE with the highest dilution in a 25-cm2 culture flask. One of the supernatants (25 ml) of the amoeba cell culture showing CPE was harvested. Viral particles were collected by centrifugation at 8,000 × g for 35 min at 4°C. The resulting pellets were resuspended and washed with 1 ml phosphate-buffered saline (PBS). The preparation was examined using scanning electron microscopy (Fig. 1A). Viral DNA was extracted using the NucleoSpin tissue XS kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany) according to the manufacturer’s instructions. A 10-μl library was prepared using a PacBio (Menlo Park, CA, USA) DNA template prep kit 1.0 (for 3 to 20 kb). SMRTbell templates were annealed using the PacBio DNA/polymerase binding kit P6. The PacBio DNA sequencing kit 4.0 and 8 single-molecule real-time (SMRT) cells were used for sequencing. FALCON software was used to assemble 1,043,248,645 raw reads into one contig with a length of 1,787,274 nucleotides. The reads were mapped to the genomic sequence using CLC Genomics Workbench software ver. 20.0.4 (Qiagen N.V., Hilden, Germany), resulting in 63 single-nucleotide insertions or deletions. The average coverage was 316.71×. These regions were confirmed via capillary sequencing, which allowed us to assemble a final genomic sequence of 1,787,268 bp. The GC content of the genome was 64.0%. Dot plot analysis (9) suggested that a part of the P. japonicus genome translocated compared with other pandoravirus genomes, although the possibility of genome rotation due to random origin or misassembly could not be excluded (Fig. 1B). Gene prediction was performed using the GeneMarkS (10) and FgenesV tools. tRNA genes were identified using the tRNAscan-SE server ver. 2.0 (11). All tools described above were run with default parameters unless otherwise specified. We identified 1,361 open reading frames (ORFs) and one tRNA (Ser). Homology searches were performed using BLASTp against the NCBI nonredundant (nr) GenBank database with an E value threshold of <10−5, and the gene annotation was manually revised. Most ORFs were highly homologous to other pandoraviruses, and 410 were putative ORFans.

FIG 1

(A) Scanning electron microscopy (SEM) image of Pandoravirus japonicus. Acanthamoeba castellanii cells were cultured in PYG medium in 75-cm2 culture flasks and infected with P. japonicus. Four days after infection, viral particles in the medium were collected at 8,000 × g for 35 min at 4°C. The virus pellet was resuspended in 5 ml of phosphate-buffered saline (PBS). After centrifugation (8,000 × g for 35 min at 4°C), the virus pellet was resuspended in 500 μl of 2% glutaraldehyde (GA) in PBS, followed by centrifugation, resuspension, and fixation with 50 μl of 2% GA in PBS, and postfixation in 2% osmium tetra-oxide for 2 h in the ice bath. Then, the specimens were dehydrated with graded ethanol and CO2 critical point drying. The dried specimens were coated by an osmium plasma ion coater. SEM was performed using a model JSM-7500F microscope (JEOL Ltd., Tokyo, Japan), and secondary electron images at 5 kV were captured at the Hanaichi UltraStructure Research Institute (Aichi, Japan). Bar, 100 nm. (B) Dot plot analysis for genome comparison of P. japonicus and P. dulcis using D-GENIES with default parameters (9).

Data availability.

The sequence data are available in the DDBJ (accession number DRA011890) and GenBank (accession number LC625835).