Draft Genome Sequence of Tokyovirus, a Member of the Family Marseilleviridae Isolated from the Arakawa River of Tokyo, Japan

Members of the Marseilleviridae family are large DNA viruses with icosahedral particles that infect Acanthamoeba cells. This report presents a new Marseilleviridae family member discovered in a water/soil sample from a river in Tokyo, named Tokyovirus, with genome size of 370 to 380 kb.

smaller particles of 200-nm diameter and with a genome size of 300 to 400 kb (1)(2)(3)(4)(5)(6)(7)(8)(9). This article reports the draft genome of a new virus belonging to the family Marseilleviridae, isolated from the coast of the Arakawa River, located in Tokyo, Japan. It was named Tokyovirus according to the rules of nomenclature for Marseilleviridae.
Water/soil samples were collected from a bank of the Arakawa River. After mud was removed by filtration (filter paper 43; Whatman PLC), samples were further filtered (0.8-m pore size, Millex-AA; Millipore). Filtered samples were concentrated by polyethylene glycol precipitation overnight at 4°C, followed by centrifugation at 1,500 ϫ g for 30 min at 4°C (10). After the supernatant was removed, the pellet was resuspended in 4 ml of peptone yeast extract-glucose (PYG) broth and then filtered again (Millex-AA; Millipore). Then, 4 ml of fresh PYG and a 1-ml suspension of amoeba cells were added to this viral solution, which was divided and cultured on 56 wells in a 96-well culture plate at 26°C. After 10 days, I found that amoeba cells in only 1 well out of 56 wells had delayed proliferation and had been almost round. The supernatant of the culture in this single well was inoculated to fresh amoeba cells of 3 wells in a 96-well culture plate. Almost all cells were rounded. The supernatant was inoculated to fresh amoeba cells in a 25 cm 2 culture flask. After 2 days, rounded amoeba cells were harvested. Then, the supernatant was stored at 4°C as an isolated virus solution. After virus cloning (11), genomic DNA of Tokyovirus (1.1 g) was prepared from PYG culture media including viral particles using NucleoSpin Tissue (Macherey-Nagel GmbH and Co. KG) according to the manufacturer's protocol. A DNA library for sequencing was prepared using the TruSeq Nano DNA LT library prep kit (Illumina, Inc.), and sequencing was performed on a HiSeq 2500 platform (Illumina, Inc.). Edena software was used to assemble 1,000,000 reads into 68 contigs with an average length of 5,481 nucleotides (nt) and a maximum contig length of 360,777 nt. The total length of the 68 contigs was 372,707 nt. Prediction of the coding region of the Tokyovirus genome was conducted using CRITICA version 1.05b and Glimmer 2 version 2.10. Prediction of tRNA was conducted using tRNAScan-SE version 1.23, according to the manufacturer's protocols. The prediction of gene function was conducted using NCBI BLASTp in the NCBI NR and NCBI COG databases.
Genome analysis showed that Tokyovirus has a 370-to 380-kb genome. The total length of the 68 contigs was 372,707 nt, which is approximately equal to the genome sizes of other Marseilleviridae members (1, 2, 4, 7). This draft sequence is predicted to have 487 coding sequences (CDSs) and 2 tRNA genes (one of them is a pseudogene). Genome comparison revealed that Tokyovirus closely resembles Marseillevirus and Melbournevirus. Tokyovirus has 47 CDSs, which are predicted to be highly similar (Ͼ90% identical) with Marseillevirus homologs.
Nucleotide sequence accession number. The draft genomic sequence of Tokyovirus has been deposited in DDBJ/ENA/ GenBank under the accession number AP017398.

FUNDING INFORMATION
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.