Here, we report the isolation and genome sequencing of a new Pacmanvirus-related isolate, Tornadovirus japonicus, from the Tamagawa River in Japan. This icosahedral virus has a genome of approximately 380 kb and 465 open reading frames, including two tRNA genes. The name "tornado"is based on its morphological features revealed by transmission electron microscopy analysis.</p

Murata, Kazuyoshi

Takemura, Masaharu

Waschestjuk, Daniel

English

ARTS repository - University of Groningen

   University of GroningenComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolatedfrom the Tamagawa River in JapanWaschestjuk, Daniel; Murata, Kazuyoshi; Takemura, MasaharuPublished in:Microbiology resource announcementsDOI:10.1128/mra.00265-24IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2024Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Waschestjuk, D., Murata, K., & Takemura, M. (2024). Complete genome sequence of Tornadovirusjaponicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan. Microbiology resourceannouncements, 13(6), Article e00265-24. https://doi.org/10.1128/mra.00265-24CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 29-04-2025 | Virology | AnnouncementComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in JapanDaniel Waschestjuk,1,2 Kazuyoshi Murata,3,4,5 Masaharu Takemura1,6AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT Here, we report the isolation and genome sequencing of a new Pacman­virus-related isolate, Tornadovirus japonicus, from the Tamagawa River in Japan. This icosahedral virus has a genome of approximately 380 kb and 465 open reading frames, including two tRNA genes. The name “tornado” is based on its morphological features revealed by transmission electron microscopy analysis.KEYWORDS Nucleocytoviricota, Pacmanvirus, tornadovirus, giant virus, acanthamoebaP acmanvirus is a dsDNA virus that infects acanthamoeba. Many Pacmanvirus genes have been reported to be similar to those of faustoviruses and kaumoebaviruses, which are relatives of the family Asfarviridae (1). To date, only two strains of pacmanvi­ruses have been isolated from water samples in Algeria (A23 and S19) (1, 2). Here, we report the successful isolation of this virus from water samples collected from the shores of the Tamagawa River in Kawasaki City, Kanagawa Prefecture, Japan (35°58911.77N, 139°65678.59E) on 22 October 2022.First, water sample was mixed with A. castllanii cell suspension in a culture solu­tion of proteose-peptone-yeast extract-glucose (PYG) medium including antibiotics as described previously (3–5). This mixture was added to a 96-well plate and incubated at 26°C. After 12 days, 10 µL of supernatant from a well that displayed cytopathic effects (CPE) of amoeba was inoculated into fresh A. castellanii cells and was cultured in 25 cm2 culture flasks to propagate the putative virus followed by virus cloning as described previously (6). To identify the viruses, negative-staining electron microscopy was performed using a JEM-2100F operated at 120 kV (JEOL Co. Ltd., Tokyo, Japan) (Fig. 1A). Cryo-electron microscopy was performed using a JEM-2200FS, operated at 200 kV (JEOL Co. Ltd.), which showed the detailed structure of the virus particles (Fig. 1B). To investigate viral formation in A. castellanii cells, transmission electron microscopy (TEM) of the virus-infected amoeba cells was performed using a JEM-1400 (JEOL Co. Ltd.) at the Hanaichi UltraStructure Research Institute (Aichi, Japan) (Fig. 1C). Although the specific morphology of the virus remains unknown, we have used here the term “tornado” to describe its unique architecture, based on the morphological features of the maturing viral particles observed in infected cells by TEM (Fig. 1C).Genomic DNA was extracted from the cloned and propagated tornadoviruses using a NucleoSpin tissue XS (Macherey-Nagel GmbH & Co. KG, Duren, Germany), according to the manufacturer's instructions. A DNA library was prepared for sequencing using a TrueSeq DNA PCR-Free (350) kit (Illumina, Inc.). Sequencing was performed using the NovaSeq 6000 platform (Illumina, Inc.). SPAdes 3.15.0 was used to assemble 28,832,452 reads (4,787,212,192 total read bases) into one contig of 378,863 nucleotides (8). Read length was 151. FastQC (v0.11.5) was used for quality control checks on raw sequence data. Open reading frames (ORFs) were detected using Prokka (9). Gene function was predicted using NCBI BLAST (10). The GC content of the genome was 38.81%. A total of 465 open reading frames were identified, including 463 coding sequences and two July 2024  Volume 13  Issue 7 10.1128/mra.00265-24 1Editor Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, BelgiumAddress correspondence to Masaharu Takemura, giantvirus@rs.tus.ac.jp.The authors declare no conflict of interest.See the funding table on p. 3.Received 15 March 2024Accepted 23 May 2024Published 11 June 2024Copyright © 2024 Waschestjuk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.tRNAs using NCBI BLAST (10). There were representative genes, which were found in previously reported pacmanviruses, including families B and X DNA polymerases, major capsid proteins, and virion packaging ATPase. Molecular phylogenetic analysis using family B DNA polymerase genes indicated that tornadoviruses are closely related to pacmanviruses with 55.6% identity (Fig. 1D).ACKNOWLEDGMENTSWe thank Macrogen Japan (Koto-ku, Tokyo, Japan) for the genome sequencing, de novo assembly, and annotation, the Hanaichi UltraStructure Research Institute (Okazaki, Aichi, Japan) for the TEM analyses, and Mr. Mitsuru Ikeda for negative-staining TEM and cryo-EM.This study was supported by grant funding from JSPS/KAKENHI (grant number 20H03078, awarded to M.T.), and ExCELLS project research (grant number 22EXC601-4, awarded to M.T.).AUTHOR AFFILIATIONS1Graduate School of Science, Tokyo University of Science, Shinjuku, Tokyo, Japan2Rijksuniversiteit Groningen, Faculty of Science and Engineering, Groningen, the Netherlands3Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Aichi, Japan4National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, Japan5Department of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, JapanFIG 1 (A) Negative-staining electron microscopy (EM) images of Tornadovirus japonicus particles. (B) Cryo-EM images of Tornadovirus japonicus particles. (C) TEM image of Tornadovirus japonicus particles during maturation of host cells. Bar: 200 nm (A–C). (D) Phylogenetic tree of B family DNA polymerase amino acid sequences. Analysis was performed by the MEGA X software (7), using Clustal W algorithm for alignment and the maximum likelihood method with 1,000 replicates for reconstruction of phylogenetic tree. Red box indicates Tornadovirus japonicus.Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 2Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.6Laboratory of Biology, Institute of Arts and Sciences, Tokyo University of Science, Shinjuku, Tokyo, JapanAUTHOR ORCIDsKazuyoshi Murata  http://orcid.org/0000-0001-9446-3652Masaharu Takemura  http://orcid.org/0000-0002-6261-3297FUNDINGFunder Grant(s) Author(s)MEXT | Japan Society for the Promotion of Science (JSPS)20H03078 Masaharu TakemuraDATA AVAILABILITYThe sequence data are available in GenBank (accession number LC801470.1) under BioProject accession numbers PRJDB17414 and SAMD00735632, and the raw reads can be found in the Sequence Read Archive (accession number DRR527376).REFERENCES1. Andreani J, Khalil JYB, Sevvana M, Benamar S, Di Pinto F, Bitam I, Colson P, Klose T, Rossmann MG, Raoult D, La Scola B. 2017. Pacmanvirus, a new giant Icosahedral virus at the crossroads between Asfarviridae and Faustoviruses. J Virol 91:e00212-17. https://doi.org/10.1128/JVI.00212-172. Geballa-Koukoulas K, Abdi S, La Scola B, Blanc G, Andreani J. 2021. Pacmanvirus S19, the second Pacmanvirus isolated from sewage waters in Oran, Algeria. Microbiol Resour Announc 10:e0069321. https://doi.org/10.1128/MRA.00693-213. Takemura M. 2016. Morphological and taxonomic properties of Tokyovirus, the first Marseilleviridae member isolated from Japan. Microbes Environ 31:442–448. https://doi.org/10.1264/jsme2.ME161074. Yoshikawa G, Blanc-Mathieu R, Song C, Kayama Y, Mochizuki T, Murata K, Ogata H, Takemura M. 2019. Medusavirus, a novel large DNA virus discovered from hot spring water. J Virol 93:e02130-18. https://doi.org/10.1128/JVI.02130-185. Takahashi H, Fukaya S, Song C, Murata K, Takemura M. 2021. Morpholog­ical and T\taxonomic properties of the newly isolated Cotonvirus japonicus, a new lineage of the subfamily Megavirinae. J Virol 95:e0091921. https://doi.org/10.1128/JVI.00919-216. Takemura M. 2022. “Genome sequence of a new “ Candidatus” phylum “dependentiae” isolate from Chiba, Japan”. Microbiol Resour Announc 11:e01123–21. https://doi.org/10.1128/mra.01123-217. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy0968. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.00219. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu15310. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 3Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.

Complete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan

   University of GroningenComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolatedfrom the Tamagawa River in JapanWaschestjuk, Daniel; Murata, Kazuyoshi; Takemura, MasaharuPublished in:Microbiology resource announcementsDOI:10.1128/mra.00265-24IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2024Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Waschestjuk, D., Murata, K., & Takemura, M. (2024). Complete genome sequence of Tornadovirusjaponicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan. Microbiology resourceannouncements, 13(6), Article e00265-24. https://doi.org/10.1128/mra.00265-24CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum. | Virology | AnnouncementComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in JapanDaniel Waschestjuk,1,2 Kazuyoshi Murata,3,4,5 Masaharu Takemura1,6AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT Here, we report the isolation and genome sequencing of a new Pacman­virus-related isolate, Tornadovirus japonicus, from the Tamagawa River in Japan. This icosahedral virus has a genome of approximately 380 kb and 465 open reading frames, including two tRNA genes. The name “tornado” is based on its morphological features revealed by transmission electron microscopy analysis.KEYWORDS Nucleocytoviricota, Pacmanvirus, tornadovirus, giant virus, acanthamoebaP acmanvirus is a dsDNA virus that infects acanthamoeba. Many Pacmanvirus genes have been reported to be similar to those of faustoviruses and kaumoebaviruses, which are relatives of the family Asfarviridae (1). To date, only two strains of pacmanvi­ruses have been isolated from water samples in Algeria (A23 and S19) (1, 2). Here, we report the successful isolation of this virus from water samples collected from the shores of the Tamagawa River in Kawasaki City, Kanagawa Prefecture, Japan (35°58911.77N, 139°65678.59E) on 22 October 2022.First, water sample was mixed with A. castllanii cell suspension in a culture solu­tion of proteose-peptone-yeast extract-glucose (PYG) medium including antibiotics as described previously (3–5). This mixture was added to a 96-well plate and incubated at 26°C. After 12 days, 10 µL of supernatant from a well that displayed cytopathic effects (CPE) of amoeba was inoculated into fresh A. castellanii cells and was cultured in 25 cm2 culture flasks to propagate the putative virus followed by virus cloning as described previously (6). To identify the viruses, negative-staining electron microscopy was performed using a JEM-2100F operated at 120 kV (JEOL Co. Ltd., Tokyo, Japan) (Fig. 1A). Cryo-electron microscopy was performed using a JEM-2200FS, operated at 200 kV (JEOL Co. Ltd.), which showed the detailed structure of the virus particles (Fig. 1B). To investigate viral formation in A. castellanii cells, transmission electron microscopy (TEM) of the virus-infected amoeba cells was performed using a JEM-1400 (JEOL Co. Ltd.) at the Hanaichi UltraStructure Research Institute (Aichi, Japan) (Fig. 1C). Although the specific morphology of the virus remains unknown, we have used here the term “tornado” to describe its unique architecture, based on the morphological features of the maturing viral particles observed in infected cells by TEM (Fig. 1C).Genomic DNA was extracted from the cloned and propagated tornadoviruses using a NucleoSpin tissue XS (Macherey-Nagel GmbH & Co. KG, Duren, Germany), according to the manufacturer's instructions. A DNA library was prepared for sequencing using a TrueSeq DNA PCR-Free (350) kit (Illumina, Inc.). Sequencing was performed using the NovaSeq 6000 platform (Illumina, Inc.). SPAdes 3.15.0 was used to assemble 28,832,452 reads (4,787,212,192 total read bases) into one contig of 378,863 nucleotides (8). Read length was 151. FastQC (v0.11.5) was used for quality control checks on raw sequence data. Open reading frames (ORFs) were detected using Prokka (9). Gene function was predicted using NCBI BLAST (10). The GC content of the genome was 38.81%. A total of 465 open reading frames were identified, including 463 coding sequences and two July 2024  Volume 13  Issue 7 10.1128/mra.00265-24 1Editor Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, BelgiumAddress correspondence to Masaharu Takemura, giantvirus@rs.tus.ac.jp.The authors declare no conflict of interest.See the funding table on p. 3.Received 15 March 2024Accepted 23 May 2024Published 11 June 2024Copyright © 2024 Waschestjuk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.tRNAs using NCBI BLAST (10). There were representative genes, which were found in previously reported pacmanviruses, including families B and X DNA polymerases, major capsid proteins, and virion packaging ATPase. Molecular phylogenetic analysis using family B DNA polymerase genes indicated that tornadoviruses are closely related to pacmanviruses with 55.6% identity (Fig. 1D).ACKNOWLEDGMENTSWe thank Macrogen Japan (Koto-ku, Tokyo, Japan) for the genome sequencing, de novo assembly, and annotation, the Hanaichi UltraStructure Research Institute (Okazaki, Aichi, Japan) for the TEM analyses, and Mr. Mitsuru Ikeda for negative-staining TEM and cryo-EM.This study was supported by grant funding from JSPS/KAKENHI (grant number 20H03078, awarded to M.T.), and ExCELLS project research (grant number 22EXC601-4, awarded to M.T.).AUTHOR AFFILIATIONS1Graduate School of Science, Tokyo University of Science, Shinjuku, Tokyo, Japan2Rijksuniversiteit Groningen, Faculty of Science and Engineering, Groningen, the Netherlands3Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Aichi, Japan4National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, Japan5Department of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, JapanFIG 1 (A) Negative-staining electron microscopy (EM) images of Tornadovirus japonicus particles. (B) Cryo-EM images of Tornadovirus japonicus particles. (C) TEM image of Tornadovirus japonicus particles during maturation of host cells. Bar: 200 nm (A–C). (D) Phylogenetic tree of B family DNA polymerase amino acid sequences. Analysis was performed by the MEGA X software (7), using Clustal W algorithm for alignment and the maximum likelihood method with 1,000 replicates for reconstruction of phylogenetic tree. Red box indicates Tornadovirus japonicus.Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 2Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.6Laboratory of Biology, Institute of Arts and Sciences, Tokyo University of Science, Shinjuku, Tokyo, JapanAUTHOR ORCIDsKazuyoshi Murata  http://orcid.org/0000-0001-9446-3652Masaharu Takemura  http://orcid.org/0000-0002-6261-3297FUNDINGFunder Grant(s) Author(s)MEXT | Japan Society for the Promotion of Science (JSPS)20H03078 Masaharu TakemuraDATA AVAILABILITYThe sequence data are available in GenBank (accession number LC801470.1) under BioProject accession numbers PRJDB17414 and SAMD00735632, and the raw reads can be found in the Sequence Read Archive (accession number DRR527376).REFERENCES1. Andreani J, Khalil JYB, Sevvana M, Benamar S, Di Pinto F, Bitam I, Colson P, Klose T, Rossmann MG, Raoult D, La Scola B. 2017. Pacmanvirus, a new giant Icosahedral virus at the crossroads between Asfarviridae and Faustoviruses. J Virol 91:e00212-17. https://doi.org/10.1128/JVI.00212-172. Geballa-Koukoulas K, Abdi S, La Scola B, Blanc G, Andreani J. 2021. Pacmanvirus S19, the second Pacmanvirus isolated from sewage waters in Oran, Algeria. Microbiol Resour Announc 10:e0069321. https://doi.org/10.1128/MRA.00693-213. Takemura M. 2016. Morphological and taxonomic properties of Tokyovirus, the first Marseilleviridae member isolated from Japan. Microbes Environ 31:442–448. https://doi.org/10.1264/jsme2.ME161074. Yoshikawa G, Blanc-Mathieu R, Song C, Kayama Y, Mochizuki T, Murata K, Ogata H, Takemura M. 2019. Medusavirus, a novel large DNA virus discovered from hot spring water. J Virol 93:e02130-18. https://doi.org/10.1128/JVI.02130-185. Takahashi H, Fukaya S, Song C, Murata K, Takemura M. 2021. Morpholog­ical and T\taxonomic properties of the newly isolated Cotonvirus japonicus, a new lineage of the subfamily Megavirinae. J Virol 95:e0091921. https://doi.org/10.1128/JVI.00919-216. Takemura M. 2022. “Genome sequence of a new “ Candidatus” phylum “dependentiae” isolate from Chiba, Japan”. Microbiol Resour Announc 11:e01123–21. https://doi.org/10.1128/mra.01123-217. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy0968. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.00219. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu15310. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 3Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.

University of Groningen

University of GroningenComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolatedfrom the Tamagawa River in JapanWaschestjuk, Daniel; Murata, Kazuyoshi; Takemura, MasaharuPublished in:Microbiology resource announcementsDOI:10.1128/mra.00265-24IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2024Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Waschestjuk, D., Murata, K., & Takemura, M. (2024). Complete genome sequence of Tornadovirusjaponicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan. Microbiology resourceannouncements, 13(6), Article e00265-24. https://doi.org/10.1128/mra.00265-24CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 01-01-2026 | Virology | AnnouncementComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in JapanDaniel Waschestjuk,1,2 Kazuyoshi Murata,3,4,5 Masaharu Takemura1,6AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT Here, we report the isolation and genome sequencing of a new Pacman­virus-related isolate, Tornadovirus japonicus, from the Tamagawa River in Japan. This icosahedral virus has a genome of approximately 380 kb and 465 open reading frames, including two tRNA genes. The name “tornado” is based on its morphological features revealed by transmission electron microscopy analysis.KEYWORDS Nucleocytoviricota, Pacmanvirus, tornadovirus, giant virus, acanthamoebaP acmanvirus is a dsDNA virus that infects acanthamoeba. Many Pacmanvirus genes have been reported to be similar to those of faustoviruses and kaumoebaviruses, which are relatives of the family Asfarviridae (1). To date, only two strains of pacmanvi­ruses have been isolated from water samples in Algeria (A23 and S19) (1, 2). Here, we report the successful isolation of this virus from water samples collected from the shores of the Tamagawa River in Kawasaki City, Kanagawa Prefecture, Japan (35°58911.77N, 139°65678.59E) on 22 October 2022.First, water sample was mixed with A. castllanii cell suspension in a culture solu­tion of proteose-peptone-yeast extract-glucose (PYG) medium including antibiotics as described previously (3–5). This mixture was added to a 96-well plate and incubated at 26°C. After 12 days, 10 µL of supernatant from a well that displayed cytopathic effects (CPE) of amoeba was inoculated into fresh A. castellanii cells and was cultured in 25 cm2 culture flasks to propagate the putative virus followed by virus cloning as described previously (6). To identify the viruses, negative-staining electron microscopy was performed using a JEM-2100F operated at 120 kV (JEOL Co. Ltd., Tokyo, Japan) (Fig. 1A). Cryo-electron microscopy was performed using a JEM-2200FS, operated at 200 kV (JEOL Co. Ltd.), which showed the detailed structure of the virus particles (Fig. 1B). To investigate viral formation in A. castellanii cells, transmission electron microscopy (TEM) of the virus-infected amoeba cells was performed using a JEM-1400 (JEOL Co. Ltd.) at the Hanaichi UltraStructure Research Institute (Aichi, Japan) (Fig. 1C). Although the specific morphology of the virus remains unknown, we have used here the term “tornado” to describe its unique architecture, based on the morphological features of the maturing viral particles observed in infected cells by TEM (Fig. 1C).Genomic DNA was extracted from the cloned and propagated tornadoviruses using a NucleoSpin tissue XS (Macherey-Nagel GmbH & Co. KG, Duren, Germany), according to the manufacturer's instructions. A DNA library was prepared for sequencing using a TrueSeq DNA PCR-Free (350) kit (Illumina, Inc.). Sequencing was performed using the NovaSeq 6000 platform (Illumina, Inc.). SPAdes 3.15.0 was used to assemble 28,832,452 reads (4,787,212,192 total read bases) into one contig of 378,863 nucleotides (8). Read length was 151. FastQC (v0.11.5) was used for quality control checks on raw sequence data. Open reading frames (ORFs) were detected using Prokka (9). Gene function was predicted using NCBI BLAST (10). The GC content of the genome was 38.81%. A total of 465 open reading frames were identified, including 463 coding sequences and two July 2024  Volume 13  Issue 7 10.1128/mra.00265-24 1Editor Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, BelgiumAddress correspondence to Masaharu Takemura, giantvirus@rs.tus.ac.jp.The authors declare no conflict of interest.See the funding table on p. 3.Received 15 March 2024Accepted 23 May 2024Published 11 June 2024Copyright © 2024 Waschestjuk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.tRNAs using NCBI BLAST (10). There were representative genes, which were found in previously reported pacmanviruses, including families B and X DNA polymerases, major capsid proteins, and virion packaging ATPase. Molecular phylogenetic analysis using family B DNA polymerase genes indicated that tornadoviruses are closely related to pacmanviruses with 55.6% identity (Fig. 1D).ACKNOWLEDGMENTSWe thank Macrogen Japan (Koto-ku, Tokyo, Japan) for the genome sequencing, de novo assembly, and annotation, the Hanaichi UltraStructure Research Institute (Okazaki, Aichi, Japan) for the TEM analyses, and Mr. Mitsuru Ikeda for negative-staining TEM and cryo-EM.This study was supported by grant funding from JSPS/KAKENHI (grant number 20H03078, awarded to M.T.), and ExCELLS project research (grant number 22EXC601-4, awarded to M.T.).AUTHOR AFFILIATIONS1Graduate School of Science, Tokyo University of Science, Shinjuku, Tokyo, Japan2Rijksuniversiteit Groningen, Faculty of Science and Engineering, Groningen, the Netherlands3Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Aichi, Japan4National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, Japan5Department of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, JapanFIG 1 (A) Negative-staining electron microscopy (EM) images of Tornadovirus japonicus particles. (B) Cryo-EM images of Tornadovirus japonicus particles. (C) TEM image of Tornadovirus japonicus particles during maturation of host cells. Bar: 200 nm (A–C). (D) Phylogenetic tree of B family DNA polymerase amino acid sequences. Analysis was performed by the MEGA X software (7), using Clustal W algorithm for alignment and the maximum likelihood method with 1,000 replicates for reconstruction of phylogenetic tree. Red box indicates Tornadovirus japonicus.Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 2Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.6Laboratory of Biology, Institute of Arts and Sciences, Tokyo University of Science, Shinjuku, Tokyo, JapanAUTHOR ORCIDsKazuyoshi Murata  http://orcid.org/0000-0001-9446-3652Masaharu Takemura  http://orcid.org/0000-0002-6261-3297FUNDINGFunder Grant(s) Author(s)MEXT | Japan Society for the Promotion of Science (JSPS)20H03078 Masaharu TakemuraDATA AVAILABILITYThe sequence data are available in GenBank (accession number LC801470.1) under BioProject accession numbers PRJDB17414 and SAMD00735632, and the raw reads can be found in the Sequence Read Archive (accession number DRR527376).REFERENCES1. Andreani J, Khalil JYB, Sevvana M, Benamar S, Di Pinto F, Bitam I, Colson P, Klose T, Rossmann MG, Raoult D, La Scola B. 2017. Pacmanvirus, a new giant Icosahedral virus at the crossroads between Asfarviridae and Faustoviruses. J Virol 91:e00212-17. https://doi.org/10.1128/JVI.00212-172. Geballa-Koukoulas K, Abdi S, La Scola B, Blanc G, Andreani J. 2021. Pacmanvirus S19, the second Pacmanvirus isolated from sewage waters in Oran, Algeria. Microbiol Resour Announc 10:e0069321. https://doi.org/10.1128/MRA.00693-213. Takemura M. 2016. Morphological and taxonomic properties of Tokyovirus, the first Marseilleviridae member isolated from Japan. Microbes Environ 31:442–448. https://doi.org/10.1264/jsme2.ME161074. Yoshikawa G, Blanc-Mathieu R, Song C, Kayama Y, Mochizuki T, Murata K, Ogata H, Takemura M. 2019. Medusavirus, a novel large DNA virus discovered from hot spring water. J Virol 93:e02130-18. https://doi.org/10.1128/JVI.02130-185. Takahashi H, Fukaya S, Song C, Murata K, Takemura M. 2021. Morpholog­ical and T\taxonomic properties of the newly isolated Cotonvirus japonicus, a new lineage of the subfamily Megavirinae. J Virol 95:e0091921. https://doi.org/10.1128/JVI.00919-216. Takemura M. 2022. “Genome sequence of a new “ Candidatus” phylum “dependentiae” isolate from Chiba, Japan”. Microbiol Resour Announc 11:e01123–21. https://doi.org/10.1128/mra.01123-217. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy0968. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.00219. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu15310. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 3Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.

University of GroningenComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolatedfrom the Tamagawa River in JapanWaschestjuk, Daniel; Murata, Kazuyoshi; Takemura, MasaharuPublished in:Microbiology resource announcementsDOI:10.1128/mra.00265-24IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2024Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Waschestjuk, D., Murata, K., & Takemura, M. (2024). Complete genome sequence of Tornadovirusjaponicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan. Microbiology resourceannouncements, 13(6), Article e00265-24. https://doi.org/10.1128/mra.00265-24CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 29-12-2025 | Virology | AnnouncementComplete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in JapanDaniel Waschestjuk,1,2 Kazuyoshi Murata,3,4,5 Masaharu Takemura1,6AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT Here, we report the isolation and genome sequencing of a new Pacman­virus-related isolate, Tornadovirus japonicus, from the Tamagawa River in Japan. This icosahedral virus has a genome of approximately 380 kb and 465 open reading frames, including two tRNA genes. The name “tornado” is based on its morphological features revealed by transmission electron microscopy analysis.KEYWORDS Nucleocytoviricota, Pacmanvirus, tornadovirus, giant virus, acanthamoebaP acmanvirus is a dsDNA virus that infects acanthamoeba. Many Pacmanvirus genes have been reported to be similar to those of faustoviruses and kaumoebaviruses, which are relatives of the family Asfarviridae (1). To date, only two strains of pacmanvi­ruses have been isolated from water samples in Algeria (A23 and S19) (1, 2). Here, we report the successful isolation of this virus from water samples collected from the shores of the Tamagawa River in Kawasaki City, Kanagawa Prefecture, Japan (35°58911.77N, 139°65678.59E) on 22 October 2022.First, water sample was mixed with A. castllanii cell suspension in a culture solu­tion of proteose-peptone-yeast extract-glucose (PYG) medium including antibiotics as described previously (3–5). This mixture was added to a 96-well plate and incubated at 26°C. After 12 days, 10 µL of supernatant from a well that displayed cytopathic effects (CPE) of amoeba was inoculated into fresh A. castellanii cells and was cultured in 25 cm2 culture flasks to propagate the putative virus followed by virus cloning as described previously (6). To identify the viruses, negative-staining electron microscopy was performed using a JEM-2100F operated at 120 kV (JEOL Co. Ltd., Tokyo, Japan) (Fig. 1A). Cryo-electron microscopy was performed using a JEM-2200FS, operated at 200 kV (JEOL Co. Ltd.), which showed the detailed structure of the virus particles (Fig. 1B). To investigate viral formation in A. castellanii cells, transmission electron microscopy (TEM) of the virus-infected amoeba cells was performed using a JEM-1400 (JEOL Co. Ltd.) at the Hanaichi UltraStructure Research Institute (Aichi, Japan) (Fig. 1C). Although the specific morphology of the virus remains unknown, we have used here the term “tornado” to describe its unique architecture, based on the morphological features of the maturing viral particles observed in infected cells by TEM (Fig. 1C).Genomic DNA was extracted from the cloned and propagated tornadoviruses using a NucleoSpin tissue XS (Macherey-Nagel GmbH & Co. KG, Duren, Germany), according to the manufacturer's instructions. A DNA library was prepared for sequencing using a TrueSeq DNA PCR-Free (350) kit (Illumina, Inc.). Sequencing was performed using the NovaSeq 6000 platform (Illumina, Inc.). SPAdes 3.15.0 was used to assemble 28,832,452 reads (4,787,212,192 total read bases) into one contig of 378,863 nucleotides (8). Read length was 151. FastQC (v0.11.5) was used for quality control checks on raw sequence data. Open reading frames (ORFs) were detected using Prokka (9). Gene function was predicted using NCBI BLAST (10). The GC content of the genome was 38.81%. A total of 465 open reading frames were identified, including 463 coding sequences and two July 2024  Volume 13  Issue 7 10.1128/mra.00265-24 1Editor Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, BelgiumAddress correspondence to Masaharu Takemura, giantvirus@rs.tus.ac.jp.The authors declare no conflict of interest.See the funding table on p. 3.Received 15 March 2024Accepted 23 May 2024Published 11 June 2024Copyright © 2024 Waschestjuk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.tRNAs using NCBI BLAST (10). There were representative genes, which were found in previously reported pacmanviruses, including families B and X DNA polymerases, major capsid proteins, and virion packaging ATPase. Molecular phylogenetic analysis using family B DNA polymerase genes indicated that tornadoviruses are closely related to pacmanviruses with 55.6% identity (Fig. 1D).ACKNOWLEDGMENTSWe thank Macrogen Japan (Koto-ku, Tokyo, Japan) for the genome sequencing, de novo assembly, and annotation, the Hanaichi UltraStructure Research Institute (Okazaki, Aichi, Japan) for the TEM analyses, and Mr. Mitsuru Ikeda for negative-staining TEM and cryo-EM.This study was supported by grant funding from JSPS/KAKENHI (grant number 20H03078, awarded to M.T.), and ExCELLS project research (grant number 22EXC601-4, awarded to M.T.).AUTHOR AFFILIATIONS1Graduate School of Science, Tokyo University of Science, Shinjuku, Tokyo, Japan2Rijksuniversiteit Groningen, Faculty of Science and Engineering, Groningen, the Netherlands3Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Aichi, Japan4National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, Japan5Department of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, JapanFIG 1 (A) Negative-staining electron microscopy (EM) images of Tornadovirus japonicus particles. (B) Cryo-EM images of Tornadovirus japonicus particles. (C) TEM image of Tornadovirus japonicus particles during maturation of host cells. Bar: 200 nm (A–C). (D) Phylogenetic tree of B family DNA polymerase amino acid sequences. Analysis was performed by the MEGA X software (7), using Clustal W algorithm for alignment and the maximum likelihood method with 1,000 replicates for reconstruction of phylogenetic tree. Red box indicates Tornadovirus japonicus.Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 2Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.6Laboratory of Biology, Institute of Arts and Sciences, Tokyo University of Science, Shinjuku, Tokyo, JapanAUTHOR ORCIDsKazuyoshi Murata  http://orcid.org/0000-0001-9446-3652Masaharu Takemura  http://orcid.org/0000-0002-6261-3297FUNDINGFunder Grant(s) Author(s)MEXT | Japan Society for the Promotion of Science (JSPS)20H03078 Masaharu TakemuraDATA AVAILABILITYThe sequence data are available in GenBank (accession number LC801470.1) under BioProject accession numbers PRJDB17414 and SAMD00735632, and the raw reads can be found in the Sequence Read Archive (accession number DRR527376).REFERENCES1. Andreani J, Khalil JYB, Sevvana M, Benamar S, Di Pinto F, Bitam I, Colson P, Klose T, Rossmann MG, Raoult D, La Scola B. 2017. Pacmanvirus, a new giant Icosahedral virus at the crossroads between Asfarviridae and Faustoviruses. J Virol 91:e00212-17. https://doi.org/10.1128/JVI.00212-172. Geballa-Koukoulas K, Abdi S, La Scola B, Blanc G, Andreani J. 2021. Pacmanvirus S19, the second Pacmanvirus isolated from sewage waters in Oran, Algeria. Microbiol Resour Announc 10:e0069321. https://doi.org/10.1128/MRA.00693-213. Takemura M. 2016. Morphological and taxonomic properties of Tokyovirus, the first Marseilleviridae member isolated from Japan. Microbes Environ 31:442–448. https://doi.org/10.1264/jsme2.ME161074. Yoshikawa G, Blanc-Mathieu R, Song C, Kayama Y, Mochizuki T, Murata K, Ogata H, Takemura M. 2019. Medusavirus, a novel large DNA virus discovered from hot spring water. J Virol 93:e02130-18. https://doi.org/10.1128/JVI.02130-185. Takahashi H, Fukaya S, Song C, Murata K, Takemura M. 2021. Morpholog­ical and T\taxonomic properties of the newly isolated Cotonvirus japonicus, a new lineage of the subfamily Megavirinae. J Virol 95:e0091921. https://doi.org/10.1128/JVI.00919-216. Takemura M. 2022. “Genome sequence of a new “ Candidatus” phylum “dependentiae” isolate from Chiba, Japan”. Microbiol Resour Announc 11:e01123–21. https://doi.org/10.1128/mra.01123-217. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy0968. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.00219. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu15310. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2Announcement Microbiology Resource AnnouncementsJuly 2024  Volume 13  Issue 7 10.1128/mra.00265-24 3Downloaded from https://journals.asm.org/journal/mra on 23 July 2024 by 129.125.148.226.

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Complete genome sequence of Tornadovirus japonicus, a relative of Pacmanvirus, isolated from the Tamagawa River in Japan

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