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. 2016 Dec 23;31(4):442-448.
doi: 10.1264/jsme2.ME16107. Epub 2016 Nov 19.

Morphological and Taxonomic Properties of Tokyovirus, the First Marseilleviridae Member Isolated from Japan

Masaharu Takemura  1
Affiliations

Morphological and Taxonomic Properties of Tokyovirus, the First Marseilleviridae Member Isolated from Japan

Masaharu Takemura. Microbes Environ. .

Abstract

Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.

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Figures

Fig. 1
Fig. 1
Transmission electron microscopy images of ultrathin sections of Tokyovirus-infected Acanthamoeba castellanii cells. (A) Comprehensive image of a Tokyovirus-infected amoeba cell at 8 h p.i. Mature virions (small black particles) are encapsulated in several intracytoplasmic vacuoles. A putative virion factory is seen in the lower-left portion of the cell. The cell nucleus is not evident in this section. Scale bar, 5 μm. (B) Enlarged image of Tokyoviruses in the intracytoplasmic vacuoles of the amoeba. The particle diameter is approximately 200 nm. Scale bar, 500 nm. (C) Transmission electron microscopy image of a typical virion factory in the cytoplasmic region of Tokyovirus-infected amoeba cells. Virion factories are distinctly classified into two portions: one in which mature virions densely co-exist (black arrow), and another portion surrounding the former (white arrow). In this section, a small part of the cell nucleus is shown (black arrowhead).
Fig. 2
Fig. 2
Fluorescent microscopy images of Tokyovirus-infected Acanthamoeba castellanii cells stained with DAPI. Staining by DAPI was performed at 8 h p.i., as described in the Materials and Methods. (A) Control amoeba cells without Tokyovirus infection. Cell nuclei were slightly stained and appeared donut-shaped. (B) Tokyovirus-infected amoeba cells. Virion factories were brightly stained. (C) Merged view of fluorescent and phase-contrast images. (D) Enlarged image of Tokyovirus-infected amoeba cells for which the virion factory is brightly stained.
Fig. 3
Fig. 3
Genome comparison of Tokyovirus and seven other Marseilleviridae family members using a dot plot analysis. (A) Dot plots based on the genomic positions of orthologous CDSs between Tokyovirus and Marseillevirus. The first part of the Tokyovirus genome exhibits collinearity with the final part of the Marseillevirus genome, but in the opposite orientation. The large central parts of both genomes exhibit clear collinearity. (B) A dot plot analysis between Tokyovirus and Melbournevirus. Almost the same collinearity was shown as that in A. (C) A dot plot analysis between Tokyovirus and Cannes 8 virus. Almost the same collinearity was shown as those in A and B. (D) A dot plot analysis between Tokyovirus and Lausannevirus. The former part of the Tokyovirus genome (position approximately 170,000 bps) exhibits weak collinearity with the Lausannevirus genome, whereas the latter part (position approximately 170,000–320,000 bps) shows some collinearity, with several inverted segments. (E) A dot plot analysis between Tokyovirus and Tunisvirus. The former part of the Tokyovirus genome exhibits weak collinearity, as with Lausannevirus. The latter part shows some collinearity with several inverted segments, similar to Lausannevirus. (F) A dot plot analysis between Tokyovirus and Insectomime virus. The former part of the Tokyovirus genome exhibits weak collinearity with the Insectomime virus genome, and the latter part shows some collinearity, but in the opposite orientation. (G) A dot plot analysis between Tokyovirus and Brazilian Marseillevirus. The former part of the Tokyovirus genome exhibits weak collinearity, as with Lausannevirus and Tunisvirus. The latter part shows some collinearity with several inverted segments, similar to Lausannevirus and Tunisvirus.
Fig. 4
Fig. 4
Venn diagram showing a comparative analysis of gene contents of Tokyovirus with six other Marseilleviridae family members. The numbers of genes showing >90% similarity are shown in the merged regions. The upper diagram shows an analysis of Tokyovirus, Marseillevirus, and Melbournevirus. The middle diagram shows an analysis of Tokyovirus, Cannes 8 virus, and Brazilian Marseillevirus. The lower diagram shows an analysis of Tokyovirus, Tunisvirus, and Lausannevirus.
Fig. 5
Fig. 5
Unrooted maximum-likelihood phylogenetic trees of B-family DNA polymerase (A), PCNA (B), and DNA-directed RNA polymerase alpha subunit (C) sequences constructed using MEGA7 software (26). The trees were reconstructed based on alignments (DNA polymerase, 1,103 sites; PCNA, 257 sites; RNA polymerase alpha subunit, 452 sites) derived from the full-length alignment in which any column containing a gap was discarded. Numbers at the branch points denote percent bootstrap values. The accession numbers of respective sequences are listed in Tables S1~S3. The letters A, B, C, and D indicate the subclades of Marseilleviridae.
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