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

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.

Continuous year-round isolation of giant viruses from brackish shoreline soils.

Giant viruses, categorized under Nucleocytoviricota, are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce, with limited information available on unrecoverable strains, viral proliferation sites, and natural hosts. Previously, the author highlighted Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. styx, isolated from brackish water soil, as potential hotspots for giant virus multiplication. This study presents findings from nearly a year of monthly sampling within the same brackish water region after isolating the three aforementioned strains. This report details the recurrent isolation of a wide range of giant viruses. Each month, four soil samples were randomly collected from an approximately 5 × 10 m plot, comprising three soil samples and one water sample containing sediment from the riverbed. Acanthamoeba castellanii was used as a host for virus isolation. These efforts consistently yielded at least one viral species per month, culminating in a total of 55 giant virus isolates. The most frequently isolated species was Mimiviridae (24 isolates), followed by Marseilleviridae (23 isolates), Pandoravirus (6 isolates), and singular isolates of Pithovirus and Cedratvirus. Notably, viruses were not consistently isolated from any of the four samples every month, with certain sites yielding no viruses. Cluster analysis based on isolate numbers revealed that soil samples from May and water and sediment samples from January produced the highest number of viral strains. These findings underscore brackish coastal soil as a significant site for isolating numerous giant viruses, highlighting the non-uniform distribution along coastlines.

Analysis of Morphological Changes in the Nucleus and Vacuoles of Acanthamoeba castellanii following Giant Virus Infection.

Acanthamoeba castellanii medusavirus is a member of the phylum Nucleocytoviricota, also known as giant viruses, and has a unique strategy of infecting Acanthamoeba castellanii and replicating viral genes in the host nucleus. Here, we show time series changes in the intracellular morphology, including the nucleus, of host cells infected with four types of giant viruses, including medusavirus, using time-lapse phase-contrast microscopy and image analysis. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) to use multiple microscopic images with different focus positions to allow a more detailed analysis of their intracellular structures. Image analysis using PKA3 revealed that as medusavirus infection progressed, the host nucleus increased in size and the number of vacuoles decreased. In addition, infected host cells are known to become smaller and rounder at later stages of infection, but here they were found to be larger than uninfected cells at earlier stages. These results suggested that the propagation mechanism of medusavirus includes the formation of empty virus particles in the host cytoplasm, packaging of the viral genome replicated in the host nucleus, and then the release of viral particles. IMPORTANCE In this study, we quantitatively revealed how long the increase in host cell size or the increase in host nucleus size occurs after infection with giant viruses, especially medusavirus. To understand the underlying mechanism, we performed image analysis and determined that the host cell size increased at approximately 6 h postinfection (hpi) and the host nucleus enlarged at approximately 22 hpi, pointing to the importance of biochemical experiments. In addition, we showed that the intracellular structures could be quantitatively analyzed using multiple phase-contrast microscopy images with different focus positions at the same time point. Hence, morphological analyses of intracellular structures using phase-contrast microscopy, which have wide applications in live-cell observations, may be useful in studying various organisms that infect or are symbiotic with A. castellanii.

"Mamonoviridae", a proposed new family of the phylum Nucleocytoviricota.

Acanthamoeba castellanii medusavirus J1 is a giant virus that was isolated from a hot spring in Japan in 2019. Recently, a close relative of this virus, named medusavirus stheno T3, was isolated in Japan. Here, we describe their morphological, genomic, and gene content similarities and also propose to create a new family, "Mamonoviridae", a new genus, "Medusavirus", and two species, "Medusavirus medusae" and "Medusavirus sthenus", to classify these two viruses within the phylum Nucleocytoviricota.

Particle Morphology of Medusavirus Inside and Outside the Cells Reveals a New Maturation Process of Giant Viruses.

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoebae infected with the medusavirus showed four types of particles, i.e., pseudo-DNA-empty (p-Empty), DNA-empty (Empty), semi-DNA-full (s-Full), and DNA-full (Full). Time-dependent changes in the four types of particles and their intracellular localization suggested a new maturation process for the medusavirus. Viral capsids and viral DNAs are produced independently in the cytoplasm and nucleus, respectively, and only the empty particles located near the host nucleus can incorporate the viral DNA into the capsid. Therefore, all four types of particles were found outside the cells. The cryo-EM of these particles showed that the intact virus structure, covered with three different types of spikes, was preserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the Empty and p-Empty particles. The results suggest that these open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. IMPORTANCE Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture. Time-course analysis and intracellular localization of the medusavirus in the infected cells suggested a new maturation process via the four types of particles. Like the previously reported pandoravirus, the viral DNA of medusavirus is replicated in the host's nucleus. However, viral capsids are produced independently in the host cytoplasm, and only empty capsids near the nucleus can take up viral DNA. As a result, many immature particles were released from the host cell along with the mature particles. The capsid structure is well conserved among the four types of particles, except for the open membrane structures in the empty particles, suggesting that they are used to exchange scaffold proteins for viral DNAs. These findings indicate that medusavirus has a unique maturation process.

Genome Sequence of a New "Candidatus" Phylum "Dependentiae" Isolate from Chiba, Japan.

Little is known about the bacterial phylum "Candidatus Dependentiae," because only three isolates have been reported. Here, I report the isolation and genome sequencing of a new member of this phylum, strain Noda2021. This is the fourth strain isolated from the phylum "Candidatus Dependentiae."

2-Dimensional genetic algorithm exhibited an essentiality of gene interaction for evolution.

Organisms consist of several genetic factors differing between species. However, the evolutionary effects of gene interactions on the evolutionary rate, adaptation, and divergence of organisms remain unknown. In a previous study, the 2-dimensional genetic algorithm (2DGA) program, including a gene interaction parameter, could simulate punctuated equilibrium under the disparity mode. Following this, we verified the effect of the number of gene interactions (gene cluster size) on evolution speed, adaptation, and divergence using the advanced 2DGA program. In this program, the population was replicated, mutated, and selected for 200,000 generations, and the fitness score, divergence, number of population, and genotype were output and plotted. The genotype data were used for evaluating the phylogenetic relations among the population. The gene cluster size 1) affected the disparity and parity mutagenesis modes differently, 2) determined the growth/exclusion rate and error threshold, and 3) accelerated or decelerated the population's speed of evolutionary advancement. In particular, when the gene cluster size expanded, the rate of increase in fitness scores decreased independently of the mutation rate and mode of mutation (disparity mode/parity mode). The mutation rate at the error threshold was also decreased by expanding the gene cluster size. Dendrograms traced the genotypes of the simulated population, indicating that the disparity mode caused the evolutionary process to enter 1) a stun mode, 2) an evolution mode, or 3) a divergence mode based on the mutation rate and gene cluster size, while the parity mode did not cause the population to enter a stun mode. Based on the above findings, we compared the predictions of the present study with evolution observed in the laboratory or the natural world and the processes of ongoing virus evolution, suggesting that our findings possibly explained the real evolution.

RNA Sequencing of Medusavirus Suggests Remodeling of the Host Nuclear Environment at an Early Infection Stage.

Viruses of the phylum Nucleocytoviricota, or nucleo-cytoplasmic large DNA viruses (NCLDVs), undergo a cytoplasmic or nucleo-cytoplasmic cycle, the latter of which involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting NCLDVs. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate its infection strategy, we performed a time course RNA sequencing (RNA-seq) experiment. All viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and often (45%) encoded putative nucleus-localized proteins. These results suggest massive reshaping of the host nuclear environment by viral proteins at an early stage of infection. Genes in other expression clusters (clusters 2 to 5) were assigned to various functional categories. The virally encoded core histone genes were in cluster 3, whereas the viral linker histone H1 gene was in cluster 1, suggesting they have distinct roles during the course of the virus infection. The transcriptional profile of the host Acanthamoeba castellanii genes was greatly altered postinfection. Several encystment-related host genes showed increased representation levels at 48 h postinfection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. IMPORTANCE Medusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family "Medusaviridae" in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA sequencing (RNA-seq) analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intranuclear environment during infection. The transcriptional profile of amoeba genes was greatly altered postinfection.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells.

Most virus-infected cells show morphological and behavioral changes, which are called cytopathic effects. Acanthamoeba castellanii, an abundant, free-living protozoan, serves as a laboratory host for some viruses of the phylum Nucleocytoviricota-the giant viruses. Many of these viruses cause cell rounding in the later stages of infection in the host cells. Here, we show the changes that lead to cell rounding in the host cells through time-lapse microscopy and image analysis. Time-lapse movies of A. castellanii cells infected with Mimivirus shirakomae, kyotovirus, medusavirus, or Pandoravirus japonicus were generated using a phase-contrast microscope. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) and used it to analyze these time-lapse movies. Image analysis revealed that the process leading to cell rounding varies among the giant viruses; for example, M. shirakomae infection did not cause changes for some time after the infection, kyotovirus infection caused an early decrease in the number of cells with typical morphologies, and medusavirus and P. japonicus infection frequently led to the formation of intercellular bridges and rotational behavior of host cells. These results suggest that in the case of giant viruses, the putative reactions of host cells against infection and the putative strategies of virus spread are diverse. IMPORTANCE Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii, which were previously unclear. It was revealed that medusavirus and Pandoravirus japonicus infection led to the formation of a significant number of elongated particles related to intercellular bridges, emphasizing the importance of research on the interaction of viruses with host cell nuclear function. Mimivirus shirakomae infection did not cause any changes in the host cells initially, so it is thought that the infected cells can actively move and spread over a wider area, emphasizing the importance of observation in a wider area and analysis of infection efficiency. These results suggest that a kinetic analysis using the phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) reveals the infection strategies of each giant virus.

Sinking of Four Species of Living Diatom Cells Directly Observed by a "Tumbled" Optical Microscope.

The study of the sinking phenomenon of diatom cells, which have a slightly larger specific gravity (~1.3) compared to that of water, is an important research topic for understanding photosynthetic efficiency. In this study, we successfully demonstrated the observation of the sinking behaviors of four different species of diatom using a homemade "tumbled" optical microscope. A homemade 1 mm3 microchamber was employed to decrease the effects of convection currents. In the microchamber, diatom cells were basically settled in a linear manner without floating, although some of the cells were rotated during their sinking. Sinking speeds of the four species of diatom cells, Nitzschia sp., Pheodactylum tricornutum, Navicula sp., and Odontella aurita, were 0.81 ± 5.56, 3.03 ± 10.17, 3.29 ± 7.39, and 11.22 ± 21.42 µm/s, respectively, based on the automatic tracking analysis of the centroids of each cell. Manual analysis of a vector between two longitudinal ends of the cells (two-point analysis) was effective for quantitatively characterizing the rotation phenomenon; therefore, angles and angular velocities of rotating cells were well determined as a function of time. The effects of the cell shapes on sinking velocity could be explained by simulation analysis using the modified Stokes' law proposed by Miklasz et al.

Morphological and Taxonomic Properties of the Newly Isolated Cotonvirus japonicus, a New Lineage of the Subfamily Megavirinae.

Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae, excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae. Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae. Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae. IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae, subfamily Megavirinae, from the fresh water of a canal in Japan, named Cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae-lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.

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.

Marseilleviridae Lineage B Diversity and Bunch Formation Inhibited by Galactose.

Marseilleviridae is a family of large double-stranded DNA viruses that is currently divided into five subgroups, lineages A-E. Hokutovirus and kashiwazakivirus, both of which belong to lineage B, have been reported to induce host acanthamoeba cells to form aggregations called "bunches". This putatively results in increased opportunities to infect acanthamoeba cells, in contrast to lineage A, which has been reported to not form "bunches". In the present study, we isolated 14 virus strains of the family Marseilleviridae from several Japanese water samples, 11 of which were identified as lineage B viruses. All 11 lineage B strains caused infected amoeba cells to form bunches. We then investigated the involvement of monosaccharides in bunch formation by amoeba cells infected with hokutovirus. Galactose inhibited bunch formation, thereby allowing amoeba cells to delay the process, whereas mannose and glucose did not. A kinetic image analysis of hokutovirus-infected amoeba cells confirmed the inhibition of bunch formation by galactose. The number of hokutovirus-infected amoeba cells increased more rapidly than that of tokyovirus-infected cells, which belongs to lineage A. This result suggests that bunch formation by infected amoeba cells is advantageous for lineage B viruses.

Draft Genome Sequence of Medusavirus Stheno, Isolated from the Tatakai River of Uji, Japan.

"Medusaviridae" is a proposed family of large double-stranded DNA (dsDNA) viruses so far represented by a sole virus isolated from a hot spring. In the present study, we report the isolation and genome sequencing of a second member of this family, medusavirus stheno, discovered from a freshwater sample with an Acanthamoeba castellanii coculture.

Medusavirus Ancestor in a Proto-Eukaryotic Cell: Updating the Hypothesis for the Viral Origin of the Nucleus.

The mechanistic evolutionary origin of the eukaryotic cell nucleus remains unknown. Among several plausible hypotheses, the most controversial is that large DNA viruses, such as poxviruses, led to the emergence of the eukaryotic cell nucleus. Several recent findings, including the discovery of a nucleus-like structure in prokaryotic viruses and prokaryotes possessing nucleus-like inner membranes, suggest genomic DNA compartmentalization not only in eukaryotes but also in prokaryotes. The sophisticated viral machinery of mimiviruses is thought to resemble the eukaryotic nucleus: DNA replicates both inside the viral factory and nucleus, which is at least partially surrounded by membranes and is devoid of ribosomes. Furthermore, several features of the recently identified Acanthamoeba castellanii medusavirus suggest that the evolutionary relationship between ancestral viral factory and eukaryotic nucleus. Notably, Ran, DNA polymerase, and histones show molecular fossils of lateral transfer of nuclear genes between the virus and host. These results suggest viral innovation in the emergence of the eukaryotic nucleus. According to these results, a new scenario explaining the origin of the eukaryotic nucleus from the perspective of viral participation is proposed. This new scenario could substantially impact the study of eukaryogenesis and stimulate further discussion about viral contributions to the evolution of the eukaryotic nucleus.

Co-Isolation and Characterization of Two Pandoraviruses and a Mimivirus from a Riverbank in Japan.

Giant viruses, like pandoraviruses and mimiviruses, have been discovered from diverse environments, and their broad global distribution has been established. Here, we report two new isolates of Pandoravirus spp. and one Mimivirus sp., named Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. isolate styx, co-isolated from riverbank soil in Japan. We obtained nearly complete sequences of the family B DNA polymerase gene (polB) of P. hades and P. persephone; the former carried two known intein regions, while the latter had only one. Phylogenetic analysis revealed that the two new pandoravirus isolates are closely related to Pandoravirus dulcis. Furthermore, random amplified polymorphic DNA analysis revealed that P. hades and P. persephone might harbor different genome structures. Based on phylogenetic analysis of the partial polB sequence, Mimivirus sp. isolate styx belongs to mimivirus lineage A. DNA staining suggested that the Pandoravirus spp. asynchronously replicates in amoeba cells while Mimivirus sp. replicates synchronously. We also observed that P. persephone- or Mimivirus sp. isolate styx-infected amoeba cytoplasm is extruded by the cells. To the best of our knowledge, we are the first to report the isolation of pandoraviruses in Asia. In addition, our results emphasize the importance of virus isolation from soil to reveal the ecology of giant viruses.

Distribution of SNSs in Mimivirus Genomes and the Classification of Mimiviruses Isolated from Japan.

Mimiviruses have been detected in various habitats. Analyses of single nucleotide substitutions (SNSs) have revealed that SNSs are mainly localized on both ends of the mimivirus genome, and mimivirus lineage A has been split into three genotype groups; therefore, mimiviruses may be classified into lineages and genotype groups based on SNSs. We isolated 9 mimiviruses from Japan and analyzed SNSs. These isolates were classified as lineage A genotype group type 2, suggesting that the local diversity of members of the family Mimiviridae isolated from Acanthamoeba spp. is lower than that of giant viruses from other families isolated in Japan.

Fifteen Marseilleviruses Newly Isolated From Three Water Samples in Japan Reveal Local Diversity of Marseilleviridae.

The family Marseilleviridae, defined as a group of icosahedral double-stranded DNA viruses with particle size of approximately 250 nm and genome size of 350-380 kbp, belongs to the nucleo-cytoplasmic family of large DNA viruses. The family Marseilleviridae is currently classified into lineages A-E. In this study, we isolated 12 or 15 new members of the family Marseilleviridae from three sampling locations in Japan. Molecular phylogenetic analysis of the MCP genes showed that the new viruses could be further classified into three groups, hokutoviruses, kashiwazakiviruses, and kyotoviruses. Hokutoviruses were closely related to lineage B, kyotoviruses were related to lineage A, and kashiwazakiviruses were also classified into lineage B but a new putative subgroup of lineage B, revealing the diversity of this lineage. Interestingly, more than two viruses with slightly different MCP genes were isolated from a single water sample from a single location, i.e., two hokutoviruses and one kashiwazakivirus were isolated from a small reservoir, five kashiwazakiviruses from the mouth of a river, and five kyotoviruses from fresh water of a river, suggesting that several milliliters of water samples contain several types of giant viruses. Amoeba cells infected with hokutoviruses or kashiwazakiviruses exhibited a "bunch" formation consisting of normal and infected cells similarly to a tupanvirus, whereas cells infected with kyotoviruses or tokyovirus did not. These results suggest the previously unrecognized local diversity of the family Marseilleviridae in aquatic environments.

Visualization of giant virus particles and development of "VIRAMOS" for high school and university biology course.

Several educational trials on handling viruses and or virology have been reported. However, given their small size, direct visualization of these viruses under a microscope has been rarely performed. The so-called "giant viruses" are larger than other viruses with a particle size greater than 200-300 nm. This enables their direct visualization under a light microscope more easily than other viruses. In this study, we developed two new types of teaching material for learning about viruses and cellular organisms using mimivirus, one of the well-known giant viruses. One teaching material involves using glass slides with enclosed mimivirus particles, and another is a paper-based teaching material, named VIRAMOS (http://tlab-edusys.azurewebsites.net/content/viramos_en.pdf). Using these, students can investigate and learn about viruses and cellular organisms. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):426-431, 2019.

Gram-Positive Bacteria-Like DNA Binding Machineries Involved in Replication Initiation and Termination Mechanisms of Mimivirus.

The detailed mechanisms of replication initiation, termination and segregation events were not yet known in Acanthamoeba polyphaga mimivirus (APMV). Here, we show detailed bioinformatics-based analyses of chromosomal replication in APMV from initiation to termination mediated by proteins bound to specific DNA sequences. Using GC/AT skew and coding sequence skew analysis, we estimated that the replication origin is located at 382 kb in the APMV genome. We performed homology-modeling analysis of the gamma domain of APMV-FtsK (DNA translocase coordinating chromosome segregation) related to FtsK-orienting polar sequences (KOPS) binding, suggesting that there was an insertion in the gamma domain which maintains the structure of the DNA binding motif. Furthermore, UvrD/Rep-like helicase in APMV was homologous to Bacillus subtilis AddA, while the chi-like quartet sequence 5'-CCGC-3' was frequently found in the estimated ori region, suggesting that chromosomal replication of APMV is initiated via chi-like sequence recognition by UvrD/Rep-like helicase. Therefore, the replication initiation, termination and segregation of APMV are presumably mediated by DNA repair machineries derived from gram-positive bacteria. Moreover, the other frequently observed quartet sequence 5'-CGGC-3' in the ori region was homologous to the mitochondrial signal sequence of replication initiation, while the comparison of quartet sequence composition in APMV/Rickettsia-genome showed significantly similar values, suggesting that APMV also conserves the mitochondrial replication system acquired from an ancestral genome of mitochondria during eukaryogenesis.