Biology subjects

Hikida, H.

Publications and source records attributed to Hikida, H..

15 recordsLinked to original sources

bioRxiv2026

Poxvirus gene repertoires are shaped by expansion-biased birth-and-death dynamics and genome architectural constraints

Poxviruses (family Poxviridae) are DNA viruses with exceptionally large genomes containing hundreds of genes. Yet the evolutionary processes contributing to their numerous gene repertoires remain insufficiently understood. Here, we inferred ortholog groups of genes across 58 poxvirus genomes and comprehensively analyzed gene gain, gene duplication, gene loss, horizontal gene transfer, and synteny conservation. Across poxviruses, coding capacity scaled linearly with genome length. Gene repertoires evolved through birth-and-death dynamics but generally expanded because gene gain cumulatively exceeded gene loss and gained genes subsequently underwent further duplication. Gene gain and duplication were concentrated in the terminal regions of most poxvirus genomes. In viral lineages whose gene repertoires expanded extensively, terminal-region expansion was accompanied by further accumulation of duplicated genes within the otherwise conserved central genomic regions. The phylogenetic placement of the bird-infecting genus Avipoxvirus, together with evidence that several horizontally acquired genes originated from mammals, raises the possibility of a mammalian origin for Avipoxvirus. Altogether, our findings reveal that poxvirus gene repertoire evolution is shaped by expansion-biased birth-and-death dynamics and genome architectural constraints.

evolutionary biology↗

bioRxiv2026

Genetic manipulation of a giant virus-associated virophage

Virophages are double-stranded DNA viruses that hyperparasitize giant viruses infecting unicellular eukaryotes. Parasitization by virophages often reduces the replication of giant viruses, thereby modulating microbial communities in the environment. However, the molecular mechanisms underlying the tripartite relationship are largely unknown due to methodological limitations. In the present study, we developed a reverse-genetics system for a Sputnik virophage that parasitizes the amoeba-infecting giant virus, mimivirus. We demonstrated that transfection of genomic DNA could recover infectious virophage particles. Transfection of genomic DNA synthesized by circular polymerase extension reaction (CPER) also resulted in the recovery of infectious viruses. As a proof of concept, we successfully modified two Sputnik genes by transfecting CPER-assembled mutant genomic DNA. Collectively, our reverse-genetics system provides a framework for assessing the functional importance of Sputnik genes and should facilitate future genetic studies of virophages. Significance statementVirophages are viruses that hyperparasitize giant viruses, which infect unicellular eukaryotes and have extremely large particles and genomes. Giant viruses modulate microbial communities not only by killing their hosts but also by altering host cellular functions. Virophages modulate the replication of giant viruses, thereby driving ecosystem dynamics. Previous studies have demonstrated their widespread distribution through isolation and metagenomic analyses. However, the functions of most virophage genes remain unknown. Due to the lack of genetic tools, the molecular mechanisms underlying the interactions between virophages and giant viruses remain largely elusive. Here, we established a virophage reverse-genetics system based on circular polymerase extension reaction. Our results demonstrate that the system can dissect virophage gene functions and will accelerate virophage genetics.

microbiology↗

bioRxiv2025

Horizontal transfer of a 180-kbp genomic fraction among the largest viral genomes

Viruses are generally considered tiny biological entities with small genomes; however, some dsDNA viruses, known as giant viruses, have large genomes that are comparable to those of small bacteria. Previous studies indicated that the genomes of giant viruses expanded from a small ancestor by a combination of gene duplication, de novo gene creation, and horizontal gene transfer (HGT). Among them, virus-to-virus HGTs are recently recognized as an important mechanism for disseminating functional genes between giant viruses. In this study, we isolated a giant virus of a member of pandoraviruses, which have the largest genome sizes reaching 2.5 Mbp. Based on the average nucleotide identity of known pandoraviruses, this new pandoravirus belongs to an existing viral species. However, its genome was approximately 200 kbp larger than those of the other strains from the same species. A genome-wide comparison identified a 180-kbp region with 168 genes missing from the other strains but found in phylogenetically distant pandoraviruses. The GC ratio and homology of the deduced amino acid sequences in this 180-kbp region suggest that the new virus horizontally acquired this region from a distantly related pandoravirus. The gene composition in the 180-kbp region further indicates that this region was already large at the time of the horizontal transfer. Our findings suggest that pandoraviruses can horizontally exchange a large portion of their genomes. This event presumably represents one mechanism for accelerating genomic evolution and gigantism in giant viruses.

microbiology↗

bioRxiv2025

Generation of a recombinant mimivirus by direct transfection of a PCR amplicon

Giant viruses are large double-stranded DNA viruses encoding hundreds of genes. These genes are considered to reprogram the host cell machinery, which, in turn, modulates the global nutrient cycles in the environment. However, the functions of giant-virus genes are largely unknown due to the lack of reverse-genetics systems. Recently, reverse-genetics systems have been developed in giant viruses infecting a free-living amoeba, Acanthamoeba castellanii. One of the methods relies on homologous recombination by plasmid transfection, but plasmid construction is generally laborious. In this study, we developed a method to generate a recombinant giant virus, mimivirus, with direct transfection of a PCR amplicon into host cells, a widely used approach in budding yeast and other organisms. Consequently, we successfully generated a recombinant mimivirus with a marker gene. Our results demonstrated that a bacterium-free method could be applied to giant viruses to generate recombinants, accelerating further experimental studies related to these viruses.

microbiology↗

bioRxiv2024

Giant virus creates subcellular environment to overcome codon-tRNA mismatch

Codon usage consonant with the cellular tRNA pool is important for efficient translation. However, many eukaryotic viruses, including amoeba-infecting mimiviruses, have codon usage that is largely deviated from that of their host, despite using the host machinery for translation. This raises the question of how these viruses cope with the mismatch between tRNA supply and demand. Here we show that Acanthamoeba castellanii mimivirus generates a subcellular area in the host cells to translate virus mRNAs. A combination of genome-wide ribosome profiling and RNA sequencing showed that ribosomes traversed along viral mRNAs with fewer pausing events than were observed on amoeba mRNAs. Frequently used codons in viral mRNAs had higher tRNA accessibility than the same type of codons in amoeba mRNAs. tRNA sequencing showed that the tRNA pool was not greatly altered during the infection even though the virus encodes tRNA genes. Instead, by in situ labelling, we found that viral mRNAs and newly synthesized proteins were localized at the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation assists the virus in overcoming the mismatch between tRNA supply and demand.

microbiology↗

bioRxiv2024

Sputnik virophage disrupts the transcriptional regulation of its host giant virus

Sputnik virophages are small double-stranded DNA (dsDNA) viruses that replicate only inside host amoebae infected with giant dsDNA viruses, mimiviruses. Sputnik infection affects mimivirus replication, but their molecular interaction remains poorly understood. Here, we performed a time-course transcriptome analysis of Acanthamoeba castellanii cells infected with Acanthamoeba polyphaga mimivirus (APMV) (hereafter referred to as Sputnik- cells) and those infected with both APMV and Sputnik 3 virophage (Sputnik+ cells). The gene expression patterns of the amoebae were similar between these two conditions, whereas the expression of APMV genes was drastically affected by Sputnik, depending on the timing of their expression. Early expressed APMV genes showed similar expression patterns in both conditions at the early stage of infection. However, at late stages, their expression levels remained higher in Sputnik+ cells than in Sputnik- cells, suggesting a prolongation of early gene expression by Sputnik. Late expressed APMV genes showed lower expression at earlier stages in Sputnik+ cells, but their expression levels reached or exceeded those in Sputnik- cells at later stages, indicating a delay of gene expression. Overall, our results demonstrated that Sputnik infection drastically alters the transcriptome of APMV rather than amoeba, likely by disturbing the transition from early to late stages of APMV infection.

microbiology↗

bioRxiv2024

Eukaryotic genomic data uncover an extensive host range of mirusviruses

A recent marine metagenomic study has revealed the existence of a novel group of viruses designated mirusviruses, which are proposed to form an evolutionary link between two realms of double-stranded DNA viruses, Varidnaviria and Duplodnaviria. Metagenomic data suggest that mirusviruses infect microeukaryotes in the photic layer of the ocean, but their host range remains largely unknown. In this study, we investigated the presence of mirusvirus marker genes in publicly available 1,901 eukaryotic genome assemblies, mainly derived from unicellular eukaryotes, to identify potential hosts of mirusviruses. Mirusvirus marker sequences were identified in 1,348 assemblies spanning 284 genera across eight supergroups of eukaryotes. The habitats of the putative mirusvirus hosts included not only marine but also other diverse environments. Among the major capsid protein (MCP) signals in the genome assemblies, we identified 85 sequences that showed high sequence and structural similarities to reference mirusvirus MCPs. A phylogenetic analysis of these sequences revealed their distant evolutionary relationships with the seven previously reported mirusvirus clades. Most of the scaffolds with these MCP sequences encoded multiple mirusvirus homologs, underscoring the impact of mirusviral infection on the evolution of the host genome. We also identified three circular mirusviral genomes within the genomic data of the oil producing thraustochytrid Schizochytrium sp. and the endolithic green alga Ostreobium quekettii. Overall, mirusviruses probably infect a wide spectrum of eukaryotes and are more diverse than previously reported. HighlightsO_LIMirusvirus signals detected in genomic data from eight eukaryotic supergroups. C_LIO_LIHabits of putative mirusvirus hosts not limited to marine environments. C_LIO_LIMajor capsid sequences from these assemblies show new mirusviral lineages. C_LIO_LIThree circular mirusvirus genomes were identified. C_LI

microbiology↗

bioRxiv2023

Complex genomes of early nucleocytoviruses revealed by ancient origins of viral aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs), also known as tRNA ligases, are essential enzymes in translation. Owing to their functional essentiality, these enzymes are conserved in all domains of life and used as informative markers to trace the evolutionary history of cellular organisms. Unlike cellular organisms, viruses generally lack aaRSs because of their obligate parasitic nature, but several large and giant DNA viruses in the phylum Nucleocytoviricota encode aaRSs in their genomes. The discovery of viral aaRSs led to the idea that the phylogenetic analysis of aaRSs can shed light on ancient viral evolution. However, conflicting results have been reported from previous phylogenetic studies: one posited that nucleocytoviruses recently acquired their aaRSs from their host eukaryotes, while another hypothesized that the viral aaRSs have ancient origins. Here, we investigated 4,168 nucleocytovirus genomes, including metagenome-assembled genomes derived from large-scale metagenomic studies. In total, we identified 780 viral aaRS sequences in 273 viral genomes. We generated and examined phylogenetic trees of these aaRSs with a large set of cellular sequences to trace evolutionary relationships between viral and cellular aaRSs. The analyses firmly establish that the origins of some viral aaRSs predate the last common eukaryotic ancestor. Inside viral aaRS clades, we identify intricate evolutionary trajectories of viral aaRSs with horizontal transfers, losses, and displacements. Overall, these results suggest that ancestral nucleocytoviruses already developed complex genomes with an expanded set of aaRSs in the proto-eukaryotic era.

evolutionary biology↗

bioRxiv2023

Gene transfer among viruses substantially contributes to gene gain of giant viruses

Horizontal gene transfers (HGTs) integrate all forms of life and viruses into a vast network of gene flow, which facilitates the transmission of genes beyond vertical inheritance and enhances genomic evolution. HGT is known to occur between closely related viruses. We hypothesized that there is frequent HGT among nucleocytoviruses, a group of diverse but evolutionarily related DNA viruses encoding hundreds to thousands of genes. However, the frequency of viral HGT (vHGT) has not been systematically investigated for nucleocytoviruses. We reconciled over 4,700 gene trees with a robust viral species tree that contains 195 reference viral genomes mainly from cultivation as a reference to infer evolutionary events, including gene gains (gene duplication, origination, and vHGT) and losses. The inferred evolutionary scenarios revealed that the genomes of these viruses have undergone numerous gene gain and loss events, with vHGT representing 28% to 42% of gene gain events in each viral order. By integrating the evolutionary paths of multiple viruses, our data suggest that vHGT is crucial for nucleocytovirus evolution.

evolutionary biology↗

bioRxiv2023

A 1.5 Mb continuous endogenous viral element region in the arbuscular mycorrhizal fungus Rhizophagus irregularis

Most fungal viruses are RNA viruses and no double-stranded DNA virus that infects fungi is known to date. A recent study detected DNA polymerase genes that originated from large dsDNA viruses in the genomes of basal fungi, suggestive of the existence of dsDNA viruses capable of infecting fungi. In this study, we searched for viral infection signatures in chromosome-level genome assemblies of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We identified a continuous 1.5 Mb putative viral region on a chromosome in R. irregularis strain 4401. Phylogenetic analyses revealed that the viral region is related to viruses in the family Asfarviridae of the phylum Nucleocytoviricota. Single-copy marker genes from Nucleocytoviricota were detected as single-copy genes in the viral region. Furthermore, this viral region was absent in the genomes of four other R. irregularis strains and had fewer signals of fungal transposable elements than the other genomic regions. These results suggest a recent and single insertion of a large dsDNA viral genome in the genome of this fungal strain, providing strong evidence of the recent infection of the fungus by a dsDNA virus.

microbiology↗

bioRxiv2023

A rapid genome-wide analysis of isolated giant viruses only using MinION sequencing

Following the discovery of Acanthamoeba polyphaga mimivirus, diverse giant viruses have been isolated. However, only a small fraction of these isolates has been completely sequenced, limiting our understanding of the genomic diversity of giant viruses. MinION is a portable and low-cost long-read sequencer that can be readily used in a laboratory. Although MinION provides highly error-prone reads that require correction through additional short-read sequencing, recent studies assembled high-quality microbial genomes only using MinION sequencing. Here, we evaluated the accuracy of MinION-only genome assemblies for giant viruses by re-sequencing a prototype marseillevirus. Assembled genomes presented over 99.98% identity to the reference genome with a few gaps, demonstrating a high accuracy of the MinION-only assembly. As a proof of concept, we de novo assembled five newly isolated viruses. Average nucleotide identities to their closest known relatives suggest that the isolates represent new species of marseillevirus, pithovirus, and mimivirus. Assembly of subsampled reads demonstrated that their taxonomy and genomic composition could be analyzed at the 50x sequencing coverage. We also identified a pithovirus gene whose homologues were detected only in metagenome-derived relatives. Collectively, we propose that MinION-only assembly is an effective approach to rapidly perform a genome-wide analysis of isolated giant viruses.

microbiology↗

bioRxiv2022

Baculovirus bv/odv-e26 is required for host behavioral manipulation by optimizing viral virulence to lepidopteran hosts

Host behavioral manipulation is a widely used strategy for parasites to enhance their transmission. Lepidopteran nucleopolyhedroviruses (NPVs) induce hyperactivity and alteration of behavioral patterns in host insect larvae. Previous studies revealed that Bombyx mori nucleopolyhedrovirus (BmNPV) does not induce hyperactivity in larval Bombyx mori, when Bm8, a homologue of the baculovirus bv/odv-e26 gene, is knocked out. Bm8 knockout also enhances pathogenicity, but the relationship between Bm8 functions and host behavioral manipulation is still unknown. This study revealed that Bm8 is dispensable for altering behavioral patterns but crucial for inducing hyperactivity. Bm8 overexpression decreased pathogenicity in B. mori larvae and delayed viral infection in cultured cells. These results suggest that Bm8 maintains larval locomotory activity and synchronizes hyperactivity induction with behavioral pattern alteration by suppressing viral virulence. Conserved amino acid residues were also identified in the coiled-coil domain of the Bm8 protein, which is required for suppressing viral virulence in cultured cells and larval hosts. Although bv/odv-e26 is previously thought to be conserved in a limited group of lepidopteran NPVs, our synteny-based bioinformatics approach discovered putative homologues in the genomes of most lepidopteran NPVs. Functional analyses revealed that phylogenetically close and distant bv/odv-e26 homologues possess suppressive activity with functionally essential common residues in their coiled-coil domains. Collectively, these findings indicate that baculovirus bv/odv-e26 is a conserved gene optimizing viral virulence for establishing behavioral manipulation in NPV-infected larval hosts. Author SummaryBaculovirus-induced host abnormal behavior has been described in various lepidopteran insects for a long time, although it is still largely unknown how the virus establishes this complex trait. We showed that baculovirus bv/odv-e26 is a factor that suppresses viral virulence and synchronizes hyperactivity and behavioral pattern alteration. We also revealed that putative bv/odv-e26 homologues are encoded by a wider range of baculoviruses than previously thought, with functionally conserved residues. We confirmed the suppressive function in both phylogenetically close and distant bv/odv-e26 homologues. These results indicate that acquiring a suppressive factor for viral virulence is a crucial event for the evolution of a host manipulation strategy by baculoviruses.

microbiology↗

bioRxiv2022

KaicoTracker: a robust and automated locomotory analysis for baculovirus-infected silkworm larvae

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen of the domestic silkworm, Bombyx mori, whose infection drastically alters larval locomotory patterns and activity. As B. mori is the only insect that has been completely domesticated, its larvae are typically inactive. In contrast, BmNPV-infected larvae exhibit vigorous movement. Recently, we developed a method for locomotory analysis of BmNPV-infected B. mori larvae, which yielded high-resolution data on BmNPV-induced host locomotory alteration. However, this method had a disadvantage when analyzing the behavior of uninfected larvae, as they were typically inactive and difficult to distinguish from the background. In addition, the analysis processes involve many manual steps, which reduces analysis throughput. We have developed KaicoTracker, a new tool for automated locomotory analysis of BmNPV-infected B. mori larvae, using Python and OpenCV. This tool is an all-inclusive package that tracks and quantifies larval locomotion and verifies the accuracy of the tracking. Larval locomotion is tracked by motion tracking function based on k-Nearest Neighbor implemented in OpenCV. Based on the tracking results, the positions of the larvae are determined, and locomotory distance, speed, duration, and frequency are calculated. The larval positions are marked on the original video, allowing tracking precision to be examined. By analyzing previously obtained data, we confirmed that this tool provides a robust analysis of inactive larvae with fewer manual steps.

microbiology↗

bioRxiv2022

Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts

Wolbachia is an extremely widespread endocellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of O. furnacalis and O. scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint. SignificanceDespite the growing number of publicly available Wolbachia genome sequences, only a few high-quality male-killer genomes exist, particularly those found in lepidopteran hosts. The complete genomes of two male-killing Wolbachia of Ostrinia moth hosts were determined in this study. The genomic data obtained here will be used to elucidate the mechanism of reproductive manipulation and the origins of this endosymbionts extraordinary diversity. Additionally, phylogenetic analysis of mitochondria and Wolbachia revealed the evolutionary history of Ostrinia hosts and Wolbachia. The inferred dynamic pattern of infection adds to our understanding of evolution and ecology of Wolbachia endosymbiont, a promising agent for biological pest control.

microbiology↗

bioRxiv2020

Reprogramming of larval locomotory pattern during baculovirus-induced host manipulation

Many parasites manipulate host behaviour to enhance their transmission. Baculovirus induces enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points. However, as ELA continues for several hours, these methods are unlikely to evaluate larval locomotion precisely during ELA. We developed a novel method to continuously trace the locomotion of Bombyx mori larvae using time-lapse imaging. This method successfully quantified the locomotory activities of larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) for 24 h. We found that both mock- and BmNPV-infected larvae periodically repeated moving and pausing with a similar frequency. In contrast, BmNPV-infected larvae showed fast and long-lasting locomotion compared with mock-infected larvae, which resulted in longer locomotory distances in infected larvae. Moreover, BmNPV-infected larvae exhibited biphasic behaviour. Initially, BmNPV-infected larvae showed longer locomotory distances, but the locomotory pattern was similar to mock-infected larvae. However, during the second phase, the locomotory pattern was drastically altered, with an extremely larger locomotory area. These results indicate that BmNPV reprograms host locomotory pattern, which is a turning point for the process of BmNPV-induced host manipulation.

microbiology↗