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.
Tracking cell motility is a useful tool for the study of cell physiology and microbiology. Although phase-contrast microscopy is commonly used, the existence of optical artifacts called "halo" and "shade-off" have inhibited image analysis of moving cells. Here we show kinetic image analysis of Acanthamoeba motility using a newly developed computer program named "Phase-contrast-based Kinetic Analysis Algorithm for Amoebae (PKA3)," which revealed giant-virus-infected amoebae-specific motilities and aggregation profiles using time-lapse phase-contrast microscopic images. This program quantitatively detected the time-dependent, sequential changes in cellular number, size, shape, and direction and distance of cell motility. This method expands the potential of kinetic analysis of cultured cells using versatile phase-contrast images. Furthermore, this program could be a useful tool for investigating detailed kinetic mechanisms of cell motility, not only in virus-infected amoebae but also in other cells, including cancer cells, immune response cells, and neurons.
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 non-tailed mimiviruses has not yet 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 to the others. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and period of 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 Mimivirinae . 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 similar to those of tupanviruses, rather than the A, B, or C lineages, and other genomic features and host specificity of cotonvirus are similar to those of the latter than the former. These results suggest that cotonvirus is a unique virus, with 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.
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