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.