Characterization of cytopathic effect of Orpheovirus evidenced morphological changes and increased motility of V. vermiformis
In order to characterize the CPE of Orpheovirus, V. vermiformis cells were infected at M.O.I. of 10 and observed up to 24 h.p.i.. Using optical microscopy, we observed that the cells became stretched into a fusiform shape at 3 h.p.i., and this effect became more evident at 9 h.p.i. and 12 h.p.i., respectively (Fig. 1a). In addition, at 12 h.p.i., some branched fusiform cells were observed (Fig. 1a). At 24 h.p.i., the cells became rounded, cell lysis was more evident and some fusiform cells were visualized (Fig. 1a). We also performed counts of normal (typical morphology of control cells), fusiform (branched and not branched) and rounded cells. We observed a decrease of normal cells, while fusiform cell counts increased at 9 h.p.i., composing ~ 40% of the total infected cells. At 24 h.p.i., most cells were rounded, but some fusiform cells were also observed (~ 20%) (Fig. 1b).
Different time points during infection (M.O.I. of 5) were selected for IF assay. IF assays using anti-orpheovirus primary antibodies at 1 h.p.i. revealed particles being endocytosed by amoebae (Fig. 2). At 3 h.p.i. and 6 h.p.i., respectively, an increase in particle amounts were visualized within the host cells. We also visualized fusiform cells at 12 h.p.i. by IF, and interestingly, we noticed viral particle polarization at one cell extremity and an increasing number of particles outside cells (Fig. 2). At 24 h.p.i., many cells were rounded and the large majority of amoebae were already lysed (Fig. 2). The CPE triggered by Orpheovirus is different from others previously described for Faustovirus, which revealed the formation of plaque forming units, and Tupanvirus, which was characterized by amoebae aggregates called bunches, as well as rounding and lysis in V. vermiformis [9, 10]. Furthermore, we observed that Orpheovirus infection induces an increase in the motility of V. vermiformis cells, especially those with a fusiform shape. This effect starts at 6 h.p.i. and became more evident at 12 h.p.i. (Additional file 1: Video S1)
Additional file 1: Video S1 Orpheovirus infection induces an increase in the motility of V. vermiformis cells, especially those with a fusiform shape. This effect starts at 6 h.p.i. and became more evident at 12 h.p.i.. This movie is not accelerated.
Orpheovirus is phagocytized, forms electron-lucent viral factories and induce cytoplasmic changes involving different organelles
Due to the large size of Orpheovirus particles (~ 1.1 μm), it was proposed that their entry into V. vermiformis cells would occur by phagocytosis, as previously described for other giant viruses, such as Pandoravirus, Mimivirus and Cedratvirus [3, 11, 12]. During early steps of the replication cycle, we visualized, by SEM, the formation of pseudopods in contact with Orpheovirus particles at the cell surface (Fig. 3a). This suggests that phagocytosis is the entry strategy used by Orpheovirus, as previously suggested [6]. IF analyses at 1 h.p.i. demonstrated that more than one particle is able to penetrate the host cell (Fig. 3b). After entry, it was observed, by TEM, that the internal particle content is released into the cell cytoplasm through an ostiole located at the apex of the viral particles (Fig. 3c).
As previously described in other giant viruses, Orpheovirus morphogenesis occurs in subcellular microenvironments called VFs, which are located in the host cell’s cytoplasm. Similar to Cedratvirus and Pandoravirus VFs, Orpheovirus VFs are large electron-lucent areas, which occupy a large part of the host cell (Fig. 4a and b), and do not exhibit well-defined zones as observed for mimiviruses [11]. We also visualized, by TEM, the formation of VFs in perinuclear regions, and unlike those described for pandoraviruses, the host nucleus remains present during the infection (Fig. 4a–c) [3]. Interestingly, the formation of VFs with the presence of particles inside blebs in advanced steps of the viral replication cycle were evidenced by TEM images (Fig. 5a and b). Induction of bleb formation has also been described during Cedratvirus getuliensis infections in A. castellanii cells, but this event requires further investigation [12].
Mitochondrial recruitment was also observed in peripheral regions and inside VFs (Fig. 5c), as well as membrane recruitment (Fig. 4a and b). The treatment of infected cells with BFA, a membrane trafficking inhibitor, at 8 h.p.i., affected both the formation of VFs and morphogenesis of new particles (Fig. 4c).
Morphogenesis dynamics of Orpheovirus particles
With analysis of TEM images of asynchronous infection of Orpheovirus (M.O.I. of 0.01), the formation of VFs that presented particles in different maturation stages was visualized (Fig. 6a), which obtained more information about the morphogenesis of new particles. The morphogenesis of Orpheovirus starts with the formation of electron-dense semicircular structures named crescents, as observed for other giant viruses, such as Faustovirus and Cedratvirus [9, 12]. These structures extend and are filled by the internal content until the formation of a mature particle (Fig. 6b–f). Recruitment of sheet-like structures in the periphery of the particles under assembly was also observed (Fig. 6e) and may be important for particle formation, which seems to be composed of several layers of proteins and membranes. Both by TEM and SEM images, it was evidenced that the mature particles have a flattening on one side (Fig. 6g and h).
In order to chronologically analyse viral genome replication and infectious particle formation, one-step growth assays were performed. An increase in genome amplification was observed at 9 h.p.i., and infectious particle detection increased at 12 h.p.i. (Fig. 6i–j). At 48 h.p.i., Orpheovirus propagation reached a plateau (Fig. 6i–j). The data corroborate those previously described [6], which suggested that the cycle of Orpheovirus in V. vermiformis is slower compared to other giant viruses, during approximately 30 h.p.i.
In addition, it was observed that Orpheovirus particles have a smaller fibril layer compared to those observed in mimiviruses (Fig. 7a and b) [13]. The analyses also revealed that mature particles were present in an outer layer, a capsid layer and an inner membrane that involve the core of the particle (Fig. 7a and b). It is noteworthy that, even in infections with low M.O.I., the presence of defective particles in different formats was evidenced (Fig. 8a-c). This finding suggests that malformed particles occur naturally, which is similar to other giant viruses [6, 10, 11].
Orpheovirus particles are released from the cell by lysis and exocytosis
After VF formation, expansion of the crescents, and complete maturation, new particles were visualized in peripheral regions in the host cell’s cytoplasm (Fig. 9a), and some are involved with membranes. Furthermore, the presence of one or more particles in the same vacuole was observed, which may have more than one membrane (Fig. 9b–d). The presence of viral particles within vacuoles has also been reported for other giant viruses such as Pithovirus and Pandoravirus, suggesting that particles are released from host cells by exocytosis [3, 14, 15]. Biological assays, including cell and particle counts in the supernatant of infected amoebae were performed over the replication cycle (M.O.I. = 5). These analyses revealed that Orpheovirus particles can be detected in the supernatant of infected cells, even at times when no cell lysis is observed (Fig. 9e and f). An increase in particle numbers was noted in the supernatant at 12 h.p.i. In addition, IF analyses performed at different times throughout the cycle also demonstrated an increase in the number of particles outside the cells at 12 h.p.i. and 24 h.p.i., respectively (Fig. 9e and Fig. 2), which reinforces exocytosis as an alternative strategy for host cell particle release (Fig. 10a), although cell lysis was also demonstrated by SEM images (Fig. 10b).