Evaluation of the interaction between uninfected amoebas and bunches

To evaluate whether bunches are able to interact with uninfected cells, 1 × 10⁶ A. castellanii cells in 25 cm² culture flasks were infected with tupanvirus (M.O.I. of 10) and at 16 h.p.i. the bunches were collected and used to inoculate uninfected A. castellanii cells in suspension in a separate 25 cm² culture flasks. After incubation for 30 min at 32 °C the non-adherent cells were removed from the culture flasks and the cells that remained adhered were counted. As a control, 16 h.p.i. APMV-infected amoebae were used in the same procedure. The experiment was performed in triplicate. The results were plotted on a column graph, and the statistical significance was calculated using a two-tailed Student’s t test, performed using Graph Pad Prism. In addition, the uninfected cells that interacted with bunches and were carried by them (the cells that did not adhere to the culture flasks again) were assayed using IF and SEM, as described above. To evaluate the importance of bunches with fresh cells for propagation of tupanvirus, 7 × 10⁶ A. castellanii cells were infected (M.O.I of 10) and at 16 h.p.i. the medium was removed; the bunches were washed and used to infect new A. castellanii cells and as a control, the bunches were also added to a flask containing fresh medium. At 36 h.p.i. the supernatants were collected and titrated as described above. Graphs were constructed using GraphPad Prism software (v7.0 for Windows).