Co-infection of phylogenetically distinct nucleocytoviruses in <i>Acanthamoeba castellanii</i> cells

Nucleocytoviruses have extraordinarily large double-stranded DNA genome, including a set of highly conserved genes for viral reproduction. Meanwhile, nucleocytoviruses supposedly acquire new genes from cellular organisms and different lineages of nucleocytovirus, leading to their evolution. Although horizontal gene transfer among nucleocytoviruses is thought to occur in cells simultaneously infected by distinct nucleocytoviruses, it remains unknown which combination of lineages can co-infect a host cell. Here, we performed co-infection experiments using cedratvirus, megavirus, mollivirus, and pandoravirus. By transmission electron microscopy, we observed Acanthamoeba castellanii cells incorporating two distinct viral species in all six combinations. Furthermore, A. castellanii cell incorporating all four viral species was observed. In these experiments, a simultaneous increase in viral particles was observed for the combination of mollivirus and pandoravirus, pandoravirus and cedratvirus, mollivirus and cedratvirus, and megavirus and cedratvirus. Furthermore, transcription levels of cedratvirus and megavirus genes in the co-infected culture were significantly lower than those in the mono-infected culture based on time-course experiments, suggesting that distinct nucleocytoviruses may compete for viral reproduction. This is the first report experimentally demonstrating that co-infection of distinct nucleocytoviruses occurs in a A. castellanii cell.