New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps.

Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses.IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.