Ten years ago, the discovery of Mimivirus, a virus infecting Acanthamoeba, initiated a reappraisal of the upper limits of the viral world, both in terms of particle size (>0.7 micrometers) and genome complexity (>1000 genes), dimensions typical of parasitic bacteria. The diversity of these giant viruses (the Megaviridae) was assessed by sampling a variety of aquatic environments and their associated sediments worldwide. We report the isolation of two giant viruses, one off the coast of central Chile, the other from a freshwater pond near Melbourne (Australia), without morphological or genomic resemblance to any previously defined virus families. Their micrometer-sized ovoid particles contain DNA genomes of at least 2.5 and 1.9 megabases, respectively. These viruses are the first members of the proposed "Pandoravirus" genus, a term reflecting their lack of similarity with previously described microorganisms and the surprises expected from their future study.

Significance Giant DNA viruses are visible under a light microscope and their genomes encode more proteins than some bacteria or intracellular parasitic eukaryotes. There are two very distinct types and infect unicellular protists such as Acanthamoeba . On one hand, Megaviridae possess large pseudoicosahedral capsids enclosing a megabase-sized adenine–thymine-rich genome, and on the other, the recently discovered Pandoraviruses exhibit micron-sized amphora-shaped particles and guanine–cytosine-rich genomes of up to 2.8 Mb. While initiating a survey of the Siberian permafrost, we isolated a third type of giant virus combining the Pandoravirus morphology with a gene content more similar to that of icosahedral DNA viruses. This suggests that pandoravirus-like particles may correspond to an unexplored diversity of unconventional DNA virus families.

Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-μm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of "omic" approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host's ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses' diversity remains to be fully explored.giant virus | permafrost | Pleistocene