New virus isolates from Italian hydrothermal environments underscore the biogeographic pattern in archaeal virus communities

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic
and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous
fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including
lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to
families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect
neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of
the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order
Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the
first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced
rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting
geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers
suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape
the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with
the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal
systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.