Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses

Abstract

African swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus.IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.

Keywords: Acanthamoeba castellanii; African swine fever virus; Asfarviridae; NCLDV; faustovirus; giant viruses; kaumoebavirus; pacmanvirus.

FIG 1

Initial data on pacmanvirus. (A) Flow cytometry dot plot showing the different viral profiles, in SSC (side scatter) versus FITC (SYBR green DNA contents), of mimivirus and faustovirus strain ST1 (unpublished data) as controls and of pacmanvirus, distinguishable at the levels of capsid shape (optical properties) and DNA components. (B) Negative staining of a culture supernatant of pacmanvirus with its particular capsid aspect.

FIG 2

Pacmanvirus infectious cycle realized on Acanthamoeba castellanii. (A) Pacmanvirus entry by typical phagocytosis and internalization into A. castellanii. (B and C) Viral particles with capsids could be detected inside phagocytotic vacuoles. (D) Two viruses without capsids clearly observed close to amoebal mitochondria. (E) At 3 h postinfection, primary virus factories (marked by asterisks) started to appear at a distinct location in the cytoplasm facing the nucleus of the amoeba. (F) At 4 h postinfection, early synthesized virions could be visualized in the area of the viral factory. (G to I) At 6 h postinfection, mature viruses occupied a large part of the amoebal cytoplasm, especially at the periphery of the virus factory.

FIG 3

Maximum likelihood tree reconstruction based on the DNA polymerase B gene. iTol visualization was used with the deletion branch option for values inferior to 0.5. We colored the Mimiviridae and extended family in blue, the pithoviruses and cedratvirus in red, some Phycodnaviridae, pandoraviruses, and Mollivirus sibericum in green, some Poxviridae in purple, the Asco-Iridoviridae in orange, and the Marseilleviridae in gray.

FIG 4

Cryo-EM 3D reconstruction of pacmanvirus A23. (A) Cryo-EM micrograph of pacmanvirus showing a distinct inner membrane (arrows). (B) Cryo-EM reconstruction of pacmanvirus at 15 Å. The capsomeres near the 5-fold axes are colored dark blue for clarity. (C) Cross section showing the distinct inner membrane in the cryo-EM density map.

FIG 5

Venn diagram representing coorthologous genes from asfarviruses, faustoviruses, kaumoebavirus, and pacmanvirus. *, same number or cluster of analysis for the group of faustovirus and asfarvirus; **, same number or cluster of analysis for the group of kaumoebavirus, faustovirus, and ASFV.