A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms.

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).

PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, "Pyrococcus abyssi".

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "Pyrococcus abyssi" previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the Fuselloviridae, isolated from Sulfolobus shibatae. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of Thermococcales isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.