Currently, the representation of viruses of Archaea within the known viruses of prokaryotes is very low, which can be partially explained by the difficulty of growing the hosts (and thus of isolating the viruses).
Hypersaline environments close to salt saturation are normally dominated by extremely halophilic Archaea and harbor the highest concentration of viruses reported for aquatic systems. Frequently, the archaeal community is dominated by the euryarchaeon Haloquadratum walsbyi as well as the recently described Nanohaloarchaea, and, depending of the analyzed system, representatives of Halorubrum and other high GC-euryhaloarchaea. These three groups represent the whole gradient of culturability, from the uncultured Nanohaloarchaea to the extremely difficult to grow Haloquadratum and the readily cultured Halorubrum. Thus, the isolation based approach for the study of haloviruses is considerable restricted by the availability of their corresponding hosts, which makes necessary the use of isolation-independent approaches
Here, we present a combination of different strategies to retrieve information on haloviruses infecting members of the Haloquadratum assemblage, since no such viruses have been cultured so far. For this purpose, we have used three types of approaches:
- Reconstruction of viral genomes from metagenomic sequences of the viral assemblage, and in silico assignment of viruses to host based on genome binning techniques and genomic signatures comparison with the corresponding cellular metagenomes.
- Direct sorting out, by means of single cell technologies, of uncultured Haloquadratum and subsequent identification of Haloquadratum virus by comparison with halovirus genomes previously immobilized on microarrays (“viriochip”).
- Incubation of virus concentrates with Haloquadratum hosts (to enrich for Haloquadratum-infecting viruses) and monitoring of the enrichment by transmission electron microscopy, viral metagenome sequencing and viriochip hybridization.
Using this polyphasic approach, we have been able to pinpoint the complete genomes of viruses infecting Haloquadratum, and to analyze their diversity, dynamics and abundance in different crystallizer ponds.