What kinds of microbes do you associate with hot springs? Maybe microbial mats? Thermus aquaticus and the discovery of Taq polymerase? Archaea, previously (and erroneously) thought to be strict extremophiles? Viruses may not be the first microbial subtype that springs to mind (pun intended) but rest assured, where cells exist, so do viruses. A recent paper published in the Journal of Virology describes a newly discovered virus, originally characterized based its genome sequence.
Traditionally new viruses (like the recently described BGHV 8) are identified via culture methods. But the harsh conditions of the hot springs – 80°C and pH 2 – make replicating the conditions difficult; additionally, the viral host may require other microbial members in coculture. Fortunately, sequencing technologies have advanced to the degree that allows viral description from bioinformatic analysis of its genome.
The new virus was isolated from Alice Spring, a high-temperature, acidic hot spring in Yellowstone National Park. To enrich the water for virus-specific genomic material, it was run through a 0.4-μM filter to eliminate free-floating celled microbes. Examining the purified particles by electron microscopy revealed a spindle-shaped virus with variable tail lengths (see figure, right).
Virus purified by the same means was sent for sequencing. First author Rebecca Hochstein, working with lead scientist Mark Young, determined that the virus has a double-stranded DNA genome approximately 70K bp in size with 96 predicted ORFs.
To be certain the DNA recovered was related to the imaged virus, the authors performed a clever trick. Purified virus was separated by SDS-PAGE, and the dominant band was analyzed by mass spectrometry. This produced bands that matched the predicted sequence of the viral major coat protein (MCP) from the DNA sequence. This and an additional experiment assured the researchers that the DNA genome indeed stemmed from the spindle-shaped virus.
Characterizing the virus in isolation can make identifying its host cell type a challenge, and 5-10 years ago this would have been almost impossible. Studies on the bacterial clustered regularly-interspace short palindromic repeats (CRISPR) system have made matching bacteria or archaea to their phage much easier. Using a CRISPR locus database, the researchers found an Acidianus hospitalis strain that contained direct repeats matching the viral genome sequence. Based on the matched host and the electron micrograph images, the virus was named Acidianus tailed spindle virus, or ATSV.
Having identified the putative host, the scientific team reasoned that the virus may be recovered from A. hospitalis cultures. Environmental samples had been simultaneously recovered from the same hot springs, and were probed with fluorescently labeled DNA probes – red for the virus, green for A. hospitalis. Using these probes for FISH (see figure, left) reveals that a subset of these archeal cells are infected with ATSV. Controls (not shown) ensured that other archaeal species did not colocalize with the ATSV probe. Finally, the authors produced infection in a closely related Acidianus strain, demonstrating that this is the host for the newly discovered ATSV.
This discovery adds depth to our knowledge of archaeal viruses, of which only 117 have been described. The methods used – starting with genomic information and using bioinformatic analyses to generate testable hypotheses – can be applied to many environments. Although the scientists used viral culture in their final experiment, a number of characteristics, including host species, were learned simply from looking at the genomic sequence. This approach may be a faster way to catalog viral diversity from understudied environments.
-- Julie Wolf