Quantifying relative virulence: when µmax fails and AUC alone just is not enough.

A challenge in virology is quantifying relative virulence (VR) between two (or more) viruses that exhibit different replication dynamics in a given susceptible host. Host growth curve analysis is often used to mathematically characterize virus-host interactions and to quantify the magnitude of detriment to host due to viral infection. Quantifying VR using canonical parameters, like maximum specific growth rate (µmax), can fail to provide reliable information regarding virulence. Although area-under-the-curve (AUC) calculations are more robust, they are sensitive to limit selection. Using empirical data from Sulfolobus Spindle-shaped Virus (SSV) infections, we introduce a novel, simple metric that has proven to be more robust than existing methods for assessing VR. This metric (ISC) accurately aligns biological phenomena with quantified metrics to determine VR. It also addresses a gap in virology by permitting comparisons between different non-lytic virus infections or non-lytic versus lytic virus infections on a given host in single-virus/single-host infections.

Transcriptomic analysis of the SSV2 infection of Sulfolobus solfataricus with and without the integrative plasmid pSSVi.

The fusellovirus SSV2 and the integrative plasmid pSSVi, which constitute a unique helper-satellite virus system, replicate in Sulfolobus solfataricus P2. In this study, we investigated the interplay among SSV2, pSSVi and their host by transcriptomic analysis. Following infection of S. solfataricus P2, SSV2 activated its promoters in a temporal and distributive fashion, starting from the transcription of ORF305. Expression of several host genes encoding DNA replication and transcription proteins was up-regulated, suggesting that SSV2 depended heavily on the host replication machinery for its replication. SSV2 gene expression appeared to follow a similar pattern in S. solfataricus P2 harboring pSSVi to that in S. solfataricus P2 lacking the plasmid. Several early genes of the virus were transcribed earlier and more efficiently in the presence of pSSVi than in its absence. These results provide valuable clues to the understanding of the three-way interactions among SSV2, pSSVi and the host.

In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that - unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus- DNA is targeted in this archaeon.

Bacteriophage observations and evolution.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.