Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus.

Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics approaches for engineering the BTV genome rely upon in vitro synthesis of capped RNA transcripts from cloned cDNA corresponding to viral genome segments. In an effort to expand the utility of BTV reverse genetics, we constructed a reverse genetics vector containing a T7 RNA polymerase promoter, hepatitis delta ribozyme sequence and T7 RNA polymerase terminator sequence. Viable virus was recovered following transfection of mammalian cells, expressing T7 RNA polymerase, with 10 plasmid constructs representing the cloned BTV-1 genome. Furthermore, the plasmid-based reverse genetics system was used successfully to isolate viable cross-serotype reassortant viruses and a mutant virus containing a defined mutation in the replicating viral genome. The new reverse genetics platform established here for BTV is likely applicable to other orbiviruses.

DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms.

The soil saprophyte Bacillus cereus forms biofilms at solid-liquid interfaces. The composition of the extracellular polymeric matrix is not known, but biofilms of other bacteria are encased in polysaccharides, protein, and also extracellular DNA (eDNA). A Tn917 screen for strains impaired in biofilm formation at a solid-liquid interface yielded several mutants. Three mutants deficient in the purine biosynthesis genes purA, purC, and purL were biofilm impaired, but they grew planktonically like the wild type in Luria-Bertani broth. Biofilm populations had higher purA, purC, and purL transcript ratios than planktonic cultures, as measured by real-time PCR. Laser scanning confocal microscopy (LSCM) of BacLight-stained samples indicated that there were nucleic acids in the cell-associated matrix. This eDNA could be mobilized off the biofilm into an agarose gel matrix through electrophoresis, and it was a substrate for DNase. Glass surfaces exposed to exponentially growing populations acquired a DNA-containing conditioning film, as indicated by LSCM. Planktonic exponential-phase cells released DNA into an agarose gel matrix through electrophoresis, while stationary-phase populations did not do this. DNase treatment of planktonic exponential-phase populations rendered cells more susceptible than control populations to the DNA-interacting antibiotic actinomycin D. Exponential-phase purA cells did not contain detectable eDNA, nor did they convey a DNA-containing conditioning film to the glass surface. These results indicate that exponential-phase cells of B. cereus ATCC 14579 are decorated with eDNA and that biofilm formation requires DNA as part of the extracellular polymeric matrix.