Understanding the evolution of large DNA viruses
Researchers at the University of Utah, USA, are utilising nanopore‑based whole genome sequencing to understand the evolutionary mechanisms employed by large DNA viruses during host‑pathogen conflict1. As with other double‑stranded DNA (dsDNA) viruses, Orthopoxvirus vaccinia, is capable of rapid adaptation despite a relatively low single nucleotide mutation rate (when compared to single‑ stranded DNA or RNA viruses)1.
Previous studies of the virus have identified two proteins, E3L and K3L, which inhibit the host response to infection2,3. It has been demonstrated that deletion of the E3L gene and subsequent passaging though human cell lines to simulate host‑pathogen conflict results in duplication of the K3L gene in long tandem arrays. Further, a single nucleotide variant (encoding an H47R amino acid change) has been shown to occur within the K3L gene of these E3L deleted strains, and encodes for enhanced pathogenicity.
According to University of Utah team, short‑read sequencing platforms can provide a population‑level view of H47R allele frequency and information on general changes in coverage at the K3L locus; however, they are unable to genotype point mutations in tandem repeats or characterise copy number changes1.
In order to understand the mechanisms by which these distinct adaptations arise during viral evolution, the team turned to the long sequencing reads offered by nanopore technology, which are capable of spanning large regions of repetitive DNA. The results revealed that, while the increase in copy number stabilises by passage 10 (to up to 15 copies), the H47R SNP accumulates from a low frequency at passage 10 to near fixation at passage 20 (Figure 9)4. The team also demonstrated that the combination of the two genetic changes also increases the fitness of the virus more than either of the changes alone. Using nanopore technology, the team were able to propose a new method of dsDNA virus evolution whereby rare beneficial variants become rapidly fixed within multiple gene copies.
In summary, the researchers stated that their work:
"…demonstrates the power of long-read sequencing to perform high resolution analyses of complex genome dynamics. […] This type of analysis provides a framework to definitively determine the sequence content of tandem gene duplications, and accurately call variants within these duplicates"4.
The team now plan to utilise nanopore long reads to examine variant phasing.
Figure: Genomes with multiple copies of the K3L gene rapidly homogenise for the H47R allele, implying increased fitness conferred by the mutation. Image courtesy of Thomas Sasani, University of Utah, USA.
This case study is taken from the Microbiology white paper.
- Sasani, T. Tracking adaptive structural variation during host‑pathogen conflict. Presentation. Available at: https://nanoporetech.com/resource-centre/tracking-adaptive-structural-variation-during-host-pathogen-conflict [Accessed: 16 February 2018].
- Beattie, E. et al. Reversal of the interferon‑sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene. J Virol 69, 499–505 (1995).
- Elde, N.C. et al. Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses. Cell 150, 831–41. doi:10.1016/j. cell.2012.05.049 (2012).
- Sasani, T.A., Cone, K.R., Quinlan, A.R., & Elde, N.C. Long read sequencing reveals poxvirus evolution through rapid homogenization of gene arrays. bioRxiv 245373 (2018).