David Evans, PhD
Dept. of Medical Microbiology & Immunology
University of Alberta
Faculty of Medicine & Dentistry
6-020 Katz Group Centre
T6G 2E1 Edmonton, AB
Office Phone : (780) 492-2109
Lab Phone: (780) 492-2106
Fax : (780) 492-7521
- Professor, Dept. of Medical Microbiology & Immunology
- Vice-Dean (Research), Faculty of Medicine & Dentistry
- Member, WHO Advisory Committee on Variola Virus
- Co-Founder ProPhysis Inc. (2014 TEC Edmonton spinoff achievement award)
My lab studies poxviruses. These are large DNA viruses, best known for causing the diseases smallpox and myxomatosis in humans and rabbits, respectively. We are currently studying different aspects of poxvirus biology using funding provided by CIHR, NSERC, the Canadian Breast Cancer Foundation, the Province of Alberta, and the private sector. How you could participate will change as these projects evolve and people come and go. Please contact me (email@example.com) for the latest news.
1.0 New methods for imaging virus replication and recombination. Poxviruses encode nearly all of the genes they need to catalyze virus DNA replication and the closely linked process of virus recombination. We’ve shown that the virus DNA polymerase catalyzes virus recombination, and you can even buy a commercial application of this recombineering technology (it’s the basis of InFusionTM cloning). Basically, we have a good idea what goes on at a molecular and biochemical level when poxviruses replicate and recombine DNA. However, it is not so easy to link these events back to the process of virus entry, replication, and assembly, as is seen at a cellular level.
Fortunately, and with generous funding from the governments of Alberta and Canada, we have been able to purchase some amazing new fluorescence microscopes. One of the things we’ve done to take advantage of these microscopes is to construct cell lines expressing DNA binding fluorescent proteins. The two tools allow us to track the growth and development of replicating poxviruses. We are using this method to follow processes like virus entry, recombinant gene production, factory movement, and factory fusion (see the movie below). We have also produced fluorescently tagged versions of several virus-encoded DNA binding proteins and are using these to study their role in virus development.
Poxvirus replication in a host cell. The cell expresses green fluorescent protein linked to a DNA binding protein. As the virus enters the cell, the reporter protein moves from the nucleus and tags the growing cytoplasmic virus factories. This movie spans approximately 10 hr in real time (Image © and courtesy of P. Paszkowski).
Super-resolution imaging of vaccinia virus infected cells. The photomicrograph shows an infected cell, approximately 24 hr after infection. The 4300 green spots are virus particles, the red fluorescence marks sites of virus replication called factories, and the blue stain identifies DNA (Image © and courtesy of M. Desaulniers)
2.0 Poxvirus genomics. The advent of next generation sequencing technologies has greatly simplified the task of sequencing these large (>200kb) viruses. What once took us over a year in the late 1990’s can now be accomplished in a few days. We have been using NGS as a tool for characterizing the viruses that make up non-clonal smallpox vaccines, as well as the old vaccine strains used in different parts of the world. We are currently using NGS methods to characterize the viruses that are produced using virus-dependent homologous and non-homologous recombination pathways.
Genomic analysis of virus recombinants. Cells were co-infected with two strains of vaccinia virus (DPP17 and TP5), which differ in sequence by about 1% due to sequence drift over many years of independent passage. After one round of passage, different progeny were cloned and sequenced. On average, each virus exhibits about 18 genetic exchanges, this is determined by counting the number of places where the coloured (DPP17) sequence switches to the white (TP05) sequence. Figure abstracted from Qin and Evans (2014).
2.0 Oncolytic viruses. Viruses can be used to treat cancer, but it is known that there are at least two big problems that must be fixed before this approach can be widely adopted. First, one has to ensure the virus is safe, and that it will attack only the cancer cells. Second, the virus infection will be rapidly eradicated by an immune response, but before that happens it is important that the infection activate (or reactivate) an immune reaction against the cancer. To address the first problem we have knocked out certain genes in vaccinia virus, creating a virus that grows only in rapidly dividing cells. These viruses are being tested as a possible treatment for bladder and breast cancer in collaboration with Dr. M. Hitt (Oncology). To address the second problem we are testing whether the oncolytic effect can be enhanced by either deleting virus genes encoding immune inhibitors, or by combining virotherapy with targeted radiotherapy using a newly purchased Xstrahl image guided micro-irradiator.
Methods: These projects provide training in cell culture, virus molecular genetics, imaging, molecular and cell biology, immunology, and enzymology.
Background preparation: A solid undergraduate training in molecular cell biology (biochemistry, cancer, cell biology, genetics, microbiology, or immunology) would be a great asset. We can teach virology to nearly anyone!
Contact information: Please contact Dr. Evans by e-mail at firstname.lastname@example.org. The laboratory can be contacted at (780) 492-1966. If you interested in a 498/499 project, or graduate training, please send a CV and recent academic transcript with your enquiry.
- Nicole Favis (Technician)
- Megan Desaulniers (Technician)
- Dr. Chad Irwin (PDF)
- Quinten Kieser (Graduate Student)
- Dr. Les Nagata (Visiting Scientist)
- Dr. Ryan Noyce (Research Associate)
- Mira Shenouda (Graduate Student)
- Brittany Umer (Graduate Student)
Selected Publication History:
A. Nucleic Acid structure
EVANS, D. H., and Morgan, A. R. (1982) Extrahelical bases in duplex DNA. J. Mol. Biol. 160, 117-122. PMID: 6294301
EVANS, D. H., and Kolodner, R. (1987) Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions. J. Biol. Chem. 262, 9160-9165. PMID: 3036850
Julien, O., Beadle, J.R., Magee, W.C., Chatterjee, S., Hostetler, K.Y., EVANS, D.H., and Sykes, B.D. (2011) Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir. J. Am. Chem. Soc. 133:2264-2274. PMID: 21280608
B. Poxvirus recombination (genetics)
EVANS, D. H., Stuart, D. and McFadden, G. (1988) High levels of genetic recombination between co-transfected plasmid DNA's in poxvirus-infected mammalian cells. J. Virology 62, 367-375. PMID: 2826801
Fisher, C., Parks, R. J., Lauzon, M., and EVANS, D. H. (1991) Heteroduplex DNA formation is associated with replication and recombination in poxvirus-infected cells. Genetics 129, 7-18. PMID: 1657705
Yao, X.-D. and Evans, D.H. (2003) High-frequency genetic recombination and reactivation of Orthopoxviruses from DNA fragments transfected into Leporipoxvirus-infected cells. J. Virology 77, 7281-7290. PMID: 12805426
Qin, L., and EVANS, D.H. (2014) Genome scale patterns of recombination between co-infecting vaccinia viruses. J. Virol. 88:5277-86. PMID: 24574414
Paszkowski, P., Noyce, R.S., and EVANS, D.H. (2016) Live-cell imaging of vaccinia virus genetic recombination. PLoS Pathogens. 12(8):e1005824. doi:10.1371/journal.ppat.1005824.
C. Poxvirus recombination (mechanism and enzymology)
Zhang, W., and EVANS, D. H. (1993) DNA strand-exchange catalyzed by proteins from vaccinia-virus infected cells. J. Virol. 67, 204-212. PMID: 8416369
EVANS, D.H., Willer, D. and Yao, X.-D. (2001) “DNA joining method”. Patent Convention Treaty (PCT) application filed March 7, 2001. Issued Germany (601 22 118.4-08) and Switzerland, UK, Sweden, and France (EU: 1263973). Issued as US Patent # 7,575,860 August 2009.
Willer, D.O., Yao, X.-D., Mann, M.J., and EVANS, D.H. (2000) In vitro concatemer formation catalyzed by vaccinia DNA polymerase. Virology 278, 562-569. PMID: 11118378
Gammon, D.B., and EVANS, D.H. (2009) Vaccinia virus recombination requires a 3'-to-5' exonuclease activity encoded by the viral DNA polymerase. J. Virology 83: 4236-50 (JV Spotlight). PMID: 19224992
Lin, Y.-C., and EVANS, D.H. (2010) Vaccinia virus particles mix inefficiently in co-infected cells and in a way that would restrict viral recombination. J. Virology 84: 2432-43 (JV Spotlight and cover illustration [November, 2010]). PMID: 20032178
D. Virus genomics
Willer, D. O., McFadden, G., and EVANS, D.H. (1999) The complete genome sequence of Shope (rabbit) fibroma virus (1999) Virology 264, 319-343. PMID: 10329561 and Cameron, C., McCaulay, C., Cao, J., Barrett, J., Hota-Mitchell, S., Willer, D.O., EVANS, D.H., and McFadden, G. (1999) The complete DNA sequence of myxoma virus. Virology 264, 298-318. PMID: 10562494
Qin, L., Upton. C., Hazes, B., and EVANS, D.H. (2011) Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine. J. Virol. 85:13049-13060. PMID: 21976639
Teferi, W., Dodd, K., Maranchuk, R., Favis, N. and EVANS, D.H. (2013) A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication. J. Virol. 87:4623-41. PMID: 23408614
Qin, L., Favis, N, Famulski, J., and EVANS, D.H. (2015) The evolution and evolutionary relationships between extant vaccinia virus strains. J. Virol. (JV Spotlight) 89:1809-24. PMID: 25410873
E. Smallpox therapeutics, virus replication
Andrei, G., Gammon, D.B., Fiten, P., De Clercq, E., Opdenakker, G., Snoeck, R., and EVANS, D.H. (2006) Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice. J Virology 80:9391-401. PMID: 16973545
Gammon, D.B., Snoeck, R., Fiten, P., Krečmerová, M., Holý, A., De Clercq, E., Opdenakker, G., EVANS, D.H., and Andrei, G. (2008) Mechanism of antiviral drug resistance of vaccinia virus: Identification of residues in the viral DNA polymerase conferring differential resistance to anti-poxvirus drugs. J. Virology 82:12520-34. PMID: 18842735
Magee, W., Aldern, K.A., Hostetler, K.Y., and EVANS, D.H. (2008) Cidofovir and (s)-HPMPA are highly effective inhibitors of vaccinia virus DNA polymerase when incorporated into the template strand. Antiviral Agents and Chemotherapy 52:586-597. PMID: 18056278
Gammon, D.B., Gowrishankar, B., Duraffour, S., Andrei, G., Upton, C., and EVANS, D.H. (2010) Vaccinia virus-encoded ribonucleotide reductase subunits are differentially required for replication and pathogenesis. PLoS Pathog. 6(7):e1000984. PMID: 20628573
Magee, W.C., Valiaeva, N., Beadle, J.R., Richman, D.D., Hostetler, K.Y., and EVANS, D.H. (2011) Inhibition of HIV-1 by octadecyloxyethyl esters of (S)-[3-Hydroxy-2-(phosphonomethoxy)propyl] nucleosides and evaluation of their mechanism of action. Antiviral Agents and Chemotherapy 55, 5063-72. PMID: 21896914