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Poxviruses: Their Impact on Human Health,
History, and Research
Don Gammon
March 18, 2011
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What is a Virus?
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What is a Virus?
• Formal definition: “an ultramicroscopic infectious agent that replicates itself only within cells of hosts; consisting of a piece of DNA or RNA wrapped in a coat of protein”
• Some viruses also have lipid membranes surrounding them (called envelopes)
• Poxviruses consist of a double-stranded DNA genome, proteins, and one or more lipid membranes
IMAGE FROM:Discovery of antivirals against smallpox.Harrison SC, Alberts B, Ehrenfeld E, Enquist L, Fineberg H, McKnight SL, Moss B, O'Donnell M, Ploegh H, Schmid SL, Walter KP, Theriot J.Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11178-92
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Poxviruses
• >60 poxviruses have been described
• Infect a wide range of hosts
• Cause acute diseases that range from benign to fatal
IMAGE FROM: Poxvirus tropism.McFadden G.Nat Rev Microbiol. 2005 Mar;3(3)Review.
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Size DOES Matter: Poxviruses are the largest of the
Mammalian Viruses
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Poxvirus “Firsts”Poxviruses were the first animal viruses to be:
• Seen microscopically
• Propagated in tissue culture
• Physically purified
• Shown to regulate gene expression
• Shown to produce inhibitors of host defence
~400 nm
VACV virions
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• Variola virus causes Smallpox
• Most infamous poxvirus
• Probably originated in Fertile Crescent (Iran & Iraq) and Nile Valley ~10,000 BC
• Deadliest viral disease in human history
• Mortality rates ~10-30% but many
survivors remain disfigured Ramses V (died 1157 BC)
Smallpox: a Tale of Tragedy & Triumph
IMAGE FROM: http://www.microbiologybytes.com/virology/Poxviruses.html
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Smallpox Pathology
Incu
batio
n P
erio
dS
ympt
omat
ic P
erio
dR
ecov
ery
http://www.who.int/emc/diseases/smallpox/slideset/
Image adapted from: http://whqlibdoc.who.int/smallpox/9241561106_chp3.pdf
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Smallpox and the Age of Exploration
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Smallpox and Spanish Domination of Mexico, Central & S. America
Cortés & Aztec Empire (1519-1521)
Pizzaro & Inca Empire (1524-1532)
http://www.ucalgary.ca/applied_history/tutor/eurvoya/aztec.html
http://www.ucalgary.ca/applied_history/tutor/eurvoya/inca.html
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Plague upon PlagueThe destruction of Central and S. American civilization
• Smallpox, 1518• Measles, 1530• Typhus? 1546• Influenza? 1558• Yellow fever, 1648
Arrival dates
By mid-17th century the population was ~1/20th that in 1492
Diego Rivera
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North America
• Encounters with explorers and trappers created an expanding wave of smallpox
• Alexander Mackenzie (1793) discovered villages “destroyed by its pestilential breath”
• Smallpox arrives in Victoria BC in 1862 and then spreads along the coast north to Alaska
Why did European settlers find a seemingly empty land?
A. Mackenzie
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Smallpox Eradication: Killing a Killer
Smallpox becomes the first and only infectious disease ever to be eradicated
1977
1979-80
19651802
1796
1718
1096-1291
10th Century
Timeline image taken from: Smallpox: anything to declare?Smith GL, McFadden G.Nat Rev Immunol. 2002 Jul;2(7):521-7
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Vaccinia virus (VACV)
• Host unknown
• Used as a Smallpox vaccine
• Encodes ~200 proteins
• Dozens of immunomodulators
• “Core” enzymes/proteins (Ex. DNA polymerase)
~400 nm
VACV virions
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Why Do We Still Care About Smallpox?
• Despite eradication of variola by 1979, threat of accidental or intentional release lingers
• Variola virus an ideal bioweapon because:
1. Highly transmissible by aerosol route2. High proportion of susceptible persons
(no vaccination)3. High morbidity and mortality (10-30%)4. Diagnosis of disease difficult- last seen
over 30 years ago5. No licensed drugs for smallpox
Mahy (2003)
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Further Challenges: Immunosuppression• Dec. 9th, 1979- WHO declares smallpox eradicated
• Reports of disseminated vaccinia in HIV-infected individuals
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– Complications of vaccination• Inadvertent inoculation• Generalized vaccinia• Eczema vaccinatum• Progressive vaccinia• Postvaccinial encephalitis
IMAGE FROM: Pathogenesis and potential antiviral therapy of complications of smallpox vaccination.Bray M.Antiviral Res. 2003 Apr;58(2):101-14. Review
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Emerging Threats: Monkeypox
2003 Outbreaks: USA & DRC
Mortality rates: 1-10 %
See: Human monkeypox: an emerging zoonotic disease.Parker S, Nuara A, Buller RM, Schultz DA.Future Microbiol. 2007 Feb;2(1):17-34. Review.
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1. Antiviral Drugs
3. Viral Immunology
4. Oncolytic Virotherapy
Poxvirus Replication & Pathogenesis;Therapeutic
Development
Research Projects
2. Basic Virology
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Cidofovir-a “New” Anti-Poxvirus Drug
• De clercq et al. (1987)-cidofovir (CDV) introduced
• Analog of dCMP
• Shown to be inhibitory to wide range of DNA viruses
• 1996 licensed for herpesvirus infections (AIDS patients)
dCMP CDV
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How Does CDV Inhibit VACV Replication?
Slow 5’-to-3’ elongation5’CTCGTCT
GAGXA
5’CTCGTCTGAGXA
Removal of drug residue by3’-to-5’ exonuclease (proofreading)
5’CTCXTCTGAGG Second round replication
Conclusions: CDV impedes DNA synthesis and is resistant to removal by viral DNA polymerase
“X” = CDV
For more information see: Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate.Magee WC, Hostetler KY, Evans DH.Antimicrob Agents Chemother. 2005 Aug;49(8):3153-62.
The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.Gammon DB, Evans DH.J Virol. 2009 May;83(9):4236-50.
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Does CDV Resistance Develop in Poxviruses?
A684VCDVR
A314TCDVR
NH2 COOH1006 a.a
VACV DNA Polymerase
Dr. G. Andrei
•G. Andrei isolated CDV-resistant (CDVR) VAC in tissue culture
•A314T and A684V a.a. substitutions identified in viral DNA polymerase gene (E9L)
Hypothesis: A314T and/or A684V substitutions are responsible for CDVR phenotype
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Plaque Reduction AssayCrystal violet staining
Virus Drug
Cultured cells
1. Culture (days)2. Fix and stain3. Count plaques
No drug
Drug
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Increasing drug concentration
Decreasing plaque numbers
Typical Plaque Reduction Assay
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Wild-typeVaccinia
Virus
0 30 60 100 1000
[CDV] (µM)
CDVR
(A314T+
A684V)
Recombinant virus are resistant to CDV
Conclusion: A314T and A684V substitution mutations in the VACV DNA Polymerase cause resistance to CDV
D. Gammon, unpublished data
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CDVR VACV Can Still Be Treated With CDV
*Infection with CDVR Virus
Cidofovir
IMAGE FROM: Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice.Andrei G, Gammon DB, Fiten P, De Clercq E, Opdenakker G, Snoeck R, Evans DH.J Virol. 2006 Oct;80(19):9391-401.
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1. Antiviral Drugs
3. Viral Immunology
4. Oncolytic Virotherapy
Poxvirus Replication & Pathogenesis;Therapeutic
Development
Research Projects
2. Basic Virology
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Poxvirus Replication
Early mRNA
Growth factors
Immune defense molecules
DNA polymerase
Intermediate transcription factors
RNA polymerase
Intermediate mRNA
Late transcription factors
Late mRNA
Late enzymes
Early transcription factors
Structural proteins
Adapted from Moss. Fundamental Virology, 2001
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Pox, Dyes, and Videotape
GFP-Cro
GFP-Cro mRNA
GFP-Cro-Expressing Cells
Viral factories
Nucleus
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1. Antiviral Drugs
3. Viral Immunology
3. Oncolytic Virotherapy
Poxvirus Replication & Pathogenesis;Therapeutic
Development
Research Projects
2. Basic Virology
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Poxviruses: Masters of Deception & Manipulation
IMAGE FROM: Poxvirus immunomodulatory strategies: current perspectives.Johnston JB, McFadden G.J Virol. 2003 Jun;77(11):6093-100. Review
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1. Antiviral Drugs
3. Viral Immunology
4. Oncolytic Virotherapy
Poxvirus Replication & Pathogenesis;Therapeutic
Development
Research Projects
2. Basic Virology
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Understanding Virus-Host Interactions May Lead to New Therapeutics
• Myxoma virus does
not replicate in normal
human cells
• Myxoma can replicate
in human cells lacking
IFN response
Normal Cell Cancer Cell
*Cancer cells often lack a functional IFN pathwayAdapted IMAGE FROM: Poxvirus tropism.McFadden G.Nat Rev Microbiol. 2005 Mar;3(3)Review.
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No Treatment Myxoma Virus Treatment
Tumour signal (luciferase) ~ 10 days afterimplantation
Tumour size
0 5 10 15 20 25 30 350
500
1000
1500
2000
2500
Day
Tum
or V
olum
e (m
m3 )
No TreatmentMyxoma Virus Treatment
Tumor Volume
Oncolytic Virotherapy with Poxviruses
C. Irwin, unpublished data
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Summary• Poxviruses are some of the largest and most complex DNA viruses
known
• Despite the eradication of Smallpox they continue to affect human health as they have done for thousands of years
• New drug strategies are needed to treat poxvirus infections in humans
• Basic research with poxviruses has furthered our understanding of our own immune system
• In the future, poxviruses may be used to treat other diseases such as cancer
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Poxvirus Resources
• Poxvirus tutorial: http://www.microbiologybytes.com/virology/Poxviruses.html
• General Virology Information: http://www.virology.net/