Download by Don Gammon

Poxviruses:
Their Impact on Human Health,
History, and Research
Don Gammon
March 18, 2011
What is a Virus?
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):1117892
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.
Size DOES Matter:
Poxviruses are the largest of the
Mammalian Viruses
Animal Virus (genome size, Kb)
Parvo (~5)
DNA polyme rase
host
RNA polyme rase
host
Papova (~5-8)
host
host
Adeno (~36)
viral
host
Herpes (~120-200)
viral
host
Pox (~130-300)
viral
viral
Mimi (~300-1,200)*
viral
viral
Bacteriophage (genome size, Kb) DNA polyme rase
X174 (5.4, ssDNA)
host
RNA polyme rase
host
Poxvirus “Firsts”
Poxviruses were the first animal viruses to be:
• Seen microscopically
• Propagated in tissue culture
• Physically purified
~400 nm
VACV virions
• Shown to regulate gene expression
• Shown to produce inhibitors of host defence
Smallpox: a Tale of Tragedy & Triumph
• 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)
IMAGE FROM: http://www.microbiologybytes.com/virology/Poxviruses.html
Recovery
Symptomatic Period
Incubation Period
Smallpox Pathology
http://www.who.int/emc/diseases/smallpox/slideset/
Image adapted from: http://whqlibdoc.who.int/smallpox/9241561106_chp3.pdf
Smallpox and the Age of Exploration
Smallpox and Spanish Domination of
Mexico, Central & S. America
Cortés & Aztec Empire
(1519-1521)
http://www.ucalgary.ca/applied_history/tutor/eurvoya/azte
c.html
Pizzaro & Inca Empire
(1524-1532)
http://www.ucalgary.ca/applied_history/tutor/
eurvoya/inca.html
Plague upon Plague
The destruction of Central and S. American civilization
Arrival dates
•
•
•
•
•
Smallpox, 1518
Measles, 1530
Typhus? 1546
Influenza? 1558
Yellow fever, 1648
Diego Rivera
By mid-17th century the population was ~1/20th that in 1492
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
A. Mackenzie
Why did European settlers find a seemingly empty land?
Smallpox Eradication: Killing a Killer
Smallpox becomes the first and only infectious disease ever to be eradicated
1965
1802
1096-1291
1796
10th Century
1979-80
1718
1977
Timeline image taken from: Smallpox: anything to declare?Smith GL, McFadden G.Nat Rev Immunol. 2002 Jul;2(7):521-7
Vaccinia virus (VACV)
• Host unknown
• Used as a Smallpox vaccine
• Encodes ~200 proteins
• Dozens of immunomodulators
~400 nm
VACV virions
• “Core” enzymes/proteins (Ex. DNA polymerase)
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:
Highly transmissible by aerosol route
High proportion of susceptible persons
(no vaccination)
High morbidity and mortality (10-30%)
Diagnosis of disease difficult- last seen
over 30 years ago
No licensed drugs for smallpox
1.
2.
3.
4.
5.
Mahy (2003)
Further Challenges: Immunosuppression
• Dec. 9th, 1979- WHO declares smallpox eradicated
• Reports of disseminated vaccinia in HIV-infected
individuals
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
Emerging Threats: Monkeypox
Mortality rates: 1-10 %
2003 Outbreaks: USA & DRC
See: Human monkeypox: an emerging zoonotic disease.Parker S, Nuara A,
Buller RM, Schultz DA.Future Microbiol. 2007 Feb;2(1):17-34. Review.
Research Projects
1. Antiviral
Drugs
2. Basic
Virology
3. Viral
Immunology
Poxvirus
Replication &
Pathogenesis;
Therapeutic
Development
4. Oncolytic
Virotherapy
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
How Does CDV Inhibit VACV Replication?
GAGXA
CTCGTCT
5’
Slow 5’-to-3’ elongation
“X” = CDV
GAGXA
CTCGTCT
5’
GAGG
CTCXTCT
5’
Removal of drug residue by
3’-to-5’ exonuclease (proofreading)
Second round replication
Conclusions: CDV impedes DNA synthesis and is resistant to
removal by viral DNA polymerase
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.
Does CDV Resistance Develop in Poxviruses?
•G. Andrei isolated CDV-resistant (CDVR) VAC in tissue
culture
•A314T and A684V a.a. substitutions identified in viral DNA
polymerase gene (E9L)
Dr. G. Andrei
NH2
COOH
1006 a.a
A314T
CDVR
A684V
CDVR
VACV DNA Polymerase
Hypothesis: A314T and/or A684V substitutions are responsible for CDVR
phenotype
Plaque Reduction Assay
Crystal violet staining
Virus
No drug
Drug
Drug
Cultured cells
1. Culture (days)
2. Fix and stain
3. Count plaques
Typical Plaque Reduction Assay
Increasing drug concentration
Decreasing plaque numbers
Recombinant virus are resistant to CDV
[CDV] (µM)
0
30
60
100
1000
Wild-type
Vaccinia
Virus
CDVR
(A314T
+
A684V)
Conclusion: A314T and A684V substitution mutations in the VACV DNA Polymerase
cause resistance to CDV
D. Gammon, unpublished data
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.
Research Projects
1. Antiviral
Drugs
2. Basic
Virology
3. Viral
Immunology
Poxvirus
Replication &
Pathogenesis;
Therapeutic
Development
4. Oncolytic
Virotherapy
Poxvirus Replication
Intermediate
mRNA
DNA polymerase
Early mRNA
Intermediate
transcription factors
Late transcription
factors
Late mRNA
RNA polymerase
Growth factors
Late enzymes
Immune defense
molecules
Early transcription
factors
Structural proteins
Adapted from Moss. Fundamental Virology, 2001
Pox, Dyes, and Videotape
GFP-Cro-Expressing Cells
Nucleus
GFP-Cro
mRNA
GFP-Cro
Viral
factories
Research Projects
1. Antiviral
Drugs
2. Basic
Virology
3. Viral
Immunology
Poxvirus
Replication &
Pathogenesis;
Therapeutic
Development
3. Oncolytic
Virotherapy
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
Research Projects
1. Antiviral
Drugs
2. Basic
Virology
3. Viral
Immunology
Poxvirus
Replication &
Pathogenesis;
Therapeutic
Development
4. Oncolytic
Virotherapy
Understanding Virus-Host Interactions
May Lead to New Therapeutics
• Myxoma virus does
not replicate in normal
human cells
Normal Cell
Cancer Cell
• Myxoma can replicate
in human cells lacking
IFN response
Adapted IMAGE FROM: Poxvirus tropism.McFadden G.Nat Rev Microbiol. 2005 Mar;3(3)Review.
*Cancer cells often lack a functional IFN pathway
Oncolytic Virotherapy with Poxviruses
No Treatment Myxoma Virus Treatment
Tumor Volume
Tumour size
2500
No Treatment
Myxoma Virus Treatment
Tumor Volume (mm3)
2000
1500
1000
500
0
0
5
10
15
20
25
30
35
Day
Tumour signal (luciferase) ~ 10 days after
implantation
C. Irwin, unpublished data
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
Poxvirus Resources
•
Poxvirus tutorial: http://www.microbiologybytes.com/virology/Poxviruses.html
•
General Virology Information: http://www.virology.net/