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):11178-92

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

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

• 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

Smallpox Pathology

Incu

batio

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dS

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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)

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

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

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

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

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

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)

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

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.

1. Antiviral Drugs

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;Therapeutic

Development

Research Projects

2. Basic Virology

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?

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.

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

Plaque Reduction AssayCrystal violet staining

Virus Drug

Cultured cells

1. Culture (days)2. Fix and stain3. Count plaques

No drug

Drug

Increasing drug concentration

Decreasing plaque numbers

Typical Plaque Reduction Assay

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

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.

1. Antiviral Drugs

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;Therapeutic

Development

Research Projects

2. Basic Virology

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

Pox, Dyes, and Videotape

GFP-Cro

GFP-Cro mRNA

GFP-Cro-Expressing Cells

Viral factories

Nucleus

1. Antiviral Drugs

3. Viral Immunology

3. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;Therapeutic

Development

Research Projects

2. Basic Virology

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

1. Antiviral Drugs

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;Therapeutic

Development

Research Projects

2. Basic Virology

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.

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

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/