Poxviruses their impact on human health history and research
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Poxviruses: Their Impact on Human Health, History, and Research. Don Gammon March 18, 2011. What is a Virus?. What is a Virus?.

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Poxviruses their impact on human health history and research

Poxviruses: Their Impact on Human Health, History, and Research

Don Gammon

March 18, 2011


What is a virus

What is a Virus?


What is a virus1

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

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.


Poxviruses their impact on human health history and research

Size DOES Matter:

Poxviruses are the largest of the Mammalian Viruses


Poxvirus firsts

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


Smallpox a tale of tragedy triumph

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


Smallpox pathology

Smallpox Pathology

Incubation Period

Symptomatic Period

http://www.who.int/emc/diseases/smallpox/slideset/

Recovery

Image adapted from: http://whqlibdoc.who.int/smallpox/9241561106_chp3.pdf


Smallpox and the age of exploration

Smallpox and the Age of Exploration


Poxviruses their impact on human health history and research

Smallpox and Spanish Domination of Mexico, Central & S. America

Cortés & Aztec Empire

(1519-1521)

http://www.ucalgary.ca/applied_history/tutor/eurvoya/aztec.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

Plague upon PlagueThe destruction of Central and S. American civilization

  • Smallpox, 1518

  • Measles, 1530

  • Typhus? 1546

  • Influenza? 1558

  • Yellow fever, 1648

Arrival dates

Diego Rivera

By mid-17th century the population was ~1/20th that in 1492


North america

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 Eradication: Killing a Killer

Smallpox becomes the first and only infectious disease ever to be eradicated

1718

1096-1291

1796

1965

1802

10th Century

1977

Timeline image taken from: Smallpox: anything to declare?Smith GL, McFadden G.Nat Rev Immunol. 2002 Jul;2(7):521-7

1979-80


Vaccinia virus vacv

Vaccinia virus (VACV)

~400 nm

VACV virions

  • Host unknown

  • Used as a Smallpox vaccine

  • Encodes ~200 proteins

  • Dozens of immunomodulators

  • “Core” enzymes/proteins (Ex. DNA polymerase)


Why do we still care about smallpox

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

Mahy (2003)


Further challenges immunosuppression

Dec. 9th, 1979- WHO declares smallpox eradicated

Reports of disseminated vaccinia in HIV-infected individuals

Further Challenges: Immunosuppression


Poxviruses their impact on human health history and research

  • 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

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.


Poxviruses their impact on human health history and research

Research Projects

1. Antiviral Drugs

2. Basic Virology

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;

Therapeutic

Development


Cidofovir a new anti poxvirus drug

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

How Does CDV Inhibit VACV Replication?

CTCGTCT

A

G

X

A

G

Slow 5’-to-3’ elongation

5’

“X”= CDV

Removal of drug residue by

3’-to-5’ exonuclease (proofreading)

CTCGTCT

A

G

X

A

G

5’

CTCXTCT

A

G

G

G

Second round replication

5’

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.


Poxviruses their impact on human health history and research

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

Drug

Cultured cells

Plaque Reduction AssayCrystal violet staining

No drug

Drug

1. Culture (days)

2. Fix and stain

3. Count plaques


Poxviruses their impact on human health history and research

Typical Plaque Reduction Assay

Increasing drug concentration

Decreasing plaque numbers


Poxviruses their impact on human health history and research

Recombinant virus are resistant to CDV

[CDV] (µM)

0

60

100

1000

30

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


Cdv r vacv can still be treated with cdv

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.


Poxviruses their impact on human health history and research

Research Projects

1. Antiviral Drugs

2. Basic Virology

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;

Therapeutic

Development


Poxvirus replication

Poxvirus Replication

Intermediate mRNA

Late transcription factors

DNA polymerase

Intermediate transcription factors

Early mRNA

Late mRNA

RNA polymerase

Growth factors

Immune defense molecules

Late enzymes

Early transcription factors

Structural proteins

Adapted from Moss. Fundamental Virology, 2001


Pox dyes and videotape

Pox, Dyes, and Videotape

GFP-Cro-Expressing Cells

Nucleus

GFP-Cro

mRNA

Viral

factories

GFP-Cro


Poxviruses their impact on human health history and research

Research Projects

1. Antiviral Drugs

2. Basic Virology

3. Viral Immunology

3. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;

Therapeutic

Development


Poxviruses masters of deception manipulation

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


Poxviruses their impact on human health history and research

Research Projects

1. Antiviral Drugs

2. Basic Virology

3. Viral Immunology

4. Oncolytic Virotherapy

Poxvirus Replication & Pathogenesis;

Therapeutic

Development


Understanding virus host interactions may lead to new therapeutics

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

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

Oncolytic Virotherapy with Poxviruses

Myxoma Virus Treatment

No Treatment

Tumor Volume

No Treatment

Myxoma Virus Treatment

Tumour signal (luciferase) ~ 10 days after

implantation

C. Irwin, unpublished data


Summary

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 Resources

  • Poxvirus tutorial: http://www.microbiologybytes.com/virology/Poxviruses.html

  • General Virology Information: http://www.virology.net/


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