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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?.

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

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  1. Poxviruses: Their Impact on Human Health, History, and Research Don Gammon March 18, 2011

  2. What is a Virus?

  3. 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

  4. 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.

  5. Size DOES Matter: Poxviruses are the largest of the Mammalian Viruses

  6. 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

  7. 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

  8. 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

  9. Smallpox and the Age of Exploration

  10. 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

  11. 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

  12. 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?

  13. 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

  14. 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)

  15. 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)

  16. Dec. 9th, 1979- WHO declares smallpox eradicated Reports of disseminated vaccinia in HIV-infected individuals Further Challenges: Immunosuppression

  17. 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

  18. 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.

  19. Research Projects 1. Antiviral Drugs 2. Basic Virology 3. Viral Immunology 4. Oncolytic Virotherapy Poxvirus Replication & Pathogenesis; Therapeutic Development

  20. 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

  21. 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.

  22. 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

  23. Virus Drug Cultured cells Plaque Reduction AssayCrystal violet staining No drug Drug 1. Culture (days) 2. Fix and stain 3. Count plaques

  24. Typical Plaque Reduction Assay Increasing drug concentration Decreasing plaque numbers

  25. 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

  26. 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.

  27. Research Projects 1. Antiviral Drugs 2. Basic Virology 3. Viral Immunology 4. Oncolytic Virotherapy Poxvirus Replication & Pathogenesis; Therapeutic Development

  28. 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

  29. Pox, Dyes, and Videotape GFP-Cro-Expressing Cells Nucleus GFP-Cro mRNA Viral factories GFP-Cro

  30. Research Projects 1. Antiviral Drugs 2. Basic Virology 3. Viral Immunology 3. Oncolytic Virotherapy Poxvirus Replication & Pathogenesis; Therapeutic Development

  31. 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

  32. Research Projects 1. Antiviral Drugs 2. Basic Virology 3. Viral Immunology 4. Oncolytic Virotherapy Poxvirus Replication & Pathogenesis; Therapeutic Development

  33. 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

  34. 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

  35. 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

  36. Poxvirus Resources • Poxvirus tutorial: http://www.microbiologybytes.com/virology/Poxviruses.html • General Virology Information: http://www.virology.net/

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