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Coronavirus Pandemic Potential and Research Response

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  1. Coronavirus Pandemic Potential and Research Response Mark R. Denison, M.D. Departments of Pediatrics, Microbiology & Immunology The Elizabeth B. Lamb Center for Pediatric Research Vanderbilt University Medical Center

  2. Denison Lab • Xiaotao Lu • Erik Prentice • Rachel Graham UNC Chapel Hill • Ralph Baric • Boyd Yount • Amy Sims • Vanderbilt University Department of Pediatrics, Department of Microbiology & Immunology • Elizabeth B. Lamb Center for Pediatric Research • NIH R01 AI26603 -15 R01 AI50083 - 01 (S1) Disclosures: none

  3. Course of the Pandemic • February 2003 “Dad why don’t you work on an ‘important’ virus” • April 2003 “Daddy, I think you are getting kind of ‘full of yourself’” • August 2003 “Dad, why haven’t you found a cure for SARS yet?”

  4. Objectives • Describe the role of basic research in SARS • Describe coronavirus life cycle and replication • Discuss genetic systems coronavirus research • Summarize research goals

  5. SARS, Public Health, and Research SARS - CoV Pandemic Potential High mortality Worldwide spread Naive population SARS - CoV Pandemic potential High mortality Worldwide spread Immune naive population

  6. SARS, Public Health, and Research SARS - CoV Pandemic Potential High mortality Worldwide spread Naive population SARS - CoV Pandemic potential High mortality Worldwide spread Immune naive population Public Health Response

  7. SARS, Public Health, and Research No Disease New models for detection and intervention of emerging infections Coronavirus replication, disease, and prevention Antivirals and vaccines for coronaviruses

  8. SARS, Public Health, and Research • Escape / mutation • Persistence • Animal reservoir No Disease New models for detection and intervention of emerging infections Coronavirus replication, disease, and prevention Antivirals and vaccines for coronaviruses

  9. SARS, Public Health, and Research • Escape / mutation • Persistence • Animal reservoir No Disease New models for detection and intervention of emerging infections Coronavirus replication, disease, and prevention Antivirals and vaccines for coronaviruses • Continued human disease • Re-emergence of epizootic, epidemic or pandemic disease • New SARS-CoV disease

  10. SARS, Public Health, and Research • Escape / mutation • Persistence • Animal reservoir No Disease New models for detection and intervention of emerging infections Coronavirus replication, disease, and prevention Antivirals and vaccines for coronaviruses • Continued human disease • Re-emergence of epizootic, epidemic or pandemic disease • New SARS-CoV disease • Need for research in biology, pathogenesis, therapeutics and vaccines

  11. SARS, Public Health, and Research • Escape / mutation • Persistence • Civet, Raccoon Dog, other? No Disease New models for detection and intervention of emerging infections Coronavirus replication, disease, and prevention Antivirals and vaccines for coronaviruses • Singapore - September 2003 • Re-emergence of epizootic, epidemic or pandemic disease • ASx infection animals / humans • Need for research in biology, pathogenesis, therapeutics and vaccines

  12. Big Questions • Is SARS-CoV still present in human populations? • Is there risk of human animal transmission? • Will SARS-CoV adapt to better survive in human populations? • Will SARS reemerge as a “seasonal disease” • Can vaccines and therapeutics for SARS be developed, tested and used?

  13. Objectives • Describe the role of basic research in SARS • Describe coronavirus life cycle and replication • Discuss genetic systems coronavirus research • Summarize research goals

  14. SARS Coronavirus (SARS-CoV) S E M RNA + N 100 nm

  15. Nidovirales Order Coronaviridae Group 1 TGEV HCoV229E Group 2 MHV BCoV HCoV-OC43 Group 3 IBV SARS-CoV Snijder et al 2003 J. Mol. Biol. (2003) 331, 991–1004

  16. Coronavirus Genome Organization MHV Genome + RNA 32 kb genes 2-7 Replicase gene 22 kb Leader AN ORF 1a S E M N ORF 1b SARS Genome + RNA 30 kb genes 2-9 Replicase gene 20 kb Leader AN M ORF 1a S E N ORF 1b

  17. Coronavirus Replicase aa 5000 1000 2000 3000 14000 6000 7000 p28 p65 p210 pol hel 3CLpro MHV PLP-2 PLP1 p213 p20 p70 pol 3CLpro hel SARS PLP2

  18. Snijder et al 2003 doi:10.1016/S0022-2836(03)00865-9 J. Mol. Biol. (2003) 331, 991–1004

  19. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  20. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  21. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  22. MHV-A59 infected DBT cells DIC/Hel

  23. Coronavirus genome and replication Genome + RNA 32 kb genes 2-7 gene 1 (replicase) Leader replicase polyprotein pol S E M N R C R C

  24. + ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA

  25. + + ( ( ) ) - ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA 3' 5' Intermediate RNA Un Genome 3' 5' RNA An

  26. + ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA 3' 5' An

  27. + ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA 3' 5' An

  28. + ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA 3' 5' Un

  29. + ( ) Coronavirus Transcription and Replication genes 2-7 Replicase gene 3' Genome 2 3 4 5 6 7 5' An RNA 3' 5' (-) strand subgenomic RNA Un (+) strand Subgenomic mRNA An Spike (S)

  30. + + ( ( ) ) - ( ) + ( ) An An An An An An Coronavirus Transcription and Replication Gene 1 Genes 2-7 RNA Proteins 3' Genomic 2 3 4 5 6 7 5' 1 An Replicase RNA 3' 5' RNA Un Leader 2 NS RNA Spike (S) 3 Subgenomic RNAs +/- ( ) 4 NS 5 E 6 M 7 N Genomic 3' 5' Replicase An RNA

  31. Coronavirus genome and replication Genome + RNA 32 kb genes 2-7 gene 1 (replicase) Leader replicase polyprotein pol S E M N R C R C

  32. Coronavirus genome and replication Genome + RNA 32 kb genes 2-7 gene 1 (replicase) Leader replicase polyprotein pol S E M N R C R C

  33. Coronavirus genome and replication Genome + RNA 32 kb genes 2-7 gene 1 (replicase) Leader replicase polyprotein pol S E M N R C R C

  34. E64-d Proteinase inhibitors result in shutoff of viral RNA synthesis at any time during MHV infection 105 [3H] cpm 104 103 1 2 3 4 6 7 8 9 10 5 Time (hours p.I) Kim et al 1995

  35. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  36. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  37. Coronavirus Life Cycle attachment entry release Replication Complexes Translation Processing RNA synthesis maturation assembly nucleus

  38. Coronavirus Replication and Targets for Inhibition • S protein, receptor • Fusion, uncoating • Replicase polyprotein expression and processing • Virus assembly and release • Novel functions (polymerase, helicase, methyltransferase, exonuclease, CoMt) • Cellular functions used by virus • Cholesterol • Membrane Trafficking • Autophagy

  39. Objectives • Describe the role of basic research in SARS • Describe coronavirus life cycle and replication • Discuss genetic systems coronavirus research • Summarize research goals

  40. Coronavirus Replication and Molecular Biology: General Themes • High theoretical mutation rate: 104 per template per replication (3 changes per genome) • RNA-RNA homologous recombination (20%) • Result: rapid adaptation, recovery from deleterious mutations, mechanisms to acquire and regain virulence. • Genome tolerates deletions, mutations, substitutions

  41. Coronavirus Diseases: General Themes • Repeat infections • Persistent infections • Disease enhancement in vaccinated animals • Trans-species transmission • Changes in virus transmission, tropism and disease - **New Coronavirus Diseases

  42. Coronavirus Vaccines: General Themes • Live attenuated, inactivated virus, protein, heterologous virus, DNA have been used • “Classical” live-attenuated vaccines have been most licensed and employed • Challenges for vaccines : poor protection, enhanced disease, altered disease, reversion to virulence, recombination • Every coronavirus has required different approaches • **Multiple pathways for SARS vaccine development

  43. Coronavirus Reverse Genetics • Infectious clones : MHV, IBV, TGEV, HCoV-229E • In vitro assembly, Vaccinia recombinants, BAC • Applicable to entire genome • Example of mutations yielding viable viruses • Gene deletions, gene duplications • Gene substitution (GFP) • Gene order rearrangement • Replicase polyprotein cleavage site deletion

  44. Genome +strand RNA (32 kb) RT / PCR cDNA clones In vitro ligation Transcribe + strand RNA Transfect cells nucleus Yount, Denison, Weiss and Baric 2002

  45. Genome +strand RNA (32 kb) RT / PCR cDNA clones In vitro ligation Transcribe + strand RNA Transfect cells nucleus Yount, Denison, Weiss and Baric 2002

  46. Genome +strand RNA (32 kb) RT / PCR cDNA clones In vitro ligation Transcribe + strand RNA Transfect cells nucleus Yount, Denison, Weiss and Baric 2002

  47. CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’ K G Y R G V K P Mutagenesis of MHV genome Replicase gene (22kb) 5’ 3’ replicase polyprotein

  48. CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’ K G Y R G V K P Mutation of CS1 Cleavage Virus In Vitro Mut 3 HNO YES Mut 4 ANO YES Mut 5 HVNO YES Mut 8 AYES YES Mut 9 HYES YES

  49. CS1 p28 p65 p93 MHV replicase cleavage site mutations block cleavage during infection Non-cleaving Cleaving wt icwt mut8 mut9 mut3 mut4 mut5 p93 p65 p28

  50. Cleavage site mutants are viable Wildtype Virus “Assembled” Wildtype Cleaving Mutant Non-Cleaving Mutant