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Transmission of 2009 H1N1 Influenza Viruses in Ferrets

Transmission of 2009 H1N1 Influenza Viruses in Ferrets. EM of CA/04/2009 – CDC/C.S. Goldsmith. Terrence Tumpey Immunology and Pathogenesis Branch Influenza Division, CDC. Animal Models: Influenza Transmission. Mice – Poor model

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Transmission of 2009 H1N1 Influenza Viruses in Ferrets

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  1. Transmission of 2009 H1N1 Influenza Viruses in Ferrets EM of CA/04/2009 – CDC/C.S. Goldsmith Terrence Tumpey Immunology and Pathogenesis Branch Influenza Division, CDC

  2. Animal Models: Influenza Transmission • Mice – Poor model • Guinea Pigs – Good transmission model, but poor model for influenza pathogenesis • Ferrets – Best model for transmission and pathogenesis (Lowen AC, S. Mubareka, J. Steel, P. Palese, PLoS Pathog. 2007 3:1470)

  3. Transmission Study: Experimental DesignRespiratory Droplet transmission Inoculated Animals

  4. Transmission Study: Experimental DesignRespiratory Droplet transmission Naive 106 PFU i.n. Inoculated Animals

  5. Transmission Study: Experimental DesignRespiratory Droplet transmission Inoculated Animals

  6. Transmission Study: Experimental Design Respiratory Droplet Transmission Contact Transmission X2 Contact Animals Inoculated Animals X2 X2

  7. Ferret Model of Respiratory Droplet Transmission Human H1N1 vs. avian H1N1 viruses Inoculated Contact ferrets (3/3) 8 6 Log10 EID50/ml 4 1918 (H1N1) (SC18 HA) 2 † † † 1 3 5 7 9 1 3 5 7 9 11 8 No transmission 6 Avian H1N1 (Duck/NY/96) Log10 EID50/ml 4 2 1 3 5 7 9 1 3 5 7 9 11 Days post inoculation/contact

  8. Ferret Model of Respiratory Droplet Transmission Human vs. avian H2N2 viruses Inoculated Contact ferrets 8 Human H2N2 (Albany/6/58) 6 Log10 EID50/ml 4 2 1 3 5 7 9 1 3 5 7 9 11 8 No transmission 6 Avian H2N2 (Mallard/NY/6750/78) Log10 EID50/ml 4 2 1 3 5 7 9 1 3 5 7 9 11 Days post inoculation/contact

  9. What influenza virus genes confer efficient transmission? = + Virus genes ? Efficient Transmission 1918 H1N1 (transmissible phenotype) Avian H1N1 (non-transmissible )

  10. Summary of Findings Two 1918 virus genes, HA and PB2, were sufficient to confer virus transmissibility in ferrets when rescued on the genetic background of an avian H1N1 virus. HA PB2

  11. Two amino acid substitutions (D190E, D225G) in HA abolishes transmissibility of the 1918 virus Nasal Wash Titers: AV18 virus Inoculated Contact ferrets* 8 7 6 5 Virus titer (log10 EID50/mL) 4 3 2 † † 1 1 3 5 7 9 1 3 5 7 9 11 Days post-inoculation Days Post-contact * Influenza sero-neg at day 0 and 18 p.c.

  12. 1918 HA and PB2 virus genes confer efficient respiratory droplet transmission Inoculated Contact ferrets 8 6 1918 HA:Duck/NY/96 Log10 EID50/ml 4 2 1 3 5 7 9 1 3 5 7 9 11 1918 HAPB2:Dk/NY/96 8 6 Log10 EID50/ml 4 2 1 3 5 7 9 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  13. PB2 • Ten amino acid differences in PB2 of the avian Dk/NY/96 virus and 1918 virus • These changes (positions 108, 114,199, 473, 475, 477, 539, 576, 627, and 702) occur in domains that are suggested to have a variety of functions including mRNA cap-binding and PB1 binding • Of particular interest is the presence of a lysine (k) residue at 627 of the 1918 PB2 protein • PB2 627K has been suggested to allow more efficient growth at the lower temperatures encountered in the upper airway of mammals (M. Hatta et al., 2007 PLoS Pathog 3)

  14. A single amino acid substitution (PB2-K627E) in PB2 abolishes transmissibility of the 1918 virus Nasal Wash Titers: 1918 (PB2-K627E) Inoculated Contact ferrets 8 7 6 Virus titer (log10 EID50/mL) 5 4 3 2 1 3 5 7 9 1 3 5 7 9 11 Days post-inoculation Days post-contact

  15. Plaque morphology of H1N1 reassortant viruses

  16. Plaque morphology of H1N1 reassortant viruses

  17. Transmission of 2009 H1N1 Influenza Viruses Do similar molecular changes influence transmission of 2009 H1N1 virus?

  18. Transmission of 2009 H1N1 in humans Study location Household Secondary Attack Rates (% ILI) Study Cauchemez et al. (NEJM 2009 361:29) U.S. 10.1 North Carolina 14.3 Doyle & Hopkins (Epidemiol Infect. 2010 21:1) New York 17.7 Lessler & Reich (NEJM 2009 361:2628) Hong Kong 5.9 Leung et al. (Epidemiol Infect. 2010 21:7) Odaira et al. (Eurosurveillance 2009 14:35) Japan 7.6 • SAR of seasonal influenza range is 10 to 40% • Transmissibility in households from previous pandemics (20-40%)

  19. Transmission of 2009 H1N1 Viruses in Ferrets Three 2009 H1N1 viruses selected: • A/California/04/2009 (uncomplicated respiratory illness) • A/Mexico/4482/2009 (severe respiratory illness) • A/Texas/15/2009 (fatal respiratory illness) • vs. • Seasonal H1N1

  20. 2009 H1N1 Viruses Spread Efficiently by Direct Contact Inoculated Contact ferrets (3/3) 8 6 Mex/4482/2009 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (1/3) 8 6 Avian H1N1 (Duck/NY/96) Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Contact Days Post-Inoculation

  21. Seasonal H1N1 vs. 2009 H1N1 RD Transmission Inoculated Contact ferrets (2/3) 8 Mex/4482/2009 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (3/3) 8 6 Brisbane/2007 (seasonal) Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  22. Seasonal vs. Texas/15/009 H1N1 Virus Transmission Inoculated Contact ferrets (2/3) 8 6 Tx/15/2009 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (3/3) 8 Solomon Is/3/06 (seasonal H1N1) 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  23. H1N1 Viruses of Swine Origin Failed to Form Significant Plaques at 33oC

  24. PB2 What is the transmission phenotype of a 2009 H1N1 PB2 (E627K) mutant virus? Glutamic acid → Lysine at position 627 PB1 PB2 PA HA NP NA M NS CA/04/2009 627E PB1 PB2 PA HA NP NA M NS CA/04/09 PB2 627K 627K

  25. E627K in PB2 Enhances RD Transmission in Ferrets Inoculated Contact ferrets (2/3) 8 CA/04/2009 6 Log10 EID50/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (3/3) 8 CA/04/09 PB2 E627K 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  26. RD Transmission of a natural 2009 H1N1 E627K isolate Inoculated Contact ferrets (3/3) NL/1132/2009 PB2 E627K Log10 PFU/ml 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  27. HA Novel HA mutation (D222G) associated with increased disease severity? • Amino acid position 222 resides in the RBS of the HA protein and may possibly influence the binding specificity and thus the cellular tropism of the virus. • HA (D222G) mutation was found with increased frequency in fatal and severe cases in Norway (Kilander et al. 2010 Eurosurveillance Vol. 15 (9). • D222G mutants were detected more frequently in viruses isolated from patients with fatal outcomes and in lung samples (Glinsky Cell Cycle 9 (5) 958-970)

  28. HA What is the transmission phenotype of a 2009 H1N1 HA (D222G) mutant virus? Aspartic acid → Glycine at 222 HA (H1 numbering) PB1 PB2 PA HA NP NA M NS CA/04/2009 222D PB1 PB2 PA HA NP NA M NS CA/04/09 HA D222G 222G

  29. The effect of HA (D222G) mutation on RD transmission Inoculated Contact ferrets (2/3), 4/6 total 8 CA/04/09 HA D222G 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (2/3) 8 CA/04/2009 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  30. HA mutation at position 219 (I219K) improves the human-receptor binding affinity of CA/04 HA 1918 HA CA/04 HA I219K CA/04 wt HA Direct glycan array binding assay of HA’s to alpha 2,6 SA human receptor

  31. HA What is the transmission phenotype of a 2009 H1N1 HA (I219K) mutant virus? Isoleucine → Lysine at 219 HA PB1 PB2 PA HA NP NA M NS CA/04/2009 219I PB1 PB2 PA HA NP NA M NS CA/04/09 HA 219K 219K

  32. A single amino acid substitution (I219K) in HA slightly enhances RD transmission of CA/04/2009 virus Inoculated Contact ferrets (3/3) 8 6 CA/04/09 HA I219K Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Inoculated Contact ferrets (2/3) 8 CA/04/2009 6 Log10 PFU/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  33. Comparative H1N1 Results in Ferrets Lung Titers Weight Loss (%) (PFU/gm) % RD Transmission Virus (log ) Lethality 10 CA/4/2009 – Wild-type 10.3 0/6 66 5.8 CA/4/2009 - D222G 11.4 0/6 66 6.3 CA/4/2009 - E627K 0/6 11.3 100 5.9 CA/4/2009 - E219K 10.1 5.4 0/6 100 Brisbane/59 (seasonal) 4.9 <1.2 0/6 100

  34. Acknowledgements Pathogenesis team CDC Atlanta

  35. Acknowledgements Centers for Disease Control and Prevention Influenza Division/IVPB Neal van Hoeven Debra Wadford Jacqueline Katz Nancy Cox Mount Sinai School of Medicine University of Washington School of Medicine The Scripps Research Institute USDA/Southeast Poultry Research Laboratory NIH Grants; 5R01 AI0506919-02 and AI058113-01 David Swayne

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