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Pathogenicity and transmissibility of the 1918 Spanish influenza pandemic virus

Pathogenicity and transmissibility of the 1918 Spanish influenza pandemic virus. Terrence Tumpey Influenza Division Centers for Disease Control and Prevention. Influenza A HA and NA Subtypes in Nature. N1. H1. N2. H2. N3. H3. H4. N4. H5. N5. H6. N6. H7. N7. H8. N8. H9. N9.

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Pathogenicity and transmissibility of the 1918 Spanish influenza pandemic virus

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  1. Pathogenicity and transmissibility of the 1918 Spanish influenza pandemic virus Terrence Tumpey Influenza Division Centers for Disease Control and Prevention

  2. Influenza A HA and NA Subtypes in Nature N1 H1 N2 H2 N3 H3 H4 N4 H5 N5 H6 N6 H7 N7 H8 N8 H9 N9 H10 H11 H12 H13 H14 H15 H16

  3. Timeline of Emergence of Influenza A Viruses in Humans Avian Influenza H9 H7 H5 H5 Hong Kong flu Asian flu H3 Spanish Influenza H2 H1 H1 1918 1957 1968 1977 1997 2006 (A/WS/33)

  4. 1918 ‘Spanish’ Influenza Pandemic • Total deaths in 1918-1919 estimated to be 20-50 million • U.S. Deaths = 550,000-675,000 • Flu deaths in Philadelphia in October 1918 = 10,959. • U.S. Military deaths to flu = 43,000 (out of ~100,000 U.S. Troop casualties in W.W.I.)

  5. U.S. Life Expectancy 1900-1960

  6. 1918 Spanish Influenza Iowa State College

  7. . . . . Lung specimens archived since 1918 . Reconstruction of the 1918 influenza virus Gene sequencing Gene reconstruction Drs. Palese, Garcia-Sastre, & Basler Virus rescue Infectious 1918 virus in BSL-3 enhanced lab CDC Atlanta

  8. 1918 autopsy cases 1918 lung block • Case 1: 21 y.o., PVT, Ft. Jackson, SC, died after 6 day course on 26 Sept. 1918 A/South Carolina/1/18 • Case 2: 30 y.o., PVT, Camp Upton, NY, died after 3 day course on 26 Sept. 1918 A/New York/1/18

  9. 1918 autopsy case 3 Johan Hultin in 1997 at the same gravesite Johan Hultin as a young man in 1951 at the Brevig gravesite 46 years later Attempt to grow live 1918 virus in 1951 Frozen cadaver lung tissue Jeffery Taubenberger and Ann Reid 1918 viral gene sequencing X FAILED

  10. Third 1918 Case: Alaska • Case 3: ~30 y.o. Inuit female from Teller Mission, Alaska, died in <5 days in Nov. 1918; Exhumation and lung biopsy in Aug. 1997. A/Brevig Mission/1/18

  11. Use the 1918 virus as a model for pandemic influenza Main Objectives • Identify properties that are responsible for the extraordinary virulence of the 1918 influenza virus • Identify genetic determinants responsible for the transmissibility of this pandemic virus

  12. The hemagglutinin (HA) and neuraminidase (NA) are the major viral surface proteins that play an important role in virulence NA PA PB2 PB1 HA HA NP NA M Lipid bilayer NS

  13. 1918 HA and NA genes enhanced the virulence of a contemporary H1N1 subtype virus 1918 HA/NA:Tx/91 rescued Texas/36/91 (Tx/91) 8 Wild-type Tx/91 8/8 dead 6 Mean lung titers Log10 EID50/ml 4 2 0/8 dead 0 0 2 6 8 Days after infection Tumpey et al. 2004 PNAS 101:3166 Kabasa et al. 2004 Nature 431:703

  14. BALB/c mouse lung pathology at 4 days following infection 1918 HA/NA:Tx/91 A/Texas/36/91 (H1N1)

  15. Reverse-genetics system for generation of influenza viruses from plasmids PB2 PB1 Transfection PB1 PB2 PA HA 293T/MDCK Cells NA NP M NS NP PA 8 plasmids expressing viral RNAs expressed from polI vectors. 4 protein-expression plasmids for viral polymerase and NP proteins expressed from pol I vectors. Recombinant influenza virus Fodor E, et. al. (1999) J. Virol. 73: 9679-9682.

  16. Reconstructed 1918 pandemic virus Negative stain EMof 1918 influenza virus EM by Cynthia Goldsmith, Infectious Disease Pathology Activity, CDC

  17. Thin section EMof MDCK cell pellets infected with the 8-gene 1918 influenza virus EM by Cynthia Goldsmith, Infectious Disease Pathology Activity, CDC

  18. 1918 recombinant viruses generated using reverse genetics Growth in MDCK cells Virus* (PFU/ml) 7 1918 9.0 X 10 7 1918:Tx/91 HA (7:1) 3.0 X 10 7 1918 HA/NA/M/NP/NS:Tx/91 P’s (5:3) 7.0 X 10 7 Contemporary H1N1 (/Tx/91) 2.3 x 10 * The identity of the 1918 and Tx/91 influenza virus genes was confirmed by RT-PCR and sequence analysis.

  19. 1918 hemagglutinin (HA) is essential for lethality in mice 100 80 Tx/91 Tx HA:1918 (7:1) 60 1918 (1) % Mouse survival 1918 (5:3) Tx/91 1918 (2) 1918 (1) 40 1918 (2) 20 0 0 2 4 6 8 10 12 14 Days after infection

  20. 1918 HA and P genes are essential for maximal replication in mouse lungs 10 * * Tx/91 8 Tx HA:1918 6 Mean lung titers log10 EID50/ml 1918 5:3 Tx/91 4 1918 (1) 1918 (2) 2 0 Day 4 after infection

  21. H5N1 versus 1918 virus in BALB/c mice Lung Titers (EID /ml) 50 Virus Subtype (log ) LD50* 10 H1N1 Tx/36/91 3.7 Not lethal H1N1 3.5 7.1 1918 2.2 6.3 A/Vietnam/1203/04 H5N1 1.7 7.7 A/Thailand/16/04 H5N1 * Expressed as the log10 PFU required to give 1 LD50

  22. Influenza transmission • Classical experimentation by Andrewes and Glover (1941) determined that human influenza virus may transmit from infected ferret to uninfected ferret. • The molecular basis of influenza virus transmission are not well understood. • The identification of molecular determinants of influenza virus transmission may provide a framework for the future identification of influenza viruses with pandemic potential.

  23. Ferret Model • Naturally susceptible to influenza virus infection • Distribution of sialic acid receptors in the respiratory tract is similar to humans • Exhibit similar symptoms to influenza virus infection as humans • Fever • Lethargy • Nasal discharge • Sneezing

  24. Inoculated Inoculated Contact Contact Inoculated Inoculated Inoculated Inoculated Contact Influenza Virus Transmission in Ferrets Inoculated Inoculated Inoculated

  25. Ferret Model of Respiratory Droplet Transmission Nasal wash virus titers Inoculated Contact ferrets 8 6 Human H3N2 Log10 EID50/ml 4 2 1 5 1 3 7 3 5 8 6 Log10 EID50/ml Avian H5N1 (HK/486/97) 4 2 9 5 3 5 1 1 7 3 Days post inoculation/contact

  26. Respiratory droplet transmission of avian H1N1 viruses Nasal Wash Titers: A/Duck/NY/15024/96 (H1N1) Inoculated Contact ferrets* 7 6 5 Virus titer (log10 EID50/mL) 4 3 2 1 1 7 1 7 9 11 5 5 3 3 Days Post-Inoculation Days Post-Contact ferrets * Influenza sero-neg at day 0 and 18 p.c.

  27. Respiratory droplet transmission of avian H1N1 viruses Nasal Wash Titers: A/duck/Alberta/35/76 (H1N1) 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 ferrets * Influenza sero-neg at day 0 and 18 p.c.

  28. 1918 virus transmission experiment Six ferrets for respiratory droplet transmission Inoculated Contacts Dose: 106 PFU of 1918 virus i.n. Untreated Slide adjacent cages together 24 hrs later Monitor disease signs and collect nasal washes daily from inoculated and contact ferrets

  29. 1918 virus inoculated ferrets 1526 1621 1500 1559 1400 1300 1/3 survived 1200 Weight change (gms) 1100 1000 900 800 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Days after infection

  30. Pathogenesis of 1918 virus in ferrets – 1918 virus spread to naïve contacts Clinical Signs Lethality 66% 33% Virus in nasal wash 3/3+ 3/3+ Max Temp Change (%) + 5.1 + 3.9 Sneezing Yes Yes Inoculated Contacts

  31. Respiratory droplet transmission of human H1N1 viruses Inoculated Contact ferrets 8 6 Log10 EID50/ml 1918 4 2 † † † 1 3 5 7 9 1 3 5 7 9 11 8 6 Texas/36/91 Log10 EID50/ml 4 2 1 3 5 7 1 3 5 7 9 11 Days Post-Inoculation Days Post-Contact

  32. Does receptor binding specificity of influenza viruses influence transmission of H1N1 viruses in mammals? Distribution of sialic acids/receptor preference • Human influenza viruses prefer αlpha 2,6 linkages • Avian influenza viruses prefer αlpha 2,3 linkages Avian Sia(2-3)Gal Human Sia(2-6)Gal

  33. Influenza virus receptors in the human airway Upper respiratory tract +++ α2,6 sialic acid +/- α2,3 sialic acid Lower respiratory tract +++ α2,6 sialic acid +++ α2,3 sialic acid

  34. Single amino acid substitutions in the 1918 HA changes the receptor binding specificity 1918 HA

  35. Properties of rescued 1918 viruses Enzymatically modified chicken red blood cells (CRBCs) Amino acid position in HA Infectivity Titer Presence or Absence of Hemagglutination (PFU/ml) Virus 190 225 α2,6 CRBCs α2,3 CRBCs Untreat. CRBCs SC 18 D D 4.8 x 107 + - + NY 18 D G 5.0 x 107 + + + AV 18 E G 5.0 x 107 - + + E G 2.2 x 107 - + + Dk/Alb (wt)

  36. Two amino acid substitutions in the 1918 HA abolishes transmissibility of the pandemic 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.

  37. Transmissibility of the 1918 NY 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-Contact ferrets Days Post-Inoculation * 2/3 seroconversion to NY 18 virus on day 18 p.c.

  38. Influenza pathogenesis and transmission of H1N1 viruses in ferrets - summary Spread to Contacts Binding preference Mortality Virus replication Sero- conversion Virus Texas/36/91 α2,6 Not lethal Yes Yes Duck/Alb/76 α2,3 Not lethal No No 1918 (Human HA) α2,6 66% Yes Yes 1918 (avian HA) α2,3 66% No No 1918 (NY HA) α2,6/α2,3 33% 1/3 2/3

  39. The 1918 HA and P genes are essential for maximal virus replication and optimal virulence. • The parental 1918 (SC18) virus and the mutant 1918 virusesare virulent in ferrets. • Two amino acid mutations that cause a switch from the human α2,6 to the avian α2,3 SA receptor binding preference resulted in a virus incapable of respiratory droplet transmission between ferrets, but that maintained its lethality and replication efficiency in the upper respiratory tract. • Poor transmission of a 1918 virus with dual α2,6/α2,3 specificity suggests that a predominant α2,6 SA binding preference is essential for optimal transmission of this pandemic virus. Summary

  40. Acknowledgements Centers for Disease Control and Prevention Influenza Division/IVPB Taronna Maines Neal van Hoeven Claudia Pappas Cynthia Goldsmith Mount Sinai School of Medicine University of Washington School of Medicine Armed Forces Institute of Pathology USDA/Southeast Poultry Research Laboratory NIH Grants; 5R01 AI0506919-02 and AI058113-01 The Scripps Research Institute

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