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 H15 H14 H13 H12 H11 H10 H3 H2 H1 H9 H8 H7 H6 H5 H4 N9 N8 N7 N6 N5 N3 N4 N2 N1 H16

3. Timeline of Emergence of Timeline of Emergence of Influenza A Viruses in Humans 191819571968197719972006 H1 H3 H2 H7 H5 H9 Spanish Influenza Asian flu Avian Influenza Hong Kong flu (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. Gene sequencing Gene reconstruction Lung specimens archived since 1918..... Virus rescue Infectious 1918 virus in BSL-3 enhanced lab CDC Atlanta Reconstruction of the 1918 influenza virus Drs. Palese, Garcia-Sastre, & Basler

8. 1918 autopsy cases 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 1918 lung block

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

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. 1.Identify properties that are responsible for the extraordinary virulence of the 1918 influenza virus 2.Identify genetic determinants responsible for the transmissibility of this pandemic virus Use the 1918 virus as a model for pandemic influenza Main Objectives

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

13. 1918 HA and NA genes enhanced the virulence of a contemporary H1N1 subtype virus Days after infection Mean lung titers Log 10 EID 50 /ml 0 2 4 6 8 0268 1918 HA/NA:Tx/91 rescued Texas/36/91 (Tx/91) Wild-type Tx/91 8/8 dead 0/8 dead 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 A/Texas/36/91 (H1N1) 1918 HA/NA:Tx/91

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

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

17. Thin section EM of 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 Virus* Growth in MDCK cells (PFU/ml) Contemporary H1N1 (/Tx/91) 2.3 x 10 7 1918:Tx/91 HA (7:1) 9.0 X 10 7 1918 3.0 X 10 7 1918 HA/NA/M/NP/NS:Tx/91 P’s (5:3) 7.0 X 10 7 * 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 0 20 40 60 80 100 02468101214 Days after infection % Mouse survival 1918 (2) Tx/91 Tx HA:1918 (7:1) 1918 (5:3) Tx/91 1918 (1) 1918 (2) 1918 (1)

20. 0 2 4 6 8 10 Tx/91 Tx HA:1918 1918 5:3 Tx/91 1918 (1) 1918 (2) * * Mean lung titers log 10 EID 50 /ml 1918 HA and P genes are essential for maximal replication in mouse lungs Day 4 after infection

21. SubtypeLD 50 * Lung Titers (log 10 ) Tx/36/91Not lethal3.7 1918 3.5 7.1 (EID 50 /ml) H5N1 versus 1918 virus in BALB/c mice * Expressed as the log 10 PFU required to give 1 LD 50 A/Vietnam/1203/04 2.2 6.3 A/Thailand/16/04 7.7 1.7 Virus H1N1 H5N1

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 Fever Lethargy Lethargy Nasal discharge Nasal discharge Sneezing Sneezing

24. Inoculated Contact Influenza Virus Transmission in Ferrets Inoculated

25. Ferret Model of Respiratory Droplet Transmission Human H3N2 13 5 Log 10 EID 50 /ml 1 35 7 2 4 6 8 Inoculated Contact ferrets Avian H5N1 (HK/486/97) Days post inoculation/contact Log 10 EID 50 /ml 1 3 5 1 35 7 2 4 6 8 9 Nasal wash virus titers

26. 1 3 5 7911 1 2 3 4 5 6 7 1 3 5 7 Days Post-Inoculation Virus titer (log 10 EID 50 /mL) Days Post-Contact ferrets Nasal Wash Titers: A/Duck/NY/15024/96 (H1N1) Respiratory droplet transmission of avian H1N1 viruses Respiratory droplet transmission of avian H1N1 viruses InoculatedContact ferrets* * Influenza sero-neg at day 0 and 18 p.c.

27. Days Post-Inoculation Virus titer (log 10 EID 50 /mL) Days Post-Contact ferrets Nasal Wash Titers: A/duck/Alberta/35/76 (H1N1) Respiratory droplet transmission of avian H1N1 viruses Respiratory droplet transmission of avian H1N1 viruses 1 2 3 4 5 6 7 8 135791357911 InoculatedContact ferrets* * Influenza sero-neg at day 0 and 18 p.c.

28. 1918 virus transmission experiment Slide adjacent cages together 24 hrs later Untreated Monitor disease signs and collect nasal washes daily from inoculated and contact ferrets Dose: 10 6 PFU of 1918 virus i.n. InoculatedContacts respiratory droplet transmission Six ferrets for respiratory droplet transmission

29. 800 900 1000 1100 1200 1300 1400 1500 -2012345678910111213141516 1918 virus inoculated ferrets 1/3 survived 1526 1621 1559 Days after infection Weight change (gms)

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 InoculatedContacts

31. Respiratory droplet transmission of human H1N1 viruses Respiratory droplet transmission of human H1N1 viruses 1918 135 Log 10 EID 50 /ml 1357 InoculatedContact ferrets Texas/36/91 Log 10 EID 50 /ml 7911 2 4 6 8 Days Post-InoculationDays Post-Contact 2 4 6 8 9 13513577911 †† †

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

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

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

35. ++- 2.2 x 10 7 GE Dk/Alb (wt) ++- 5.0 x 10 7 GEAV 18 +++ 5.0 x 10 7 GDNY 18 +-+ 4.8 x 10 7 DDSC 18 Untreat. CRBCs α2,3 CRBCs α2,6 CRBCs (PFU/ml) 225190Virus Presence or Absence of Hemagglutination Infectivity Titer Amino acid position in HA Properties of rescued 1918 viruses Enzymatically modified chicken red blood cells (CRBCs)

36. Days post-inoculationDays Post-contact Two amino acid substitutions in the 1918 HA abolishes transmissibility of the pandemic virus InoculatedContact ferrets* 1 2 3 4 5 6 7 8 Virus titer (log 10 EID 50 /mL) 135791573911 Nasal Wash Titers: AV18 virus * Influenza sero-neg at day 0 and 18 p.c. ††

37. Days Post-Inoculation Virus titer (log 10 EID 50 /mL) Days Post-Contact ferrets Transmissibility of the 1918 NY virus * 2/3 seroconversion to NY 18 virus on day 18 p.c. 1 2 3 4 5 6 7 8 135791357911 InoculatedContact ferrets* †

38. Virus Binding preference Mortality YesNot lethalYes Influenza pathogenesis and transmission of H1N1 viruses in ferrets - summary Virus replicationSero-conversion Spread to Contacts α 2,3Not lethal Yes66% No Texas/36/91 1918 (Human HA)Yes NoDuck/Alb/76 No66%1918 (avian HA)No α 2,6 α 2,3 α 2,6 1/333%1918 (NY HA)2/3α 2,6/α 2,3

39. Summary The 1918 HA and P genes are essential for maximal virus replication and optimal virulence. The parental 1918 (SC18) virus and the mutant 1918 viruses are 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.

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