1. Update on Influenza A(H5N1) Activity in Asia Nancy J. Cox, Ph.D. Chief, Influenza Branch National Center for Infectious Diseases Centers for Disease Control and Prevention

3. Pandemic Influenza: The Ever Present Threat

4. Virologic and Epidemiologic Criteria for a Pandemic Novel HA subtype, naïve populations Causes morbidity and mortality in humans Easily transmissible from person to person

5. Direct Interspecies Transmission of Avian Viruses to Humans Exposed to Infected Poultry 1997: 18 cases of human respiratory illness caused by HP avian H5N1; 6 deaths – Hong Kong 1998/9: 8 cases of human respiratory illness caused by H9N2; no deaths – China, HK 2003: 2 additional HP H5N1 cases in humans; 1 death – China, HK 2003: >80 cases of infection by HP H7N7 avian viruses; 1death - ND 2004: 28 cases of human respiratory illness by HP H5N1 avian viruses; 20 deaths – Vietnam and Thailand

7. HEMAGGLUTINATION INHIBITION REACTIONS OF INFLUENZA H5 VIRUSES * Serology antigens REFERENCE ANTISERA GOATSHEEPFERRET CHICKFERRET REFERENCE ANTIGENSTERN/SAHK/213HK/213XPR8HK/213GSE/HKHK/156RB POC 1.A/TERN/S. AFRICA/61128080 20640320160 2.A/HONG KONG/213/200312802560>512032012803202560 3.A/HONG KONG/213/2003 X A/PR/8/346402560>512032012803202560 4.A/GOOSE/HK/437-4/99320 640801280320 5.A/HONG KONG/156/976403201280801280320 6.A/RB POCH/HONG KONG/281/2002106401602016020320 7.A/GOOSE/VIETNAM/113/2001208032040 160 80 TEST ANTIGENS 8.A/CHICKEN/KOREA/ES/03403208010802040 9.A/CHICKEN/VIETNAM/NCVD-7/20031016040580540 10.A/CHICKEN/VIETNAM/NCVD-8/20032080 20320160 11.A/CHICKEN/VIETNAM/NCVD-13/2003104020516080 12.A/MUSKOVY DUCK/VIETNAM/NCVD-14/200358020580540 13.A/DUCK/VIETNAM/NCVD-25/2003580401016080 14.A/MUSKOVY DUCK/VIETNAM/NCVD-28/200358020580540 15.A/DUCK/VIETNAM/NCVD-29/2003516040580540 16.A/CHICKEN/VIETNAM/NCVD-30/2003101604051601040 17.A/CHICKEN/VIETNAM/NCVD-31/20031016040580520 18.A/MUSCOVY DUCK/VIETNAM10160405805 19.A/CHICKEN/VIETNAM1016040580540 20A/VIETNAM/1204/20041016040580540 21A/VIETNAM/1194/20041016040580540 22A/VIETNAM/1203/200410160405160580

8. Goose/Guangdong/96* Duck/Meat/Anyang/2001 Hong Kong/156/97* Hong Kong/483/97 Goose/Hong Kong/437-4/99* Goose/Vietnam/113/2001* Goose/Guangdong/96 Teal/Hong Kong/2978/2002 Duck/Vietnam/NCVD25/2003 Muscovy Duck/Vietnam/NCVD9/2003 Chicken/Vietnam/NCVD4/2003 stjude Chicken/Hong Kong/YU357/2003 Chicken/Korea/ES/2003 stjude Chicken/Hong Kong/NT71/2003 Duck/China/319-2/2003 stjude Chicken/Hong Kong/YU250/2003 stjude Pheasant/Hong Kong/NT123/2003 Rosy-Billed Pochard/Hong Kong/821/2002 stjude Chicken/Hong Kong/NT47/2003 lim Vietnam/3212/2004 Thailand/16/2004 Muscovy Duck/Vietnam/NCVD21/2003 Vietnam/1203/2004 stjude Pigeon/Hong Kong/WF32/2003 Hong Kong/213/2003* Duck/Vietnam/NCVD30/2003 Chicken/Vietnam/NCVD11/2003 Muscovy Duck/Vietnam/NCVD20/2003 Muscovy Duck/Vietnam/17/2004 Muscovy Duck/NIVR3/2003 lim Vietnam/3218/2004 Vietnam/1194/2004 Muscovy Duck/Vietnam/4/2004 nucleotides 0 3 Scale Evolutionary Relationships Among Influenza A (H5N1) Hemagglutinin (HA1) Genes in South Asia: 1996- 2004 Human H5N1 Isolates

9. Evolutionary Relationships Among Influenza A(H5) NA Genes ckvnncvd-152003 ckvnNCVD-162003 ckvnNIVR-2exe12003 mudkvnncvd-232003 mudkvnNCVD-172003 ckvnncvd-312004 ckvnNCVD-112003 mudkvnncvd-142003 ckvnNCVD-102003 dkvnNCVD-192003 ckvnncvd-302003* dkvnncvd-292003 ckvnNCVD-62003 mudkvnNIVR-3exe12003 dkvnNCVD-242003 mudkvnNCVD-182003 mudkvnncvd-212003 dkvnNCVD-222003 ckvnncvd-72003 ckvnnivr-22003 mudkvnncvd-282003 mudkvnnivr-32003 vn11942004 vn12032004 vn12042004 th66352004 rbpoch82102 ckkoes2003 dkmtay2001 gogd196 gogd397 ckhk317501 envhk4371099 envhk437899 dkhkww38100 dkhkww46100 gohkww49100 dkhk2986100 dkhk380501 gs-hk-76.1-2001 gohk3014500 govn11301na govn32401na hk21303e2 tealhk2978102 mudkvnNCVD-22003 dkvnncvd-12002 ckvnNCVD-132003 ckvnncvd-82003 mudkvnNCVD-122003 dkvnncvd-252003 ckvnNCVD-32003 ckvnNCVD-42003 ckvnNCVD-52003 mudkvnNCVD-92003 phehknt26100 qulhksf55000 qulhk17213099 hk53297 hk48597 hk48297 hk15697 hk48197 swcot148899 paul73 0.02 20 AA deletion (49-68) *3 additional AA deletion at 37-39 No AA deletion T83K, S95N, S105G, N270D, Y253H, E382G A79T, S189N 69

10. Development of Reagents and Methods for Identification/Detection of H5N1 Viruses Develop updated WHO kit for identification of H5N1 viruses (antiserum with high antibody titer and inactivated antigen for HI tests) Develop rapid detection methods for H5 using real time PCR (positive RNA controls and primer sequences)

11. H5N1 Vaccine Development Modified Fodor Vector system –8 plasmid approach Cloning and characterization –Selected clones sequence identical to RNA Removal of polybasic peptide –Original: QRERRRRKKR*GLFG –Engineered: QRETRR*GLFG Regulatory authority compliance –No animal derived ingredients

12. Generation of H5N1 vaccine with modified HA using plasmid-based reverse genetics N1 NA Mod. H5 HA RERRRKKRRETR PR8 h.g. donor Reassortant Modified H5N1 Vaccine PA PB1 HA PB2 NP NA M NS HP avian virus Bi-directional plasmids expressing both mRNA and vRNA Transfect Vero cells

13. Virus Recovery and Analysis Recovery of rg-VN/1203/PR8 reassortant (2:6) Cell culture system –Certified Vero cells –Certified cell culture medium –Amplification in eggs Characterization –Nucleotide sequence analysis: HA and NA –Yield in eggs (goal is approx. 1024 HAU/ml) Reference virus must regulatory requirements for derivation of reference strain for inactivated vaccine production

14. Safety and Immunogenicity Chick embryo lethality Mice –LD 50 study and/or virus lung titer following IN inoculation Chickens –Standard pathotyping (USDA SEPRL) Ferrets –Lung virus titer following IN inoculation (LD 50 study/virulence) Immunogenicity –HI cross test using post infection ferret serum (infected with WT & rg-VN1203/PR8) Protective efficacy in mouse model –Homologous challenge with wt A/Vietnam/1203/2004 –Heterologous challenge with A/HK/213/2003 or other viruses

15. Summary: Avian Influenza A(H5N1) as of 2/17/04 Poultry outbreaks caused by HPAI H5N1 viruses reported in Cambodia, China, Hong Kong, Laos Indonesia, Japan, S. Korea, Thailand and Vietnam Human cases reported by Vietnam (20;14 d) and Thailand (8; 6 d); no human cases reported elsewhere - but expected Most cases had exposure to sick/dead birds; family clusters raise questions about person-to-person transmission

16. Summary continued H5N1 viruses from birds and humans in VN are genetically and antigenically closely related Human isolates from VN & Thailand and 1 group of VN avian isolates resistant to adamantanes, but sensitive to oseltamivir (Tamiflu) Candidate vaccine reference strain produced with H5N1 virus from 2003 human case is not an optimal antigenic match to 2004 H5N1 viruses; might provide some protection Construction by reverse genetics of new vaccine reference strains in 2 U.S. labs and one in U.K.

17. Summary continued Need genetic and antigenic comparisons of H5N1 viruses from more countries for understanding H5N1 spread and for developing vaccine strains Culling infected birds/proper disposal necessary to reduce risk of human infection; human exposure continues in developing countries with backyard flocks constituting majority of poultry (e.g., China has 13 billion birds; ¾ of farms have < 100 birds) Poor or nonexistent human influenza surveillance in countries affected by poultry outbreaks

18. Summary continued Unprecedented human exposure to H5N1 highly pathogenic viruses Threat to global health: circulation of avian H5N1 and human H3N2 viruses in the region, so reassortment is possible as is adaptation through mutation Unlikely H5N1 viruses eradicated soon; eradication of backyard flocks difficult; infections in wild birds documented International efforts coordinated by WHO/FAO: assist in culling, surveillance and disease control efforts As outbreak continues, need to consider H5N1 vaccine production: trigger points; target populations; quantity?

19. Acknowledgements Members of the Influenza Branch The WHO National Influenza Centers The WHO Collaborating Centers in London, Tokyo and Melbourne The WHO Regional Offices WHO Headquarters in Geneva

21. 20-47 million illnesses 18-42 million clinic visits Up to 730,000 hospitalizations 89,000 to 207,000 deaths Likely U.S. Impact of an Influenza Pandemic: Estimates from a CDC Model

22. Seroprevalence of H5 and H9 Antibody in Hong Kong Poultry Workers, 1997-98 10%Poultry workers 0%General urban population H5 % Positive 23% 5% H9Group

23. Strengthen Global and Domestic Surveillance Support, strengthen WHO’s Global Influenza Network to improve the “early warning system”; interactions with NIHE in Hanoi Support, strengthen U.S. influenza sentinel physician, virologic, and mortality surveillance systems Develop contingency plans for enhancing surveillance when a new subtype is detected Develop surveillance methods to monitor hospitalized cases of influenza in the U.S. and mortality in children