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

Pandemic Influenza: The Ever Present Threat

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

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

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/ A/HONG KONG/213/ > A/HONG KONG/213/2003 X A/PR/8/ > A/GOOSE/HK/437-4/ A/HONG KONG/156/ A/RB POCH/HONG KONG/281/ A/GOOSE/VIETNAM/113/ TEST ANTIGENS 8.A/CHICKEN/KOREA/ES/ A/CHICKEN/VIETNAM/NCVD-7/ A/CHICKEN/VIETNAM/NCVD-8/ A/CHICKEN/VIETNAM/NCVD-13/ A/MUSKOVY DUCK/VIETNAM/NCVD-14/ A/DUCK/VIETNAM/NCVD-25/ A/MUSKOVY DUCK/VIETNAM/NCVD-28/ A/DUCK/VIETNAM/NCVD-29/ A/CHICKEN/VIETNAM/NCVD-30/ A/CHICKEN/VIETNAM/NCVD-31/ A/MUSCOVY DUCK/VIETNAM A/CHICKEN/VIETNAM A/VIETNAM/1204/ A/VIETNAM/1194/ A/VIETNAM/1203/

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: Human H5N1 Isolates

Evolutionary Relationships Among Influenza A(H5) NA Genes ckvnncvd ckvnNCVD ckvnNIVR-2exe12003 mudkvnncvd mudkvnNCVD ckvnncvd ckvnNCVD mudkvnncvd ckvnNCVD dkvnNCVD ckvnncvd * dkvnncvd ckvnNCVD mudkvnNIVR-3exe12003 dkvnNCVD mudkvnNCVD mudkvnncvd dkvnNCVD ckvnncvd ckvnnivr mudkvnncvd mudkvnnivr vn vn vn th rbpoch82102 ckkoes2003 dkmtay2001 gogd196 gogd397 ckhk envhk envhk dkhkww38100 dkhkww46100 gohkww49100 dkhk dkhk gs-hk gohk govn11301na govn32401na hk21303e2 tealhk mudkvnNCVD dkvnncvd ckvnNCVD ckvnncvd mudkvnNCVD dkvnncvd ckvnNCVD ckvnNCVD ckvnNCVD mudkvnNCVD phehknt26100 qulhksf55000 qulhk hk53297 hk48597 hk48297 hk15697 hk48197 swcot paul AA deletion (49-68) *3 additional AA deletion at No AA deletion T83K, S95N, S105G, N270D, Y253H, E382G A79T, S189N 69

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)

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

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

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 HAU/ml) Reference virus must regulatory requirements for derivation of reference strain for inactivated vaccine production

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

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

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.

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

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?

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

20-47 million illnesses 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

Seroprevalence of H5 and H9 Antibody in Hong Kong Poultry Workers, %Poultry workers 0%General urban population H5 % Positive 23% 5% H9Group

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