Update on Influenza A(H5N1) Activity in Asia

Nancy J. Cox, Ph.D.Chief, Influenza Branch

National Center for Infectious DiseasesCenters 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 ANTISERAGOAT SHEEP FERRET FERRET CHICK FERRET FERRET

REFERENCE ANTIGENS TERN/SA HK/213 HK/213XPR8 HK/213 GSE/HK HK/156 RB POC

1. A/TERN/S. AFRICA/61 1280 80 80 20 640 320 1602. A/HONG KONG/213/2003 1280 2560 >5120 320 1280 320 25603. A/HONG KONG/213/2003 X A/PR/8/34 640 2560 >5120 320 1280 320 25604. A/GOOSE/HK/437-4/99 320 320 640 80 1280 320 3205. A/HONG KONG/156/97 640 320 1280 80 1280 320 3206. A/RB POCH/HONG KONG/281/2002 10 640 160 20 160 20 3207. A/GOOSE/VIETNAM/113/2001 20 80 320 40 160 80 80

TEST ANTIGENS

8. A/CHICKEN/KOREA/ES/03 40 320 80 10 80 20 409. A/CHICKEN/VIETNAM/NCVD-7/2003 10 160 40 5 80 5 4010. A/CHICKEN/VIETNAM/NCVD-8/2003 20 80 80 20 320 160 16011. A/CHICKEN/VIETNAM/NCVD-13/2003 10 40 20 5 160 80 8012. A/MUSKOVY DUCK/VIETNAM/NCVD-14/2003 5 80 20 5 80 5 4013. A/DUCK/VIETNAM/NCVD-25/2003 5 80 40 10 160 80 8014. A/MUSKOVY DUCK/VIETNAM/NCVD-28/2003 5 80 20 5 80 5 4015. A/DUCK/VIETNAM/NCVD-29/2003 5 160 40 5 80 5 4016. A/CHICKEN/VIETNAM/NCVD-30/2003 10 160 40 5 160 10 4017. A/CHICKEN/VIETNAM/NCVD-31/2003 10 160 40 5 80 5 2018. A/MUSCOVY DUCK/VIETNAM 10 160 40 5 80 5 8019. A/CHICKEN/VIETNAM 10 160 40 5 80 5 4020 A/VIETNAM/1204/2004 10 160 40 5 80 5 4021 A/VIETNAM/1194/2004 10 160 40 5 80 5 4022 A/VIETNAM/1203/2004 10 160 40 5 160 5 80

Goose/Guangdong/96*

Duck/Meat/Anyang/2001

Hong Kong/156/97*Hong Kong/156/97*Hong Kong/483/97Hong Kong/483/97

Goose/Hong Kong/437-4/99*

Goose/Vietnam/113/2001*

Goose/Guangdong/96

Teal/Hong Kong/2978/2002Duck/Vietnam/NCVD25/2003

Muscovy Duck/Vietnam/NCVD9/2003Chicken/Vietnam/NCVD4/2003

stjudeChicken/Hong Kong/YU357/2003Chicken/Korea/ES/2003

stjudeChicken/Hong Kong/NT71/2003

Duck/China/319-2/2003

stjudeChicken/Hong Kong/YU250/2003

stjudePheasant/Hong Kong/NT123/2003

Rosy-Billed Pochard/Hong Kong/821/2002stjudeChicken/Hong Kong/NT47/2003

limlimVietnam/3212/2004Vietnam/3212/2004Thailand/16/2004Thailand/16/2004

Muscovy Duck/Vietnam/NCVD21/2003

Vietnam/1203/2004Vietnam/1203/2004

stjudePigeon/Hong Kong/WF32/2003Hong Kong/213/2003*Hong Kong/213/2003*

Duck/Vietnam/NCVD30/2003Chicken/Vietnam/NCVD11/2003

Muscovy Duck/Vietnam/NCVD20/2003Muscovy Duck/Vietnam/17/2004

Muscovy Duck/NIVR3/2003limlimVietnam/3218/2004Vietnam/3218/2004

Vietnam/1194/2004Vietnam/1194/2004

Muscovy Duck/Vietnam/4/2004

nucleotides0 3

Scale

Evolutionary Relationships Among Influenza A (H5N1) Hemagglutinin (HA1) Genes in South Asia: 1996-

2004Human H5N1 IsolatesHuman H5N1 Isolates

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 AAdeletion at 37-39

No AA deletion

T83K, S95N, S105G, N270D, Y253H, E382G

A79T, S189N

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

Generation of H5N1 vaccine with modified HA using plasmid-based reverse genetics

N1 NA

Mod. H5 HA

RERRRKKR RETR

PR8 h.g. donor

Reassortant Modified H5N1 Vaccine

PAPB1 HAPB2

NP NA M NS

HP avian HP avian virusvirus

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. 1024 HAU/ml)

• Reference virus must regulatory requirements for derivation of reference strain for inactivated vaccine production

Safety and Immunogenicity

• Chick embryo lethality• Mice

– LD50 study and/or virus lung titer following IN inoculation• Chickens

– Standard pathotyping (USDA SEPRL) • Ferrets

– Lung virus titer following IN inoculation (LD50 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

• 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

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

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