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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