# Presentation: 472724

## 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**

## Slide 2

## Pandemic Influenza: The Ever Present Threat

**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

**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

## Slide 6

## HEMAGGLUTINATION INHIBITION REACTIONS OF INFLUENZA H5 VIRUSES

- * Serology antigens

**REFERENCE ANTISERA**

**GOAT**

**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**

**160**

**2.**

**A/HONG KONG/213/2003**

**1280**

**2560**

**>5120**

**320**

**1280**

**320**

**2560**

**3.**

**A/HONG KONG/213/2003 X A/PR/8/34**

**640**

**2560**

**>5120**

**320**

**1280**

**320**

**2560**

**4.**

**A/GOOSE/HK/437-4/99**

**320**

**320**

**640**

**80**

**1280**

**320**

**320**

**5.**

**A/HONG KONG/156/97**

**640**

**320**

**1280**

**80**

**1280**

**320**

**320**

**6.**

**A/RB POCH/HONG KONG/281/2002**

**10**

**640**

**160**

**20**

**160**

**20**

**320**

**7.**

**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**

**40**

**9.**

**A/CHICKEN/VIETNAM/NCVD-7/2003**

**10**

**160**

**40**

**5**

**80**

**5**

**40**

**10.**

**A/CHICKEN/VIETNAM/NCVD-8/2003**

**20**

**80**

**80**

**20**

**320**

**160**

**160**

**11.**

**A/CHICKEN/VIETNAM/NCVD-13/2003**

**10**

**40**

**20**

**5**

**160**

**80**

**80**

**12.**

**A/MUSKOVY DUCK/VIETNAM/NCVD-14/2003**

**5**

**80**

**20**

**5**

**80**

**5**

**40**

**13.**

**A/DUCK/VIETNAM/NCVD-25/2003**

**5**

**80**

**40**

**10**

**160**

**80**

**80**

**14.**

**A/MUSKOVY DUCK/VIETNAM/NCVD-28/2003**

**5**

**80**

**20**

**5**

**80**

**5**

**40**

**15.**

**A/DUCK/VIETNAM/NCVD-29/2003**

**5**

**160**

**40**

**5**

**80**

**5**

**40**

**16.**

**A/CHICKEN/VIETNAM/NCVD-30/2003**

**10**

**160**

**40**

**5**

**160**

**10**

**40**

**17.**

**A/CHICKEN/VIETNAM/NCVD-31/2003**

**10**

**160**

**40**

**5**

**80**

**5**

**20**

**18.**

**A/MUSCOVY DUCK/VIETNAM**

**10**

**160**

**40**

**5**

**80**

**5**

**80**

**19.**

**A/CHICKEN/VIETNAM**

**10**

**160**

**40**

**5**

**80**

**5**

**40**

**20**

**A/VIETNAM/1204/2004**

**10**

**160**

**40**

**5**

**80**

**5**

**40**

**21**

**A/VIETNAM/1194/2004**

**10**

**160**

**40**

**5**

**80**

**5**

**40**

**22**

**A/VIETNAM/1203/2004**

**10**

**160**

**40**

**5**

**160**

**5**

**80**

## Evolutionary Relationships Among Influenza A (H5N1) Hemagglutinin (HA1) Genes in South Asia: 1996-2004

**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**

**Scale**

**Evolutionary Relationships Among Influenza A (H5N1) Hemagglutinin (HA1) Genes in South Asia: 1996-2004**

- Human 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**

**20 AA deletion**
**(49-68)**

***3 additional AA**
**deletion at 37-39**

**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:       **Q****R****E****RRRRKKR*****GLFG**
**Engineered: ****Q****R****ET****RR*****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**

**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. 1024 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; 3⁄4 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

- 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%

- H9

- Group

## 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