Avian Influenza

Jen-Ren Wang, Ph. D. (王貞仁 )

Dept. of Medical Laboratory Science and Biotechnology

National Cheng Kung University

Influenza viruses

Orthomyxoviridae Influenzavirus A, B, CGenome consists of 8 single-

stranded RNA segmentsEnveloped virus with

hemagglutinin and neuraminidase spikes

(Linda Stannard, University of Cape Town, S.A.)

Prevalence of human influenza viruses

B

H3N2

H2N2

H1N1H1N1

H3N8

H2N?

1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

YearHampson, et al. MJA 2006

Pandemics in this century

Year Virus subtype Deaths

1918-1919 Spanish " flu" (H1N1) 20-40 million

1957-1958 Asian " flu " (H2N2) 1-2 million

1968-1969 Hong Kong " flu " (H3N2) 700,000

1977-1978 Russian " flu " (H1N1) ? Benign pandemic

Polypeptide

Nucleotide Length (Nt) Function

PB2 2341 Transcriptase: cap binding

PB1 2341 Transcriptase: elongation

PA 2233 Transcriptase: protease activity

HA 1778 Hemagglutinin

NP 1565Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/ cytoplasmic transport of vRNA

NA 1413 Neuraminidase: release of virus

M11027

Matrix protein: major component of virion

M2 Integral membrane protein – ion channel

NS1

890

Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein

NS2Non-structural: nucleus+cytoplasm, function

unknown

Influenza virus A genome

Influenza virus A HA and NA

Hemagglutinin(HA)

Mediating virus binding to cell receptorPromoting release of virus RNP through membrane fusionPosttranslational proteolytic activation of the precursor HA0 into HA1 and HA2

Allows for penetration through RNPs mucous layer to epithelial cellsFacilitates release of virions by cleaving sialic acid residues thus preventing aggregation

Neuraminidase(NA)

Molecular determinants of host range restriction

Human Viruses:• N-acetylsialic acid α

2,6 linked to galactose

• HA226Leu

Avian Viruses:• N-acetylsialic acid α

2,3 linked to galactose

• HA226Gln

Pigs provide a mixing pot thus allowing the passage of avian virus to humans.

Ecology of influenza A viruses and interspecies transmission

Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Avian Influenza Infections in Humans

• 1997: In Hong Kong, avian influenza A (H5N1) infected both chickens and humans. This was the first time an avian influenza virus had ever been found to transmit directly from birds to humans.

• 1999: In Hong Kong, cases of avian influenza A H9N2 were confirmed in 2 children.

• 2003: Two cases of avian influenza A (H5N1) infection occurred among members of a Hong Kong family that had traveled to China.

• 2003: Avian influenza A (H7N7) infections among poultry workers and their families were confirmed in the Netherlands

• 2003: H9N2 infection was confirmed in a child in Hong Kong.• 2004: H5N1 infections in Asia• 2005-present: H5N1 infections in Asia, Europe, others

Avian Influenza (H5N1)

• Chicken: characterized by sudden onset, severe illness, and rapid death, with a mortality that can approach 100%.

• Human cases– fever – sore throat– cough– fatal cases: severe respiratory distress

secondary to viral pneumonia

Avian Influenza (H5N1)

• Ducks may be infected without illness

- 1999-2002, no mortality in ducks

- 2002, started causing mortality in ducks• Transmitted long distance by migratory birds• Pig have been infected (China, Indonesia;

asymptomatic)• Infect domestic cats, tigers• Continue to evolve

Influenza A virus (H9N2)

• Caused increased morbidity and mortality of chickens and decreased egg production

• 2 children were infected with respiratory disease

• Contains 6 genetic segments that are similar to those of H5N1

• 1999, 2003

Influenza A virus (H7N7)

• 2003, Netherlands

• Acute conjunctivitis

• 430 (380 conjunctivitis, 85 ILI, 56 other)

• 87 H7 positive conjunctival swab, 10 H7 positive throat swab

• One fatal case

• Human-to-human transmission reported

Human disease caused by interspecies transmission of avian influenza viruses

without prior reassortment

Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Country 

2003 2004 2005 2006 2007 Total

cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths

Azerbaijan 0 0 0 0 0 0 8 5 0 0 8 5

Cambodia 0 0 0 0 4 4 2 2 1 1 7 7

China 1 1 0 0 8 5 13 8 3 2 25 16

Djibouti 0 0 0 0 0 0 1 0 0 0 1 0

Egypt 0 0 0 0 0 0 18 10 20 5 38 15

Indonesia  0 0 0 0 20 13 55 45 31 27 106 85

Iraq 0 0 0 0 0 0 3 2 0 0 3 2

Lao People's Democratic Republic

0 0 0 0 0 0 0 0 2 2 2 2

Nigeria 0 0 0 0 0 0 0 0 1 1 1 1

Thailand 0 0 17 12 5 2 3 3 0 0 25 17

Turkey 0 0 0 0 0 0 12 4 0 0 12 4

Viet Nam 3 3 29 20 61 19 0 0 7 4 100 46

Total 4 4 46 32 98 43 115 79 65 42 328 200Total number of cases includes number of deaths.WHO reports only laboratory-confirmed cases.

http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_09_10/en/print.html

Confirmed human cases of H5N1 reported to WHO

Seasonality of avian H5N1 viruses from domestic poultry in mainland in China

Li et al., 2004 Nature 430, 209–213.

The genotypes of H5N1 reassortants(1999-2001)

Guan et al., 2002 Proc Natl Acad Sci U S A 99, 8950–8955.

The genotypes of H5N1 influenza reassortants

Li et al., 2004 Nature 430, 209–213

Phylogenetic relationships of the (A) HA and (B) NP genes of influenza A viruses isolated in Indonesia and Vietnam.

Smith GJ, et al. Virology. 2006. 5;350(2):258-68.

Definition of avian influenza

• Intravenous pathogenicity index (IVPI) in 4-8 week-old chickens greater than 1.2, or cause more than 75% mortality within 10 days or nucleotide sequencing of H5 or H7 has demonstrated the presence of multiple basic amino acids at the cleavage site of the haemagglutinin.

• New proposed definition: IVPI in 6-week-old chickens greater than 1.2 or any influenza A virus of H5 or H7 subtype.

Birds are examined at 24-hour intervals for 10 days. At each observation, each bird is scored 0 if normal, 1 if sick, 2 if severely sick, 3 if dead. (The judgement of sick and severely sick birds is a subjective clinical assessment. Normally, ‘sick' birds would show one of the following signs and ‘severely sick' more than one of the following signs: respiratory involvement, depression, diarrhoea, cyanosis of the exposed skin or wattles, oedema of the face and/or head, nervous signs. Dead individuals must be scored as 3 at each of the remaining daily observations after death [when birds are too sick to eat or drink, they should be killed humanely and scored as dead at the next observation].)

The intravenous pathogenicity index (IVPI) is the mean score per bird per observation over the 10-day period. An index of 3.00 means that all birds died within 24 hours, and an index of 0.00 means that no bird showed any clinical sign during the 10-day observation period.

Office International des Epizooties (OIE)

Host protease-dependent activation of influenza virus

S

SCOOH

HA１

NH2

S

SCOOH

HA

NH2

HA２

From Lance Jennings

Virus Activating Protease

Ubiquitous Protease present in Golgi Body

FurinPlasmin

Fowl Plague

Fatal Systemic Infection

All tissues and organs

HPAI

Specific Secretory ProteaseTrypsin

Tryptase ClaraFXa

Plasmin

Subclinical

Localized Infection

Respiratory & Alimentary Tract ｓ ‘Epithelial cells’

LPAI

ResponsibleProteases

Structure of HADiseases in Chickens

Tissue TropismVirus

HA1

HA2

- - - - - - R / - - -

HA1

HA2

R R R R E K R / - - -

Avian Influenza Viruses

R J Webby, et al. 2004

NS gene of Influenza A

27 57 530 718 864

NS1

NS2

poly-A binding protein II (PABII) binding domain (223-237)

Influenza A virus NS1 protein

RNA-binding domain

(1-73)

Effector domain

(73-237)

1 19 34 36 38 73 137 146 186 216 221 223 237

RNA-binding domain (19-38)

Nuclear localization signal (34-38, 216-221)

Nuclear export signal (137-146)

30 kDa subunit of Cleavage and polyadenylation specific factor (CPSF) binding site (186)

PDZ domain ligand (228-231)

Virulence & cytokine response Importance of NS1 in Influenza A virus

• A D92E mutation in NS1 strongly affect the virule

nce of influenza virus, eg: the H5N1 avian flu viru

s (Seo et al. 2004) .

• C-terminal PDZ domain ligand in NS1 act as a po

tential virulence determinant (Krug et al. 2006) .

Nat.Struct.Mol.Biol. 2006

E92 and del 80-84 may affect RNA binding affinity

• Cytokine-resistance

H5N1 of Hong Kong outbreak

NS1 gene

PDZ ligand motif in NS1 as a potential virulence determinant

PDZ domain

Regulating the activity and trafficking of membrane proteins

Maintaining cell polarity and morphology

Organizing postsynaptic density in neuronal cells

Large-scale sequence analysis of avian influenza isolates Obenauer et al. science. 2006

1997

2003

1918

Role of PB2 genes

• A E627K mutation in PB2 strongly affect the virulence of influenza virus, eg: the 1918 flu virus and the H5N1 avian flu virus (Gillis et al. 2005).

PB2 gene

Schematic diagram of chimeric and single amino acid PB2 mutants, with their virulence in mice (MLD50)

Hatta M, et al. Science. 2001. 7;293(5536):1840-2.

mutation at position 627 in the PB2 protein influenced the outcome of infection in mice

Mechanisms of pathogenesis of human H5N1 disease

Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Generation of pandemic influenza virus strains

Nichol, et al. PNAS 2000

Development of a vaccine

• Grow in eggs• Grow in MDCK cells• H5N1 is so deadly in chicken embryos, a new

technique, known as “reverse genetics”, is required to prepare the prototype H5N1 virus for vaccine production.

• The virus can also be genetically modified so that it is no longer lethal to chicken embryos.

Vaccine of high pathogenic avian influenza virus

Hoffmann, et al. 2000