avian influenza jen-ren wang, ph. d. ( 王貞仁 ) dept. of medical laboratory science and...
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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