1. Avian Influenza Jen-Ren Wang, Ph. D. (王貞仁)
Dept. of Medical Laboratory Science and Biotechnology
National Cheng Kung University

2. Influenza viruses Orthomyxoviridae Influenzavirus A, B, C
Genome consists of 8 single-stranded RNA segments
Enveloped virus with hemagglutinin and neuraminidase spikes
(Linda Stannard, University of Cape Town, S.A.)

3. Prevalence of human influenza virusesB
H3N8
H3N2
H2N2
H2N?
H1N1
H1N1
Year
Hampson, et al. MJA 2006

4. Pandemics in this century
Year Virus subtype Deaths
Spanish " flu" (H1N1) million
Asian " flu " (H2N2) million
Hong Kong " flu " (H3N2) ,000
Russian " flu " (H1N1) ? Benign pandemic

5. Influenza virus A genome
Polypeptide
Nucleotide Length (Nt)
Function
PB2
2341
Transcriptase: cap binding
PB1
Transcriptase: elongation PA
2233
Transcriptase: protease activity HA
1778
Hemagglutinin NP
1565
Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/ cytoplasmic transport of vRNA NA
1413
Neuraminidase: release of virus M1
1027
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
NS2
Non-structural: nucleus+cytoplasm, function
unknown
Except for proteins in the envelope, there were still several in influenza virus genome. PB1,2,PA protein have the functions of transcriptase. NP was Nucleoprotein. M1 was the major component of virion and NS1 protein has the effects on RNA transport, splicing, and translation. Using these protein, the influenza virus can infect and replicate in cells.

6. Influenza virus A HA and NA
Hemagglutinin(HA)
Neuraminidase(NA)
Mediating virus binding to cell receptor
Promoting release of virus RNP through membrane fusion
Posttranslational proteolytic activation of the precursor HA0 into HA1 and HA2
Allows for penetration through RNPs mucous layer to epithelial cells
Facilitates release of virions by cleaving sialic acid residues thus preventing aggregation
First, let’s summary the function of influenza HA and NA protein. First, HA mediates virus binding to cell receptor,
promotes release of virus RNP through membrane fusion, and actives posttranslational proteolysis of the precursor HA0 into HA1 and HA2. Second, Neuraminidase allows for penetration through RNPs mucous layer to epithelial cells and help the release of virions by cleaving sialic acid residues thus preventing aggregation. So, both of these two protein are two most important antigenicity surface glycoproteins in influenza virus.

7. Molecular determinants of host range restriction
Avian Viruses:
N-acetylsialic acid α2,3 linked to galactose
HA226Gln
Human Viruses:
N-acetylsialic acid α2,6 linked to galactose
HA226Leu
By antigenic shift and drift, influenza viruses change their characteristics to infect other species. However, there are still species barriers to stop the spread of viruses. These barriers includes the linkage of N-acetylsialic acid to galactose and hemagglutinin amino acid residues. If the avian influenza virus overcome these barriers, it could infect humans through pigs or directly infect humans.
Pigs provide a mixing pot thus allowing the passage of avian virus to humans.

8. Ecology of influenza A viruses and interspecies transmission
Malik Peiris, et al. Clin Micro Rev 20:243, 2007

9. 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

10. 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

11. Avian Influenza (H5N1) Ducks may be infected without illness
, 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

12. 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

13. 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

14. Human disease caused by interspecies transmission of avian influenza viruses without prior reassortment
Malik Peiris, et al. Clin Micro Rev 20:243, 2007

15. Confirmed human cases of H5N1 reported to WHO
Country
2003
2004
2005
2006
2007
Total
cases
deaths
Azerbaijan 8 5
Cambodia 4 2 1 7
China 13 3 25 16
Djibouti
Egypt 18 10 20 38 15
Indonesia  55 45 31 27
106 85
Iraq
Lao People's Democratic Republic
Nigeria
Thailand 17 12
Turkey
Viet Nam 29 61 19
100 46 32 98 43
115 79 65 42
328
200
Total number of cases includes number of deaths. WHO reports only laboratory-confirmed cases.

16. Seasonality of avian H5N1 viruses from domestic poultry in mainland in China
Li et al., 2004 Nature 430, 209–213.

17. The genotypes of H5N1 reassortants(1999-2001)
Guan et al., 2002 Proc Natl Acad Sci U S A 99, 8950–8955.

18. The genotypes of H5N1 influenza reassortants
Li et al., 2004 Nature 430, 209–213

19. Phylogenetic relationships of the (A) HA and (B) NP genes of influenza A viruses isolated in Indonesia and Vietnam.
Smith GJ, et al. Virology ;350(2):

20. 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.

21. Birds are examined at 24-hour intervals for 10 days
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)

22. Host protease-dependent activation of influenza virus
NH2
NH2
HA１ S S
Virus Activating Protease HA S S
HA２
COOH
COOH
From Lance Jennings

23. Ubiquitous Protease present in Golgi Body Specific Secretory Protease
Avian Influenza Viruses
Ubiquitous Protease present in Golgi Body
Furin
Plasmin
Fowl Plague
Fatal Systemic Infection
All tissues and
organs
HPAI
Specific Secretory Protease
Trypsin
Tryptase Clara
FXa
Subclinical
Localized Infection
Respiratory &
Alimentary Tractｓ
‘Epithelial cells’
LPAI
Responsible
Proteases
Structure of HA
Diseases
in Chickens
Tissue Tropism
Virus
HA1
HA2
R / - - -
R R R R E K R / - - -
Viral factors determining pathogenicity of avian influenza viruses
1. HA cleavage site
Single or multiple basic amino acids:
Susceptible to different host proteases
2. PB2 (polymerase) gene
Amino acid change (E627 > K) among
A/HK/97(H5N1) viruses --- mouse pathogenicity
3. NS1 gene
(type 1 IFNs and TNF-a antagonism)
Amino acid change (F92 > E or D?)
--- Pathogenicity in pigs
LPAI to HPAI in chickens?
Passages of a LPAI H7 virus in CEC resulted in a multi-basic cleavage motif in HA
1) Insertion of a gene fragment of host cellular 28s r-RNA into the cleavage site
2) Insertion of a fragment of the homologous NP gene into the cleavage site and/or a few point mutations near the cleavage site
A similar change was documented during natural transmission of a LPAI virus among chickens in Chile
R J Webby, et al. 2004

24. NS gene of Influenza A
NS1
NS2

25. Influenza A virus NS1 protein
RNA-binding domain
(1-73)
Effector domain
(73-237)
RNA-binding domain (19-38)
Nuclear localization signal (34-38, )
Nuclear export signal ( )
30 kDa subunit of Cleavage and polyadenylation specific factor (CPSF) binding site (186)
poly-A binding protein II (PABII) binding domain ( )
PDZ domain ligand ( )

26. Virulence & cytokine response Importance of NS1 in Influenza A virus
A D92E mutation in NS1 strongly affect the virulence of influenza virus, eg: the H5N1 avian flu virus (Seo et al. 2004) .
C-terminal PDZ domain ligand in NS1 act as a potential virulence determinant (Krug et al. 2006) .

27. NS1 gene E92 and del 80-84 may affect RNA binding affinity
H5N1 of Hong Kong outbreak
Nat.Struct.Mol.Biol. 2006
E92 and del may affect RNA binding affinity
Cytokine-resistance

28. PDZ ligand motif in NS1 as a potential virulence determinant
Large-scale sequence analysis of avian influenza isolates
Obenauer et al. science. 2006
1997
PDZ domain
Regulating the activity and trafficking of membrane proteins
Maintaining cell polarity and morphology
Organizing postsynaptic density in neuronal cells
2003
1918

29. 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).

30. PB2 gene

31. Schematic diagram of chimeric and single amino acid PB2 mutants, with their virulence in mice (MLD50)
mutation at position 627 in the PB2 protein influenced the outcome of infection in mice
Hatta M, et al. Science ;293(5536):

32. Mechanisms of pathogenesis of human H5N1 disease
Malik Peiris, et al. Clin Micro Rev 20:243, 2007

33. Generation of pandemic influenza virus strains
In this figure, let’s summarize the models for the generation of pandemic influenza virus strains. In the classical genetic reassortment model (A), avian and human viruses bind respectively to NeuAca2,3Gal and NeuAca2,6Gal (a2,3 and a2,6) linkages in the pig trachea, setting the stage for the emergence of a reassortant that infects lots of the human population. The segments in the center of each particle represent the viral genome. The black HA gene comes from an avian virus. In the adaptation model (B), avian viruses acquire the ability to replicate efficiently in humans during adaptation to the NeuAca2,6Gal linkage in pigs. This change is mediated by a mutation in the HA gene. Third, an avian influenza virus is transmitted directly to humans, where it reassorts with a human virus (C) or acquires the ability to recognize the NeuAca2,6Gal linkage after direct introduction from birds (D), leading to efficient replication in humans.
Nichol, et al. PNAS 2000

34. 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.

35. Vaccine of high pathogenic avian influenza virus
Current vaccines are produced from virus grown in fertile hens’ eggs and inactivated by either formaldehyde or B-propiolactone. They consist of whole virus, detergent-treated split product, or purified haemagglutinin and neuraminidase surface antigen formulations of the three virus strains currently recommended by WHO. About 50 countries have government-funded national immunisation programs. However, when trying to make vaccine of H5N1 influenza virus A, the virus strain was too virulent for eggs to survive and replicate in them. In order to decrease its virulence, a scientist remove the additional basic amino acid of high pathogenic virus H5, then other protein genome was came from H1N1 virus strain. This vaccine was in clinical trial, but the efficiency of the recombinant virus vaccine still need to be determinated.
Hoffmann, et al. 2000