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Avian Influenza

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, C Genome consists of 8 single-stranded RNA segments

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Avian Influenza

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  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 viruses B H3N8 H3N2 H2N2 H2N? H1N1 H1N1 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 Year Hampson, et al. MJA 2006

  4. Pandemics in this century Year Virus subtype Deaths 1918-1919Spanish " flu" (H1N1) 20-40 million 1957-1958Asian " flu " (H2N2) 1-2 million 1968-1969Hong Kong " flu " (H3N2) 700,000 1977-1978Russian " flu " (H1N1)? Benign pandemic

  5. Influenza virus A genome

  6. Influenza virus A HA and NA Hemagglutinin(HA) Neuraminidase(NA) • Allows for penetration through RNPs mucous layer to epithelial cells • Facilitates release of virions by cleaving sialic acid residues thus preventing aggregation • 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

  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 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 - 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

  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 Total 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

  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. 2006. 5;350(2):258-68.

  20. Definition of avian influenza • Intravenous pathogenicity index(IVPI) in 4-8 week-old chickensgreater than 1.2, or cause more than 75% mortality within 10 daysor nucleotidesequencing of H5 or H7 has demonstrated the presence ofmultiple basic amino acidsat the cleavage site of the haemagglutinin. • New proposed definition: IVPI in 6-week-old chickens greater than 1.2 orany influenza A virus of H5 or H7 subtype.

  21. 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. Virus Tissue Tropism Diseases in Chickens Structure of HA Responsible Proteases LPAI Respiratory & Alimentary Tractｓ ‘Epithelial cells’ Subclinical Localized Infection Specific Secretory Protease Trypsin Tryptase Clara FXa Plasmin HA1 HA2 - - - - - - R / - - - HA1 HA2 R R R R E K R / - - - HPAI All tissues and organs Fowl Plague Fatal Systemic Infection Ubiquitous Protease present in Golgi Body Furin Plasmin Avian Influenza Viruses R J Webby, et al. 2004

  24. 27 57 530 718 864 NS1 NS2 NS gene of Influenza A

  25. 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) poly-A binding protein II (PABII) binding domain (223-237) PDZ domain ligand (228-231)

  26. Virulence & cytokine responseImportance 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 H5N1 of Hong Kong outbreak Nat.Struct.Mol.Biol. 2006 • E92 and del 80-84 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. 2001. 7;293(5536):1840-2.

  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 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 Hoffmann, et al. 2000

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