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Genetic evolution of H5N1 Jen-Ren Wang, Ph. D. Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University Department of.

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Presentation on theme: "Genetic evolution of H5N1 Jen-Ren Wang, Ph. D. Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University Department of."— Presentation transcript:

1 Genetic evolution of H5N1 Jen-Ren Wang, Ph. D. Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University Department of Pathology, National Cheng Kung University Hospital National Health Research Institutes Tainan Virology Laboratory for Diagnosis and Research Department of Health, Taiwan Centers for Disease Control contract Virology Laboratory

2 World Health Organization 18/Mar/2008

3 Webster, et al. N ENGL J MED :

4 Webster G, et al. N ENGL J MED :

5 Genetic reassortment of H5N1 in Asia from 1999 to 2005 Peiris M. J. S. et al., 2007 Clin Microbiol Rev 20(2), 243–267.

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

7 Clade I Clade II, subclade I Smith GJ, et al. Virology ;350(2):

8

9 (Z) (Z+V) (Z) (Z+G) (Z+G+W) Genotypes of H5N1 influenza reassortants in Asia during Chen et al., 2006 PNAS 103,

10 Smith, G. J. D. et al. (2006) Proc. Natl. Acad. Sci. USA 103, Emergence and predominance of an H5N1 variant in China since late 2005 Collection dateNo. of FJ-like viruses 2005 July–September1/33 (3) October–December72/136 (53) 2006 January–March90/113 (80) April–June103/108 (95) Total266/390 (68)

11 Smith, G. J. D. et al. (2006) Proc. Natl. Acad. Sci. USA 103, Antigenetic analysis of different H5N1 lineages FJ-like GD06, QH-like, Mixed/VNM2 IDN GY2 VTM GY1

12 Phylogenetic analysis of H5N1 HA gene from Europe, Africa, and Asia Salzberg SL, et al. EID (5):

13

14 H5N1 pathology/disease Cell tropism: HA, NA High viral replication: PA, PB1, PB2, NP Host immune response: NS

15 HA cleavability determinates the tissue tropism Horimoto T, et al. Nature Reviews of Microbiology :

16 Effect of HA and NA on replication of influenza virus Influenza viruses bind to sialic acid present on cell surface through the receptor-binding site in the HA molecules followed by receptor-mediated endocytosis during viral entry (Hanson et al., Virology 1992). The NA cleaves the Neu5Ac from the HA molecule to release the progeny virus from the cell membrane and to prevent aggregation of progeny virions (Rogers et al., Virology 1989). This NA enzymatic activity, however, also cleaves the receptor from the target cells. Therefore, the balance between the receptor-binding activity of the HA and the neuraminidase activity of the NA is critical for efficient virus replication in host cells (Kaverin et al., Virology 1998).

17 The NA stalk was correlated with the efficiency of virus replication Castrucci MR, et al. Journal of Virology : NA Stalk

18 poly-A binding protein II (PABII) binding domain ( ) 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) PDZ domain ligand ( ) Inhibition of PKR activation and regulation of vRNA synthesis ( )

19 NS1 as IFN-α/β antagonist Influenza A virus IRF-3/IRF-7 + P300 and CREB-binding protein dsRNA NS1 cytoplsam IFN/ISRE AAUAAA Antiviral pre- mRNAs AAUAAA5’ cap AAAAA…… … nucleus NS1 CPSF 30k PABII X X NS1 AAUAAA5’ cap X NS1 splicingsnRNA U6 PKR RIG-I X NS1 X NS1 binds to dsRNA and RIG-I as IFN-α/β antagonist NS1 binds to snRNA U6, poly-A mRNA, CPSF, PABII and PKR against antiviral genes expression Yuan L. et al., Virology, 1995 Zhongying C. et al., EMBO, 1999 Yun Q. et al., J.Virol., 1994 Nemeroff M. et al., Mol.Cell., 1998 Mibayashi M et al., J. Virol., 2007

20 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

21 Importance of NS1 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). Amino acid 89 in the NS1 protein as being critical for binding to p85 (Hale et al. 2006). Phe-103 and Met-106 residues in NS1 is critical for CPSF binding (Kochs et al. 2007).

22 Nat.Struct.Mol.Biol  E92 and del may affect RNA binding affinity Cytokine-resistance H5N1 of Hong Kong outbreak NS1 gene

23 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). Majority of avian viruses have PB2 E627 except Qinghai lineage, it may be one adaptation of virus to mammalian host.

24 PB2 gene

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


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