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

World Health Organization 18/Mar/2008

Webster, et al. N ENGL J MED. 2006. 355:2174-2177.

Webster G, et al. N ENGL J MED. 2006. 355:2174-2177.

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

Phylogenetic relationships of the HA and NP genes of influenza A viruses isolated in Indonesia and Vietnam HA NP Smith GJ, et al. Virology. 2006. 5;350(2):258-68.

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

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

Genotypes of H5N1 influenza reassortants in Asia during 2003-2005 (Z+V) (Z+G) (Z) (Z) (Z) (Z+G+W) Chen et al., 2006 PNAS 103, 2845-2850.

Emergence and predominance of an H5N1 variant in China since late 2005 Collection date No. of FJ-like viruses 2005     July–September 1/33 (3)     October–December 72/136 (53) 2006     January–March 90/113 (80)     April–June 103/108 (95) Total 266/390 (68) Smith, G. J. D. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 16936-16941

Antigenetic analysis of different H5N1 lineages GY1 IDN FJ-like GY2 VTM GD06, QH-like, Mixed/VNM2 Smith, G. J. D. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 16936-16941

Phylogenetic analysis of H5N1 HA gene from Europe, Africa, and Asia Salzberg SL, et al. EID. 2007. 13(5):713-718.

Salzberg SL, et al. EID. 2007. 13(5):713-718.

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

HA cleavability determinates the tissue tropism Horimoto T, et al. Nature Reviews of Microbiology. 2005. 3:591-600.

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).

The NA stalk was correlated with the efficiency of virus replication Castrucci MR, et al. Journal of Virology. 1993. 67:759-764.

Influenza A virus NS1 protein RNA-binding domain (1-73) Effector domain (73-237) 1 19 34 36 38 73 123-127 137 146 186 216 221 223 237 RNA-binding domain (19-38) Nuclear localization signal (34-38, 216-221) Inhibition of PKR activation and regulation of vRNA synthesis (123-127) 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)

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 5’ cap AAAAA……… nucleus CPSF 30k PABII X splicing snRNA U6 PKR RIG-I Nemeroff M. et al., Mol.Cell., 1998 Yuan L. et al., Virology, 1995 Zhongying C. et al., EMBO, 1999 Yun Q. et al., J.Virol., 1994 Mibayashi M et al., J. Virol., 2007 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

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

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).

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 80-84 may affect RNA binding affinity Cytokine-resistance

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.

PB2 gene

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.