Genetic evolution of H5N1

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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 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. 2006.
3552174-2177.
4
Webster G, et al. N ENGL J MED. 2006.
3552174-2177.
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Genetic reassortment of H5N1 in Asia from 1999 to
2005
Peiris M. J. S. et al., 2007 Clin Microbiol Rev
20(2), 243267.
6
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. 5350(2)258-68.
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Clade I
Clade II, subclade I
Smith GJ, et al. Virology. 2006. 5350(2)258-68.
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Smith GJ, et al. Virology. 2006. 5350(2)258-68.
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Genotypes of H5N1 influenza reassortants in Asia
during 2003-2005
(Z)
(ZV)
(ZG)
(Z)
(Z)
(Z)
(ZGW)
Chen et al., 2006 PNAS 103, 2845-2850.
10
Emergence and predominance of an H5N1 variant in
China since late 2005

Smith, G. J. D. et al. (2006) Proc. Natl. Acad.
Sci. USA 103, 16936-16941
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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
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Phylogenetic analysis of H5N1 HA gene from
Europe, Africa, and Asia
Salzberg SL, et al. EID. 2007. 13(5)713-718.
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Salzberg SL, et al. EID. 2007. 13(5)713-718.
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Cell tropism HA, NA
Host immune response NS
H5N1 pathology/disease
High viral replication PA, PB1, PB2, NP
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HA cleavability determinates the tissue tropism
Horimoto T, et al. Nature Reviews of
Microbiology. 2005. 3591-600.
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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).

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The NA stalk was correlated with the efficiency
of virus replication
NA Stalk
Castrucci MR, et al. Journal of Virology. 1993.
67759-764.
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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)
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NS1 as IFN-a/ß antagonist
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-a/ß
  antagonist
• NS1 binds to snRNA U6, poly-A mRNA, CPSF, PABII
  and PKR against antiviral genes expression

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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
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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).

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NS1 gene
H5N1 of Hong Kong outbreak
Nat.Struct.Mol.Biol. 2006

• E92 and del 80-84 may affect RNA binding affinity
• Cytokine-resistance

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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.

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PB2 gene
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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. 7293(5536)1840-2.