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noisymaniacalBiotechnology

Feb 20, 2013 (4 years and 6 months ago)

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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

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

Smith GJ,
et al.

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

HA

NP

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.

(Z)

(Z+V)

(Z)

(Z)

(Z)

(Z+G)

(Z+G+W)

Genotypes of H5N1
influenza
reassortants in Asia
during 2003
-
2005

Chen
et al
., 2006 PNAS 103, 2845
-
2850.

Smith, G. J. D. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 16936
-
16941

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

FJ
-
like

GD06, QH
-
like, Mixed/VNM2

IDN

GY2

VTM

GY1

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

High viral replication: PA, PB1, PB2, NP

Host immune response: NS

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.

NA Stalk


poly
-
A binding protein II (PABII) binding domain (223
-
237)

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)

Nuclear export signal (137
-
146)

30 kDa subunit of Cleavage and polyadenylation specific factor
(CPSF) binding site (186)

PDZ domain ligand (228
-
231)

Inhibition of PKR activation and regulation of vRNA synthesis (123
-
127)

NS1 as IFN
-
α/β antagonist

Influenza A virus

IRF
-
3/IRF
-
7

+

P300 and
CREB
-
binding
protein

dsRNA

NS1

NS1

NS1

cytoplsam

IFN/ISRE

AAUAAA

Antiviral pre
-
mRNAs

AAUAAA

5’ cap

nucleus

NS1

CPSF

30k

PABII

X

X

NS1

NS1

NS1

AAUAAA

5’ cap

X

NS1

NS1

NS1

splicing

snRNA U6

PKR

RIG
-
I

NS1



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


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.

2006

1997

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).

Nat.Struct.Mol.Biol.

2006


E92 and del 80
-
84
may affect RNA binding
affinity



Cytokine
-
resistance


H5N1 of Hong Kong outbreak

NS1 gene

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)

Hatta M, et al. Science. 2001. 7;293(5536):1840
-
2.

mutation at
position 627 in
the PB2 protein
influenced the
outcome of
infection in mice