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 Ho

spital • National Health Research Institutes Tainan Virology Laborator

y for Diagnosis and Research • Department of Health, Taiwan Centers for Disease Control con

tract 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 IClade 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

GY2VTM

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 tropismHorimoto 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 antagonistInfluenza A virus

IRF-3/IRF-7

+P300 and

CREB-binding protein

dsRNA

NS1

NS1

NS1

cytoplsam

IFN/ISRE

AAUAAAAntiviral pre-

mRNAs

AAUAAA5’ cap AAAAA………

nucleus

NS1CPSF 30kPABII

X

X

NS1

NS1NS1

AAUAAA5’ cap

X NS1

NS1NS1splicing snRNA 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

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 inf

luenza virus, eg: the H5N1 avian flu virus (Seo et al. 2004) .

• C-terminal PDZ domain ligand in NS1 act as a potential vir

ulence determinant (Krug et al. 2006) .

• Amino acid 89 in the NS1 protein as being critical for bindi

ng to p85 (Hale et al. 2006).

• Phe-103 and Met-106 residues in NS1 is critical for CPSF b

inding (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