VIZIER CONFERENCE

Marseille, 27.04.2007

Molecular Mechanisms of
Pathogenicity and Interspecies
Transmission of Avian Influenza Virus

Hans Dieter Klenk

Institut für Virologie
Philipps Universität Marburg
Germany
The Impact of the Spanish Influenza 1918

U.S. Life Expectancy

By age
70

60

50

40

30
20-40 million deaths worldwide 1900 ‘30 ‘50 ‘70 ‘90
 Influenza A Virus

PB1
PB2
PA

HA

NP

Orthomyxoviridae NA

Segmented,
M1
negative stranded
RNA genome M2

NS1

NS2
 Life Cycle of Influenza Virus
Receptor binding

Neuraminic acid

Endocytosis

RNP, M1
Budding
mRNA

Fusion Translation of
internal proteins
mRNA synthesis
RNA replication
RNP formation

Insertion of envelope proteins

into plasmamembrane

Translation and processing

of envelope proteins
 Influenza – a zoonosis
Major natural reservoir of influenza viruses: feral aquatic birds
Contains many viruses defined by 16 HA and 9 NA subtypes

Order Anseriformes (waterfowl) (ducks, geese and swans)

Order Charadriiformes (shorebirds and gulls)

Interspecies transmission of influenza viruses

- Occasional transmissions with

H13
H3 outbreaks of various severity
H4
H1
H3 H1´
- Transmission may involve
H7 H3 intermediate host

- On rare occasions, adaptation to

new species and establishing of
stable virus lineages

H5 H1 - Transmission to humans of
H2
H6 antigenically new virus can initiate
H7 H3
H9 B?
pandemic
 Pathogenicity of Influenza A Viruses
Human Influenza Avian Influenza
Apathogenic Pathogenic
(H1-H16) (H5, H7)

Enteric Infection

E. Munch
Self portrait Fowl Plague
1918

Respiratory infection
 Outbreaks of Highly Pathogenic Avian Influenza
Year Subtype Country Year Subtype Country

1878-1935 * Italy-enzootic 1985 H7N7 Australia

1890-1930 * Germany-enzootic 1991 H5N1 England
1922 * England 1992 H7N3 Australia
1923-1945 * Egypt-enzootic 1994-95 H5N2 Mexico
1924-1925 * USA 1995 H7N3 Australia
1927 * Indonesia 1995 H7N3 Pakistan
1929 * England 1996-ongoing H5N1 Asia, Europe, Africa
1929 * USA 1997 H7N4 Australia
1959 H5N1 Scotland 1997 H5N2 Italy
1963 H7N3 England 1999 H7N1 Italy
1966 H5N9 Canada 2002 H7N3 Chile
1975 H7N7 Australia 2003 H7N7 Netherlands
1979 H7N7 England 2004 H7N3 Canada
1983-84 H5N2 USA 2004 H5N2 USA
1983-84 H5N8 Ireland

*presumably H7 Alexander, 1986

Subbarao et al., 2006
 Avian Influenza Virus Infections in Man

Year Country Virus Cases Death Disease Man-man

Transmission
1996 UK H7N7 1 0 Conjunctivitis -
1997 Hongkong H5N1 18 6 Influenza (ARDS) -
1999 HK (China) H9N2 7 0 Influenza -
2003 Hongkong H5N1 2 1 Influenza (ARDS) -
2003 NL H7N7 83 1 Conjunctivitis (ARDS) +
2004/5/6 East Asia H5N1 ca. 300 ca.190 Influenza (ARDS) ?
 H5N1 April 2007

Outbreaks: Indonesia, Thailand, Vietnam

Japan, Nigeria, Egypt, Middle East, and others

since 2003: 291 human cases, 172 deaths in 12 countries

ca. 200 Mio. killed birds

Host Range of H5N1
Determinants of Host Specitivity and Pathogenicity
of Influenza-A-Viruses

Viral Components Mechanism Effect

Hemagglutinin Receptor specificity Host tropism, cell tropism

Proteolytic activation Organ tropism
Budding polarity Organ tropism
Antigenic variability Immune escape

Neuraminidase Receptor specificity Host tropism, cell tropism

Antigenic variability Immune escape

Polymerase Replication rate Host tropism

NS1 Interferon antagonism Immune suppression

Apoptosis antagonism Cell death
The Cleavage Site of HA Determines the Pathogenicity of Avian
Influenza Viruses

H1-H16 H5, H7

R
X
R
K/R
R
trypsin-
like protease furin

apathogenic pathogenic
virus virus
local systemic
infection infection

Klenk et al., Virology 1975 Kawaoka and Webster, PNAS 1988

Bosch et al., Virology 1981 Stieneke-Gröber et al., EMBO J. 1992
Garten et al., Virology 1981 Böttcher et al., J. Virol. 2006
Generation of Vaccine Strains by Genetic Manipulation
of HA Cleavage Site
Decreasing pathogenicity

Inactivated vaccines
R (pandemic H5N1 vaccine)
X
R
K/R
R trypsin-like
furin protease

Live vaccines

Stech et al., R V
Nature Medicine, 2005
elastase
trypsin-like
protease
 PROTEASES CLEAVING AT MONOBASIC
CLEAVAGE SITES

Plasmin Lazarowitz et al., Virology 56, 172 (1973)

Goto and Kawaoka, PNAS 95, 10224 (1998)

Factor X Gotoh et al., EMBO J. 9, 4189 (1990)

Tryptase Clara Kido et al., J. Biol. Chem. 267, 13573 (1992)

Novel trypsin-like lung proteases Böttcher et al., J. Virol. 80,

9896-9898 (2006)
TMPRSS2 (Transmembrane Protease,
Serine S1 Family Member 2)
catalytic domain

N- cyto. D TM LDLa SRCR Pro H D S -C

Cyto. D: cytoplasmic domain, TM: transmembrane domain, LDLa: LDL receptor class A domain,
SRCR: Group A Scavenger receptor domain, Pro: pro domain, Catalytic domain: serine protease domain.

• Type II transmembrane serine proteases (TTSPs)

• Trypsin-like protease
• Highly expressed in prostate and prostate cancer cells but also expressed in lung,
kidney and pancreas
• Involved in regulation of epithelial sodium channel (ENaC) important for airway
surface liquid regulation and so for mucociliary clearance
• Synthesized as a full-length protein of 70kDa
• Autocatalytic cleavage and secretion (in prostate cells)

Ø TMPRSS2 was cloned into a human expression vector

Crossing the Species Barrier
 The receptor specificity of influenza
A viruses is a host determinant

α
2,
6
-N
AN
A
α 2,3 - NANA

Avian viruses bind to α 2,3 – NANA Human viruses bind to α 2,6 – NANA
prevalent in avian tissues prevalent in human tissues
How are avian viruses
transmitted to humans?
 Cell tropism of human and avian influenza
viruses in human airway epithelium (HTBE cultures)
A/Memphis/14/96 (H1N1) A/mallard/Alberta/119/98(H1N1)

1. In human airway epithelium, human influenza viruses preferentially infect

non-ciliated cells, whereas avian influenza viruses preferentially infect ciliated
cells.
2.The different cell tropism of these viruses depends on their receptor
specificity and on predominant expression of 6-linked receptors on non-
ciliated cells and 3-linked ones on ciliated cells.
3.Ciliated cells are the entry site of avian influenza viruses into the human
respiratory tract Matrosovich et al., PNAS 2004
Adaption to a New Host
The earliest pandemic strains from 1918, 1957, and 1968
have adapted to human receptor (α 2,6-NANA) by
mutations in the RBS

α 2,3-NANA α 2,6-NANA α 2,6-NANA

Matrosovich et al., 2000

SC35 SC35M
H7N7 H7N7

Adaptation of an avian influenza virus

to a mammalian host

Gabriel et al., PNAS 2005

 SC35 SC35M
333T 701D 714S 333I 701N 714R
Reassortants
13L 678S
PB2 13P 678N
PB2
PB1 PB1
615K 615N
340G PA 340R PA
319N HA 319K HA
328A NP 328S NP
NA NA
Minigene-Based Activity Assay for Polymerase
Complexes of Recombinant Viruses

pPolI

10h later

293T
Luciferase
 Polymerase Activities in
avian cells mammalian cells
mRNA mRNA

RNA expression [%]

RNA expression [%] cRNA cRNA
vRNA vRNA

Relative Polymerase Activity [%]

0 100 200 300 400 500
0

M LD 50 [lo g 10 pfu]
2

Æ SC35M has a higher polymerase activity in mammalian cells than SC35 which correlates with
the increased pathogenicity of SC35M in mice
Æ SC35 has a higher polymerase activity in avian cells than SC35M which correlates with
higher pathogenicity of SC35 in chicken embryos
 Mutations observed in the SC35/SC35M polymerase resemble
mutations thought to promote H5N1 adaptation to man
___________________________________________________________________

PB1 Leu 13 Pro PB2 Glu 627 Lys

(Ser 678 Asn) Asp 701 Asn
Ser 714 Arg(Ile)

PA Lys 615 Asn(Arg) NP Asn 319 Lys

___________________________________________________________________

Gabriel et al., PNAS 102, 18590-95 (2005)

de Jong et al., Nature Med. 12, 1203-1207 (2006)
 SC35 SC35M
H7N7 H7N7

Adaptation of an avian influenza virus

to a mammalian host

Host
Host
factors
factors
PB1 PA PB2
NP
Host Host
factors factors

Adaptation to mice is mediated by

mutations in the polymerase

Working hypothesis: Polymerase mutations mediate adaptation to host factors

Gabriel et al., PNAS 2005
 PB2 host adaptation
residues 701 and 714
interact with bipartite
NLS (736-KRKR-739
…752-KRIR-755) that
mediates binding to
importin α5

Tarendeau et al.,
Nature Struct. Mol. Biol., 2007
Conclusions
_______________________________________________________________________
Hemagglutinin and polymerase are key determinants of interspezies transmission, host
adaptation, and pathogenicity

Host adaptation
- HA adapts to receptors of the new host by mutations in the receptor binding site:
altered receptor specificity
- Mutations in the polymerase mediate adaptation to host factors:
enhanced replication efficiency

Pathogenicity
- Proteolytic cleavability of HA determines spread of infection
- Interaction of polymerase with host factors modulates replication efficiency

Host adaptation mutations: markers of emerging pandemic virus.

_______________________________________________________________________
ACKNOWLEDGEMENTS

Gülsah Gabriel
Jürgen Stech
Mikhail N. Matrosovich
Tatyana Y. Matrosovich
Eva Böttcher
Jennifer Uhlendorff
Björn Keiner
Wolfgang Garten

Institut für Virologie

Philipps-Universität
Marburg