Pathogenicity

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 70

By age

60

50

40

20-40 million deaths worldwide

30 1900

‘30

‘50

‘70

‘90

Influenza A Virus

PB1 PB2 PA HA NP

Orthomyxoviridae Segmented, negative stranded RNA genome

NA

M1 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

H3 H4 H3 H7

- Occasional transmissions with

H13

outbreaks of various severity H1 H1´ H3

- Transmission may involve intermediate host - On rare occasions, adaptation to new species and establishing of stable virus lineages

H5 H6 H7 H9

H1 H2 H3 B?

- Transmission to humans of antigenically new virus can initiate pandemic

Pathogenicity of Influenza A Viruses Human Influenza

Avian Influenza Apathogenic (H1-H16)

Pathogenic (H5, H7)

Enteric Infection

E. Munch Self portrait 1918

Fowl Plague

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

1996 UK 1997 Hongkong 1999 HK (China) 2003 Hongkong 2003 NL 2004/5/6 East Asia

Virus

Cases

Death

Disease

Man-man Transmission

H7N7 H5N1 H9N2 H5N1 H7N7 H5N1

1 18 7 2 83 ca. 300

0 6 0 1 1 ca.190

Conjunctivitis Influenza (ARDS) Influenza Influenza (ARDS) Conjunctivitis (ARDS) 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 Proteolytic activation Budding polarity Antigenic variability

Host tropism, cell tropism Organ tropism Organ tropism Immune escape

Neuraminidase

Receptor specificity Antigenic variability

Host tropism, cell tropism Immune escape

Polymerase

Replication rate

Host tropism

NS1

Interferon antagonism Apoptosis antagonism

Immune suppression Cell death

The Cleavage Site of HA Determines the Pathogenicity of Avian Influenza Viruses H1-H16

H5, H7

R trypsinlike protease

R X K/R R furin

apathogenic virus

pathogenic virus

local infection

systemic infection

Klenk et al., Virology 1975 Bosch et al., Virology 1981 Garten et al., Virology 1981

Kawaoka and Webster, PNAS 1988 Stieneke-Gröber et al., EMBO J. 1992 Böttcher et al., J. Virol. 2006

Generation of Vaccine Strains by Genetic Manipulation of HA Cleavage Site Decreasing pathogenicity

R X K/R R

Inactivated vaccines (pandemic H5N1 vaccine)

R trypsin-like protease

furin

Live vaccines Stech et al., Nature Medicine, 2005

R trypsin-like protease

V elastase

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 A AN -N

α 2,3 - NANA

Avian viruses bind to α 2,3 – NANA prevalent in avian tissues

Human viruses bind to α 2,6 – NANA 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 nonciliated 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

Matrosovich et al., 2000

α 2,6-NANA

SC35

SC35M

H7N7

H7N7 Adaptation of an avian influenza virus to a mammalian host

Gabriel et al., PNAS 2005

SC35 333T 701D 714S 13L

678S

615K 340G 319N 328A

PB2 PB1 PA HA NP NA

SC35M 333I 701N 714R 13P

678N

615N 340R 319K 328S

PB2 PB1 PA HA NP NA

Reassortants

Minigene-Based Activity Assay for Polymerase Complexes of Recombinant Viruses

pPolI

10h later

293T

Luciferase

mRNA cRNA vRNA

RNA expression [%]

RNA expression [%]

Polymerase Activities in avian cells mammalian cells mRNA cRNA vRNA

Relative Polymerase Activity [%] 0

100

200

300

400

500

0

M LD 50 [lo g 10 pfu]

1 2 3 4 5 6 7

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

PA

Leu 13 Pro (Ser 678 Asn)

Lys 615 Asn(Arg)

PB2

NP

Glu 627 Lys Asp 701 Asn Ser 714 Arg(Ile) 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 factors

NP

Host factors

PB1 PA PB2 Host factors

Host 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