Determinants of Pathogenicity and Host Range of … · Determinants of Pathogenicity and Host Range...
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Determinants of
Pathogenicity and Host
Range of Influenza Viruses
Hans Dieter Klenk
Institut für Virologie
Philipps Universität Marburg
Summer School on Influenza
Siena, August 1-5, 2011
Emerging Infections: Zoonoses
Virus Disease Year Natural Host
Influenza A Virus Spanish Influenza 1918 Birds
Asian Influenza 1957
Hongkong Influenza 1968
Mexican Influenza 2009
„Bird Flu“ 1997
Marburg Virus Hemorrhagic Fever 1967 Bats
Ebola Virus Hemorrhagic Fever 1976 Bats
HIV AIDS 1981 Apes
Hendra Virus Encephalitis 1994 Bats
Nipah Virus Encephalitis 1996 Bats
Sin Nombre Virus Hemorrhagic Fever 1993 Mice
SARS Coronavirus SARS 2002 Bats
Man (Marburg-Virus)
ManMan
AnimalAnimal
Zoonoses: 2 types
(Influenza-Virus)
Zoonoses
Type 1 High case-fatality rate (< 100%)Local and temporal restriction of outbreaksRestricted human to human transmissionVirus does not adapt to man
Type 2 High case numbersPandemic spreadHuman to human transmissionVirus adapts to man
Discovery of Influenza-Viruses
W.S. Smith, C.H. Andrewes, P.P. Laidlaw
1901E. Centanni
1955W. Schäfer
1931R.E. Shope
1933
human
avianporcine
Influenza – a Zoonosis
Influenza – a zoonosisMajor 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)
- Occasional transmissions with
outbreaks of various severity
- Transmission may involve
intermediate host
- On rare occasions, adaptation to
new species and establishing of
stable virus lineages
- Transmission to humans of
antigenically new virus can initiate
pandemic
Interspecies transmission of influenza viruses
H3
H7
H1
H1´
H3
H5
H6
H7
H9
H1
H2
H3
B ?
H3
H4
H13
Pathogenicity of Influenza A Viruses
Human Influenza Avian Influenza
E. Munch
Self portrait
1918
Respiratory infection
Enteric Infection
Pathogenic
(H5, H7)
Apathogenic
(H1-H16)
Fowl Plague
1900 1910 1920 1930 1940 1950 1960 19801970 1990 2000 2010
A/H1N1
A/H2N2
A/H5N1
A/H1N1
A/H3N2
A/H1N1
Human Influenza A Outbreaks
The Impact of the Spanish Influenza 1918
20-40 million deaths worldwide
70
60
50
40
30
1900 „30 „50 „70 „90
U.S. Life ExpectancyBy age
Influenza H5N1 virus causes severe multisystem disease in chickens and humans
• pneumonia
• severe systemic infection
• diarrhoea
• Encephalitis
• Lymphopenia, thrombocytopeni, hypoglycaemia
• death
05/02/04 10/02/04
Jeremy Farrar, Oxford, HCMC
1900 1910 1920 1930 1940 1950 1960 19801970 1990 2000 2010
?1946
“non-pandemic”
19571977
Brisbane/59/2007
Human
Classical swine
Fort Dix
Human H3N2
avian
Triple reassortant
Eurasian swine
avian
1918 Pandemic
Human and swine H1N1 viruses
New H1N1
Pig – the mixing vessel
C. Scholtissek, 1990
Wilhelm Busch, 1832-1908
H1N1 Pandemic 2009, Europe
Deaths 500 – 4.000
ARDS-cases 5000 – 75.000
ECDC, Jan. 2010
ARDS/H1N1v Patients UKGM Nov. 2009 – Jan. 2010
Patient Sex Age Comorbidity Coinfection IC Influenza- Course
Treatment therapy
I.N. w 21 - - Ventilation Oseltamivir Discharged
K.C. m 40 - - ECMO Oseltamivir Discharged
K.G. w 56 ? - ECMO Oseltamivir
S.M. m 53 cor. sclero. - ECMO Zanamivir. Discharged
A.O. m 51 Lung fibrosis Klebsiella ECMO Zanamivir Lungtranspl.
H.K. w 52 - Klebs./HSV1 ECMO Zanamivir Discharged
M. Sch.w 19 - - ECMO Zanamivir Discharged
M. St. m 21 - Candida ECMO Zanamivir Discharged
A. A. m 45 - mdr. Pseudom. ECMO Zanamivir
L. M. w 30 - - ECMO Zanamivir Discharged
T. S. w 39 - - ECMO Zanamivir Discharged
V. E. m 39 - HSV1 ECMO Zanamivir Discharged
J. E. w 25 - - ECMO Zanamivir Discharged
Courtesy: S. Herold, Gießen – C. Aepinus,
Marburg
Extra-Corporal Membrane Oxygenation
Uniklinik Gießen, Jan 2010
H1N1v
HA
NA
M1
PB1
PB2
PA
NS2
NP
M2
NS1
PB1 F2
Orthomyxoviridae
Segmented, negative stranded RNA
genome
Influenza A Virus
RNP, M1
mRNA
Budding
Endocytosis
Fusion
Translation and processing
of envelope proteins
Translation of
internal proteins
Insertion of envelope proteins
into plasmamembrane
mRNA synthesis
RNA replication
RNP formation
Receptor binding
Life Cycle of Influenza Virus
Neuraminic acid
The Polymerase
- Role in Host Adaptation -
Gabriel et al., PNAS 2005
Gabriel et al., J Virol 2007
NPPB1 PB2
PA
Adaptation to Mice is Mediated
by Mutations in the
Polymerase Complex
Host
factors
Host
factors
Host
factors
Host
factors
Adaptation of an Avian Influenza Virus to a Mammalian Host
SC35H7N7
SC35MH7N7
SC35-PB2
NLS
D701 NLSD701
Importin a1
SC35M-PB2
Importin a1
NLS
N701
Cytoplasm Nucleus
NLSN
701
SC35-PB2
Importin a1
SC35M-PB2
Importin a1
Cytoplasm Nucleus
NLS NLS
NLS NLS
Differential interaction with importin a regulates nuclear import of PB2
Mammalian Cells Avian Cells
Gabriel et al., PLoS Pathogens, 2008
Effects of Importin-α Knockout and Silencing on Replication and Pathogenicity
Differential Use of Importin- α Isoforms Governs Cell Tropism and Host Adaptation of Influenza Viruses
α1 α3 α4 α5 α7
SC 35 = = =
SC 35 M = = =
A/FPV/Rostock/1/34 (H7N1) = = =
A/Thai/KAN-1/04 (H5N1) (human iso.) = = =
A/Victoria/3/75 (H3N2) = = =
A/Sachsen-Anhalt/101/09 (H1N1 pdm) = =
Gabriel et al., Nature Com., 2011
Compartment Borders are Favorite Cellular Sites for Adaptation Processes
Neuraminic acid
RNP, M1
mRNA
Budding
Endocytosis
Fusion
Translation and processing
of envelope proteins
Translation of
internal proteins
Insertion of envelope proteins
into plasmamembrane
mRNA synthesis
RNA replication
RNP formation
Receptor binding
- Receptor Binding of HA at Plasma Membrane
- Importin Binding of Pol at Nuclear Envelope
Conclusion
PB1 PB2 PA NP Adaptive mutations ---------- ------------------------------------ --------- ----------- (avian-mammalian) L13P E627K D701N S714R(I) K615R N319K 2009 A(H1N1) 13P E627 D701 S714 K615 N319
Host Range Markers of New Influenza A (H1N1) Polymerase
Determinants for avian specificity have been preserved
Adaptive PB2 mutations increase polymerase
activity of A/Hamburg/05/2009 (H1N1)
10
100
397
335 293
627
761
565
906
0
200
400
600
800
1000
1200
moc
kW
T62
770
171
4
627-
701
627-
714
701-
714
Tripel
rela
tive a
cti
vit
y (
%)
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14
we
igh
t lo
ss
[%
]
days p.i.
PBS
H1N1v
H1N1v-PB2-E627K
H1N1v-PB2-D701N
H1N1v-PB2-S714I
H1N1v-PB2-E627K-D701N
H1N1v-PB2-E627K-S714I
H1N1v-PB2-D701N-S714I
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
su
rviv
al [%
]
days p.i.
PBS
H1N1v
H1N1v-PB2-E627K
H1N1v-PB2-D701N
H1N1v-PB2-S714I
H1N1v-PB2-E627K-D701N
H1N1v-PB2-E627K-S714I
H1N1v-PB2-D701N-S714I
Mouse pathogenicity of PB2 mutants of H1N1v
Summary
- The influenza virus polymerase is an important determinant
of host range, tissue tropism and pathogenicity
- Interspecies transmission depends on adaptation of
polymerase subunits to importin-α
- The H1N1v polymerase has retained genetic markers of
avian viruses
- Introduction of mammalian markers enhances
pathogenicity for mice
- The pathogenetic and pandemic potential of H1N1v may
not be exhausted yet
Effector DomainRBD
20 41 108 110 126 217
ISG-15 K
Effector DomainRBD
217219
SUMO1 K
221
ISG-15 NS1 SUMO-1
Importin a binding and nuclear
transport of NS1 disrupted
Dimerization of NS1 impaired
Virus replication reduced
Chen et al., PNAS 2010
Tang et al., J. Immunol. 2010
Stability of NS1 enhanced
Virus replication enhanced
Xu et al., J. Virol. 85, 1086-1098 (2011)
ISG-15ylation and SUMOylation of NS1
219
C.
Strain dependent SUMOylation of
NS1
A, B: Truncated C-terminus of NS1 of 2009 pdm H1N1 is not SUMOylated
C: C-Terminal elongation does not restore SUMOylation
(De)-SUMOylation of NS1: Effect on Pathogenicity of 2009 pdm H1N1?
The Hemagglutinin
- Role in Pathogenesis and Host Specificity -
The Cleavage Site of HA Determines the Pathogenicity of Avian Influenza Viruses
R
H1-H16
R
X
K/R
apathogenic
virus
pathogenic
virus
local
infection
systemic
infection
trypsin-
like proteasefurin
R
H5, H7
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
Serine Proteases HAT and TMPRSS2
TMPRSS2 (Transmembrane protease, serine S1 family member 2)
HAT (Human airway trypsin-like protease)
-CH D SSRCRLDLRAN- TM
S - S
H D SN- SEA
S - S
-CTM
Trypsin-like substrate specificity
Expression in human airways
(HAT: predominantely trachea; TMPRSS2: nasal cavity, trachea, bronchi, lung)
Type II transmembrane serine proteases
Synthesized as zymogens, autocatalytic activation
Physiological role in the airway epithelium remains to be determined
TMPRSS2
HAT
??
Subcellular localization of HA cleavage
HAT cleaves HA at the cell surface
TMPRSS2 cleaves HA intracellularly
HAT activates newly synthesized HA and
HA of incoming virus
TMPRSS2 cleaves newly synthesized HA
but is not capable to activate HA of
incoming virus
Cleavage of HA by TMPRSS2 and HAT
differs in subcellular localisation and can
take place at different steps of viral
replication:
nicht
infiziert
S. aureusInfluenzavirus
S. aureus
Influenzavirus
Bakterielle Protease und Influenzapneumonie
Tashiro et al., Nature 1987
Inhibition of Viral Spread of A/Hamburg/5/2009 (H1N1) by Peptidomimetics
I-1: Bzls-dArg-Pro-4-amidinobenzylamid
I-2: Bzls-dCha-Pro-4-amidinobenzylamid
I-3: Bzls-dCha-Lys(Dec)-4-amidinobenzylamid
Bzls = Benzylsulfonyl
Cha = Cyclohexylalanin
Dec = decanoyl
- + + + + Dox
I-1 I-2 I-3
A/H
am
bu
rg/0
9V
SV
MDCK-
HAT
MDCK-
TMPRSS2
MDCK-
TMPRSS2
Inhibitor
Böttcher-Friebertshäuser et al. (2010), J Virol
S
HN
O O
N
O
NH
NH2HN
O HN NH2
NH
S
HN
O O
N
OO H
N NH2
NH
S
HN
O O
NH
OO H
N NH2
NH
NH
O
CH38
I-2
I-3
I-1
Viral spread in HAT-expressing cells suppressed by
all applied inhibitors
Inhibition of viral spread in TMPRSS2-expressing
cells seems to require cellular uptake of the inhibitor
Virus spread in MDCK cells is retarded and reduced to ~300 fold by inhibitors
R
10µM 25µM 50µM Ø
MI-0299
MI-0701
MI-0702
Inhibitor
Lu, Steinmetzer & Garten, unpublished
5x107 1,5 x106 4,5 x108
MI-299 MI-701
PFU/ml
Plaque reduction test
Influenzavirus A/chick/Rostock/ 34 (H7N1)
Inhibitors
No inhibitor
control
FPV
Spread
immuno-
Stained
18 h p.i.
Time (hrs)
Δ
10
30
50
120
0 10 20 30 40 50 60 70
Δ
ΔΔ
Re
lea
se
d v
iru
s p
art
icle
s
MI-299
MI-701
w/o
HAU
25µM
Inhibition of Fowl Plague Virus (FPV) Replication by Peptidomimetics
Generation of Vaccine Strains by Genetic Manipulation of HA Cleavage Site
Decreasing pathogenicity
Inactivated vaccines
(pandemic H5N1 vaccine)
Stech et al.,
Nature Medicine,
2005
R
trypsin-like
protease
RX
K/RR
furin
Live vaccines
R
trypsin-like
protease
V
elastase
RX
K/RR
furin
V
elastaseGabriel et al.
Vaccine 2008
R
R
Receptor specificity of H1N1v
HA polymorphism in position 222 (225, H3 numb.)
- Major variant, 222D. Substitutions G,E,N.
- 222G found in 7-10% of sequences in fatal and severe cases, but not in
clinically mild cases. The mutants seem to occur sporadically with no
evidence of sustained transmission.
- 222E – No apparent correlation with disease severity; transmissible
virus.
- 222N – Not enough data.
Amino acid 222
- Correlates with the virus host species. Avian viruses have 222G, human and
swine viruses have 222D/E. Propagation of human viruses in hen‟s eggs often
leads to mutations D222G/N.
- Mutation D222G increases binding to 3-linked receptors of human viruses
(Gambaryan et al., 1997,1999; Glaser et al., 2005; Stevens et al., 2006), including
H1N1pdm (Yang, Carney & Stevens, PLoS Curr Influenza 2010).
- Mutation D222G decreased airborne transmission of the 1918 virus in ferrets
(Tumpey et al., 2007).
Can mutations D222G/E in H1N1pdm
change viral cell tropism and replication
efficiency in human respiratory tract ?
Human and avian viruses target different types of
cells
Seasonal human virus Avian virus
<5 % of infected ciliated cells >70
Cell tropism of H1N1pdm in HTBE cultures
222D: 222G:
Moldova/G186/09, Cyprus/S2487/09 Lviv/N6/09 (fatal),
Hamburg/5/09 Norway/3206-3/09 (fatal)
222E: Hamburg/5/09-e (egg-derived)
Dakar/37/09
3-5 % of infected ciliated cells 20-30
Receptor specificity, glycoarraysD222G mutation increases binding to 3-linked Sia
2-6
2-3
2-6
2-3
- Mutation D222G alters receptor specificity
and cell tropism of H1N1v in human
respiratory epithelium
- Mutation D222G may cause (or reflect
severe disease resulting form H1N1v
replication in the LRT (Shinya et al., 2006; van Riel et al., 2007; Nell et al, 2010)
- Position 222 mutants and other mutations
with altered receptor specificity have to be
closely monitored
Conclusions
ACKNOWLEDGEMENTS
University of Tokushima
Hiroshi Kido
Yuushi Okumura
Universität Marburg
Joanna Baron
Volker Czudai
Björn Keiner
Folker Schwalm
Wolfgang Garten
Eva Böttcher-Friebertshäuser
Catharina Freuer
Carolin Tarnow
Mikhail Matrosovich
Tatyana Matrosovich
Jennifer Uhlendorff
Stephan Becker
Markus Eickmann
Torsten Steinmetzer
Heinrich Pette Institut Hamburg
Gülsah Gabriel
Kawasaki Medical
School
Masanobu Ohuchi
Imperial College
London
Ten Feizi
Yan Lin
Robert A. Childs
MRC NIMR Mill Hill
Alan Hay
Stephen Warton
Rodney Daniels
Deutsche
Forschungsgemeinschaft
SFB 593, SPP 1190
European Commission
STREP FLUPOL
Pasteur Institute Shanghai
Ke Xu
Bing Sun
Universität Würzburg
Christoph Klenk
University of Oxford
George Brownlee
Ervin Fodor