Topics
- Variation of receptor specificity of influenza viruses in
different avian species
- Receptor specificity and cell tropism of H1N1pdm and of
its mutants associated with severe cases of infection
3- and 6-linked Sia receptors in different species
2-3
2-3
2-3
2-3
2-3
2-6
2-6
2-3
2-6
2-3
2-3
2-6
Characterization of receptor-binding specificity of avian
influenza viruses, 2002  present
Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana
Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria
Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al.,
Alexandra Gambaryan
M.P.Chumakov Institute of
Poliomyelitis, Moscow
Nikolai Bovin
Institute of Bio-organic
Chemistry, Moscow
A panel of sialylglycopolymers (Nikolai Bovin’s lab)
Receptor specificity of avian viruses
Fuc
Fuc
(H9 and H7)
Molecular features that determine binding of avian viruses to sulfated
and fucosylated receptors
Fuc
193
193
Su
222
Fuc
222
Sia
Sia
Ionic bond between sulfate (type II core) and
conserved lysine/arginine in position 193
(H5, H7, some H13)
No sterical conflicts between fucose and
amino acid in position 222
(gull viruses, some poultry viruses)
Crystal structure of H7 HA: Russell, Gamblin, Skehel et al.
- Receptor specificity of influenza viruses is not limited
to recognition of the type of Sia-Gal linkage.
- Receptor specificity of avian viruses is not uniform.
Viruses of gulls and poultry are closer to each other than
to duck viruses. This may facilitate virus exchange
between gulls and poultry.
- Expression of distinct 3-linked sequences varies
depending on the animal species, tissue and cell type.
Avian viruses may differ by their tissue and cell tropism
in humans.
- 6-linked Sia are present on epithelial cells in
galliformes; evolution of viruses in these species may
increase binding to receptors in humans.
Further studies are needed to
characterize role of receptor specificity
of avian viruses in their host-range,
tissue tropism and pathogenicity
Alan Hay
Ten Feizi
The neoglycolipid-based carbohydrate microarrays
(Ten Feizi lab, Imperial College London, UK)
- Sequence-defined natural and synthetic
oligosaccharides are coupled to synthetic lipid
to allow efficient immobilization on solid
supports.
- Neoglycolipids are spotted on nitrocellulosecoated glass slides.
- The lipid-linked probes have some lateral
mobility. Clustering of glycolipids allows their
optimal multivalent presentation that mimics
presentation at the cell surface in vivo.
Liu et al., Biol.Chem. 2009
Examples of glycan probes
Examples of binding profiles
Virus 1
2-3
Virus 2
2-6
2-8
2-3
2-6
2-8
80 sialic acid-containing oligosaccharide probes with differing backbone types, chain
lengths and branching patterns
2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors)
6 neutral probes (negative control; gray)
Binding profiles
H1N1pdm (Hamburg/5/09)
2-3
2-6
H1N1 seasonal (Memphis/14/96)
2-8
2-3
2-6
2-8
H3N2 pandemic (Aichi/2/68)
2-3
2-6
2-8
-H1N1pdm, similar to 1918, 1957 and 1968 pandemic viruses,
preferentially binds to 6-linked Sia
- H1N1pdm differs from seasonal H1N1 virus by a broader binding
specificity and by low-avidity binding to 3-linked Sia
No binding of H1N1pdm to 2-3-probes in several assays
1.
2.
Blue – 2-3 probes, red: 2-6 probes
Yang, Carney & Stevens, PLoS Curr Influenza 2010
Maines et al., Science 2009
Binding to the glycoarray of recombinant HA protein
2-3-fetuin
Kass, 1/uM Neu5Ac
3.
2-6-fetuin
50
40
30
20
10
0
Ham/09
(H1N1v)
Mem/96 (H1N1) HK/68
(H3N2) duck/98 (H1N1)
Binding of soluble probes, 3- and 6-fetuin, to the solid-phase immobilized virus
Neoglycolipid-based arrays:
Utilization of whole virus particles and clustered glycolipid probes
could facilitate detection of low-affinity polyvalent interactions
H1N1pdm
H1N1 seasonal
Biological significance of binding to 2-3 Sia ?
6- and 3-linked Sia in human respiratory tract
Binding:
Lectins 2-6 (green), 2-3 (red)
Viruses
Nasal and tracheo-bronchial epithelial
cells mainly express 6-linked Sia. Binding
to 6-linked receptors seems to be
essential for virus replication in the upper
respiratory tract and efficient human-tohuman transmission.
3-linked Sia are mainly present in
bronchioles and alveoli. Binding to 3linked receptors could facilitate virus
replication in the lower respiratory tract
and increase pathogenicity.
Shinya et al. 2006
van Riel et al., 2007
Can low-avidity binding of H1N1pdm
to 3-linked Sia contribute to viral
capacity to cause
severe disease in humans ?
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.
Two of five HA sequences
from the victims of the
1918 pandemic had
mutation D222G
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 ?
Cultures of differentiated human tracheo-bronchial epithelial
cells (HTBE) - an in vitro model of human airway epithelium
Immuno-staining of cilia in fixed culture
Primary airway epithelial cells grow on membrane
supports at air-liquid interface
Cross-section of 6-wk-old culture
Beating of cilia in live culture
6- and 3-linked sialic acids in HTBE cultures
2-6
Sambucus nigra agglutinin
SNA
2-3
Maackia amurensis lectin
MAL-1
Expression pattern agrees with that in vivo (Baum and Paulson, 1990;
Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007)
Human and avian viruses target different types of cells
Red, viral antigen; gray, cilia of ciliated cells
Seasonal human virus
Avian virus
<5 % of infected ciliated cells >70
Cell tropism of H1N1pdm in HTBE cultures
222D:
Moldova/G186/09, Cyprus/S2487/09
Hamburg/5/09
222E:
Dakar/37/09
3-5
% of infected
222G:
Lviv/N6/09 (fatal),
Norway/3206-3/09 (fatal)
Hamburg/5/09-e (egg-derived)
ciliated cells
20-30
Receptor specificity, glycoarrays
D222G mutation increases binding to 3-linked Sia
2-6
2-3
2-6
2-3
Binding to resialylated soluble 3- and 6-fetuin
D222G mutation decreases binding to 6-linked Sia
Tropism, % ciliated cells
Circulation in humans
3-5
Yes
20-30
No
Replication kinetics in HTBE cultures
Hamburg/5/09 (H1N1pdm) vs its D222G mutant
wt (222D)
D222G
*
*p=0.0001
Sharon Brookes
Ian Brown
Selection of receptor-binding variants during
replication and transmission in pigs
Inoculum:
Egg-grown A/California/7/09
wt + D222G + Q223R
wt + 222G
wt (222D)
Transmission
Direct infection
3 - 6 dpi
wt + 222G
222G
1 and 2 dpi
4 and 7 dpi
Middle lung
lobe tissues
Figure modified from
Brookes et al., 2010
Expression of 3- and 6-Sia in pigs is similar to that in humans
(van Riel et al., 2007; Van Poucke et al., 2010; Nelli et al., 2010)
Humans
Pigs
2,6 (green); 2,3 (red)
Shinya et al. 2006
Nelli et al. BMC Veterinary Research, 2010
I. Mutation D222E has marginal effect on receptor
specificity and does not affect viral cell tropism in HTBE
cultures. This is consistent with apparent unrestricted
circulation of the variant in humans and with lack of
correlation of 222E with severe disease.
II. Mutation D222G reduces viral replication efficiency in
HTBE cultures and prevents transmissibility in
experimentally infected pigs. These findings could
explain why the mutants do not transmit in humans.
III. Mutation D222G alters viral receptor specificity and
cell tropism in human epithelium, provides the mutant
with replicative advantage in the LRT of pigs.
Potential explanations of association of D222G
with severe disease:
- Mutation causes severe disease owing to propensity
of the mutant to reach and infect the LRT
or/and
- Mutation emerges as a consequence of severe
disease owing to the virus replication in the LRT
It is important to closely monitor position 222
mutants and other potential mutants with
altered receptor specificity and cell tropism
Institute of Virology, Philipps University, Marburg, Germany
Tatyana Matrosovich
Hans-Dieter Klenk
Markus Eickmann
Jennifer Uhlendorff
M.P.Chumakov Institute of
Poliomyelitis, Moscow, Russia
Alexandra Gambaryan
Collaborators
Carbohydrate laboratory,
Institute of Bio-organic
Chemistry, Moscow, Russia
Nikolai Bovin
Alexander Tuzikov
Galina Pazynina
Glycosciences laboratory,
Faculty of Medicine, Imperial
College, London, UK
Ten Feizi
Yan Liu
Robert Childs
Angelina Palma
Wengang Chai
Maria Campanero-Rhodes
Yibing Zhang
D.I.Ivanovsky Institute of
Virology, Moscow, Russia
Svetlana Yamnikova
Dmitri Lvov
Natalia Lomakina
Influenza Division, CDC,
Atlanta, GA, USA
Alexander Klimov
Amanda Balish
St.Jude Children’s Research
Hospital, Memphis, TN, USA
Robert Webster
Scott Krauss
Erasmus Medical Center,
Rotterdam, The Netherlands
Ron Fouchier
Vincent Munster
Members of EU Concortium
FLUPATH
Ilaria Capua
Giovanni Cattoli
Jill Banks
Division of Virology,
MRC NIMR,
Mill Hill, London, UK
Alan Hay
Steve Warton
Rod Daniels
Vicky Gregory
VLA,Weybridge, Addlestone,
Surrey, UK
Ian Brown
Sharon Brookes
Alejandro Nunez
Bhudipa Choudhury
Stephen Essen
Derek Clifford
Marek Slomka
Fanny Garson
Bethany Nash
Amanda Hanna
Rebecca Gardner
Richard Irvine
Members of EU Concortium
ESNIP-2
Emanuela Foni
Gaelle Kuntz-Simon
Michel Bublot
Jaime Maldonado Garcia
Willie Loefflen
Kristien Van Reeth
Thanks to:
WHO Global Influenza Network
Grant support:
FLUPATH, EU
Wellcome Trust, UK
SFB 593, Germany
LOEWE UGLMC, Hesse, Germany
von Behring-Röntgen-Stiftung, Germany
Marburg
Virus binding to selected 2-3 Sia sequences
V
V
% infected ciliated cells
3-5
20-30