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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. 2-3. 2-3. 2-3. 2-6. 2-3. 2-3. 2-6. 2-3. 2-6. 2-6. 2-3. 2-3. - PowerPoint PPT PresentationTRANSCRIPT
- Variation of receptor specificity of influenza viruses in - Variation of receptor specificity of influenza viruses in
different avian speciesdifferent avian species
- Receptor specificity and cell tropism of H1N1pdm and of - Receptor specificity and cell tropism of H1N1pdm and of
its mutants associated with severe cases of infectionits mutants associated with severe cases of infection
Topics Topics
3- and 6-linked Sia receptors in different species3- and 6-linked Sia receptors in different species
2-3
2-32-3
2-6
2-62-32-3
2-6
2-3
2-32-3 2-6
Characterization of receptor-binding specificity of avian Characterization of receptor-binding specificity of avian influenza viruses, 2002 influenza viruses, 2002 present present
Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana Alexandra Gambaryan, Nikolai Bovin, Mikhail Matrosovich, Svetlana Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria Yamnikova, Dmitri Lvov, Alexander Klimov, Robert Webster, Ilaria
Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al., Capua, Jiovanni Cattoli, Ron Fouchier, Vincent Munster, Jill Banks et al.,
Alexandra GambaryanAlexandra Gambaryan
M.P.Chumakov Institute of M.P.Chumakov Institute of Poliomyelitis, MoscowPoliomyelitis, Moscow
Nikolai BovinNikolai Bovin
Institute of Bio-organic Institute of Bio-organic Chemistry, MoscowChemistry, Moscow
A panel of sialylglycopolymers (Nikolai Bovin’s lab)A panel of sialylglycopolymers (Nikolai Bovin’s lab)
Receptor specificity of avian viruses Receptor specificity of avian viruses
(H9 and H7)
Fuc
Fuc
Molecular features that determine binding of avian viruses to sulfated Molecular features that determine binding of avian viruses to sulfated and fucosylated receptorsand fucosylated receptors
No sterical conflicts between fucose and No sterical conflicts between fucose and amino acid in position 222 amino acid in position 222 (gull viruses, some poultry viruses)(gull viruses, some poultry viruses)
Ionic bond between sulfate (type II core) and Ionic bond between sulfate (type II core) and conserved lysine/arginine in position 193 conserved lysine/arginine in position 193 (H5, H7, some H13)(H5, H7, some H13)
193193
222222
SuSu
193193
222222
FucFuc
FucFuc
SiaSia
SiaSia
Crystal structure of H7 HA: Russell, Gamblin, Skehel et al. Crystal structure of H7 HA: Russell, Gamblin, Skehel et al.
- Receptor specificity of influenza viruses is not limited - Receptor specificity of influenza viruses is not limited
to to recognition of the type of Sia-Gal linkage. recognition of the type of Sia-Gal linkage.
- Receptor specificity of avian viruses is not uniform. Receptor specificity of avian viruses is not uniform.
Viruses of gulls and poultry are closer to each other than Viruses of gulls and poultry are closer to each other than
to duck viruses. This may facilitate virus exchange to duck viruses. This may facilitate virus exchange
between gulls and poultrybetween gulls and poultry. .
- Expression of distinct 3-linked sequences varies Expression of distinct 3-linked sequences varies
depending on the animal species, tissue and cell type. depending on the animal species, tissue and cell type.
Avian viruses may differ by their tissue and cell tropism Avian viruses may differ by their tissue and cell tropism
in humans.in humans.
- 6-linked Sia are present on epithelial cells in 6-linked Sia are present on epithelial cells in
galliformes; evolution of viruses in these species may galliformes; evolution of viruses in these species may
increase binding to receptors in humans. increase binding to receptors in humans.
Further studies are needed to Further studies are needed to
characterize role of receptor specificity characterize role of receptor specificity
of avian viruses in their host-range, of avian viruses in their host-range,
tissue tropism and pathogenicitytissue tropism and pathogenicity
Ten FeiziTen Feizi
Alan HayAlan Hay
The neoglycolipid-based carbohydrate microarrays The neoglycolipid-based carbohydrate microarrays (Ten Feizi lab, Imperial College London, UK)(Ten Feizi lab, Imperial College London, UK)
- Sequence-defined natural and synthetic Sequence-defined natural and synthetic
oligosaccharides are coupled to synthetic lipid oligosaccharides are coupled to synthetic lipid
to allow efficient immobilization on solid to allow efficient immobilization on solid
supports. supports.
- Neoglycolipids are spotted on nitrocellulose-Neoglycolipids are spotted on nitrocellulose-
coated glass slides.coated glass slides.
- The lipid-linked probes have some lateral The lipid-linked probes have some lateral
mobility. Clustering of glycolipids allows their mobility. Clustering of glycolipids allows their
optimal multivalent presentation that mimics optimal multivalent presentation that mimics
presentation at the cell surface in vivo.presentation at the cell surface in vivo.Liu et al., Biol.Chem. 2009Liu et al., Biol.Chem. 2009
Examples of binding profilesExamples of binding profiles
80 sialic acid-containing oligosaccharide probes with differing backbone types, chain 80 sialic acid-containing oligosaccharide probes with differing backbone types, chain
lengths and branching patternslengths and branching patterns
2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors)2-3, 2-6, 2-8-linked sialyl epitopes and their combinations (depicted by different colors)
6 neutral probes (negative control; gray) 6 neutral probes (negative control; gray)
Examples of glycan probesExamples of glycan probes
2-3 2-6 2-8 2-3 2-6 2-8
Virus 1 Virus 2
Binding profilesBinding profiles
H1N1pdmH1N1pdm (Hamburg/5/09) (Hamburg/5/09) H1N1 seasonalH1N1 seasonal (Memphis/14/96) (Memphis/14/96) H3N2 pandemicH3N2 pandemic (Aichi/2/68) (Aichi/2/68)
2-3 2-6 2-8 2-3 2-6 2-8 2-3 2-6 2-8
-H1N1pdm, similar to 1918, 1957 and 1968 pandemic viruses, H1N1pdm, similar to 1918, 1957 and 1968 pandemic viruses, preferentially binds to 6-linked Siapreferentially binds to 6-linked Sia
- H1N1pdm differs from seasonal H1N1 virus by a broader binding - H1N1pdm differs from seasonal H1N1 virus by a broader binding specificity and by low-avidity binding to 3-linked Siaspecificity and by low-avidity binding to 3-linked Sia
No binding of H1N1pdm to 2-3-probes in several assaysNo binding of H1N1pdm to 2-3-probes in several assaysBlue – 2-3 probes, red: 2-6 probes
Maines et alMaines et al.,., Science 2009 Science 2009 Yang, Carney & Stevens, PLoS Curr Influenza 2010Yang, Carney & Stevens, PLoS Curr Influenza 2010
1.1. 2.2.
0
10
20
30
40
50
Ham/09(H1N1v)
Mem/96 (H1N1) HK/68 (H3N2) duck/98 (H1N1)
Kas
s, 1
/uM
Neu
5Ac
2-3-fetuin 2-6-fetuin
Binding of Binding of soluble probes, 3- and 6-fetuin,soluble probes, 3- and 6-fetuin, to the solid-phase immobilized virus to the solid-phase immobilized virus
3.3.
Binding to the glycoarray of Binding to the glycoarray of recombinant HA proteinrecombinant HA protein
Neoglycolipid-based arrays:Neoglycolipid-based arrays:Utilization of whole virus particles and clustered glycolipid probes Utilization of whole virus particles and clustered glycolipid probes
could facilitate detection of low-affinity polyvalent interactionscould facilitate detection of low-affinity polyvalent interactions
H1N1pdm H1N1 seasonalH1N1pdm H1N1 seasonal
Biological significance of binding to 2-3 Sia ?Biological significance of binding to 2-3 Sia ?
6- and 3-linked Sia in human respiratory tract6- and 3-linked Sia in human respiratory tract
Shinya et al. 2006 van Riel et al., 2007Shinya et al. 2006 van Riel et al., 2007
Binding:Binding:
Lectins Lectins 2-6 (green), 2-3 (red) 2-6 (green), 2-3 (red) Viruses Viruses
Nasal and tracheo-bronchial epithelial Nasal and tracheo-bronchial epithelial cells mainly express 6-linked Sia. cells mainly express 6-linked Sia. Binding Binding to 6-linked receptors seems to be to 6-linked receptors seems to be essential for virus replication in the upper essential for virus replication in the upper respiratory tract and efficient human-to-respiratory tract and efficient human-to-human transmission.human transmission.
3-linked Sia are mainly present in 3-linked Sia are mainly present in bronchioles and alveoli. bronchioles and alveoli. Binding to 3-Binding to 3-linked receptors could facilitate virus linked receptors could facilitate virus replication in the lower respiratory tract replication in the lower respiratory tract and increase pathogenicity.and increase pathogenicity.
Can low-avidity binding of H1N1pdm Can low-avidity binding of H1N1pdm
to 3-linked Sia contribute to viral to 3-linked Sia contribute to viral
capacity to causecapacity to cause
severe disease in humans ?severe disease in humans ?
HA polymorphism in position 222 HA polymorphism in position 222 (225, H3 numb.)(225, H3 numb.)
- Major variant, Major variant, 222D222D. Substitutions . Substitutions G,E,N. G,E,N.
- 222G222G found in 7-10% of sequences in fatal and severe cases, but not in clinically found in 7-10% of sequences in fatal and severe cases, but not in clinically mild cases. mild cases. The mutants seem to occur sporadically with no evidence of The mutants seem to occur sporadically with no evidence of sustained transmission.sustained transmission.
- 222E222E – No apparent correlation with disease severity; – No apparent correlation with disease severity; transmissible virus.transmissible virus.
- 222N – Not enough data. 222N – Not enough data.
Two of five HA sequences Two of five HA sequences
from the victims of the from the victims of the
1918 pandemic had 1918 pandemic had
mutation D222Gmutation D222G
Amino acid 222 Amino acid 222
- Correlates with the virus host species. Avian viruses have 222G, human and swine viruses 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 have 222D/E. Propagation of human viruses in hen’s eggs often leads to mutations D222G/N.D222G/N.
- Mutation D222G increases binding to 3-linked receptors of human virusesMutation D222G increases binding to 3-linked receptors of human viruses (Gambaryan et al., (Gambaryan et al.,
1997,1999; Glaser et al., 2005; Stevens et al., 2006),1997,1999; Glaser et al., 2005; Stevens et al., 2006), including H1N1pdmincluding H1N1pdm ((Yang, Carney & Stevens, Yang, Carney & Stevens,
PLoS Curr Influenza 2010)PLoS Curr Influenza 2010)..
- Mutation D222G decreased airborne transmission of the 1918 virus in ferretsMutation D222G decreased airborne transmission of the 1918 virus in ferrets (Tumpey et al., (Tumpey et al.,
2007)2007)..
Can mutations D222G/E in H1N1pdm Can mutations D222G/E in H1N1pdm
change viral cell tropism and replication change viral cell tropism and replication
efficiency in human respiratory tract ?efficiency in human respiratory tract ?
Cultures of differentiated human tracheo-bronchial epithelial Cultures of differentiated human tracheo-bronchial epithelial
cells (HTBE) - an cells (HTBE) - an in vitroin vitro model of human airway epithelium model of human airway epithelium
Immuno-staining of cilia in fixed cultureImmuno-staining of cilia in fixed culture
Beating of cilia in live cultureBeating of cilia in live culture
Cross-section of 6-wk-old culture
Primary airway epithelial cells grow on membrane Primary airway epithelial cells grow on membrane supports at air-liquid interfacesupports at air-liquid interface
6- and 3-linked sialic acids in HTBE cultures6- and 3-linked sialic acids in HTBE cultures
Sambucus nigraSambucus nigra agglutinin agglutinin Maackia amurensisMaackia amurensis lectin lectin SNASNA MAL-1 MAL-1
2-62-6 2-32-3
Expression pattern agrees with that Expression pattern agrees with that in vivoin vivo ((Baum and Paulson, 1990; Baum and Paulson, 1990;
Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007Gagneux et al., 2003; Shinya et al., 2006; Nicholls et al., 2007; Yao et al., 2007) )
Human and avian viruses target different types of cellsHuman and avian viruses target different types of cellsRedRed, viral antigen; , viral antigen; graygray, cilia of ciliated cells, cilia of ciliated cells
Seasonal human virus Avian virusSeasonal human virus Avian virus
<5 <5 % of infected ciliated cells % of infected ciliated cells >70 >70
Cell tropism of H1N1pdm in HTBE culturesCell tropism of H1N1pdm in HTBE cultures
222D: 222D: 222G: 222G:
Moldova/G186/09, Cyprus/S2487/09Moldova/G186/09, Cyprus/S2487/09 Lviv/N6/09 (fatal), Lviv/N6/09 (fatal),
Hamburg/5/09Hamburg/5/09 Norway/3206-3/09 (fatal)Norway/3206-3/09 (fatal)
222E: 222E: Hamburg/5/09Hamburg/5/09-e-e (egg-derived)(egg-derived)
Dakar/37/09Dakar/37/09
3-5 3-5 % of infected ciliated cells % of infected ciliated cells 20-3020-30
Receptor specificity, glycoarraysReceptor specificity, glycoarrays D222G mutation increases binding to 3-linked SiaD222G mutation increases binding to 3-linked Sia
2-6
2-3
2-6
2-3
Binding to resialylated soluble 3- and 6-fetuinBinding to resialylated soluble 3- and 6-fetuinD222G mutation decreases binding to 6-linked SiaD222G mutation decreases binding to 6-linked Sia
Tropism, Tropism, % ciliated cells% ciliated cells 3-5 20-30 3-5 20-30Circulation in humans Yes NoCirculation in humans Yes No
Replication kinetics in HTBE culturesReplication kinetics in HTBE culturesHamburg/5/09 (H1N1pdm) vs its D222G mutantHamburg/5/09 (H1N1pdm) vs its D222G mutant
wt (222D) D222G
*
*p=0.0001
Sharon Brookes Ian BrownSharon Brookes Ian Brown
Selection of receptor-binding variants during Selection of receptor-binding variants during replication and transmission in pigsreplication and transmission in pigs
Inoculum:Inoculum: Egg-grown A/California/7/09 Egg-grown A/California/7/09
Direct infectionDirect infection
TransmissionTransmission
Middle lung Middle lung
lobe tissueslobe tissues
1 and 2 dpi1 and 2 dpi
wt + 222Gwt + 222G
wt (wt (222D222D) ) wt + 222G wt + 222G
4 and 7 dpi4 and 7 dpi
222G222G
wt + D222G + Q223Rwt + D222G + Q223R
3 - 6 dpi3 - 6 dpi
Figure modified from Figure modified from Brookes et al., 2010Brookes et al., 2010
Expression of 3- and 6-Sia in pigs is similar to that in humans 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)(van Riel et al., 2007; Van Poucke et al., 2010; Nelli et al., 2010)
Humans Pigs Humans Pigs
Shinya et al. 2006 Shinya et al. 2006 N Nelli et al. BMC Veterinary Research, 2010elli et al. BMC Veterinary Research, 2010
2,6 (green); 2,3 (red)2,6 (green); 2,3 (red)
I.I. Mutation D222EMutation D222E has marginal effect on receptor has marginal effect on receptor
specificity and does not affect viral cell tropism in HTBE specificity and does not affect viral cell tropism in HTBE
cultures.cultures. This is consistent with apparent unrestricted This is consistent with apparent unrestricted
circulation of the variant in humans and with lack of circulation of the variant in humans and with lack of
correlation of 222E with severe disease. correlation of 222E with severe disease.
II. Mutation D222GII. Mutation D222G reduces viral replication efficiency in reduces viral replication efficiency in
HTBE cultures and prevents transmissibility in HTBE cultures and prevents transmissibility in
experimentally infected pigs.experimentally infected pigs. These findings could These findings could
explain why the mutants do not transmit in humans. explain why the mutants do not transmit in humans.
III.III. Mutation D222G alters viral receptor specificity and Mutation D222G alters viral receptor specificity and cell tropism in human epithelium, provides the mutant cell tropism in human epithelium, provides the mutant with replicative advantage in the LRT of pigs.with replicative advantage in the LRT of pigs.
Potential explanations of association of D222G Potential explanations of association of D222G with severe disease:with severe disease:
- Mutation - Mutation causes severe diseasecauses severe disease owing to propensity owing to propensity of the mutant to reach and infect the LRTof the mutant to reach and infect the LRT
or/andor/and- Mutation - Mutation emerges as a consequence of severe emerges as a consequence of severe diseasedisease owing to the virus replication in the LRT owing to the virus replication in the LRT
It is important to closely monitor position 222 It is important to closely monitor position 222
mutants and other potential mutants with mutants and other potential mutants with
altered receptor specificity and cell tropismaltered receptor specificity and cell tropism
Hans-Dieter KlenkHans-Dieter Klenk
Markus EickmannMarkus Eickmann Jennifer UhlendorffJennifer UhlendorffTatyana MatrosovichTatyana Matrosovich
Institute of Virology, Philipps University, Marburg, GermanyInstitute of Virology, Philipps University, Marburg, Germany
M.P.Chumakov Institute of M.P.Chumakov Institute of Poliomyelitis, Moscow, RussiaPoliomyelitis, Moscow, RussiaAlexandra GambaryanAlexandra Gambaryan
Carbohydrate laboratory, Carbohydrate laboratory, Institute of Bio-organic Institute of Bio-organic Chemistry, Moscow, RussiaChemistry, Moscow, RussiaNikolai BovinNikolai BovinAlexander TuzikovAlexander TuzikovGalina PazyninaGalina Pazynina
D.I.Ivanovsky Institute of D.I.Ivanovsky Institute of Virology, Moscow, RussiaVirology, Moscow, RussiaSvetlana YamnikovaSvetlana YamnikovaDmitri LvovDmitri LvovNatalia LomakinaNatalia Lomakina
Influenza Division, CDC, Influenza Division, CDC, Atlanta, GA, USA Atlanta, GA, USA Alexander KlimovAlexander KlimovAmanda BalishAmanda Balish
St.Jude Children’s Research St.Jude Children’s Research Hospital, Memphis, TN, USAHospital, Memphis, TN, USARobert WebsterRobert WebsterScott KraussScott Krauss
Erasmus Medical Center,Erasmus Medical Center,Rotterdam, The NetherlandsRotterdam, The NetherlandsRon FouchierRon FouchierVincent MunsterVincent Munster
Members of EU Concortium Members of EU Concortium FLUPATHFLUPATHIlaria CapuaIlaria CapuaGiovanni Cattoli Giovanni Cattoli Jill BanksJill Banks
VLA,Weybridge, Addlestone, VLA,Weybridge, Addlestone, Surrey, UKSurrey, UKIan BrownIan BrownSharon BrookesSharon BrookesAlejandro NunezAlejandro NunezBhudipa ChoudhuryBhudipa ChoudhuryStephen EssenStephen EssenDerek CliffordDerek CliffordMarek SlomkaMarek SlomkaFanny GarsonFanny GarsonBethany NashBethany NashAmanda HannaAmanda HannaRebecca GardnerRebecca GardnerRichard IrvineRichard Irvine
Members of EU Concortium Members of EU Concortium ESNIP-2ESNIP-2Emanuela FoniEmanuela FoniGaelle Kuntz-SimonGaelle Kuntz-SimonMichel BublotMichel BublotJaime Maldonado GarciaJaime Maldonado GarciaWillie LoefflenWillie LoefflenKristien Van ReethKristien Van Reeth
Glycosciences laboratory, Glycosciences laboratory, Faculty of Medicine, Imperial Faculty of Medicine, Imperial College, London, UKCollege, London, UK Ten FeiziTen FeiziYan LiuYan LiuRobert ChildsRobert ChildsAngelina PalmaAngelina PalmaWengang ChaiWengang ChaiMaria Campanero-RhodesMaria Campanero-RhodesYibing ZhangYibing Zhang
Division of Virology, Division of Virology, MRC NIMR, MRC NIMR, Mill Hill, London, UKMill Hill, London, UKAlan HayAlan HaySteve WartonSteve WartonRod DanielsRod DanielsVicky GregoryVicky Gregory
CollaboratorsCollaborators
Thanks to:Thanks to:
WHO Global Influenza NetworkWHO Global Influenza Network
Grant support:Grant support:
FLUPATH, EUFLUPATH, EU
Wellcome Trust, UK Wellcome Trust, UK
SFB 593, GermanySFB 593, Germany
LOEWE UGLMC, Hesse, GermanyLOEWE UGLMC, Hesse, Germany
von Behring-Röntgen-Stiftung, Germany von Behring-Röntgen-Stiftung, Germany
Marburg
Virus binding to selected 2-3 Sia sequencesVirus binding to selected 2-3 Sia sequences
V
V
% infected ciliated cells% infected ciliated cells 3-5 20-303-5 20-30