1
Viral characteristics of H5N1 influencing mutation/reassortment events with
pandemic potential
Ruben Donis, Ph.D.Influenza Division, NCIRD, CCID
Centers for Disease Control and PreventionAtlanta, Georgia, United States
FAO-OIE-WHO Scientific Consultation on Avian Influenza at the Human-Animal Interface
Inherent characteristics predisposing to reassortment & mutation
• Genome Structure–8 Interchangeable RNA molecules: Reassortment
• Conservation of critical compatibility features in type A viruses
• RNA polymerase without proof-reading–PB1 fidelity: frequent mutations
• Numerous envelope alleles: 16 HA and 9 NA–Broad range of biological activities: host, tropism,
shedding, immune evasion
• Short generation time–Accelerate evolution, evade host defenses
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• Viral host range–Established in multiple avian and mammalian host species,
including humans• Multiple options for interspecies transmission and reassortment• Multiple HA and NA alleles
– Reduced interference for co-infection – Expand circulation of reassorted “internal genes”
–Viruses with broad host range: dual infection and reassortment• “Mixing vessel” species such as swine
–Gap of knowledge: host range of animal influenza strains
• Mucosal infection–Limited immunologic memory, favors immune evasion–Repeat infections, co-infection, reassortment and mutation
Inherent characteristics predisposing to mutation & reassortment
• Genome Structure–Segmentation and reassortment
• Avian-Avian: in nature• Avian-Mammalian: in nature and experimental
• RNA polymerase without proof-reading–PB1 fidelity: frequent mutations
Inherent characteristics predisposing to mutation & reassortment
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Avian-Avian Reassortment in NatureObenauer et al. 2006
• North American AI isolates, n=169
• Extensive gene reassortment– HA and NA most mobile
• Linkage of segments– Stressed the importance of protein sequences in the analysis:
“proteotypes”– H6 North American viruses (example):
• Linkage of HA and NA proteotypes• Linkages of PB1-PA-M-NS and HA-NA-M • Proteotype combinations persist over years• Postulate that functional interactions responsible for linkage
– “Hitchhiking” effects not ruled out completely
Obenauer, J. C., et al. 2006. Large-scale sequence analysis of avian influenza isolates. Science 311:1576-80.Finkelstein, D. B., et al. 2007. Persistent host markers in pandemic and H5N1 influenza viruses. J Virol 81:10292-9.
Proteotypes Identify Associations
• Proteotype association between HA and M
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Avian-Avian ReassortmentDugan et al (2008)
• North American AI isolates; n=167• Extremely frequent reassortment • Little clear linkage among specific internal gene segments
Tre
e i
nco
ng
ruen
ce in
dex
Dugan, V. G., et al. 2008. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathog 4:e1000076.
H5N1 Eurasian Lineage: Extensive Reassortment with Avian Viruses
• China: Harbin, HKU and St. Jude studies
• Vietnam: Wan et al (2008) PLoS in press– Vietnam 2007, clade 1-2
reassortant genotypes
Conclusions: 1. High frequency reassortment between avian viruses2. Contribution to host range expansion?
Wen XF et al, 2008. Evolution of Highly Pathogenic H5N1 Avian Influenza Viruses in Vietnam between 2001 and 2007. PLoS One In Press
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Reassortment Between Avian And Mammalian Viruses
• Natural– Reassortment of swine, avian and human viruses– Triple reassortant SIV: 1998 USA
• Experimental– Reassortment avian H5N1 and human H3N2
• Chen et al (2008)
H3N2 Swine 3X reassortant
HA NA PB2 PB1 PA NP M NS
Chen, L. M., et al. 2008. Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog 4:e1000072.
H5N1
Reassortant Genotypes
human/avian combinatorial26
or or or or or or
63 combinations
63 reassortant viruses
H3N2 107.2 PFU/mL
HA NA PB2 PB1 PA NP M NS
107.6 PFU/mLH5N1
SYSTEMATIC
6
>6 log10 >4 log10 > 2 log10 < 2 log10
45%
22%
12%
20%
>106 PFU/mL
Replication in Cell Culture
Growth of 63 human-avian H5N1 reassortant viruses
63
Virulence in mice (i.n. route)
HA NA PB2 PB1 PA NP M NS
101.8 PFU H5N1LD50
>2.8 log10 >3.8 log10 >4 log10 >5 log10
102.8 PFU8%
103.8 PFU26%
13%
53%Replication of 38 reassortant
genotypes38
Lethality of human-avian H5N1 reassortant viruses
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Plaque size(mm)
RescueefficiencyHA NA PB2 PB1 PA NP M NS
4-57.6
NP Gene InteractionsChen et al. 2008
ND<2.0
1.54.3
3.5-56.9
ND<2.0
Conclusions: 1. Human NP needs cognate M and NS2. RNP reconstitution not beneficial
Plaque size(mm)
RescueefficiencyHA NA PB2 PB1 PA NP M NS
4-57.6
<0.53.1
4-56.9
Polymerase Gene Interactions
7.4 2-3
Conclusions: 1. Avian PB2 prefers cognate PA2. Functional interaction
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Polymerase Gene Reassortment
• Lowest mouse LD50 among 38 reassortants
• Resemble 1957 and (in part) 1968 pandemic strains
HA NA PB2 PB1 PA NP M NSH5N1 HU/AV reassortant
Conclusions: 1. Avian HA-NA may benefit from cognate PB1 in vivo2. Contribution to host range expansion?
Chen, L. M., et al. 2008. Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog 4:e1000072.
Influenza Interactome vs. Reassortment
2NS1(NS2)
(D)nM1(M2)
4+NA
Dn+++NP
D3HA
I1+PA
ID1+PB1(F2)
DD1PB2
NS(NS2)
M1 (M2)NANPHAPAPB1
(F2)PB2
Physical PPI: direct (D) or indirect (I)
Func
tiona
l (+)
Reassortment phenotype (Chen et al: avian/human) (Li et al: equine/human)
I
+
Gap of knowledge: fitness loss/gain by reassortmentLi, C., et al. 2008. Compatibility among polymerase subunit proteins is a restricting factor in the reassortment between equine H7N7 and human H3N2 viruses. J Virol. In press.
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• Genome Segmentation–Reassortment
• RNA genome and polymerase lacking proof-reading–PB1 fidelity: frequent mutations–Critical role of selection
Inherent characteristics predisposing to mutation & reassortment
Mutations: focus on HA
• Diverse selection forces drive evolution – Receptor specificity, antigenic drift, etc are driven by the host
– Expansion of viral host range, increased risk for humans
– Mutations in the remaining 7 genes also influence host range
• Consequence of mutations depends on the subtype of the HA and strain variations in the RBS structure– Examples of RBS changes in H9, H7 and H5
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Stevens, J. et al., 2006, Nature Rev Microb 4(11):857-64
H5 Eurasian LineageStevens et al (2008)
α2-6
A/Vietnam/1203/2004
Wildtype α2-6
A/Indonesia/5/2005
From Stevens, J., et al. 2008. Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381:1382-94.
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turkey/Virginia/4529/2002
Netherlands /230/2003
rhea/N Carolina/39482/1993
• North American H7: mutations near receptor binding site?• Eurasian H7: ?
H7 HA North American & EurasianBelser et al. (2008)
α2-6α2-6
α2-6
?Belser, J. A., et al 2008. Contemporary North American influenza H7 viruses possess human receptor specificity: Implications for virus transmissibility. Proc Natl AcadSci U S A 105:7558-63.
H9 HA Eurasian LineageWan et al (2008)
guinea fowl/HK/WF10/99quail/HK/88
• Emergence of avian viruses with changes at position 226 of HA– Gln to Leu change increases α2-6 sialoside & reduces α2-3 binding
α2-6α2-6
Gap of knowledge: What are the selection forces for RBS switch?Gap of knowledge: RBS profiles for a broad range of viruses
From: Wan, H., et al. 2008. Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PLoS ONE 3:e2923.
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RBS of Avian and Mammalian Viruses
0
10000
20000
30000
40000
50000
1 3 5 7 9 1113 151719 21 2325 2729 31 333537 39 4143 4547 49 5153
Bin
ding
α2-6α2-3
Gap of knowledge: sialoside receptor expression in different species
Sialic Acid Structural Diversity
• Determine sialic acid receptor structures in upper and lower airway
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Sialic Acids in Animals
Acknowledgements
• Limei Chen• James Stevens • Todd Davis• Sara Jackson• Yumi Matsuoka• Amanda Balish• Hong Zhou • Laurie Kamimoto• Cathy Smith• Michael Shaw• Joe Bresee• Jacqueline Katz• Alexander Klimov• Ann Moen• Tim Uyeki• Terry Tumpey• Tony Marfin• Sharon Daves• Dan Jernigan• Nancy Cox • SRP Core, ARB, IACUC• MVVB, VSDB, IPB, • Influenza Division
• WHO GIP Surveillance Network• David Swayne; USDA, ARS, SEPRL • James Paulson, TSRI/CFG• Ola Blixt, TSRI/CFG• Ian Wilson, TSRI • Gavin Smith, HKU• Malik Pieris, HKU• Hien Nguyen, NIHE, Vietnam• Daniel Perez, U Maryland• Richard Webby, St Jude CRH• Aloke Chakraborty, NIV, India• Tung Nguyen, NCVD, Vietnam• Yuelong Shu, China CDC• Catherine Gerdil, Sanofi• Ervin Fodor, U Cambridge• Erich Hoffmann, St. Jude• Kanta Subbarao, NIH• Patrick Blair, NAMRU2• Ken Earhart, NAMRU3• Jeffrey Tjaden, NAMRU3• Armen Donabedian, BARDA
CDC Beyond CDC
Glycan microarrays were obtained from the Consortium for Functional Glycomics (funded by GM062116), and from the Centersfor DiseaseControl that were produced for the CDC with the CFG glycan library.
• Robin Robinson, BARDA• Michael Perdue, BARDA• Ben Schwartz, NIP• Roland Lewandowski, NIH