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

2

• 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