Transmission of 2009 H1N1 Influenza
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Transmission of 2009 H1N1 Influenza Viruses in Ferrets. EM of CA/04/2009 – CDC/C.S. Goldsmith. Terrence Tumpey Immunology and Pathogenesis Branch Influenza Division, CDC. Animal Models: Influenza Transmission. Mice – Poor model

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Transmission of 2009 H1N1 Influenza Viruses in Ferrets

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Transmission of 2009 h1n1 influenza viruses in ferrets

Transmission of 2009 H1N1 Influenza

Viruses in Ferrets

EM of CA/04/2009 – CDC/C.S. Goldsmith

Terrence Tumpey

Immunology and Pathogenesis Branch

Influenza Division, CDC


Transmission of 2009 h1n1 influenza viruses in ferrets

Animal Models: Influenza Transmission

  • Mice – Poor model

  • Guinea Pigs – Good transmission model, but poor model for influenza pathogenesis

  • Ferrets – Best model for transmission and pathogenesis

(Lowen AC, S. Mubareka, J. Steel, P. Palese, PLoS Pathog. 2007 3:1470)


Transmission study experimental design respiratory droplet transmission

Transmission Study: Experimental DesignRespiratory Droplet transmission

Inoculated Animals


Transmission study experimental design respiratory droplet transmission1

Transmission Study: Experimental DesignRespiratory Droplet transmission

Naive

106 PFU i.n.

Inoculated Animals


Transmission study experimental design respiratory droplet transmission2

Transmission Study: Experimental DesignRespiratory Droplet transmission

Inoculated Animals


Transmission study experimental design

Transmission Study: Experimental Design

Respiratory Droplet Transmission

Contact Transmission

X2

Contact Animals

Inoculated Animals

X2

X2


Transmission of 2009 h1n1 influenza viruses in ferrets

Ferret Model of Respiratory Droplet Transmission

Human H1N1 vs. avian H1N1 viruses

Inoculated

Contact ferrets (3/3)

8

6

Log10 EID50/ml

4

1918 (H1N1)

(SC18 HA)

2

1

3

5

7

9

1

3

5

7

9

11

8

No transmission

6

Avian H1N1

(Duck/NY/96)

Log10 EID50/ml

4

2

1

3

5

7

9

1

3

5

7

9

11

Days post inoculation/contact


Transmission of 2009 h1n1 influenza viruses in ferrets

Ferret Model of Respiratory Droplet Transmission

Human vs. avian H2N2 viruses

Inoculated

Contact ferrets

8

Human H2N2

(Albany/6/58)

6

Log10 EID50/ml

4

2

1

3

5

7

9

1

3

5

7

9

11

8

No transmission

6

Avian H2N2

(Mallard/NY/6750/78)

Log10 EID50/ml

4

2

1

3

5

7

9

1

3

5

7

9

11

Days post inoculation/contact


Transmission of 2009 h1n1 influenza viruses in ferrets

What influenza virus genes confer efficient transmission?

=

+

Virus genes ?

Efficient

Transmission

1918 H1N1

(transmissible phenotype)

Avian H1N1

(non-transmissible )


Transmission of 2009 h1n1 influenza viruses in ferrets

Summary of Findings

Two 1918 virus genes, HA and PB2, were sufficient to confer virus transmissibility in ferrets when rescued on the genetic background of an avian H1N1 virus.

HA

PB2


Transmission of 2009 h1n1 influenza viruses in ferrets

Two amino acid substitutions (D190E, D225G) in HA abolishes transmissibility of the 1918 virus

Nasal Wash Titers: AV18 virus

Inoculated

Contact ferrets*

8

7

6

5

Virus titer (log10 EID50/mL)

4

3

2

1

1

3

5

7

9

1

3

5

7

9

11

Days post-inoculation

Days Post-contact

* Influenza sero-neg at day 0 and 18 p.c.


Transmission of 2009 h1n1 influenza viruses in ferrets

1918 HA and PB2 virus genes confer efficient respiratory droplet transmission

Inoculated

Contact ferrets

8

6

1918 HA:Duck/NY/96

Log10 EID50/ml

4

2

1

3

5

7

9

1

3

5

7

9

11

1918 HAPB2:Dk/NY/96

8

6

Log10 EID50/ml

4

2

1

3

5

7

9

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

PB2

  • Ten amino acid differences in PB2 of the avian Dk/NY/96 virus and 1918 virus

  • These changes (positions 108, 114,199, 473, 475, 477, 539, 576, 627, and 702) occur in domains that are suggested to have a variety of functions including mRNA cap-binding and PB1 binding

  • Of particular interest is the presence of a lysine (k) residue at 627 of the 1918 PB2 protein

  • PB2 627K has been suggested to allow more efficient growth at the lower temperatures encountered in the upper airway of mammals (M. Hatta et al., 2007 PLoS Pathog 3)


Transmission of 2009 h1n1 influenza viruses in ferrets

A single amino acid substitution (PB2-K627E) in PB2 abolishes transmissibility of the 1918 virus

Nasal Wash Titers: 1918 (PB2-K627E)

Inoculated

Contact ferrets

8

7

6

Virus titer (log10 EID50/mL)

5

4

3

2

1

3

5

7

9

1

3

5

7

9

11

Days post-inoculation

Days post-contact


Transmission of 2009 h1n1 influenza viruses in ferrets

Plaque morphology of H1N1

reassortant viruses


Transmission of 2009 h1n1 influenza viruses in ferrets

Plaque morphology of H1N1

reassortant viruses


Transmission of 2009 h1n1 influenza viruses in ferrets

Transmission of 2009 H1N1 Influenza Viruses

Do similar molecular changes influence transmission

of 2009 H1N1 virus?


Transmission of 2009 h1n1 influenza viruses in ferrets

Transmission of 2009 H1N1 in humans

Study

location

Household Secondary Attack Rates (% ILI)

Study

Cauchemez et al.

(NEJM 2009 361:29)

U.S.

10.1

North Carolina

14.3

Doyle & Hopkins

(Epidemiol Infect. 2010 21:1)

New York

17.7

Lessler & Reich

(NEJM 2009 361:2628)

Hong Kong

5.9

Leung et al.

(Epidemiol Infect. 2010 21:7)

Odaira et al.

(Eurosurveillance 2009 14:35)

Japan

7.6

  • SAR of seasonal influenza range is 10 to 40%

  • Transmissibility in households from previous pandemics (20-40%)


Transmission of 2009 h1n1 influenza viruses in ferrets

Transmission of 2009 H1N1 Viruses

in Ferrets

Three 2009 H1N1 viruses selected:

  • A/California/04/2009 (uncomplicated respiratory illness)

  • A/Mexico/4482/2009 (severe respiratory illness)

  • A/Texas/15/2009 (fatal respiratory illness)

  • vs.

  • Seasonal H1N1


Transmission of 2009 h1n1 influenza viruses in ferrets

2009 H1N1 Viruses Spread Efficiently by Direct Contact

Inoculated

Contact ferrets (3/3)

8

6

Mex/4482/2009

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (1/3)

8

6

Avian H1N1

(Duck/NY/96)

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Contact

Days Post-Inoculation


Transmission of 2009 h1n1 influenza viruses in ferrets

Seasonal H1N1 vs. 2009 H1N1 RD Transmission

Inoculated

Contact ferrets (2/3)

8

Mex/4482/2009

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (3/3)

8

6

Brisbane/2007

(seasonal)

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

Seasonal vs. Texas/15/009 H1N1 Virus Transmission

Inoculated

Contact ferrets (2/3)

8

6

Tx/15/2009

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (3/3)

8

Solomon Is/3/06

(seasonal H1N1)

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

H1N1 Viruses of Swine Origin Failed to Form

Significant Plaques at 33oC


Transmission of 2009 h1n1 influenza viruses in ferrets

PB2

What is the transmission phenotype of a

2009 H1N1 PB2 (E627K) mutant virus?

Glutamic acid → Lysine at position 627

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/2009

627E

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/09

PB2 627K

627K


Transmission of 2009 h1n1 influenza viruses in ferrets

E627K in PB2 Enhances RD Transmission in Ferrets

Inoculated

Contact ferrets (2/3)

8

CA/04/2009

6

Log10 EID50/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (3/3)

8

CA/04/09

PB2 E627K

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

RD Transmission of a natural 2009 H1N1 E627K isolate

Inoculated

Contact ferrets (3/3)

NL/1132/2009

PB2 E627K

Log10 PFU/ml

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

HA

Novel HA mutation (D222G) associated with

increased disease severity?

  • Amino acid position 222 resides in the RBS of the HA protein and may possibly influence the binding specificity and thus the cellular tropism of the virus.

  • HA (D222G) mutation was found with increased frequency in fatal and severe cases in Norway (Kilander et al. 2010 Eurosurveillance Vol. 15 (9).

  • D222G mutants were detected more frequently in viruses isolated from patients with fatal outcomes and in lung samples (Glinsky Cell Cycle 9 (5) 958-970)


Transmission of 2009 h1n1 influenza viruses in ferrets

HA

What is the transmission phenotype of a

2009 H1N1 HA (D222G) mutant virus?

Aspartic acid → Glycine at 222 HA (H1 numbering)

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/2009

222D

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/09

HA D222G

222G


Transmission of 2009 h1n1 influenza viruses in ferrets

The effect of HA (D222G) mutation on RD transmission

Inoculated

Contact ferrets (2/3), 4/6 total

8

CA/04/09

HA D222G

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (2/3)

8

CA/04/2009

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

HA mutation at position 219 (I219K) improves the

human-receptor binding affinity of CA/04 HA

1918 HA

CA/04 HA I219K

CA/04 wt HA

Direct glycan array binding assay of HA’s to alpha 2,6 SA human receptor


Transmission of 2009 h1n1 influenza viruses in ferrets

HA

What is the transmission phenotype of a

2009 H1N1 HA (I219K) mutant virus?

Isoleucine → Lysine at 219 HA

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/2009

219I

PB1

PB2

PA

HA

NP

NA

M

NS

CA/04/09

HA 219K

219K


Transmission of 2009 h1n1 influenza viruses in ferrets

A single amino acid substitution (I219K) in HA slightly

enhances RD transmission of CA/04/2009 virus

Inoculated

Contact ferrets (3/3)

8

6

CA/04/09 HA I219K

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Inoculated

Contact ferrets (2/3)

8

CA/04/2009

6

Log10 PFU/ml

4

2

1

3

5

7

1

3

5

7

9

11

Days Post-Inoculation

Days Post-Contact


Transmission of 2009 h1n1 influenza viruses in ferrets

Comparative H1N1 Results in Ferrets

Lung Titers

Weight

Loss (%)

(PFU/gm)

% RD

Transmission

Virus

(log

)

Lethality

10

CA/4/2009 – Wild-type

10.3

0/6

66

5.8

CA/4/2009 - D222G

11.4

0/6

66

6.3

CA/4/2009 - E627K

0/6

11.3

100

5.9

CA/4/2009 - E219K

10.1

5.4

0/6

100

Brisbane/59 (seasonal)

4.9

<1.2

0/6

100


Transmission of 2009 h1n1 influenza viruses in ferrets

Acknowledgements

Pathogenesis team

CDC Atlanta


Transmission of 2009 h1n1 influenza viruses in ferrets

Acknowledgements

Centers for Disease Control and Prevention

Influenza Division/IVPB

Neal van Hoeven

Debra Wadford

Jacqueline Katz

Nancy Cox

Mount Sinai School of Medicine

University of Washington School of Medicine

The Scripps Research

Institute

USDA/Southeast Poultry

Research Laboratory

NIH Grants; 5R01 AI0506919-02 and AI058113-01

David Swayne


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