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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

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

  • 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


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


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


What influenza virus genes confer efficient transmission?

=

+

Virus genes ?

Efficient

Transmission

1918 H1N1

(transmissible phenotype)

Avian H1N1

(non-transmissible )


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


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.


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


PB2 droplet transmission

  • 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)


A single amino acid substitution ( droplet transmissionPB2-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


Plaque morphology of H1N1 droplet transmission

reassortant viruses


Plaque morphology of H1N1 droplet transmission

reassortant viruses


Transmission of 2009 H1N1 Influenza Viruses droplet transmission

Do similar molecular changes influence transmission

of 2009 H1N1 virus?


Transmission of 2009 H1N1 in humans droplet transmission

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 Viruses droplet transmission

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


2009 H1N1 Viruses Spread Efficiently by Direct Contact droplet transmission

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


Seasonal H1N1 vs. 2009 H1N1 RD Transmission droplet 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


Seasonal vs. Texas/15/009 H1N1 Virus Transmission droplet 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


H1N1 Viruses of Swine Origin Failed to Form droplet transmission

Significant Plaques at 33oC


PB2 droplet transmission

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


E627K droplet transmission 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


RD Transmission of a natural 2009 H1N1 E627K isolate droplet transmission

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


HA droplet transmission

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)


HA droplet transmission

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


The effect of HA ( droplet transmissionD222G) 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


HA mutation at position 219 (I219K) improves the droplet transmission

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


HA droplet transmission

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


A single amino acid substitution (I droplet transmission219K) 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


Comparative H1N1 Results in Ferrets droplet transmission

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


Acknowledgements droplet transmission

Pathogenesis team

CDC Atlanta


Acknowledgements droplet transmission

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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