Transmission of 2009 H1N1 Influenza Viruses in Ferrets

Terrence TumpeyImmunology and Pathogenesis Branch

Influenza Division, CDC

EM of CA/04/2009 – CDC/C.S. Goldsmith

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)

Ino

cula

ted

Ani

ma

lsTransmission Study: Experimental Design

Respiratory Droplet transmission

Ino

cula

ted

Ani

ma

ls

Naive

Transmission Study: Experimental DesignRespiratory Droplet transmission

106 PFU i.n.

Ino

cula

ted

Ani

ma

lsTransmission Study: Experimental Design

Respiratory Droplet transmission

Ino

cula

ted

Ani

ma

ls

Co

nta

ct A

nim

als

Transmission Study: Experimental Design

Contact Transmission

X2

X2

X2

Respiratory Droplet Transmission

Avian H1N1(Duck/NY/96)

Lo

g10

EID

50/m

l

Days post inoculation/contact

Ferret Model of Respiratory Droplet TransmissionFerret Model of Respiratory Droplet TransmissionHuman H1N1 vs. avian H1N1 virusesHuman H1N1 vs. avian H1N1 viruses

1 3 5 1 3 57 7 9 119

2

4

6

8

1918 (H1N1)(SC18 HA)

Lo

g10

EID

50/m

l

Inoculated

No transmission

† †2

4

6

8

1 3 5 1 3 57 7 9 119

†

Contact ferrets (3/3)

Human H2N2(Albany/6/58)

Lo

g10

EID

50/m

l

Inoculated Contact ferrets

Avian H2N2(Mallard/NY/6750/78)

Lo

g10

EID

50/m

l

2

4

6

8

1 3 5 1 3 57 7 9 119

Days post inoculation/contact

Ferret Model of Respiratory Droplet TransmissionFerret Model of Respiratory Droplet TransmissionHuman vs. avian H2N2 virusesHuman vs. avian H2N2 viruses

2

4

6

8

1 3 5 1 3 57 7 9 119

No transmission

What influenza virus genes confer What influenza virus genes confer efficient transmission?efficient transmission?

1918 H1N1 (transmissible phenotype)

Avian H1N1(non-transmissible )

=Virus genes ?

+

EfficientTransmission

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.

Summary of Findings

HAPB2

Days post-inoculation Days Post-contact

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

Contact ferrets*

1

2

3

4

5

6

7

8

Vir

us ti

ter

(lo

g 10

EID

50/

mL

)

1 3 5 7 91 5 739 11

Nasal Wash Titers: AV18 virus

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

† †

Inoculated

1918 HAPB2:Dk/NY/96

Lo

g10

EID

50/m

l

Inoculated Contact ferrets

Lo

g10

EID

50/m

l

Days Post-Inoculation Days Post-Contact

1 3 5 1 3 57 7 9 119

2

4

6

8

2

4

6

8

1 3 5 1 3 57 7 9 119

1918 HA:Duck/NY/96

1918 HA and PB2 virus genes confer 1918 HA and PB2 virus genes confer efficient respiratory droplet transmissionefficient respiratory droplet transmission

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)

Days post-inoculation Days post-contact

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

Contact ferrets

Vir

us ti

ter

(lo

g 10

EID

50/

mL

) Inoculated

2

3

4

5

6

7

8

1 3 5 1 3 57 7 9 119

Nasal Wash Titers: 1918 (PB2-K627E)

Plaque morphology of H1N1 reassortant viruses

Plaque morphology of H1N1 reassortant viruses

Do similar molecular changes influence transmission Do similar molecular changes influence transmission of 2009 H1N1 virus? of 2009 H1N1 virus?

Transmission of 2009 H1N1 Influenza VirusesTransmission of 2009 H1N1 Influenza Viruses

Household Secondary Attack Rates (% ILI)

Odaira et al. (Eurosurveillance 2009 14:35)

North Carolina

Study location

U.S.

Japan

Transmission of 2009 H1N1 in humans

Cauchemez et al. (NEJM 2009 361:29)

Doyle & Hopkins (Epidemiol Infect. 2010 21:1)

Lessler & Reich (NEJM 2009 361:2628)

New York

Leung et al. (Epidemiol Infect. 2010 21:7)

Hong Kong

Study

14.3

10.1

7.6

17.7

5.9

• SAR of seasonal influenza range is 10 to 40%• Transmissibility in households from previous pandemics (20-40%)

Three 2009 H1N1 viruses selected:

1. A/California/04/2009 (uncomplicated respiratory illness)

2. A/Mexico/4482/2009 (severe respiratory illness)

3. A/Texas/15/2009 (fatal respiratory illness)

vs.

Seasonal H1N1

Transmission of 2009 H1N1 Viruses in Ferrets

2009 H1N1 Viruses Spread Efficiently by Direct Contact

Inoculated Contact ferrets (1/3)

Lo

g10

PF

U/m

lL

og

10 P

FU

/ml

Days Post-Inoculation Days Post-Contact

Inoculated Contact ferrets (3/3)

Mex/4482/2009

Avian H1N1(Duck/NY/96)

1 3 5 1 3 57 7 9 11

1 3 5 1 3 57 7 9 11

2

4

6

8

2

4

6

8

Seasonal H1N1 vs. 2009 H1N1 RD Transmission

Brisbane/2007(seasonal)

Inoculated Contact ferrets (3/3)

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

Inoculated Contact ferrets (2/3)

Mex/4482/2009

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

Days Post-Inoculation Days Post-Contact

Seasonal vs. Texas/15/009 H1N1 Virus Transmission

Solomon Is/3/06(seasonal H1N1)

Inoculated Contact ferrets (3/3)

2

4

6

8

Lo

g10

PF

U/m

l

Inoculated Contact ferrets (2/3)

Days Post-Inoculation Days Post-Contact

Tx/15/2009

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

1 3 5 1 3 57 7 9 11

H1N1 Viruses of Swine Origin Failed to Form Significant Plaques at 33oC

What is the transmission phenotype of a 2009 H1N1 PB2 (E627K) mutant virus?

PB2 PA HA NP NA M NSPB1

CA/04/09 PB2 627K

CA/04/2009

Glutamic acid → Lysine at position 627

PB2 PA HA NP NA M NSPB1

627K

627E

PB2

E627K in PB2 Enhances RD Transmission in Ferrets

Inoculated Contact ferrets (3/3)

CA/04/09 PB2 E627K

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

Inoculated Contact ferrets (2/3)L

og

10 E

ID50

/ml

1 3 5 1 3 57 7 9 11

2

4

6

8

CA/04/2009

Days Post-Inoculation Days Post-Contact

Inoculated Contact ferrets (3/3)

1 3 5 1 3 57 7 9 11

NL/1132/2009PB2 E627K

RD Transmission of a natural 2009 H1N1 E627K isolate L

og

10 P

FU

/ml

Days Post-Inoculation Days Post-Contact

Novel HA mutation (D222G) associated with increased disease severity?

HA

• 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)

CA/04/09 HA D222G

CA/04/2009PB2 PA HA NP NA M NSPB1

PB2 PA HA NP NA M NSPB1

222D

What is the transmission phenotype of a 2009 H1N1 HA (D222G) mutant virus?

Aspartic acid → Glycine at 222 HA (H1 numbering)

222G

HA

1 3 5 1 3 57 7 9 11

2

4

6

8

CA/04/2009

The effect of HA (D222G) mutation on RD transmission L

og

10 P

FU

/ml

Inoculated Contact ferrets (2/3)

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

Days Post-Inoculation Days Post-Contact

CA/04/09 HA D222G

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

HA mutation at position 219 (I219K) improves the human-receptor binding affinity of CA/04 HA

CA/04 HA I219K 1918 HA

CA/04 wt HA

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

CA/04/09 HA 219K

CA/04/2009PB2 PA HA NP NA M NSPB1

PB2 PA HA NP NA M NSPB1

219I

What is the transmission phenotype of a 2009 H1N1 HA (I219K) mutant virus?

Isoleucine → Lysine at 219 HA

219K

HA

1 3 5 1 3 57 7 9 11

2

4

6

8

CA/04/2009

A single amino acid substitution (I219K) in HA slightly enhances RD transmission of CA/04/2009 virusL

og

10 P

FU

/ml

Inoculated Contact ferrets (2/3)

Inoculated Contact ferrets (3/3)

Days Post-Inoculation Days Post-Contact

CA/04/09 HA I219K

1 3 5 1 3 57 7 9 11

2

4

6

8

Lo

g10

PF

U/m

l

WeightLoss (%)

% RD Transmission

Lung Titers

(log10)

Brisbane/59 (seasonal) 100<1.2

(PFU/gm)

Comparative H1N1 Results in Ferrets

CA/4/2009 - D222G 666.3

Virus

4.9

11.4

CA/4/2009 – Wild-type 665.810.3

Lethality

0/6

0/6

0/6

CA/4/2009 - E219K 1005.410.1 0/6

CA/4/2009 - E627K 1005.911.3 0/6

CDC Atlanta

Acknowledgements

Pathogenesis team

Acknowledgements

Mount Sinai School of Medicine

USDA/Southeast PoultryResearch Laboratory

Centers for Disease Control and Prevention

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

University of Washington School of Medicine

The Scripps ResearchInstitute

Influenza Division/IVPB

David Swayne

Neal van HoevenDebra Wadford Jacqueline KatzNancy Cox