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
(Lowen AC, S. Mubareka, J. Steel, P. Palese, PLoS Pathog. 2007 3:1470)
• Ferrets – Best model for transmission and
pathogenesis
Inoculated Animals
Transmission Study: Experimental Design
Respiratory Droplet transmission
Transmission Study: Experimental Design
Respiratory Droplet transmission
Inoculated Animals
106 PFU i.n.
Naive
Inoculated Animals
Transmission Study: Experimental Design
Respiratory Droplet transmission
Transmission Study: Experimental Design
Respiratory Droplet Transmission
Contact Transmission
Contact Animals
Inoculated Animals
X2
X2
X2
Ferret Model of Respiratory Droplet Transmission
Human H1N1 vs. avian H1N1 viruses
Inoculated
Contact ferrets (3/3)
Log10 EID50/ml
8
6
4
1918 (H1N1)
(SC18 HA)
2
†
1
3
5
7
†
†
9
1
3
5
7
9
11
Log10 EID50/ml
8
No transmission
6
Avian H1N1
4
(Duck/NY/96)
2
1
3
5
7
9
1
3
5
Days post inoculation/contact
7
9
11
Ferret Model of Respiratory Droplet Transmission
Human vs. avian H2N2 viruses
Inoculated
Log10 EID50/ml
8
Contact ferrets
Human H2N2
6
(Albany/6/58)
4
2
1
3
5
7
9
1
3
5
7
9
11
Log10 EID50/ml
8
No transmission
6
Avian H2N2
4
(Mallard/NY/6750/78)
2
1
3
5
7
9
1
3
5
Days post inoculation/contact
7
9
11
What influenza virus genes confer
efficient transmission?
+
=
Virus genes ?
1918 H1N1
(transmissible phenotype)
Avian H1N1
(non-transmissible )
Efficient
Transmission
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
Virus titer (log10 EID50/mL)
Nasal Wash Titers: AV18 virus
Inoculated
8
Contact ferrets*
7
6
5
4
3
2
† †
1
1
3
5
7
9
Days post-inoculation
* Influenza sero-neg at day 0 and 18 p.c.
1
3
5
7
9
Days Post-contact
11
1918 HA and PB2 virus genes confer
efficient respiratory droplet transmission
Inoculated
Log10 EID50/ml
8
Contact ferrets
6
1918 HA:Duck/NY/96
4
2
1
3
5
7
9
1
3
5
7
11
1918 HAPB2:Dk/NY/96
8
Log10 EID50/ml
9
6
4
2
1
3
5
7
9
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
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)
A single amino acid substitution (PB2-K627E) in PB2
abolishes transmissibility of the 1918 virus
Virus titer (log10 EID50/mL)
Nasal Wash Titers: 1918 (PB2-K627E)
Inoculated
8
Contact ferrets
7
6
5
4
3
2
1
3
5
7
Days post-inoculation
9
1
3
5
7
9
Days post-contact
11
Plaque morphology of H1N1
reassortant viruses
Plaque morphology of H1N1
reassortant viruses
Transmission of 2009 H1N1 Influenza Viruses
Do similar molecular changes influence transmission
of 2009 H1N1 virus?
Transmission of 2009 H1N1 in humans
Study
Cauchemez et al.
(NEJM 2009 361:29)
Doyle & Hopkins
(Epidemiol Infect. 2010 21:1)
Lessler & Reich
(NEJM 2009 361:2628)
Leung et al.
(Epidemiol Infect. 2010 21:7)
Odaira et al.
(Eurosurveillance 2009 14:35)
•
•
Study
location
Household Secondary
Attack Rates (% ILI)
U.S.
10.1
North Carolina
14.3
New York
17.7
Hong Kong
5.9
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
in Ferrets
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
2009 H1N1 Viruses Spread Efficiently by Direct Contact
Inoculated
Log10 PFU/ml
8
Contact ferrets (3/3)
6
Mex/4482/2009
4
2
1
5
7
1
Inoculated
8
Log10 PFU/ml
3
3
5
7
9
11
Contact ferrets (1/3)
6
Avian H1N1
(Duck/NY/96)
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
Seasonal H1N1 vs. 2009 H1N1 RD Transmission
Inoculated
Contact ferrets (2/3)
Log10 PFU/ml
8
Mex/4482/2009
6
4
2
1
3
5
7
1
Inoculated
3
5
7
9
11
Contact ferrets (3/3)
Log10 PFU/ml
8
6
Brisbane/2007
(seasonal)
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
Seasonal vs. Texas/15/009 H1N1 Virus Transmission
Inoculated
Log10 PFU/ml
8
Contact ferrets (2/3)
6
Tx/15/2009
4
2
1
3
5
7
Inoculated
1
3
5
7
9
11
Contact ferrets (3/3)
Log10 PFU/ml
8
6
Solomon Is/3/06
(seasonal H1N1)
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
H1N1 Viruses of Swine Origin Failed to Form
Significant Plaques at 33oC
PB2
What is the transmission phenotype of a
2009 H1N1 PB2 (E627K) mutant virus?
Glutamic acid → Lysine at position 627
PB1
CA/04/2009
CA/04/09
PB2 627K
PB2
PA
HA
NP
NA
M
NS
PA
HA
NP
NA
M
NS
627E
PB1
PB2
627K
E627K in PB2 Enhances RD Transmission in Ferrets
Log10 EID50/ml
8
Inoculated
Contact ferrets (2/3)
CA/04/2009
6
4
2
1
5
7
1
Inoculated
8
Log10 PFU/ml
3
3
5
7
9
11
Contact ferrets (3/3)
CA/04/09
PB2 E627K
6
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
RD Transmission of a natural 2009 H1N1 E627K isolate
Inoculated
Log10 PFU/ml
8
Contact ferrets (3/3)
7
NL/1132/2009
PB2 E627K
6
5
4
3
2
1
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
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)
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
CA/04/2009
CA/04/09
HA D222G
HA
NP
NA
M
NS
NP
NA
M
NS
222D
PB1
PB2
PA
HA
222G
The effect of HA (D222G) mutation on RD transmission
Inoculated
Contact ferrets (2/3), 4/6 total
Log10 PFU/ml
8
CA/04/09
HA D222G
6
4
2
1
Log10 PFU/ml
8
3
5
7
1
Inoculated
3
5
7
9
11
Contact ferrets (2/3)
CA/04/2009
6
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
HA mutation at position 219 (I219K) improves the
human-receptor binding affinity of CA/04 HA
CA/04 HA I219K
1918 HA
Direct glycan array binding assay of HA’s to alpha 2,6 SA human receptor
HA
What is the transmission phenotype of a
2009 H1N1 HA (I219K) mutant virus?
Isoleucine → Lysine at 219 HA
PB1
PB2
PA
CA/04/2009
CA/04/09
HA 219K
HA
NP
NA
M
NS
NP
NA
M
NS
219I
PB1
PB2
PA
HA
219K
A single amino acid substitution (I219K) in HA slightly
enhances RD transmission of CA/04/2009 virus
Inoculated
Log10 PFU/ml
8
Contact ferrets (3/3)
6
CA/04/09 HA I219K
4
2
1
5
7
1
Inoculated
8
Log10 PFU/ml
3
3
5
7
9
11
Contact ferrets (2/3)
CA/04/2009
6
4
2
1
3
5
7
Days Post-Inoculation
1
3
5
7
9
Days Post-Contact
11
Comparative H1N1 Results in Ferrets
Virus
Lung Titers
Weight
(PFU/gm)
Loss (%)
(log10)
Lethality
% RD
Transmission
CA/4/2009 – Wild-type
10.3
5.8
0/6
66
CA/4/2009 - D222G
11.4
6.3
0/6
66
CA/4/2009 - E627K
11.3
5.9
0/6
100
CA/4/2009 - E219K
10.1
5.4
0/6
100
4.9
<1.2
0/6
100
Brisbane/59 (seasonal)
Acknowledgements
Pathogenesis team
CDC Atlanta
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
David Swayne
NIH Grants; 5R01 AI0506919-02 and
AI058113-01