Transmission of 2009 H1N1 Influenza Viruses in Ferrets
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Transcript of Transmission of 2009 H1N1 Influenza Viruses in Ferrets
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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
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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)
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Ino
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Respiratory Droplet transmission
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Naive
Transmission Study: Experimental DesignRespiratory Droplet transmission
106 PFU i.n.
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Ino
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Ani
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Respiratory Droplet transmission
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Co
nta
ct A
nim
als
Transmission Study: Experimental Design
Contact Transmission
X2
X2
X2
Respiratory Droplet Transmission
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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)
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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
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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
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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
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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
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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
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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)
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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)
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Plaque morphology of H1N1 reassortant viruses
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Plaque morphology of H1N1 reassortant viruses
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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
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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%)
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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
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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
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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
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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
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H1N1 Viruses of Swine Origin Failed to Form Significant Plaques at 33oC
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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CDC Atlanta
Acknowledgements
Pathogenesis team
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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