transmission of 2009 h1n1 influenza viruses in ferrets
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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