Download 45.Inter- and intra-herd sequence variability of foot-and-mouth disease viruses recovered during the 2007 UK outbreak

Appendix 45
Summary and Conclusion
 Aim
 2007 UK outbreak: Inter- & intra- herd FMDV variability
Inter- and intra-herd sequence variability of
foot-and-mouth disease viruses recovered
during the 2007 UK outbreak
 M&M
 44 field samples, 39 animals, 8 IP
 FG sequencing strategy: Cottam et al, 2008
 Statistical parsimony methods (TCS)
 Results




Begoña Valdazo-Gonzalez, Nick J. Knowles, Jemma Wadsworth, Donald P. King
Molecular Characterisation and Diagnostics Group
Institute for Animal Health
United Kingdom
50 nt changes along the genome
Intra-herd clustering
Probably chain of transmission events
Different degree of inter- and intra-herd variability
 Discussion and conclusions
 Further knowledge epidemiological dynamics of FMDV
 Different evolutionary processes
 Acute vs chronically infected animals
FMDV evolution and diversity
FMDV molecular characterization
* putative functions
X
X
XX
X
X
X
X X
X
5x
Protease
VPG
L
VP1
X
X
X
Population diversity Cell-to-cell
infection
5’
VP1
VP1 5x
VP2 VP3
VP1
3x
VP3
VP2
VP2
VP3
VP2
VP3
2x
2x
VP3
VP2
VP1 VP1
X
X
X
5’UTR
VP3 2x VP2
X
X
X
X
AAAA
1C
VP3
1D
VP1 2A
2B
2C
3A
3B
Within-host
pathways
2A
L
1B/RNA?
1
3C
2
3C
3C
3
3C
4
3C
Kilobases
5
3C
6
7
640 nt
1%
Type O
ASIA
- Recombination
EA-3
- Host immune response
!(
(!
Animal-animal
transmission
(!
(!!(!(
!(!(
!(
!(
ME-SA
EURO-SA
EA
EA
SEA
-4
-2
A-Iran-05
CATHAY
ISA-1
ISA-2
EURO-SA
A11/GER/29 (AGB)
Background: 2001 UK outbreak
?
WA
EA-1
AFRICA
Outbreak
epidemiology
Farm-to-farm
transmission
3D
3C
VP1 region
Type A
- Natural selection
3’UTR
3C
AAA (n)
0
- High replication rates
- Large population size
100.000 viral copies/10 hours
(
!( !(!(
!(!(
!(!(
!(
!(
!(!(
!( !(!( !( !(
!(!(!(!(!(!(!(!(!(!(!( !(
!(!(!(!(!( !(!(!(!(!(!(!(!(!(!( !(!(!( !(
!( (!!( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !( !(!(!( !( !( !(!(!(!(!(!(!(!(
!( !( !( !( !(!(!(!( !(!( !( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !( !(!(!(!(!(!(!(!(!(
!(
!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !( !( !(!( !(!(!(!(
!( !( !(!(!(!(
!(!(!(!(!(!( !(
!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(
!( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !((! !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !(
!(
!( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !( !( !(!(
!(!(!(!(!(!(
!(
!( !(
!( !(!( !(!( !(
!( !(!(!( !(
!( !(!(!( !( !(!(!(!(!(!( !( !( !(!(!(!(!(
!( !( !(
!(!(!( !(!(!(!(!( !(!(!(!(!( !(
!( !( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(
!(
!(
!( !(
!(
!(
!( !(
!(!( !( !(!(
!(!(!(!(!(
!( !(
!( !(!( !(
!( !( !(!(!(!( !(
!(!(
!( !(
!(
!( !( !( !( !(!( !( !(!(!(!( !(
!(!(!(!(!(!(!(!(!(!( !(!( !(
!(!(!( !( !(!( !(!(!(
!( !(!(!(
!( !(
(
!
(
!
!(!(!(
!(
!(
!(
!(
!(!(!( !(!(!(
!(!( !(!( !(!(!(
!(!(!(!( !(!(!( !(
!(!(!(!(
!( !(!(!(!(!(!(!(
!(!(!(!(!(!( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !(!(!(!(!( !(!(
!(
!( !( !( !(!(!(!(!(!(!(!(!(!(
!( !(
!(
!( !( !( !(!(!(!(!(!(!(!(!(!(
!(
!(!( !(
(
!
!(!(!(
!(
!(
!(
!(!(!(!(
!( !(
!(!(!( !(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!(!( !( !(!(!(
!(!( !(!(!(!(!(!(!(!(!(!(!(!(!(!( !(
!(
!(
!(!(
!( !( !(!(!(!(!(!(!(!(!(!(!(!(!( !(
!( !( !(!( !(
!(
!(
1B
VP2
5x
New viral variants
- High error rate of virus polymerase
10-3–10-4 misincorporations
nucleotide
1A
VP4
Polymerase
Poly(C)
Primary cleavages
Secondary cleavages
Intra-cellular
dynamics
Membrane-binding
Genome-linked
(VPg)
Protease
Carboxy-terminal
self-cleaving
NTP binding*
Capsid
8
A/IRN/41/2003
A/IRN/7/2004
A/PAK/5/2006
A/SAU/15/2005
A/SAU/16/2005
A/IRN/30/2005
A/IRN/22/2005
A/IRN/27/2005
A/IRN/40/2005
A/IRN/50/2005
A/IRN/53/2005
A/IRN/36/2005
A/IRN/24/2005
A/IRN/34/2005
A/IRN/25/2005
A/PAK/1/2006
A/PAK/3/2006
A/IRN/44/2005
A/IRN/7/2005
A/IRN/4/2005
A/IRN/1/2005
A/IRN/2/2005
A/IRN/5/2005
A/IRN/31/2005
A/TUR/2/2006
A/TUR/3/2006
A/TUR/1/2006
A/IRN/38/2005
A/IRN/39/2005
A/IRN/26/2005
A/IRN/33/2005
A/IRN/42/2005
A/IRN/43/2005
A/IRN/55/2005
A/IRN/29/2005
A/IRN/28/2005
A/IRN/51/2005
A/IRN/54/2005
A/IRN/13/2005
A/IRN/18/2005
A/IRN/10/2005
A/IRN/14/2005
A/IRN/16/2005
Background: 2007 UK outbreak
IAH2
IP6b
AY593815
IAH1
IP1b(2)
MAH
IP3c
IP1b(1)
M4
IP4b
WINDSOR
IP2b
HEATHROW
IP7
IP5
• Detected by sero-surveillance
• After IP3 and IP4
• Seropositive cattle and sheep
• No acute clinical signs
• Evidence of healed lesions
IP2c
8 8
EGHAM
6 6 3b
3b
3c 3c
7 7 4 4
5 5
X
X
X
M3
FIELD EPI DATA
1
Pirbright
WOKING
X
X
IP1b(2)
IP1b(1)
IP2c
IP2b
M25
IP5
IP4b
IP3c
IP3b
2b 2b
2
1b 1b
Infection profiles of farms
J
I
Statistical parsimony analysis (TCS)
GUILDFORD
IP8
X
KEY
1c
Likelihood of being infectious
X
30-Sep-07
23-Sep-07
02-Sep-07
09-Sep-07
19-Aug-07
Date
FMD confirmed
Preclinical (lab only)
TIME
26-Aug-07
12-Aug-07
F
C
E
O
N
L
K
B
A
10 km
GODALMING
Likelihood of infection
29-Jul-07
Putative ancestor virus
Field epidemiological data
Genetic data
IP6b
IP7
X
2c
ALDERSHOT
M
G
D
05-Aug-07
4
22-Jul-07
3
16-Sep-07
SEQUENCE DATA
IP5
Sampled virus
Putative ancestor virus
Nt change
Aa change
His to Arg
Asp to Gly
X
Location
Date of cull
Number of animals
Est. age of oldest lesion
Relationship
between sequences
IP8
IP3b
No evidence of infection
“New tools and challenges for progressive control”
Open Session of the EuFMD Research Group, Vienna (Austria) 29 September ‐ 1 October 2010
Lesion age estimation
Incubation period (<14 days)
Most likely date of infection
(2-5 days before clinical disease)
Appendix 45
Objectives:
Material & Methods
 Detailed investigation of the inter- and intra- herd consensus sequence
variability during the 2007 UK outbreak
(Ryan et al. 2008)
5’ UTR
Original clinical sample
• Epithelium suspension
• Blood
• Oesophageal/pharyngeal scrapings
1. To develop application tools for fine-scale
molecular FMDV epidemiology
FMDV
3’ UTR
AAAA
Poly C
~700b
RNA extraction
• TRIZOL
• RNeasy Mini Kit (QIAGEN)
 Analytical models to integrate molecular
and field epidemiology data
Reverse Transcription
• Oligo-dT primer (Rev 6)
 Estimation of undisclosed FMD circulation in
endemic regions
 Give clues about how virus persistence is
maintained and could be blocked
~700b
24 tagged
primer pairs for
amplification
cDNA clean up
PCR
DNA clean up




10 GenBank sequences
2. To increase the knowledge about FMDV
evolution
Evolution rates
Sites and importance of recombination
Identification of ordered structures
Contribution of quasi-species to evolution
Cycle sequencing reaction
- 44 clinical samples
- 39 animals
- 8 infected premises (IP)
- Up to 7 per location
34 new sequences
Statistical parsimony methods (TCS)
IP3
IP2
IP1b
Results & Discussion
IP2b
IP2c
IP3b
Ethanol Precipitation
ABI PRISM 3730 DNA Analyzer
24 inner and 1 outer primer pairs for sequencing
Up to 8 coverage/site
Data analysis
IP6
IP3c
IP4 IP5
IP7
IP6b IP6a
IP8
Results & Discussion
 Full genome
 Full genome
 50 substitutions
†62 considering ambiguities
Exclusive: 12
60
- 12 sites, 9 isolates
6, 4 esophageal/pharyngeal
scrapings
50
Cumulative Substitutions
Not exclusive: 4
- 3 sites, 4 isolates
* Blood, IP·3
Polyprotein
 42 substitutions
†54 considering ambiguities
 7 out of 12 non synonimous
5, 4 esophageal/pharyngeal
scrapings
40
30
20
10
25 synonymous
0
17 non synonymous
0
1000
2000
3000
4000
5000
6000
Genome position
Results & Discussion
Results & Discussion
 2001 UK outbreak (Cottam et al., 2006)
 Poliprotein
9 NS substitutions
 23 full genome sequences
 191 nt substitutions
 7 months
 Nt substitution rate:
2.26 X 10-5/site/day
7 NS substitutions
30
Cumulative Substitutions
25
20
15
NS
S
10
5
 Future work
 Mathematical analysis
 dN/dS
 Recombination
 Analytical models
Cottam et al. 2006
0
0
1000
2000
3000
4000
5000
6000
7000
8000
Genome position
“New tools and challenges for progressive control”
Open Session of the EuFMD Research Group, Vienna (Austria) 29 September ‐ 1 October 2010
7000
8000
Appendix 45
Results & Discussion
Results and Discussion
IAH2
IP6b
IP1b(2)
AY593815
MAH
IP2b
(95)
IP3b
IP2b
IAH2 & IAH3
IP1b (7)
IP1b (9)
MAH
No. of genomes = 10
IP2c
IP3c
IP3b
(1153 &1170) (648b, 649b)
IP3b (650a)
IP5 (1426a)
&
IP3b (642, 643, 644, 645,
IP4b (800)
646, 647, 648a & 649a)
IP5 (1426b)
IP1b (11)
IP5 (1425)
IP4b (805)
IP5 (1421a)
IP5 (1421b)
M3
M3
1b 1b
IP5 (1418a-h)
No. of genomes = 44
WOKING
X
X
X
2c
KEY
X
1c
IP2b
(95)
0-1
2
IP2b
(92b)
2
IP2b
IP5
0-6
1-12
4
IP4
0
IP2b
(91 & 96)
IP1b (7)
IP1b (9)
2
0-3
1
1
IP3c
0
IP3c
IP3b
(1153 &1170) (648b, 649b)
IP3b (650a)
IP5 (1426a)
&
IP3b (642, 643, 644, 645,
IP4b (800)
646, 647, 648a & 649a)
IP5 (1426b)
IP2b
(92a, 97)
IP6
2
0
IP5 (1425)
IP2c (132, 150
& 158 a,b,d)
IP2c (158b)
IP2c (158d)
IP2c (158c)
IP5 (1421a)
IP4b (805)
IP5 (1421b)
IP6a & b
(1484, 1485 & 1486)
No evidence of infection
IP8
0-5
IP7 (1709b)
 Institute for Animal Health
 Staff involved in the data and specimen collection from the 2007
UK outbreak
 Nigel Ferris
IP7 (1684)
IP8 (2366)
 Geoff Hutchings
IP7 (1704)
 Department for Environment, Food and Rural Affairs (SE2938)
Complete genome sequence of virus
HS- lab virus
HS+ lab virus
IP1b
IP2b
IP2c
IP3b
IP3c
IP4b
IP5
IP6b
IP7
IP8
1
IP7 (1701)
IP7 (1694,
1679 & 1609a)
IP3b (650b)
IP7
Inter- herd variability: 0-4
Intra- herd variability: 0-12
IP7 (1693)
IP1b (11)
X
Acknowledgements
IP3b
2
IP2c
0
IP2b
(93b)
IP2b
(93a)
IP1b (7A, 13 & 32)
& IP2b (94)
GUILDFORD
GODALMING
Preclinical (lab only)
2
No. of genomes = 44
X
2c
FMD confirmed
0
IP5 (1418a-h)
IP5 (1419a)
Possible intermediate virus
Nucleotide substitution that is silent
Nucleotide substitution causing a change in amino acid
Nucleotide substitution causing an amino acid change (His to Arg)
important for heparan sulphate binding (cell culture adaptation)
Nucleotide substitution causing an amino acid change (Asp to Gly)
associated with, but not critical for, heparan sulphate binding
Ambiguous nucleotide substitution
IP5 (1419b)
IP5
• Detected by sero-surveillance after IP3 and IP4
• Seropositive cattle and sheep
• No acute clinical signs
• Evidence of healed lesions
• Samples: esophageal/pharyngeal scrapings sheep
M25
ALDERSHOT
10 km
X
IP1b
WOKING
X
X
2b
1b 1b
GUILDFORD
GODALMING
Ambiguous nucleotide substitution
MAH
Pirbright
2b
Results & Discussion
IAH2 & IAH3
M25
ALDERSHOT
IP5 (1419b)
Nucleotide substitution that is silent
Nucleotide substitution causing a change in amino acid
Nucleotide substitution causing an amino acid change (His to Arg)
important for heparan sulphate binding (cell culture adaptation)
Nucleotide substitution causing an amino acid change (Asp to Gly)
associated with, but not critical for, heparan sulphate binding
X
X
X
2b 2b
Possible intermediate virus
3c 3c
X
5 5
X
Complete genome sequence of virus
IP5 (1419a)
3b EGHAM
IP7 (1704)
Pirbright
HS- lab virus
HS+ lab virus
IP1b
IP2b
IP2c
IP3b
IP3c
IP4b
IP5
IP6b
IP7
IP8
6
3b
7 7 4 4
X
X
HEATHROW
8 8
6
X
5 5
IP8 (2366)
IP7 (1709b)
IP6a & b
(1484, 1485 & 1486)
WINDSOR
HEATHROW
8 8
EGHAM
6 6 3b
3b
3c 3c
7 7 4 4
IP7 (1684)
IP7 (1694,
1679 & 1609a)
IP3b (650b)
M4
WINDSOR
IP7 (1701)
IP7 (1693)
IP2c (132, 150
& 158 a,b,d)
IP2c (158b)
IP2c (158d)
IP2c (158c)
M4
IP7
IP2b
(92a, 97)
IP2b
(91 & 96)
IP2b
(93a)
IP1b (7A, 13 & 32)
& IP2b (94)
Present work
IP8
IP4b
Cottam et al., 2008
IP2b
(93b)
Cottam et al., 2008
IP3c
IP1b(1)
IAH1
IP2b
(92b)
 Biotechnology and Biological Sciences Research Council
“New tools and challenges for progressive control”
Open Session of the EuFMD Research Group, Vienna (Austria) 29 September ‐ 1 October 2010
10 km