Nick J. Knowles, Eleanor M. Cottam, Jemma Wadsworth, Katja Ebert, Donald P. King and
David J. Paton
Institute for Animal Health, Pirbright Laboratory, Ash Road,
Pirbright, Woking, Surrey, GU24 0NF, UK.
Conclusions� VP1 sequencing is useful for estimating relationships
between viruses in different epidemics
� However, the resolution (~64o nt) is too low for discriminating closely related viruses within the same epidemic
� Complete genome sequences (~8200) can discriminate between viruses infecting different animals and can therefore be used for tracing the course of an epidemic
IntroductionWhen an outbreak of FMD occurs in a FMD-free
country it is important, from an epidemiological point of view, to:
� Trace the origin of the introduced virus
� Establish transmission routes within the new epidemic
� Predict unrecognised infected premises
� Analyses of complete genome sequences can possibly achieve these aims
-AAAAAAnCCCn-pk-pk-pk
Poly(C) tract & pseudo-knots Poly(A) tail
1)
FMDV genome
-AAAAAAnCCCn-pk-pk-pk
Poly(C) tract & pseudo-knots Poly(A) tail
2)
FMDV genome
-AAAAAAnCCCn-pk-pk-pk
Poly(C) tract & pseudo-knots Poly(A) tail
M13-F M13-R
3)
4)
FMDV genome
New method needed…� Which will allow us to sequence a complete FMDV
genome in less that 24 h without prior knowledge of the serotype…
� …and which preferably does not require excessive numbers of primers.
Timescale: Sample to Tree� Sample processing
� RNA extraction
� RT-PCR
� Visualization of amplicons
� Amplicon clean-up
� Cycle sequencing
� Ethanol precipitation
� Automated sequencing
� Genome sequence assembly
� Phylogenetic analysis
Less than 24 h
(a)
(c)
(f)(e)
(d)
(b)Wood lane, Normandy
(a) – stream running through fields
(b) – drooling observed within a herd of 38 beef cattle held at pasture
(c) – Recent 3 day tongue lesion
(d) – Older 8 to 9 day old tongue lesion (first signs of disease on July 29th?)
(e) and (f) – 5 to 6 day interdigital foot lesions
Outbreaks in August
•6 Aug: IP2 (3 locations)
•3 contact herds culled
•24 Aug: PZs lifted
•8 Sep: SZ lifted
IP6
•Confirmed 21 September
•34 cattle
•2 locations
•2 out of 32 cattle at 1
location with 2-4 day
lesions
•2 virus +ve
•All seronegative
IP5
•Detected 16 September. No
acute signs.
•17 out of 22 cattle with 2-3 week
old lesions. All seropositive,
virus negative.
•12 out of 16 sheep seropositive;
10 with old lesions. 2 pigs –no
lesions; seronegative, virus
negative.
•Confirmed 17 September.
Single location.
X
ALDERSHOT
XX
WOKING
X
XGUILDFORD
X
XX
M3
M25
IP8
•Confirmed 29 September
•134 cattle, 16 sheep
•4 locations (3 PZ, 1 SZ,
just outside PZ)
•54 cattle at infected site,
estimated maximum 3-4
days.
•15/54 cattle viraemic
•Other sites no signs,
initial serology negative
IP7
•Confirmed 24 September
•16 cattle
•Single location
•14 with acute signs
•1-4 day lesions
•15 virus +ve
•2 seropositive (with 4 day
lesions)
IP4
•Confirmed 15 Sep
•54 cattle (location B)
•743 pigs (location A)
•Clinical findings in cattle only
•Laboratory –cattle positive, pigs
negative
•Culling completed 16 Sep
IP3
•Confirmed 12 Sep 2007
•281 cattle
•8 pigs
•8 locations
•Clinical findings
•2 locations positive in lab
•Culling completed 16 Sep
Outbreaks in August
•3 Aug: IP1 (3 locations)
10 km
GODALMING
Outbreaks in August
•Close to Pirbright site
X
FMD confirmed
Preclinical (lab only)
No evidence of infection
KEY
29
-Ju
l -07
19
-Au
g-0
7
12
-Au
g-0
7
05
-Au
g-0
7
26
-Au
g-0
7
02
-Sep
-07
09
-Sep
-07
16
-Sep
-07
23
-Sep
-07
Date
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP1b(2)
IP1b(1)
IP6b
IP7
22
-Ju
l -07
15
-Ju
l -07
IAH2
IAH1
AY593815
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP6b
IP1b(1)
IP1b(2)
IP7
MAH
30
-Sep
-07
IP8
*
IP8
29
-Ju
l -07
19
-Au
g-0
7
12
-Au
g-0
7
05
-Au
g-0
7
26
-Au
g-0
7
02
-Sep
-07
09
-Sep
-07
16
-Sep
-07
23
-Sep
-07
Date
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP1b(2)
IP1b(1)
IP6b
IP7
22
-Ju
l -07
15
-Ju
l -07
IAH2
IAH1
AY593815
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP6b
IP1b(1)
IP1b(2)
IP7
MAH
30
-Sep
-07
IP8
*
IP8
10 km
5
7
6
8
1b
43b
3c
2b
ALDERSHOT
HEATHROWWINDSOR
XXXX
WOKING
XX
M4
XXGUILDFORD
EGHAM
XX
XXXX
M3
M25
1b
2b
2c
5
4 3c
3b6
7
8
Pirbright
X
FMD confirmed
Preclinical (lab only)
No evidence of infection
KEY
GODALMING
10 km
aa / c
odo
n in
Substitu
ted
AY
593
815
aa /
cod
on
159 C C C C C C C C C C C C C C A 5' UTR - - -
170 C C C C T T T T T T T T T T T 5' UTR - - -
262 T T T T T T T T T T T T T C T 5' UTR - - -
792 G G G G G G G A G G G G G G G 5' UTR - - -
913 A A A A A A A A T T T A T T T 5' UTR - - -
1083 C C A A A A A A A A A A A A A 5' UTR - - -
1181 G G A G G G G G G G G G G G G Lb 2 E (gaa) K (aaa)
1951 C C C C C T C C C C C C C C C VP4 - - -
2184 G A G G G G G G G G G G G G G VP2 78 C (tgc) Y (tac)
2377 T T T T C C C C C C C C C C C VP2 - - -
2446 C C C C C C C C T T T T T T T VP2 - - -
2558 A A A A A A G A A A A A A A A VP2 203 I (att) V (gtt)
2772 A A G G A A A A A A A A A A A VP3 56 H (cac) R (cgc)
2784 A A G G G G G G G G G A G G G VP3 60 D (gac) G (ggc)
3043 A A A A A A A G A A A A A A A VP3 - - -
3157 C C C C C C C C C T C C C C C VP3 - - -
3433 T T T T C C C C C C C C C C C VP1 - - -
3862 C C C C C C C C C C C C C C T VP1 - - -
3994 G G G G G G G G G G G G G A A 2B - - -
4045 T T T T T T Y T T T T T T T T 2B - - -
4407 A A A A A A A A A A A A A A G 2B 152 E (gag) G (ggg)
4639 A A A A A A A A G A A A G G G 2C - - -
4927 C C C C C C C C C C C T C C C 2C - - -
5152 C C C C C C C C C C C Y C C C 2C - - -
5335 C C C C T T T T T T T T T T T 2C - - -
5462 G G G G G G G G G G G G G A A 3A 32 D (gac) N (aac)
5473 A A A A A A A A G G G G G G G 3A - - -
5492 A A C C C C C C C C C C C C C 3A 42 I (atc) L (ctc)
5592 C T T T T T T T T T T T T T T 3A 75 T (acg) M (atg)
5599 T C T T T T T T T T T T T T T 3A - - -
5655 A A A A A A A A A A A A G A A 3A 96 K (aaa) R (aga)
5808 A A A A A A A A A A A A G A A 3A 147 E (gag) G (ggg)
6175 T T T T T T T T C T T T C C C 3C - - -
6616 A A A A A A A A A A A A A A G 3C - - -
6673 T T T T T T T T C C C T C C C 3C - - -
7021 C C T T T T T T T T T T T T T 3D - - -
7162 C C C C C C C C T C C C T T T 3D - - -
7729 C C C C C C C C T T T C T T T 3D - - -
7750 T T T T T T T T C C C T C C C 3D - - -
8149 A A G G G G G G G G G G G G G 3' UTR - - -
8152 C C C C T T T T T T T T T T T 3' UTR - - -
8176 A A A A A A A A A A A G A A A 3' UTR - - -
8180 A A A A A A T A T T T T T T T poly(A) - - -
UK
G/1
421/2
00
7 (
IP5)
UK
G/1
484/2
00
7 (
IP6)
UK
G/1
679/2
00
7 (
IP7)
UK
G/2
366/2
00
7 (
IP8)
Ge
ne
Cod
on n
o.
in g
en
e†
UK
G/7
/200
7 (
IP1b
)
UK
G/9
3/2
007 (
IP2
b)
UIK
G/1
50/2
007 (
IP2c)
UK
G/6
43/2
007
(IP
3b)
UK
G/1
153/2
00
7 (
IP3c)
UK
G/8
00/2
007
(IP
4b)
Positio
n*
AY
593
815
IAH
1
IAH
2
MA
H
UK
G/7
B/2
00
7 (
IP1b
)
*, numbered according to AY593815; the following positions were not sequenced: 1-17and 363-466†, only listed where an amino acid substitution has occurred.
MAH
B (IP1b)
IAH2 & IAH3
IAH1
AY593815
AY593816
IP1b (7)
IP2b (93)
IP2b (93)
IP2c (150 & 158)
IP2c (158)
IP2c (158)
IP2c (158)
IP1b (9)
IP3b (642, 643,
644 & 645)
IP4b (800)
IP4b (805)
IP3c (1153)
IP5 (1421)
IP5 (1421)
IP6b (1484)
IP7 (1679)IP2b (91)
IP8 (2366)IP2b (92)
IP2b (92)IP2b (92)
IP2b (92)
IP2b (95)
IP2b (94)
No. of genomes = 26
IP1b (11)
HS- lab virus
HS+ lab virus
IP1a
IP2b
IP2c
IP3b
IP3c
IP4b
IP5
IP6b
IP7
IP8
Nucleotide substitution that is silent
Nucleotide substitution causing a change in amino acid
Ambiguous nucleotide substitution
Complete genome sequence of virus
Possible intermediate virus
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
Cow A Cow B+2 Cow B+3
Cow A+3
IAH2
Cow A+2
Cow A+1
Cow B+1
Cow B
Cow B (2 days later)?
Cow B+2Cow B+3
Indirect contact
Inoculation
Nick Juleff, Bryan Charleston, Jemma Wadsworth
Conclusions� VP1 sequencing is useful for estimating relationships
between viruses in different epidemics
� However, the resolution (~64o nt) is too low for discriminating closely related viruses within the same epidemic
� Complete genome sequences (~8200) can discriminate between viruses infecting different animals and therefore be used for tracing the course of an epidemic
Acknowledgements� John Gloster
� Andrew Shaw
� Rebecca Rowlands
� Lynnette Goatley
� Sushila Maan
� Narender Maan
� Peter Mertens
� Yanmin Li
� Eoin Ryan
� Nicholas Juleff
� Nigel Ferris
� Geoff Hutchings
� John Wilesmith
� Dan Haydon
� Bryan Charleston