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