Transcript Identification of a ninth foot-and

WRLFMD
Identification of a Ninth Footand-Mouth Disease Virus Type O
Topotype and Evidence for a
Recombination Event in its
Evolution
Nick J. Knowles, Paul R. Davies,
Rebecca J. Midgley and Jean-François Valarcher
Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking,
Surrey, GU24 0NF, UK
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WRLFMD
FMDV O in Africa
TUNISIA
MOROCCO
ALGERIA
LIBYA
EGYPT
WESTERN
SAHARA
MAURITANIA
MALI
NIGER
ERITREA
CHAD
SUDAN
SENEGAL
THE GAMBIA
GUINEA-BISSAU
BURKINA
FASO
GUINEA
SIERRA-LEONE
LIBERIA
COTE
D'IVOIRE
DJIBOUTI
TOGO
NIGERIA
ETHIOPIA
CENTRAL
AFRICAN REPUBLIC
BENIN
GHANA
CAMEROON
KENYA
RWANDA
CONGO
Endemic?
Sporadic
Not known
or no FMD
SOMALIA
UGANDA
EQUATORIAL
GUINEA
GABON
D.R.
CONGO
BURUNDI
ANGOLA
TANZANIA
ANGOLA
ZAMBIA
MALAWI
MOZAMBIQUE
ZIMBABWE
NAMIBIA
BOTSWANA
SWAZILAND
IAH
SOUTH
AFRICA
LESOTHO
MADAGASCAR
WRLFMD
Previous studies
• Few studies on the European FMDV serotypes in Africa:
–
–
–
–
–
–
Knowles et al., 1998 (type A)
Samuel et al., 1999 (type O)
Samuel & Knowles, 2001 (type O)
Sangare et al., 2001 (type O)
Sahle et al., 2004 (type O)
Bronsvoort et al., 2004 (types O & A)
• Distinct genetic lineages of both FMDV-O and FMDV-A circulate in
East and West Africa and viruses occurring in North Africa may be
introduced from a variety of sources (Europe, South America, Middle
East and West Africa).
• Outbreaks of FMD types O and A in southern Africa are very rare
and are usually introduced from outside the continent.
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FMDV O Topotypes - 2001
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Samuel, A.R. & Knowles, N.J. 2001. Foot-and-mouth disease type O viruses exhibit genetically
and geographically distinct evolutionary lineages (topotypes). J. Gen. Virol. 82: 609-621.
WRLFMD
RT-PCR & Sequencing
Capsid
5’ UTR L
VPg
Non-structural proteins
P1
P2
P3
3’ UTR
IRES
AAAAAAAAAn
VP4 VP2 VP3 VP1 2A2B
Poly C
O-1C244F
O-1C272F
O-1C283F
2C
3A 3B
3C
3D
NK61
NK61
NK61
Primers for RT-PCR an sequencing of FMDV O RNA.
Genome location
Designation
IAH
Sequence (5' to 3')
Gene
Position within the gene
O-1C244F
GCAGCAAAACACATGTCAAACACCTT
VP3
244-269
O-1C272F
TBGCRGGNCTYGCCCAGTACTAC
VP3
272-294
O-1C283F
GCCCAGTACTACACACAGTACAG
VP3
283-305
NK61
GACATGTCCTCCTGCATCTG
2B
58-77
NK72
GAAGGGCCCAGGGTTGGACTC
2A/2B
34-48; 1-6
O-1C499F
TACGCGTACACCGCGTC
VP3
499-515
O-1C583F
GACGGYGAYGCICTGGTCGT
VP3
583-602
WRLFMD
VP1
(complete)
IAH
O2/Brescia/ITL/47
O1/Kaufbeuren/FRG/66
O3/VEN/51
O/ISA/1/74
1000
ISA-2
O/JAV/5/72
O/ISA/1/62
1000
ISA-1
O/ISA/9/74
961
O/ISA/8/83
O/HKN/14/82
1000
O/HKN/6/83
Cathay
732
O/PHI/7/96
1000
745
O/PHI/5/95
503
O/TAW/81/97
O/CAM/2/98
1000
O/VIT/7/97
993
SEA
O/TAI/189/87*
984
725
O/TAI/4/99
O/A/CHA/58
O/IND/R2/75*
ME-SA 220 639
O1/Manisa/TUR/69
320
O/IND/53/79
522
O/IRQ/30/2000
1000
O/TAW/2/99
932
987
O/SAR/1/2000
973
O/UKG/12/2001
O/MAU/19/2000
724
O/BKF/1/2002
1000
O/NGR/11/2001
497
O/ALG/2/99
723
996
O/CIV/8/99
O/CAR/6/89
WA
676
182
1000
O/GHA/5/93
O/CAR/16/2000
1000
230
O/CAR/17/2000
O/ERI/1/96
1000
O/ETH/8/94
EA-1? 806
O/SUD/25/2004
1000
186
O/SUD/16/2004
594
O/SUD/26/2004
819
O/SUD/30/2004
O/KEN/102/60
EA-1 604
O/UGA/5/96
988
O/KEN/83/79
997
O/KEN/2/95 [K10/95]
67
O/UGA/3/72
EA-2?
O/TAN/3/96
O/BUN/3/2003
1000 O/BUN/7/2003
832
O/BUN/6/2003
680
O/KEN/5/2002
385
510
O/KEN/7/2002
O/KEN/3/2002
O/UGA/5/2002
415
654
O/UGA/4/2004
843
996 641 O/UGA/18/2004
EA-2 968
O/UGA/1/2004
870
O/UGA/3/2004
882
949
O/UGA/6/2004
967 O/UGA/8/2004
597 O/UGA/5/2004
965
562
O/UGA/7/2004
O/UGA/6/2002
1000
O/UGA/3/2002
O/TAN/7/98
513
1000
O/MAL/1/98
993
O/MAL/2/98
778
O/ZAM/1/2000
1000
O/ZAM/2/2000
O/RWA/2/2004
1000
O/RWA/3/2004
Euro-SA
996
574
10%
WRLFMD
VP1
(145-642)
IAH
Euro-SA
O3/VEN/51
822
O/ANG/10/74
O/ANG/1/75
O1/Kaufbeuren/FRG/66
O2/Brescia/ITL/47
O/ISA/1/74
1000
ISA-2
O/JAV/5/72
O/ISA/1/62
1000
O/ISA/9/74
ISA-1
840
O/ISA/8/83
O/HKN/14/82
520
O/HKN/6/83
1000
O/PHI/7/96
Cathay
1000
664
O/PHI/5/95
524
O/TAW/81/97
O/CAM/2/98
1000
O/VIT/7/97
995
SEA
O/TAI/189/87*
931
O/TAI/4/99
905
O/A/CHA/58*
O/IND/R2/75*
723
ME-SA 310
O1/Manisa/TUR/69
393
O/IND/53/79
586
O/IRQ/30/2000
1000
O/TAW/2/99
953
967
997 O/SAR/1/2000
O/UKG/12/2001
O/KEN/102/60
O/NGR/1/88
280
O/MAU/19/2000
855
O/BKF/1/2002
1000
O/NGR/11/2001
996
WA
573
O/ALG/2/99
643
979
O/CIV/8/99
354
O/CAR/6/89
368
653
O/GHA/5/93
1000 O/CAR/16/2000
O/CAR/17/2000
O/UGA/3/72
O/TAN/3/96
O/BUN/3/2003
1000 O/BUN/7/2003
778
O/BUN/6/2003
644
201
O/KEN/5/2002
543
O/KEN/7/2002
367
1000 O/TAN/7/98
156
783 O/MAL/1/98
865
O/MAL/2/98
EA-2 832
1000 O/ZAM/1/2000
O/ZAM/2/2000
O/KEN/3/2002
963
O/UGA/5/2002
531
O/UGA/4/2004
887
O/UGA/18/2004
900 696
860 O/UGA/1/2004
O/UGA/3/2004
916
968
O/UGA/6/2004
737
786 O/UGA/8/2004
561
559 O/UGA/5/2004
O/UGA/7/2004
1000 O/UGA/3/2002
O/UGA/6/2002
1000 O/RWA/2/2004
O/RWA/3/2004
107
O/ETH/15/01* [OVI]
O/ERI/1/96
243
1000
O/ERI/1/96 [OVI]
643
1000 O/ETH/8/94
O/ETH/8/94 [OVI]
O/ETH/16/01* [OVI]
1000
933
O/ETH/22/01* [OVI]
O/ETH/1/95 [OVI]
315
436
999 O/ETH/3/90 [OVI]
O/ETH/8/90 [OVI]
654
539
O/ETH/12/90 [OVI]
463
767
997 O/ETH/2/93 [OVI]
O/ETH/24/94 [OVI]
O/ETH/19/77 [1983]* [OVI]
771
O/ETH/5/95 [OVI]
O/ETH/1/79
[OVI]
908
EA-3 829
O/ETH/3/79 [OVI]
675
O/ETH/9/92 [OVI]
O/ETH/30/94 [OVI]
743
O/ERI/2/96 [OVI]
981
O/ETH/3/96 [OVI]
898
1000 O/SUD/16/2004
O/SUD/25/2004
502
O/SUD/26/2004
760
O/SUD/30/2004
O/UGA/5/96
988
O/KEN/83/79
EA-1
998
1000 O/KEN/2/95
O/KEN/10/95 [OVI]
1000
946
542
10%
WRLFMD
VP1
(478-642)
Euro-SA
522
O3/VEN/51
401
ISA-2
ISA-1
883
408
Cathay
357
SEA
987
O/CAM/2/98
951
615
512
293
247
WA
131
IAH
644
110
141
SEA
EA-1
O1/Kaufbeuren/FRG/66
O2/Brescia/47
1000 O/ISA/1/74
O/JAV/5/72
O/ISA/1/62
O/ISA/9/74
785
O/ISA/8/83
O/HKN/14/82
O/HKN/6/83
797
O/TAW/81/97
588
O/PHI/5/95
813
O/PHI/7/96
711
O/VIT/7/97
O/MAU/19/2000
O/BKF/1/2002
756
O/NGR/11/2001
773
694 O/ALG/2/99
O/CIV/8/99
O/CAR/6/89
O/GHA/5/93
1000 O/CAR/16/2000
O/CAR/17/2000
O/TAI/189/87*
998 O/SUD/16/2004
O/SUD/25/2004
436
O/SUD/26/2004
679
O/TAI/4/99
O/SUD/30/2004
O/ERI/1/96
O/ETH/8/94
O/KEN/102/60
EA-1
169
O/TAN/7/98
995
O/MAL/1/98
EA-2
971
43
94
O/MAL/2/98
O/UGA/5/96
844
EA-1
O/KEN/83/79
993
O/KEN/2/95 [K10/95]
O/IND/53/79
ME-SA
78
O/IND/R2/75*
630
O1/Manisa/TUR/69
ME-SA 81
O/A/CHA/58
223
O/IRQ/30/2000
12
810
O/TAW/2/99
31
871
903 O/SAR/1/2000
O/UKG/12/2001
O/UGA/3/72
EA-2 268
O/TAN/3/96
4
998O/RWA/2/2004
O/RWA/3/2004
O/KEN/5/2002
O/KEN/7/2002
49
165
EA-2 86
O/BUN/3/2003
980
599 O/BUN/7/2003
77
O/BUN/6/2003
335
996 O/UGA/6/2002
O/UGA/3/2002
O/KEN/3/2002
143
989 O/ZAM/1/2000
O/ZAM/2/2000
268
O/UGA/4/2004
504
O/UGA/5/2002
543 O/UGA/18/2004
O/UGA/8/2004
3
553 O/UGA/7/2004
11
27 O/UGA/5/2004
190
707 O/UGA/1/2004
O/UGA/3/2004
O/UGA/6/2004
10%
EA-1
769
WRLFMD
Scanning analysis of VP1
SimPlot - Query: EA-2 O/UGA/3/2002
FileName: N:\evd\MEG\db\FMDV\O\Crete-k2.fas
100 %
99 %
WA1
EA2
SEA
MES
EUR
CAT
IS1
IS2
EA1
98 %
97 %
96 %
95 %
94 %
93 %
92 %
91 %
90 %
89 %
88 %
87 %
86 %
Similarity (%)
85 %
84 %
83 %
82 %
81 %
80 %
79 %
78 %
77 %
76 %
75 %
74 %
73 %
72 %
71 %
70 %
69 %
68 %
67 %
66 %
65 %
0
IAH
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320 340
Position
360
380
400
420
440
460
480
500
520
540
560
580
600
620
640
Window : 160 bp, Step: 20 bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0
WRLFMD
Scanning analysis of VP1
SimPlot - Query: EA-2
FileName: D:\ClustalX-data\Picornaviridae\Aphthovirus\Topotype\TOPOVP1X.FAS
100
99
WA
EA-1
SEA
ME-SA
Euro-SA
ISA-2
ISA-1
Cathay
KEN02-05
98
97
96
95
94
93
92
91
90
89
88
Similarity (%)
87
86
85
84
83
82
81
80
79
78
77
76
75
74
73
72
71
70
20
IAH
40
60
80
100
120
140
160
180
200
220
240
260
280
300 320 340
Position (bp)
360
380
400
420
440
460
480
500
520
540
560
580
600
620
640
Window: 140 bp, Step: 10 bp, GapStrip: On, J-C Correction: Off
WRLFMD
Occurrence of FMDV O Topotypes
in Africa
TUNISIA
MOROCCO
ALGERIA
LIBYA
EGYPT
WESTERN
SAHARA
MAURITANIA
MALI
NIGER
ERITREA
CHAD
SUDAN
SENEGAL
THE GAMBIA
GUINEA-BISSAU
BURKINA
FASO
GUINEA
SIERRA-LEONE
COTE
D'IVOIRE
LIBERIA
DJIBOUTI
TOGO
NIGERIA
ETHIOPIA
CENTRAL
AFRICAN REPUBLIC
BENIN
GHANA
CAMEROON
KENYA
RWANDA
CONGO
WA
SOMALIA
UGANDA
EQUATORIAL
GUINEA
GABON
D.R.
CONGO
BURUNDI
ANGOLA
TANZANIA
EA-1 & EA-2
EA-2
ANGOLA
ZAMBIA
MALAWI
EA-3
MOZAMBIQUE
ZIMBABWE
ME-SA & WA
NAMIBIA
ME-SA
BOTSWANA
Euro-SA
IAH
SWAZILAND
SOUTH
AFRICA
LESOTHO
MADAGASCAR
WRLFMD
FMDV O Topotypes - 2004
East Africa 1
(EA-1)
West Africa
(WA)
O/ALG/1/99
O/CIV/8/99 O/UGA/5/96
O/KEN/2/95
O/KEN/83/79
South-east Asia
(SEA)
O/TAI/4/99
O/CAM/2/98
O/GHA/5/93
O/TAW/81/97
O/VIT/7/97
Middle East-South Asia
(ME-SA)
O/PHI/7/96
O/IRQ/30/2000
Cathay
O/HKN/14/82
O1/Manisa/TUR/69
O/A/CHA/58
O/ISA/8/83
O/BUN/7/2003
Indonesia-1
(ISA-1) O/ISA/9/74
O/KEN/5/2002
O/KEN/7/2002
O/UGA/3/2002 O/KEN/3/2002
O/ISA/1/62
O/TAN/7/98 O/MAL/1/98
East Africa 2
(EA-2)
O/JAV/5/72
1%
IAH
Indonesia-2
(ISA-2)
O/ISA/1/74
O3/VEN/51
O2/Brescia/ITL/47
O1/Kaufbeuren/FRG/66
Europe-South America
(Euro-SA)
Future work
• We are completing the complete capsid
sequencing of 9 African FMDV O isolates
including representatives of all indigenous
topotypes
• We have an on-going study of FMDV O in Africa
• We have completed a study of FMDV A in Africa
(~80 VP1 sequences)
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WRLFMD
Conclusions
• Two previously unrecognised genetic lineages of FMDV O
were identified in East Africa, each having a distinct
geographic distribution.
• Recombination near the 3’ end of VP1 may have played
a role in the evolution of the EA-2 topotype.
• Alternatively, it may be that all the African lineages
evolved after the introduction of Asian FMD viruses into
Africa and more mutations have subsequently
accumulated in certain parts of the capsid-coding region
while other parts may have remained more conserved.
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WRLFMD
Recommendations
• Future phylogenetic analyses for molecular
epidemiological purposes should be based on complete
VP1 sequences.
• More extensive molecular epidemiological studies of the
European FMDV serotypes in Africa should be
undertaken (this is in progress in our laboratory for
serotypes O and A).
• More extensive genome analyses need to be performed
to access the role of recombination in the natural
evolution of FMD viruses.
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WRLFMD
Acknowledgements
• Nigel Ferris & Geoff Hutchings
– for viruses
• Rahana Dwarka & Wilna Vosloo
– for sequences
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