identification of a ninth foot- and-mouth disease virus type o topotype and evidence for a...

Identification of a Ninth Identification of a Ninth Foot-and-Mouth Disease Foot-and-Mouth Disease

Virus Type O Topotype and Virus Type O Topotype and Evidence for a Evidence for a

Recombination Event in its Recombination Event in its EvolutionEvolution

Nick J. Knowles, Paul R. Davies,Nick J. Knowles, Paul R. Davies,Rebecca J. Midgley and Jean-François ValarcherRebecca J. Midgley and Jean-François Valarcher

Institute for Animal Health, Pirbright Laboratory, Ash Road, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF, UKWoking, Surrey, GU24 0NF, UK

WRLFMD

IAH

EGYPTLIBYAALGERIA

MOROCCO

NIGERMAURITANIA

WESTERNSAHARA

MALI

CHADSUDAN

D.R. CONGO

ANGOLA

NAMIBIA

SOUTHAFRICA

BOTSWANA

ETHIOPIA

SOMALIA

NIGERIAGUINEA

TUNISIA

ERITREA

CENTRALAFRICAN REPUBLIC

MADAGASCAR

TANZANIA

GABON

BURKINA FASO

GHANA

COTED'IVOIRE

ZAMBIA

ZIMBABWE

CAMEROON

TOGO

BENIN

UGANDA

KENYA

MOZAMBIQUE

LESOTHO

SWAZILAND

CONGO

SENEGAL

THE GAMBIA

GUINEA-BISSAU

SIERRA-LEONE

LIBERIA

EQUATORIAL GUINEA

ANGOLA

RWANDA

BURUNDI

MALAWI

DJIBOUTI

Endemic?

Sporadic

Not knownor no FMD

FMDV O in AfricaFMDV O in Africa

WRLFMD

IAH

Previous studiesPrevious studies

• Few studies on the European FMDV serotypes in Africa:Few studies on the European FMDV serotypes in Africa:– Knowles Knowles et alet al., 1998 (type A)., 1998 (type A)– Samuel Samuel et alet al., 1999 (type O)., 1999 (type O)– Samuel & Knowles, 2001 (type O)Samuel & Knowles, 2001 (type O)– Sangare Sangare et alet al., 2001 (type O)., 2001 (type O)– Sahle Sahle et alet al., 2004 (type O)., 2004 (type O)– Bronsvoort Bronsvoort et alet al., 2004 (types O & A)., 2004 (types O & A)

• Distinct genetic lineages of both FMDV-O and FMDV-A Distinct genetic lineages of both FMDV-O and FMDV-A circulate in East and West Africa and viruses occurring in circulate in East and West Africa and viruses occurring in North Africa may be introduced from a variety of sources North Africa may be introduced from a variety of sources (Europe, South America, Middle East and West Africa).(Europe, South America, Middle East and West Africa).

• Outbreaks of FMD types O and A in southern Africa are very Outbreaks of FMD types O and A in southern Africa are very rare and are usually introduced from outside the continent.rare and are usually introduced from outside the continent.

WRLFMD

IAH

FMDV O Topotypes - 2001FMDV O Topotypes - 2001

WRLFMD

0.1

685685991991

10001000

781781

913913

10001000

638638

10001000

10001000

1000100010001000

998998

999999

824824

412412488488

10001000

958958

O/A/CHA/58O/A/CHA/58

O1/ManisaO1/Manisa/TUR/69/TUR/69

O/ALG/1/99O/ALG/1/99O/CIV/8/99O/CIV/8/99

O/GHA/5/93O/GHA/5/93

O/KEN/83/79O/KEN/83/79

O/KEN/2/95 (K10/95)O/KEN/2/95 (K10/95)

O/UGA/5/96O/UGA/5/96

O1/Kaufbeuren/FRG/66O1/Kaufbeuren/FRG/66

O3/VEN/51O3/VEN/51

O2/Brescia/ITL/47O2/Brescia/ITL/47

O/ISA/1/74O/ISA/1/74

O/JAV/5/72O/JAV/5/72

O/ISA/1/62O/ISA/1/62O/ISA/9/74O/ISA/9/74


O/HKN/14/82O/HKN/14/82

O/PHI/7/96O/PHI/7/96

O/TAW/81/97O/TAW/81/97

O/CAM/2/98O/CAM/2/98

O/VIT/7/97O/VIT/7/97

O/TAI/4/99O/TAI/4/99O/IRQ/O/IRQ/30/200030/2000

801801

502502

East Africa (EA)

West Africa (WA)

Middle East-South Asia (ME-SA)

South-east Asia (SEA)

Europe-South America (Euro-SA)

Indonesia-2 (ISA-2) Indonesia-1 (ISA-1)

Cathay

Unrooted Neighbor-joining tree based on a comparison of complete VP1 gene sequences (639-642 nucleotides)

IAHSamuel, 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.

RT-PCR & SequencingRT-PCR & Sequencing

AAAAAAAAAn

L P1 P2 P35’ UTR 3’ UTR

VPgVP4 VP2 VP3 VP12A2B 2C 3A3B 3C 3DPoly C

Capsid Non-structural proteins

IRES

WRLFMD

Primers for RT-PCR an sequencing of FMDV O RNA.

Designation Sequence (5' to 3')Genome location

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

NK61NK61NK61

O-1C244F O-1C272F O-1C283F

IAH

VP1VP1(complete)(complete)

10%

O2/Brescia/ITL/47O1/Kaufbeuren/FRG/66

O3/VEN/51O/ISA/1/74

O/JAV/5/72O/ISA/1/62


O/HKN/14/82O/HKN/6/83

O/PHI/7/96O/PHI/5/95O/TAW/81/97

O/CAM/2/98O/VIT/7/97

O/TAI/189/87*O/TAI/4/99

O/A/CHA/58O/IND/R2/75*

O1/Manisa/TUR/69O/IND/53/79

O/IRQ/30/2000O/TAW/2/99

O/SAR/1/2000O/UKG/12/2001

O/MAU/19/2000O/BKF/1/2002

O/NGR/11/2001O/ALG/2/99

O/CIV/8/99O/CAR/6/89

O/GHA/5/93O/CAR/16/2000O/CAR/17/2000

O/ERI/1/96O/ETH/8/94

O/SUD/25/2004O/SUD/16/2004

O/SUD/26/2004O/SUD/30/2004

O/KEN/102/60O/UGA/5/96

O/KEN/83/79O/KEN/2/95 [K10/95]

O/UGA/3/72O/TAN/3/96

O/BUN/3/2003O/BUN/7/2003O/BUN/6/2003

O/KEN/5/2002O/KEN/7/2002

O/KEN/3/2002O/UGA/5/2002

O/UGA/4/2004O/UGA/18/2004

O/UGA/1/2004O/UGA/3/2004

O/UGA/6/2004O/UGA/8/2004O/UGA/5/2004O/UGA/7/2004

O/UGA/6/2002O/UGA/3/2002

O/TAN/7/98O/MAL/1/98

O/MAL/2/98O/ZAM/1/2000O/ZAM/2/2000

O/RWA/2/2004O/RWA/3/2004

973

574996

1000

9611000

5031000

7321000

1000

984993

639

9871000

522

320

220

996497

1000724

676

723

1000

1000

1000

819594

1000806

230

997988

604

186

8321000

510

680

870641

562597967

882

996843

415

1000

949

654

9931000

1000

778

965

1000

513

968

385

67

182

932

725

745

Cathay

WA

EA-2

ME-SA

ISA-2

SEA

Euro-SA

EA-1

ISA-1

EA-1?

EA-2?

WRLFMD

IAH

VP1VP1(145-642)(145-642)

10%

O/ANG/10/74O/ANG/1/75O1/Kaufbeuren/FRG/66

O2/Brescia/ITL/47O/ISA/1/74

O/JAV/5/72O/ISA/1/62


O/HKN/14/82O/HKN/6/83


O/TAW/81/97O/CAM/2/98O/VIT/7/97

O/TAI/189/87*O/TAI/4/99

O/A/CHA/58*O/IND/R2/75*

O1/Manisa/TUR/69O/IND/53/79

O/IRQ/30/2000O/TAW/2/99

O/SAR/1/2000O/UKG/12/2001

O/KEN/102/60O/NGR/1/88

O/MAU/19/2000O/BKF/1/2002

O/NGR/11/2001O/ALG/2/99

O/CIV/8/99O/CAR/6/89O/GHA/5/93

O/CAR/16/2000O/CAR/17/2000


O/BUN/3/2003O/BUN/7/2003O/BUN/6/2003778

O/KEN/5/2002O/KEN/7/2002

O/TAN/7/98O/MAL/1/98

O/MAL/2/98783

O/ZAM/1/2000O/ZAM/2/2000

O/KEN/3/2002O/UGA/5/2002

O/UGA/4/2004O/UGA/18/2004

O/UGA/1/2004O/UGA/3/2004860


561

O/UGA/3/2002O/UGA/6/2002

O/RWA/2/2004O/RWA/3/2004

O/ETH/15/01* [OVI]O/ERI/1/96

O/ERI/1/96 [OVI]O/ETH/8/94

O/ETH/8/94 [OVI]1000

O/ETH/16/01* [OVI]O/ETH/22/01* [OVI]

O/ETH/1/95 [OVI]O/ETH/3/90 [OVI]

O/ETH/8/90 [OVI]O/ETH/12/90 [OVI]654


O/ETH/19/77 [1983]* [OVI]O/ETH/5/95 [OVI]



O/ERI/2/96 [OVI]O/ETH/3/96 [OVI]

O/SUD/16/2004O/SUD/25/2004

O/SUD/26/2004O/SUD/30/2004

1000

O/UGA/5/96O/KEN/83/79

O/KEN/2/95O/KEN/10/95 [OVI]

O3/VEN/511000

5241000

1000

997953

1000

979573

1000855

1000

1000

543

644

1000

1000

865

696

559

786

916

900

887531

1000

968

1000

1000

1000

999436

997

771

760502

1000

822

542

946

1000

8401000

5201000

931995

723

586393

310

653

643

368

996

280

367

963

737

832

201

643

243

539

315

463

933

675908

767

981743

898

829

998988

107

156

354

967

905

664 Cathay

WA

EA-2

ME-SA

ISA-2

SEA

Euro-SA

EA-1

ISA-1

EA-3

WRLFMD

IAH

VP1VP1(478-642)(478-642)

10%

O1/Kaufbeuren/FRG/66O2/Brescia/47

O/ISA/1/74O/JAV/5/72


O/ISA/8/83O/HKN/14/82

O/HKN/6/83O/TAW/81/97


O/VIT/7/97O/MAU/19/2000

O/BKF/1/2002O/NGR/11/2001

O/ALG/2/99O/CIV/8/99

O/CAR/6/89O/GHA/5/93

O/CAR/16/2000O/CAR/17/2000

O/TAI/189/87*O/TAI/4/99

O/SUD/16/2004O/SUD/25/2004

O/SUD/26/2004O/SUD/30/2004

O/ERI/1/96O/ETH/8/94

O/KEN/102/60O/TAN/7/98

O/MAL/1/98O/MAL/2/98

O/UGA/5/96O/KEN/83/79

O/KEN/2/95 [K10/95]O/IND/53/79

O/IND/R2/75*O1/Manisa/TUR/69O/A/CHA/58

O/IRQ/30/2000O/TAW/2/99

O/SAR/1/2000O/UKG/12/2001


O/RWA/2/2004O/RWA/3/2004

O/KEN/5/2002O/KEN/7/2002

O/BUN/3/2003O/BUN/7/2003O/BUN/6/2003

O/UGA/6/2002O/UGA/3/2002

O/KEN/3/2002O/ZAM/1/2000O/ZAM/2/2000

O/UGA/4/2004O/UGA/5/2002


O/UGA/1/2004O/UGA/3/2004

O/UGA/6/2004

O3/VEN/51

993

401522

1000

785883

813588

797987

408

O/CAM/2/98951

694773

756615

644

512

1000

247

711

110

679436

998

769

971995

169

844

43

630

903871

810223

81

268

31

998

599980

165

996

77

49

989

7072711

3

190

553

543

504268

143

335

86

4

12

78

94

131

141

293

357

Cathay

WA

EA-2

ME-SA

ISA-2

SEA

Euro-SA

EA-1

ISA-1

SEA

EA-1

EA-1

EA-2 EA-1

ME-SA

EA-2

WRLFMD

IAH

Scanning analysis of VP1Scanning analysis of VP1WA1EA2SEAMESEURCATIS1IS2EA1

SimPlot - Query: EA-2 O/UGA/3/2002FileName: N:\evd\MEG\db\FMDV\O\Crete-k2.fas

Window : 160 bp, Step: 20 bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0

Position640620600580560540520500480460440420400380360340320300280260240220200180160140120100806040200

Sim

ilarit

y (%

)

100 %

99 %

98 %

97 %

96 %

95 %

94 %

93 %

92 %

91 %

90 %

89 %

88 %

87 %

86 %

85 %

84 %

83 %

82 %

81 %

80 %

79 %

78 %

77 %

76 %

75 %

74 %

73 %

72 %

71 %

70 %

69 %

68 %

67 %

66 %

65 %

WRLFMD

IAH

Scanning analysis of VP1Scanning analysis of VP1WAEA-1SEAME-SAEuro-SAISA-2ISA-1CathayKEN02-05

SimPlot - Query: EA-2FileName: D:\ClustalX-data\Picornaviridae\Aphthovirus\Topotype\TOPOVP1X.FAS

Window: 140 bp, Step: 10 bp, GapStrip: On, J-C Correction: Off

Position (bp)64062060058056054052050048046044042040038036034032030028026024022020018016014012010080604020

Sim

ilarit

y (%

)

100

99

98

97

96

95

94

93

92

91

90

89

88

87

86

85

84

83

82

81

80

79

78

77

76

75

74

73

72

71

70

WRLFMD

IAH

Occurrence of FMDV O Occurrence of FMDV O Topotypes in AfricaTopotypes in Africa

EGYPTLIBYAALGERIA

MOROCCO

NIGERMAURITANIA

WESTERNSAHARA

MALI

CHADSUDAN

D.R. CONGO

ANGOLA

NAMIBIA

SOUTHAFRICA

BOTSWANA

ETHIOPIA

SOMALIA

NIGERIAGUINEA

TUNISIA

ERITREA

CENTRALAFRICAN REPUBLIC

MADAGASCAR

TANZANIA

GABON

BURKINA FASO

GHANA

COTED'IVOIRE

ZAMBIA

ZIMBABWE

CAMEROON

TOGO

BENIN

UGANDA

KENYA

MOZAMBIQUE

LESOTHO

SWAZILAND

CONGO

SENEGAL

THE GAMBIA

GUINEA-BISSAU

SIERRA-LEONE

LIBERIA

EQUATORIAL GUINEA

ANGOLA

RWANDA

BURUNDI

MALAWI

DJIBOUTI

WA

EA-1 & EA-2

EA-2

EA-3

ME-SA & WA

ME-SA

Euro-SA

WRLFMD

IAH

FMDV O Topotypes - 2004FMDV O Topotypes - 2004

1%

O/A/CHA/58

O1/Manisa/TUR/69

O/ALG/1/99 O/CIV/8/99

O/GHA/5/93

O/KEN/83/79O/KEN/2/95

O/UGA/5/96

O/BUN/7/2003

O/KEN/5/2002

O/KEN/7/2002

O/KEN/3/2002O/UGA/3/2002

O/MAL/1/98O/TAN/7/98

O1/Kaufbeuren/FRG/66

O3/VEN/51 O2/Brescia/ITL/47O/ISA/1/74

O/JAV/5/72

O/ISA/1/62

O/ISA/9/74

O/ISA/8/83

O/HKN/14/82

O/PHI/7/96

O/TAW/81/97

O/CAM/2/98

O/VIT/7/97

O/TAI/4/99

O/IRQ/30/2000

East Africa 1 (EA-1)

West Africa (WA)

Middle East-South Asia (ME-SA)

South-east Asia (SEA)

Europe-South America (Euro-SA)

Indonesia-2 (ISA-2)

Indonesia-1 (ISA-1)

Cathay

East Africa 2 (EA-2)

WRLFMD

IAH

Future workFuture work

• We are completing the complete capsid We are completing the complete capsid sequencing of 9 African FMDV O isolates sequencing of 9 African FMDV O isolates including representatives of all indigenous including representatives of all indigenous topotypestopotypes

• We have an on-going study of FMDV O in We have an on-going study of FMDV O in AfricaAfrica

• We have completed a study of FMDV A in We have completed a study of FMDV A in Africa (~80 VP1 sequences)Africa (~80 VP1 sequences)

IAH

ConclusionsConclusions

• Two previously unrecognised genetic lineages of Two previously unrecognised genetic lineages of FMDV O were identified in East Africa, each having a FMDV O were identified in East Africa, each having a distinct geographic distribution.distinct geographic distribution.

• Recombination near the 3’ end of VP1 may have Recombination near the 3’ end of VP1 may have played a role in the evolution of the EA-2 topotype.played a role in the evolution of the EA-2 topotype.

• Alternatively, it may be that all the African lineages Alternatively, it may be that all the African lineages evolved after the introduction of Asian FMD viruses evolved after the introduction of Asian FMD viruses into Africa and more mutations have subsequently into Africa and more mutations have subsequently accumulated in certain parts of the capsid-coding accumulated in certain parts of the capsid-coding region while other parts may have remained more region while other parts may have remained more conserved. conserved.

WRLFMD

IAH

RecommendationsRecommendations

• Future phylogenetic analyses for molecular Future phylogenetic analyses for molecular epidemiological purposes should be based on epidemiological purposes should be based on complete VP1 sequences.complete VP1 sequences.

• More extensive molecular epidemiological studies More extensive molecular epidemiological studies of the European FMDV serotypes in Africa should of the European FMDV serotypes in Africa should be undertaken (this is in progress in our be undertaken (this is in progress in our laboratory for serotypes O and A).laboratory for serotypes O and A).

• More extensive genome analyses need to be More extensive genome analyses need to be performed to access the role of recombination in performed to access the role of recombination in the natural evolution of FMD viruses.the natural evolution of FMD viruses.

WRLFMD

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AcknowledgementsAcknowledgements

• Nigel Ferris & Geoff HutchingsNigel Ferris & Geoff Hutchings– for virusesfor viruses

• Rahana Dwarka & Wilna VoslooRahana Dwarka & Wilna Vosloo– for sequencesfor sequences

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identification of a ninth foot- and-mouth disease virus type o topotype and evidence for a...

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