The Molecular Epidemiology of BTV

Peter P. C. Mertens

European Community Reference Laboratory for Bluetongue virus

Institute for Animal Health Pirbright UK

Development of techniques

• Rapid sequencing from full length RT-PCR products

• Reference collection• Identify targets• Generate databases• Link data to specific isolates• Sources of Information

Isolates of Bluetongue virus type 1 (BTV-1)in the dsRNA virus collection at IAH Pirbright

related to

sample ALG2006/04

Hafsa MADANIMinistere de

L’agriculture et du DeveloppementRural, InstitiutNational de la

Medicine eterinaire[A89/06 6]

[IAH/KC2/BHK1]

No10

Shelf D2,box D2 γ

rows G1-H10

(added to collection9th Nov 2006)

Isolated from whole sheep blood at IAH

Pirbright

AlgeriaBerrouaghia

(23rd/24th Aug 2006)

BTV Type 1(Type

confirmed by Seg-2 RT-PCR and

sequencing)

ALG2006/06

[S2:AJ585122S3: DQ186792S6:AJ586695]

Vero, C6/36, KC cells

(E2/BHK7)[IAH-BHK2]

(Ref. Strain)

Shelf B2,

boxB2β,

rows E1-F10

(17/Mar/1982

NKSupplied by

OVI S. Africa*

BTV Type 1(Type

confirmed by Seg-2 RT-PCR and

sequencing)

RSArrrr/01

(41010B4F)

References & accession

numbers

Comments

* Original sample supplied to the collection by

person / institution

[IAH, ISIS sample number]

Original sample

material,

Adapted to which cell

types

(Passage history of sample)

[IAH passage history]

Suppliers Isolate

Designation

Position of storage in reference freezers

(Date added to collection)

Isolated from which host species

e.g cow, sheep, etc

(Date of virus

isolation, where, by

whom)

Place of original

sample egcountry town

[grid reference]

(date original

sample was taken)

Virus Species

& Serotype

(identification method)

*IAH dsRNA virus

collection number

( ICTVdbisolate

Accession Number)

Family: Reoviridae, Genus: Orbivirus, Species: Bluetongue virus, serotype 1 (BTV-1)

From : www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-1.htm

ALG2006/06Algeria

Berrouaghia

(23rd/24th

Aug 2006)

[S2:AJ585122

S3: DQ186792

S6:AJ586695]

Web pages for BTV Virus Isolates

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm

Virus isolates by countrywww.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/virus-nos-by-country.htm

Phylogenetic trees

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/orbivirus-phylogenetic-trees.htm

Accession numbers for individual BTV genome segments

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/orbivirus-accession-numbers.htm

BTV Seg-2 specific primers

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-S2-primers.htm

Conserved termini of dsRNA viruses

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/CPV-RNA-Termin.htm

B

12

3

46

5

78

9

10

B

12

3

46

5

78

9

10

B

12

3

46

5

78

9

10

B

12

3

46

5

78

9

10

The Genome segments of Bluetongue Virus

Types 1-24 analysed by polyacrylamide gel electrophoresis

The Genome segments of Bluetongue Virus

Types 1-24 analysed by agarose gel electrophoresis

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M

1

23

456

789

10

4 Kb

3 Kb

1 Kb

S-2

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M

1

23

456

789

10

4 Kb

3 Kb

1 Kb

S-2

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M

1

23

456

789

10

4 Kb4 Kb

3 Kb3 Kb

1 Kb1 Kb

S-2

Variable SegmentsConserved segments

The outer surface of the BTV virus particle VP and VP5 - Encoded by segments 2 and 6

(Cryo-EM reconstruction)

VP2 trimers encoded by Seg-2

VP5 trimers encoded by Seg-6

0.1

BTV-3

BTV-2 ChinaAF135218(p)

BTV-21 RSA

BTV-6 RSA

BTV-14 RSA

BTV-13 D00153BTV-8 RSA

BTV-18 RSA

BTV-19 RSA

BTV-7 RSA

BTV-1 RSA

BTV-9 Bulgaria

BTV-9 RSA

BTV-5 RSA

BTV-15 RSA

BTV-12 RSA

BTV-2 RSA

BTV-2 Italy

BTV-2 India

BTV-1 Greece

BTV-1 Malaysia

BTV-23 IndiaBTV-23UO4200

BTV-23L46685

BTV-16 Nigeria

BTV-16 RSA

BTV-11 M17437

BTV-4 ArgentinaBTV-4 Sudan

BTV-4 TurkeyBTV-4 RSA

BTV-17AF017278

BTV-17S72158

BTV-10L29026

BTV-1 Australia M21844

BTV-20 RSA

BTV-24 RSA

Comparison of All 24 serotypes of Bluetongue virus (BTV), genome S-2 NT sequences (Full length 2.9Kb)

www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv-seg-2.htm

BTV-2

BTV-1

BTV-8

BTV-16

BTV-4

BTV-9

Generated by S. Maan, A. Samuel and P.P.C.Mertensavailable from http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv-seg-2.htm

Comparison of segment 2 (VP2 gene) from all 24 serotypes of bluetongue virus (BTV) serotypes

0.1

BTV-11/S.Africa

BTV-11/Roy et al.M17437

BTV-17/S72158/California

BTV-17/S.AfricaBTV-17/AF017278//Colorado/USA

BTV-20/S.Africa

BTV-4/S.Africa

BTV-24/S.AfricaBTV-10/S.Africa

BTV-10/L29027

BTV-15/S.AfricaBTV-12/S.Africa

BTV-22SA

BTV-9/Bosnia

BTV-9/S.Africa

BTV-5/S.Africa

BTV-5/Nigeria

BTV-7/S.Africa

BTV-19/S.Africa

BTV-18/S.Africa

BTV-8/S.Africa-SM

BTV-8/Nigeria

BTV-23/S.Africa

BTV-23/Indonesia

BTV-1/S.AfricaBTV-1ndiaA

BTV-1GreeceBTV-2/S.Africa BTV-2

India BTV-3/ZimbabweBTV-3/X55801

BTV-16/S.AfricaBTV-16/Nigeria

BTV-13/S.Africa

BTV-13/Roy et al.D00153

BTV-21/S.Africa

BTV-6/S.Africa

BTV-14/S.Africa

BTV-14/Camaroon

18

8

14

23

10

20 1716 3

13 4 11

5

21

6

129

2

221

715 19

24

Serological relationships between BTV serotypes

Strong

weak

very weak

0.1

13SA

5SA

14Cam3SA

6SA16SA

21SA7SA

19SA

20SA10SA

24SA4SA4Gre

11SA17SA

12SA

22Nig

8Nig18SA

9SA 1SA

2Ind1Gre

1Ind

23Ind

2SA

All BTV genome S6 nucleotide sequences (1.6Kb)

European BTV serotypes

18

8

14

23

10

20 1716 3

13 4 11

5

21

6

129

2

221

715 19

24

Serological relationships between BTV serotypes

Strong

weak

very weak

Relationships based on VP5 sequences

BTV serotype specific RT-PCR (Seg-2)

BT24/783/R CATAAGCTCCAACTTCAACBT24/313/F TGGATTTATCTACACGATT24

BT23/653/R GTTTAACATGCATACTCAGBT23/459/F GCTTAGACCTGGCGATAAG23

BTV-22/682R GCTTATACCTCGCATCATCGBTV-22/311F GACGCGTTGGATAGGATAT22

BT21/754/R GCCTCCACACAGCGAAGACAGBT21/116/F GAGGAATGGCTGAACTGG21

BT20/812/R GTACGTCGTCAGCAATCTGBT20/135/F CATTACTCGATAGATTACC20

BT19/841/R GTCATCGTCGAGTGCGTGBT19/190/F TGTGCTCAAGCAAGCGCGTAT19

BT18/828/R GTACTCAGATAATAGTCGAGBT18/160/F GTCTTATCATATAGAGCCAG18

BT17/800/R T GAAACCGTGTAAAGTTCATBT17/146/F GCGAATGCTGCCAACGCTG17

BT16/832/R GATAGCGCCTGCGCACGCAAGBT16/165/F GTGAGTGTCGTCATGTACG16

BT15/793/R GTATGCGTGCCAATCGCCTAGBT15/103/F GAGACCGACTGACCATGACG15

BT15/806/R GTGAGACATATAATGTTCAAGBT15/343/F GTGGCAGAACGCAGAGGCAG15

BT14/805/R CTCCGCTTCATCCAGCTCBT14/121/F GAAGGTTAGCTTAGGTTTG14

BT13/800/R GAACATCATCAATTCCAGAATGBT13/157/F CGAGGAAAGCGGATACCAC13

BT12/776/R CTACGATCATATGATAACTCBT12/111/F GGATCACAATATAGATGTG12

BT12/500/R CATAATAATACGGCATAACBT12/226/F GCTGGCGTTACGCGCGGCG12

BT11/770/R GTCATCGTATAGTATCATBT11/147/F TGTATTGTTAAGGCTAGG11

BT10/803/R GTGAAACCTAATGAAATTGBT10/183/F GATGTTCCACATCTTACAG10

BT10/665/R GGTGCATGTATGTCTCATGBT10/400/F TGCGTCCAGAATATGGT10

BT9/852/R CCACATARTGAYGAATGATAGATBT9/287/F GCACACTGCTCTCGCGGATAG9

BT8/500/R CCGATGTGCGCATTCCTCTCBT8/101/F GAATGATGGTCATAGCGAG8

BT8/623/R GTTGCTTAGAGAGAGCGCGGBT8/280/F GAGAACAATGGATCGCTCG8

BT8/765/R CCACTGTCTATGTGCGTAABT8/134/F GCCCACACGCGCTCAAGG8

BT7/853/R GATAGATAAGCGAATCGAGBT7/83/F GATGTATCCATAGCGGCAT7

BT6/853/R CAAAGGGAACCTCGCGCGTAATCBT6/301/F GGTGGTATGTATAGAGGAAG6

5NIG/791/R TATATGCTCACAGATATCT5NIG/123/F TACGGATCATAAATGGATGG5

BT5/562/R GATGTCCTACAGCTCTCAGBT5/191/F GTGGCGCAAGAGTGTGCGT5

BT4/775/R GTATCAACCTGACCGCGTCGBT4/67/F GCTTAACTATAAACCAACGAGG4

BT4/675/R TCACGGAAGGATGTACATACGBT4/56/F * GGTTGATGTGCCTAAACTAG4

BT3/772/R GCGTATAATATTATTGGBT3/156/F CACCACATCGTACCACGC3

BT2/845/R GGTKGAAACAACRTTMAAATTBT2/302/F GCGGAACCBGTDGATGAAG2

BT2-TRi GTAAGTTGAACAGATCGCGGACCTGBT2-TFi GTTAAAACAGGATCGCGATGGATG2

BT1-TR GTAAGTMTRATAGYGCGCGGABT1-TF GTTAAAATAGTRKCGCGATGGATGAG1

Reverse PrimerForward PrimerSerotype of primers

21 22 22 23 2421 22 22 23 24RNA controls – Seg7RNA controls – Seg7

1 2 2 3 4 4 5 5 6 7 8 8 8 9 Serotype

Serotyping. Confirmed by typing primers against S2 as being type 8. There is a faint band for BTV1 but it is completely of the wrong size.

1 2 2 3 4 4 5 5 6 7 8 8 8 9 Serotype

Serotyping. Confirmed by typing primers against S2 as being type 8. There is a faint band for BTV1 but it is completely of the wrong size.

10 10 11 11 12 13 14 15 15 16 17 18 19 2010 10 11 11 12 13 14 15 15 16 17 18 19 20

Identification of NET2006/01 as BTV-8

M 1 2 3 4 5 6 C C C

M 7 8 9 10 11 12 C C C

M 1 13 14 15 16 17 18 C

M 19 20 21 22 23 24 C C C

M 1 2 3 4 5 6 C C C

M 7 8 9 10 11 12 C C C

M 1 13 14 15 16 17 18 C

M 19 20 21 22 23 24 C C C

Identification of MOR2006/06 as BTV-1

2231-BTV-4

2302-BTV-15

2309-BTV-4

RT-PCR based typing of 3 Israeli isolates 2006 using 24 serotype specific primers

1-24

Right sized products are encircled which indicate virus type (see table 1 for details of PCR products)

2000 bp

1650 bp

723BT15/806/R GTGAGACATATAATGTTCAAG

BTV-15/2/p565F GAATCACATTATGAATCGACG

15-8

684BTV-15/2/p793R GTATGCGTGCCAATCGCCTAG

BTV-15/2/p565F GAATCACATTATGAATCGACG

15-7

2232BT15/806/R GTGAGACATATAATGTTCAAG

BTV-15/2/p62F GATACGTGTTGGACTGAAATATCG

15-6

2193BTV-15/2/p793R GTATGCGTGCCAATCGCCTAG

BTV-15/2/p62F GATACGTGTTGGACTGAAATATCG

15-5

2109BT15/806/R GTGAGACATATAATGTTCAAG

BTV-15/2/p103F GAGACCGACTGACCATGACG

15-4

1350BTV-15/2/p793R GTATGCGTGCCAATCGCCTAG

BT15/343/F GTGGCAGAACGCAGAGGCAG

15-3

2070BTV-15/2/p793R GTATGCGTGCCAATCGCCTAG

BTV-15/2/p103F GAGACCGACTGACCATGACG

15-2

1389BT15/806/R GTGAGACATATAATGTTCAAG

BT15/343/F GTGGCAGAACGCAGAGGCAG

15-1

Product sizeRVS PrimerFWD PrimerType Used to design primers

Design of novel Seg-2 primers to confirm identifica tion of BTv-15 by RT-PCR

Confirmation of Israel isolate as BTV-15 (no 2302) using multiple BTV-15 specific primer pairs in RT-PCR assay

Right sized products in all lanes confirm it as type 15 (see table 2 for details of PCR products)

2000 bp

1650 bp

2302-BTV-15

Number 2231/BHK1/S30659: BTV-4Number 2302/BHK1/S324521: BTV-15Number 2309/BHK1/S32390: BTV-4

Typing Results:

Molecular epidemiology of European BTV

isolates based on Genome segment 2

Comparison of NET2006/01 Seg-2

to the 24 BTVreference strains

08

AJ5

8512

9 B

TV

8 R

SA

rrrr

08

AJ5

8518

3 B

TV

8 K

EN

01

AJ5

8518

4 B

TV8

NIG

1982

07

Net

AJ585144 BTV23 RSArrrr 23

AJ585139 BTV18 RSArrrr 18

AJ585110 BTV1 RSAvvvv 01

AJ585123 BTV2 RSArrrr 02

AJ585124 BTV3 RSArrrr 03

AJ585137BTV 16 R

SA

rrrr 16A

J585134 BTV

13 RS

Arrrr 13

AJ585142 B

TV

21 RS

Arrrr 21

AJ585127 B

TV

6 RS

Arrrr 06

AJ585135B

TV14 R

SA

rrrr 14

AJ58

5128

BTV

7 R

SArr

rr 0

7

AJ585140 BTV19 R

SArrrr 1

9

AJ585126 BTV5 RSArrrr 05

AJ585130 BTV9 RSArrrr 09

AJ585131 BTV10 RSArrrr 10

AJ585145 BTV24 RSArrrr 24AJ585125 BTV4 RSArrrr 04

AJ585141 BTV20 RSArrrr 20

AJ585138 BTV17 R

SArrrr 17

AJ585132 B

TV11 R

SA

rrrr 11

AJ585136 B

TV15 R

SA

rrrr 15

AJ585133 B

TV12 R

SA

r rr r 12A

J585143 BTV

22 RS

Arrrr 22

0.05

BTV-8 reference strain( RSArrrr/08 )

0.01

BTV-8 Nigeria 1982 (NIG1982/07)

BTV -8 Netherlands 2006 ( NET2006/01)

0.01

BTV-8 Kenya (KEN----/01)

NJ tree showing comparisons of nucleotide sequences of Seg2 from the BTV 8 strains.See: www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm

South African BTV-8 Seg-2 partial sequences provided

by Dr. AC. Potgieter(OVI Onderstepoort)

BTV-8 South Africa (06/92) (RSA1992/01)

BTV-8 South Africa (16/87) (RSA1987/01)BTV-8 South Africa (34/98B) (RSA1998/01)

Seg-2 comparison of NET2006/01 to the other BTV-8 strains

(BTV-8 vaccine strain)

BTV-1 South African vaccine strain RSAvvvv/01

BTV-1 Morocco -MOR2006/06

BTV-1 segment 2 tree showing east-west split

BTV-1 Australia -AUS1981/01

BTV-1 Algeria -ALG2006/01

BTV-1 – Cameroon- CAR1982/01

BTV-1 NigeriaNIG1982/01

BTV-1 SA reference strain RSArrrr/01

BTV-1 Sudan -SUD1987/01

BTV-1 China - AF135217

BTV-1 Malaysia MAY1987/01

BTV-1 Greece -GRE2001/01

BTV-1 India -IND1992/01

0.1

Eastern Group

Western Group

http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV1-segment2-tree.htm

BTV-1RSArrrr/01

BTV-1/IND1999/01

BTV-2/RSArrrr/02

BTV-2/IND1982/01

BTV-18/RSArrrr/18

BTV-8/RSArrrr/08BTV-8/NIG1982/07

BTV-23/RSArrrr/23

BTV-23/IND1997/01

BTV-3/RSArrrr/03

BTV-3/ZIM2002/01

BTV-16/RSArrrr/16

BTV-16/NIG1982/10

BTV-13/RSArrrr/13

BTV-13/D00153

BTV-21/RSArrrr/21

BTV-6/RSArrrr/06

BTV-14/RSArrrr/14

BTV-14/CAR1982/04

BTV-20/RSArrrr/20

BTV-4/RSArrrr/04

BTV-4/CYP1969/01

BTV-11/RSArrrr/11

BTV-11/M17437

BTV-17/RSArrrr/17BTV-17/S72158

BTV-24/RSArrrr/24

BTV-10/RSArrrr/10BTV-10/L29027

BTV-12/RSArrrr/12

BTV-12/NIG1982/09

BTV-22/RSArrrr/22

BTV-22/NIG1982/11 BTV-15/RSArrrr/15

ISR2006/11 BTV-15 IsraelBTV-15/ZIM2003/08

BTV-9/BOS2002/02

BTV-9/RSArrrr/09

BTV-5/RSArrrr/05

BTV-5/NIG1982/03

BTV-7/RSArrrr/07

BTV-19/RSArrrr/19

BTV-4&

BTV-16

BTV-9

BTV-1

BTV-4

The 10 introductions of different BTV strains into Europe and north Africa into since 1998

BTV-2&BTV-4

BTV-8

BTV-1

BTV- type 8?

1410 Ppair may cross react withBTV-4 ISR

BT24/783/R CATAAGCTCCAACTTCAACThe bases in red matches seq of BTV-4 Israel

BT24/313/F TGGATTTATCTACACGATTThe bases in red match BTV-4 Israel sequence

24

582BT23/653/R GTTTAACATGCATACTCAGBT23/459/F GCTTAGACCTGGCGATAAG23

1113BTV-22/682R GCTTATACCTCGCATCATCGBTV-22/311F GACGCGTTGGATAGGATAT22

1914BT21/754/R GCCTCCACACAGCGAAGACAGBT21/116/F GAGGAATGGCTGAACTGG21

2031BT20/812/R GTACGTCGTCAGCAATCTGBT20/135/F CATTACTCGATAGATTACC20

1953BT19/841/R GTCATCGTCGAGTGCGTGBT19/190/F TGTGCTCAAGCAAGCGCGTAT19

2004BT18/828/R GTACTCAGATAATAGTCGAGBT18/160/F GTCTTATCATATAGAGCCAG18

1521BTV-17/2/p653R GCGCGACTTTCCTAAACGBTV-17/2/p146F GCGAATGCTGCCAACGCTG17

1974 (-Nig)BTV-16/2/p924R GCCGAAGGTGCGATCTGGCCGBTV-16/2/p266F GCGATAAGAGATAMTTGGAT16B

1389BT15/806/R G TGAGACATATAATGTTCAAGBT15/343/F GTGGCAGAACGCAGAGGCAG15

2052BT14/805/R CTCCGCTTCATCCAGCTCBT14/121/F GAAGGTTAGCTTAGGTTTG14

1422BTV-13/2/p631R TGCTTAATGCTCAAACTCGBT13/157/F CGAGGAAAGCGGATACCAC13A

1995BT12/776/R CTACGATCATATGATAACTCBT12/111/F GGATCACAATATAGATGTG12

1569BT11/770/R GTCATCGTATAGTATCATBT11/147/F TGTATTGTTAAGGCTAGG11

1860BT10/803/R GTGAAACCTAATGAAATTGBT10/183/F GATGTTCCACATCTTACAG10

1128 (9C)BTV-9/2/p867R GGAAAACTGATTGGCAATBTV-9/2/p491F GGSAATATATTTCTRATGG9

1197BT8/500/R CGATGTGCGCATTCCTCTCBT8/101/F GAATGATGGTCATAGCGAG8

2310BT7/853/R GATAGATAAGCGAATCGAGBT7/83/F GATGTATCCATAGCGGCAT7

1656BT6/853/R CAAAGGGAACCTCGCGCGTAATCBT6/301/F GGTGGTATGTATAGAGGAAG6

20045NIG/791/R TATATGCTCACAGATATCT5NIG/123/F TACGGATCATAAATGGATGG5

2124BT4/775/R GTATCAACCTGACCGCGTCGBT4/67/F GCTTAACTATAAACCAACGAGG4

1500BTV-3/2/p840R TGAGATCGTCGTCTGAAGATBTV-3/2/p340F GAACTAATGAGGACAGAT3C

653 (2A)BTV-2/2/p416R TACTAAAKATATACTTCTCCGTBTV-2/2/p198F TATCAGTTAATAGTGCATT2

1686 (1A)BTV-1/2/p968R TYATACGTTGAGAAGTTTTGTBTV-1/2/p406F GAGAACARGAARAATATAT1

Product sizeRVS PrimerFWD PrimerSerotype

Detection of ‘reassortant’ strains by Comparisons of multiple BTV genome

segments

• Genome segment 2 encoding VP2

• Genome segment 5 Encoding NS1

AF530067/BTV-16

16-RSA/RSArr/16

16RSA/vaccine

16Turkey/1016Turkey/TUR00/01

16Turkey/TUR00/02

AF135222/16

16/DPP965 /L4663

BTV-16Nigeria

0.1

Comparison of BTV-16 genome S-2 NTsequences(Full length 2.9 Kb)

East

West

BTV-2 ITL2002/07 (AJ585160)

BTV-2 SAD2001/02 (AJ585162)BTV-2 TUN2000/01 (AJ585156)

BTV-2 FRA2001/03 (AJ585154)

BTV-2 RSAvvvv/02 (AJ585157)BTV-2 RSArrrr/02 (AJ585123)

BTV 16 TUR2000/10 (AJ585148)BTV 16 ITL2002 (DQ191260)

BTV 16 RSAvvvv/16 (AJ585149)BTV 16 RSArrrr/16 (AJ585137)

BTV-2 BTV-16

0.05

Comparison of genome segment 2 (VP2 - gene) nt sequencesfrom Mediterranean BTV isolates of type 2 and type 16

(Western)(Eastern)

No. of Taxa : 24Data File : N:\wrl-meg\ClustalX-data\Reoviridae\Orbivirus\BTV\net.megData Title : NS1 partialData Type : Nucleotide (Coding)Analysis : Phylogeny reconstructionTree Inference : ==============================Method : Neighbor-JoiningPhylogeny Test and options : Bootstrap (1000 replicates; seed=64238)

Include Sites : ==============================Gaps/Missing Data : Pairwise DeletionCodon Positions : 1st+2nd+3rd+Noncoding

Substitution Model : ==============================Model : Nucleotide: Kimura 2-parameterSubstitutions to Include : d: Transitions + TransversionsPattern among Lineages : Same (Homogeneous)Rates among sites : Uniform rates

No. of Sites : 482No Of Bootstrap Reps = 1000

ITL2001/01 BTV2TUN2000/01 BTV2FRA2001/03 BTV2ITL2002/07 BTV2

SPA2005/05 BTV4GRE1999/15 BTV4

NET2006-12BTV4 VACBTV9 VAC

BTV1 VACCSPA2005/01 BTV2BTV2vacc NS1ITL2002 DQ017960

BTV2 america NS1GRE200107

SAD2004/03 BTV16SAD2004/04 BTV16ISR2001/18 BTV16BTV16 VACCINE NS1 DQ017961

TUR2000/03 BTV9BUL1999/01 BTV9TUR2000/10 BTV16BOS2002/01 BTV9

GRE1999/13 BTV16

5953

92

261624

99

98

100

100

8575

98

9796

68

88

100

0.02

NS1 482 nt (3’ end of gene)

No. of Taxa : 24Data File : N:\wrl-meg\ClustalX-data\Reoviridae\Orbivirus\BTV\net.megData Title : NS1 partialData Type : Nucleotide (Coding)Analysis : Phylogeny reconstructionTree Inference : ==============================Method : Neighbor-JoiningPhylogeny Test and options : Bootstrap (1000 replicates; seed=64238)

Include Sites : ==============================Gaps/Missing Data : Pairwise DeletionCodon Positions : 1st+2nd+3rd+Noncoding

Substitution Model : ==============================Model : Nucleotide: Kimura 2-parameterSubstitutions to Include : d: Transitions + TransversionsPattern among Lineages : Same (Homogeneous)Rates among sites : Uniform rates

No. of Sites : 482No Of Bootstrap Reps = 1000

ITL2001/01 BTV2TUN2000/01 BTV2FRA2001/03 BTV2ITL2002/07 BTV2

SPA2005/05 BTV4GRE1999/15 BTV4

NET2006-12BTV4 VACBTV9 VAC

BTV1 VACCSPA2005/01 BTV2BTV2vacc NS1ITL2002 DQ017960

BTV2 america NS1GRE200107

SAD2004/03 BTV16SAD2004/04 BTV16ISR2001/18 BTV16BTV16 VACCINE NS1 DQ017961

TUR2000/03 BTV9BUL1999/01 BTV9TUR2000/10 BTV16BOS2002/01 BTV9

GRE1999/13 BTV16

5953

92

261624

99

98

100

100

8575

98

9796

68

88

100

0.02

NS1 482 nt (3’ end of gene)

BTV-1

European Field Strains 1998 onwardsVaccine StrainsWestern Group

Eastern Group

Phylogenetic analysis of 482 nucleotides from the downstream end of the BTV-NS1 gene of European field and vaccine strains

BTV-16

BTV-8

(Western)

BTV-16

BTV-16

BTV-2

0.1

SAD2004/03

RSAvvvv16SAD2004/04

TUR2000/10

GRE1999/13

ITL2002

RSAvvvv02

TUN2000/01ITL2002/07

ITL2001/01

FRA2001/03

Comparison of BTV-16 and BTV-2, Seg-5 (NS1) partial nt sequences (498nt)

BTV-16

Generation of reassortant BTV-16

Strain – containing NS1 gene from BTV-2 vaccine

VP2

NS1

Conclusions

• The outbreak of BTV-16 in Sardinia 2004 was caused by the BTV-16 vaccine

• The BTV-16 field strain from Turkey 2000 has an NS1 Gene indistinguishable from that of the BTV-9 field strain from Bulgaria 1999, suggesting that the same gene was acquired by reassortment

• The Italy 2002 strain of BTV-16 is derived from the Turkey 2000 strain of BTV16 but has acquired the NS1 gene from the BTV-2 vaccine used in Italy, by genome segment reassortment

Sources of Information• References• Maan S., Maan N.S, Samuel A.R., Rao S, Attoui, H., & Mertens P.P.C (2006) Analysis and

Phylogenetic Comparisons of Full-Length VP2 Genes of the Twenty-Four Bluetongue Virus serotypes. Journal of General Virology (in Press).

• Anthony S. , Jones H., Darpel K.E, Elliott H., Maan S., Samuel A., Mellor P. S. and Mertens P. P. C. (2006) A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes. Journal of Virological Methods (in Press).

• Shaw A.E., Monaghan, P., Alpar, H.O., Anthony, S., Darpel, K.E., Batten, C.A., Carpenter, S., Jones, H., Oura, C.A.L., King, D.P., Elliot, H., Mellor P.S., Mertens, P.P.C. (2006) Development of a real time RT-PCR assay to detect bluetongue virus genome segment 1 (submitted to Journal of Virological Methods).

• Reference collection www.iah.bsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm• BTV Accession. Nos. www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv_sequences.htm• BTV strain Distribution www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv-serotype-distribution.htm• BTV Seg-2 Trees www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/orbivirus-phylogenetic-trees.htm

AcknowledgementsStephan Zientara, Emmanuel Breard, Corinne Sailleau (Maisons Alfort)

Christiaan Potgieter, Truske Gerdes (Onderstepoort South Africa)

Kiki Nomikou, Olga Mangana, (Greek Veterinary Lab Athens)

Martin Beer, Bernd Hoffman, Eugene van Rooij, Chris De Clercq, Piet van Rijn

Mehdi Al Harrak (Biopharma Morocco)

All of the members of the Arbovirus Department, IAH Pirbright

I. A. H. PirbrighI. A. H. Pirbright

Also many other Colleagues in:

Israel Argentina Australia Morocco Sardinia Italy SpainTurkey Cyprus India Bulgaria Peru Sicily Netherlands Germany Belgium Greece Morocco Algeria Australia USA

Supported by Funding from The European Commission, BBSRC, Defra, Wellcome Trust & Commonwealth Fellowships