Bluetongue is a disease of domestic animals

….causing high morbidity and often mortality

Coronitis

Oedema & salivation

Hyperemia

Oedema face

Bluetongue in Sheep and Cattle

• First described in 18th century• Thought to be confined to Africa• 1924 Cyprus, 70% mortality• 1943-47 Palestine and Turkey• 1948 first recognized in USA• by 1951 reported in Israel and Asia• 1956 Southern Europe (17,900 sheep

killed in 4 months (75% mortality))• Now described as an ‘emerging disease’

and includes 24 serotypes

Domestic Livestock

Wildlife reservoir

Blood feeding vector

Transmission of BTV from

wild to domesticated animals is by biting midges

Historically BTV has been endemic in warmer climates

Europe/Africa/Middle East

C. imicola (Old)

C. obsoletus

C. pulicaris (New)N.America

C. sonorensis

Europe/Africa/Middle East

C. imicola (Old)

C. obsoletus

C. pulicaris (New)

C. insignis

AmericaCentral & South

Europe/Africa/Middle EastEurope/Africa/Middle

BTV & C. imicola in the Mediterranean Basin 1998-2003

P Mellor, IAH

BTV "cores" have a much improvedresolution offering scope for study.

Structure of the BTV virion and core

BTV is an complex virus and is poorly resolved by negative staining.

The outer capsid; two major proteins

VP2 & VP5

The core; two major proteins

(VP3 and VP7) three minor proteins(VP1, VP4 and VP6)

VP1

VP2VP3

VP4

VP5

VP6

VP7

The genometen dsRNAsegments

BTV – the structural components and their location

Note that BTV lacks lipid containing envelope

RNA fingerprinting allowed a comparison between two RNA molecules

Type 10 Type 11

Fingerprints of gene 2 of two BTV serotypes

All other segments are highly conserved among different types

Fingerprints of gene 8 of two serotypes

• Isolates of single serotype (e.g. BTV-11) from different states in one year (1973)

• Isolates from same state (e.g. Colorado) but in different years (1963-1975)

• Isolates from a different animal (wild, domestic) and vectors

Oligonucleotide fingerprint analysis of natural viruses

All showed sequence differencesConsiderableEvolution : Mutation

Sugiyama et al, 1981,1982; Rao etal, 1983

Infection of cultured cellswith two types

Our analysis showed that mixing of different genomes occurs between different viruses and could

lead to new virulent strains

BTV Type X

BTV Type Y

Swapping of RNA segments occurs to

generates new viruses

Different mixes of RNA segments can lead to enhanced pathogenicity

Segment variationcan lead to emerging pathogenic strains

Long incubation in cattle

Potential danger of the generation of reassortants with wild virulent strains

Vaccine strain

Virulent strain Reassorted andVirulent strain

10 20 30 40 50 60 70 80 90 100BTV-10 MEEFVIPVFSERDIPYSLLNHYPLAIQIDVKVDDEGGKHNLIKIPESDMIDVPRLSIIEALNYRPKRNDGVVVPRLLDITLRAYDNRKSAKNA-KGVEFMBTV-11 TE S VRTN IANVDEG DVV V V AAKT I D AM S RBTV-17 Y DE A SR TN IE VE VV I K T VM KA I D T S R IBTV-13 L IT FDKR VGR DYV ELARPEG WSG DVTH DRR F IKVQP RDID K VE E L I MSIAC M RMMKR D D VBTV-2 D LG IYG NY EH KG EFL NTG YPSQ R DVS MFAY IKDEG RD K M T L IV S KG I VIE NSNSHBTV-1 D LG IYK GF EH KG EFT DSST IQSV R DVT L MNAY IKSEGMRT W N V F L V G GKRAVIDSSRHKS H

110 120 130 140 150 160 170 180 190 200BTV-10 TDTKWMKWAIDDKMDIQPLKVTLDN HCSVNHQLFNCIVKARSANADTIYYDYYPLENGAKRCNHTNLDLLRSLTTTEMFHILQGAAYALKTTYELVAHBTV-11 NA R AI D NA P V S F RDKV K G M C SS ITNBTV-17 NAR R H Y V NA F DYK NM L A SI SS YBTV-13 S LE M Q S V DMKEDHSTVQYDMFSAKLHVDSRKADT S NILALETKEGAQ H VHTNIWNHMIRNHL AV ESC IF P K TVN BTV-2 S VQ M K S Q ISI EE SR V S Q IDAKK LS HLEAI DAE A L RGQ CNH RMDLL AA EI I P Q IVBTV-1 ER VQ MMK S A G D QTQKIA S H V ID KK MS HIE I DSL G L RTM WNH VRV S AA EIP DI V

•210 220 230 240 250 260 270 280 290 300

BTV-10 SERENMSESYQVGTQRWIQLRKGTKIGYRGQPYERFISSLVQVIIKGKIPDEIRTEIAELNRIKDEWKNAAYDRTEIRALELCKILSAIGRKMLDVQEEPBTV-11 N TE T AP VHNR R VR R K EA S V R Q QT P VDD R E RT QF S K NTHBTV-17 GSLE T V QPK H TR R NS LS M SVN AN Q RA T T GR S T NTHBTV-13 RTPD DF I NPQFLT RNQQ FLGDDA KKTAKG LVN VV I N A DA R K QGN E H KS NL VNLE BTV-2 AST DNFEL R DV T R HRVQMGDEA TKLMER RLTVQ NV RK QS EQ EA RTR ATGR PAH NSQD R R I QEABTV-1 A RDR QPFRP D TL NFSR Q VQMNHNS KMVEG AHLV R T EL D TK DE CNR RSR PG K Y V TV QEK

•310 320 330 340 350 360 370 380 390 400

BTV-10 KDEMALSTRFQFKLDEKFIRTDQEHVNIFKVGGSATDDGRFYALIAIAGTDTQQGRVWRTNPYPCLRGALIAAECELGDVYFTLRQTYKWSLRPEYGQREBTV-11 D D KK S I N AP HE A R Q H RV QD RTBTV-17 D N A S G R P E A K V S RV T HBTV-13 RD L H D AKN RNV AQKSQRN QD V MV AS NNS WS K YK SW E V EG KP DBTV-2 V DS L RA RLN S RNK EHKSHKK ED V LR AS YNS I WS T TK SW D V SS IPBTV-1 A AN I E I N RQH S RLK EHRNQRR ED I LM AS FNT WS T S TK SMM LW D V T IPY

•410 420 430 440 450 460 470 480 490 500

BTV-10 RPLEDNKYVFARLNLFDTNLAVGDEIIHWRYEVYQPKETTHDDGYICVSQKGDDE LLCEVDEDRYKEMFDRMIQGGWDQERFKLHNILT EPNLLTIBTV-11 V N S I S E QVV K IDG A Y N KTERE G V KIS EK T L YSV D BTV-17 Q N N I S Q KASA Y S M RHEAEE KIN K E L D BTV-13 LDRQYE IVG V LEAEP TKVL E LISKLY VSNHEGNQCDLHP EGE IVTKF DT SD IQTI NE K ND MFKM KD G P LYBTV-2 ER TE I SKI YEAGPSSKV E QL KRERVVTLERGNPCDLYP EDDEVIITKF DAK S VGEI D ND E MYKL QEKG VBTV-1 KSR QE IYG V YVAEP TK E KLN QIKDITYEQGNPCDLFP DEA IVTKF DVA GQ VNDL N M K KSQG V

510 520 530 540 550 560 570 580 590 600BTV-10 DFEKDAYLGARSELVFPPYYDKWINSPMFNARLKIARGEIATWKADDPWSNRAVHGYIKTSAESLEYALGPYYDLRLQLFGDTLSLGQRQSAVFEHMAQQBTV-11 NI F L S F Q Y R TH G R SA NK V F V A T P Q F T I Y A K S Q QS BTV-17 NS L D F S R TK G S K N R PL DFV LF DE K E QYLS LBTV-13 L IK DRV RV F Q TYV I PCEV VGER NI YVK THRPLKADCI LMR HMSQ M VS Q TS IK TP SIHQSL RDBTV-2 TK YNT V L D G VA SKMR IET N S MIK TLKPMTDDPV LQR T AR I PG M RS NRT T TFDAKVSELBTV-1 K TSNEGVAM E F IA K R KH QRRN MVK TLSPIAFAPIVLQRLT ARF I PAIM QA RQ G TYD EISKI

610 620 630 640 650 660 670 680 690 700BTV-10 DDFSTLT DYTKGRTVCPHSGGTFYTFRKVALIILSNYERLDPSLHEGREHETYMHPAVNDVFRRHVLEMKDFSQLICFVFDYIFEKHVQLRNAKEARRIBTV-11 E PV S A DV AL MLMA S D M DH T SIGGANQKRI R II R D DMR BTV-17 PA QLR DA AL FLIG K S D M QR V STGGTYQKR SC T I RE DT YBTV-13 ASYAEILSRRRENLDYKSQCPIVTNL LLEKFFL IFTTMEKHYWEMDDDETE E KIDPSKFEVEGTLH V VMVHL RF RRF TVD S WBTV-2 P YEKVVSRFGVIKKPTRPCVTLTGRYILEKYSL LIDILKYHTEV NPQ EFT RIDPQ KFNGNTLS LN TVV IV LH RNYV SIY Y BTV-1 EGYAEILQRRGIVQIPKKPCPTVTAQYTLERYALFLINILEQHIIQSTD DVM S RVDYKLEV GENII I IVI FL RRRTV GVY S YM

710 720 730 740 750 760 770 780 790 800BTV-10 IYLIQNTSGAYRLDVLREAFPNFLKHVMNLRDVKRICDLNVINFFPLLFLVQDNISYWHRQWSIPMILFDQVIRLIPVEVGAYANRFGLKSFFNFIRFHPBTV-11 L V SLGEPQ SV S SRYFLK K Q S L I AV Y DT K I T BTV-17 V SLT TQ S ST FQRLLM KEI FVR L M H V DT K F M T BTV-13 LH RSA R E SRF A S DGLRI EF KVR IMLL L F TG A E AV V FYADK MI A Y TCILELMM FBTV-2 SR RSST A MSIIEFY T ARLIS A EPTYVK AL L I G MI K LL YTDRVKV L SSN Q FV YLEYMF F BTV-1 VTR RDAQ QN IN IT F T GY LSRVKEATI QEI YL L F S I T K V LF YAHELKV L S ND CS V YIEYMV F

810 820 830 840 850 860 870 880 890 900BTV-10 GDSKKRQDADDTHKEFGSICFEYYTTTKISQGEIDVPVVTSKLDTLKLHVASLCAGLADSLVYTLPVAHPKKSIVLIIVGDDKLEPQIRSEQIVNKYYYSBTV-11 A K LIS N AN A GVHT T I LS V C HV V S F BTV-17 EL K I E I VVA N NVHT M T M V RV LS V C HT SR NBTV-13 SYDTRNENLSEDVRACIGPIIN LD T N G QTSI ST ALLYETYLS I G FSEAILWY IT S CLIALE S ALTS EL IDK KRRFPLBTV-2 SLADRTSKV ESMIKVSKEMVN MK T E GVNLN ST SLLYDIYLS V G VS GV WY IT Y CV A E C RVPARL CDRLKLRFPL BTV-1 SKAFRTSKL EVQPKIAREMLK IN FE G NLN T QLLYETYLA I G S GI WY IT N CL A E S ERVPAS ASH KLRFPL

•910 920 930 940 950 960

BTV-10 RRHISGVVSICVNQGGQLKVHSMGITRHRICDKSILKYKCKVVLVRMPGHVFGNDELMTKLLNVBTV-11 K V IG ND Y S V E F R K Y BTV-17 K C I VTIG NS R TS VK V F H I YBTV-13 SN LK I Q S RP RTFS VTQ VK V K TL R R D I IQT Y L IBTV-2 AQ LK I V QI EE GFD YTE VT V K L HV DI LKFH ML BTV-1 VK LK I I Q DEE KFT Y E VS V K NL M DI LKFS ML

• •

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-10

L29026

BTV-1 Australia M21844

BTV-20 RSA

BTV-24 RSA

Comparison of All 24 serotypes of Bluetongue virus segment 2 (2.9Kb)

Generated by S. Maan, A. Samuel and P.P.C.Mertens , IAH, 2003

Map of the Mediterranean Basin showing the possible

routes of entry of the BTV serotypes.

BTVBTV--22

BTVBTV--99

BTVBTV--4 & 164 & 16

BTVBTV--11

BTV-9 from Eastinto Greece, the Balkans & Italy

BTV-1 from EastBTV-4 &16 from Mid East

BTV-2 from Sub-Saharan Africa

A. Samuel and P.P.C.Mertens, IAH

I Prichard, Geelong

Gnat transmitted Orbiviruses

Bluetongue Virus(BTV)

24 SerotypesBluetongue disease

of sheep

Epizootic haemorrhagic disease virus

(EHDV)8 Serotypes

Epizootic haemorrhagic disease of deer

African horsesickness virus(AHSV)

9 SerotypesAfrican horsesickness

disease of horse

Comparisons of the three major structural proteinsof BTV-10, EHDV-1 and AHSV-4

Sequence comparisons VP3 VP7 VP5

BTV-10 EHDV-1 BTV-10 EHDV-1 BTV-10

(%) (%) (%) (%) (%)

AHSV-4 Identity 58 57 44 46 45

Similarity 76 75 67 67 64

EHDV-1 Identity 79 - 64 - 62

Similarity 90 81 77

BTV VP5

PHLYOGENETIC ANALYSIS OF VP2

• Greatest variability is apparent within the VP2 sequences

• Evolutionary association between the 3 orbiviruses are apparent

• The serogroups are maintained as monophyletic clusters

BTV-11

BTV-17

BTV-10

AHSV-6

AHSV-3

AHSV-4

EHDV-1

BTV-3

BTV-13

BTV-2

BTV-1auBTV-1sa

BTV-23au

100

100 54

78

92

100

87

100

VP 215% divergence

The outer capsid; two major proteins

VP2 & VP5

The core; two major proteins

(VP3 and VP7) three minor proteins(VP1, VP4 and VP6)

The genometen dsRNAsegments

Four major proteins in

different molar ratios make up the Orbivirus

capsids

French & Roy, 1990;French et al,1990;

Loudon & Roy, 1991

Assembly of heterologous particles using different BTV serotypes allowed the determination of

functionally compatible proteins

BTV 10 VP3 + BTV 17 VP7 BTV 10 VP3 + BTV 17 VP7+ BTV 2 VP2 + BTV 1 VP5

CLP VLP

VP3 EHDV

VP7 BTV

EHDV-1 VP3 plus BTV-10 VP7

Native EHDV Cores

encapsidated into CLPs

VP1VP3

VP4

VP7

EHDV minor proteins can also be

Assembly of Heterologous Recombinant Particles using core proteins of BTV/EHDV

VP7

VP3

VP6

VP4

VP1

Particle assembly

The surface of the virus core is formed of 780 copies of the VP7 protein organized into 260 trimers

core is the transcriptional heart of the virus

Ability or not to assemble heterologousparticles may reside in atomic detail of

interaction

The VP7 trimers are arranged as T=13 lattice

RGD site Upperdomain

Lowerdomain

C-terminal end Helix 5

Helix 8

Helix 6

VP7 trimer

Comparison of the Top Domains of AHSV/BTV VP7

BTVAHSV

Construction of chimeric VP7 proteins

VP7 genes were spliced together to form VP7 mosaic proteins that exchanged the upper and lower domains of AHSV and BTV

AHSV BTVBTV BAB

ABA

UPPER DOMAIN

Amino terminal lower Carboxy terminal lower

Both of the chimeric VP7 proteins form trimers

Reconstruction of BTV Particle by Cryo-EM

Resol~23 Å

810 Å

VP2

Purified VP2

Incubation with cells at 37OC.

Stain

The most variable VP2 is the attachment and entry protein of the virus

110 kDa

Neutralisation titres

Antigen dilution giving 50% plaque reduction of BTV-10

VP2 alone

Virus-like particles

1:500

1:100,000

0

20

40

60

80

100

120

% R

ecip

roca

l clin

ical

sym

ptom

s

Antigen (n=4)

1000 µgVP2

100 µgVP2

50 µg VP2+

25 µg VP5

saline500 µgVP2

Effect of antigen dose and composition on protection against challenge

1

10

100

1000

21 28 40 49 63 77 91

105

117

20010050100

Days

Neu

tral

izin

g A

b tit

re challenge

Dose(µgs)

Outcome= all

protected

Long term protection against BTV challenge after NAb titres have reduced

BTV-1

Vaccinate Boost

On homologous

serotypes

On hetorologous

serotypes

Challenge after 14 months

Monitor neutralizing antibodies

Monitor CRI

BTV-2

BTV-17

BTV-10

BTV-13

BTV-1

BTV-2

BTV-17

BTV-10

BTV-13

LIVE

BTV

Serotype specific

protection

Crossprotection

to some other

serotypes

Recombinant particles lacking the genome- ideal for an efficient and safe vaccine

VLP offers a novel technology for safe vaccine development

Rotavirus VLPs

Bovine HumanPorcine

SARS VLPs

Assembly of M, E & S proteins

Jonathon Diprose

Grimes et al, Nature 1998

• Surveillance is key to the discovery and tracking of emerging disease.

• However, an active response can only be based on fundamental research that explains how the emergent virus differs from those already circulating.

• Based on common structural features it may be possible to have a “base” VLP style vaccine to which the sero-dominant epitopes of an emerging strain are added.

• Such a vaccine could complete all regulatory hurdles in the base form and so be ready for rapid roll-out, incorporating relevant antigens, should an outbreak occur.

A universal response to an emerging virus

B. V. Venkataram Prasad (Bayler College, Texas)

Elizabeth A. Hewat(Grenoble, France)

David Stuart and group(University of Oxford)

Peter MertensPhillip mellor(Pirbright Laboratory, IAH)

Collaborators

Baltus Erasmus(Onderstepoort, SA)