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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)
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