Virus Research, 15 (1990) 213-230

Elsevier 213

VIRUS 00560

Expression of the outer capsid protein, VP2, from a full length cDNA clone of genome segment

2 of bluetongue serotype 1 from South Africa, using both Sp6 and vaccinia expression systems and a comparison of the nucleic acid sequence of this segment with those of other serotypes

A.M. Wade-Evans and P.P.C. Mertens AFRC Insiitute for Animal Health, Pirbright Laboratory, Pirbright, Wok& Surrey, lJ. K.

(Accepted 9 November 1989)

Genome segment 2 of bluetongue virus serotype 1 from South Africa (BTV-1SA) was purified from a preparation of all ten dsRNA segments. This dsRNA was used as a template to make a f~-length DNA copy of segment 2, which was then cloned into pUC19. The cDNA insert was transferred into a bacterial expression vector (pGEM; PROMEGA) and, by means of in vitro transcription and translation systems, used to synthesise a polypeptide of similar size to VP2 (as analyzed by PAGE). The cDNA insert was also transferred into a vaccinia virus vector using homologous recombination. The resulting recombinant virus when transfected into TK- cells produced a protein that co-migrated with VP2 of bluetongue virus. Immunoprecipitation of these polypeptides, synthesised by in vitro and in vivo techniques, using BTV-1SA antisera, confirmed that they were virus specific.

Nucleotide sequence analysis of the cDNA demonstrated that genome segment 2 is 2940 base pairs in length. The positive sense ( + ve) RNA strand contains an open reading frame, coding for a pol~~tide of 961 amino acids, which is flanked by 3’ and 5’ terminal non-coding regions of 37 and 17 nucleotides, respectively. Compari- son with published data shows that genome segment 2 of BTV-ISA is identical in these characteristics to segment 2 of BTV-1 from Australia (BTV-1AUS) but differs

Correspondence fo: A.M. Wade-Evans, AFRC Institute for AnimaI Health, Pirbright Laboratory, Pirbrigbt, Woking, Surrey GU24 ONF, U.K.

0168-1702/90/$03.50 6 1990 Elsevier Science Publishers B.V. (Biomedical Division)

214

from isolates of the five American serotypes of BTV (BTV-2, -10, -11, -13 and -17). However, there is a higher level of homology, in both the nucleotide and the amino acid sequence of genome segment 2 and protein VP2 respectively, between the two isolates of BTV-1 and the American isolate of BTV-2, than there is between BTV-2 and the other American serotypes. The significance of this similarity is discussed.

Bluetongue virus; Genome segment 2; VP2; Sequence homology; Vaccinia virus recombinant

Introduction

Bluetongue virus is the prototype member of the Orbiuirus genus within the Reoviridae (Matthews, 1982). The virus mainly causes disease in sheep but can also infect cattle and goats. The virus particle is composed of a two-shelled protein coat, containing a genome made up of ten segments of dsRNA. The inner protein layer contains six separate proteins (VP& VP3, VP4, VP6/VP6a and VP7) surrounded by an outer capsid layer, which is composed of two major protein components (VP2 and VP5) and, in at least some cases, is associated with small amounts of protein NS2 (Verwoerd, 1969; Martin and Zweerink, 1972; Verwoerd et al., 1972; Gorman et al., 1983; Huismans, 1979; Van Dijk and Huismans, 1980; Mertens et al., 1987a).

Bluetongue viruses are classified into serotypes on the basis of their reaction in serum-neutralisation tests (Gorman et al., 1983). Twenty-four serotypes have so far been identified (Knudson and Shope, 1985). The neutralising antibodies, which are used in these neutralisation-tests, react with the major components of the outer capsid layer of the virus particle, VP2 and VP5 (Huismans and Erasmus, 1981; Huismans et al., 1983,1987; Appleton and Letchworth, 1983; Grubman et al., 1983; Kahlon et al., 1983; Inumaru and Roy, 1987; Purdy et al., 1985; Letchworth and Appleton, 1983). Proteins VP2 and VP5 of BTV are encoded by genome segments 2 and 5, respectively (Mertens et al., 1984; Pedley et al., 1988; Appleton and Letchworth, 1983). The question as to whether both major outer capsid proteins appear to play some part in the specificity of this reaction is as yet unresolved (Kahlon et al., 1983; Cowley and Gorman, 1989; Mertens et al., 1989). Even if VP5 does have a role to play in neutralisation, VP2 seems likely to be more significant, as demonstrated by the ability of gel-purified VP2 to elicit neutralising antibodies, to block cell attachment and to protect sheep from BTV infection (Ht.&mans et al., 1983, 1987; Inumaru and Roy, 1987). Although cross-hybridisation studies demon- strate that both of these segments (unlike the remainder of the genome) contain serotype specific RNA sequences, segment 2 was found to be the most variable among different BTV serotypes (Huismans and Cloete, 1987; Kowalik and Li, 1987; Mertens et al., 1987b). This conclusion has been confirmed by full nucleotide sequence analyses of these BTV genome segments (Ghiasi et al., 1987; Wade-Evans et al., 1988; Gould, 1988; Gould and Pritchard, 1988; Fukusho et al., 1987; Yamaguchi et al., 1988a, b; Purdy et al., 1985, 1986).

215

A possible method of demonstrating unequivocally the role of each of these outer capsid proteins in neutralisation and protection, is to use expression vectors that will allow them to be synthesised individually, in vivo. Thus their functions can be studied ~dependently of each other and any other viral proteins. ~fication of the same proteins from virus particles involves the risk of contamination, however small, with other viral proteins. To this aim we have transferred full-length clones of segments 2 and 5 into vaccinia virus and Sp6 vectors (Wade-Evans et al., unpub- lished data). The results obtained from expression of the segment 2 cDNA clone are discussed in this paper.

The ancestry of ‘new’ American serotypes of BTV has been the subject of much speculation and is of considerable epidemiological interest (Barber, 1979). Data presented by Yamaguchi et al. (1988a), for genome segment 2 of BTV-2, suggest that this serotype is less closely related to other established serotypes of BTV from North America, than they are to one another (Purdy et al., 1985; Ghiasi et al., 1987; Fukusho et al., 1987). To analyse the possible relationships between BTV serotypes isolated from distinct geographical locations, we have compared the sequence of genome segment 2 from BTV-lSA, with published data for the same segment from BTV-1AUS (Gould, 1988) and the North American isolates of BTV-2, -10, -11, -13 and -17 (Ghiasi et al., 1987; Purdy et al., 1985; Yamaguchi et al., 1988a). The implications of the results obtained concerning the possible ancestry of BTV serotypes in North America (in particular serotype 2) is discussed.

Materials and Methods

Growth and ~~i~cution of virus and viral RNA

The South African isolate of bluetongue virus, serotype 1 (BTV-1SA) used is identical to that described in previous publications (Mertens et al., 1984, 1987a, b; Mertens and Sangar, 1985; Pedley et al., 1988; Wade-Evans et al., 1988). The virus was grown in BHK cells and virus particles or cores were purified as described by Mertens et al. (1987a).

Double-stranded RNA was extracted from BTV-1SA cores, separated into in- dividual genome segments, and isolated from, polyacrylamide gels according to the method of Mertens et al. (1984).

Cloning and sequencing of genome segment 2

The cloning method adopted was essentially similar to that of Gubler and Hoffman (1983), with modifications as described in detail by Wade-Evans et al. (1988). The cDNA clones obtained were transferred to pUC19 and analysed by direct plasmid sequencing using DNA extracted from bacterial (TGl) mini-preps as described by Chen and Seeburg (1985). The sequence was confirmed in both orientations and verified from several independently derived clones (see Results).

216

Probes and Northern blots

DNA restriction fragments to be used as probes were labelled with [(Y-~~P] by the oligonucleotide method of Feinberg and Vogelstein (1983).

Northern blots were performed as described by Mertens et al. (1987b).

In vitro transcription/translation of segment 2 cDNA

A full-length copy of BTV-1SA genome segment 2 (see Results for construction of the full-length cDNA) was unidirectionally transferred to pGem2 by a EcoRl/BamHl double digest. The pGem construct was linear&d with BamHl

prior to transcription, so that a run-off transcript could be generated using Sp6 polymerase and the method described by Melton et al. (1984).

The Sp6 transcript was translated in a reticulocyte lysate system as described by the manufacturers (Amersham). The resulting protein products were labelled with [ 35S] and immunoprecipitated as described previously (Wade-Evans et al., 1988).

BTV genomic dsRNA preparations were denatured and translated in vitro as described by Mertens et al. (1984).

Synthesis of peptides and production of anti-peptide antisera

All peptides were synthesised and antisera were raised in female New Zealand white rabbits as described previously (Wade-Evans et al., 1988).

Construction of vaccinia recombinants and expression of VP2 in mammalian cells

The cDNA copy of genome segment 2 was inserted into the shuttle vector pGS20, downstream of the vaccinia ~7.5 promoter and transferred, by homologous recombination into vaccinia virus as described by Mackett et al. (1984), thus generating TK- recombinants that were capable of expressing the bluetongue virus protein, VP2, in 143 osteosarcoma cells (Wade-Evans et al., unpublished data).

Cell monolayers infected with the recombinant virus were washed twice in methionine-free medium (30 min each wash), then incubated in fresh medium containing [35S]methionine for 4 h. The cells were washed twice in cold PBS and then lysed in NP40 buffer (Wade-Evans et al., 1988). Cell extracts were stored at - 70 o C prior to immunoprecipitation. All immunoprecipitations were performed as described by Wade-Evans et al. (1988).

Enzyme-linked immunosorbent assay system

96-well trays were coated for 16 h at 4°C with 5 pg/ml of peptide in coating buffer as described by Anderson (1987). The plates were then washed 4 times in PBS and incubated with 50 ~1 of the serum samples for 1 h at 37 o C, with shaking. The plates were again washed 4 times in PBS. Fifty microlitre of l/2000 dilution of Protein A conjugated to peroxidase (DAKO) was added to each well and the plate

217

incubated for 1 h at 37” C with shaking. The plates were washed 4 times in PBS. O-phenylenediamine (OPD - Sigma) was added as a substrate and the results read on a multiskan (Titertek) at 492 nm after the addition of 1.25 M sulphuric acid to the wells.

Results

A full-length DNA copy of genome segment 2 of BTV-1SA was constructed by ligating together the inserts of three partial clones (2F45, B89 and B33; Fig. 1) using two unique enzyme sites (Axy 1 and Pfl Ml, marked in Fig. 1 as Ax and P, respectively). The three partial clones were used separately to probe a Northern blot of all ten segments of BTV-1SA genomic RNA and were found to hybridise only to segment 2 (Fig. 2), demonstrating that none of these clones contain sequences homologous to, or derived from, other BTV-1SA genome segments. The presence of both terminal sequences, which had been previously determined by direct RNA sequencing of genome segment 2 (Mertens and Sangar, 1985) and which were used to synthesise the oligonucleotide primers for cloning, suggested that collectively the sequences of these clones overlapped to include a full length copy of segment 2. The sequence of the assembled full-length cDNA (shown in Fig. 3) was confirmed by analysis of a number of partial length, independently derived, clones (according to the sequencing strategy shown in Fig. 1). To confirm that the full-length cDNA clone codes for VP2, the insert was placed down-stream of an Sp6 promoter, in pGEM, and transcribed in vitro (Melton et al., 1984) and then translated using a rabbit reticulocyte lysate system to produce a major protein product, which co- migrated during PAGE with VP2 from virus particles of BTV-ISA. This protein was also immunoprecipitated by antisera raised against purified BTV-1SA virus par- ticles, thereby confirming its viral specificity (Fig. 4).

The deduced amino-acid sequence for VP2 of BTV-1SA (Fig. 3) was used to define the sequences of two synthetic peptides; ,,,GNVLTIDFEKDA,,, (VPZX),

Fig. 1. Sequencing strategy of cDNA clones. The boxed regions represent the three cDNA clones; 2F45, 2B89 and 2B33; used to generate a full length DNA copy of genome segment 2 of BTV-ISA. The arrows

indicate the strategy adopted to determine the complete sequence of 2940 nuoleotides. Restriction enzyme sites are indicated as follows: A, Act 1; Ax, Axy 1; P, Pfl Ml.

218

Fig. 2. Confirmation of the origin of the cDNA clones. Unlabelled dsRNA (track 1) and ten-fold less 3’ end-labelled dsRNA (track 2) were transferred to nylon filters and probed with the cDNA insert from p2F45 labelled with [cY-~‘P]~ATP. The insert hybrid&d solely to segment 2. Hybridisation with the

inserts from p2B89 and p2B33 gave identical results (data not shown).

chosen because of its conserved nature between serotypes and ,,,ERLKI- FEHRNQRRDEDRFYILLMIA,, ( VPZY), chosen because this region has been identified as a neutralisation epitope on VP2 of BTV-1AUS (Gould et al., 1988). Antiserum raised against peptide VP2X failed to immunoprecipitate VP2 translated in vitro from BTV RNA (Fig. 4), but the antisera raised against peptide VP2Y did recognise VP2 synthesized in this way. The failure of VP2X to react with the VP2 protein was not due to a poor immune response to the peptide in the rabbit as can be seen from the ELISA results obtained for both peptides (Table 1). However, these antisera both failed to immunoprecipitate VP2 synthesized in vivo by a vaccinia virus recombinant containing BTV-1SA genome segment 2 (Fig. 5) and did not recognise VP2 synthesised in BTV-1SA infected BHK21 cells (by im- munofluorescence - data not shown). Neither of these peptide antisera reacted significantly with BT’V virus particles, either in an ELISA or in serum neutralisation (data not shown).

The complete sequence of segment 2 of BTV-1SA and the deduced amino-acid sequence of VP2 were compared to those published for BTV-1AUS (Gould et al.,

219

1* v* M D E L G I PVYKRGFP

GTTAAAATAATAGCGCGATGGATGAACTAGGCATCCCAGTTTATAAGAGAGGATTTCCCG 10 20 30 40 50 60

H T* S* VI1 Q

LLRGYEFII DVGTKIESV ~C~CCTGCTTCGTGGTTACGAGTTCATAATAGATGTTGG~CC~GATA-GTGTTG

70 SO 90 100 110 120

L S* GGRHDVTKI PEHNAYD I K Q E GAGGACGTCATGATGTAACGAAAATACCAGAAATGAATGCATATGACATCAAGCAGGAGGAGA

130 140 150 160 170 180

G M vr2 V V RTALWYNPI R N D G F V L P R

~CA~CCGAACCGCATTATGGTATAATCCGATAAGAAATGATGGTTTTGTATTGCCGCGAG 190 200 210 220 230 240

G K I D .E* V L D I TLRGYD ERRAVVESTR TGCTGGATATCACATTGAGGGGTTACGATGAAAGACGGGGCGGTTGTTG~GTACGAGAC

250 260 270 260 290 300

E R* HKSFHTNDQWVQWMMKDSMD ACAAGAGTTTCCATACGAATGACCAGTGGGTGCAGTGGATGATGAAAGACTCGACTCGATGGACG

310 320 330 340 350 360

D* T* Q* K* I

A Q PLKVGLD TQVWNVAH S L H CTCAGCCTTTAAAGGTTGGGTTAGATACTCAAGTATGGAR

370 380 390 400 410 420

C K* s* I NSVVEIDS KKADTMAYHVEP ACTCGGTAGTCGAAATCGATTCAG~GGCTGATACTATGGCTTACCATGTAGAGCCGA

430 440 450 460 470 480

s* L* L I E D A S KGCLHTRTMMWNHLV TAGAGGACGCGTCAAAGGGGTGTTTGCATACGAGAACCATC

490 500 510 520 530 540

V H* s* I L* T* L R I ETFHAAQ E V H I LFKPTYD GAATAGAAACATTTCATGCGGCGCAGGAGGAGGTGCATATACTCTTTAAACCTACTTATGACA

550 560 570 580 590 600

D IVVHAERRDRSQP FRPGDQT TCGTGGTCCACGCTGAAAGGAGAGATCGTAGTCAACCGTTTAGGCCAGGGGATCAGACAT

610 620 630 640 650 660

S 9* E L I NFGRGQ KVAHNHNSYDKN TAATTAATTTTGGGAGAGGTCAGAAGGTOGCAATGAACCACAATTCATATGATAAGATGG

670 680 690 700 710 720

A* L E VEGLTHLVI RGKTPEVI R D D TTGAGGGATTAACACATTTAGTGATTAGAGGGAAAACTCC

Fig. 3. Nucleotide and deduced amino acid sequence of genome segment 2 of BTV-1SA: comparison with

the sequence from BTV-IAUS. The deduced amino acid sequence for BTV-1SA is printed in single letter

code above the nucleotide sequence. The amino acid changes between BTV-1SA and BTV-1AUS are

indicated in the line above the BTV-1SA amino acid sequence. The amino acids accompanied by an

asterisk represent non-conservative changes. The position of the two peptide sequences are shown by

boxes around the relevant amino acids. The hypervariable regions vrl-6 mentioned in the text are

underlined.

220

730 740 750 760 770 780

T* K* R* Y* IASLDEI CNRWIQS RiDPGE TTGCGAGCTTGGATGAGATATGTAATAGGTGGATAGGTG~TACAGAGTAGG~CGACCCCG~GAGA

790 800 810 820 830 840

v* V M* Q" I KAY E L C K Y L STIGRKSLDR TAAAAGCATATGAATTATGTAAATATTTATTTATC~CGATTGGTCG~GTCCCTAGATCGAG

850 860 870 880 890 900

A* E K E P E DEANLSI RFQEAIDN AGAAGGAACCAGAGGACGAGGCGAATCTATCGATCAGATTTCAAGAGGCAATCGACAATA

910 920 930 940 950 960

s* VPPY Doratide K F R Q HDP[ERLKIPEHRNQRR AGTTCCGACAACATGATCCTGAGCGCCTGAAGATATTTGAGCATAGGMTCAACGTAGAGAG

970 980 990 1000 1010 1020

A DEDRFYILLMIAjGSDTFNTR ATGAGGACCGATTCTATATTCTCTTGATGATTGCAGGCTCCGATACGTTT~TACACGAG

1030 1040 1050 1060 1070 1080

G T v w w s NPYPCLRRKL I A S E T K TGTGGTGGTCGAATCCATATCCATGTTTAAGAAGGAAGTTAATCGCGTCGCGAAGC

1090 1100 1110 1120 1130 1140

L vr3

LGDVYSMMRSWYDWSVRPTY TAGGTGACGTTTATTCGATGATGCGTTCATGGTATGATTGGAGTGTTCGCCC~CCTATA

1150 1160 1170 1180 1190 1200

1* s* A P Y E KTREQE K Y I YGRVNLF CGCCTTACGAGAAAACGAGAGAACAGGAAAAA TATATTTATGGGCGGGTTAACCTGTTTG

1210 1220 1230 1240 1250 1260

Y T* Q Q* DFVAEPGIKI IHWEY-KLNHS ACTTTGTCGCGGMCCTGGMTTATTCATTATTCATTGGGMTATMGCTGMTCATTCCA

1270 1280 1290 1300 1310 1320

I*K D E* vr4

L* F D E T R E I TYAQGNPCDYYPEDDD CCCGGGAGATAACCTATGCGCAAGGGAACCCATGTGATTATTACCCAGAGGATGATGATG

1330 1340 1350 1360 1370 1380

A* V D L v I VTKFDDVAYGQ MINEMIN TAATAGTCAC~AAGTTCGATGATGTCGCGTATGGCC~TGATC~TGAGATGAT~TG

1390 1400 1410 1420 1430 1440

D R Q GGWNQEQF KMHKILKS E(G N V GGGGTTGGAATCAAGAACAGTTCAAGATGCATAAAATTTTAAAATCGGAAGGTAATGTAATGTTC

1450 1460 1470 1480 1490 1500

VPPX Peptide R* S* A* L T I D F E K D A) K LTTNEGVTMP TAACGATAGATTTTGAAXUA GATGCAAAGCTAACMCCAATGAAGGCGTAACGATGCCAG

1510 1520 1530 1540 1550 1560

Fig. 3 (continued).

221

D EYFNKWI IAPMFNAKLRIKH AATATTTCAATRAGTGGATAATCGCTCGCTCCGATGTTCAACGCTAAGCTGCGTATAAAACATG

1570 1580 1590 1600 1610 1620

G R* N* E E IAQRQ8DDPHVKRTLSPI RAGAGATTGCGCAACGGCAAAGTGATGATGATCC~TGGT~CGTACTCTATCACCTATAG

1630 1640 1650 1660 1670 1680

F* A A D P IVLQRLTLAR FYDIRP CCGCAGATCCAATCGTATTGCAAAGGTTGACTTTGGTTGACTTTGGCGAGGTTTTACGACATTCGTCCCG

1690 1700 1710 1720 1730 1740

I M A G A L I G Q G L SRQQAQSTYDEEI CTTTAATAGGACAAGGCCTTTC~CGGCAACAGGCAGGCACAGTCCACTTACGATG~G~TAT

1750 1760 1770 1780 1790 1800

I E Q* SKQAGYAEILKRRGIVQIPK CGAAACAAGCGGGATATGCGGAUTATTGGTCCCCAAGA

1810 1820 1830 1840 1850 1860

Rf A* F* K P C PTVTAQY TLELYSLSLI AACCTTGTCCAACTGTGACAGCTCAGTATACCCTGGAACTGTATAGCTTGTCATTAATA

1870 1880 1890 1900 1910 1920

E I I* Q* S* T* D V*M s* N I LQQHVAR DCDEEAI Y E H P ACATCTTACAACAGCATGTAGCACGAGACTGCGACGAGGAGGC~TATAC~G~TCCGA

1930 1940 1950 1960 1970 1980

R V* “” K* V H* N* I KADYELEI F G E S I V D I SQ:: AAGCAGATTATGAACTTGTATTTGGCGAGAGCATTGTGGATATCTCTC~GTGATCG

1990 2000 2010 2020 2030 2040

I F P L T* G VLVFDLI F E R R R R V R .D V Y E S TTCTAGTTTTTGACTTGATATTTGAGAGAAGAAGGAGGGT~GA~TGTGTAT~TCGC

2050 2060 2070 2080 2090 2100

M V D* A* Q* Q* N I T* R Y I I T R I RRMRGK ERLNVIA GGTACATAATTACGCGCATTAGGAGGATG~GAGGTAAAGCGTGATCGCGG

2110 2120 2130 2140 2150 2160 qre

F Y* H* S* R* V K* E* T*I I E FFPT?GiiLNGLNSA;;;Q AGTTTTTCCCAACCTATGGRAGTCTTCTAAATGGATTAAACAGCGCGCGTACGTAGTACAGG

2170 2180 2190 2200 2210 2220

E F V* S N I DIMYLNFLPLYPLAGDNMIY ATATTATGTATCTAAACTTTCTCCCATTGTACTTTTTAGCAGGCGATAACATGATATATACT

2230 2240 2250 2260 2270 2280

T* K V F A* L K* S HRQWSI PLLLYTHEVMVIP CTCATAGGCAGTGGTCTATTCCTTTACTTCTATACACTCATG~GTGATGGTGATCCCAT

2290 2300 2310 2320 2330 2340

Fig. 3 (continued).

222

S v s* I LEVGSYNDRCGLIAYLEYMV

F T* 5 V*Q P F F P SKAIRLSKLNEAHAKIA

1* K*I F E I* II* R E M L KYYANTTVYDGGDNSN

I L VVTTRQLLYETYLASLCGGF TTGTGACGACGARGCAGCTACTATAT~~~TACT~~~~TTCGTT~~~~GGGGGTTTTT

2530 2540 2580

s* N* L 1. rl r. I v w Y L P IT B P K R C I VA I

s Ii K EVSDERVPASIRAGRI R L R F AGGTATCTGATG~GGGTTCCGGCT~~~~~G~~~~GCGTAT~GGCT~GATTTC

2650 2660 2690 2700

V* K I* v* V Z* Z* K

H* R* F T V Y SZGIVSFLVCKKNLLK

M* D V YKCEIILLKFSGHVFGNDER ATAAGTGCGAAATTATATTACTGAAG;;;;CGGGGCMX;TTTTGGW&-AAATGC

2830 2840 2880

LTKLLNV* TGA~CTCCT~CGTAT~TCT~~~~GACCG~~~CCGCG~~~~~CTTAC

2890 2900 2940

Fig. 3 (continued).

1988; Fig. 3). Seventy-four percent homology was found at the nucleotide level, which increased to 89% when the ammo acid sequences were compared (including conservative ammo acid changes). These comparisons and those made with the published sequences for other BTV serotypes are shown in Table 2. As might be expected, BTV-1SA shows lower levels of homology in segment 2, with the Ameri- can isolates, which are of different serotypes, than it does with BTV-1AUS. However, when considering the relationships between different serotypes, greater similarity was detected between the BTV-1 isolates and BTV-2, than between BTV-2 and the other American isolates (Table 2).

223

TABLE 1

Results from ELISA using peptides VPZX and VP2Y as antigen

Plate A 1

A 0.05 B 0.05 c 0.04 D 0.05 E 0.58 F 0.79 G 0.06 H 0.04

Plate B 1

A 0.05 B 0.04 c 0.04 D 0.05 E 0.04 F 0.04 G 0.68 H 0.04

2 3 4 5 6 0.04 0.04 0.08 0.05 0.04 0.04 0.13 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.87 0.85 0.63 0.44 0.28 0.86 0.78 0.56 0.42 0.31 0.05 0.09 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04

2 3 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.79 0.68 0.05 0.05

4 0.05 0.04 0.04 0.06 0.05 0.04 0.55

5 6 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.41 0.32 0.04 0.04

7 0.04 0.04 0.04

0.16 0.16 0.04

7

0.04

8 9 10 11 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.09 0.05 0.04 0.05 0.09 0.06 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04

8 9 10 11 12 0.04 0.04 0.04 0.07 0.05 0.05 0.05 0.04 0.04 0.05 0.04 0.04 0.04 0.06 0.04 0.04 0.04 0.04 0.04 0.08 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.12 0.08 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.03

12 0.07 0.04 0.04

Plate A was coated with peptide VP2X as described in the Materials and Methods. The antisera were diluted across the plate using doubling dilutions fin PBS/M~el/O.l% Tween-20) starting at a dilution of l/10. Plate B was coated with peptide VP2Y using the same method. The following antisera were used: A, normal rabbit sera; B, anti-bluetongue rabbit sera; C, pre-immune rabbit sera; D, anti-VP5 peptide sera (control); E, anti-VP2X sera; F, anti-VP2X sera (another rabbit); G, anti-VP2Y sera; H, normal rabbit sera. The OD of each plate was read at 492 nm on a Multiskan and the results as shown above printed out using a BBC computer.

TABLE 2

Nucleotide and amino acid sequence comparisons between serotypes

Nucleotide 1SA 1AUS 2 13 10 11 17

ISA - 74 57.8 52.8 49.5 48.5 48.9 1AUS - 99.3 B 60.4 52.5 50.4 48.7 50.6 2 - - 53.6 49.5 50.2 50.4

13 - - 49.2 50.1 50.0 10 67.4 68.8 11 68.5

Amino acid ISA 1AUS 2 13 10 11 17

1SA - 89.4 73.0 63.9 59.2 58.5 59.1 1AUS _ 99.4 B 75.4 64.9 62.3 61.5 62.1 2 66.4 60.6 59.5 61.5

13 - 61.4 60.2 60.2 10 - 83.9 84.7 11 84.6

Values given in the above Tabfe represent % homologies between serotypes. a represents the homology between the sequences of two independent isolates of BTV-1AUS (Gould, 1988; Yamaguchi et al., 1988b). These vahtes include conservative amino acid changes.

224

TABLE 3

Comparison of segment 2 non-coding and coding regions

Length

1SA

1AUS 2

13

10 11

17

Non-coding

5’ Region

17

17 17

21

19 19

19

3’ Region

37

37 37

34

36 36

36

Coding

Nucleotides

2940

2940 2943

2935

2926 2926

2923

Amino acids

961

961 962

959

956 956

955

Although the genome segment 2 of BTV-2 contains three more base pairs in its coding region than either of the isolates of BTV-1 (and therefore VP2 is one amino acid larger) the 3’ and 5’ non-coding regions are of the same lengths in all three viruses (Table 3). BTV-2 differs in these respects from isolates of the other American serotypes (BTV-10, -11, -13 and -17). Apart from the clear similarities between viruses of the same serotype, from these comparisons and those published for the American isolates of different BTV serotypes (Yamaguchi et al., 1988a; Ghiasi et al., 1987), it appears that these viruses could be divided into three sub-groups: BTV-lSA, BTV-1AUS and BTV-2; BTV-10, BTV-11 and BTV-17; and BTV-13.

Discussion

The full length cDNA clone of genome segment 2 was used to express VP2 protein both in vitro (using the Sp6 promoter and rabbit reticulocyte lysate system) and in vivo (using the ~7.5 vaccinia early promoter and TK- cells). The protein produced, in both cases, con&rated with VP2 from virus particles (Figs. 4, 5). The regions of VP2 represented by peptides VP2X and VP2Y do not appear to be exposed on the outer surface of the protein when it is expressed in vivo, since antibodies raised against them do not recognise VP2 expressed by the vaccinia recombinants in TK- cells. However antiserum to VP2Y did immunoprecipitate VP2 synthesized in a rabbit reticulocyte lysate system indicating that the conforma- tional structure of this protein is dependent on its mode of synthesis. The antibodies against VP2Y also failed to react with virus particles in either an ELISA or a serum neutralisation test, suggesting that VP2 synthesized in the vaccinia expression system may adopt the correct conformation as opposed to the in vitro synthesized protein. However, these results indicate that the region of VP2 identified by Gould et al. (1988) as a neutralising epitope (VP2Y) may not be exposed on the surface of virus particles and may therefore not be the actual site of neutrahsation. It seems likely that mutations within this region (321-346) may affect interaction of BTV

225

Ml234 M5 6 7 8

Fig. 4. Analysis of the in vitro translation products from the cDNA clone of genome segment 2 and from viral dsRNA. Sp6 transcripts were synthesised in vitro from pGem2, Seg2 as described by Wade-Evans et al. (1988). dsRNA was denatured using methyl mercury as described by Mertens et al. (1984) to generate single-stranded RNA that could be used as a template for translation. These transcripts were translated in a rabbit reticulocyte lysate system using [‘?i]methionine and then i~un~r~ipitat~ by: normal rabbit serum (tracks 1 and 5); anti-BTV-1SA virus serum (tracks 2 and 6); anti-VPZX serum (tracks 3 and 8); and anti-VF2Y serum (tracks 4 and 7). Tracks l-4 are immunoprecipitations of translates generated using the Sp6 system and tracks 5-8 are the results obtained using viral RNA as the template.

The lanes marked M represent a translation of all ten RNA segments of BTV-1SA.

virus particles with neutralising monoclo~ antibodies by exerting a confo~ational constraint upon the true neutralising epitope. Gould et al. (1988) observed than au ammo acid change at position 328 of VP2 also affected the binding of a second distinct monoclonal antibody, to a different epitope, again indicating that this region (321-346) may influence the conformation of VP2.

Antibodies to VP2, expressed using the vaceinia virus system, have been raised in rabbits and are currently being used in neutralisation tests in order to establish if neutralising antiserum can be raised by this protein acting as an immunogen.

Comparison of the amino acid sequences of VP2 from North American serotypes 10, 11 and 17, by Ghiasi et al. (1987) identified six regions within the protein that were hypervariable. They postulated that these may represent antigenic sites based on hydropathy and secondary structure predictions (labelled vrl-6 in Fig. 3). However, the region 321-346 is not one of these “possible sites”. If these regions are

226

M 12345

Fig. 5. Immunoprecipitation of BTV-1SA VP2 synthesized in vivo using vaccinia virus recombinants. TK- cells infected with vaccinia virus recombinant, 27,,, were labelled with [ 3sS]methionine and lyxd in

NP40 buffer for 30 min at 4OC. The lysates were precleaned with Immunoprecipitin (BRL) and then

incubated for 16 h at 4OC with normal rabbit serum (track l), anti-BTV-1SA virus serum (track 2), anti-VP5 peptide serum (track 3), anti-VPZX peptide serum (track 4) and anti-VP2Y peptide serum (track

5). The marker lane indicated by M is the translation products from viral RNA.

looked at in detail by comparing the amino acid sequences of VP2 from BTV-1SA and BTV-lAUS, only three remain variable whilst the other three are fairly well conserved. These later three, which appear to be variable between different sero- types, but conserved within a single serotype, may therefore be good candidates for serotype specific neutralising epitopes. Peptides have been synthesised to these regions, to which antisera are being raised. These antisera will be used to help identify which of these regions, if any, represent neutralising epitopes.

We have also demonstrated that genome segment 2 of BTV-2 shows greater homology to the two, non-American isolates of serotype 1 (BTV-1SA and BTV-

221

1AUS) than to any of the other American isolates (BTV-10, BTV-11 and BTV-17). Although this level of homology was lower than detected between the BTV sero- types 10, 11 and 17, it appears to indicate that BTV-2 represents an introduction of a new serotype into the U.S.A., rather than a serotype which has evolved from those already present on the continent, as has also been suggested by Collison et al. (1985).

Now that a valuable library of sequence data is being developed, many useful comparisons can be made of proteins from different serotypes of BTV. The VP2, outer capsid protein is the most divergent protein of bluetongue virus and has been identified as the major serotype-specific antigen. It seems likely that the origin of new isolates of bluetongue virus can be predicted by comparative analyses of their VP2 and genome segment 2 sequences. However, because of the possible involve- ment of protein VP5 in determination of virus serotype (Cowley and Gorman, 1989; Mertens et al., 1989), comparative analyses of genome segment 5 from the range of BTV isolates compared here, may provide additional valuable information on the inter-relationships among BTV serotypes.

Acknowledgements

I would like to thank Dr T. Doe1 and his group for the provision of the peptides used in this study, Len Pullen and John Eveleigh for the immunisation of the rabbits, Karen Austin for her technical assistance and Dr C. Bostock and Dr A. Gould for useful discussion.

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(Reoeived 14 July 1989; revision received 27 October 1989)