31

Ykus Research, 17 (1990) 31-52 Elsevier VIRUS 00599

Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences A.R. Gould and L.I. Pritchard C.S.I.R. O., Austrafian Animal He&h Laboratory Geelong, Victoria, A~tralia (Accepted 15 May 1990)

Sequence data from the gene segments coding for the capsid protein, VP3, of all eight Australian bluetongue virus serotypes were compared. The high degree of nucleotide sequence homology for VP3 genes amongst BTV isolates from the same geographic region supported previous studies (Gould, 1987; 1988b, c; Gould et al., 1988b) and was proposed as a basis for “ topotyping” a bluetongue virus isolate (Gould et al., 1989). The complete nucleotide sequences which coded for the VP2 outer coat proteins of South African BTV serotypes 1 and 3 (vaccine strains) were determined and compared to cognate gene sequences from North American and Australian BTVs. These VP2 comparisons demonstrated that BTVs of the same serotype, but from different geographical regions, were closely related at the nucleotide and amino acid levels. However, close inter-relationships were also demonstrated amongst other BTVs irrespective of serotype or geographic origin. These data enabled phylogenic relationships of the BTV serotypes to be analysed using VP2 nucleotide sequences as a determinant. Bluetongue virus; VP2 sequence; Phylogeny; Topotype;

Nucleotype

Introduction Bluetongue virus (BTV) is the type member of the Orbiuirus genus, which is one of the six genera in the family Reoviridae. A total of 24 serotypes of BTV have been

Correspondence to: A.R. Gould, C.S.I.R.O., Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia. 0168-1702/~/$03.50

0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

32

identified on the basis of serum neutralization tests and at present eight of these have been identified in Australia (Gould, 1988~). Verwoerd et al. (1972) and Martin and Zweerink (1972) showed that orbiviruses have a double layered capsid, in which a diffuse outer layer comprising viral proteins VP2 and VP5 surrounds an inner core particle comprising VP7 and VP3. Within the inner core, minor proteins VPl, VP4 and VP6 and ten double-stranded RNA molecules are encapsidated. More recently, Hyatt and Eaton (1988) have shown that portions of both VP7 and the non-structural protein NSl were accessible at the surface of the virion (Eaton et al., 1988; Hyatt and Eaton, 1988). Orbiviruses can be grouped into one of thirteen serogroups by standard serological tests which detect viral coded group-reactive antigens. However, nucleotide sequence analyses of the group-reactive capsid protein, VP3 (Gould, 1987) suggested that not only could the gene sequences themselves be used to group orbiviruses in a manner which reflected serogrouping but also to determine the geographic origin of a BTV isolate. This ability to ‘topotype’ or assign an isolate to a geographic region was independent of the serotype of an isolate. These observations were extended by hybridization studies (Gould, 1988~) and sequence analyses (Gould, 1988b; Gould et al., 1988b, 1989) to delineate at least three well defined geographic regions which could be identified by sequence based analyses of either structural or non-structural BTV genes. These regions were Australia, South Africa and North America. Hybridization and sequence analyses done in other laboratories have generally supported these observations (Kowalik and Li, 1987; Mertens et al., 1987; Ritter and Roy, 1988). Although heterologous hybridizations between the VP3 genes of Australian BTV isolates indicated that they formed a distinct geographic grouping, with the exception of BTVlS, we wished to obtain sequence data for both serogroup and serotype specific BTV proteins from all of the known Australian BTV serotypes as a preliminary step in establishing a polymerase chain reaction (PCR) based diagnostic procedure capable of topotyping a BTV isolate (Gould et al., 1989). To this end partial nucleotide sequences from both VP3 and VP2 genes of Australian and South African BTV were deduced by rapid cloning and sequencing procedures. Analysis of these data was found to reveal novel relationships which are discussed in relation to VP2 and BTV evolution.

Materials and Methods Viruses and nucleic acid preparation The viruses and maintenance of virus stocks was as described in Gould (1988~); while the isolation and preparation of double-stranded (ds) RNA segments from infected BHK-21 cells was as described by Eaton and Gould (1987). Viruses from Australia are designated as BTV (serotype) AUS, while those from North America or South Africa as BTV (serotype) US or SA-VACC, respectively. All South African isolates were vaccine strains obtained from the Commonwealth Serum Laboratories, Parkville, Victoria, which had been attenuated by repeated egg and tissue culture

33

passage at the Ondersterpoort Veterinary Research Laboratories, South Africa. The specific passage history of BTVlSA-VACC was as follows: BTVl (Biggersburg strain) was passaged in chick embryo 60 times, followed by once in BHK cells for plaque purification and then twice in BHK cells. BTV3SA-VACC was the type 3 Cyprus strain which had been passaged 50 times in chick embryo, once in BHK cells, plaque purified and passaged once again in both L cells and BHK cells. BTV9SA-VACC (University) was passaged 70 times in chick embryo, once in BHK cells and plaque purified followed by two further passages in BHK cells.

The transcription of ds RNA segments into complementary DNA (cDNA) followed by homopolymeric tailing and cloning into Pstl cut, dG-tailed pBR322 was as described previously (Gould, 1987). Sequencing of clones was done using the dideoxynucleotide chain termination techniques (Sanger and Coulson, 1978; Sanger et al., 1977) after sub-cloning into the appropriate Ml3 vectors (Vieiera and Messing, 1982) or by direct plasmid sequencing (Chen and Seeburg, 1985). Direct sequencing on either BTV specific mRNA (Air, 1979; Gould and Symons, 1982) or purified ds RNA (Bassel-Duby et al., 1986) was done as described using synthetic ohg~~x~ucleotid~ synthesized on an Applied Biosystems DNA Synthesizer. To derive nucleotide sequence data covering the neutr~g epitope of VP2 (Gould et al, 198Sa) for all of the BTV serotypes which were not generated by ‘shotgun’ cloning methodologies using Sau3Al digestion of ds cDNA, cDNA was transcribed from BTVgAUS, BTV21AUS and BTV23AUS ds RNA segments using the primers 5’-GCA’ITCCGTTGCAATTAACG, 5’-ATCGAACAGGTTCACTCGGC and 5’GTCTGACGCTCCAATCATAG, respectively. These primers in conjunction with a universal 5’-VP2 specific oligonucleotide 5’-AGTGTCGCGATGGATGAGTTAGGC were used to perform a polymerase chain reaction (PCR; Saiki et al., 1988) on their respective cDNAs using the conditions recommended by Perkin Elmer-Cetus with the GeneAmp DNA reagent kit. The PCR products were purified by agarose gel electrophoresis and sequenced using the modified l7 polymerase, Sequenase (USB).

Cloning and sequencing of Australian BTV VP3 sequences Previously reported sequence comparisons of Australian BTVl (BTVlAUS) and BTV9AUS VP3 sequences have shown them to be closely related (z 95%) at both the nucleotide and amino acid levels (Gould, 1987). Randomly sequenced portions of each of the BTVAUS serotypes (3, 15, 16, 20, 21 and 23) VP3 genes were compared to those previousiy determined for BTVlAUS and BTV9AWS VP3 sequences (Gould, 1987) (Fig. 1). A partial VP3 sequence from South African BTV3 (BTV3SA-VACC) which also overlapped with BTV3AUS RNA3 was also included

34 BTV3AUSR3

471-586

n.p.

77.4X

BTVlNSR3

77.4%

BTWSAR3

96.6%

BTVlAUSR3

76.9%

BTV3SAR3

81 .n,

BTVlSAUSR3

30

60

GAT CAT

CGT GGA ATG GAA TTT

GCG GAA CCA GAA GTG TTG

GGC GTG GAA TTC

AAG AAC

GTG

Asp

Arg

Ata

Gty

Vat

His

Gty

Uet

Gtu

Phe

Gtu

Pro

Glu

Vat

Leu

Vat

GLu

Phc

Lys

Asn

90 CTG CCC GTG CTA ACC GCT GAA CAT ACA

GCA ATG ATA

CAG MT

GCT TTA

GA1

CGA TC

Leu

Ala

Gin

Ala

Asp

Gly

Pro

Val

Leu

Thr

Ala

Glu

BTV3SARJ

His

“.p.

Thr

Met

Ile

439-586

Asn

Leu

87.8%

BTVlNSR3

95.6%

BTWSAR3

77.0%

BTVlAUSR3

80.9%

BTVlSAUSR3

30 .GA

60

TCC

TTT

ATA

TTA

CAT CAT ATT

CCG ACC AGA GAT CAT CGT GGT ATG GAG GTC GCC GAA

Sw

Phe

XI+

Leu

His

Pro

Asp

Ite

Thr

Arg

Asp

His

Arg

Gly

Met

Gtu

Vel

Ale

90

Glu

120

CCA GAG GTA TTC

GGA GTT

GAG TTC

AAA MT

GTG TTA

CCT

GTA CTG ACT

GCC GAG CAT

CGC

Pro

Gty

GLU Phe

Lys

Val

Pro

Val

Ala

At-g

GLu

Val

Leu

GCG AT0

ATT

CAA AAC GCA TTC

GAT GGA TC

Ate

ile

Gin

Asp

Met

Asn

Val

Ala

Leu

BTWAUSR3

n.p.

Asn

Leu

Leu

Thr

Glu

His

Gly 3t2-442

80.2%

BTVlNSR33

79.4%

BTVPSAR3

97.7%

BTVlAUSR3

80.4%

RTVlSAUSR3

30

60

GAT CGA GlA

CTA

CGG GTA GAC ACA TAC TAT

GAG GAG ATA

TCA

CAG GTG GliT

CAT

GTT ATC

Asp

Leu

Arg

Glu

Ser

Gin

Asp

Val

Ike

Arg

Val

Val

Asp

Thr

Tyr

Tyr

Glu

Ile

Val

GLy

90

120

ACG GAA GAT GAA CCG GAG AAG TTC

TAC TCG ACG ATC ATC AAA AM

GTC AGA TTT

ATA

CGC

Thr

Tyr

Val

Ite

Arg

GLu

CGA AAA G~x..b’s

Asp

Glu

Pro

Glu

Lys

Phe

Ser

Thr

Ile

Ik

tys

Lys

Arg

Phe

CGA TC .GLY

Fig. 1. Partial nucleotide sequences of the RNA segment 3 from Australian BTV serotypes 3, 9, 15, 16, 20, 21 and 23. Additional sequence data from each VP3 gene were generated (not shown) and are available on request. The nudeotide sequence number is shown immediately above each sequence while its deduced translation product is shown below the sequence. Also given is the nucleotide position (n.p.) number aligned to the sequence of BTVlAUS VP3 (Gould, 1987) and the percent homology to various BTV RNA3 segments. Also shown is a partial VP3 sequence from BTV3SA for comparison to the homologous BTV3AUS region.

35 n.p.

BTVl5AUSR3

722-933

78.9%

BTVlNSR33

79.3%

BTWSAR3

80.1%

BTVlAUSR3

60

30 ..G

ATE

GGA TGG CTG GAG AGA TTA

CGT CAA AGA AAG AGA ATC ACA TAT

TCG CM

GAG GTG

Ile

Gly

Gly

Ser

Gtu

Vat

Trp

Leu

Glu

Arg

Leu

GLn Arg

LyS

Are

Ile

Thr

Tyr

Gin

120

90 CTG ACC GAT TTC

AGA AGA CM

WCC ACA ATA

TGG WT

TTG

GCT TTA

CAG CTA CCC CIA

MC

Leu

Thr

Asp

Phs

Arg

Asp

Trp

Leu

Ata

Leu

Gtn

Leu

Pro

Val

Am

CC1

WC

GTT

GTG TGG GAT GTC CCA AGA AGT TCC ATT

GCA MC

TTA

ATT

ATG MC

AK

GCG

Pro

Gln

Vat

Vat

Pro

Arg

Ala

Leu

Ile

Met

Am

Ite

Ate

ACG TGT

TTG

CCC ACA GGA GAG TAT

ATC

GCT CGA A

Thr

Leu

Pro

It&

Ala

Arg

Gin

Thr

Ite

Vet

180

150

Trp

Asp

Vat

SW

Ser

Ile

Asn

210

Cys

Thr

GIy

Gtu

Tyr

n.p.

ETVlkAUSR3

Arg

439-586

76.5%

BTVlNSR33

80.3%

BTV9SAR3

96.4%

mVwISR3

77.2X

ETV-MUSR3

30 .GA

60

TCC

TTC

ATA

TTA

CAT

OAT ATC

CC0 ACG AGA OAT CAT

CGC GGG ATG

GM

GTT

GCG GAA

Ser

Phe

I te

Leu

His

Asp

Pro

Arg.

GLu

Vat

Ate

Ite

Thr

Are

Asp

His

Gty

Ret

Gtu

120

90 CCA GM

GTG TTA

GGC GTG GAA TTC

AAG AAC ETA

CTG CCC GTG TTA

ACG GCC GAA CAC AGA

Pro

Val

Gly

Lys

Leu

Thr

Gtu

Leu

Vat

Glu

Phe

Asn

Vat

Pro

Vat

Leu

Ala

Gtu

His

150

Arg

180

GCA ATG ATA

CA0 MT

GCT TTA

GAT GGA TCA ACG CCA ACC GCA CAG CAG CTT

MT

GAC GTT

Ats

Gtn

Atei

Leu

Asp

Gly

Sar

Am

Asp

Uet

Ile

Asn

Thr

Pro

Thr

Ats

Gln

Gtn

Leu

Vat

210 AGG AAA ATC

TAC TTA

GCC TIC

AT6

TTT

CCA EGA CM

CTC ATC

ATT

GAT C

Arg

Tyr

Ala

Met

Phe

Pro

Leu

Ile

Asp

Lys

Ile

Leu

Leu

Gty

Gtn

Ile

Fig. 1 (continued).

for comparison. It was apparent that all Australian BTV VP3 sequences, with the exception of BTVISAUS (see later), were very closely related at both the nucleotide ( 2 95% homology) and amino acid level (2 98% homology) and distinct from those BTVs present in either South Africa or North America (Ghiasi et al., 1985; Gould, 1987).

36

lS93-1701

““P.

75.43

BTVlNSR3

78.8%

BTWSAR3

95.4%

BTVlAUSR3

81.5%

ETV15AUSR3

60

30 &GA TTC

GET hGA ATT

AAC CAG ATT

ATA

AAT GAG GAT

TTA

CAT

TCG GTG TTC

TCG TTA

CCG

Gty

Ala

1Ie

Sle

Am

Gtu

Asp

Lcu

His

Ser

Ser

Leu

Pro

Phe

Arg

Ite

Asn

Gln

Vai

GAT GAT ATG

TTT

MC

GCG TTA

CTT

CCC GAT TTA

ATA

GCC GGC CCC CAT

Asp

Phe

Asn

Ale

Leu

Pro

fte

Ate

Phe

w

Asp

Wet

Leu

BTV2lAUSR3

n.p.

Asp

Leu

312-462

Gty

Ala

C

His

75.43

BTVK’USRJ

79.3%

BTWSAR3

95.4%

BTVlAUSR3

77.9X

BTV15AUSR3

30

40

GAT CGA GTA CTG CGA GTA MC

ACA

TAT

TAT

GAG GAG ATG TCA CAG GTG GGT GA1

GTT

ATC

Asp

Arg

Vet

Thr

Tyr

Tyr

Gtu

Vat

tie

AC0

CM

GAT GAA CCG GAG AAG TTC

TAT

TCG ACG ATC Ate

Thr

GLu

Asp

Tyr

Ser

Leu

Arg

Vat

Asp

Gto

Met

Ser

GLn

Vat

Gty

Asp

90

Glu

Pro

Glu

I.ys

Phe

120

Thr

Ike

t le

AAA AM

GTT AGA TTT

ATA

CGC

Lys

Yal

Ile

Arg

Lys

Arg

Phe

GGG AAA GtiA it Gly

Lys

GIy

Fig. 1 (continued).

For comparative purposes we have accumulated partial VP2 sequence data from all known Austria BTV serotypes 3, 9, 15, 16, 20, 21 and 23 and deduced the complete sequences for BTVISA-VACC and BTV3SA-VACC (Figs. 2, 3 and 4). These and a partial sequence from BTV9SA VP2 were compared to BTVlAUS VP2 (Could, 1988a) and those previously deduced for BTVXJS, BTVlOUS, BTVIlUS, BTV13US and BTV17US (Yamaguc~ et al., 1988b; Roy, 1989 and references therein). Comparisons between BTVIA US and BTVlSA-VACC

VP2 gene segments

Analyses of the nucleotide and amino acid sequences coding for the VP2 proteins of BTVIAUS and BTVlSA-VACC were very similar. They both coded for proteins of 961 amino acids which had high + ve charges at neutral plli, while 194 amino acids were ~srnat~h~. Their homology rose from - 80% to - 90% at the amino acid 1evel when conservative substitutions were allowed for (Table 1). At the

37 n.p.

STV23AUSR3

761-1086

78.7%

BTVlNSR3

8115%

BTWSARJ

98.2%

BTVlAUSR3

75.7%

BTVl5AUSR3

50 ..f

60

ACT

TAT

TCG CAA GAG GTA CT1

ACT

GA1 TTT

AGA AGG WG

GA1 ACC ATT

TGG GTG CTG

Thr

Tyr

Ser

Gin

Thr

Asp

Arg

Asp

Trp

GCT CTG CA4

TTG

CCC GTG AAT CCA CAA GTA GTT

TGG GAC GTT

CCT CGG AGT

TCA ATT

GCT

Ats

Leu

Gin

Leu

Pro

Trp

Pro

Scr

Ala

MC

CTT

ATT

Am

Leu

Glu

Val

Leu

Phe

Arg

Gin

Thr

Ile

Vel

120

90 Pro

GLn Val

Leu

Val

Asn

Vat

Asp

Vsl

Arg

Ser

Ite

ATG MT

ATA

GCG ACG TGC TTA

Ile

Met

Ile

Ala

Thr

Cys

Let8 Pro

AGO ATT

TCT

TCA ATC ACG CTC ACT

CM

CGA ATC ACA ACG ACC GGG CCC TTT

GCG ATA

TTG

Arg

Ile

Ser

Ser

Gin

Arg

Leu

ACT Thr

150

Asn

180 CCA ACG GGA GAA TAC ATA Thr

Gly

Glu

Tyr

Ile

CCA CCA MC

CCA

Al8

Pro

Pro

Am

210

Ilc

Thr

Leu

Thr

240

llc

Thr

GGA TCA ACG CCA ACC GCA CAG ‘ZAG CTT

MT

Gly

Am

Thr

Thr

Gly

Pro

Phc

Ala

Ile

CAT WT

AGG AAA ATC

TIC

TTA

GCC CTG

Asp

Arg

Tyr

Leu

Ala

270

Pro

Thr

Ale

Gin

ATG TTT

CCA GGA CM

ATT

ATA

CTT

GAT C

Uet

Pro

ile

Ite

Leu

Asp

Phe

Ser

Thr

Gly

Gin

Gin

Leu

300 Val

Lys

Ile

Leu

Fig. 1 (continued).

nucleotide level, their initiation and termination codons coincided at nucleotides 18-20 and 2901-2903, respectively (Fig. 2), while both segments had a total of 2940 base pairs which were approximately 77% homologous, a figure apparently lower than the 90% observed between BTVlOUS and a partial sequence from BTVlOSA (Huismans et al., 1987; Purdy et al., 1985). When the VP2 genes from either the field (BTVlSA-F; Wade-Evans and Mertens, 1990) or vaccine strains of BTVlSA-VACC (this paper) were compared, they were found to have 59 amino acid differences randomly spread throughout the protein. Of these, only 18 were conservative substitutions. The ratio of non-conservative to conservative amino acid differences amongst the 59 amino acids was approximately 2-fold higher than for any other comparative differences observed between other BTV gene products (Gould et al., 1988b; and references therein).

Nucleotide sequence

of BTV3SA VP2 gene segment

The gene segment which coded for VP2 for BTV3SA-VACC was 2935 nucleotides in length, coded for a Mr112175 protein of 959 amino acids and had 5’- and 3’ non-coding regions of 21 and 37 nucleotides, respectively (Fig. 3). At neutral pH the

38

Fig. 2. The complete nucleotide sequence of BTVlSA vaccine strain RNA segment 2 and its deduced amino acid sequence. The numbering refers to the nucleotide sequence position, the termination triplet is marked * * *, while a neutralizing epitope of BTVlAUS (Gould et al., 1988a) is underlined. The numbers below the deduced amino acid sequence give their position number.

VP2 protein had a charge of +23.0, while the hydrophylicity profile, charge distribution as well as the total number and general positions of cysteine residues (19) were very similar to the VP2 proteins previously sequenced (not shown).

39

Fig. 4. Partial nucleotide sequences for the VP2 genes of Australian BTV serotypes 3, 9, 15,16,20, 21 and 23 as well as BTV9SA are shown, along with their deduced amino acids. Nucleotide or ammo acid sequences derived from clones were aligned with known VP2 sequences using the diagon and alignment programmes of Queen and Kom (1984). The presumed nucleotide positions are given above the sequences. Unknown data are indicated by a row of dots while the amino acids in the equivalent position to the neutralizing epitope of BTVlAUS are underfmed. The presumed te~ation codon for BTV23AUS VP2isgivenas***.

Comparisons between the VP2 sequences between BTV9A US and BTV9SA- VACC

of BTV3AUS

and BTV3SA-VACC,

and

As with the BTVlSA-VACC and BTVlAUS VP2 sequence comparisons, similar homologies at both the nucleotide and amino acid levels were seen when comparisons were done for VP2 gene segments of two other South African BTVs (BTV3SAVACC and BTVS)SA-VACC) and their Australian counterparts. For VP2 proteins of

a common serotype nucleotide sequences were - 70% homologous (Figs. 2, 3 and 4), while at the amino acid level, their homologies were - 70% rising to 80-90% if conservative ammo acid substitutions were allowed for (Table 1). Therefore for at least three of the serotypes in common between Australia and South Africa (BTVl, BTV3 and BTV9) there appears to be a consistently high level of sequence homology between these serotype specific proteins. Comparison

of BTVISA-VACC

and BTV3SA-VACC

VP2 sequences

Comparison of the complete VP2 sequences of BTVlSA-VACC and BTV3SAVACC revealed them to have dissimilar lengths of coding and non-coding regions

42

Fig. 4 (continued)

(Figs. 2 and 3). Several discrete, highly conserved amino acid sequences were found when their amino acid sequences were compared. These regions were similar to those observed between the VP2 coding sequences of BTVlAUS and BTVlOUS (Figs. 2 and 3, and Gould 1988a). The overall hydropathy profiles of the two proteins were very similar (not shown) although the amino acid homology was only

Fig. 4 (continued).

44% (Table l), a figure similar to that observed between BTVlAUS (see later). Comparisons sequences

between

Australian,

South African

and North

and BTVlOUS

American

BTV

VP2

Comparisons were made between BTV VP2 nucleotides and deduced amino acid sequences from North America (Roy, 1989; and references therein), Australia and South Africa (Figs. 2, 3 and 4). Relationships were apparent at both levels of comparison. BTVlOUS, BTVllUS, BTV17US and BTV20AUS were as closely related to each other as were BTV isolates of the same serotype from different geographic regions, i.e., BTVl, BTV3 or BTV9 from Australia or from South Africa (Fig 4, Table 1). A similar relationship was observed between BTV3SA-VACC or BTV3AUS, BTV13US and BTV16AUS wherein these VP2 segments were approximately 70% identical at the nucleotide level and having amino acid homologies of between 80-908 if conservative substitutions were allowed for (Table 1). This latter example was of interest as it included examples of the inter-relationships observed between BTV VP2 sequences of the same or different serotype from different geographical locations. A somewhat more distant relationship was de-

44 TABLE Sequence

1 homology

between

VP2 genes and their translation

products

BTV

SEROTYPE

VP2

HOMOLOGY

The VP2 sequences Figs. 2, 3 and 4 and their predicted translation products were compared to those previously published (Gould, 1988a; Roy, 1989). Percent homologies were calculated for each equivalent region as aligned using the programmes of Queen and Kom (1984) and summed for the total number of comparisons. The upper numbers for the VP2 amino acid homologies were calculated directly for homologous alignments while those immediately below were corrected allowing for conservative amino acid substitutions. Significantly high homologies are highlighted by being boxed. A - denotes where no overlapping consensus sequences were available.

termined to exist between BTVlSA or BTVlAUS and BTV2US as had been previously suggested (Roy, 1989) while BTV16AUS was observed to have a higher degree of amino acid homology with many serotypes from different geographic regions than was usually observed, i.e., BTV2US and BTV17US being 42.1% while BTVZUS and BTV16AUS was 59.2% (Table 1). A comparison of all of the available VP2 nucleotide sequences was done using either the DNAMETRO or DNAPARS analysis programs of Feldenstein (1984) to determine the phylogenic relationships between the BTV serotypes from different geographical regions. Both of these analyses gave the same single most parsimoni-

Fig. 5. Phylogenic associations of BTV serotypes from different geographic locations of the world. Equivalent regions of the VP2 gene from each BTV serotype (216 nucleotides) from nucleotide 975 to 1190 (relative to BTVlAUS VP2) were used to calculate the inter-relationships shown. The site chosen for comparisons included two presumably important regions; the variable neutralization epitope, amino acids 328-335 and the highly conserved region, amino acids 357-371 (see text). The single most parsimonious, uprooted tree shown was rooted at node 14 and generated by using the DNADIST and KITSCH programmes. The distance between nodes is proportional to the calculated genetic distances. All South African BTV isolates shown were vaccine strains with the exception of lSA-F which refers to the field strain of BTVlSA of Wade-Evans and Mertens (1990). The IAUS-F or lAUS-V refer to two Australian isolates of BTVl which were either avirulent or virulent, respectively. ous, unrooted tree. The interrelationships between the BTV serotypes observed in Table 1 were graphically represented (Fig. 5) using DNADIST to compute genetic distance matrices for use in the KITSCH program (Feldenstein, 1989) by a comparison of nucleotides 975-1190, an important peptide region of VP2 as it covered a hyper-variable virus-neutralization site for BTV (Gould et al., 1988a) as well as one of the most highly conserved VP2 peptide regions (Yamaguchi et al., 1988b; Gould, 1988a). These inter-relationships were also evident when Diagon matrix plots were done either between related or non-related VP2 sequences at the nucleotide or amino acid levels (Fig. 6).

Conservation of VP2 amino acid sequences It was previously shown that 11 conserved amino acid sequences were present between the VP2 proteins of BTVlAUS and BTVlOUS (Gould, 1988a). However, a

Fig. 6. Comparkms of BTWSA, BTvf3US and BTV17US VF2 gene segments and their predicted translation products. The VP2 nucleotide sequences of BTWSA, ETVl3K.E and 3TV17US (Ray, 1989) were compared using the dot-matrix method d Queen and Kern (MM) using a window apertive of 14 having a homology of 90% The sequences of BTWSA were plotted on the X axis while those of BTV13US or BTVl7US were on the Y axis.

more extensive comparison was done using dot matrix plots (Queen and Korn, 1984) having a window aperture of 6 and a 90% homology value for all of the available VP2 sequences and has shown that this number can be reduced to seven. The most highly conserved ammo acid sequences were located at positions 357-371 and at the carboxy-terminus at ammo acids 945-961 (using BTVlAUS VP2 as a reference protein). The former peptide along with the conserved motif at ammo acids 279-292 flank the previously defined neutrahzing epitope present on VP2 spanning ammo acids 330-338 (Gould et al., 1988a). Other conserved ammo acid regions were at positions 69-81, 108-120, 512-528 and 7322748. Of the nine

47

eysteine residues previously reported as conserved only six (at positions 154, 281, 365, 854,931 and 940) were conserved in all serotypes examined. Discussion This paper provides the complete nucleotide sequences of the outer coat protein VP2 for two South African vaccine strains of BTV, serotypes f and 3. These data permit comparisons to be made for the first time concerning VP2 sequences from BTV isolates of the same and different serotypes originating from different geographical regions. As the deduced VP2 protein sequences were compared it was obvious that although these proteins appeared to be highly variable, many important features were conserved including hydropathy profiles, charge distribution and cyst&e residues? indicating that the basic structure of these proteins was similar and that as three ~mensio~al entities presenting neutralizing epitopes, restrictions were placed on their variability, By computer-aided comparisons, it was possible to map seven peptide regions that were conserved across all BTV serotype VP2s. Some amino acids within these sequences were rigorously maintained while at other positions conservative substitutions were evident. It is suggested that these regions play crucial roles in either folding or protein-protein interactions. Previous attempts involving nucleic acid hybridizations have delineated relationships amongst BTVs isolated world-wide (Huismans and Bremmer, 1981; German et al., 1982; Gould, 1988~; Kowalik and Li, 1987; Huismans et al., 1987; Mertens et al., 1987; Huismans and Cloete, 1987; Ritter and Roy? 1988). Although it has generally been assumed that both VP2 and VP5 gene segments were serotype specific, both strong and weak heterologous hyb~~zations between cognate segments from different BTV serotypes have been observed from which relationships could be inferred (Huismans and Cloete, 1987; Mertens et al., 1987; Ritter and Roy, 1988; Gould, 1988c). However, caution is needed when weak signals are interpreted as suggesting relationships between viruses, as differences in hyb~~~tion methodologies and/or the stringency of duplex formation may confuse interpretations. It has been shown that the coding and non-coding regions of the same gene have differences in their degree of sequence conservation, which in heterologous hybrid formation unduly influenced the apparent sequence homology between gene segments (Gould et al., 198813).A more objective analysis is provided by nucleotide or amino acid sequence comparisons which have revealed several important features amongst BTV isolates. The first was that irrespective of whether an isolate was newly isolated from the field or propogated for at least a decade in the laboratory, gene and amino acid sequences such as those coding for VP3 or VP7 (Ghiasi et al., 1985; Gould, 1987; Gould et al,, 1989) varied by as little as 2-5s between serotypes, provided they originated from the same geographic region. Secondly, when comparisons were done between cognate genes from different BTV serotypes, and from different geographical regions, they were found to code for proteins which had very few amino acid differences (Ghiasi et al., 1985; Gould, 1987; Gould et al., 1988b, Kowalik and Li, 1989). However at the nucleotide level, the genes for both structural and non-struct-

48

ural proteins varied such that BTVs of differing serotype but of the same topotype, consistently had the same percent nucleotide differences when comparisons were made from one geographic region to another. These differences were - 20% between Australian and South African or North American BTVs and 11% between North American and South African BTVs, while BTVlSAUS genes were - 20% different from those of all other geographic regions examined (Gould, 1987; Gould, unpublished results; Gould et al, 1988; Ghiasi et al., 1985). Moreover, recent sequence data (Grubman and Samal, 1989; Hall et al., 1989) for NSl and NS2 from BTV17US and BTVlOSA, respectively, directly support the proposed topotyping of BTV. The nucleotide sequences of these genes had exactly the sequence divergences predicted for these different geographical isolates (Gould, 1987; Gould et al., 1988b). BTV13US which was initially isolated in 1967 from bovine blood, has been described as being distinct from previous BTV isolates in the United States (BTVlOUS, BT’VllUS and BTV17US) by both genetic and molecular studies (Sugiyama et al., 1982; Roy et al., 1985; Fukusho et al., 1987). Recently, Kowalik and Li (1989) reported the complete nucleotide sequence for the major capsid protein, VP7, of BTV13US. When comparisons were made to BTVlOUS VP7, the nucleotide sequences were 79.6% conserved while at the amino acid level they were 94% conserved. Similar degrees of homology were found with BTVlAUS VP7 (Gould et al., 1989, and unpublished data) or BTVlOUS VP7 gene segments (Yu et al., 1988). Thus it may be possible that BTV13US represents another distinct topotype of BTV from those previously described in the United States of America. If this indeed were the case, then the same sequence divergence should be maintained for the remaining structural and non-structural genes of this isolate. Similarly, BTV2US has been reported as a recent introduction into the United States (Collisson and Barber, 1985) and it will be of interest to see if this isolate has the same topotype as BTV13US or represents an incursion from a separate geographical region. Thus rDNA probes or nucleic acid sequence data may be used to delineate the geographic origin or topotype of a new BTV isolate. To date we propose that there exist at least four distinct topotypes of BTV for which the type members are BTVlAUS, BTVlSA, BTVlOUS, and BTVlSAUS which represent the geographic regions Australia, South Africa, North America and an as yet undefined region, respectively. The above observations appear not to apply to the RNA segments which code for the outer coat proteins VP2 or VP5, where perhaps different selection pressures (probably immunological) operate. From hybridization studies it appears that not only are VP2 or VP5 gene sequences serotype specific at high stringency but are also topotype specific. That is, although the VP2 gene sequences for both BTVlAUS and BTVlSA-VACC code for serologically indistinguishable proteins (Della-Porta et al., 1983), at the nucleotide level sufficient differences exist such that they do not cross-hybridize at the high stringencies used to serogroup the orbiviruses (Gould, 1988~; Kowalik and Li, 1987; Huismans et al., 1987; Fig. 4 and Table 1). Similar observations have been made for hybridizations involving BTV9 and BTV3 from both Australia and South Africa (not shown) which are supported by the sequence

49

data presented in this paper (Table 1). However, when VP2 sequences from the same serotype isolated from the same geographic region were compared either by hybridization (Gould, 1988c) or sequence analyses (Gould, 1988a; Yamaguchi et al., 1988) they were found to have very similar sequences. Indeed, a comparison between virulent and avirulent BTVl isolates from Australia had VP2 segments which had only 10 nucleotide differences (Gould and Eaton, submitted) while a comparison of the vaccine strain (this paper) and avirulent field isolate of BTVlSA-F (Wade-Evans and Mertens, 1990) were 2 94% homologous at the nucleotide level. VP2 sequence data comparisons between BTV serotypes revealed that there existed relationships between certain BTV serotypes irrespective of geographic origin. For those BTV serotypes which were closely related by both nucleotide and amino acid sequence homologies (i.e., BTV3, BTV13, and BTV16 or BTVlO, BTVll, BTV17 and BTV20) we would like to propose the nomenclature of a BTV “nucleotype”. Within a nucleotype, VP2 nucleotide or amino acid sequence homologies were - 70% while for comparisons done between nucleotypes, homologies dropped to - 55% (Table 1). Also, within a nucleotype both 5’ and 3’ noncoding regions were highly conserved in both length and sequence as were the toal gene segment lengths. However, when comparisons were made between different nucleotypes, these sequences and total base pair length parameters were variable. Hybridization studies (Mertens et al., 1987; Huismans and Cloete, 1987) have indicated that the BTVlO-BTVll-BTV17-BTV20 nucleotype should also probably include BTV4SA and BTV24SA. These relationships could also be seen when amino acid comparisons were made within the previously described neutralization epitope of BTV (Gould et al., 1988a). Those serotypes which were related, i.e., within a nucleotype, had a pattern of similar and variable amino acids across the span of this epitope (Figs. 2-4; Gould 1988a; Yamaguchi et al., 1988b). However, if comparisons were made between non-related nucleotypes at this site, then very few if any amino acids were conserved. Further relationships between BTV serotypes will be delineated upon the accumulation of additional VP2 sequence data and help to define a more complete evolutionary scheme for BTV. While accumulation of sequence data for the other structural and non-structural BTV genes will help to define the number and location of topotypes. Such nucleotide sequence studies have been important not only for studying influenza virus and reovirus lineages (Wiener and Joklik, 1989; Donis et al., 1989; Cox et al., 1989) but also for analyses of topotypes and geographic distribution of yellow fever virus (Deubel et al., 1986) St. Louis encephalitis virus (Trent et al., 1981) and Thogoto virus (Calisher et al., 1987). As with BTV, a single topotype of all Kunjin virus isolates in Australia has recently been observed (Flynn et al., 1989) a finding which was similar to that reported for Murray Valley encephalitis in Australia (Coelen and Mackenzie, 1988) but different to that found for SLE in the United States (Trent et al., 1981). Acknowledgements

The authors would like to express their appreciation to Drs P. Mertens and A. Wade-Evans for access to their nucleotide sequence data for the field strain of BTVlSA prior to publication.

50

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52 Sugiyama, K., Bishop, D.H.L. and Roy, P. (1982) Analyses of the genomes of bluetongue viruses recovered from different states of the United States and at different times. Am. J. Epidemiol. 115, 332-347. Trent, D.W., Grant, J.A., Vomdam, A.V. and Monath, T.P. (1981) Genetic heterogeneity among Saint Louis encephalitis virus isolates of different geographic origin. Virology 114, 319-332. Verwoerd, D.W., Els, H.J., DeVilliers, EM. and Huismans, H. (1972) Structure of the bluetongue virus capsid. J. Virol. 10, 783-794. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268. Wade-Evans, A.M. and Mertens, P.P.C. (1990) Expression of the outer capsid protein, VP2, from a full-length cDNA clone of genome segment 2 of BTVlSA using Sp6 and vaccinia expression systems. Virus Res. 15,213-230. Wiener, J.R. and Joklii, WK. (1989) The sequences of the reovirus serotype. 1, 2, and 3 Ll genome segments and analysis of the mode of divergence of the reovirus serotypes. Virology 169, 194-203. Yamaguchi, S., Fnkusho, A. and Roy, P. (1988a) Complete sequence of VP2 gene of the bluetongue virus serotype 1 (BTV-1). Nucleic Acids Res. 16, 2725. Yamaguchi, S., Fukusho, A. and Roy, P. (1988b) Complete sequence of neutralization protein VP2 of the recent US isolate Bluetongue virus serotype 2: its relationship with VP2 species of other US serotypes. Virus Res. 11, 49-58. Yu, Y., Fukusho, A., Ritter, D.G. and Roy, P. (1988) Complete nucleotide sequence of the group-reactive antigen VP7 of bluetongue virus. Nucleic Acids Res. 16, 1620. (Received 8 February 1990; revision received 14 May 1990)