AU4933793A - Recombinant antigens and vaccines of bovine ephemeral fever virus - Google Patents

Description

RECOMBINANT ANTIGENS AND VACCINES OF BOVINE EPHEMERAL FEVER VIRUS TECHNICAL FIELD This invention relates to antigens of bovine ephemeral fever virus (BEFV) and vaccines against BEFV. More particularly, the invention relates to glycoprotein G of BEFV and vaccines comprising glycoprotein G.

BACKGROUND ART

Bovine ephemeral fever virus (BEFV) is an arthropo -borne rhabdovirus which causes an acute febrile infection in cattle and water buffalo. The disease occurs in most tropical and sub-tropical regions of

Africa, Asia, the Middle-East and Australia, where seasonal epidemics can have significant economic consequences.

BEFV virions are bullet-shaped and contain 5 structural proteins: L (Mr = 180 Da); G (Mr = 81 kDa); N (Mr = 52 kDa); Ml (Mr = 43 kDa); and M2 (Mr = 29 kDa) (Walker et al., (1991) J. Gen. Virol . 72, 67-74). As for rabies virus and vesicular stomatitis virus, the BEFV membrane glycoprotein (G) can be removed from virions by treatment with non-ionic detergent. The G protein presents type-specific and neutralising antigenic sites. Six neutralisation sites have been identified by competitive binding of G protein monoclonal antibodies (Cybinski et al . , (1990) J. Gen. Virol . 72, 2065-2072. The virion G protein also protects cattle from experimental infection with virulent BEFV as described in Australian Patent No. 636907. The above mentioned Australian Patent No.

636907 discloses that in addition to the virion 81 kDa G protein, a 90 kDa non-structural glycoprotein (G_NS) is synthesised in BEFV-infected cells. G_NS appears in similar abundance to the virion G protein in infected cells but has not been detected in virions. Non- glycosylated precursors and core-glycosylated intermediates of each BEFV glycoprotein have been identified. BEFV infection is part of a cycle whereby infected cattle may be bitten by insects such as mosquitoes and sandflies which may then transmit the infection to healthy animals. BEFV replicates in cells of the insect vector and it is hypothesised that the G_Ng protein may be present on the surface of virus produced in the insects although this has yet to be established.

While Australian Patent No. 636907 teaches that BEFV G protein is effective as a vaccine component, known methods of producing the protein are unsuitable for the large scale production of vaccine. Virus must be cultured in a host organism, harvested, and proteins isolated therefrom. The overall process yields low levels of protein immunogen and there is the additional disadvantage of having to initially culture the infectious agent. Similarly, any use of G_NS protein as a vaccine component would be limited by the difficulties in producing the protein.

SUMMARY OF THE INVENTION It is an object of this invention to provide a method of producing BEFV G and/or G_NS protein which does not require the culturing of BEFV.

It is also an object of this invention to provide alternative BEFV vaccines to known BEFV vaccines. According to one aspect of this invention, there is provided an isolated DNA molecule comprising a nuσleotide sequence encoding bovine ephemeral fever virus (BEFV) G protein and/or G_NS protein.

A preferred BEFV G protein encoding sequence is a sequence corresponding to nucleotides 21 to 1889 of SEQ

ID No:l. A preferred BEFV G_NS protein encoding sequence is a sequence corresponding to nucleotides 1969 to 3726 of SEQ ID No:l (as hereinafter defined).

Sequences having about 70% or greater identity with the foregoing sequences which encode proteins having the immunogenic properties of BEFV G protein and/or G_NΞ protein are also within the scope of the invention.

In another aspect of the invention, there is provided a DNA vector which includes a sequence encoding BEFV G protein and/or G_NS protein.

In further aspects of the invention there are provided BEFV G protein and/or G_NS protein expressed from the DNA molecules of the invention.

The invention includes within its scope immunogenic fragments of the G protein and/or G_NS protein encoded by the respective genes. Immunogenic fragments include mature G protein or G_NS protein after cleavage of signal peptide or even smaller fragments which may be encoded by portions of the G protein or G_NS protein genes.

A preferred BEFV G protein amino acid residue sequence is a sequence corresponding to SEQ ID No:2 (as hereinafter defined). A preferred BEFV G_NS protein amino acid sequence is a sequence corresponding to SEQ ID No:3 (as hereinafter defined).

In yet another aspect of this invention, there is provided a method for the production of BEFV G protein and/or G_NS protein, said method comprising the steps of:

I) preparing an expression vector incorporating a DNA sequence comprising a sequence encoding bovine ephemeral fever virus (BEFV) G protein and/or G_NS protein;

II) transforming a host cell with the expression vector prepared in step I;

III) culturing said host cell under conditions in which G protein and/or G_NS protein is expressed; and

IV) harvesting said expressed G protein and/or G_NS protein from the culture prepared in step III. In other aspects of the invention there are provided vaccines for protecting animals against BEFV comprising BEFV G protein or an immunogenic fragment thereof. In a still further aspect of the invention, there is provided a vaccine for protecting an animal against BEFV, said vaccine comprising a non-pathological virus which expresses BEFV G protein. The term "non-pathological" virus denotes a virus which replicates within the vaccinated animal but does not ellicit a deleterious disease state. Typically, the non-pathological virus is vaccinia virus.

The vaccines of the invention can also include BEFV G_NS protein and/or BEFV N antigen.

In still further aspects of the invention, there are provided methods of protecting animals against BEFV infection, the methods comprising administering to the animals the aforementioned vaccines. In regard to a preferred embodiment of this invention, and as discussed in greater detail hereinafter, a 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which, are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]₇) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and 6 potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and 8 potential glycosylation sites, and appears to correspond to the 90 kDa non-structural G_NS protein discussed above. A database search described in Walker et al., (1992) Virology 191 , 49-61, the entire contents of which are incorporated herein by cross-reference, indicated that both the G and G_NS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with the virion G proteins of sigma virus and vesicular stomatitis virus serotypes. The invention also includes within its scope not only the G protein gene and the G_NS protein gene which each have a specific nucleotide sequence as described hereinafter but also RNA molecules having about 70% or greater identity with the specific sequences discussed hereinafter.

The invention also includes within its scope nucleic acid molecules that can hybridise with the G protein gene and G_NS protein gene. Such hybrids may include RNA hybrids as determined by Northern hybridisation methods or DNA hybrids.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be more fully described with reference to the drawings in which:

Figure 1 depicts maps of BEFV cDNA clones used for nucleotide sequence analysis, in which arrows indicate the location of terminal sequences obtained from these clones and from subclones generated by progressive exonuclease digestion or by removal of restriction fragments, and in which the origin of the kilobase scale is the start of the N gene for which the complete nucleotide sequence is known;

Figure 2 depicts hydropathy plots of the BEFV G and G_NS proteins;

Figure 3 depicts the results of Northern hybridisations using ³²P-labelled BEFV cDNA probes to detect BEFV G, G_NS and L mRNA wherein in lane A RNA from uninfected BHK-21 cells was probed with a mixture of clones m5 (G gene), m3 (G_NS gene) and m85 (L gene) and wherein RNA from BEFV-infected BHK-21 cells was probed with clone m5, clone m3 or clone m85 in lanes B, C and D respectively;

Figure 4 is a map of plasmid pTP41; Figure 5 is a map of plasmid pTP42; Figure 6 depicts the results of neutralising antibody assays wherein the symbols (+), (o), (A) and ( ) represent results for rW-G, rW-G_NS, rW-G + rW-G_NS and W-NYBH respectively; and Figure 7 depicts the results of blocked ELISA wherein in each set of results the filled bar represents the result for rW-G, the finely cross-hatched bar represents the result for rW-G_KS, the coarsely cross- hatched bar represents the result for rW-G + rW-G-_s, and the stippled bar represents the result for W-NYBH.

BEST MODE AND OTHER MODES OF CARRYING

OUT THE INVENTION The methodology used in relation to preparation of the recombinant antigens of this invention can be all based on conventional procedures well known in the art.

Initially purified virus can be prepared which can be treated with detergent and extraction of the desired glycoprotein as discussed in Australian Patent No. 636,909. The purified peptides can then be prepared for determination of sequence analysis after reduction and alkylation as described by Stone et al. (1989) A Practical Guide to Protein and Peptide Purification for Microsequencing (P T Matsudaira, ed.), Academic Press, San Diego, pp 31-47, and further purification by HPLC. Genomic RNA can be purified in any suitable manner as described in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbour Laboratories, New York. For example, purified virus can be sedimented from density gradients obtained from glycerol, caesium chloride, sucrose or other suitable compound useful in a density gradient. The virus can then be resuspended in appropriate buffer containing a detergent and a ribonuclease inhibitor. Subsequently the purified virus can be digested with a suitable proteinase to strip nucleoprotein away from the genomic RNA whereafter the genomic RNA can be extracted with a suitable solvent.

It is preferred in regard to the method of this invention that full length genomic RNA from a wild type virus is utilised and to this end preparation of total cytoplasmic RNA can be initiated wherein appropriate host cells are infected with virus substantially depleted of defective-intefering (DI) particles which have large deletions in their genome. In the process using a density gradient as described above, the band corresponding to the wild type virus will have a greater density than the band corresponding to a DI particle which therefore can be discarded.

Subsequently, total cytoplasmic RNA can also be extracted and this can be carried out by infection of host cells with DI particle depleted virus. Actinomycin D or any other suitable inhibitor of transcription of cellular mRNA can be added to the infected cells after initial infection. At a suitable post infection time the host cells can be harvested for RNA extraction and thus the cells can be washed before being lysed with a suitable buffer containing a detergent and ribonuclease inhibitor as described above as well as a proteinase before extraction with a suitable solvent and subsequent extraction with a DNAase free of RNAase to digest cellular DNA. cDNA clones can then be obtained from the genomic RNA as well as mRNA included in the total cytoplasmic RNA which can be both used as independent templates. In this step the cDNA insert(s) can be inserted into a cloning site of a suitable cloning vector such as a plasmid cloning vector such as pUC18 or a cosmid vector. cDNA clones derived from mRNA can then be analysed by sequence comparisons with known sequences of other rhabdoviruses to obtain approximate location of the inserts corresponding to the clones in the genome. A primer based on this information can then be designed to generate clones of a substantial length from a genomic RNA template.

Five large clones (g233, g208, g310, g236 and g207) were obtained from a genomic DNA template and a synthetic oligonucleotide corresponding to the 3' coding region of the BEFV N protein mRNA was also obtained for use as a primer. Sequence analysis of these clones together with the obtaining of relevant sub-clones and also clones from BEFV mRNA and the compilation of overlapping sequences lead to the analysis of structures of genes encoding both protein G and protein G_NS.

Clones containing the complete coding regions of each glycoprotein gene can be prepared by subcloning of gene fragments as is well known in the art. Preferably, complete coding regions are obtained by PCR amplification as described hereinafter.

In relation to commercial production of each of the abovementioned glycoproteins, it will be appreciated that any suitable expression vector can be adopted to express each of the glycoproteins. Typically, the glyproteins are expressed in a eukaryote system: for example, mammalian cells using a poxvirus such as vaccina virus, papilloma virus or retrovirus vectors or alternatively yeast cells harbouring suitable vectors. A suitable expression system for use in commercial production of the glycoproteins is the use of a baculovirus vector to infect an insect host cell such as Spodoptera frugiperda.

Adjuvants for use with the vaccines of the invention are known in the art. Such adjuvants include QuilA or other saponins, ISCOMS or other adjuvants as described in Vanselow (1987) Vet. Bull . 57 , 881-886. It will also be appreciated that the antigens of the invention including G protein, G_NS protein and N protein can be obtained from a suitable expression system as a fusion protein whereby the antigen is coupled to β- galactosidase, glutathione-S-transferase or other suitable carrier proteins.

In order that the invention described herein be more fully understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limitations upon the invention.

EXAMPLE 1 PURIFICATION AND PEPTIDE SEQUENCE ANALYSIS OF THE VIRION G PROTEIN

The BB7721 strain of BEFV was used as a source of G protein. The virus was isolated in 1968 from a cow with clinical bovine ephemeral fever at Charters Towers (146°16'E, 20°5'S), Australia (Doherty et al. (1969) Australian J. Sci . 31, 365-366) and has the same origin as the current Australian attenuated vaccine strain 919 (Tzipori and Spradbrow (1973) Australian Vet . J. 49, 183- 187). The virus was passaged in calves and in suckling mice before adaptation to cell culture. After an unknown number of passages in BHK-21 cells, the virus was plaque cloned 3 times in Vero cells. After 2 further passages in Vero cells, a DI particle-depleted stock of virus was prepared by sedimentation velocity centrifugation. This stock was used to inoculate cultures for preparation of purified virus and viral mRNA. Virus was grown in BHK-21 cells as described previously (Walker et al., (1991) J. Gen. Virol . 72, 67-74).

Purified virus was treated with 0.5% Triton X- 100 and the glycoprotein purified from the detergent- soluble fraction by wheat germ lectin-Sepharose affinity chromatography and size-exclusion HPLC. To obtain peptides suitable for amino acid sequence analysis, approximately 55 μg of the purified glycoprotein was reduced and alkylated as described by Stone et al. (1989) in the above cited reference and digested at 24°C for 24 h with 3 μg endoproteinase Glu-C (Boehringer-Mannheim) in 2 M urea, 100 mM ammonium bicarbonate pH 8.3. Peptides were purified by 2 cycles of reverse phase HPLC on a Brownlee Aquapore RP-300 C-8 column (Applied Biosystems Inc.). Initial separation was conducted using a linear gradient of 0-75% acetonitrile in 0.1% aqueous heptafluorobutyric acid (HFBA) . Selected peptides were then applied to the same column and eluted in a 0-75% linear acetonitrile gradient in 0.1% aqueous trifluoroacetic acid (TFA). The purified peptides were sequenced using an Applied Biosystems amino acid sequencer. Four resolved peptide fractions were obtained in quantities suitable for amino acid sequence analysis. Two fractions contained unique peptides (G163 and G164) that produced unequivocal sequence data. One fraction contained 2 peptides (G141a and G141b) in 2:1 molar ratio from which individual sequences could be deduced with certainty. One fraction (G101) contained 2 peptides in equimolar quantities from which unique peptide sequences could be determined only by comparison with sequences deduced from nucleotide sequence data. Amino acid sequences of peptides G116, G164, G141a and G141b are shown in Table I. Corresponding sequences deduced from nucleotide sequence analysis of the G protein gene are reported below.

TABLE I PEPTIDE AMINO ACID SEQUENCES

PEPTIDE AMINO ACID SEQUENCE

G141a (E) K I Y N V P V N C G E

G141b (E) C I T V K S F R S E

G163 (E) K L C L S L P D S X R V X X D C N I

G164 (E) X W Y F X T C I E

In Table I, the standard one letter amino acid code has been used. Residues which could not be assigned with confidence are represented as X. The symbol C has been used to represent carboxymethyl cysteine or glutamic acid which have similar retention times as PTH-amino acids on the reverse phase system used. The initial E residue was assumed to be present because of the specificity of endoproteinase Glu-C. The C residue in peptide G164 differs from the deduced amino acid sequence of the BEFV G gene. EXAMPLE 2 CDNA CLONING AND SEQUENCING OF G AND G_B GENES

Genomic RNA was prepared from the virus purified as described by Walker et al (1991) J. Gen. Virol. 72, 67-74). The virus was sedimented from selected sucrose gradient fractions at 70,000 rpm for 10 min at 4°C (Beckman TLA-100 rotor), resuspended in RNA Extraction Buffer, digested with 2 mg/ml Proteinase K (Boehringer Mannheim) at 37°C for 30 min and extracted with phenol/chloroform/isoamyl alcohol as described by Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbour Laboratories, New York.

For preparation of total cytoplasmic RNA, cells were infected at a multiplicity of 10 pfu/cell with DI particle-depleted virus. At 9 h post-infection, 0.3 μg/ml actinomycin D was added and at 15 h post-infection the cultures were harvested for RNA extraction. Monolayers were washed twice with ice-cold PBS, lysed with equal volumes of RNA Extraction Buffer and Proteinase K Digestion Buffer, extracted with phenol/chloroform/isoamyl alcohol and treated with RQ DNase I (Promega) as described in the above mentioned Sambrook et al., (1989). Synthesis of cDNA was conducted using the cDNA

Synthesis System Plus kit (Amersham) according to the manufacturer's instructions. First-strand synthesis from total cytoplasmic RNA was initiated with an oligo (dT)₁₈ primer (Amersham). Synthesis from genomic RNA template was initiated with the synthetic oligonucleotide primer N.1B (5'AGTTTTGCATCAGGGAGTGCAAGA3' ) which corresponded to a sequence known to be located in the 3'terminal coding region of BEFV N protein mRNA. Double-stranded cDNA was filled by using T4 DNA polymerase to obtain blunt-ends and cloned into the S-nal site of pUClδ. cDNA clones longer than 350-400 nucleotides were subcloned after unidirectional deletion by using the Erase-a-Base kit (Promega) according to the manufacturer's instructions or after selection deletion of restriction fragments. The size of the clones was determined by agarose gel electrophoresis or by complete nucleotide sequence analysis (clones g207, m5, m3, G1.A6 and G2.C1). cDNA clones were sequenced using T7 DNA polymerase (Sequenase Version 2.1, United States Biochemicals) according to the manufacturer's instructions and Ml3 forward and reverse sequencing primers (Pharmacia). All sequences were confirmed on both DNA strands.

Five large clones (g233, g208, g310, g236 and g207) containing inserts of approximately 2.0 kb, 2.3 kb, 3.9 kb, 5.1 kb and 7.0 kb respectively (Figure 1) were obtained from cDNA clones of BEFV genomic DNA. Nucleotide sequence analysis confirmed that one end of each clone corresponded to the 3' end of the N protein mRNA. Sequence analysis of the other end of the clones g310 and g233 indicated that these contained putative polyadenalylation (CATG[A]₇) and consensus (AACAGG) sequences and were likely to be located at two distinct gene junctions. Subclones of clone g207 were obtained by progressive unidirectional deletion using Exo III and a panel of overlapping clones terminating 2.4 kb - 5.2 kb downstream of the N gene were sequenced. Subclone g207.17 contained putative consensus and polyadenylation sequences and appeared to bridge a third gene junction. Subclones of g207 were also obtained by deletion of BamHI and Sacl fragments and were sequenced. A compilation of overlapping subclone sequences indicated that the region between the putative consensus sequence in clone g310 and the putative polyadenylation sequence in clone g207.17 contained a 1758 base pair open reading frame (ORF) which mapped between 3.6 kb and 5.4 kb downstream of the N gene (Figure 1). This region was identified subsequently as the G_NS protein gene (see below).

Two cDNA clones were obtained from BEFV mRNA by όligo (dT) priming (Figure 1). Clone m5 contained a 1.55 kb insert and nucleotide sequence analysis indicated that one end mapped to the gene junction identified in clone g233. The sequence of the other end corresponded to that of a g207 subclone (g207.45-4). Overlapping subclones of m5 were obtained by ExolII digestion and the complete sequence of the insert was determined. A compilation of overlapping sequences of g233, g208, m5 and the g207 subclones indicated that the region between the putative consensus sequence in clone g233 and the putative polyadenylation sequence in clone g207.17 contained a 1869 base pair ORF which mapped between 1.65 kb and 3.55 kb downstream of the N gene and immediately upstream of the G_NS gene (Figure 1). This region was identified subsequently as the G protein gene (see below) . Clone m3 contained a 0.5 kb insert. Nucleotide sequence analysis of the complete clone indicated that it mapped in the G_HS ORF (Figure 1) . Both clones m3 and m5 appeared to have initiated at A rich regions within the G_κs and G genes and did not represent full length mRNA clones.

EXAMPLE 3 SUBCLONES OF GLYCOPROTEIN GENES

Clones containing the complete coding regions of each glycoprotein gene were obtained by PCR amplification from clone g207 described in Example 2 by using the PCR primer pairs G1.1B (5 'GGCCAGATCTACAATGTTCAAGGTCCTCATAATT3' )/Gl.2B(5'GGCCAGAT CTTTAATGATCAAAGAACCTATCATCACCAG3 * ) ; and G2.1AB ( 5 'GGCCGGATCCATCATGTTCCTGCAACTATTTAATATCG3 ' )/ G2.2AB (5'GGCCGGATCCGCTCAATAATCCAACTTAAAG3' ) . PCR reactions were conducted in 50 μl Tag polymerase Buffer (Boehringer Mannheim) containing 10 ng plasmid DNA, 100 ng each primer DNA, 250mM dNTPs, lμl Perfect Match (Promega), 1.5 mM MgC12 and 5 units Tag DNA polymerase. Reactions were conducted using the temperature cycle program: 95°C/90s, 37°C/30s, 50°C/30s, 72°C/180s (4 cycles); 95°C/90s, 57°C/60s, 72°C/180s (30 cycles) and 95°C/90s, 60°C/60s, 72°C/5 min (1 cycle). The PCR products were filled with T4 DNA polymerase and purified by LGT Agarose (BRL) gel electrophoresis and exrraction with phenol and phenol/chloroform before cloning in the Smal site of pUC118.

Clones G1.A6 (G gene) and G2.C1 (G_NS gene) were used to obtain subclones by progressive unidirectional deletion using ExolII. Analysis of overlapping subclones allowed completion of the bidirectional sequence of the G and G_NS genes and the associated non-coding regions (Figure 1). One sequence discrepancy (A-> G at position +27 from the putative initiation codon) was detected in PCR clone G1.A6. However, independent bidirectional analysis of the corresponding sequence in clones g207 and g310 indicated that the substitution is likely to have occurred during PCR. The substitution did not change the deduced amino acid sequence of the G protein. The sequence of a 3789 nucleotide region of the

BEFV genome located 1.65 kb downstream of the N gene polyadenylation signal is shown as (+) sense DNA in Sequence ID No:l. The region contains two long ORFs (1869 and 1758 nucleotides), each bounded by a consensus sequence (AACAGG) and a common polyadenylation sequence (CATG[A]₇). The ORFs are separated by an intergenic sequence of 53 nucleotides which features a sequence similar to the consensus sequence (AACAG) followed by the unusual pallindrome (T)₆(A)₁₀. The polyadenylation signal of the second gene is followed by an intergenic region of 37 nucleotides and another consensus sequence (AACAGG) which marks the start of another long ORF immediately downstream (not shown). The 5' and 3' non-coding regions of each putative mRNA are very short, comprising 8, 0, 6 and 1 nucleotides excluding consensus and polyadenylation sequences. At position 1490 in the first ORF, a sequence similar to the common polyadenylation signal (CATG[A]₆) is followed by a sequence similar to the consensus sequence (AACAGA). The significance of these sequences is not evident but they are not adjacent to initiation or termination codons.

Amino acid sequences deduced from the consecutive ORFs are shown in SEQ ID No:2 and SEQ ID No:3. The first ORF encodes the BEFV virion G protein. The G protein sequence consists of 623 amino acids with a calculated pi of 6.191 and molecular weight of 72,278 Da. The calculated molecular weight is similar to that of the 71 kDa glycoprotein precursor detected in BEFV-infected cells (Walker et al., (1991) J. Gen. Virol . 72, 67-74). As can be seen from SEQ ID No:2, the protein contains amino acid sequences corresponding to all 4 peptides derived by endoproteinase Glu-C digestion of the BEFV virion G protein (Table I): G141a corresponds to residues 17 to 28; G141b to residues 210 to 220; G163 to residues 349 to 367; and G164 to residues 90 to 99. The first three of these peptides were identical to the deduced sequence. Peptide G164 differed in one residue from the deduced sequence. This may have resulted from a single base transversion which would change the serine codon (TCT) to cysteine (TGT). No similar peptide sequences were detected in the protein encoded in the second ORF. The hydropathic!ty profile of the G protein (Figure 2), calculated by the method of Kyte and Doolittle (1982) J. Mol . Biol . 157, 105-132, displays hydrophobic domains bounded by basic residues (lysine or arginine) corresponding to the signal peptide and transmembrane regions of other rhabdovirus glycoproteins. The transmembrane domain is relatively short (16 residues) and is adjacent to a second shorter hydrophobic region (13 residues) bounded by lysine residues which may also by associated with the viral membrane. Six putative glycosylation sites were identified in the deduced sequence of the BEFV G protein (SEQ ID No:2). One of these is located in the COOH-terminal domain and is unlikely to be glycosylated in vivo

The second ORF, SEQ ID No:3 encodes the BEFV G_NS non-structural glycoprotein. The G_NS protein sequence consists of 586 amino acids with a calculated molecular weight of 68,806 Da and a pi of 8.144. The calculated molecular weight is similar to that reported for the 67 kDa glycoprotein precursor detected in BEFV-infected cells (Walker et al., (1991) J. Gen. Virol . 72, 67-74). The hydropathicity plot of G_NS protein also features hydrophobic domains bounded by acidic and basic residues which are likely to constitute signal peptide and transmembrane domains (Figure 2). Eight potential N- glycosylation sites were identified in the G_NS protein and all appear to be located in the external domain (Sequence ID No:3).

The putative signal domain of the BEFV G protein, residues 1 to 12 of SEQ ID No:2, conforms with those recognised in the signal peptides of othe eukaryote membrane proteins, comprising an N-terminal charged region, a central hydrophobic core and a more polar domain approaching the peptidase cleavage site. It is known that the peptidase cleavage site of vesicular stomatitis virus (VSV) serotypes (other than Chandipura virus) and rabies virus is followed by a basic residue (lysine or arginine) which is subsequently located at the N-terminus of the mature glycoprotein. Similar potential cleavage sites have been recognised in the putative signal domains of the G proteins of other viruses. Two potential peptidase cleavage sites can be identified in the BEFV G protein corresponding to lysine residues at positions 13 and 18. A stretch of 16 hydrophobic amino acids at residues 539-554 appears to constitute the transmembrane domain of the BEFV G protein. This region is bounded by basic residues (R and K) which are characteristic of other rhabdovirus transmembrane segments. In addition, it is preceded by the sequence VXGWF(X)S which also precedes the transmembrane domains of VSV Indiana, VSV New Jersey and Chandipura viruses. However, it has been suggested that a minimum segment of 21 amino acids may be required to span the non-polar region of the cytoplasmic membrane, suggesting that a second hydrophobic region comprising residues 522-534 may also be involved. If so, the transmembrane domain of the BEFV G protein would comprise 32 amino acids including 2 charged residues (K₅₃₅ and R₅₃₈ ) .

The BEFV G_NS gene is located immediately downstream of the G gene. Although there is no direct evidence that this gene encodes the G_Ng protein, the structure predicted from the deduced amino acid sequence corresponds to that expected for G_NS. The predicted size of the G and G_NS gene products (72,278 daltons and 68,806 daltons) correspond closely to those of non-glycosylated precursors (Mr=71 kDa; Mr=68 kDa) detected in BEFV- infected cells (Walker et al., (1991) J. Gen. Virol . 72, 67-74). The G_NS gene product has the structural characteristics of a rhabdovirus glycoprotein including a signal domain, hydrophobic transmembrane domain and 8 potential N-glycosylation sites (SEQ ID No:3) The signal domain comprises a primarily hydrophobic segment at the N-terminus which appears to terminate at arginine (residue 15). The putative transmembrane domain comprises a stretch of 19 uncharged amino acids (residues 544-562) bounded by the highly charged sequences (QNKEYWNEE) and (RKNRREK). The large number of potential glycosylation sites on the G_HS gene product during maturation. The function of the G_MS protein is not yet known.

EXAMPLE 4 Identification of G, G-,-. and L mRNA

BEFV cDNA inserts were cut from plasmids by restriction endonuclease digestion and purified by LGT Agarose (BRL) gel electrophoresis and by binding to Geneclean II (BIO 101, La Jolla, Ca. ) according to the manufacturer's instructions. The purified inserts were used to prepare P-labelled DNA probes by random hexanucleotide priming using the Multiprime DNA labelling system (Amersham). Northern hybridisations were conducted on RNA transferred from formamide-agarose gels to nylon membranes (Hybond N Plus, Amersham) according to methods described by Sambrook et al. (1989) in the above cited reference.

Total RNA was purified from BEFV-infected cells at 15 h post-infection and reacted in northern hybridizations with probes prepared from cDNA clones m5 and m3. A 1.8 kb cDNA clone (m85) which was known to be derived from the 3' end of the BEFV L gene was also included for reference (Figure 3). Clone m5 detected a 2.2 kb band corresponding to G mRNA. Clone m3 detected a smaller 2.0 kb band corresponding to G_NS mRNA. Clone m85 detected a 6.5 kb band corresponding to L mRNA. All probes failed to react with total RNA prepared from uninfected cells. Each mRNA appeared to represent a full-length polyadenylated transcript of the corresponding gene. There was no evidence that a smaller RNA was transcribed from the G gene, terminating at the CATG(A)₆ at positions 1521 to 1530. EXAMPLE 5

Plasmid insertion vectors and recombinant vaccinia viruses expressing G and G_B Both BEFV glycoprotein genes were subcloned into the BamHI site of vaccinia virus transfer vector pFN243, G as a Bgrlll fragment derived from pUCllβ clone G1.A6 and G_NS as BamHI fragment from pUCllδ clone G2.H4. Maps of the two plasmids pTP41 and pTP42 are presented in Figures 4 and 5. In Figure 4, the symbol "B/B" denotes the point of fusion of the ends of the linearised pFN243 and the G gene fragment while in Figure 5 the symbol "B" denotes the BamHI sites reformed after insertion of the G_NS gene at the BamHI site of pFN243. In pFN243, the vaccinia virus thymidine kinase region is interrupted by a fowlpox early-late promoter and a multiple cloning site. Human TK 143B cells infected with vaccinia virus strain NYBH were used to construct recombinant vaccinia viruses (rW-G and rW-G_NS) by homologous recombination. rW were plaque purified three times on 143B cells selecting recombinants by screening for expression of β- glactosidase.

Expression of G protein was determined by western blots, and immunifluorescence using monoclonal antibodies against G. In western blots rW-G expressed G migrates at the same molecular weight as G from BEFV infected cells and in the immunofluor-escence assay rW-G infected cells stain at the same intensity as BEFV infected cells with a panel of monoclonal antibodies to BEFV G protein. At present time we have no G_NS specific antisera. To test expression of recombinant G_NS we vaccinated rabbits with rW-G_κα. In the immunofluorescence assay these rabbit sera stain G_NS in BEFV infected cells and in western blots detect a protein of the appropriate size in lysates of BEFV infected cells.

EXAMPLE 6 Vaccination of cattle and challenge with BEFV

Eighteen six month old Hereford cattle were vaccinated intradermally by day 1, revaccinated on day 15 and challenged on day 29. Six animals were vaccinated with rW-G, six with rW-G_NS, three animals received a combination of rW-G and rW-G_KO and three control animals were vaccinated with a wild type vaccinia virus (NYBH). Blood samples were collected and the rectal temperature determined prior to vaccination, revaccination and challenge. After challenge this was done twice a day for nine days (day 30-38). Additional blood samples were collected on day 39 and 45.

Animals vaccinated with rW-G alone or in combination with rW-G_HS developed neutralising antibodies (see Table II and Fig. 6) Titres continued to rise after revaccination. Vaccination with rW-G_NS or wild type vaccinia virus did not induce the production of neutralising antibodies. The neutralising antibody response in rW-G vaccinated animals was confirmed by the blocking ELISA (Fig. 7).

At day 29 cattle were challenged with 5 ml of blood from a BEFV infected cow. Protection of vaccinated animals was evaluated by monitoring viremia (Table III). Virus was isolated from the blood samples of 4 out of 6 rW-G_NS and 2 out of 3 NYBH vaccinated animals. No virus was isolated from animals vaccinated with rW-G alone or rVV-G in combination with rW-G„ . TABLE II BOVINE EPHEMERAL FEVER VIRUS NEUTRALISATION ASSAY RESULTS

TABLE III BOVINE EPHEMERAL FEVER VIRUS ISOLATION ( IMMUNIFLUORESCENCE)

DAY

VACCINE ANIMAL 29 30 31 32 33 34 35 36 37 38 39 rW-G 2

8

9

12

17

77 rVV-G^ 3 +/^■ +/- 4 nd +/^■ 11 14 15 +/^■ 16 rW-G + 1 rVV-G 5 8

W NYBH 6

10 nd

13

(-) both samples negative, (+) both samples positive, (+/-) one postive and one negative, (nd) not done

While a number of embodiments of the invention have been presented above, it will be apparent that a number of variations can be made thereto without departing from the broad ambit of the invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.

It will also be appreciated that the term "bovine ephemeral fever virus" used herein also includes within its scope Malakal virus, Puchong virus, Berrimah virus, Kimberley virus and Adelaide River virus.

DEPOSIT OF MATERIAL ASSOCIATED WITH

THE INVENTION A sample of g207 plasmid DNA was deposited with the Australian Government Analytical Laboratories, P.O.

Box 385, Pymble NSW 2073, Australia on 14 September 1992 and given the Accession No. N92/44230

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANTS: Commonwealth Scientific and Industrial Research Organization (all states except US) Walker, Peter John (US only) Riding, George Alfred (US only) McWilliam, Sean Michael (US only) Boyle, David Bernard (US only) Hertig, Christian (US only)

(ii) TITLE OF INVENTION: Recombinant antigens and vaccines of bovine ephemeral fever virus

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Cullen & Co

(B) STREET: Level 12, 240 Queen Street

(C) CITY: Brisbane

(D) STATE: Queensland

(E) COUNTRY: Australia

(F) POSTAL CODE: 4000 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 2DD

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: MS-DOS version 5.1

(D) SOFTWARE: WordPerfect version 5.1

(2) INFORMATION FOR SEQ ID No: 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3789 base pairs

(B) TYPE: nucleotide

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to genomic DNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: bovine ephemeral fever virus

(B) STRAIN: BB7721 (vii) IMMEDIATE SOURCE:

(B) CLONE: g207 ( ix) FEATURE :

(A) NAME/KEY: consensus sequence

(B) LOCATION: 7..12

(C) IDENTIFICATION METHOD: by similarity to an established consensus sequence

(ix) FEATURE:

(A) NAME/KEY: coding sequence

(B) LOCATION: 21..1889

(C) IDENTIFICATION METHOD: experimentally

(D) OTHER INFORMATION: encodes bovine ephemeral fever virus G protein

(ix) FEATURE:

(A) NAME/KEY: polyadenylation site

(B) LOCATION: 1893..1903

(C) IDENTIFICATION METHOD: by similarity to an established consensus sequence

(ix) FEATURE:

(A) NAME/KEY: consensus sequences

(B) LOCATION: 1957..1962

(C) IDENTIFICATION METHOD: by similarity to an established consensus sequence

(ix) FEATURE:

(A) NAME/KEY: coding sequence

(B) LOCATION: 1969..3726

(C) IDENTIFICATION METHOD: by similarity with known sequences

(D) OTHER INFORMATION: encodes bovine ephemeral fever virus G_NS protein

(ix) FEATURE:

(A) NAME/KEY: polyadenylation site

(B) LOCATION: 3731..3741

(C) IDENTIFICATION METHOD: by similarity to an established consensus sequence

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Walker, Peter J. Byrne, Keren A. Riding, George A. Cowley, Jeff A. Wang, Yonghong McWilliam, Sean

(B) TITLE: The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes

(C) JOURNAL: Virology

(D) VOLUME: 191

(F) PAGES: 49-61

(G) PUBLICATION: NOVEMBER 1992

(xi) SEQUENCE DESCRIPTION: SEQ ID No: 1:

TTTGTCAACA GGACCTCACA ATGTTCAAGG TCCTCATAAT TACCTTGCTA GTCAATAAGA 60

TTCATTTGGA GAAAATTTAC AATGTTCCGG TGAATTGTGG AGAGCTTCAT CCGGTAAAAG 120

CTCATGAGAT CAAATGTCCA CAACGTTTAA ATGAATTATC ACTTCAAGCC CATCATAATC 180

TTGCAAAGGA TGAACATTAT AATAAGATTT GTAGACCACA GTTGAAAGAT GACGCTCATC 240

TAGAAGGATT CATTTGTAGG AAGCAAAGGT GGATAACTAA ATGTAGTGAA ACCTGGTATT 300

TTTCTACATC TATAGAATAT CAGATATTAG AGGTAATACC AGAATATTCC GGTTGCACAG 360

ATGCAGTTAA GAAATTAGAT CAGGGTGCAT TAATTCCCCC TTATTACCCT CCTGCTGGAT 420

GCTTTTGGAA TACTGAAATG AATCAAGAAA TAGAATTCTA TGTCCTAATA CAACATAAGC 480

CTTTCTTAAA TCCTTATGAT AATTTAATTT ATGATTCTCG ATTTTTAACT CCTTGTACTA 540

TTAATGATAG TAAAACAAAG GGATGTCCTC TAAAAGACAT AACCGGAACA TGGATACCAG 600

ATGTAAGAGT CGAAGAAATA AGTGAGCACT GCAATAATAA ACATTGGGAA TGCATTACAG 660

TTAAATCATT TAGGAGTGAA TTAAATGATA AAGAGAGATT ATGGGAAGCT CCAGATATCG 720

GGCTAGTCCA CGTAAATAAA GGATGTTTGT CGACATTTTG TGGGAAAAAT GGCATCATTT 780

TTGAGGATGG AGAATGGTGG AGTATAGAAA ACCAAACAGA ATCTGACTTC CAAAATTTTA 840

AAATTGAAAA ATGCAAGGGT AAAAAACCAG GTTTTAGGAT GCACACAGAT AGAACAGAGT 900

TTGAAGAATT AGATATTAAG GCTGAATTAG AACATGAAAG ATGCTTAAAC ACTATCAGTA 960

AAATACTTAA TAAAGAAAAT ATAAATACAT TAGATATGTC TTACTTGGCT CCCACGAGAC 1020

CAGGAAGGGA TTATGCATAT TTATTTGAGC AAACAAGTTG GCAAGAAAAA CTCTGTTTGT 1080

CCTTACCAGA CTCGGGTAGA GTTTCCAAGG ATTGTAATAT TGATTGGAGA ACATCAACAA 1140

GAGGGGGAAT GGTGAAAAAG AATCATTATG GGATAGGATC CTATAAAAGA GCTTGGTGTG 1200

AATACAGACC TTTTGTTGAC AAGAACGAAG ATGGCTATAT TGATATACAA GAATTGAATG 1260

GACATAACAT GTCTGGGAAT CATGCTATAT TGGAAACAGC GCCGGCAGGA GGAAGCTCCG 1320

GAAATAGGCT GAATGTAACT CTAAACGGAA TGATCTTCGT GGAACCAACT AAATTATATT 1380

TGCATACAAA ATCTCTATAT GAGGGGATAG AGGATTATCA AAAATTGATC AAATTCGAGG 1440

TAATGGAGTA TGATAATGTG GAAGAGAACT TGATTAGGTA TGAGGAGGAT GAGAAATTTA 1500

AACCAGTAAA TTTAAATCCA CATGAAAAAA GTCAAATCAA CAGAACTGAT ATCGTAAGAG 1560

AAATTCAAAA AGGAGGGAAA AAGGTTTTAT CTGCTGTTGT AGGTTGGTTT ACAAGCACGG 1620

CAAAGGCAGT AAGATGGACA ATTTGGGCTG TTGGTGCAAT AGTCACCACA TATGCTATCT 1680

ACAAATTGTA TAAAATGGTC AAGTCCAACT CATCTCACAG TAAGCATAGG GAAGCTGACC 1740

TAGAAGGACT TCAATCAACA ACAAAAGAAA ATATGAGGGT GGAAAAGAAT GACAAGAATT 1800

ATCAAGATTT AGAATTAGGA TTATATGAAG AGATTAGAAG CATCAAAGGT GGAAGTAAAC 1860

AAACTGGTGA TGATAGGTTC TTTGATCATT AACATGAAAA AAACCATTTG TGTCATGTGT 1920

TGACAAAACA GTTTTTTAAA AAAAAAATCA AGAGGCAACA GGGCAATCAT GTTCCTGCAA 1980

CTATTTAATA TCGTATTAAT ATACGGTGTG AGAACTTCCC AGTCAACTTG GATTAACTAC 2040

CCTGAGAATT GCACATCTAT CAGCCTGCAA GATGGATTGA GAGAATTATG CGGAGGTGAT 2100

CAGTTGATGA ACATCCGAAA TCAGCTTTTG GATGATACAT ATAAAGAAAT AGGAGAAATA 2160

TGTACCCCAA ATTATAGTAT GGAGAAAAAA TCAGAAGGGT ATCGATGCGC ATCGATAAAG 2220

AAAAAGGTAA TTTGTAAAAT GCTGGAAAAT TTCGATCATG AAGTGACATA TATAAGTGAA 2280

TCACACCCAA TAGACAAGGC CAAATGCCAT GAGCTAATAA TTAATAAAGA CTTGCTTAAC 2340

AATATTGAAG AGCCTTATTA TCCTCCTCCA AAGTGTGATT CCTC"AAATC TTCAGTTAGT 2400

GAGCTCGAAT TCATCAAATT GATCAATTAT GATGTTATCC TAGATCCGGT GGGATTTCAG 2460

AATGAAGATA ATTATTTATT TCAATTTGAT AAAACTAACC CTATCCCTAT TGATTATATA 2520

TACCAATCAG AGTTTTGTCA ATCAAAAAAT TGGATATGCC ATGGGGATAA ATCTTACATC 2580 CCGTTAGAAA TATTTAAAGG TGATAATCAG GCTAGTATTA GACTTGAGTT GATTAAGCTT 2640 AGCATTATAT ATGATAGCAA TTTTGGAGAA TTACCAATTA GAGATGCCTG TAGGTTACAC 2700 TACTGCGGAA AGCCAGCAAT TAAATTATTC AATGGTGCCA TCATAAAGAT CAAAGAGTCA 2760 CCTATAGTCT TAGGACTACC ATCTTGCAAT AGGAGTAGAA TAGAAATGCC AGAGACTAAT 2820 TTGGCTAAGA AAAGATATTC AAATGTTGGC CCTGTTTTAT TAACTACATT GAATAAGAGA 2880 TTCGAACTTT GCAAAAAGAT TAAAAAGAAC TTGGAATTAA AACAGCCAAT ACCAATTAAC 2940 AATCTTCATT ATCTAGCTCC ATTTGAGCCA GGCAAGCATC CTGCCTTAGT GTATCGATTA 3000 GTTAGTACAA CAATCAATCA ATCCCTAAGA AATAAAGTTG TACCTGTTAG TATGCTGTCT 3060 ATGAGTATGT GTGAGTACAT AACAGGTCAA ATTATTGAAG ATGGAATCAA AAGGAATTTA 3120 ACTGATGAAG ATACTGTCAT TATATTGGCC AATAACAAAG AGATCAAATG GAAAGATCTC 3180 AAGGGTAGAG AGAATTGGTA TCAAGAACAG GCTAATCCAA ATAT/vATAGA TAAAAACCCA 3240 GATCACTTGT CTTATTACTG GTACAATGGA GTTATGAGGA GAGAAGATAA GTTTACCTAC 3300 CCATCTCGCT ATATCCTTCA AACATTAAAG AAAATATATA CTGACACTGA AAGAGAATCC 3360 AGAATATCAT TTTTCAAGTT TAGATTAGAA AGAAACATAA CCAAAACGGA AGTAATTAAA 3420 TTTAGGGATA TAGAAGAATC ATCTGACCAA GATCACTCAC AATCAGTCAA TAAAACACTA 3480 GAAGGCGATG ATTACTGGAA TTGGGTTGAA GAAACCACAA GTGACAAAAA TAAGACGGAT 3540 GGGTCTAGAG GAGATGAGAA ACAAACCATA CAGAACAAAG AATATTGGAA TGAAGAATCA 3600 TCCATCTGGG GGATTTCAAC TATAATCACA GTTCTCGGCA TTTATTATAT ATATAGGAAA 3660 AATAGAAGAG AAAAGATCTT CTTGAATATG AAACACAGAG TACAAAGATT CTTTAAGTTG 3720 GATTATTGAG CATGAAAAAA ATAATAACAA TAATTTAC C CGATCAATTT TAAATTGCAA 3780 CAGGCAATG 3789

(2) INFORMATION FOR SEQ ID No:2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 623 amino acid residues

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no (vi) ORIGINAL-SOURCE:

(A) ORGANISM: bovine ephemeral fever virus

(B) STRAIN: BB7721 (vii) IMMEDIATE SOURCE:

(B) CLONE: g207 (ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 175..177

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 264..266

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites ( ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 416..418

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 438..440

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 507..509

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 563..565

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(xi) SEQUENCE DESCRIPTION: SEQ ID No:2:

Met Phe Lys Val Leu lie lie Thr Leu Leu Val Asn Lys lie His Leu

5 10 15

Glu Lys lie Tyr Asn Val Pro Val Asn Cys Gly Glu Leu His Pro Val 20 25 30

Lys Ala His Glu lie Lys Cys Pro Gin Arg Leu Asn Glu Leu Ser Leu 35 40 45

Gin Ala His His Asn Leu Ala Lys Asp Glu His Tyr Asn Lys lie Cys 50 55 60

Arg Pro Gin Leu Lys Asp Asp Ala His Leu Glu Gly Phe lie Cys Arg 65 70 75 80

Lys Gin Arg Trp lie Thr Lys Cys Ser Glu Thr Trp Tyr Phe Ser Thr 85 90 95

Ser lie Glu Tyr Gin He Leu Glu Val He Pro Glu Tyr Ser Gly Cys 100 105 110

Thr Asp Ala Val Lys Lys Leu Asp Gin Gly Ala Leu He Pro Pro Tyr 115 120 125

Tyr Pro Pro Ala Gly Cys Phe Trp Asn Thr Glu Met Asn Gin Glu He 130 135 140 Glu Phe Tyr Val Leu He Gin His Lys Pro Phe Leu Asn Pro Tyr Asp 145 150 155 160

Asn Leu He Tyr Asp Ser Arg Phe Leu Thr Pro Cys Thr He Asn Asp 165 170 175

Ser Lys Thr Lys Gly Cys Pro Leu Lys Asp He Thr Gly Thr Trp He 180 185 190

Pro Asp Val Arg Val Glu Glu He Ser Glu His Cys Asn Asn Lys His 195 200 205

Trp Glu Cys He Thr Val Lys Ser Phe Arg Ser Glu Leu Asn Asp Lys 210 215 220

Glu Arg Leu Trp Glu Ala Pro Asp He Gly Leu Val His Val Asn Lys 225 230 235 240

Gly Cys Leu Ser Thr Phe Cys Gly Lys Asn Gly He He Phe Glu Asp 245 250 255

Gly Glu Trp Trp Ser He Glu Asn Gin Thr Glu Ser Asp Phe Gin Asn 260 265 270

Phe Lys He Glu Lys Cys Lys Gly Lys Lys Pro Gly Phe Arg Met His 275 280 285

Thr Asp Arg Thr Glu Phe Glu Glu Leu Asp He Lys Ala Glu Leu Glu 290 295 300

His Glu Arg Cys Leu Asn Thr He Ser Lys He Leu Asn Lys Glu Asn 305 310 315 320

He Asn Thr Leu Asp Met Ser Tyr Leu Ala Pro Thr Arg Pro Gly Arg 325 330 335

Asp Tyr Ala Tyr Leu Phe Glu Gin Thr Ser Trp Gin Glu Lys Leu Cys 340 345 350

Leu Ser Leu Pro Asp Ser Gly Arg Val Ser Lys Asp Cys Asn He Asp 355 360 365

Trp Arg Thr Ser Thr Arg Gly Gly Met Val Lys Lys Asn His Tyr Gly 370 375 380

He Gly Ser Tyr Lys Arg Ala Trp Cys Glu Tyr Arg Pro Phe Val Asp 385 390 395 400

Lys Asn Glu Asp Gly Tyr He Asp He Gin Glu Leu Asn Gly His Asn 405 410 415

Met Ser Gly Asn His Ala He Leu Glu Thr Ala Pro Ala Gly Gly Ser 420 425 430

Ser Gly Asn Arg Leu Asn Val Thr Leu Asn Gly Met He Phe Val Glu 435 440 445 Pro Thr Lys Leu Tyr Leu His Thr Lys Ser Leu Tyr Glu Gly He Glu 450 455 460

Asp Tyr Gin Lys Leu He Lys Phe Glu Val Met Glu Tyr Asp Asn Val 465 470 475 480

Glu Glu Asn Leu He Arg Tyr Glu Glu Asp Glu Lys Phe Lys Pro Val 485 490 495

Asn Leu Asn Pro His Glu Lys Ser Gin He Asn Arg Thr Asp He Val 500 505 510

Arg Glu He Gin Lys Gly Gly Lys Lys Val Leu Ser Ala Val Val Gly 515 520 525

Trp Phe Thr Ser Thr Ala Lys Ala Val Arg Trp Thr He Trp Ala Val 530 535 540

Gly Ala He Val Thr Thr Tyr Ala He Tyr Lys Leu Tyr Lys Met Val 545 550 555 560

Lys Ser Asn Ser Ser His Ser Lys His Arg Glu Ala Asp Leu Glu Gly 565 570 575

Leu Gin Ser Thr Thr Lys Glu Asn Met Arg Val Glu Lys Asn Asp Lys 580 585 590

Asn Tyr Gin Asp Leu Glu Leu Gly Leu Tyr Glu Glu He Arg Ser He 595 600 605

Lys Gly Gly Ser Lys Gin Thr Gly Asp Asp Arg Phe Phe Asp His 610 615 620

(2) INFORMATION FOR SEQ ID No:3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 586 amino acid residues

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: bovine ephemeral fever virus

(B) STRAIN: BB7721 (vii) IMMEDIATE SOURCE:

(B) CLONE: g207 (ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 27..29 (C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites (ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 68..70

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 274..276

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 350..352

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 383..385

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 476.. 78

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 501..503

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(ix) FEATURE:

(A) NAME/KEY: N-glycosylation site

(B) LOCATION: 521..523

(C) IDENTIFICATION METHOD: by similarity with known N- glycosylation sites

(xi) SEQUENCE DESCRIPTION: SEQ ID No:3: Met Phe Leu Gin Leu Phe Asn He Val Leu He Tyr Gly Val Arg Thr

5 10 15

Ser Gin Ser Thr Trp He Asn Tyr Pro Glu Asn Cys Thr Ser He Ser 20 25 30

Leu Gin Asp Gly Leu Arg Glu Leu Cys Gly Gly Asp Gin Leu Met Asn 35 40 45

He Arg Asn Gin Leu Leu Asp Asp Thr Tyr Lys Glu He Gly Glu He 50 55 60

Cys Thr Pro Asn Tyr Ser Met Glu Lys Lys Ser Glu Gly Tyr Arg Cys 65 70 75 80

Ala Ser He Lys Lys Lys Val He Cys Lys Met Leu Glu Asn Phe Asp 85 90 95

His Glu Val Thr Tyr He Ser Glu Ser His Pro He Asp Lys Ala Lys 100 105 110

Cys His Glu Leu He He Asn Lys Asp Leu Leu Asn Asn He Glu Glu 115 120 125

Pro Tyr Tyr Pro Pro Pro Lys Cys Asp Ser Ser Lys Ser Ser Val Ser 130 135 140

Glu Leu Glu Phe He Lys Leu He Asn Tyr Asp Val He Leu Asp Pro 145 150 155 160

Val Gly Phe Gin Asn Glu Asp Asn Tyr Leu Phe Gin Phe Asp Lys Thr 165 170 175

Asn Pro He Pro He Asp Tyr He Tyr Gin Ser Glu Phe Cys Gin Ser 180 185 190

Lys Asn Trp He Cys His Gly Asp Lys Ser Tyr He Pro Leu Glu He 195 200 205

Phe Lys Gly Asp Asn Gin Ala Ser He Arg Leu Glu Leu He Lys Leu 210 215 220

Ser He He Tyr Asp Ser Asn Phe Gly Glu Leu Pro He Arg Asp Ala 225 230 235 240

Cys Arg Leu His Tyr Cys Gly Lys Pro Ala He Lys Leu Phe Asn Gly 245 250 255

Ala He He Lys He Lys Glu Ser Pro He Val Leu Gly Leu Pro Ser 260 265 270

Cys Asn Arg Ser Arg He Glu Met Pro Glu Thr Asn Leu Ala Lys Lys 275 280 285

Arg Tyr Ser Asn Val Gly Pro Val Leu Leu Thr Thr Leu Asn Lys Arg 290 295 300 Phe Glu Leu Cys Lys Lys He Lys Lys Asn Leu Glu Leu Lys Gin Pro 305 310 315 320

He Pro He Asn Asn Leu His Tyr Leu Ala Pro Phe Glu Pro Gly Lys 325 330 335

His Pro Ala Leu Val Tyr Arg Leu Val Ser Thr Thr He Asn Gin Ser 340 345 350

Leu Arg Asn Lys Val Val Pro Val Ser Met Leu Ser Met Ser Met Cys 355 360 365

Glu Tyr He Thr Gly Gin He He Glu Asp Gly He Lys Arg Asn Leu 370 375 380

Thr Asp Glu Asp Thr Val He He Leu Ala Asn Asn Lys Glu He Lys 385 390 395 400

Trp Lys Asp Leu Lys Gly Arg Glu Asn Trp Tyr Gin Glu Gin Ala Asn 405 410 415

Pro Asn He He Asp Lys Asn Pro Asp His Leu Ser Tyr Tyr Trp Tyr 420 425 430

Asn Gly Val Met Arg Arg Glu Asp Lys Phe Thr Tyr Pro Ser Arg Tyr 435 440 445

He Leu Gin Thr Leu Lys Lys He Tyr Thr Asp Thr Glu Arg Glu Ser 450 455 460

Arg He Ser Phe Phe Lys Phe Arg Leu Glu Arg Asn He Thr Lys Thr 465 470 475 480

Glu Val He Lys Phe Arg Asp He Glu Glu Ser Ser Asp Gin Asp His 485 490 495

Ser Gin Ser Val Asn Lys Thr Leu Glu Gly Asp Asp Tyr Trp Asn Trp 500 505 510

Val Glu Glu Thr Thr Ser Asp Lys Asn Lys Thr Asp Gly Ser Arg Gly 515 520 525

Asp Glu Lys Gin Thr He Gin Asn Lys Glu Tyr Trp Asn Glu Glu Ser 530 535 540

Ser He Trp Gly He Ser Thr He He Thr Val Leu Gly He Tyr Tyr 545 550 555 560

He Tyr Arg Lys Asn Arg Arg Glu Lys He Phe Leu Asn Met Lys His 565 570 575

Arg Val Gin Arg Phe Phe Lys Leu Asp Tyr 580 585

Claims (21)

1. An isolated DNA molecule comprising a nucleotide sequence encoding bovine ephemeral fever virus (BEFV) G protein and/or G_HS protein.

2. The DNA molecule of claim 1, wherein said sequence encoding BEFV G protein has a sequence corresponding to nucleotides 21 to 1889 of SEQ ID No:l (as herein defined), or sequences having about 70% or greater identity therewith which encode proteins having the immunogenic properties of said BEFV G protein.

3. The DNA molecule of claim 1, wherein said sequence encoding BEFV G_NS protein has a sequence corresponding to nucleotides 1969 to 3726 of SEQ ID No:l (as herein defined), or sequences having about 70% or greater identity therewith which encode proteins having the immunogenic properties of said BEFV G_NS protein.

4. A DNA vector which includes a sequence encoding BEFV G protein or G_NS protein.

5. The DNA vector of claim 4, wherein said vector is plasmid g207 deposited with the Australian Government

Analytical Laboratories under accession number N92/44230.

6. BEFV G protein or an immunogenic fragment thereof, wherein said BEFV protein G or fragment thereof is the expression product of all or a portion of the DNA molecule of claim 2.

7. The BEFV G protein of claim 6 wherein the amino acid residue sequence of said G protein corresponds to SEQ ID No:2 (as herein defined).

8. BEFV G_NS protein or an immunogenic fragment thereof wherein said BEFV G_NS protein or fragment thereof is the expression product of all or a portion of the DNA molecule of claim 3.

9. The BEFV G_NS protein of claim 8 wherein the amino acid residue sequence of said G_NS protein corresponds to SEQ ID No:3 (as herein defined).

10. A method for the production of BEFV G protein or G_NS protein, said method comprising the steps of:

I) preparing an expression vector incorporating a DNA sequence comprising a sequence encoding bovine ephemeral fever virus (BEFV) G protein and/or G_NS) protein; II) transforming a host cell with the expression vector prepared in step I;

III) culturing said host cell under conditions in which G protein and/or G_Ng protein is expressed; and

IV) . harvesting said expressed G protein and/or G_NS protein from the culture prepared in step III.

11. A vaccine for protecting an animal against BEFV, said vaccine comprising BEFV G protein or an immunogenic fragment thereof together with an adjuvant, wherein said BEFV G protein or fragment thereof is the expression product of all or a portion of the DNA molecule of claim 2.

12. The vaccine of claim 11, wherein said vaccine further comprises BEFV G_HS protein or an immunogenic fragment thereof, wherein said BEFV G_NS protein or fragment thereof is the expression product of all or a portion of the DNA molecule of claim 3.

13. A vaccine for protecting an animal against BEFV, said vaccine comprising a non-pathological virus which expresses BEFV G protein.

14. The vaccine of claim 13, wherein said non- pathological virus is vaccinia virus.

15. The vaccine of claim 13, wherein said non- pathological virus is rVV-G.

16. A method of protecting an animal against BEFV infection, said method comprising administering to said animal a vaccine according to claim 11.

17. A method of protecting an animal against BEFV infection, said method comprising administering to said animal a vaccine according to claim 12.

18. A method of protecting an animal against BEFV infection, said method comprising administering to said animal a vaccine according -co claim 13.

19. A method of protecting an animal against BEFV infection, said method comprising administering to said animal a vaccine according to claim 14.

20. A method of protecting an animal against BEFV infection, said method comprising administering to said animal a vaccine according to claim 15.

21. The method according to claim 20 wherein a non- pathological virus which expresses BEFV G_NS protein is administered in combination with said non-pathological virus which expresses BEFV G protein.