AU665195B2 - Immunogenic peptides or polypeptides of the BVD virus and related viruses, vaccines incorporating them or expressing them, methods and means of production thereof - Google Patents

Description

1' 665195

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Rhone Merleux ADDRESS FOR SERVICE: DAVIES COLI.ISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: C C, C C, t t C Immunogenic peptioes or polypeptides of the vaccines incorporating them or expressing production thereof BVD virus and related viruses, them, methods and means of

I

The following statement is a full description of this invention, including of performing it known to me/us:the best method t C 4,VC It l t I.I t Pit I; N'1 I 0 la- Immunogenic peptides or polypeptides of the BVD virus and related viruses, vaccines incorporating them or expressing them, methods and means of production thereof.

The present invention relates to new immunogenic peptides or polypeptides of the bovine diarrhoea virus and related viruses, in particular the Border disease virus, to vaccines incorporating them or expressing them, as well as to methods and means of production thereof.

The bovine diarrhoea virus (BVD) is an infectious single-stranded RNA-containing enveloped virus which is related to the conventional hog cholera virus and to the Border disease virus, the three, viruses forming the Pestivirus genus which belongs to the Togaviridae family.

The BVD virus is universally distributed in bovine populations and manifests itself by a very wide range of clinical symptoms associated with respiratory or enteric congenital diseases (bovine viral diarrhoea or mucosal S. disease).

BVD virus isolates may be classified into two distinct categories or biotypes according to their effects in cell culture: cytopathogenic and noncytopathogenic.

Acute infection of seronegative animals is t" usually benign or subclinical. On the other hand, intrauterine infection of the foetus, in approximately the I* first four months after the start of the pregnancy, by a t 1 noncytopathogenic strain may not only produce abortions, still births or the birth of weak calves, but also the birth of calves having persistent viremia. This period S. 30 corresponds to an absence of immunity in the foetus. When the immune system then becomes competent, it recognises i the virus as its own and a situation of immunotolerance is established. These animals will not be able to survive Sa subsequent infection by a cytopathogenic strain of homologous BVD virus.

Maintenance of the noncytopathogenic virus within the bovine population is ensured by its slow i 1 i I 2 dissemination following acute infection of seronegative animals and especially by its continuous excretion by animals having a persistent viremia. See J. Brownlie et al., Ann. Rech. Vet. (1987) 18:157-166.

Vaccines containing attenuated viruses and killed viruses have been developed in the past, with no satisfactory results. See P. Saurat et al. "La Maladie des Muqueuses" (Mucosal Disease) (1972): 229-251, l'Expansion scientifique frangaise, Paris, and A.L. Fernelius et al., Am. J. Vet. Res. (1971) 32:1963-1979. Attenuated viruscontaining vaccines may be responsible for the death of certain inoculated animals, especially animals with persistent viremia. Killed virus-containing vaccines, for their part, require multiple inoculations and only provide an immunity which is relatively low and not lasting.

Research therefore appears to be orientated towards other types of vaccines, synthetic vaccines and recombinant live vaccines. For this to be achieved, S* 20 antigenic determinants must first be sought out.

The genome of the viral strain Osloss, of cytopathogenic biotype, has been cloned and completely sequenced by Renard et al. (Patent Application EP-A-0,208,672 of 8 July 1985).

S 25 The Applicant found that the open reading frame (ORF) of the genomic sequence of BVD Osloss, 12,408 amino acids (aas).

The structural proteins are encoded by the 30 portion of the genome and they are in particular a nonglycosylated protein of 270 amino acid-, called according to the nomenclature proposed by Collet et al.

(Virology 165:200-208, 1988), and two glycosylated proteins called gp62 and gp53 according to this same nomenclature. In the following text, another nomenclature is used for these three structural proteins, which will be C for p20, gEl for gp62 and gE2 for gp53.

:1 7W I l.i'""iwl--,x6 l ;i:li 3 r ir r ei s r r r i r r r e r r r r R.O. Donis et al. (Neutralizing Monoclonal Antibodies to Bovine Viral Diarrhoea Virus Bind to the 56 K to 58 K Glycoprotein J. Gen. Virol. (1988), 69:77- 86) have shown the presence of neutralising epitopes in a 56-58-kD glycosylated envelope protein.

A neutralising peptide epitope which happens to be situated at the splicing site, between the glycoproteins gEl and gE2, is also known from Patent Application EP-A-0,236,977.

The objective of the present invention is to demonstrate other neutralising epitopes and to obtain the peptides or polypeptides containing them, and to produce vaccinal compositions, including synthetic vaccines and recombinant live vaccines, against the bovine diarrhoea virus and related viruses.

Another objective of the invention is to provide such vaccines which are effective against the two biotypes of the BVD virus.

In accordance with the invention, neutralising 20 epitopes have been located in a region encoding the portion of the BVD/Osloss virus genome and, from information regarding the location, DNA constructs comprising a fraction of the BVD genome were produced, which fraction contains at least one nucleotide sequence 25 corresponding to at least one of these epitopes. More specifically, the sequences relating to these epitopes were in particular located in the first half of the portion of the viral genome (transcribed into DNA) which encodes gE2. Epitopes were also located in the sequence encoding gEl.

The subject of the invention is therefore a nucleotide sequence substantially corresponding to the BVD virus genome fraction encoding gE2 or a sequence present therein, and comprising the first 273 amino acids of gE2, and a nucleotide sequence corresponding to the BVD virus fraction encoding gEl or a sequence present therein, as well as any new nucleotide sequence containing them, separately or together, and which comprises n i:: -4means permitting their expression or which is associated with such means.

A nucleotide sequence substantially corresponding to the first 273 amino acids of the portion of the genome of the Osloss strain Liess, 1967, Dtsch. Tierdrtztl.

Wochenschr., 74:46-49), encoding gE2 is represented, by way of example, in the attached list of sequences, under the reference SEQ ID No: 1. For this same strain, a sequence encoding gEl is represented under the reference SEQ ID No: 3.

By way of example, such sequences may be the following: initiation codon for translation sequence encoding gEl sequence encoding gE2 stop signal (a complete sequence is represented, by way of example, in the list of sequences, under the reference SEQ ID No: 2); initiation codon for translation sequence encoding 0 gEl the first 273 codons of gE2 stop signal (for example SEQ ID No: 4); S- initiation codon for translation sequence encoding the \C end of gEl the first 273 codons of gE2 stop signal; S- initiation codon for translation sequence encoding the first 273 codons of gE2 stop signal; initiation codon for translation sequence encoding C S't I sequence encoding gE2 stop signal (for example SEQ ID No:2) S c c• The subject of the invention is also the nucleotide fragments belonging to the said genome portion encoding gE2, or to the portion encoding gEl, and whose translational products are recognised by neutralising antibodies, the said fragments being associated with means permitting their expression.

Of course, the abovementioned nucleotide sequences B include all equivalent sequences, that is to .A 950301,q;\oper\ejh,18147.059,4 say, which assume the essential properties of the sequence. By way of example, this would be the case for a sequence which encodes an identical amino acid sequence, but which would use other specific codons by degeneracy of the code.

This would also be the case for a sequence encoding an amino acid sequence which is no longer identical but similar, taking into account the similarities between amino acids.

Of course, the sequences may be derived from different Pestivirus strains or may consist of hybrid sequences resulting from the assembling of genomic fragments derived from different strains. The abovementioned sequences therefore include any differing sequence maintaining recognition by the same neutralising antibodies.

These sequences may be RNA or DNA sequences which are positively oriented in the 3' direction.

i The subject of the invention is also the peptides 20 or polypeptides corresponding to the translation of these f i nucleotide sequences.

:These peptides or polypeptides may be prepared by Svarious methods which are also part of the invention.

Thus, in a first embodiment, the relevant nucleotide sequences according to the invention may be introduced into viral or eukaryotic expression vectors.

Various known techniques for constructing these vectors are described in Patent Application EP-A-0,208,672.

:i 30 As viral expression vectors, the Baculovirus system (US-A-4,745,051) may be used in particular, by integrating the relevant nucleotide sequence inside the Baculovirus genome. An example of a construct in this system will be described later.

The eukaryotic hosts may be animal or yeast, in particular Saccharomyces cerevisiae, cell cultures. The transfer vectors for yeasts advantageously comprise markers which permit the selection of useful i -6 recombinants, for example, via resistance to antibiotics or other known means of selection (Broach J. et al., Meth. Enz. (1983) 101: 307). For the promoters, see also Hess et al., J. Adv. Enz. Reg. (1968) 7:149, and Holland et al., Biochemistry (1968) 17:4900 or Itzeman et al., J. Biol. Chem. (1980) 255:2073.

The animal cells are preferably known mammalian cell lines such as HeLa, CHO or BHK, cells of insects, for example Spodoptera frugiperda (deposit ATCC CRL 1711, Sf9) (especially for the Baculovirus system) and, in general, lines whose use, for the expression of substances to be administered to animals, has been recognised by the health authorities, will be preferred.

Viral promoters such as those of the SV40 virus (Fiers et al., Nature, (1978) 273:113) and of the CMV virus or human cyto-megalovirus (McGregor and Caskey, Nucleic Acids Res. 17:2365, 1989), or alternatively, that of the polyhedrin gene of the Baculovirus AcNPV or Autographa I californica nuclear polyhedrosis virus (Hooft van 20 Iddekinge et al., 1983, Virology 131: 561-565) will be used as promoter in these cellular constructs.

The subject of the invention is therefore also the eukaryotic hosts which permit the expression of these peptides or polypeptides, prefeably in a glycosylated 25 state.

~The peptides or polypeptides according to the invention may also be manufactured by customary peptide synthesis (see for example Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co, San Francisco (1969) S 30 or Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) or Patents US-A-3,862,925; 3,842,047; 3,972,859 and 4,105,602).

In order rapidly to manufacture small amounts of peptides intended for exmaple for searching out immunological reactions with antibodies, monoclonal or otherwise, an in vitro synthesis via translation in an acellular system (lysate of rabbit reticulocytes), supplemented or not with microsomal membranes, may also be used 1 20 -7depending on whether it is desired to obtain a glycosylated or nonglycosylated peptide.

The subject of the invention is also the vaccines containing, in an effective vaccinal composition, or expressing, such peptides or polypeptides, especially the peptides derived from a sequence encoding gEl and gE2 or gEl and at least the first half of gE2 and preferably the peptides derived from a sequence encoding gEl gE2, or C gEl gE2. The peptides or polypeptides may be unconjugated or conjugated with #itdams and the vaccines may possess customary adjuvants.

These recombinant viruses may also be used for the expression of peptides or polypeptides according to the invention in virus cultures on cells.

Brief description of the drawing.

Figure 1 shows, on thl map of the BVD Osloss genome, the position of the cDNA clones 63, 36 and 183, spanning the r gion encoding the structural proteins of BVD, the restriction sites used below for the cloning of the gEl fragment, the open reading frame and the structural proteins El and E2; Figure 2 shows the hydrophobicity profile of the Sopen reading frame of the BVD Osloss genome encoding the structural proteins and the location of the potential sites for glycosylation and the cysteine residues on the open reading frame; "igure 3 shows the position of the entire restriction sites used below as well as the strategy followed for preparing the fragment E1/830; Figures 4 and 3 show.' the location of the three main fragments, El, E1/830. and El/E2, the cloning of which is ~described below, in the portion of the BVD Osloss genome encoding the structural proteins; i Figure 5 shows the restriction map of the transfer vector pJVP1OZ; the promoters of the P10 gene and of the polyhedrin gene (PH) as well as the. direction of transcription which they initiate are indicated by the arrows; S indicates the cloning site; and Figure 6 shows the kinetics of expression of the glycoproteins in the Sf9 cells infected with AcNPV-E1/E2-; L 1 p _I 1 i:v 1 1 1 1 i j 1 i b"i -21 Figure 7 shows the location of C,E1 and E2 fragments.

1. Location of the regions encoding the glycoproteins.

A Location of the region encoding gEl.

The portion of the viral genome relating to the membrane proteins is represented in Figure 1. The protein gEl (glycosylated protein of :;6-58 kD; nonglycosylated protein of 48 kD) is a membrane protein of the BVD virus.

The following data have permitted its location: a) A portion of the open reading frame is very rich in potential glycosylation sites and in cysteine, which is typical of glycosylated membrane proteins (see Figure 2).

b) A rabbit serum directed against the fusion protein p-gal-C63 decreases the size of the lysis plaques probably by recognising epitopes at the surface of the virus.

c) The beginning of the protein gEl was determined by the presence of a characteristic signal sequence (von Heijne N.A.R. 14:4683-4690, 1986) starting from the amino acid 250 and preceded by a tripeptide I Arg-Lys-Lys which may be the splicing signal of the first protein (see Figure 2).

d) The end of the protein was determined from the size expected for the nonglycosylated protein. It corresponds to the location of a potential membrane helix.

To summarise, the size of the nonglycosylated gEl protein will be: S. with the signal sequence (uncleaved): 453 amino acids; without the signal sequence: 425 amino acids.

SB Location of the region encoding gE2.

This region contains 4 potential glycosylation sites (see Figure the end of the protein was determined, on the one hand, from the size expected for the nonglycosylated protein and, on the other hand, on the basis of analysis of the hydrophobicity profile, the sequence encoding gE2 being followed by a highly hydrophobic, highly conserved region in known Pestiviruses il u J L i.P-.li~Lc~ni-VTlii-"" 22 9 whose role is still poorly defined.

2. Preparation of the Iragment E1/830.

A Cloning of the sequence encoding gEl.

The sequence encoding gEl was isolated from the clone 63 (C63) from the cDNA library described in Patent Application EP-A-0,208,672 (the clone is designated pCT63 therein), in the plasmid pSP65 (vector described by Melton D.A. et al., 1984, in Nucleic Acids Res. 12:7035- 7056). Clones of this type can be easily obtained either during the construction of a cDNA library from viral RNA as described in Patent Application EP-A-0,208,672, or by synthesis, or alternatively by amplification (Polymerase Chain Reaction) from amplimers chosen from the sequence given in the abovementioned patent application. This stage was carried out as follows: a) A fragment [BglI-BanII] containing practically all the sequence encoding gEl (see Figure 1, was isolated from the clone 63.

b) A synthetic oligonucleotide was synthesised I 20 whose sequence comprises: an EcoRI 5' end; an initiation codon ATG for translation; the first 5 codons of the gEl sequence; a BglI restriction site; a BanII restriction site; the last 6 codons of the gEl sequence; a stop codon TAG fo:: translation; a BamHI 3' end.

c) This synthetic oligonucleotide was cloned 30 between the EcoRI and BamHI sites of the plasmid pBR322 S (Bolivar et al. 1977, Gene 2: 95-113) whose inner BglI and BanlI sites had been deleted beforehand; the [BglI- BanII) fragment isolated in a) was then inserted between the corresponding sites of the oligonucleotide cloned into pBR322. The resulting fragment can be schematically represented as follows: [EcoRI ATG sequence encoding gEl STOP codon-BamHI] Bi *1 4 C 9* 9 5* 10 It is 1376 base pairs (bp) in size. Its nucleotide sequence has the reference SEQ ID No: 3 in the list of sequences attached.

d) This [EcoRI BamHI] fragment was then transferred into the plasmid pSP65 which contains the promoter for the SP6 bacteriophage RNA polymerase, to give the plasmid pSP65-gEl and thus permits the transcription in vitro of the inserted sequence.

B Cloning of a sequence encoding a portion of gE2 into the plasmid The clone 36 (C36), from the abovementioned library (pCT36), whose position on the map is represented in Figure 1i is used.

A PvuII-NdeI fragment comprising the end of the sequence encoding gEl and 273 codons of the sequence encoding gE2, was isolated from this clone 36 using restriction enzymes. The position of the restriction sites as well as the strategy followed are illustrated in Figure 3.

20 An oligonucleotide was synthesised comprising: an NdeI 5' end, a stop codon TAG for translation, a BamHI 3' end.

This oligonucleotide was added to the PvuII-NdeI 25 fragment.

The resulting fragment was then digested with BglII and BamHI and inserted into the plasmid pSP65-gE1 described in A.

The final fragment can thus be schematically represented as follows: [EcoRI ATG sequence encoding gEl 830 nucleotides of gE2 STOP BamHI]. It is 2200 bp in size. Its nucleotide sequence is given in the list of sequences under the reference SEQ ID No: 4.

3. Transcription/translation in vitro.

The fragment obtained in cloned into the plasmid pSP65, was transcribed in vitro and then translated, ir. an acellular system (rabbit reticulocyte i 11 lysate). In fact, two translations were performed, one in the presence of dog pancreas microsomal membranes, the other in the absence of microsomal membranes.

Analysis of the translational product shows, in the absence of microsomal membranes, an estimated molecular weight of 80,000 daltons.

When the translation is performed in the presence of microsomal membranes which lead to the maturation and to the glycosylation of the protein, a first glycosylated protein of 62 kD and a second protein of about 40 kD are obtained. The 62-kD protein is the protein gEl as shown by similar experiments performed using the clone 63 from the cDNA library as startir material. The protein of about 40 kD corresponds to the beginning of the protein gE2.

4. Immunoprecipitation of the translational products.

The various synthesised proteins were immunoprecipitated by means of two types of neutralising monoclonal antibodies: the 62-kD glycosylated protein is recognised by the first group, whereas the 40-kD protein corresponding to gE2 contains at least one epitope recognised by the second group of monoclonal antibodies.

All the antibodies also immunoprecipitate the 80-kD protein observed in the absence of microsomal membranes, which shows that the glycosylation is not indispensable for recognition by these antibodies.

The tests carried out with the constructs resulting in gEl lead to an immunoprecipitation only in the presence of the first group of antibodies, as is the case for the experiments carried out with a construct incorporating gEl extended by the first 32 codons of gE2 and therefore possessing the epitope described in Patent Application EP-A-0,236,977.

5. Preparation of the fragment E1/E2.

The StuI and Aval restriction sites used to extend the fragment E1/830 and to construct the completes fragment E1/E2, are indicated in Figure 3. The strategy ^Ii l 31 b. x: Pr 12 followed was as follows: a) Isolation of the [StuI-AvaI] fragment from the clone 183 derived from the cDNA library (where it is designated by pCT183) identified above (C183 in Figure 3).

a 1b) Synthesis of an oligonucleotide whose sequence comprises a StuI 5' end, an Aval restriction site, a stop codon TGA for translation and a BamHI 3' end.

c) Cloning of the oligonucleotide between the StuI and BamHI restriction sites of the plasmid containing the fragment E1/830. The StuI site of this plasmid is situated in the E1/830 fragment, 361 nucleotides upstream of the Ndel site used in the preparation of E1/830, and the BamHI site corresponds to the 3' end of the fragment cloned into pSP65 (see Figure 3).

d) Digestion of the resulting plasmid by StuI and Aval.

e) Cloning of the fragment isolated in a) into the plasmid obtained in d).

O 20 Following this procedure, the E1/830 fragment was extended by 277 nucleotides in length; the resulting Y fragment is 2487 nucleotides in length and comprises the complete sequences encoding the two glycoproteins El and E2. This E1/E2 fragment can be schematically represented as follows: [EcoRI ATG sequence encoding El sequence encoding E2 TGA BamHI]. Its location is illustrated in Figure i' 1 4 by comparison with the two fragments El and E1/830.

Its sequence is attached below: SEQ ID No:2.

6 30 6. Expression in the Baculovirus system.

Integration of the El/E2 fragment into the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome was carried out via the transfer vector pJVP1OZ Vialard et al., 1990, Journal of Virology 64: 37-50) whose restriction map is illustrated Sin Figure The vector pJVP10Z contains: hI 26 -13 an Nhel cloning site placed between the promoter and the polyadenylation site of the AcNPV polyhedrin gene; the gene for resistance to ampicillin and the origin of replication of the plasmid pUC8 Messing, 1983, Metl. Enzymol. 101:20); the lacZ gene encoding p-galactosidase placed under the control of the P10 promoter of AcNPV and followed by the polyadenylation signal of the SV40 virus.

Detection of the recombinants may be performed more easily by adding a p-galactosidase-indicator substrate into cover agarose, resulting in blue staining of the recombinant plaques.

The E1/E2 fragment was cloned into the NheI restriction site of the plasmid pJVP1OZ and the Srecombinant plasmid pJVP1OZ-E1/E2 was used to cotransfect insect cells (Sf9, Spodoptera frugiperda cells, see J.

Vialard et al. mentioned above) together with purified DNA from wild AcNPV. Purification of a recombinant virus 20 AcNPV-E1/E2 was then carried out by plating the cotransfection supernatant in Petri dishes and isolating the viral plaques by covering with agarose; two criteria make it possible to differentiate the recombinant plaques from those generated by the wild virus: the expression of i p-galactosidase (see above) and the absence of polyhedrin visible, on the other hand, in the cells infected by the wild virus.

7. Detection of the neutralising epitopes.

The glycoproteins expressed in the Baculovirus S 30 system may be detected in a sandwich ELISA involving a t mixture of two neutralising monoclonal antibodies as capture antibodies (12B1 and 204B11), and the nonneutralising monoclonal antibody 23G11 as staining antibody.

12B1 anti-gEl 204B11 anti-gE2

I

Since the two capture antibodies were directed U3 wJ against distinct epitopes, their reactivity was VT OI

FI

i- 14 individually tested by carrying corresponding to the following scheme: Captor Sample 12B1 or Sf9/AcNPV-E1/E2 204B11 out an ELISA Stain 23G11* The results of this test are collated in Table 1.

Table 1: Reactivity, in ELISA, of the two neutralising monoclonal antibodies 12B1 and 204B11 on the proteins expressed by the recombinant virus AcNPV-E1/E2.

sandwich ELISA: captors 12B1 or 204B11; stain 23G11* (antibody dilution: 4000 x).

Sf9/AcNPV-E1/E2: Sf9 .cells (Spodoptera frugiperda) infected with the recombinant AcNPV-E1/E2 and harvested 72 hours after the infection; Sf9: uninfected cells.

The values are expressed as OD x 1000 (OVER OD 2).

Captor Dilution of Sf9/AcNPV-E1/E2 Sf9 cells 12B1 3 x OVER 72 9 x 1558 49 27 x 1078 44 81 x 259 48 204B11 3 x OVER 46 9 x OVER 36 27 x 1915 32 81 x 602 These results show that the expressed proteins are captured by the two types of neutralising antibodies tested; it is important to note that these two monoclonal antibodies are different in two respects: a. Transcription/translation/immunoprecipitation experiments have shown that they were directed against distinct epitopes, one situated in the glycoprotein gEl,

I

ii i 15 the other in gE2; their ability to capture the proteins produced in Baculovirus shows, as a result, that the two proteins are not only expressed, but that they are expressed, in addition, in a conformation permitting their recognition by the neutralising monoclonal antibodies.

b. Both neutralising antibodies were developed using different immunising antigens as starting material: 204B11 (or BD204B11) is derived from the strain BD/Moredun, 12B1 (or NY12B1) from the strain New York; 216E7 (or AV216E7, anti-gE2), derived from the strain Aveyron, also specifically recognises the proteins expressed in Baculovirus which are derived from the Osloss strain.

15 8. Kinetics of expression of the glycoproteins.

The level of expression of the glycoproteins in Sf9 cells infected by the recombinant AcNPV-E1/E2 was r still rising 72 hours after the infection; a kinetics of S.k 20 infection spanning a longer period (11 days) was therefore performed and the level of glycoproteins expressed was estimated over time from the values obtained by HI ELISA. The ELISA is of the sandwich type which is carried out according to the same scheme as that described in 7, in which the neutralising antibodies 12B1 and 204B11 are used individually as captors and 23G11* as stain. The results are illustrated in Figure 6: S- 204B11:A, B, C curves obtained with the monoclonal antibody 204B11 which is used as captor, and for i 30 three dilutions of infected Sf9 cells (4 x, 12 x and 36 x).

S12B1:B, C curves obtained with the monoclonal antibody 12B1 used as captor, for two infected cell dilutions (12 x and 36 x).

The level of expression of the proteins recognised in ELISA, regardless of the antibody used as Scaptor, reaches a plateau 4 days after the infection (99 Shours), a plateau which persists particularly up to the 16 seventh day after the infection (167 hours). The parallelism observed between the curves obtained with both types of captors confirms the presence of both epitopes in the proteins expressed, epitopes which are maintained at the same stability during the entire kinetics of infection.

9. Location of the expressed proteins The neutralising monoclonal antibodies 204B11, 12B1 and 216E7 were used to detect the glycoproteins by the technique of indirect immunofluorescence. For this, the recombinant AcNPV-El/E2-infected Sf9 cells were subjected to various fixation conditions: in 95% acetone, 20 minutes at in 4% formalin, 10 minutes at room temperature; 10 minutes in 4% formalin, followed by minutes in 0.1% Triton X-100.

The fixation conditions in acetone are too drastic to permit precise location of the expressed proteins: the membranes are made permeable and the 20 fluorescence is intense but distributed throughout the fcytoplasm. In contrast, when the cells are fixed with formalin, the fluorescence is visible in the form of 'i granules located on the entire surface of the infected cells.

25 The addition of a Triton X-100 treatment stage to the formalin fixation, renders the cytoplasmic membrane fragile without making the nuclei permeable, which imparts a more intense fluorescence than previously, in granular form, and entering into the cytoplasm at certain Ct t :i t 30 points.

It is important to emphasise that the two antibodies 12B1 (NY; anti-gEl) and 204B11 (BD; anti-gE2) give images which are quite comparable, and that the same applies to 216E7 (AV; anti-gE2). These results therefore confirm the presence of both glycoproteins which are expressed at the surface of the infected cells and which may be associated with each other.

i ii i !I I- 17 The expressed glycoproteins are recognised by neutralising monoclonal antibodies which differ in two respects. On the one hand, they are directed against distinct epitopes, one of which is situated in the first glycoprotein and the other in the second; the results therefore show that the two proteins are both present in the cells infected by the recombinant, and they follow an expression curve which is quite similar. On the other hand, they differ with respect to the viral strains used as immunising antigens: 12B1: BVD/New York strain (Rh8ne M6rieux, France); 204B11: BD/Moredun strain (Rhone M6rieux, France); 216E7: Aveyron strain (Rhbne M6rieux, France).

This surprising observation is important within the framework of the use of expressed glycoproteins (derived from the Osloss strain) for vaccination purposes, given the similarity of recognition of these 20 proteins by antibodies derived from viral strains as serologically divergent as Aveyron and New-York. It should be noted for example that not only does the antibody 216E7 not neutralise the New-York virus, but it does not recognise it in immunofluorescence.

Finally, the pictures observed in immunom fluorescence under various cell fixation conditions clearly show that the glycoproteins, which are revealed by the neutralising monoclonal antibodies, are expressed at the surface of the infected cells.

It should be noted that the glycoproteins gEl and gE2 are expressed at a sufficient level for being Sobserved in gel electrophoresis after Coomassie blue Sstaining. Their positions on the gel correspond to the molecular weights expected after cleavage and glycosylation, which suggests proper maturation of the proteins expressed in this system.

El/E2: Immunisation of rabbits and analysis of the antibodies induced.

L t 7- I 1 -18 18 A lysate of Sf9 insect cells (Spodoptera frugiperda) infected by the recombinant AcNPV-E1/E2 was used in rabbit immunisation trials carried out according to the following procedure: Material: immunising antigen: Sf9 cells infected with AcNPV- El/E2; control antigen: uninfected Sf9 cells; 6 rabbits: 3 antigen doses, 1 control for each dose: rabbit nos. 1, 2, 3: inoculation of the infected cells with an amount of 20 x 10 6 4 x 106 and 0.8 x 106 cells per inoculation, respectively.

Rabbit nos. 4, 5, 6: inoculation of the control cells with an amount of 20 x 106,. 4 x 106 and 0.8 x 10 6 cells per inoculation, respectively.

Immunisation: day 0 1st inoculation; day 28 2nd inoculation; day 56 3rd inoculation; day 85 4th inoculation; H 20 day 110 5th inoculation.

C Serological analysis: tThe sera were collected before the first inoculation and then on days 38, 66, 99 and 120. The BVDspecific antibodies are then analysed by competition j 25 ELISA with respect to neutralising monoclonal antibodies, and by seroneutralisation against three viral strains: Aveyron, New-York and noncytopathogenic Osloss.

Nomenclature used for the successive samplings: r r C 1' C: P i- i 'ri i c ii i 9 ii :i Rabbit Inoculated Samplings j No. dose Day 38 Day 66 Day 99 Day 120 1 20 x 10" CI S1/P38 S1/P66 S1/P99 S1/P120 2 4 x 106 CI S2/P38 S2/P66 S2/P99 S2/P120 3 0.8 x 106 CI S3/P38 S3/P66 S3/P99 S3/P120

I

32 1i -19 Rabbit Inoculated Samplings No. dose Day 38 Day 66 Day 99 Day 120 4 20 x 106 CTe S4/P38 S4/P66 S4/P99 S4/P120 4 x 106 CTe S5/P38 S5/P66 S5/P99 S5/P120 6 0.8 x 10 6 CTe S6/P38 S6/P66 S6/P99 S6/P120 .4.

I

4* I ,1 4, r C 'I 4- 4- 4i Ii 4 I 4 '44 CI AcNPV-El/E2-infected Sf9 cells; CTe Uninfected Sf9 cells.

a) Results obtained by competition ELISA.

The sera collected after the 2nd and 3rd inoculations of crude cellular lysates' (P38 and P66) were analysed for their ability to compete with a neutralising monoclonal antibody, in this case BD204B11, with respect to its binding to the E1/E2 antigen produced in the insect cells. The results are collated in Table 2.

From these values, the percentage competition of sera 1 to 3 (CI antigen) was calculated relative to the initial OD (control without serum), after correction according to their respective controls by rabbits 4 to 6 (CTe antigen), in other words: 20 %age competition (OD CTe OD CI) x 100 (I) OD TSS where CI AcNPV-El/E2-infected Sf9 cells; CTe uninfected Sf9 cells; TSS control without serum.

25 The values calculated are collated in Table 3.

Table 2: Reactivity, in ELISA, of the sera collected from the 6 rabbits on days 38 and 66.

Fixation of the antigen E1/E2 via the antibody

S

NY12B1 (anti-gEl) which is used as captor (ascite diluted 4000 Competing antibody: BD204B11 (anti-gE2; purified and labelled monoclonal antibody, used at a 1000 x dilution). The values are expressed as OD x 1000. The nomenclature for the samples is that described above.

i:

I

I i ;ii. i ;i -7 i 4 p x i i s u

I

iii i-i r:i i-'

II

'i is 1 i,;i i ii a 1 r j r r r Il'i;' 1 Dilution of the samples 375 x 75 x 15 x 1st series of samples (P38): Dose:. 20 x 106 cel./inj.

Si (CI) 1076 936 727 S4 (CTe) 965 903 780 Dose: 4 x 106 cel./inj.

S2 (CI) 971 812 555 (CTe) 993 847 597 Dose: 0.8 x 105 cel./inj.

S3 (CI) 1006 822 608 S6 (CTe) 929 809 571 2nd series of samples (P66): 64. Dose: 20 x 106 eel. /inj.

Si (CI) 1001 957 912 S4 (CTe) 1064 1014 897 ~*Dose: 4 x 106' cel./inj.

S2 (CI) 973 773 406 (CTe) 1083 957 800t S 20 Dose: 0.8 x 106 cel./inj.

S3 (CI) 1012 889 765 S6 (CTe)' 1052 983 939

C

(TSS (control without serum): 1103 Table 3: Level -of competition cf the sera collected on days 38 and 66, with respect to 'the monoclonal antibody BD2O4B1 (expressed in and calculated from the values in Table 2, according to formula I Ki::i 1 21 Dilution of the 375 x 75 x samples 15 x P38 Sl 0 0 4.8 S2 2 3.2 3.8 S3 0 0 0 P66 Sl 5.7 5.2 0 S2 10 16.7 35.7 S3 0 8.5 15.7 b) Seroneutralisations.

The presence of neutralising antibodies in the four series of samples, P38, P66, P99 and P120 was tested with respect to the three Pestivirus strains cultured on OCK cells: noncytopathogenic Osloss; 15 New York; Aveyron.

The results are collated in Table 4.

Table 4: Seroneutralisation titres of sera collected on days 38, 66, 99 and 120, with respect to Osloss NC (OsNC), New York (NY) and Aveyron (AV) strains (100 pfu/well).

The values are expressed as dilution of serum for which 50% neutralisation is obtained for the viruses tested.

25 0: no observable effect; I :ii su r ,wa9 b r~ar surcr n or D P C u*~*a u or o or os r rruu r r uu o* a a ouuo r, r 1 sil; r r I I c -i I 22

S

Rabbit no. 1 2 3 4 5 6 OsNc strain: P38 4 16 16 0 0 0 P66 32 256 64 0 0 0 P99 4096 4096 4096 0 0 0 P120 16384 4096 16384 0 0 0 NY strain: P38 4 4 4 0 0 0 P66 4 4 0 0 0 0 P99 1024 16 '256 0 0 0 P120 1024 16 1024 0 0 0 AV strain: P38 0 8 4 0 0 0 P66 0 32 4 0 0 0 P99 1024 512 256 0 0 0 P120 256 1024 1024 0 0 0 c) Conclusions The viral proteins expressed in insect cells Iz the recombinant Baculovirus AcNPV-El/E2 induce in rabbits, the appearance of neutralising antibodies: with respect to the three Pestivirus strains tested, that is to saycyniopathogenic Osloss, New York and Aveyron ec:u.

in the three rabbits immunised with different antigen doses; immediately after the'first sampling, that is to say, after two injections of antigen, with a low but significant seroneutralising titre compared with the control rabbits; the titre increases on subsequent samplings and becomes very significant fjrom the third sampling onwards, and this with respect to the three viral strains.

The antibodies induced in rabbits by the, 23 inoculation of extracts of cells expressing the recombinant antigens El/E2 therefore neutralise at least three very different Pestivirus strains, namely Osloss NC (BVD), New York (BVD) and Aveyron (Border disease).

11. Extension of the constructs E1/E2 Fiqure 7 illustrates the portion of the BVD/Osloss genome encoding the structural proteins C, El and E2, with the presumed limits of the three coding sequences and the location of the previous constructs in the El/E2 regions.

Using as starting material the. total RNA isolated from OCK cells infected with the cytopathogenic viral strain BVD/Osloss, the C-encoding sequence was amplified by PCR using the following two amplimers Ampli A and ampli B in Figure 3): Ampli A: located at the level of the initiation codon strand) for translation, in position 384 in the BVD/Osloss genome sequence: GGAATTCCCATG GAG TTG ATT ACA AAT -3' EcoRI NcoI Ampli B: located at 640 bp downstream of the beginning strand) of the El sequence, in position 1760 in the BVD/Osloss genome; sequence: ,.CT TTC TAG AGA ATT GGT CAT -3' XbaI The amplified fragment can therefore be schematically represented as follows: ti [EcoRI end ATG included in NcoI 750 bp encoding C 640 bp corresponding to the beginning of 1 XbaI end] This fragment was inserted at the 5 end of the three constructs gEl, gE1/830 and gE1/1100, at the level of the XbaI restriction site (see Fiqure 7).

II

-24- ANNEX: LIST OF SEQUENCES.

SEQ ID NO 1 TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 894 base pairs NUMBLR OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 1-894 PROPERTIES: encodes the first half of the structural glycoprotein gE2 G3ii Val Leu Gin Gly Ile Lau Trp Lou Ile Laeu l10 Thr Gly Ale Gin 1b CAA GTG TTG CAA GGC ATA CTG IGG TTG ATA CTC-ATA ACA GGG GCA CAA 4 Gly Leu Pro Val Cyi Lys Pro Gly Phi, Tyr Tyr Ala Ile Ala Lys Asn 32 *GGG CTC CCA GTT TGC AAA CCC GGC TTT TAC TAC GCC ATA GCC AAA AAT 9 Asn Glu Ile Gly Pro Leu G I Ala Thr Gly Leu Thr Th r Gin Trp TYr 48 AAT GAG ATC GGC CCT CTT GOO GCT ACG GGC CTC ACC ACT CAG TOG TAT 144 O lv Tyr Ser Asp GlIy Met Az-g Levt Gln Asp Thr Gly Val Val Val TrD 64 GAA TAC TCG GAT CGG ATG CGG CTG CAG GAC ACG GGA GTT GTA GTG TOG 192 Cys Lys Gly ClIy Glu Ilie Lys Tyr Leu Ile Thr Cys Glu Arg Glu Ala TGT AAA GGT GGA GAG AIC AAA TAT CTA ATT ACA TOT GAG AGO GAA GCC 240 ArV Tyr Leu Ala Ile Leu His Thr Ar; Ala Lou Pro Thr Ser Vatl Vali 96 AGO TAT CTG GCC ATT CTA CAC ACG AGA GCC CTG CCG ACO TCT CTA GTA 28 Phe GCIu Lys Ile Ile Asp Gly Lys Glu Gln Glu Asp Val Val CIO3 Met -1 TIT CAA AAA ATC ATA GAT GGA AAA GAA CAA GAG GAC GTA GTCG AA ATG 3361 Asp Aso Asn Ph. Glu Lau Gly Laeu Cys Pro CYs Aso Ala Lys Pro Lau 128 OA G A CT AAC TTT GAA CTC GIGT CTT TGC CCG TOT GAT OCT AAA CCC TTG 384 Val Arg Gly Lys Phe ASrI Thr Thr Lou Lou Asii GiY Pro Ala Pho G.i.n 1.44 *.GTA AG(; GGA AMA TTT MAT ACA ACA CTT, CTG AAT 000 CCA GCC TTC CAG 432 Met Val Cys Pro Ile Gly Trp Thi. Gh' Thr Vali Ser Leu CYs His Trp 160 ATG GTT TGC CCT ATA GGA TGG ACA GGA ACT GTG ACT CIG TOT CAC TOG 480 Ser Asi, Lys Aso Thr Leu Ale Met Thr Vali Vali Arg Thr Tyr Lys Arg 176 TCC AAT AAG GAT ACC TTA GCC ATG ACC OTT GTA CCA ACA TAC MAG AGO 528 His Arg Pro Phe Pro Ph. Arg Gin Gly Cys Ile Th? GlnLys Val 119 192 CAC AGO CCT TTC CCC TTT AGG CMA GGC TGC ATT ACC CAGi AAM GTC ATC 576 Oh' Oh' As P Leu Tyzr Asp Cys Ala Lau GlY Gly Asn Trp Thr Cys Vali 208 GGG GOA GAC CTC TAC GAC TOT GCC TTG GGA GGG AAC, TGG ACT TOT GTA 624 Pro Gly Asp Ile Le~u Arg Tyr Val Asp G1ly Pro Val G1u Ser Cys L~ys 224 CCG GGG GAC XTA CTA CGA TAT GTA GAT GGG CCT GTC GAG TCT TGC AAG 672 Trp Cys Gly Tyr Lys Phe His Lys Se r Glu Gly L e.U Pro W. s Phe Pro 240 T.CQ TGT GGT TAC AA.G TTT CAT AAA AGT GAG GGT CTG CCA CAC TTC CCA '/ZU Ili Gly Lys Cys [.ys Leu Lys Asn Glu Ser Gly Tyr Arg Gin Val Asp 256 ATT GGC AAG TGC AAG CTG AAG AAT GAA AG2' GGC TAC AGA CAA GTA GAT 768 Glu Thr Sor Cys Asn Arg Asp Gly Val A 'la Ile Val Pro Th r Gly Ser 272 GAG ACC TCT TGC MAC AGA GAC GOT GTG 4CT ATA GTA CCA ACT GGT TCG 816 Val Lys CYs Lys Ile Gly Asp Thr Val Val Gin Val Ile Ala Met Asp 288 GTG A kA TGC MAG ATA GGG GAC ACA GTG GTG CAA GTC ATA GCA ATG GAT 864 Asp Lys Lau Gly Pro Met Pro Cys Arg Pro23 GAT AAG CTA GGG CCT ATC CCT TGC AGA CCA 894 39 -26- SEQ ID NO :2 TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 2487 base pairs NUMBER OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 10-2479 PROPERTIES: encodes the structural glycoproteins gEl and gE2 Met GIy Lys Leu Glu Lys Ala Lau Leu Ala Trp Ala Val 13 GAA TTC ACAT, GC AAA CTA GAG AAA r7CC CTG TTG GCA TGG GCA GTA 48 EcoRI co Ile Ala Lau Val 1,eu Phe Gin Val Ala Val. Gly Glu Asn Ile Thr Gin 29 ATA GCC TTG GTT TTG TTT CAA GTC GCA GTG GGA GAG MAC ATA ACA CAA 96 TrD Aszi Lau Gln Asp Asn Gly Thr Glu Gly Ile Gln Arg Ala MeL Phe Gin Arp Gly Val Msn Arg Ser Lau His Cly Ile Trp Pro Glu Lys Ile 61 CAA AGA GGC GTA AAT AGA AGT CTG CAT GGG ATC TGG CCA GAG MAA ATC 192 Cys Thr GXy Val. Pro Ser His Lau Ala Thr Asp Thr Glu Lou Lys Ala 77 TGT ACA GGT GTC CCC TCC CAC TTG GCC ACT GAT ACA GAA CTG AAG GCA 240 110 His Gly Met Met Asp Ala Ser Giu Lys Th r Asn Ty r Thr Cys CYS 93 ATT CAT GGT ATG ATG GAT GCT AGC GAG MAG ACA AAT TAC ACA TGC TUCC 288 Arg Lau Gln Arg His Ulu Tro Asn Lys His Gly Trp Cys Asn Trp Tyr 109 AGG CTC CAA CGC CAT GAG TGG AAC MAG CAT GGT TGG TGC MAT 2TGG TAC 336 Asn Ile Glu Pro Trp Ile Val. Leu Met Asn Lys Thr GIn Ala Asn Lou 125 AAT ATT GAA CCT TGG ATT GTT CTC ATG AAT AAA ACC CAA GCC kAC CTT 384 Ala Glu Gly Gin Pro Pro Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp 141 GCT GAG GGT CAG ZECA CCA AGG GAG TGT GCC GTT ACA TGC CGG TAT GAC 432 Arg Asp Bar Asp Lou Asn Val Val Thr Gin Ala Arg Asn Sar Pro Thr 157 CGA GAT AGT GAC CTA MAT GTA GTA ACA. CAA GCT AGG MAC AGC CCC AC?. 480 Pro Lou Thr Gly Cys Lys Lys Gly Lys Asn Pjh, S*r Phe Ala Gly Val 113 CC?. TIG ACA GGC TGC MAG AAA GGC AAG AAC !CTC TCC TTT GCA GGT CGTG 528 Lau Val Gin Gly Pro Cys Asn Phe Glu Ile Ala Val Ser Asp Val Lou 199 TTG GTA CMA GGG CCT TGC AAC TTT GA?. ATA GCT OTA AGT GAT GTG CTG 576 Phe Aro Glu His Asp Cys Thr Ser Val Ile Gin Gly Thr Ala His Tyr 205 TTT AG?. GAG CAC GAT TOC ACA AGT GTG ATT CAA GGC ACG GC CAC TAT 624 p.; 27 Lqu Val Asp Gly Met Thr Asn Soy Leu G1U Sor Ala Arg GIn Oly Thr 221 CTG r.PTA GAC GGG ATG ACC AAT TCT CTA GMAA AT CC AGG CM GGG ACC 672 Ala Lys Leu Thr Thr Trp Leu Gly Arg G.n Leu Lys Lys Leu GlY Lys 2 37 OCA AAG TTA ACT ACT TGG TTG OCT AGG CAG CTT AMG AAA CTA GGG AAG 720 Lys Lieu Glu Asn LYs Ser Lys Thr Trp Phe. Gly Ala Tyr Ala Ala Sgr 253 MAA CTG GAA AAC AG ACT AAG ACA TGG TTT GGG GOCA TAT GCA GCC TCT 768.

Pro Tyr Cys Giu Vl Glu Arg Arg Leu Gly Tyr le Trp Tyr Thr Lys 269 CCC TAC TGC GAG GTA GAA CGG AGG CTT GGT TAC ATC TGO TAT ACA MG 16 Asn Cys Thr Pro Ala Cys Lau Pro Lys Asn Thr Lys 1e Val Gly Pro 285 AAT TOC ACC CCT GCC TOT TTA CCA AAA AAT ACA AAG ATC OTT CGC CCC 064 Gly Arg Phe Asp Thr Asn Ala Glu Asp Gly Lys 119 Leu His Glu Met. 301 GGT AGO TTC GAC ACC MT GCG GAG GAT GOT MAA ATA CTG CAT GAG ATG 912 Gly Gly His Lou Ser Glu Val Lau Leu Lou Ser Val Vol Val Leu Ser 317 COG CC CAC TTG TCA GAG GTG CTA CTA CTC TCA GTG GTA GTG CTT TCC 960 Asp Pho Ala Pro Olu Thr Ala Ser Val Val Tyr Leu Ile Leu His Phe 333 GAT TTC GCT CCA GAG ACA CC AGT GTG GTA TAT TTA ATT CTA CAT TTC 1008 Ser lie Pro Gin Gly His Thr Asp lie His Asp Cys Asp LyS Asn Gin 349 TCC ATC CCA CAA GGA CAC ACT GAG ATA CAT GO TOT GAT AM AAC CAA 1056 Leu Asn Leu Thr Va1 Gly Leu Thr Thr Ala Glu Val Ile Pro Gly Ser 365 CTX AAC CTC ACC GTA GGA CTC ACA ACA GCT GAA GTA ATA CCT GGG TCA 1104 Va lTrp ASn Leu Gly Lys Tyr Val Cys lie Arg Pro Asp Trp Trp Pro 301 OTT TGG AAT TG r3GC AAA TAT OTT TGT ATA AGA CCA GAT TOG TOG CT 1152 Tyr Glu Thr Ala Thr Phe Lqu Val Phe Glu Giu Val Gly Gin Val lie 397 TAT GAG ACA GCC ACG'TTC OTA GTG TTT GAA GAG GTG GOT CAA GTG ATC 1200 Arg Iie Val Lou Arg Ala Leu Arg Asp Leu Thr Arg Ile Tro Thr Ala 413 AGG ATA GTC TTG AGG OCT TTA AGA GAT CTA ACG CCC ATT TGG ACC OCT 1248 A la Thr Thr Thr Ala Phe Lou Val Cys Leu Val Lys Val Val Arg Gly 429 GOT ACO ACT ACT OCA TTC CTG GTA TOT CTG GTG AAG OTG GTG AGA GCC 1296 Gin Val Leu Gin Gly Ile Lou Trp Lou 110 Leu lie Thr Gly Ala Gin 445 CAA GTG TTG CAA GGC ATA CTG TOG TTG ATA CTC ATA ACA GOO GCA CAA 1344 GlY Lou Pro Val Cys Lys Pro Gly Ph* Tyr Tyr Al. Ile Ala Lys Asi 461 GG rTO CCA OTT TGC AAA CCC GGC TTT TAC TAC GCC ATA 0CC AM MT 1392 Asn Glu Il Gly Pro Lou Gly Ala Thr GlY Leu Thr Thr Gin Tro Tyr 471 AAT GAG ATC GGC CCT CTT GGG OCT ACG GCC CTC ACC ACT CAG TGG TAT 1440 Olu Tyr Ser Asp Gly Met Arg Lou Gin Asp Thr Oly Vol Val Val Trp 493 GAA TAC TOG GAT GGG ATG COG CTG CAG GAC ACG GOA GTT GTA GTG TGG 1488 Cys LYS GLY GlY Glu 110 LYS Tyr Lou lie Thr CYS Giu Arg Glu Ala 509 TOT AMh GGT GGA GAG ATC MA TAT CTA ATT ACA TOT GAG AGO GA CC 1536

A

28 a.

w

I

I I

CI

A ro

AG

Phe

ITTT

Asp G AT Veil

GTA

Met

ATC

Ser

TCC

His

CAC

Gly Q Q0 Pro cCO I rP

TOG

Ile AlT Glu

GAG

Val.

Asp

GAT

Glu

GAG

Lou Met

ATG

His

CAC

Lou

CTC

.eu

CTA

TAT

GAA

Asp)

GAT

AGG

OTT

As n

AAT

A rg

AGO

Gly Cly Cy s

TOT

Gly

GC

Thr AC C Ly s

AAA

Lys

AAG

Gly

OGG

Lys

MNG

L-e u

TIA

His

CAC

Leu

CTG

Ser E.eu NA CTG 000 L~ys Ile AAA ATO Asn Phe AAC TTT Gly Lys GGA AAM Cys Pro TGC COT Lys Asp AAG GAT Pro Ph.

CCT TTC Asp Leu GAC CIC Asp Ile GAC ATA Gly Tyr GOT TAC LYS Cys AAG OC Ser Cys TOT C Cys Lys TGC AAG Leu Gly CTA GGC Pro Val CCG CIA Asn Lys AAC AAG Lys Gly AAA CG Arg Asp COG GAT Giy Gly G0C GOT Ile

AT

I Ie

ATA

Glu

C'AA

Pho

TIT

116 AlA Th r

NCO

Pro

CCC

Tyr1

TAC

Leu

CTA

Ly s

AAC

Ly s

AAG

Asn

AAC

Ile

ATA

Pro

CT

Glu

GMA

Ty r

TAT

Glu

GAG

Ty r

TAC

Arg

AGO

Leu

CTA

Asp

GAT

Leu

CTC

Asn

AAT

Gly

GOA

Lau T TA Phe

ITTT

Asp

OAO

Ara

CGA

Phe

TIT

Lou

CTG

AGA

Cly G00 Met

ATG

Ly s

MAG

Tyr

TAT

Tyr

TAC

Ph.

TC

Ty r

TAC

His Thr CAC ACG Oly t.y s GCA A Gly Lau OT CIT Thr Thr ACA ACA Trp Thr TOG ACA Ala Met GCC AIG Arg Gin AGO CAA Cys Al 1a TGT GC Tyr Val TAT GTA His Lys CA T AAA Lys Asn e\AG XAA Asp Gly CA C GCI Aso Thr CAC ACA Pro Cys CCT C Thr Ala XCG GCA Glu Pro GAG OCT Gin Tyr CAA TAT Ala Glu OCT GAG Val Lau GTG CTC A rg

AGA

Olu

GAA

Cy s

C

Lou C TT O ly

GGA

Th r

ACC

Gly

CC

Lou

ITO

ASP

GAT

Set

AGT

Glu G AA Val

GTG

Val 010 A rg

AGA

Cys

TOT

A rg

AGO

Trp

TOG

So r

TC

T rp

TG

Gly Nla Leu GCC OTG Gin Glu Chh GAG Pro cys CCC TOT Lou Msn CTG AAT Thr Val ACT GIG Val Val GTT GTA Cys Ile TGC AT7 Gly Cly GGA CG Gly Pro Olu Gly GAG GOT Ser Oly OCT ATA Val Cmn GTG CMA Pro Tyr CCA TAT Thr Phe ACC TTC Asp Asn GAT AAT Phe Asp ITT GAC Leu Lou CTA CTG Lou Lou TTA CTG P r o P 1o

CCC

A s

GAC

Aso

GAT

Gly

GGG

Ser

AGT

A rg

CGA

Thr

ACC

Xsn

AAC

Val 010 Leu

CTG

Tyr

TAO

Val

GTA

VIal

OTC

Glu

GMA

Asn

AAC

Ty r

TAT

Leu

CTA

Val

GTG

Val

GTT

Thr

ACC

Val

GTA

Ala

OCT

Pro

CCA

Lo u 010 Thr

ACA

Gin

CAG

I rp

TG

Glu

GAG

Pro

CCA

A rt AG A Pro

CCA

Ile

ATA

119

ATC

Ty r

TAO

Ptie

TTC

Glu

GAG

Ile

ATA

Th f

ACA

Se

TCT

Vali

GIG

Lys

AAA

Ala cc Cys

TOT

Ty r

TAC

Lys

AAA

Thi

ACT

Ser

TCT

His

CAC

Gin

CAA

Thr

ACT

Ala

GCA

Ile

AT

Thr

ACA

Gin C A Ile

ATO

Val.

GIG

Ty r

TAT

Val

GTA

GIlu

GAA

Pro

CCC

Phe

TC

His

CAC

AAG

Val cys

TOT

Cys5

C

Phe

ITO

Vol

GTA

Cly

GOT

Met Al 0 Pro

CCC

Ly s

MA

Gln

CAA,

Thr

ACT

Val

OTT

Met

ATC

Vail

CIA

Met

ATG

Leou

TIC

GC1in

CAC

Tr

TOG

Arg

AGO

Ile

ATC

Val

GTA

Lys

AAO

Pro

CCA

Asp

GAT

Se 1,

TCC

Asp

GAT

Se r AGl 1 hr

ACA

Ty r

TAC

ASP

GAkC Ala

GCA

Ilie

ATC

525 1584 541 16.42 557 1680 573 1728 589 1776 605 1824 621 1872 637 1920 653 1Y6 8 669 2016 685 2064 101 2112 717 2160 733 2208 749 2256 165 2304 781 2352 .797 2400 813 2448 $is3 2.487 Giu Gin Met Thr Ser Gly TCA GAA CAA ATO ACC TCG GGA 000 CCC TOA GO C -29- SEQ ID NO :3 TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 1376 base pairs NUMBER OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 10-1371 PROPERTIE3S: encodes the structural'glycoprotein gEl Hot Gly Lys Leu, Cli Lys Ale Lou Leu Ala Trp Ala Val 13 GAA TT.q ACC ATG GGC AAA CIA GAG AAA GCC CTG TTG CCA TOG GCA GIA 48 EcoRI W 0 Ile Ala Leu Val Leu Phe Gin Val Ala Val Gly Clu Asn 119 Thr Gin 29 ATA GCC TTG OTT TO TTT CAA GTC GCA GTG GGA GAG MAC ATA ACA CAA 96 Trp Asii Leu GIn Asp Asn Gly Thr Glii Gly 110 Gin Arc Al& Met. Phe TGG AAC TTA CAA GAC AAT GGG ACG GAA GOA ATA CAA COG GCC ATG TTC 144 GIn Proi Gly Val Asn Arg Ser Leu His Gly Ile 'Irp Pro Glu Lys Ilie 61 CAA AGA GGC GTA AAT AGA AGT C TG^ CAT OGG ATC TOOC CCA GAG AAA ATC 192 Cys Thr Gly Val P ro Ser HIS Leu Ala Thr Aso Thr G Iu Leu Lys Ala 77 IGT ACA GOT GTC CCC 'ICC CAC TTC GCC ACT GAT ACA GAA CTG AAG GCA 240 l1e HI~S Gly Met. Met Asp Ala ar Ciii Lys Thir Asn Tyr Thr Cys Cys 93 ATT CAT GOT AIG ATG GAT OCT ACC GAG AAG ACA AAT TAC ACA TCC T C 288 Arg Leu Gin Arc His Ciii Trp Asn Lys HIs Gly Trp Cys Asn Trp, Tyr 109 AGO CTC CAA CGC CAT GAG TOO AAC AAG CAT GOT TOO TGC MAT TOG TAC 336 Asn Ile G Iu Pro Tro Ile Val Lau Met Asn Lys Thz- Gin Ale Asn Lau 125 MAT ATT GAA CCT TOG ATT OTT CTC ATG AAT AAA ACC CAA GCC AAC CTT 384 Ala Giu Cly Gin Pro Pro Arg Clii Cys Ala Val Thr Cys Arp Tyr- Asp 141 OCT GAG GOT CAG CCA CCA AGO GAG TOT GC C.,OT ACA TCC COG TAT GAC 432 Arc Aso Ser Asp Lou Asn Val Val Thr Gin Ala Arg Asn Ser Pro Thr 157 CGA GAT ACT GAC CTA AAT GTA GTA ACA CAA OCT AGO MAC AGC CCC ACA 480 Pro Lou Thr Ci'i Cys Lys Lys Gly Lys Asn Phe Ser Ph, Ale Gly Val 173 CCA TTG ACA CCC TG AAG AAA CCC AAG AAC TIC TCC TTT CCA GOT GTG 528 Lou Val Gin Cly Pro Cys Asn Phe Glu Ilie Ala Val Sar Asp Val Leu 169 TTC GTA CAA GGG CCT TCC MAC TTT GAA ATA OCT GTA ACT GAT GIG CTG 576 Pho Arg Glu His ASjP Cys Thr $or Val Ile Gin Gly Thr Ala His Tyr 205 TTT AGA GAG CAC GAT TGC ACA ACT GIG AT? CAA GGC ACG OCT CAC TAT 624 2/6 y 30 Leu

GCA

Lys

AAA

Pro

CCC

Asn

AAT

Gly

GGIT

CGGG

Asp

GAT

Se r

TCC

Leu

CTA

Va I

GTT

Tyr

TAT

A ru

AGG

Al a

GCT

CAA

Gly

GGG

Vail G T A

AAG

Lou

CTC

Ty r

TAC

Cys

TGC

A rg

AGG

Gly

GGC

Phe

TTC

Ile

ATC

Asn

AAC

T rP

TG

Glu

GAG

Ile

ATA

Thr

ACCG

Val1 Leu

CTC

Asp

GAC

Le u

TIA

G I u,

GAA

Cys

?GC

Th r

ACC

Phe

TTC

His

CAC

Ala

CCT

Pro

CCA

Leu

CTC

Asn

AAT

Th r

ACA

Val

GTC

Tb r

ACT

Leu

TTG

Pro

CCA

cly cGGc Thr

ACT

As n

AAC

Glu

GAG

Pro

CCT

Aso~ GA C Leu

TTG

Pro

CCA

Gin

CAA

Th r AC C Leu

TTG

Al a

GCC

Lou

TTG

Th r

ACT

Gin

CAA

Val

GTT

Met

ATG

Th v

ACT

AAG

ValI

GTA

Ala

GCC

Th r

ACC

se r

TCA

Giu

GAG

Gly

GGA

Val

GTA

Gly

GGC

Th r

ACG

A rg

AGO

Ala

GCA

Gly,

GC

Cy s

TGC

Th r Asn ACC AAT TrP LOU TOG TTG Ser LYS AGiT AAG Glu Arg GMA CGG Cys Leu TGT TTA Asn Ala AAT GCG Giu Val GAG GTG Thr Ala ACA GCC His Thr CAC ACT Gly Leu GGA CTC Lys Tyr AAA TAT Phe Leu TTC CTA Ala Leu OCT TTA Phe Leu TTC CTG Ile Leu ATA CTG Lys Pro AAA CCC Sor

TCT

G I Y

GGT

Th r

ACA

A rp

AGG

Pro

CCA

G Iu

GAG

Loeu

CTA

See-

ACT

As p

GIAC

Thr

ACA

Val

GTT

Val

GTG

Arg

AGA

Val

GTA

T rp

TG

Gly

GGC

~sr a a Leu, Giu Se CTA GAA AG Arg Gin Le AGG CAG CT Trp Phe GI TCG TTT CC Lou MlY TY CTT 0G TA Lys Asn Th AAA AAT AC.

Asp Gly Ly GAT GGT AA.

Leu Leu Se, CTA CTC TC, Val Val TY GIG GTA TA Ile His Asi ATA CAT GAC The- Ala Gli ACA GCT GAi Cys Ile Ar~ TGT ATA AGd ?he Glu GI~ TTT GAA CAC Asp Leu Th~ GAT CTA AC( Cys Leu Va: TOT CTG GT( Lou Ilie Lei TTG ATA C

STOP

TAG GAT CC aarhx 1.

T

y a r

C

r

A

S

r r r G1

U

5± Ala

GCC

Ly s

AAG

Al a

GCA

ATC

AAG

Ile

ATA

Val

C.TG

Leu

TTA

Cy s

TGT

Val

GTA

Pro

CCA

Vei

GTG

Arg

COC

Ly s

AAG

Ile

ATA

Argj

AGG

L-ys

AAA

Tyr

TAT

T rp

TG

Ile

ATC

Leu

CTG

Val

CGTA

1ie

AT?

Asp

GAT

Ile

ATA

Asp

CAT

Gly

GGT

Ile

AT

Val1

GIG

G 1,n

CAA

Leu

CTA

Ala

GCA

Tyr

TAT

ValI

OITT

His

CAT

Val

GTG

Leou

CTA

L~ys

AAA

Pro

CCT

Trp,

TGG

Gin

CAA

Trrp

TG

Val.

GTG

Gly

GG

Gly

COO

Ala

GCC

T hr

ACA

Gly

GGC

Giu

GAG

L e

CIT

HIS

CAT

As n

A.C

01 y

GGG

Ti P

TGG

Val

GTG

Tb r

ACC

A rg

AGA

Ih r

ACC

LYS

AAG

Ser TC T Ly s

AAG

Pro

CCC

Met

ATG

Ser

TCC

TC

Gin

CAA

S e r

TCA

Pro

CCT

Ile

ATC

Ala

GCT

Gly 221 672 237 720 253 768 269 816 285 864 30.1 912 3117 960 333 1008 349 1056 365 1104 38 1 1152 39? 1200 413 1240 429 1296 445 1344 454 1376

A

t i 4

A,

a S U Ut U a ft Ar Thr Gly Ala Gin ACA GGG GCA CAA i r

I.

31 SEQ ID NO 4 TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 2210 base pairs NUMBER OF STRANDS: single CONFIGURATION: linear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 10-2206

I

PROPERTIES: encodes the structural glycoprotein gEl and the f irst portion of the structural glycoprotein gE2 0 AA

ATA

Ti-p

T(,G

Gin

CAA

Cys 1T Ile AlT Arg

AGG

Asri AAiT Alit

GCT

A rg

CCA

Pro

CCA

Leu T 7 Phe

TTT

TIC

Alit

GCC

Asn

AAC

Arg

AGA

Thr

ACA

HIS

CAT

Lou

CTC

Ile

ATT

Glu

GAG

Asp

GAT

-Lou

TTG

Val1

GTA

A rg

AGA

ACC

Leu

TIC

Le u

TTA

G; I y

C

Gly

GT

Gly Gin

CAA

Glu G AA Gly

GT

Ser

AGT

Th r

ACA

Gin

CAA

Glu

GAG

Met~

ATG

Val1

GTT

G In

CAA

Val

GTA

Val

GTC

Met

ATG

Arg

CGC

Pro

CCT

Gin

CAG

Asp

GAC

G1?

GGC

Gly

GGG

Hii s

CAC

Cl y

C

Le u

TTG

Asor.

GAC

As n

AAT

Pro

CCC

Met

ATG

Hi~s

CAT

T rp

TGG

Pro

CCA

Loeu

CTA

Cys

TGC

Pro

CCT

Asp

CAT

Ly s

AAA

Phe

TTT

As n

AAT

A ro

AGA

Sal,

TCC

Asp

GAT

Glu

GAG

Ile AlT Pro

CCA

Asn

AAT

Ly s

AAG

CYS

TGC

Cy s

TOO

Leu

CTA

Gin

CAA

Gly 000G Sal, AC r His

CAC

Ala

GCT

I rp

TG

Val

ITT

A rt

AGG

Val1

GTA

I-y S

A

Asn

AAC

Th r ACAk Glu

GAG

Val

GTC

T hr

ACO

LeU

CTC

TTG

Se r

ACC

Asn

AAC

rLeu

CTC

Giu

GAG

Val1

GTA

C

Phe

TTT

Se r

ACT

Ly s

AAA

Ala

GCA

Glu

GAA

His

CAT

Alita

GCC

Giu

GAG

Ly s

AAG

Met

ATG

Cy s

TGT

Th r

ACA

Ly s

AAG

Giu

GAA

Val

GTG

Al a

OICC

V'9 I

GTG

01?

GGA

Gly

GGG

Thr

ACT

L.Ys

AAG

His

CAT

Asn

AAT

Ala

CCC

Gin

CAA

Asri

AAC

Ile

ATA

Ile

ATT

ILeu CI .3 Gly

GGA

Ile

ATA

Ile

ATC

Asp

CAT

Ttir

ACA

Oly

GGT

Ly s

AAA

Val

OTT

Al a

GCT

Phe

TC

Al a

GCT

Gin

CAA

'Lou

TIG

Giu

GAG

Gi1n

CAA

T rp

TOG

Thr

ACA

Asn

AAT

T rp

TOG

Thr

ACC

Th r

ACA

A rg

AGO

Se r

TCC

Vol

GTA

Gly

GC

x1.a

C'CA

Asn

AAC

A rg

COG

Pro

CCA

Ciu

GAA

Tyr

TAC

Cy s

IC

Gln

CAA

CYS

TC

As n

AAC

Ph e

ITTT

Se r

AG.T

Th r

ACG

T rp

TG

Ile

ATA

Al a

GCC

Ciu

GAG

Le u

CIG

Tb r ACAk Asn

AAT

Ala

GCC

A rg

CG

Se r

AGC

Ala

GCA

Asp

GAT

Al a

GCT

Al t 0 CA Thr

ACA

Met

ATG

LYS

AAA

Lys

AAG

Cys

TGC

TOG

Asn

AAC

Tyr

TAT

Pro

CCC

Gly Val

GIG

Hi s

CAC

Val 0 TA Gl n

CAA

Phe

TTC

Ile

AIC

AlIo G CA Cys

TGC

Tyr

TAC

LOU

CIT

As C)

GAC

Tb r

ACA

Val1 G TG Lou

CIG

Tyr

TAT

13 48 29 96 144 61 192 .7' 240 93 288 109 336 125 384 141 432 15.7 480 173 52_8 189 576 205 624 r 4/6 -32 Le u

CTG

C'CA

E y s

AAA

Pro ccc Asn

AAT

Gly

CGT

Gly

GGG

As 1)

GAT

r TCC Leu

CTA

Va I G7T Ty r

TAT

FE AGG FCCL Ala

OCT

CCC

AAT

Gly

;AA

Cys Vail

GTA

AAG

La u

CTG

Tyr

TAC

Cys

TGC

A ra

AG

Gly

GCC

Phe

TTC

l1e

ATC

As n

AAC

TOO

Glu

GAG

11e

ATA

Thr

ACU

Val

GTG

Leu

CTC

Glu

GAG

Ty r

TAC

AAA

Aspr

GAC

TLeu

'XTA

Glu

GAA

Cys

TGC

Thr

ACC

Phe

TTC

His

CAC

Ala

OCT

Pro

CCA

Lou

CTC

Asn

AAT

Thr

ACA

Val

OTC

Th r

ACT

Lou

TTG

P~ro

CCA

Ile

ATC

Se r

TCG

Cly

GT

Gly

GGG

Tb r

ACT

Asn

AAC

Glu

G.AG

Pro

CCT

Asp

GAC

Leu

TTG

Pro C CA Gln

CAA

Thr

ACC

Leu

TTG

Ala

GCC

Lou

TTG

Thr

ACT

GIn

CAA

Val

GTT

Gly

GGC

Aso

GAT

Gly

GGA

Met

ATG

Thr

ACT

Ly s

AAG

Val1

GTA

Ala

GCC

Tb r

ACC

Se 1, T CA Glu

GAG

Gl

GTA

Tb r

ACG

A rg

AGO

Ala C CA Gly

GGC

Cys

TGC

Pro

CT

Gly

GGG

Glu

GAG

Thri

ACC

Trp

TCG

Ser

ACT

Clu

GAA

Cys

TGT

As n

A.AT

Glii

GAG

T hr

ACA

His

CAC

Gly

GGA

Ly s

A

Phe

TTC

Al.a

OCT

Phe

TTC

Ile

ATA

Ly s

AAA

Lou

CTT

Met

ATC

110

ATC

As n

AAT

Lou

TTG

Lys

AAG

Ara

CG

Lou

TTA

Ala

GC

Val

GTG

Th r Lau

CTC

Tyr

TAT

Lou

CTA

Leu

TTA

Lau Leu

CTG

Pro

CCC

CG1y

CCC

Arg

CGG

Ly s

AAA

Sol,

TCT

Gly

GGT

Th r 'kCA Ara

AGG

Pro

CCA

G AG Lau 1,TA or

AGT

Aso

GCC

Th r

ACA

Val

GTT

Val1

G.TG

A rg

AGA

Val

GTA

T rp

TGC

Gly

GGC

Ala

CT

Lou

CTC

Ty r

TAT

Le u

CTA

A ra

AGO

Trp

TGG

CTT

AAA

Asp

GAT

Lau

CTA

Val1 GT C Ile

ATA

Thr

ACA

Cys

TGT

P rhe

TTT

Aso

GAT

Cy s

TOT

Leu

TTG

Phe

TTT

Th r

ING

Gin

CAG

Leu Glu

C.AA

G In

CAG

Phe

TTT

Gly

GOT

Asn

MAT

Gly

OGT

Leu

CTC

Val

GTA

His CA T Al a

GCT

Ile

ATA

Ci u

GA

Lou

CTA

La u

CTG

Ile ATh Ty r ThO GI Y

GCC

Aspi

GAO

Ile

ATT

Ser Ala Ara Gin AGT GCC AGG CAA Lau Iys Lyq Leu CTT AAG AAA CTA Cly Ala Tyr Ala GGG GCA TAT C.CA Tyr Ile Trp Tyr TAC ATC TGG TAT Thr Lys Ile V. I ACA AAG ATC CTT Lys Ile Lou His AAA ATA OTG CAT Ser Val Vail Val T GTG GIA GTG Tyr Lou Ile Leu TAT TTA AT? CTA Aso Cys Asp Lys GAC TOT GAT AA clii Val I Ie. P ro CIAA GTA ATA CCT A rg Pro ASP Trp AGA CCA GAT TGG Glu Val Gly Gin GAG GTG CCT CAA Thr Arg Ile Trp ACG CGC ATT TGG Val Lys Val Val GTG AAG GTG GIG Lau Ile Thr GIy CTC ATA ACA GGG Tyr Ala Ile Ala TAC GCC ATA GCC LOu Thr Thr GIn OTC ACC ACT CAG Thr Uly Val Val ACG GGA OTT GTA Thr Cys Glu'Arq ACA TOT GAG AGO Gly Thr GGG ACC Gly Lys GG G AAG Ala Ser GCC TCT Th j Lys ACA AAG Gly Pro GGC CCC clu Met GAG ATG Lou Ser CTT TCC His Phe CAT TTC Asn GI i ANC CAA GlIy Soer GOO TCA Trp Pro TOG CCT Val 110 GTG ATO Thr Al a ACC OCT Ara G Iy AGA GC Ala Gin GCA CAA Lys Asn AAA AAT Trp Tyr TOG TAT Val Tro GTG TGG Glu Ala GAA 0GCC 672 237 720 253 768 269 816 285 864 301 9 12 317 960 333 1008 349 1056 365 1104 381 1152 39, 1200 4 13 1248 429 1296 445 1344 461 1392 477 1440 493 1488 509 1536 r 33 A rg

ANGO

rr h64 T TT Asp

GAT

Val

GTA

Met

ATC

Ser

TCC

His

CAG

Gly

GGG

Pro cG T'rp

TGG

Ile A TT C'iu

GAG

Vail

GIG

Asp

GAT

Tyr Leu Ala TAT CTG GCC 0111 Lys Ile GAA AAA ATC Asp AsIn Phe GAT AAC TTT Arg Oly Lys AGO GGA AAA Val Cys Pro GTT TG CCT Asri Lys Asp AAT MOG GAT Ar.0 Pro Phe AGO CCT TIC Gly Asp Leu GGA GAC CIC Gly Asp Ile GGG GAG ATA Cys Gly TYr TGT G4OT TAG Gly Lys Cys -GCOC AAG 1OC Thr Ser Cys ACC TCT TG Lys Cys Lys AAA TGC AAG Lys Leu Gly AAG CTA GG Ile

ATT

Ile

ATA

G).u

GAX

Phe

TTT

11e

ATA

Th r

ACC

Pro

CCC

Ty r

TAC

L eu

CIA

Lys

AAG

Lys

AAG

Asn

A.C

Ile AlA Pro Leu

CTA

A sp

GAT

Le u

CIG

Asn

AAT

Gly

OGA

Leu

ITA

Phe

TTT

Asp

GAG

Ara

CGA

Phe

TT

Leu

CIG

Arg

AGA

Gly

GG

Met H*~is

CAC

01 y

CIGA

G1 Y

GOT

Th r

ACA

Trp

TG

Ala c0CC Aro

AGG

Cy s

TOT

Ty r

TAT

His

CAT

Lys

AAG

Asp

GAC

ASp3

GAC

Pro Thr

ACG

Lys

AAA

Le u

CTT

Th r

ACA

Th r

ACA

Met

ATG

Gln

CAA

Al a 0ICC Val1

GTA

Ly s

AAA

Asn

AAT

Oh'

GT

Th r

ACA

Cys Ara

AGA

Glu

GMA

Gy s

TG

Leu

CT

GGA

ACC

Gly G GC Leu

TTG

As P

GAT

Ser

AGT

GMu Val

GTG

Val

GIG

A rg Ale

GCC

Gin

CAA

Pro Ccc Leu

CTG

Th r

ACT

GTT

cy s

TGC

Cly

GGA

Gly Giu

GAG

Soc

AGT

Ala

OICT

Val

GIG

Pro Leu

CG

C; 1 u

GAG

GY S

TOT

Asn

AM'

Val

GTG

Val

GTA

Ile AlT Gly GGG0 Pro

CCT

Gly

GOT

Gly

G

Ile

ATA

G In

CAA

Tyr

TAT

Pro

CG

Asp

GAG

As p

GAT

Gly

CG

Ser

AGT

Arg

CGA

Thr

ACC

Asn

AAG

Val

GTG

Leu C TG Ty r

TAG

Val

OTA

Val

GIC

Gly

GOC

Thr

AG

Val

GTA

Alai

GCT

Pro C CA Leu

CTG

Th r

ACA

Gin

GAG

I rp

TG

Glu

GAG

Pro C CA Arao

AGA

Pro

CCA

Ile

ATA

MeL

ATG

Be r IdT V,1I

GTG

Ly s

AAA

Ala

GCC

Cy s

TOT

Tyr

TAC

Ly s

AAA

Th r

ACT

Ser

TCT

H is

GAG

Gin

GAA

Thr

ACT

Ala~

GCA

Prto

CCA

Val Va~l OTA GTA Glu MeL OAA ATG Pro Leu CCC TT Phe Gin TIC CAG Hi.s Trp CAG TG Lys Ara AAG AGG Vail Ile GTC ATC Gys Val TGT GTA Cys Lys TG MAG Phe Pro TIC CGA Val Asp GTA OAT Oly Ser GGT TCG Met Asp ATO GAT TAG GAT 525 1584 541 1632 1680 573 1728 589 1776 605 1824 621 18e72 637 1920 653 1968 669 2016 2064 701 2112 717 2160 2208 *4t4 C C C Cr t C t 4: 4

CCC

CC C

C

C

SC.'

t 4: C 4: 4 4144

CC

.4 C 4: C

C

CCI ATG CCT TGC AGA CGA

U-

K

pT 34 ANNEX: LIST OF SEQUENCES.

SEQ ID NO TYPE OF SEQUENCE: nucleotide sequence LENGTH OF SEQUENCE: 3 224base pairs NUMBER OF STRANDS: single CONFIGURATION: J.inear TYPE OF MOLECULE: cDNA for genomic RNA ORIGIN: BVD Osloss REGION TRANSLATED: 9 -3218 PROPERTIES: encodes the structural proteini C GA. ATT CCC FC-)-R Z Lyz; Gin.*LysL.

AAA CKA A:A.

14 48 96 46 144 62 1582 576 240 624 222 36 384 742 42 768 Asp C AC Lys

AAA

Ser

TCT

Val1

CTC

L, ys

AA-A

Ser

TCT

Gly

GGT

Lys

AAG

Lys Glu

GAA

Gly

GOO

Lys

AA-A

Lys

A.AG

His

C-AC

Lau

CTG

Pro

CCT

Leu

CTG

L eu

CTG

S et

ACT

Gly

GA

Met

ATG

Lys

A.A-A

S er

AGC

Leu

CTA

Gly

G

Ar q

AGO

Pro

CCG

Lys

AAA

Asn

A-AC

Leu

TTG

Leu

TTG

P=o

CCA

Pro

CCA

Cly

GA

Pr o

CCC

Cys

TOT

Lau

TITO

Ala

GCC

Xs5n

A.AT

Al a

GCG

met

ATO

Pro

CCA

Lys

AA-A

Lys

A-AA

Al a

GCX

Met Gin Leu A-TO GAG TTG Pro Ala Gly CCC CCT GCA Phe Giy Gin TTT GGC GAG His Lys Arq CAT AAA A-GA Lys Arg Gly A-AA A-GA OCT Ile Tyr Leu ATC TA-C CTG Val Tv= Ni-s GTC TAT CAT Gin Thr Thr GAG A-CA ACT Tyr His.Ile TAC CA-C ATT Thr Lys Tyr A-CA AA-A TAT Cys Pro Len TGC CCT CTA Thr Axg Lys A-CA A-GA A-AG Lys Ile Thr A.AG ATA ACT Asp Ala Thr OAT OCT ACG Gly Lys Ile COG A-AA ATC Asn Lys Pro A-AT A-AA CCT Trp Ala Val TOG GC GTA

ATT

Val1

CTG

Ar g

A-GA

Gly

CCC

Asp

GA-C

L ys

A-AA

Ar g

A-CA

Ar g

AGA

Tyr

TAT

His

CAT

Tr p

TOG

Lys

A-AC

Pro

CCT

Ile

A-TA

Lys

AAG

Gin Ile

A-TA

A-CA

Gin

GAC

Cly

OCA

Gin

GAC

Cys

TC

Pro

CCG

Al a

CCT

Ar g

AGG

Val1

GTO

Gin

CA-A

Val

GTT

Gin

CAA

Lys Val1

GTG

S et

ACT

Gin

GAG

Al a

CC

A-AT

Ciu

CA-A

Val.

GTG

Ar g

CC

Ar g

A-CC

Gly

CCC

Pro

CCA

Ile

ATT

Cys

TC

Lys

A-AC

Ser

TCA

Gin

CAA

Gin

GAG

Val

GTA

Lys

A-AG

S ex

TCA

Leu,

TTC

Ile Thr Asn Gin Leu Lau

GAA

Pro

CCA

Val C TT Ciu

CAA

S er

TCG

Pro

CCC

Leu

TTG

Oly Ile

ATA,

Val1

OTA

S er

A-C

Lys

A-AA

Ser

TCO

Asp

GAT

Asn

A-AT

Ax g, Cc Va I

OTT

CTT

Val1

GTA

His

CAT

Val.

GTA

Gly

OCT

Lau

TA

Clu

GAG

Ar g

A-GA

As p

GAT

Leu

CTC

Cys

TOC

Pro

CCX

Gin

GAG

Gly

OT

ACC

Lys

A-AC

'Len

TTO

'rTA Tyr

TAT

Pro

CCC

Pro CCPf As n

A-AC

?he If TC Phe

TTC

Val1

GTA

Cly

GGA

Lys

AAA

Ser

TCC

Asp

GAT

Lys

AAA

Val

GTC

Gln

CA-C

Lys

AAA

Phe

TI'?

Tyr

TAC

A-sn

AAC

Gin

CAC

Thr

ACT

Sex

A-C

Tyr

TAC

P he

TTT

Thz

ACT

Cys

TGC

Tr p

TG

Asp

GA-C

A= g

AGO

As p

GAT

Lys

AAA

As p

GAC

Leu

CTA

Gin

CAA

Lys Thr Tyr AAA ACA TA-C Gin Ala Cly CAAk OCA GOT Ala Thr Leu CC ACG CTA A-sn Leiu Ala A-AT CTG CC Lys Cly Pro A-AC GGA CCC Gin Asp Tyr CAC CAT TAC Gin 02-u Ala CAC GAA CC Gly Ser Asp GGT ACT CAT Ile Ile Val -A-TA A-TA OTT Val His A-sn GTC CA-C AC' Thr Lys Ala A-CA A-A-A GC Len Gin Lys CTC GA.A A-AG Set Lys Thr ACT A-AG A-CC Tyr Gin Val.

TAT CAC OTA Oly Len Tyr COT TTC TAC Gin Lys Aa GAO AAA CC Val Ala Val OTC CCA OTO 3 47 29 77 239 93 287 189 335 125 383 141 4-11 157 479 173 527 189 575 205 221 672 237 719 253 767 269 815 270 816 864 302 912 318 960 334 1008 350 1056 366 1104 382 1152 398 1200 414 1248 430 1296 *446 1344 5 462 1392 478 1440 526 542 1632 558 1680 4, 574 1728 590 1776 4 606 1824 622 1872 Gly Glu Asn GGA CG AAC Ile Gin Arg ATA CA.A CG Ile Trp Pro ATC TOO CCA Asp Thr Glu~ GAT ACA GAA Thr Asn Tyr ACA AAT TAG Gly Trp Cys GGT TGG TGC Lys Thr Gin AAA ACC CAA Val, Thr :.Cys OTT Ad~A TGC Ala Arg Asn OCT AGO AAC Phie Set Phe TTC TCC TTT Ala Val Set GCT GTA ACT Gin Gly Thr CAA GGC ACG Ser Ala Arg AGT GCC AGO Leo Lys Lys CTT AAG A.AA Gly Ala Tyr GOG GCA TAT Tyr Ile Tzp TAC ATC, TGO Thr Lys Ilie ACA AAG ATC Lys Ile Leu AAA ATA CTO Ser Val Val TCA OTC GTA Tyr Leu Ile TAT TTA ATT Asp Cys Asp GAC TGT GAT Glu Val Ile GAA OTA ATA Arg Pro Asp AGA CCA GAT lie AT A Al a

GCC

Glu

GAG

Leu

CTG

Thr

ACA

Asn

AAT

Ala

GCC

Ar g

CG

AGC

Ala~

GCA

As p

OAT

Al a

GCT

Gin

CAA

Leu

CTA

Ala

GCA

Tyr

TAT

GTT

His

CAT

Val

GTG

CTA

Lys

AAA

Pro

CCT

Trp

TOO

Thr

ACA

Met

ATG

Lys

AAA

Lys

A.AG

Cys

TGC

Trp

'GG

As n

AAC

Tyr

TAT

Pro ccc Gly (7GT Val

GTG

His

CAC

Gly 000 Gly

GO

Ala

GCC

Thr

ACA

Gly

GOC

Glu

GAG

Leu

CTT

His

CAT

Asn

AAC

Giy

CG

Trp

TG

Gin

CAA

P he

TTC

Ile

ATC

Ala

OCA

Cys

TOG

Tyr

TAC

Leu

CTT

As p

GAC

Thr

ACA

Val1

OTG

Leo

CTG

Tyr

TAT

Thr

ACC

Lys

AAG

S er

TCT

Lys

AAG

Pr o

CCC

Met

MTG

Set

TC

Phe

TTC

Gin

CAA

S ex

TCA

Pro

CT

Tzr p

TG

Gin

CAA

cys

TOT

Ile

ATT

Ar g

AGG

As n

AAT

Al a

GCT

Ar g

CGA

Pro

OCA

Lau

TTG

Phe

T?

Lau crc Al a

GCA

Lys

AA.A

Pr o

CCC

Asn

AAT

Gly

GT

Gly

G

As p

OAT

Sex

TC

Leu

CTA

Val

OTT

Tyr

TAT

As n

AAC

Ar g

AGA

Thr

ACA

His cATr Lau

CTC

Ile

AT?

Glu

GAG

Asp

OAT

Leo

TTG

Val1

GTA

Ar g

AGA

Val

OTA

Lys

A.AG

Lau

CTG

Tyr

TACI

Gys

TOC

AGO

Gly

GCC

Phe

TTC

Ile

ATC

AAC

Trp

TGG

Glu

GAG

Leu T TA Oly

GGC

Oly

GT

Gly

GGT

Gin

CAA

Glu

GAA

Gly

GT

S er

ACT

Thr

ACA

Gin CA A Glu

GAG

As p

GAC

Leu

TTA

Glu

GAA

Cys,

TGC

Thr

ACO

Phe

*TTLC

His

CAC

Ala

GCT

Pro

CCA

Lea-,

CTC

Asn

AAT

Thr

ACA

Gin

CAA

Val1

GTA

Va 1

GTC

Met

ATG

Ar g

CG

Pro

CCT

Gin

CAG

As p

GAG

Gly

GC

Oly 000 His

CAC

Gly 000 Thr

ACT

As n

AAC

Glu

GAG

Pro

CCT

As p

GAC

Leu

TTG

Pro

CCA

Gin

CAA

lTnr

ACC

Leu

TTG

Al1a

GCC

Asp Asn Gly Thr Glu 01 GAC AAT GGG ACG GAA GG~ Asn Arg qset Lau His G1l AAT AGA AOT CTfG CAT GOC Pro ger His L eu Ala Th3 CCC TCC CAC TTG 0CC AC~ Met Asp Ala Ser Glu L ATO OAT OCT AOC GAG AA( His Glu Trp Asn Lys Hi CAT GAO TOG AAC AAG CA Trp Ile Val Leu Met Asr TOO ATT GTT CTC ATG AMl Pro Pro Arg Glu Cys Alz CGA OGA AGO GAG TOT GCC Leu Asn Vai Val Thr GIX- CTA AMT GTA OTA ACA -CA.) C ys Lys Lys Gly Ly's Asn TGC AAG AAA G00 AAO AAC Pro Cys Asn Phe Glu Ile CCT TG AAC TTT GAA ATA Aso Cys Thr Ser Val Ile GAT TGC ACA ACT OTG AT'T Met Thr Asn Sex Lau G' A% G ACC AAT TOT CTA GAA Thr Tzp Leu Gly Arg Gin ACT TO TTG GOT AGO CG Ly's Ser Lys Thr Trp Phe A.AO AG T AAG ACA TOG TTT Val Gbu Arg Arg Leu Gly OTA OAA COG AGG CTT OCT Ala Ci's Leu Pro Lys Asn GCC TOT TTA CCA AAA AAT Thr Asn Ala Glu Asp Guy ACO AMT GCG GAG OAT OT Ser Glu Vai Lau Leu Leu TCA GAG OTG CTA OTA CTC Glu Thr Al1a Ser Val Val GAG ACA G CC AGT? OTC GTA Giy His- Thr Asp Ile His GGA GAG ACT GAG ATA CAiT Val Gly Lau Thx Thr Ala GTA OGA OTC ACA ACA OCT Gly Lys Tyr Val Cys Ile GGC AAA TAT OTT TOTr ATA Thr Phe Leu Val ?he Glu ACO TTC CTA OTO TTT GAA

C,

2 a63 381 911 317 959 333 1007 349 1055 365 1103 381 1151 397 1199 413 12 47 429 1255 4145 1343 461 1391 477 1439 493 1487 509 1535 525 1583 541 1631 557 1679 573 1727 589 1775 605 1823 621 1371 637 1919 638 Glu Val GlY 1920 GAG GTG GGT Gin Val lfe CAA 070 ATC 4 1968 670 2016 686 2064 702 2112 718 2160 734 2208 750 2256 -766 2304 782 2352 82 2408 2814 428 2830 294 2828 292 2978 Thr

ACC

Val1

GTG

Leu

CTC

Tryr

TAC

Leu

CTC

Thr

ACC

Thr

ACA

Leu

CTO

01 o

GAG

Cys

TOT

Asn

AAT

Val 070 Val

GTA

Ile

ATT

Gly

OGG

Pro

CCT

Gly

GOT

Gly ac Ile

ATA

Gin

CAA

Tyr

TAT

Phe

TTC

Ar g

CC

Lys

AAG

Ile

ATA

Al a

GCC

Thr

ACC

Gly

GA

Cys

TOT

Pro

CCC

As p

GAC

Asp

GAT

Gi y 000 ,S er

AGT

Ar g

CGA

Thr

ACC

Asn

AAC

Val1

GTC

Leu

CTG

Tyr

TAC

Val1

GTX

Val

OTC

01 o

GAA

Asn

AAC

Ile

AT

Val1 G70 Thr

ACA

I I P

ATA

Thr

ACT

Val1

OTT

-GIL~

GAG

Thr

ACG

Val1

OTA

Al a

OCT

Pro

CCA

Leu

CTG

Thr

ACA

Gin

CAG

Trp

TOG

Glu

GAG

Pro

CCA

Ar g

AGA

Pro

COCA.

Ile

ATA

Ile

ATC

Tyr

TAC

Trp

TGG

Val1

GTG

Gly Ala

GCC

Gin

CAC

Val

CPA

Arg

AGO

S et

TCT

Val1

GTO

Lys

AAA

Al a

CC

Cys

TOT

Tyr

TAC

Lys Thr

ACT

S er

TCT

His,

CAC

Gin

CAA

Thr

ACT

Al a

GCA

Ile

AT

Phr

ACA

Thr

ACC

Ar g Al a

GCA

Lys

AAA

Trp

TOO

Val1

GTG

Glb

GAA

Val

GTA

Glu

GAA

Pro

CCC

Phe

TTC

His

CAC

Lys

A.AG

Val1

OTC

Cys

TOT

Cys

TOG

P he T-1C Val

OTA

Gly

GGT

Met

ATC

Pro

CCC

Lys

AAA

Al a

OCT

Oly

GCC

Gin

CAA

As n

AAT

Tyr

TAT

Tr p

TOG

Ala 0CC Val1

GTA

Met

ATG

Leu

TTO

Gin

CAC

Tr p

TG

Ax g

AGO

Ile

ATC

Val

OTA

ELys

AAG

Pro

CCA

P

GAT

S er

TCG

Asp

CAT

Ser

ACT

Thr

ACA

Ar g

AGO

Al a

OCT

Gin

CAA

Gly 000 As n

AAT

GAA

Cys

TGT

Ar g

AGO

Phe

TTT

Asp

GAP

Val1

OTA

Met

ATO

Ser

TCC

Hi.5

CAC

Oly

G

Pro CCa Trp

TOO

Ile

AT

Glu

GAG

Val GT1' Asp

GAP

Glu

GAO

Leu Ie -VaJ. Leu ATA GTC TTG Thr Thr Thr ACG ACT ACT Val Lau Gin 070 TTG CAA Leu 2ro Val CTC CCA OTT Gu Ile Cly GAO ATC 0C Tyr Ser Asp TAC TCO CAT Lys Gly Gly AAA GOT GGA' Tyr Leo Ala TAT CTG 0CC Glu Lys Ile GAA A"A A'rC Asp Asn Phe GAT AAC TTT Arg Gly Lys AGO GGA AAA Val Cys Pro OTT TGC CCT Asn Lys Asp AA1,T AAO OAT Arg Pro Phe AGO CCT TTC Gly Asp Lau GGA GAC CTC Cly Asp Ile 000 CAC ATA Cys Gly Tyr TOT COT TAC Gly Lys Cys GOC AAG C Thr Ser Cys ACC TCT TOO Lys Cys Lys A.AA TOC AAG Lys Lau Gly AAG CPA GGC Gly Pro Vai 000 CCC OTA Lys Asn Lys AAO AAC AAG Ar g

AGO

Al a

GCA

Gly

GC

C';s

TOC

Pro

CT

Gly 000 Glu

GAO

Ile

AT

Ile

AT).

Gbu

GAA

Phe

TTT

Ile

AT).

Phr

ACG

Pro

CCC

Tyr

TAC,

Leu

CPA

Lys

AAG

L.ys

AAG

As n

AAC

Ile

ATA

Pro

OCT

Glu

GAA

Tyr

TAT

Ala

OCT

Phe

TTC

Ile

AT).

Lys

AAA)

Leu

CTT

Met ArC Ile

ATC

Leu

CT).

p sn

OAT

Leu

CTC

Asn

AAT

Giy

OGA

Leu

TTA

P he.

777 Asp

CAC

Ar g

CGA

Phe

TTT

Leu 070 Arg

'AGA

Cly

G

Met

ATO

Lys

AAO

Tyr

TAT

Leo

TTA

Leu

CTG

Leu

CTG

Pro

CCC

Gly 000 Ar g

COG

Lys His

CAC

Gby

'GA

Gly

COT

Thr

ACA

Trp

TOO

Al a 0CC Ar g

AIGO

Cys

TGT

Tyr

TAT

His

CAT

Lys

AAO

Asp

CAC

As p

GAO

Pro

OCT

Thr

ACO

GAG

Ar c, AGA 1 Val

GTA

Trp

TOO

Gly

C

Al a

OCT

Leu 070 Tyr

TAT

Thr

ACO

Lys

AAA

Leu

OTT

Pbr

ACA.

Thr

ACA

Met

ATG

Gin

CAA.

Ala

CC

Val1

OTA

Lys

AAA)

As n

AAT

Gly

GOT

Thr

ACA.

Cys

TGC

Al a

OCA

Pro

O'CT

AspD

OAT

Cys

TOT

Leo TT0 Phe

TTT

T hr

ACO

Gin

CAG

Leu

CT).

).rg

ACGA

G1 o

GA).

Cvs

C

Leu.

CT

Gly

GO).

Thr

ACC

Cly

GC

Leu TT0 Asp

GAT

S et

ACT

Glu

CAA

Val1

GPO

V.1l

GPO

Ar g

AGA.

cys

TOT

Ar 9

AGG

Le a

CT).

Leu

CTG

Ile

ATA

Tyr

TAC

Gly 0C Asp

GAC

Ile

ATT

Ala

CC

Gin

CA).

Pro

CCG

Leo

CTG

Thy

ACT

Val1

OTT

Cys

TC

Gly

GO).

Gly

OGO

Glu

GAG

8cr

ACT

Ala

OCT

Val

GTO

Pro

CCA

Thr

ACC

Asp

GAT

665 2015 685 2063 701 2111 717 2159 733 2207 749 2255 765 2303 781 2351 797 2299 813 2447 829 2495 845 2543 861 2591 877 2639 893 2687 909 2735 925 2782 941 2831 957 2879 973 2927 989 2975.

1005 3023 2-'

I.

.i 1006 3024 1022 3072 1054 3168 1070 3216 Asn Tyr Phe AAT TAT TTC Asp Leu Glu GAC CTA GAG Leu Vai Ile CTG GTG ATA Leu Val Thr CTG OTT ACA S-ro? TGA GA TCC 6 oj ilir Gin

CAA

Ile

ATC

Va1

GTO

Tyr

TAT

Gin

CAA

Thr

ACT

Va1

GTT

Met

ATO

Tyr

TAC

Asp

GAC

Ala

GCA

Ile

ATC

Leu

TTA

His

CAC

Leu

CTG

Ser

TCA

Lys

AAA

Arg

CGO

Gly

GGC

Glu

GAA

Giy

GG

Asp

CAT

Giy

OGT

Gin

CAA

Tyr

TAG

Phe

TTC

Tyr

TAC

Thr

ACC

Gin

CAA

Ala

OCT

Va1

GTG

Ser

TCG

Tyr

TAT

Olu

GAG

Leu

CTC

Gly

OGA

Trp Ser

PCC

Trp

TGG

Giy

GGG

TPT

Le

OTA

Leu

TTA

Pro

CCC

1021 3071 1037 3119 1053 3167 1069 3215 r 1::i

I

'I

.i

I:

1 1~

Claims (16)

1. Nucleotide sequence substantially corresponding to the BVD virus genome fraction encoding the glycoprotein gE2.

2. Nucleotide sequence substantially corresponding to the BVD virus genome fraction encoding the glycoprotein gEl or a portion thereof encoding translational products which are recognised by neutralizing antibodies to said gEl.

3. Nucleotide sequence containing the sequence according to claim 1 or the sequence according to claim 2 or the sequence according to claim 2. t C t

4. Nucleotide sequence comprising a characterised in that it contains: initiation codon for translation sequence encoding gE2 stop signal. Nucleotide sequence comprising a characterised in that it contains: initiation codon for translation stop signal.

6. Nucleotide sequence comprising a characterised in that it contains: initiation codon for translation BVD genome fraction, sequence encoding gEl BVD genome fraction, sequence encoding gE2 BVD genome fraction, sequence encoding C sequence encoding gEl sequence encoding gE2 stop signal.

7. Fragments of the nucleotide sequence according to one of claims 1 to 3 and whose translational products are recognised by at least one neutralising monoclonal antibody of the BVD virus.

8. Peptide corresponding to the sequence according to any one of claims 1 to 7. 950904,q:\oper\ejh,18147/92.rsp,38 L i. -39- i

9. Peptide according to claim 8, characterised in that it is glycosylated. Recombinant virus incorporating and expressing the sequence according to any one of claims 1 to 7.

11. Virus according to claim 10, characterised in that it is a Baculovirus.

12. Yeast expression vector incorporating and expressing the sequence according to any one of claims 1 to 7.

13. Eukaryotic host cell comprising the virus according to claim 10 or 11.

14. Eukaryotic host cell according to claim 13, characterised in that it is chosen from mammalian cells and insect cells. c t Eukaryotic host cell according to claim 14, characterised in that it is Spodoptera frugiperda.

16. Yeast comprising the veccor according to claim 12.

17. Yeast according to claim 16, characterised in that it is i Saccharomyces cerevisiae.

18. Vaccine containing, in an appropriate vehicle, or expressing, peptides corresponding to a sequence encoding gEl and gE2. S19. Vaccine containing, in an appropriate vehicle, or expressing, peptides corresponding to a sequence encoding gEl and the first half of gE2. r 20. Vaccine, containing, in an appropriate vehicle, or expressing, peptides corresponding to a sequence encoding C, y 0904,q: rjh, 814 2.p 950904,q:\oper\ejh,18147/92.rsp,39 j

40- i gEl and gE2. 21. Vaccine containing, in an appropriate vehicle, or expressing, at least one of the peptides according to one of claims 8 or 9. 22. A nucleotide sequence according to any one of claims 1 to 7, a peptide according to claims 8 or 9 or a vaccine according to any one of claims 18 to 21, substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 4th day of September, 1995 RHONE MERIEUX c t by its Patent Attorneys DAVIES COLLISON CAVE 94 1 T 0 RA 950904,q prej 18147 l 2. A4 4. ABSTRACT Immunogenic peptid~as or polypeptides of the BVD vi.rus and related viruses, vaccines incorporating them or expressing them, methods and means of production thereof. The nucleotide sequences substantially corres- ponding to all3 or to a portion of the BVD virus genonie fractions encoding the glycoproteins gE2 and gEl respec- tively# or to combinations or fragments thereof, are used for the production of paptides and recombinant viruses incorporating or axpressing thA~tn paptide. Figure 1. '.44 AAAt** 6* 4 6 4. *6 I, St 4 S. 4 4* 4 'I