IBV SPIKE PROTEIN (2) Background of the invention

1. Field of the invention This invention relates to the spike protein of infectious bronchitis virus (IBV) and to a reco binant DNA method of preparing it. IBV is a virus which causes respiratory disease in the fowl, and is of particular importance in relation to poultry.

2. Description of the prior art IBV is a virus of the type Coronaviridae. It has a single- stranded RNA genome, approximately 20 kb in length, of positive polarity, which specifies the production of three major structural proteins: nucleocapsid protein, membrane glycoprotein, and spike glycoprotein. The spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus. The spike protein is believed likely to be responsible for immunogenicity of the virus, partly by analogy with the spike proteins of other coronaviruses and partly by in vitro neutralisation experiments, see, for example, D. Cavanagh t al- , Avian Pathology IS, 573-583 (1984). Although the term "spike protein" is used to refer to the glycoproteinaceous material of the spike, it has been characterised by D. Cavanagh, Journal of General Virology , 1187-1191; 1787-1791; and 2577-2583 (1983) as comprising two or three copies each of two glycopolypeptides,

51 (90,000 daltons) and S2 (84,000 daltons). The polypeptide components of the glycopolypeptides SI and S2 have been estimated after enzymatic removal of oligosaccharides to have a combined molecular weight of approximately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the

52 polypeptide. Thus, the protein comprises an extra cellular domain, a transmembrane domain and a cytoplasmic anchor domain.

European Patent Application Publication No. 218625A NRDC (and equivalent US Patent 5,032,520 and corresponding application in Japan) discloses the cloning of cDNA sequences coding for the spike protein precursor as well as sequences coding specifically

for the SI and S2 polypeptides. Such a DNA molecule which codes for an IBV spike protein will hereinafter be referred to as "spike DNA" for brevity. The disclosed spike DNA codes for the whole spike protein, i.e. all 3 domains. Summary of the invention

It has now been found that it is unnecessary to clone the whole spike cDNA disclosed in European Patent 218625A in order to obtain an im unological response: a truncated "spike DNA" considerably shorter in length may be cloned and expressed to produce a polypeptide that will generate an immunological response.

Thus, the present invention relates to a DNA molecule which codes substantially for a truncated IBV spike protein polypeptide. The truncated IBV spike protein polypeptide produced as a result of the cloning and expression of a DNA molecule of the present invention is characterised in lacking the transmembrane domain and cytoplasmic anchor region of the native IBV spike protein.

A DNA molecule according to the invention is shown in the Sequence Listing ⁽ SEQ ID NO: 1). This DNA molecule was obtained as a result of research on the M41 strain of IBV, but it is expected that similarly truncated spike protein of cDNA of other IBV serotypes and strains such as Beaudette, M42, 6/82, Connecticut isolate A5968, Arkansas and Holland strains H120, H52, Ma5, D207, D212, D3128 and D3896, whether or not exhibiting a high degree of homology with M41 , will express IBV spike protein.

In referring to a DNA molecule defined as coding substantially for a truncated IBV spike protein it will be appreciated that it is intended not to exclude DNA flanking sequences, which may be, for example, cDNA to flanking sequences in the IBV RNA genome (other than transmembrane sequences) or may be foreign sequences derived from other genes, such as leader sequences that may assist in driving expression of the truncated polypeptide or may be a short sequence of plasmid DNA. Also, it is not intended that the DNA molecule should necessarily code for

amino acids extending right up to the 5 ¹ - terminus or 3'- truncated end. It may be possible to obtain expression of the truncated spike protein lacking say, up to 5 or even 10 of the amino acids (30 nucleotides) at either end. The invention also includes a vector containing the above defined DNA molecule, including a cloning vector such as a plasmid or phage or expression vector, preferably a pox virus vector, and a host containing the vector. Mammalian cells containing the above-defined DNA molecule, whether as naked DNA or contained in a vector, are also included. Further, the invention includes isolated biosynthetic truncated spike protein polypeptide and its expression from mammalian cells. Brief description of the drawings

Figures 1-17 show plasmid constructs of use in the preparation of DNA molecules of the present invention. Description of the preferred embodiments

SEQ ID NO: 1 shows the complete nucleotide sequence of a cDNA molecule of the invention obtained from IBV genomic RNA M41 strain. The IBV RNA of other strains is believed to be fairly similar to that of M41 , and therefore oligonucleotides derived from DNA of the present invention can be used as primers for sequencing RNA of other serotypes thus enabling truncated cDNA for all or virtually all other serotypes to be prepared using methods described hereinafter. Obviously, those serotypes in which the entire IBV spike protein cDNA has a high degree of nucleotide sequence homology with IBV M41 strain are slightly preferred, as giving a wider choice of potential oligonucleotides.

The vectors included in the invention are cloning and expression vectors. The DNA molecule of the present invention is conveniently multiplied by insertion in a prokaryotic vector, for example pBR322, and cloning in an appropriate host such as a bacterial host, especially E. coli . Alternatively, using appropriate different vectors it could be multiplied in (say) Bacillus species, or a yeast. For expression, mammalian cells can be transfected by the calcium phosphate precipitation method or transformed by a viral vector. Viral vectors include

retrov ruses and poxviruses such as fowlpox virus or vaccinia virus.

A DNA molecule of the present invention may be prepared by first obtaining full length IBV spike DNA in a suitable plasmid. European Patent 218625A NRDC predicts the probable transmembrane domain of the spike protein and indicates the region of DNA coding for it. A suitable endonuclease restriction site near the beginning of the DNA sequence coding for the transmembrane domain, can then be identified. Using the desired endonuclease, the IBV spike DNA may be cleaved and the truncated DNA molecule coding for the extracellular domain, introduced into a viral vector as described below. Care is needed to ensure either that the chosen restriction site is a unique one in the spike DNA, or that a cloning procedure such as described in the Example is devised to compensate. In the Example, a two-step cloning process was used to overcome a second ££yl site in the M41 spike DNA molecule. Alternatively, once it is known where the sequence coding for the transmembrane domain begins, the truncation can be brought about using Polymerase Chain Reaction (PCR) cloning or by using oligonucleotide site-directed utagenesis. In the latter method, a stop codon is inserted at the desired position.

The truncated IBV spike DNA can be introduced into the viral vector as follows. The DNA is inserted into a plasmid containing an appropriate non-essential region of poxvirus DNA, such as the thymidine kinase gene of vaccinia virus or into any suitable non-essential region of fowlpox virus, e.g. as described in European Patent 353851A, so that the insert interrupts the NER sequence. A poxvirus promoter, e.g. the vaccinia virus p7.5K promoter, which is usable in vaccinia virus or avipoxviruses, or a fowlpox virus promoter as described in our prior patent applications publication Nos. WO89/03879, W090/04638 and W091/02072, is also introduced into the NER sequence in such a position that it will operate on the inserted truncated spike DNA sequence. When an intergenic NER is used a "marker" gene with its own promoter e.g. the lac Z gene will be inserted along with the sequence coding for the truncated spike protein. When the

poxvirus and the plasmid recombinant DNA are co-transfected into a mammalian cell, homologous recombination takes place between the poxvirus NER, such as TK in vaccinia virus, or a said non-essential region of fowlpox virus and the same gene or region present in the plasmid. Since the truncated IBV spike DNA has thereby interrupted the poxvirus gene, viruses lacking the gene expression product, such as TK, are selected. If the NER used is an intergenic region, viruses expressing the truncated spike protein will be identified by the co-expression of the "marker" gene e.g. blue plaques colonies if lac Z is the marker gene. Once such a recombinant virus vector has been thus constructed it can be used to introduce the truncated IBV spike DNA directly into the desired host cells without the need for any separate step of transfecting plasmid recombinant DNA into the cells. In order to improve the expression of the truncated spike protein it may be preferable to replace part or all of the untranslated leader sequence upstream of the spike gene. The leader sequence is the region between the TAATTATT of the promoter sequence and the ATG initiation codon of the gene. By replacing part or all of the native FPV IBV leader sequence with leader sequences derived from other related viruses such as poxviruses it may be possible to initiate stronger translation in FPV.

Such leader sequences could be derived from: (i) part or all of the sequences found downstream of other poxviral promoters e.g. the vaccinia virus p7.5 promoter (ii) part or all of the leader sequences from foreign genes that have been shown to be well expressed in cells infected by the appropriate recombinant poxviruses or (iii) synthetic sequences shown to promote efficient translation in poxvirus-infected cells. The replacement of appropriate sequences can be accomplished using PCR cloning or by inserting synthetic oligonucleotides. The choice of leader sequence to be used and the method of insertion is well within the ability of skilled man. The Example 2 hereinafter illustrates how the procedure could be performed.

The invention therefore further relates to a vector wherein containing part or all of a sequence found downstream of a poxvirus promoter, not being the poxvirus promoter of use in the vector, between the promoter and the IBV DNA Molecule. With a view ultimately to obtaining expression of the recombinant virus ia vivo, the preferred poxvirus is fowlpox virus. It may be that the inserted truncated IBV DNA contains a sequence, which, in the fowlpox vector, leads to premature termination of transcription. In this case, the truncated spike DNA would have to be modified slightly by one or two nucleotides, thereby to allow transcription to proceed along the full length of the gene.

The vector can be introduced into any appropriate host by any method known in recombinant DNA technology. Hosts include E. coli . Bac llus spp, animal cells such as avian or mammalian cells and yeasts. The method of introduction can be transformed by a plasmid or cosmid vector, or infection by a phage or viral vector etc. as known in recombinant DNA technology.

The following Examples illustrate the invention. All temperatures are in °C.

EXAMPLE 1 I. Preparation of a full length IBV spike protein cDNA from M41 strain

Example 2 of European Patent Application Publication No. 218625 (NRDC) describes the preparation of cDNA coding for the spike protein precursor of IBV strain M41. It describes therein the preparation of plasmids pMB276 and pMB250 containing the entire M41 spike protein cDNA sequence.

An initial step in the preparation of a DNA molecule encoding a truncated IBV spike protein was to join pMB276 and pMB250 to produce a full length clone of the IBV M41 spike gene.

1. Joining PMB276 and PMB250 at a shared Ndel site to produce a full length clone of the IBV M41 spike gene (in PMB374) Plasmids pMB276 and pMB250 were digested with Ndel (20 units) in 50mM tris-HCl pH 8.0, lOmM MgCl ₂ , 50mM NaCl , final volume 20μl.

SUBSTITUTE H

The digested DNA was then phenol-extracted with an equal volume of TE-saturated phenol, ether extracted twice with an equal volume of water-saturated ether, then ethanol- precipitated. The precipitated DNA was resuspended in 15μl water. Then 2.5μl of each digest were ligated together in a total volume of 10μl in 50mM tris-HCl pH 7.5, OmM MgCl ₂ , 10mM DTT, ImM ATP, 1 unit T4 DNA ligase at 4 ^β C overnight. Ligated DNA, in lμl of the ligation mix, was transformed into competent E. coli DH5 and transformed bacteria were selected on agar plates containing tetracycline. Transformant colonies were grown in L broth plus tetracycline and DNA was isolated therefrom using a standard procedure described by Holmes and Quigley (1981), Analytical Biochemistry 114: 193-197. Following digestion of the isolated DNA with Ndel and agarose gel electrophoresis, it was apparent that, of 48 clones screened, one (no. 17) had inherited the desired fragments from the parental plasmids, viz. a fragment of circa 6kbp from pMB276, Fig. 1 and a fragment of about 4kbp from pMB 250, Fig. 2. The desired recombinant plasmid would also have a fragment, following £s_ l digestion, equivalent to the length of pBR322 (Pi±l sites flank the M41 spike cDNA). Analysis of clone 17 showed that it did not have a pBR322-sized Pstl fragment, indicating that the two Ndel fragments had ligated together in the wrong relative orientation. Clone 17 DNA was therefore digested with Ndel and religated (using procedures described above) to allow isolation of recombinants with the two Ndel fragments in the correct orientation. Analysis of Ps_il-digested DNA from a number of clones showed that about 50% had religated to give the correct orientation. One of these clones was saved, as pMB374, Fig, 3. 2. Cloning the IBV M41 spike gene under control of the vaccinia virus P7.5 promoter to make pGSS2

The IBV M41 spike protein gene was cut out of pMB374 by digestion of the plasmid with J_£hl11 1, see Fig. 3, in lO M tris-Hcl pH 7.4, lOmM MgCl ₂ , 50mM NaCl , lOmM (J-mercaptoethanol , at 65°C in a final volume of 20μl. The DNA was made blunt-ended

by the addition of 0.025mM dATP, dCTP, dGTP, dTTP and 5 units of Klenow polymerase, followed by incubation for In at room temperature. The digestion products were electrophoresed on an agarose gel using standard procedures as described by Maniatis ≤±jil. , (1982) in "Molecular cloning: a laboratory manual" (Cold Spring Harbor Laboratory) and a 5kb fragment, containing the spike gene was purified using "Geneclean II" (Bio 101) as per supplier's instructions. The purified DNA was then cloned into the Smal site of pGS20 (from Dr. G. L. Smith, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford 0X1 3RE, as described in Mackett, Smith & Moss (1984), J. Virol. 4£, 857-864) to make pGSS2, see Fig. 4. II. Truncation of M41 spike gene

It was desired to truncate the spike protein gene so that the protein would not carry a transmembrane segment and a cytoplasmic domain. This could be conveniently achieved by cutting the gene at the Stvl site (position 4384 in pGSS2). As there is another Stvl site within the gene, a two step process was devi ed. This involved the transfer of the spike protein gene sequences (without the p7.5 promoter) to pUC19 (Yanisch-Perron, Vieira & Messing, 1985, Gene 33., 103-119). Finally the truncated spike gene was transferred to the fowlpoxvirus expression plasmid, pEFL29. These steps are described below.

1. Transfer of the Styl fragment within the spike gene from PGSS2 to PUC19 to make pUC/M41Stv pGSS2 was digested with Stvl . the DNA was made blunt-ended with Klenow polymerase and the 1.95kb fragment (2430-4384) was recovered and purified. This fragment was ligated into pUC19 digested with Smal. Reco binants carrying the inserted fragment were isolated and the orientation of the inserted fragment was checked by digestion of their plasmid DNA with Mlul and Ba Hl . The required recombinant had a small Mlul/BamHl fragment of 480 bp (and not 1480bp) and was given the title pUC/M41Sty, Fig. 5 (note that ligation of the blunt-ended Stvl fragment into the Smal site restores the Stvl sites but not the Smal site).

2. Cloning the N-terminal part of the spike gene from pGSS2 into pUC/M41 Stv to give pUC/M41 Bam-Stv (containing spike sequences from the N-terminus to the C-terminal Styl site)

Plasmids pGSS2, Fig. 4, and pUC/M41Sty, Fig. 5, were both digested with BamHl and Afl2 and fragments of 2.2kb and 3.8kb, respectively, were recovered. The purified fragments were ligated together and reco binants were isolated. The required recombinant (titled pUC/M41 Barn-Sty) , Fig. 6, had the 0.85kb BamHl/Af12 fragment of pUC/M41Sty replaced by a 2.2kb fragment from pGSS2, Fig. 6.

3. Transfer of the truncated spike gene from pUC/M41 Bam-Stv into the fowlpoxyirus expression vector. pEFL29. to give PEFS17

The entire truncated spike gene sequences were cut out of pUC/M41 Barn-Sty using BamHl and EcoRl . Following repair of the ends of the DNA with Klenow polymerase, the 3.3kb fragment was isolated, purified and blunt-end ligated into pEFL29, Fig. 7, digested with Sjal • Recombinants were screened by digestion with BamHl/Bgl2 to check that the spike gene insert was in the correct orientation relative to the p7.5 promoter in pEFL29. Correct recombinants were titled pEFS17, Fig. 8. (The derivation of pEFL29 is described below).

4. Derivation of pEFL29

A DNA fragment containing the fowlpoxvirus 4b promoter driving a Jjtc_Z reporter gene was cut out of plasmid pNM4b30 (see the relevant fowlpox virus promoter patent specification (WO89/03879), page 35, Table 2) using Ej&Rl and Nry . The fragment was end-repaired and was then blunt-end ligated into the end-repaired BgJ2 site of a plasmid containing part of the terminal BamHl fragment of fowlpoxvirus (pB3ME, described in Boursnell et al., 1990, J. Gen. Virol. 21, 621-628) to create plasmid pEFLlO.

The vaccinia virus p7.5 promoter was then introduced, on a 300bp EcoRl (end-repaired) DNA fragment from pGS20 (see above), into the S££l site of pEFLlO. A recombinant with the

p7.5 promoter in such an orientation that transcription from it is initiated in the opposite direction to that from the fowlpoxvirus 4b promoter, identified by restriction analysis using BamHl. was titled pEFL29. 5. Isolation of recombinant fowlpoxyirus expressing the truncated IBV M41 spike gene

Chick embryo fibroblasts (CEFs), at 80% confluence, were infected with the Duphar "Poxine" strain of fowlpoxvirus at a multiplicity of infection (m.o.i.) of 1. At 4h post-infection, pEFSU DNA (lOμg per 25cm ² flask) was introduced to the cells using the 'Lipofectin' method (BRL) under manufacturer's instructions. Five days post-infection, when there was complete cytopathic effect, the cells were harvested. Virus, released from the cells by freeze/thawing three times, was used at various dilutions to infect CEFs which were then overlaid with agarose to allow plaques to form. When plaques were visible the plates were overlaid with X-gal agarose- Two days later, blue plaques were picked and virus was released by freeze/thawing. The virus was titrated again, overlaid with X-gal agarose and blue plaques were picked again. This procedure was repeated three more times. Finally two plaques (fpl74Pxim and fp!74Pxll21) were chosen for further characterisation.

Ill- Characterisation of the fowlpoxyirus/EFS17 recombinant viruses 1. (a) Plaque hybridisation analysis

These viruses were propagated and plaques on CEFs were once more obtained. The agarose overlay was removed and a nitrocellulose filter was applied to the cell sheet. A piece of 3MM filter paper, soaked in 20X SSC, was applied to the nitrocellulose filter. The nitrocellulose filter was then removed and baked at 80°C in a vacuum oven. The filters were then probed with a 3-p-radiolabelled probe specific for the IBV M41 spike protein gene, to verify that the recombinant fowlpox viruses carried the IBV M41 spike protein gene.

2. (b) Radio-immunoprecipitation assay (RIPA)

CEFs were infected with the fpEFS17 recombinant viruses (or with a control 'poxine'/lacZ recombinant virus or mock-infected) at a m.o.i. of 10. At 24h post-infection the tissue culture medium was replaced with methionine-free medium to 'starve' the cells (i.e. to deplete the cells of their intracellular methionine pool ⁾ for lh. The cells were then labelled with

³ ^S-methionine (lOOμCi) for 3h. Then they were harvested, washed and lysed in RIPA buffer (the RIPA procedures used are described in detail in "Antibodies: a laboratory manual", Harlow and Lane (1988), Cold Spring Harbor Laboratory, New York). A polyclonal serum raised in rabbits against purified IBV M41 spike protein was added to the clarified extracts and immune complexes were precipitated with protein-A/Sepharose. The protein-A/Sepharose was washed thrice with RIPA buffer, then resuspended in SDS-PAGE sample buffer and boiled for 3 min. The samples were then applied to a 5-10% gradient SDS-PAGE gel and electrophoresed. The gel was fixed and exposed by f1uorograph .

RIPA analysis showed that cells infected with the fpEFS17 recombinant viruses, but not those infected with control 'poxine'/lacZ recombinant nor uninfected cells, synthesised a new protein ⁽ apparent molecular weight about 160K). The band appeared 'fuzzy', characteristic of an extensively glycosylated protein such as the spike protein. When the infected cells were 'starved' and labelled in the presence of tunicamycin, an inhibitor of N-linked glycosylation, a faint band was seen at about 120K (the predicted size of the unmodified primary translation product) but most of the new product appeared as two closely migrating bands of 90-95K, suggesting that the unglycosylated protein was unstable and was being cleaved by protease activity.

EXAMPLE 2

The Example below describes the replacement of the untranslated IBV spike sequences with sequences derived from part of the leader downstream of the p7.5 promoter, by cloning synthetic oligonucleotides between the BamHl site in the leader and a Spel site near the 5' end of the IBV spike coding sequence. The complete leader is then cloned upstream of the truncated IBV spike gene from pEFS 17 to give pEFS 20.

In summary the 83 base pair BamHI-Spel fragment (SEQ ID NO 3) in pEFS17 is replaced with a synthetic leader based on p7.5 (SEQ ID NO 4) using the oligonucleotides MAS-H7 and MAS-H8 (SEQ ID 5 and 6 respectively).

1) Replacing non-transl ted leader from IBV in pGSS2 with leader seguences from the vaccinia virus P7.5 promoter Plasmid pGSS2 (Fig. 4) was digested with BamHl (1059) and Spel (3358), and fragments of lOkb and 2.2kb (Fig. 9 were recovered. To anneal synthetic oligonucleotides MAS-H7 and MAS-H8, 50 p ol of each were mixed in 10μl water. They were then boiled for 3 minutes and allowed to cool slowly to room temperature. The annealed oligonucleotide duplex (0.2 to 5 pmol) was then ligated to the 10 kb BamHI-Spel fragment from pGSS2. The required recombinant, pGSS3 (Fig. 10), had retained the BamHl and Spel sites but had deleted a 2.2 kb Spel fragment relative to pGSS2. 2) Replacing the deleted 2.2 kb Spel fragment from DGSS2 into PGSS3 to make PGSS4

The 2.2 kb Spel fragment from pGSS2 (Fig 11) was ligated into pGSS3 linearised with Spel. The presence and orientation of the inserted Spel fragment in the resultant recombinant, pGSS4 (Fig. 12), was verified by digestion with Spel, Aflll, Mlul or BamHl/Sal1.

3) Cloning the IBV spike gene with the new leader from pGSS4 into the expression vector. PEFL29

Plasmid pGSS4 was digested with Ba Hl and EcoRl, repaired with Klenow polymerase then a 4.9 kb fragment (Fig. 13) was recovered and ligated into pEFL29 (Fig. 7) digested with Smal. The presence and orientation of the spike gene insert in the desired recombinant, pEFS19 (Fig. 14), was checked by digestion with BamHl, EcoRl, Styl or BamHI/Styl .

4) Combining the new leader and 5'-terminus of the IBV spike gene (from pEFSI9) with the C-terminus of the truncated spike gene (from pEFS17)

Plasmids pEFS17 and pEFSI9 were digested with Ncol and Bglll then 3 kb (Fig. 15) and 11.8 kb (Fig. 16) fragments, respectively, were recovered and ligated together. The required recombinant pEFS20 (Fig. 17) was checked by digestion with Kpnl,

Ba Hl, Styl and BamHI/Styl.

Recombinant fowlpox viruses were derived, using pEFS20, and analysed as described above in Example l.III for pEFS17.

- n- -

SEQUENCE LISTING

Cl) GENERAL INFORMATION:

(i) APPLICANT: British, Technology Group Ltd (ii) TITLE OF INVENTION: IBV Spike Protein (2) (iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: British Technology Group Ltd

(B) STREET: 101 Ne ington Causeway

(C) CITY: London

(E) COUNTRY: U.K.

(F) ZIP: SE1 6BU

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

( i) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9203509.6

(B) FILING DATE: 19-FEB-1992

( iϋ) ATTORNEY/AGENT INFORMATION: (A) NAME: Percy, R K

(C) REFERENCE/DOCKET NUMBER: 135324

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 017 403 6666

(B) TELEFAX: 071 403 7568

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3281 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Infectious bronchitis virus

(B) STRAIN: M41

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..3281

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATG TTG GTA ACA CCT CTT TTA CTA GTG ACT CTT TTG TGT GTA CTA TGT

48 Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys

1 5 10 15

AGT GCT GCT TTG TAT GAC AGT AGT TCT TAC GTT TAC TAC TAC CAA AGT

96 Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gin Ser

20 25 30

GCC TTT AGA CCA CCT AAT GGT TGG CAT TTA CAC GGG GGT GCT TAT GCG

144 Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala

35 40 45

GTA GTT AAT ATT TCT AGC GAA TCT AAT AAT GCA GGC TCT TCA CCT GGG

192 Val Val Asn lie Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly

50 55 60

TGT ATT GTT GGT ACT ATT CAT GGT GGT CGT GTT GTT AAT GCT TCT TCT

240 Cys He Val Gly Thr He His Gly Gly Arg Val Val Asn Ala Ser Ser

65 70 75 80

ATA GCT ATG ACG GCA CCG TCA TCA GGT ATG GCT TGG TCT AGC AGT CAG

288 He Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gin

85 90 95

TTT TGT ACT GCA CAC TGT AAC TTT TCA GAT ACT ACA GTG TTT GTT ACA

336 Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr

100 105 110

CAT TGT TAT AAA TAT GAT GGG TGT CCT ATA ACT GGC ATG CGT CAA AAG 384

His Cys Tyr Lys Tyr Asp Gly Cys Pro He Thr Gly Met Arg Gin Lys 115 120 125

AAT TTT TTA CGT GTT TCT GCT ATG AAA AAT GGC CAG CTT TTC TAT AAT

432 Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gin Leu Phe Tyr Asn

130 135 140

TTA ACA GTT AGT GTA GCT AAG TAC CCT ACT TTT AAA TCA TTT CAG TGT

480 Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gin Cys

145 150 155 160

GTT AAT AAT TTA ACA TCC GTA TAT TTA AAT GGT GAT CTT GTT TAC ACC

528 Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr

165 170 175

TCT AAT GAG ACC ACA GAT GTT ACA TCT GCA GGT GTT TAT TTT AAA GCT

576 Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala

180 185 190

GGT GGA CCT ATA ACT TAT AAA GTT ATG AGA GAA GTT AAA GCC CTG GCT

624 Gly Gly Pro He Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala

195 200 205

TAT TTT GTT AAT GGT ACT GCA CAA GAT GTT ATT TTG TGT GAT GGA TCA

672 Tyr Phe Val Asn Gly Thr Ala Gin Asp Val He Leu Cys Asp Gly Ser

210 215 220

CCT AGA GGC TTG TTA GCA TGC CAG TAT AAT ACT GGC AAT TTT TCA GAT

720 Pro Arg Gly Leu Leu Ala Cys Gin Tyr Asn Thr Gly Asn Phe Ser Asp

225 230 235 240

GGC TTT TAT CCT TTT ATT AAT AGT AGT TTA GTT AAG CAG AAG TTT ATT

768 Gly Phe Tyr Pro Phe He Asn Ser Ser Leu Val Lys Gin Lys Phe He

245 250 255

GTC TAT CGT GAA AAT AGT GTT AAT ACT ACT TTT ACG TTA CAC AAT TTC

816 Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe

260 265 270

ACT TTT CAT AAT GAG ACT GGC GCC AAC CCT AAT CCT AGT GGT GTT CAG

864 Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gin

275 280 285

AAT ATT CAA ACT TAC CAA ACA CAA ACA GCT CAG AGT GGT TAT TAT AAT

912 Asn lie Gin Thr Tyr Gin Thr Gin Thr Ala Gin Ser Gly Tyr Tyr Asn

290 295 300

TTT AAT TTT TCC TTT CTG AGT AGT TTT GTT TAT AAG GAG TCT AAT TTT

960 Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe

305 310 315 320

ATG TAT GGA TCT TAT CAC CCA AGT TGT AAT TTT AGA CTA GAA ACT ATT

1008 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr He

325 330 335

AAT AAT GGC TTG TGG TTT AAT TCA CTT TCA GTT TCA ATT GCT TAC GGT

1056

Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser He Ala Tyr Gly

340 345 350

CCT CTT CAA GGT GGT TGC AAG CAA TCT GTC TTT AGT GGT AGA GCA ACT

1104

Pro Leu Gin Gly Gly Cys Lys Gin Ser Val Phe Ser Gly Arg Ala Thr

355 360 365

TGT TGT TAT GCT TAT TCA TAT GGA GGT CCT TCG CTG TGT AAA GGT GTT

1152 Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val

370 375 380

O 93/17109 _ „. _,

TAT TCA GGT GAG TTA GAT CTT AAT TTT GAA TGT GGA CTG TTA GTT TAT 1200

Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400

GTT ACT AAG AGC GGT GGC TCT CGT ATA CAA ACA GCC ACT GAA CCG CCA

1248 Val Thr Lys Ser Gly Gly Ser Arg He Gin Thr Ala Thr Glu Pro Pro

405 410 415

GTT ATA ACT CGA CAC AAT TAT AAT AAT ATT ACT TTA AAT ACT TGT GTT

1296 Val He Thr Arg His Asn Tyr Asn Asn He Thr Leu Asn Thr Cys Val

420 425 430

GAT TAT AAT ATA TAT GGC AGA ACT GGC CAA GGT TTT ATT ACT AAT GTA

1344 Asp Tyr Asn He Tyr Gly Arg Thr Gly Gin Gly Phe He Thr Asn Val

435 440 445

ACC GAC TCA GCT GTT AGT TAT AAT TAT CTA GCA GAC GCA GGT TTG GCT

1392 Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala

450 455 460

ATT TTA GAT ACA TCT GGT TCC ATA GAC ATC TTT GTT GTA CAA GGT GAA

1440 He Leu Asp Thr Ser Gly Ser He Asp He Phe Val Val Gin Gly Glu

465 470 475 480

TAT GGT CTT ACT TAT TAT AAG GTT AAC CCT TGC GAA GAT GTC AAC CAG

1488 Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gin

485 490 495

CAG TTT GTA GTT TCT GGT GGT AAA TTA GTA GGT ATT CTT ACT TCA CGT

1536

Gin Phe Val Val Ser Gly Gly Lys Leu Val Gly He Leu Thr Ser Arg

500 505 510

AAT GAG ACT GGT TCT CAG CTT CTT GAG AAC CAG TTT TAC ATT AAA ATC 1584

- \-λ-

Asn Glu Thr Gly Ser Gin Leu Leu Glu Asn Gin Phe Tyr He Lys He 515 520 525

ACT AAT GGA ACA CGT CGT TTT AGA CGT TCT ATT ACT GAA AAT GTT GCA

1632 Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser He Thr Glu Asn Val Ala

530 535 540

AAT TGC CCT TAT GTT AGT TAT GGT AAG TTT TGT ATA AAA CCT GAT GGT

1680 Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys He Lys Pro Asp Gly

545 550 555 560

TCA ATT GCC ACA ATA GTA CCA AAA CAA TTG GAA CAG TTT GTG GCA CCT

1728 Ser He Ala Thr He Val Pro Lys Gin Leu Glu Gin Phe Val Ala Pro

565 570 575

TTA CTT AAT GTT ACT GAA AAT GTG CTC ATA CCT AAC AGT TTT AAT TTA

1776 Leu Leu Asn Val Thr Glu Asn Val Leu He Pro Asn Ser Phe Asn Leu

580 585 590

ACT GTT ACA GAT GAG TAC ATA CAA ACG CGT ATG GAT AAG GTC CAA ATT

1824 Thr Val Thr Asp Glu Tyr He Gin Thr Arg Met Asp Lys Val Gin He

595 600 605

AAT TGT CTG CAG TAT GTT TGT GGC AAT TCT CTG GAT TGT AGA GAT TTG

1872 Asn Cys Leu Gin Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu

610 615 620

TTT CAA CAA TAT GGG CCT GTT TGT GAC AAC ATA TTG TCT GTA GTA AAT

1920 Phe Gin Gin Tyr Gly Pro Val Cys Asp Asn He Leu Ser Val Val Asn

625 630 635 640

AGT ATT GGT CAA AAA GAA GAT ATG GAA CTT TTG AAT TTC TAT TCT TCT

1968 Ser He Gly Gin Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser

645 650 655

ACT AAA CCG GCT GGT TTT AAT ACA CCA TTT CTT AGT AAT GTT AGC ACT

2016 Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr

660 665 670

GGT GAG TTT AAT ATT TCT CTT CTG TTA ACA ACT CCT AGT AGT CCT AGA

2064 Gly Glu Phe Asn He Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg

675 680 685

AGG CGT TCT TTT ATT GAA GAC CTT CTA TTT ACA AGC GTT GAA TCT GTT

2112 Arg Arg Ser Phe He Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val

690 695 700

GGA TTA CCA ACA GAT GAC GCA TAC AAA AAT TGC ACT GCA GGA CCT TTA

2160 Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu

705 710 715 720

GGT TTT CTT AAG GAC CTT GCG TGT GCT CGT GAA TAT AAT GGT TTG CTT

2208 Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu

725 730 735

GTG TTG CCT CCC ATT ATA ACA GCA GAA ATG CAA ACT TTG TAT ACT AGT

2256 Val Leu Pro Pro He He Thr Ala Glu Met Gin Thr Leu Tyr Thr Ser

740 745 750

TCT CTA GTA GCT TCT ATG GCT TTT GGT GGT ATT ACT GCA GCT GGT GCT

2304 Ser Leu Val Ala Ser Met Ala Phe Gly Gly He Thr Ala Ala Gly Ala

755 760 765

ATA CCT TTT GCC ACA CAA CTG CAG GCT AGA ATT AAT CAC TTG GGT ATT

2352 He Pro Phe Ala Thr Gin Leu Gin Ala Arg He Asn His Leu Gly He

770 775 780

ACC CAG TCA CTT TTG TTG AAG AAT CAA GAA AAA ATT GCT GCT TCC TTT

2400 Thr Gin Ser Leu Leu Leu Lys Asn Gin Glu Lys He Ala Ala Ser Phe

785 790 795 800

AAT AAG GCC ATT GGT CGT ATG CAG GAA GGT TTT AGA AGT ACA TCT CTA

2448 Asn Lys Ala He Gly Arg Met Gin Glu Gly Phe Arg Ser Thr Ser Leu

805 810 815

GCA TTA CAA CAA ATT CAA GAT GTT GTT AAT AAG CAG AGT GCT ATT CTT

2496 Ala Leu Gin Gin He Gin Asp Val Val Asn Lys Gin Ser Ala He Leu

820 825 830

ACT GAG ACT ATG GCA TCA CTT AAT AAA AAT TTT GGT GCT ATT TCT TCT

2544 Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala He Ser Ser

835 840 845

GTG ATT CAA GAA ATC TAC CAG CAA CTT GAC GCC ATA CAA GCA AAT GCT

2592 Val He Gin Glu He Tyr Gin Gin Leu Asp Ala He Gin Ala Asn Ala

850 855 860

CAA GTG GAT CGT CTT ATA ACT GGT AGA TTG TCA TCA CTT TCT GTT TTA

2640 Gin Val Asp Arg Leu He Thr Gly Arg Leu Ser Ser Leu Ser Val Leu

865 870 875 880

GCA TCT GCT AAG CAG GCG GAG CAT ATT AGA GTG TCA CAA CAG CGT GAG

2688

Ala Ser Ala Lys Gin Ala Glu His He Arg Val Ser Gin Gin Arg Glu

885 890 895

TTA GCT ACT CAG AAA ATT AAT GAG TGT GTT AAG TCA CAG TCT ATT AGG

2736 Leu Ala Thr Gin Lys He Asn Glu Cys Val Lys Ser Gin Ser He Arg

900 905 910

TAC TCC TTT TGT GGT AAT GGA CGA CAT GTT CTA ACC ATA CCG CAA AAT 2784

Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr He Pro Gin Asn 915 920 925

GCA CCT AAT GGT ATA GTG TTT ATA CAC TTT TCT TAT ACT CCA GAT AGT

2832 Ala Pro Asn Gly He Val Phe He His Phe Ser Tyr Thr Pro Asp Ser

930 935 940

TTT GTT AAT GTT ACT GCA ATA GTG GGT TTT TGT GTA AAG CCA GCT AAT

2880 Phe Val Asn Val Thr Ala He Val Gly Phe Cys Val Lys Pro Ala Asn

945 950 955 960

GCT AGT CAG TAT GCA ATA GTA CCC GCT AAT GGT AGG GGT ATT TTT ATA

2928 Ala Ser Gin Tyr Ala He Val Pro Ala Asn Gly Arg Gly He Phe He

965 970 975

CAA GTT AAT GGT AGT TAC TAC ATC ACA GCA CGA GAT ATG TAT ATG CCA

2976

Gin Val Asn Gly Ser Tyr Tyr He Thr Ala Arg Asp Met Tyr Met Pro

980 985 990

AGA GCT ATT ACT GCA GGA GAT ATA GTT ACG CTT ACT TCT TGT CAA GCA

3024 Arg Ala He Thr Ala Gly Asp He Val Thr Leu Thr Ser Cys Gin Ala

995 1000 1005

AAT TAT GTA AGT GTA AAT AAG ACC GTC ATT ACT ACA TTC GTA GAC AAT

3072 Asn Tyr Val Ser Val Asn Lys Thr Val He Thr Thr Phe Val Asp Asn

1010 1015 1020

GAT GAT TTT GAT TTT AAT GAC GAA TTG TCA AAA TGG TGG AAT GAC ACT

3120 Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr

1025 1030 1035 1040

AAG CAT GAG CTA CCA GAC TTT GAC AAA TTC AAT TAC ACA GTA CCT ATA

3168 Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro He

1045 1050 1055

~ 2

CTT GAC ATT GAT AGT GAA ATT GAT CGT ATT CAA GGC GTT ATA CAG GGT

3216 Leu Asp He Asp Ser Glu He Asp Arg He Gin Gly Val He Gin Gly

1060 1065 1070

CTT AAT GAC TCT TTA ATA GAC CTT GAA AAA CTT TCA ATA CTC AAA ACT

3264

Leu Asn Asp Ser Leu He Asp Leu Glu Lys Leu Ser He Leu Lys Thr

1075 1080 1085

TAT ATT AAG TGG CCA AG

3281 Tyr He Lys Trp Pro 1090

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1093 amino acids

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

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Leu Val Thr Pro Leu Leu Leu Val Thr Leu Leu Cys Val Leu Cys 1 5 10 15

Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr Gin Ser 20 25 30

Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala Tyr Ala 35 40 45

Val Val Asn He Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser Pro Gly 50 55 60

Cys He Val Gly Thr He His Gly Gly Arg Val Val Asn Ala Ser Ser 65 70 75 80

He Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser Ser Gin

85 90 95

Phe Cys Thr Ala His Cys Asn Phe Ser Asp Thr Thr Val Phe Val Thr 100 105 110

- %-\-

His Cys Tyr Lys Tyr Asp Gly Cys Pro He Thr Gly Met Arg Gin Lys 115 120 125

Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gin Leu Phe Tyr Asn 130 135 140

Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Phe Lys Ser Phe Gin Cys 145 150 155 160

Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Tyr Thr

165 170 175

Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190

Gly Gly Pro He Thr Tyr Lys Val Met Arg Glu Val Lys Ala Leu Ala 195 200 205

Tyr Phe Val Asn Gly Thr Ala Gin Asp Val He Leu Cys Asp Gly Ser 210 215 220

Pro Arg Gly Leu Leu Ala Cys Gin Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240

Gly Phe Tyr Pro Phe He Asn Ser Ser Leu Val Lys Gin Lys Phe He

245 250 ^• 255

Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His Asn Phe 260 265 270

Thr Phe His Asn Glu Thr Gly Ala Asn Pro Asn Pro Ser Gly Val Gin 275 280 285

Asn He Gin Thr Tyr Gin Thr Gin Thr Ala Gin Ser Gly Tyr Tyr Asn 290 295 300

Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320

Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr He

325 330 335

Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser He Ala Tyr Gly 340 345 350

Pro Leu Gin Gly Gly Cys Lys Gin Ser Val Phe Ser Gly Arg Ala Thr 355 360 365

Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys Gly Val 370 375 380

Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400

Val Thr Lys Ser Gly Gly Ser Arg He Gin Thr Ala Thr Glu Pro Pro

405 410 415

Val He Thr Arg His Asn Tyr Asn Asn He Thr Leu Asn Thr Cys Val 420 425 430

Asp Tyr Asn He Tyr Gly Arg Thr Gly Gin Gly Phe He Thr Asn Val 435 440 445

Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460

He Leu Asp Thr Ser Gly Ser He Asp He Phe Val Val Gin Gly Glu 465 470 475 480

Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val Asn Gin

485 490 495

Gin Phe Val Val Ser Gly Gly Lys Leu Val Gly He Leu Thr Ser Arg 500 505 510

Asn Glu Thr Gly Ser Gin Leu Leu Glu Asn Gin Phe Tyr He Lys He 515 520 525

Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser He Thr Glu Asn Val Ala 530 535 540

Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cys He Lys Pro Asp Gly 545 550 555 560

Ser He Ala Thr He Val Pro Lys Gin Leu Glu Gin Phe Val Ala Pro

565 570 575

Leu Leu Asn Val Thr Glu Asn Val Leu He Pro Asn Ser Phe Asn Leu 580 585 590

Thr Val Thr Asp Glu Tyr He Gin Thr Arg Met Asp Lys Val Gin He

-2 -

595 600 605

Asn Cys Leu Gin Tyr Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu 610 615 620

Phe Gin Gin Tyr Gly Pro Val Cys Asp Asn He Leu Ser Val Val Asn 625 630 635 640

Ser He Gly Gin Lys Glu Asp Met Glu Leu Leu Asn Phe Tyr Ser Ser

645 650 655

Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr 660 665 670

Gly Glu Phe Asn He Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg 675 680 685

Arg Arg Ser Phe He Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val 690 695 700

Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu 705 710 715 720

Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu

725 730 735

Val Leu Pro Pro He He Thr Ala Glu Met Gin Thr Leu Tyr Thr Ser 740 745 750

Ser Leu Val Ala Ser Met Ala Phe Gly Gly He Thr Ala Ala Gly Ala 755 760 765

He Pro Phe Ala Thr Gin Leu Gin Ala Arg He Asn His Leu Gly He 770 775 780

Thr Gin Ser Leu Leu Leu Lys Asn Gin Glu Lys He Ala Ala Ser Phe 785 790 795 800

Asn Lys Ala He Gly Arg Met Gin Glu Gly Phe Arg Ser Thr Ser Leu

805 810 815

Ala Leu Gin Gin He Gin Asp Val Val Asn Lys Gin Ser Ala He Leu 820 825 830

Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Phe Gly Ala He Ser Ser 835 840 845

- 21-

Val He Gin Glu He Tyr Gin Gin Leu Asp Ala He Gin Ala Asn Ala 850 855 860

Gin Val Asp Arg Leu He Thr Gly Arg Leu Ser Ser Leu Ser Val Leu 865 870 875 880

Ala Ser Ala Lys Gin Ala Glu His He Arg Val Ser Gin Gin Arg Glu

885 890 895

Leu Ala Thr Gin Lys He Asn Glu Cys Val Lys Ser Gin Ser He Arg 900 905 910

Tyr Ser Phe Cys Gly Asn Gly Arg His Val Leu Thr He Pro Gin Asn 915 920 925

Ala Pro Asn Gly He Val Phe He His Phe Ser Tyr Thr Pro Asp Ser 930 935 940

Phe Val Asn Val Thr Ala He Val Gly Phe Cys Val Lys Pro Ala Asn 945 950 955 960

Ala Ser Gin Tyr Ala He Val Pro Ala Asn Gly Arg Gly He Phe He

965 970 975

Gin Val Asn Gly Ser Tyr Tyr He Thr Ala Arg Asp Met Tyr Met Pro 980 985 990

Arg Ala He Thr Ala Gly Asp He Val Thr Leu Thr Ser Cys Gin Ala 995 1000 1005

Asn Tyr Val Ser Val Asn Lys Thr Val He Thr Thr Phe Val Asp Asn 1010 1015 1020

Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr 1025 1030 1035 1040

Lys His Glu Leu Pro Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro He

1045 1050 1055

Leu Asp He Asp Ser Glu He Asp Arg He Gin Gly Val He Gin Gly 1060 1065 1070

Leu Asn Asp Ser Leu He Asp Leu Glu Lys Leu Ser He Leu Lys Thr 1075 1080 1085

-2Z-

Tyr He Lys Trp Pro 1090

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 83 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Infectious bronchitis virus

(B) STRAIN: M41

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 7..57

(D) OTHER INFORMATION: /function= "IBV LEADER SEQUENCE"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 58..83

(D) OTHER INFORMATION: /function= "IBV SPIKE CODING SEQUENCE"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGATCCCCGATCCCCTAGTCTTTAATTTAATTAAGTGTGG TAAGTTACTGGTAAGAGATG 60

TTGGTAACAC CTCTTTTACT AGT 83

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Infectious bronchitis virus

(B) STRAIN: M41

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 7..20

(D) OTHER INFORMATION: /function= "VACCINIA P7.5 LEADER SEQUENCE"

(ix) FEATURE:

-2 ^« \-

(A) NAME/KEY: misc_feature

(B) LOCATION: 21..46

(D) OTHER INFORMATION: /function= "IBV SPIKE CODING SEQUENCE"

( i) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GGATCCAATC AATAGCAATC ATGTTGGTAA CACCTCTTTT ACTAGT 46

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GATCCAATCA ATAGCAATCA TGTTGGTAAC ACCTCTTTTA 40

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CTAGTAAAAG AGGTGTTACC AACATGATTG CTATTGATTG 40