WO1995000172A1 - Recombinant deletion feline herpesvirus vaccine and method for the preparation thereof - Google Patents

Abstract

A recombinant, deletion virus of feline herpesvirus (FHV-1) is described. Portions of genes encoding polypeptide precursors of gE and gI are deleted and the remaining DNA is operably linked to DNA encoding a marker for the recombinant deletion FHV-1 virus. The recombinant, deletion FHV-1 is used in a method and vaccines for vaccinating cats against feline viral rhinotracheitis which is a major disease of cats.

Description

RECOMBINANT DELETION FELINE

HERPESVIRUS VACCINE AND METHOD

FOR THE PREPARATION THEREOF

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to a recombinant, deletion feline herpesvirus (FHV-1) vaccine and to a method for producing the recombinant, deletion FHV-1 vaccine. In particular the present invention relates to a vaccine wherein a marker DNA replaces a portion of the DNA encoding precursor polypeptides of gl and gE of FHV-1. (2) Prior Art

Feline viral rhinotracheitis (FVR) is caused by feline herpesvirus-1 (FHV-1) . Feline herpesvirus (FHV-1) is a member of the genus Alphaherpesvirinae . This widespread virus is responsible for 40-45% of all respiratory infections of Felidae (the cat family) . All cats, receiving veterinary care are vaccinated against this disease. Several existing odified-live (MLV) and inactivated vaccines (IV) against the disease have either residual virulence (MLV) or suffer from a lack of immunogenicity. Furthermore, none of the existing parenterally administered MLV or IV can protect vaccinated cats from infection with virulent virus (as opposed to the disease) when exposed to it. This automatically leads to latent infections, which are epidemiologically very important because of the ease and frequency by which latent FHV-1 is reactivated and spread by asymptomatic, latently infected carriers.

Several million domesticated cats that are kept as house pets in the U.S. receive annual vaccinations for FVR. The need for an improved vaccine is substantial.

As typical of other herpesviruses, numerous glycoproteins are synthesized by FHV-1 and incorporated into cellular membranes and the virion envelope. These glycoproteins have been shown to be involved in membrane attachment, penetration of the virion into cells via cell-to-cell spread, complement binding, virus neutralization and immune destruction of infected cells. It has been previously demonstrated that there is a significant host immune response to viral glycoproteins during FHV-1 infection (Maes, R. , et al., J. Virol. 51:259-262 (1984)). The temporal development of immunity against FHV-1 glycoproteins in cats inoculated with FHV-1 on the oral, nasal and conjunctival mucosa has also been defined. Thus, the concurrent detection of virus-neutralizing antibody and glycoprotein-specific immunoprecipitins implied that FHV-1 glycoproteins were important in the induction of virus-neutralizing antibodies to FHV-1 in cats (Burgener, D. and R. Maes, American J. Vet. Res. 49:1673-1676 (1988); and Rota, P., et al. , Virology 154:168-179 (1986)).

Glycoproteins present in the envelope of herpesviruses play an important role in induction of humoral, cell-mediated and nonspecific defense mechanisms. The genome of herpes simplex virus-type 1 (HSV-1) codes for at least 10 antigenically distinct glycoproteins: gB, gC, gD, gE, gG, gH, gl, gJ, gK and gL (Spear, P., Glycoproteins specified by herpes simplex viruses. In "The Herpesviruses" (B. Roizman, ED.) Vol. 3 pp 315-356. Plenum, New York (1984); and Hutchinson, L., et al., J. Virol. 66: 2240-2250 (1992)). These glycoproteins have been well characterized and are fairly conserved among related herpesviruses. This conversation is not surprising since gB, as well as glycoproteins D, H, K and L, have been shown to be essential for production of enveloped viruses (Spear, P., Glycoproteins specified by herpes simplex viruses. In "The Herpesviruses" (B. Roizman, ED.) Vol. 3 pp 315- 356. Plenum, New York (1984); Hutchinson, L. , et al., J. of Virology Vol. 66, 2240-2250 (1992); and MacLean, C, et al., J. Gen. Virology. 72: 897-906 (1991)).

Immunological and biochemical studies on the polypeptides of FHV-1 has indicated the presence of at least 7 glycoproteins. In studies involving ¹⁴C and ³H glycosamine, (Maes, R. , et al., J. of Virology 51: 259- 262 (1984)) have identified a group of closely migrating glycoproteins with molecular weight ranging from 103-107 kd. Three additional glycoproteins (85, 68 and 59 Kd) were also identified. Two glycoproteins with MW's of 107 and 75 Kd were detected in the culture medium harvested from FHV-1 infected cells. Similar protein profiles have been observed by Fargeaud and Limcumpao (Fargeaud, D. , et al., Arch. Virology 80: 69-82 (1984); Limcumpao, J. , et al., Arch. Virology 111: 165-176 (1990)). In addition,, Horimoto has identified a 60 Kd protein that is capable of hemagglutination (Horimoto, T., et al., Jpn. J. Vet. Sci. 5: 607-612 (1989)). Based on the complete nucleic acid sequences of the genomes of HSV-1, VZV and EHV-1, a glycoprotein gene cluster in the U_s region appears to be conserved throughout herpesviruses (McGeoch, D., et al., J. Mol. Biol. 181: 1-13 (1985): Davison, A., EMBO 12: 2203-2209 (1984); Telford, E. , et al., Virology 189: 304-316 (1992); Elton, D., et al., Am. J. Vet. Res. 52: 1252- 1257 (1991); and Audonnet, J. , et al., J. of Gen. Virology 71: 2969-2978 (1990)). In addition, partial DNA sequencing of the U_s region of PRV, MDV and BHV-1 have revealed minor differences in the genetic organization of the U_s gene cluster (Petrovskis, E. , et al., J. of Virology 60: 1166-1169 (1986); Ross, L. and Binns, M., J. of Gen. Virology 72: 939-947 (1991)). These variations range from the lack of a gD homolog in VZV to additional glycoprotein genes in MDV and EHV-1. The majority of U_s glycoprotein genes have been shown to be dispensable for replication of the virus in cell culture (Mettenleiter, T. , et al., Virology 179: 498-503 (1990)) . In the case of HSV-1, 11 of the 12 U_s genes can be deleted (Longnecker, R. , et al., Proc. Natl. Acad. Sci. USA. 84: 4303-4307 (1987)). Glycoprotein D has been shown to be the only U_s glycoprotein essential for virion production. Through continuous passage in tissue culture, many of the U_s genes of animal herpesvirus have been naturally deleted and resulted in attenuated avirulent strains (Kimman, T., et al., J. of Gen. Virology 73: 243-251 (1992); Petrovskis, E., et al., J. of Virology 60: 1166-1169 (1986)). Avirulant strains of pseudorabies virus (i.e. (Bartha) and EHV-1 (i.e. KyA) containing deletions in the U_s glycoprotein genes have been generated and their protective immunity is well documented (Mettenleiter, T., et al., Virology 179: 498-503 (1990); Vandeputte, J. , et al.. Am. J. Vet. Res., 51: 1100-1106 (1990); van Oirschot, J. , et al., Am. J. Vet. Res. 52: 1056-1060 (1991); ardley, R. , et al., Res. Vet. Sci. 50: 178-184 (1991)). Interestingly, all these vaccines strains contain deletions in the gE gene and have reduced neurovirulence (Petrovskis, E. , et al., J. of Virology 60: 1166-1169 (1986); Flowers, C. and O'Callaghan, D., Virology 190:307-315 (1992)). Animal studies with the Bartha strain of pseudorabies virus (Card, J. , et al., J. of Virology 66: 3032-3041 (1992)) have demonstrated that this virus is unable to infect certain neurons that were readily susceptible to the parent strain containing functional gE. Genetic analyses of DNA isolated from currently available vaccine strains of FHV-1 (Solvay and Fermenta) have indicated the lack of genetic alterations (i.e. deletions) within the gE gene.

OBJECTS

It is therefore an object of the present invention to provide a recombinant, deletion FHV-1 vaccine which eliminates portions of the gl and gE encoding genes. Further, it is an object of the present invention to provide a recombinant, deletion FHV-1 vaccine which includes a marker gene encoding a product or containing DNA which can be identified. These and other objects will become increasingly apparent by reference to the following description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A, IB, IC and ID show the genomic organization of the FHV-1 unique short genes encoding a putative protein kinase and glycoproteins gG, gD, gl and gE. Figure 1A shows the 134 Kb genome represented as two unique sequences (U_L and U_s) and two inverted repeats (IR_S and TR_S) flanking the U_s region. Figure IB shows the Sail and EcoRI restriction maps of FHV-1 (C-27) . Figure IC shows a detailed restriction map of the unique short region along with the positions and transcriptional direction of the genes encoding the putative PK, gG, gD, gl and gE. Figure ID shows the black boxes (1-6) representiong the hybridization probes used to map the U_s transcripts.

Figure 2 shows the nucleotide sequence and predicted amino acid sequences of the FHV-1 polypeptides, gG, gD, gl and gE and part of the putative threonine/serine kinase. Cis-acting sites are (CAAT, TATA) in boxes and polyadenylation sites are in bold. Potential N-linked glycosylation sites are bracketed by two lines. Direct repeats of the sequence GGG GCT GTG GGG ACG A are indicated with a partitionary line.

Figure 3 represents a schematic drawing of the donor plasmid, pB-gal gl gE. In this construct the Bet- galactosidase gene under the control of the cytomegalovirus immediate early promoter is flanked with FHV-1 sequences. These FHV-1 sequences include the 5' terminus of the gene encoding a precusor polypeptide of gl and the 3• terminus of the gene encoding a precusor polypeptide of gE.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a recombinant, deletion feline herpesvirus type 1 (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding precursor polypeptides of gl and gE, wherein the foreign DNA serves as an identification marker for the recombinant deletion FHV-1. Further, the present invention relates to a method for vaccinating against feline viral rhinotracheitis (FVR) which comprises: vaccinating the feline with a recombinant, deletion feline herpesvirus (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding precursor polypeptides of gl and gE to vaccinate against FVR, wherein the foreign DNA serves as an identification marker for the recombinant, deletion FHV-1. Finally, the present invention relates to a vaccine for feline viral rhinotracheitis (FVR) which comprises: a recombinant, deletion feline herpesvirus (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding precursor polypeptides of gl and gE in a dosage for vaccination against FVR, wherein the foreign DNA serves as an identification marker for the recombinant, deletion FHV-1.

The vaccines can be intranasally administered. Intramuscular injection is recommended for cats housed in multiple cat households since the virus is contained within the tissue of the cat and thus is not likely to be shed.

The vaccine preferably contains between about 10⁵ and 10⁷ PFU of the recombinant, deletion virus per dosage unit. Higher or lower amounts can be used depending upon the antigenicity of the vaccine. The vaccine is of the live virus type.

Restriction mapping studies of FHV-1 have indicated that the U_s region is approximately 8.0 Kb. The nucleotide sequence of a 6.2 Kb portion of this region has been determined. Analyses of this sequence has identified 5 open reading frames capable of encoding polypeptides homologous to HSV-1 Pk, gG, gD, gl and gE. Homologs to these gene products have been found in a number of alphaherpesviruses (i.e. HSV-1, EHV-1, PRV, VZV, BHV-1 and MDV) . Comparison at the amino acid level has indicated that the U_s gene products of FHV-1 are most similar to those of EHV-1, except for glycoprotein D which shows more homology with gD of BHV-1. Hydropathic analysis has shown that FHV-1 glycoprotein G, D, I and E exhibit features typical of membrane- bound glycoprotein: a hydrophobic signal sequence at the N- terminus, potential N-linked glycosylation sites and a hydrophobic transmembrane domain near the C-terminus. Although glycoprotein G of FHV-1 has features displayed by membrane proteins, it maybe a secreted protein. Glycoprotein G homologs of the varicelloviruses (EHV-1 and PRV) have been reported to be secreted and extensive homology exist these glycoproteins and gG of FHV-1. Northern analyses of the FHV-1 U_s genes have indicated the likelihood of numerous co-terminal transcripts.

Example 1 The nucleotide sequence of the 6.2 Kb fragment from the unique short region of the FHV-1 genome contains 5 open reading frames (ORFs) . Two of which encoding precursor polypeptides of gl and gE which are described in this Example.

Material and Methods Viral and bacterial strains.

FHV-1 (strain C-27) was obtained from the American Type Culture Collection and propagated in Crandell Reese Feline Kidney (CRFK) cells as described previously (Maes, R. , et al, J. Virol. 51:259-262 (1984)). Infectious cellular lysates were used as the source for viral DNA Escherichia coli strain JM 101 and JMlOl were grown in LB medium and used to propagate recombinant M13 mp 18 and mpl9 clones. All of these materials are available from Michigan State University, East Lansing, Michigan. Cloning and DNA Sequencing

The complete nucleotide sequences for both strands of a 6,208 bp portion of the larger 14.5 Kb Sail B fragment

(Rota, P., et al., Virology 154: 168-179 (1986)) have been determined (Figure 2). The 4.3 Kb EcoRI-EcoRI fragment and the adjacent 1.9 Kb EcoRI-Sall fragment located at the right terminus of the Sail B fragment were chosen for DNA sequence analysis (Figure 1) . Hybridization analysis have indicated these two restriction fragments contain solely unique DNA.

For rapid generation of sequencing data, four individual M13 libraries were created using HAEIII, RSAI, TAQI and SAU3A restriction digestions of the 4.3 Kb EcoRI- EcoRI and 1.9 Kb EcoRI-Sall fragments. Single stranded DNA from recombinant M13 phage was isolated according to Ausubel, F. , et al, Current Protocols in Molecular Biology, John Wiley and son. New York) and sequenced using standard dideoxynucleotide chain termination reactions with the modified T7 polymerase, sequenase (US Biochemical, Cleveland, Ohio) . ³⁵S thio dATP (Amersham, Arlington Heights, Illinois) was used as the label and dITP was used to resolve band compressions. Synthetic oligonucleotides, along with the Universal and 17'mer M13 primers were used to obtain sequencing information from both strands. Reaction products were electrophoretically separated and visualized by autoradiography of dried 8% acrylamide/7.0 M urea gels using Kodak X-AR film (Rochester, New York) . Analyses of Sequence data

DNA sequences were compiled on a VAX computer using version 6.2 and 7.0 of the University of Wisconsin package UWGCG (University of Wisconsin, Madison, Wisconsin) . Hydrophilicity analysis of individual predicted translation products were generated by the method of (Kyte and Doolittle, J. of Mol. Biol. 157:105- 132 (1982)). Amino acid homology searches of the Swissprot (Release 18.0, 5/91) data bases were conducted using the FASTA program (UWGCG). The GAP, LINEUP, PILEUP programs were used to generate multiple alignments between FHV-1 U_s predicted polypeptides and homologs found in related herpesviruses.

Example 2 The specific aim of this invention is the construction of deletions in two non-essential glycoprotein genes (gE and gl) of the selected parent strain of FHV-1 in function of creating a gene deleted vaccine against feline viral rhinotracheitis (FVR) . The deletions selected can significantly reduce or eliminate the neurovirulence of the FHV-1 strain, while maintaining full immunogenicity, thus overcomming the shortcomings of currently available vaccines. A donor plasmid in which the CMV Immediate Early/ beta- galactosidase gene is flanked with DNA sequences specific for the gl and gE genes is used to construct the deletion mutant.

This Example involves construction of a recombinant strain of FHV-1 in which the genes encoding the polypeptides of glycoprotein I and E have been replaced with the "marker" gene, beta-galactosidase. This recombinant strain contains the gene encoding beta- galactosidase under the control of the Immediate Early promoter of cytomegalovirus (CMV) .

The complete nucleic acid sequence of the gene encoding FHV-1 glycoproteins I and E are set forth in Figure 2 and Example 1. These two genes are located next to each other with gE downstream from the gl gene. Both are located in the 14.5 Kb Sail B fragment. The construction of a mutant strain of FHV-1 in which the gl and gE genes have been deleted and replaced with beta- galactosidase involves the following steps. 1. Construction of a donor plasmid in which the CMV Immediate Early/ beta-galactosidase gene is flanked with DNA sequences specific for the gl and gE genes. The 1.3 Kb Xbal-BamHI restriction fragment encoding the 5* region of the gl gene was cloned into the plasmid, BLUESCRIPT II -SK (Stratagene, La Jolla, CA) . This recombinant plasmid was purified and used as a host for the next cloning event. The 0.95 Kb EcoRV- Sall fragment containing the 3 ' portion of the glycoprotein E gene was cloned into the recombinant plasmid. This plasmid was the host for the final cloning event, the insertion of the CMV immediate early promoter/ Beta galactosidase gene into the PstI site. The final product of these cloning events resulted in a donor plasmid in which the beta galactosidase gene is flanked by portions of the gl and gE genes of FHV-1 (Figure 3) .

2. Rescue of the construct made in section 1 into the genome of FHV-1. Feline herpesvirus-1 genomic DNA and plasmid DNA are transfected into feline kidney cells and recombinant viruses are identified via a colorimetric reaction involving X-gal. Blue plaques are purified three times.

3. Genetic and immunological characterization of the gl and gE deletion strain of FHV-1. The deletion of the gl and gE genes is verified via restriction analysis. To investigate the protein profile of the deletion recombinant, SDS-PAGE is used with anti-FHV-1 antisera and ¹⁴C-labeled glycosamine proteins.

4. Clinical trials with SPF cats are then performed to determine the protective nature of the deletion strain.

The vaccines of the present invention are easily differentiated from wild type virus because of the deletion. The deletion can be detected by various means such as ELISA or DNA hybridizations.

It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims.

APPENDIX I

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Roger K. Maes and Stephen J. Spatz

(ii) TITLE OF INVENTION: Recombinant Deletion

Feline Herpesvirus Vaccine and Method For the Preparation Thereof

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Ian C. McLeod

(B) STREET: 2190 Commons Parkway

(C) CITY: Okemos

(D) STATE: Michigan

(E) COUNTRY: USA

(F) ZIP: 48864

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 Kb storage

(B) COMPUTER: Acer

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 08/082,841

(B) FILING DATE: June 24, 1993

(C) CLASSIFICATION:

(vϋ) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: Ian C. McLeod

(B) REGISTRATION NUMBER: 20,931

(C) REFERENCE/DOCKET NUMBER: MSU 4.1-167

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (517) 347-4100

(B) TELEFAX: (517) 347-4103

(C) TELEX: None

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6208

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE:

(A) Description: DNA

(iii) HYPOTHETICAL: No

(iv) ANTI-SENSE: No

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Feline herpesvirus

(B) STRAIN: FHV-1

(C) INDIVIDUAL ISOLATE: C-27 (G) CELL TYPE: N/A

(ix) FEATURE:

(A) NAME/KEY:

(B) LOCATION:

(C) IDENTIFICATION METHOD: Sequencing

(D) OTHER INFORMATION:

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

GAA TTC CCA GGC GAC CCG ACT TCT AAA TTA ACT ATA GAC TTT ATT CAT 48 Glu Phe Pro Gly Asp Pro Thr Ser Lys Leu Thr He Asp Phe He His

5 10 15

TAT GCC TCA TGT GTA AGA CAG CCT TAT ACA CGA TAC GAT TGT ATG TCA 96 Tyr Ala Ser Cys Val Arg Gin Pro Tyr Thr Arg Tyr Asp Cys Met Ser

20 25 30

AAA TAC GAT TTG CCG CTA GAT GGG GAG TTT GTA GTG CAT AAG ATG TTG 144 Lys Tyr Asp Leu Pro Leu Asp Gly Glu Phe Val Val His Lys Met Leu

35 40 45

ACT TTC GAT GCC AAG TTC CGA CCA TCG GCC GCT GAA ATT CTA AAC TAT 192 Thr Phe Asp Ala Lys Phe Arg Pro Ser Ala Ala Glu He Leu Asn Tyr

50 55 60

CCA ATG TTT CGT GAT ACA TAG TTACATCAT TATCTATGGG TGGGACTTTC 242 Pro Met Phe Arg Asp Thr 65 70

CACCAAGACA GTATAAAGTA TTTGGGACTC CACAATAGCA TTCATCCTTC TTACCCTTCT 302

ACGAGTAAGA ACTTCAATCA CTCAACTTGG AAGAAAA ATG GGA AAT CGT ATA 354

Met Gly Asn Arg He 5 CAT ATT TTA ATA TGC ATT GCA GCA TTC TAC ATA ACC ATC GCG GCT GCT 402 His He Leu He Cys He Ala Ala Phe Tyr He Thr He Ala Ala Ala

10 15 20

AGG AAT GCC CCA ATG GAT CTC TGT TAC GCC GAC CCC AGA GAT ACA TCA 450 Arg Asn Ala Pro Met Asp Leu Cys Tyr Ala Asp Pro Arg Asp Thr Ser

25 30 35

CCA CAA CCC ATA GGA CAT CCT AAT TAT AAA CAA GTG AAT ATA ACG ATC 498 Pro Gin Pro He Gly His Pro Asn Tyr Lys Gin Val Asn He Thr He

40 45 50

CAC TAC CCC GCA CCA AAG TGG GGA TAT GTT GAA CAT TCC AGT GGA TGT 546 His Tyr Pro Ala Pro Lys Trp Gly Tyr Val Glu His Ser Ser Gly Cys 55 60 65 GAA TTA CGT TTA TTG GAC CCG AGA GTT GAT GTG TCT CTT CAA GAT CAC 594

Glu Leu Arg Leu Leu Asp Pro Arg Val Asp Val Ser Leu Gin Asp His

70 75 80 85

CAG AGA AGG GCA GAC GCT ACG ATT GCT TGG ACT TTT GAT CTC GGA ACA 642

Gin Arg Arg Ala Asp Ala Thr He Ala Trp Thr Phe Asp Leu Gly Thr

90 95 100

TGT CAA ATA CCT ATC GCG TAT AGA GAA TAT TAT AAC TGT ACT GGG AAT 690 Cys Gin He Pro He Ala Tyr Arg Glu Tyr Tyr Asn Cys Thr Gly Asn

105 110 115

TTA ATA CCC TCC CCA GAA ACT TGC GAA GGG TAT TCC GCG ACC TCC ATA 738 Leu He Pro Ser Pro Glu Thr Cys Glu Gly Tyr Ser Ala Thr Ser He

120 125 130

CGC TTC GAA GGT CTA ACC ATC TAT ACC TTG GTA AAT ATA AGT CTA CTC 786 Arg Phe Glu Gly Leu Thr He Tyr Thr Leu Val Asn He Ser Leu Leu

135 140 145

CTT CAA CCA GGA ATA TTC GAT TCC GGG AGT TTC CTG TAT TCA TTT ATA 834 Leu Gin Pro Gly He Phe Asp Ser Gly Ser Phe Leu Tyr Ser Phe He 150 155 160 165

TAT GGT CAA AAT AGA TAC AAT GGA CGT ATT ATA GTT CAT GTA GAA AAA 882 Tyr Gly Gin Asn Arg Tyr Asn Gly Arg He He Val His Val Glu Lys

.170 175 180

AAT ACT GAT TAT CCC TGC AAA ATG TAT CAT GGA CTC ATG GCT CCA TTT 930 Asn Thr Asp Tyr Pro Cys Lys Met Tyr His Gly Leu Met Ala Pro Phe

185 190 195

GAC CAT CAT CCC CAA AGC CAC GTT GAA ACT CCG AAT GAT AAG AAT CAT 978 Asp His His Pro Gin Ser His Val Glu Thr Pro Asn Asp Lys Asn His

200 205 210

CGT AGA GGG CGG GGA TGT TTT CCC GAA TTG GTG GAA CCT GTT CTA TGG 1026 Arg Arg Gly Arg Gly Cys Phe Pro Glu Leu Val Glu Pro Val Leu Trp

215 220 225

GTT AAT ATC AGC AGT GAT CTT ATT GGT GGT CCA CCT TTC GAC TAT AAT 1074 Val Asn He Ser Ser Asp Leu He Gly Gly Pro Pro Phe Asp Tyr Asn 230 235 240 245

CAT GAA GAT GAG GCT GAT ATT GAG AGT GAT GAG CTC CCG GAG GAG ATA 1122 His Glu Asp Glu Ala Asp He Glu Ser Asp Glu Leu Pro Glu Glu He

250 255 260

TAC ATA ACT ACT CAG ATT GTC GTG CGA CTA ATA TGT TTG TTC CGA GAG 1170 Tyr He Thr Thr Gin He Val Val Arg Leu He Cys Leu Phe Arg Glu

265 270 275

AGC CCC TCA GTC AAA GTT CTT GGT TCT CAA AGT CTA CTG GTT GGT AGT 1218 Ser Pro Ser Val Lys Val Leu Gly Ser Gin Ser Leu Leu Val Gly Ser

280 285 290

TTA GGT TTC CAG ATA ATT ACT CAA CCC TGG CAA CTG AAG CAG AAT GAA 1266 Leu Gly Phe Gin He He Thr Gin Pro Trp Gin Leu Lys Gin Asn Glu

295 300 305

AGT TAT GAT GGA CTA AGA AAT GCC TCT CTT GAA CCC CGA CAC CTT GAC 1314 Ser Tyr Asp Gly Leu Arg Asn Ala Ser Leu Glu Pro Arg His Leu Asp 310 315 320 325

TCC AGT AAC GAT CGT GAT CTA CTA GAT GAA ACT GAA ATG ATT GGA TCG 1362 Ser Ser Asn Asp Arg Asp Leu Leu Asp Glu Thr Glu Met He Gly Ser

330 335 340

ATT ATT ACG ACT CCA CCA CCA ACC CAT CCA AAA GGT GTC AAT GGG GGT 1410 He He Thr Thr Pro Pro Pro Thr His Pro Lys Gly Val Asn Gly Gly

345 350 355

TTC CTC CAA GAT CTA CCA ATT ATC GAG CCT ACG ACC GAA CCA TGC TTA 1458 Phe Leu Gin Asp Leu Pro He He Glu Pro Thr Thr Glu Pro Cys Leu

360 365 370

GTA CAT ACA AAG ATC ATT GGG ATC GGA ACA GTA GTC GTT GTA TTT TTG 1506 Val His Thr Lys He He Gly He Gly Thr Val Val Val Val Phe Leu 375 380 385

TTA TTT ATT CTC ATA TCC CTA TGT GTT TAT ACT TGC GTT CTA CGA TCC 1554 Leu Phe He Leu He Ser Leu Cys Val Tyr Thr Cys Val Leu Arg Ser 390 395 400 405

CGC ATC GGT ATG GTA GAT CGC GCC TAT GTG AAA CAA GTA CGA TTT AAT 1602 Arg He Gly Met Val Asp Arg Ala Tyr Val Lys Gin Val Arg Phe Asn 410 415 420 TCC AAT CCA TCA TAT CAA CAG TTG ACA AGA TAC CCC CAA CCA TAA TAA 1650 Ser Asn Pro Ser Tyr Gin Gin Leu Thr Arg Tyr Pro Gin Pro 425 430 435

ACTGATTAAA TTTAATTAAA GTCTCATATG TGGGGCTGTG GGGACGAGGG GCTGTGGGGA 1710

CGAGGGGCTG TGGGGACGAG GGGCTGTGGG GACGAGGGGC TGTGGGGACG AGGGGCTGTG 1770

GGGACGAGGG GCTGTGGGGA CGAGGGGCTG TGGGGACGAG GGGCTGTGGG GACGAGGGGC 1830

TGTGGGGACG AGGGGCTGTG GGGACGAGGG GCTGTGGGGA CGAGGGGCTG TGGGACGATT 1890

ACAACCGATA AATGTCGTAT ATGAAATGTG GTGTTAACAT AACACGGATT TTTAAGCACA 1950

CCACATGACA CACCCCCACG ATAACGGTTA AATCACCAGC TATGTGAACT GCCCTCCATT 2010

CTACTCAAAT GAGTGGTGGT GTGTGGCATA TTAGAACCAT TTCGTCTAAT G ATG 2064

Met

ACA CGT CTA CAT TTT TGG TGG TGT GGA ATC TTT GCG GTC CTG AAA TAT 2112 Thr Arg Leu His Phe Trp Trp Cys Gly He Phe Ala Val Leu Lys Tyr

5 10 15

CTG GTA TGT ACT TCA AGC CTT ACG ACC ACG CCA AAA ACA ACT ACG GTT 2160 Leu Val Cys Thr Ser Ser Leu Thr Thr Thr Pro Lys Thr Thr Thr Val

20 25 30

TAT GTG AAG GGA TTT AAT ATA CCT CCA CTA CGC TAC AAT TAT ACT CAA 2208 Tyr Val Lys Gly Phe Asn He Pro Pro Leu Arg Tyr Asn Tyr Thr Gin

35 40 ' 45

GCC AGA ATC GTG CCA AAA ATT CCC CAG GCG ATG GAT CCG AAG ATA ACA 2256 Ala Arg He Val Pro Lys He Pro Gin Ala Met Asp Pro Lys He Thr 50 55 60 65

GCT GAA GTA CGT TAT GTA ACA TCA ATG GAT TCA TGT GGG ATG GTG GCA 2304 Ala Glu Val Arg Tyr Val Thr Ser Met Asp Ser Cys Gly Met Val Ala

70 75 80

TTG ATA TCA GAG CCG GAT ATA GAC GCT ACT ATT CGA ACC ATA CAA CTA 2352 Leu He Ser Glu Pro Asp He Asp Ala Thr He Arg Thr He Gin Leu

85 90 95

TCT CAA AAA AAA ACA TAT AAC GCG ACT ATA AGT TGG TTT AAG GTA ACC 2400 Ser Gin Lys Lys Thr Tyr Asn Ala Thr He Ser Trp Phe Lys Val Thr

100 105 110

CAG GGT TGT GAA TAC CCT ATG TTT CTT ATG GAT ATG AGA CTT TGT GAT 2448 Gin Gly Cys Glu Tyr Pro Met Phe Leu Met Asp Met Arg Leu Cys Asp

115 120 125

CCT AAA CGG GAA TTT GGA ATA TGT GCT TTA CGG TCG CCT TCA TAT TGG 2496 Pro Lys Arg Glu Phe Gly He Cys Ala Leu Arg Ser Pro Ser Tyr Trp 130 135 140 145

TTG GAA CCT TTA ACA AAG TAT ATG TTC CTA ACA GAC GAT GAA CTG GGT 2544 Leu Glu Pro Leu Thr Lys Tyr Met Phe Leu Thr Asp Asp Glu Leu Gly

150 155 160

TTG ATT ATG ATG GCC CCG GCC CAA TTT AAT CAA GGA CAA TAT CGA AGA 2592 Leu He Met Met Ala Pro Ala Gin Phe Asn Gin Gly Gin Tyr Arg Arg

165 170 175 GTT ATA ACC ATC GAT GGT TCC ATG TTT TAT ACA GAT TTT ATG GTA CAA 2640 Val He Thr He Asp Gly Ser Met Phe Tyr Thr Asp Phe Met Val Gin 180 180 190

CTA TCT CCA ACG CCA TGT TGG TTC GCA AAA CCC GAT AGA TAC GAA GAG 2688 Leu Ser Pro Thr Pro Cys Trp Phe Ala Lys Pro Asp Arg Tyr Glu Glu 195 200 205

ATT CTA CAT GAA TGG TGT CGA AAT GTT AAA ACT ATT GGC CTT GAT GGA 2736

He Leu His Glu Trp Cys Arg Asn Val Lys Thr He Gly Leu Asp Gly

210 215 220 225

GCT CGT GAT TAC CAC TAT TAT TGG GTA CCC TAT AAC CCA CAA CCT CAC 2784

Ala Arg Asp Tyr His Tyr Tyr Trp Val Pro Tyr Asn Pro Gin Pro His

230 235 240

CAT AAA GCC GTA CTC TTA TAT TGG TAT CGG ACT CAT GGC CGA GAA CCC 2832 His Lys Ala Val Leu Leu Tyr Trp Tyr Arg Thr His Gly Arg Glu Pro 245 250 255 CCA GTA AGA TTC CAA GAG GCC ATT CGA TAT GAT CGT CCC GCC ATA CCG 2880 Pro Val Arg Phe Gin Glu Ala He Arg Tyr Asp Arg Pro Ala He Pro

260 265 270

TCT GGG AGT GAG GAT TCG AAA CGG TCC AAC GAC TCT AGA GGA GAA TCG 2928 Ser Gly Ser Glu Asp Ser Lys Arg Ser Asn Asp Ser Arg Gly Glu Ser

275 280 285

AGT GGA CCC AAT TGG ATA GAC ATT GAA AAT TAC ACT CCT AAA AAT AAT 2976 Ser Gly Pro Asn Trp He Asp He Glu Asn Tyr Thr Pro Lys Asn Asn 290 295 300 305

GTG CCT ATT ATA ATA TCT GAC GAT GAC GTT CCT ACA GCC CCT CCC AAG 3024 Val Pro He He He Ser Asp Asp Asp Val Pro Thr Ala Pro Pro Lys

310 315 320

GGC ATG AAT AAT CAG TCA GTA GTG ATA CCC GCA ATC GTA CTA AGT TGT 3072 Gly Met Asn Asn Gin Ser Val Val He Pro Ala He Val Leu Ser Cys

325 330 335

CTT ATA ATA GCA CTG ATT CTA GGA GTG ATA TAT TAT ATT TTG AGG GTA 3120 Leu He He Ala Leu He Leu Gly Val He Tyr Tyr He Leu Arg Val

340 345 350

AAG AGG TCT CGA TCA ACT GCA TAT CAA CAA CTT CCT ATA ATA CAT ACA 3168 Lys Arg Ser Arg Ser Thr Ala Tyr Gin Gin Leu Pro He He His Thr

355 360 365

ACT CAC CAT CCT TAA GTCCACATTC CAATCGAGTT GGTAGGGAAG ATATGAAGTG 3223 Thr His His Pro 370 GGCGGTACCA ACCATCATAA AATAGGTTGG AGTCTGGACC ACGTTCACTA CTTTTGAGTG 3283

TAAAGGACCA CAGCATAATA CTTAAT ATG TCG TCG ATA GCC TTC ATC TAT 3333

Met Ser Ser He Ala Phe He Tyr 5 ATA TTG ATG GCG ATT GGA ACA GTT TAT GGG ATT GTG TAT CGT GGA GAT 3381 He Leu Met Ala He Gly Thr Val Tyr Gly He Val Tyr Arg Gly Asp

10 15 20

CAT GTA AGT CTT CAT GTT GAT ACA AGC TCC GGC TTT GTA ATA TAT CCA 3429 His Val Ser Leu His Val Asp Thr Ser Ser Gly Phe Val He Tyr Pro 25 30 35 40

ACA CTG GAG AAT TTT ACG ATC TAC GGC CAT CTA ATC TTT CTC GAC GAC 3477 Thr Leu Glu Asn Phe Thr He Tyr Gly His Leu He Phe Leu Asp Asp

45 50 55

CAA CCA TTA CCA GTA AAC AAT TAT AAT GGA ACC CTC GAG ATT ATA CAT 3525 Gin Pro Leu Pro Val Asn Asn Tyr Asn Gly Thr Leu Glu He He His

60 65 70

TAC AAC CAT CAC TCT TCT TGC TAT AAA ATC GTT CAA GTA ATA GAA TAT 3573 Tyr Asn His His Ser Ser Cys Tyr Lys He Val Gin Val He Glu Tyr

75 80 85

TCA TCA TGT CCA CGT GTA CGC AAT AAT GCT TTC CGG TCC TGT CTC CAC 3621 Ser Ser Cys Pro Arg Val Arg Asn Asn Ala Phe Arg Ser Cys Leu His

90 95 100

AAG ACC TCT ATG CAC CAA TAC GAT CAG CTT TCC ATA AAC ACA TCC GTT 3669 Lys Thr Ser Met His Gin Tyr Asp Gin Leu Ser He Asn Thr Ser Val 105 110 115 120

GAA ACG GGG ATG TTA TTG ACA ATA ACA TCT CCG AAA ATG GAA GAT GGT 3717 Glu Thr Gly Met Leu Leu Thr He Thr Ser Pro Lys Met Glu Asp Gly

125 130 135

GGA ATC TAC GCA CTG CGG GTA AGA TTT AAC CAT AAT AAC AAA GCT GAT 3765 Gly He Tyr Ala Leu Arg Val Arg Phe Asn His Asn Asn Lys Ala Asp

140 145 150

GTA TTT GGC CTT TCG GTG TTT GTT TAC TCA TTC GAT ACG CGT GGT CAT 3813 Val Phe Gly Leu Ser Val Phe Val Tyr Ser Phe Asp Thr Arg Gly His

155 160 165

CGA CAT CAT GCG GAC GAA AAT TTG AAT GGT GAA ATT CTT ACT ACT CCA 3861 Arg His His Ala Asp Glu Asn Leu Asn Gly Glu He Leu Thr Thr Pro

170 175 180

TCA CCG ATG GAA ACA TAT GTT AAA GTT AAC ACA CCA ATA TAT GAT CAT 3909 Ser Pro Met Glu Thr Tyr Val Lys Val Asn Thr Pro He Tyr Asp His 185 190 195 200

ATG GTG ACA ACT CAA ACA ACT TCT AAT AAA TCG ATG GAG TCT GAA CCA 3957 Met Val Thr Thr Gin Thr Thr Ser Asn Lys Ser Met Glu Ser Glu Pro 205 210 215

TCA AAT ACA TCA ATA TCA TGC CAT ACA TTT CAA AAT GAC CCG AAT GAG 4005 Ser Asn Thr Ser He Ser Cys His Thr Phe Gin Asn Asp Pro Asn Glu

220 225 230

GGT GAG ACT TTA TAT ACA CAC TTA TTG AAC ATC GCT GGA AAT ATA ACA 4053 Gly Glu Thr Leu Tyr Thr His Leu Leu Asn He Ala Gly Asn He Thr

235 240 245

TAT GAT GAC ATG GTT ATG GAT GGC ACC ACA TTG AAA CCC AGA TTA ATC 4101 Tyr Asp Asp Met Val Met Asp Gly Thr Thr Leu Lys Pro Arg Leu He

250 255 260

GAT ATG GGA CTT AAC TTG TCT GTT ACA TCT TCC TTT AAA AAT GGA AAC 4149 Asp Met Gly Leu Asn Leu Ser Val Thr Ser Ser Phe Lys Asn Gly Asn 265 270 275 280

CAC GCA AAA ATG GAC ACC AGA CAG AAA GGT GGG TTT TGT TAT AGT AAT 4197 His Ala Lys Met Asp Thr Arg Gin Lys Gly Gly Phe Cys Tyr Ser Asn

285 290 300

CTC AAT CGC AGT TTT ACT ACT CTT GCG GTC ATC GGA TCC ATC ATC AAT 4245 Leu Asn Arg Ser Phe Thr Thr Leu Ala Val He Gly Ser He He Asn

305 310 315

AGT GCA ATA CGC AAG CAT ATA ATG GTC TGT GCT GGG CGG CGG ATC TAT 4293 Ser Ala He Arg Lys His He Met Val Cys Ala Gly Arg Arg He Tyr

320 325 330

ATA CCA AAC AAC GAT GGG CGA CCA TCA ACG GAA ATG ACA CGG TTT ACT 4341 He Pro Asn Asn Asp Gly Arg Pro Ser Thr Glu Met Thr Arg Phe Thr

335 340 345

CGC CAG ACT AAA CCA TCG AAT TCC ACC CCA ACC GAT GGC GTC TCT AGA 4389 Arg Gin Thr Lys Pro Ser Asn Ser Thr Pro Thr Asp Gly Val Ser Arg 350 355 360 365

AGT CAG TTA ACC GTA ATT AAC GAA GAA ACC TAA TATATTTATA AACAAATAAA 4442 Ser Gin Leu Thr Val He Asn Glu Glu Thr 370 375

ATACTTTTCA AAATGGATAT CTGGTCATGT GTAATGTTGA CGCATAGTGG GTGGTGACCT 4502

AAGATTATAT TAAAATGTAG AAGGTTTTAT GCCCAGTTCA CAGTATCTAC TGTGACCTAC 4562

CCCGGGGTGG TAATAACAAT ACTATCGAAT AGCCAACA ATG GGA CTG CTT GTT 4615

Met Gly Leu Leu Val 5 ACC ATC CTC GTG ATA TTA TTG ATT GTT ACT TCA TCA AGT TCT ACT ATT 4663 Thr He Leu Val He Leu Leu He Val Thr Ser Ser Ser Ser Thr He

10 15 20

CAT CAA GTA ACG ATG ACA GAA GGT GCC GCA CTT TTA GTC GAT GGG GAT 4711 His Gin Val Thr Met Thr Glu Gly Ala Ala Leu Leu Val Asp Gly Asp

25 30 35

GGG ATC GAC CCA CCT TTA AAC AAA ACT TCA CAT TTT TTG CGA GGT TGG 4759 Gly He Asp Pro Pro Leu Asn Lys Thr Ser His Phe Leu Arg Gly Trp

40 45 50

ACA TTT CTA GAG ACT CCG AAA GGA TGT ACA GGA GAG GTG AGT GTT CTA 4807 Thr Phe Leu Glu Thr Pro Lys Gly Cys Thr Gly Glu Val Ser Val Leu

55 60 65

AAA GTA TGT ATA GAT CGT GGG GTA TGT CCG GAT GAT ATC GTT ATA AAT 4855 Lys Val Cys He Asp Arg Gly Val Cys Pro Asp Asp He Val He Asn 70 75 80 85

AAG AGA TGT GGT CAC AAA ATG CTT GAA ACC CCA CTA GCG TTG GGC GAA 4903 Lys Arg Cys Gly His Lys Met Leu Glu Thr Pro Leu Ala Leu Gly Glu

90 95 100

TTT GGA ATT TCT AAT AGT TCT CTC ATC AGA ACC AAA GAC GTA TAT TTC 4951 Phe Gly He Ser Asn Ser Ser Leu He Arg Thr Lys Asp Val Tyr Phe

105 110 115

GTG AAT AAG ACC GTG TTT CCA ATT CTC ACA CCC GAA AAA AGT GGC CTT 4999 Val Asn Lys Thr Val Phe Pro He Leu Thr Pro Glu Lys Ser Gly Leu

120 125 130

GGT ATT CAG GGG GCC ACT ACG AAT ATA TCC GGG ATA TAT ACC CTG CAT 5047 Gly He Gin Gly Ala Thr Thr Asn He Ser Gly He Tyr Thr Leu His

135 140 145

GAG CAC GGT GAT AAT GGA TGG AGT CAT CAA TCT ACA TTT TTT GTG ACC 5095 Glu His Gly Asp Asn Gly Trp Ser His Gin Ser Thr Phe Phe Val Thr 150 155 160 165

GTA AAG GCA AAA CAT CCC GGA CCA TCG TTA ACC CCA GCA CCG GTT CAC 5143

Val Lys Ala Lys His Pro Gly Pro Ser Leu Thr Pro Ala Pro Val His

170 175 180

TTA ATA ACA CCA CAT CGC CAT GGG GCA CAT TTC CAC GTA AGA AAC TAT 5191 Leu He Thr Pro His Arg His Gly Ala His Phe His Val Arg Asn Tyr

185 190 195

CAT TCG CAT GTC TAC ATT CCG GGA GAT AAG TTC TTA TTA GAA ATG CAC 5239 His Ser His Val Tyr He Pro Gly Asp Lys Phe Leu Leu Glu Met His

200 205 210

CTC AAA TCA GAT ATC TAT GAT CCA GAA TTT TCA GCA ACA ATA GAC TGG 5287 Leu Lys Ser Asp He Tyr Asp Pro Glu Phe Ser Ala Thr He Asp Trp

215 220 225

TAT TTT ATG GAG ACT GAT ATA AAA TGC CCA GTT TTT AGA ATT TAT GAA 5335 Tyr Phe Met Glu Thr Asp He Lys Cys Pro Val Phe Arg He Tyr Glu 230 235 240 245

ACT TGT ATA TTT CAC CCC CAT GCC GCA TCC TGT CTA CAT CCG GAA GAT 5383 Thr Cys He Phe His Pro His Ala Ala Ser Cys Leu His Pro Glu Asp

250 255 260

CCC TCA TGC AGT TTT ACA TCA CCA CTT CGA GCG GTA TCT TTA ATT AAT 5431 Pro Ser Cys Ser Phe Thr Ser Pro Leu Arg Ala Val Ser Leu He Asn

265 270 275

AGA TTT TAT CCA AAA TGC GAT CAC AGA TAT GCC GAT TGG ACA TCC AGA 5479 Arg Phe Tyr Pro Lys Cys Asp His Arg Tyr Ala Asp Trp Thr Ser Arg

280 285 290

TGT ATC AAC ACT CCA AGT ATA AAT CAT ATG CCA TAT ATC GAA CAG CCG 5527 Cys He Asn Thr Pro Ser He Asn His Met Pro Tyr He Glu Gin Pro

295 300 305

GCC AAT AAC GTG GAT CTA AAG TTT ATC AAT GTA CCC ACC AAC GCT TCT 5575 Ala Asn Asn Val Asp Leu Lys Phe He Asn Val Pro Thr Asn Ala Ser 310 315 320 325

GGG TTG TAC GTA TTC ATA CTT CGT TAT AAT GGA CAT CCG GAA GAA TGG 5623 Gly Leu Tyr Val Phe He Leu Arg Tyr Asn Gly His Pro Glu Glu Trp

330 335 340

ACC TAT ACA CTC ATA TCA ACA GGA GCT AAA TTT TTG AAT GTG ATT AGG 5671 Thr Tyr Thr Leu He Ser Thr Gly Ala Lys Phe Leu Asn Val He Arg

345 350 355

GAT CTG ACA CGC CCA CGT CTT GGT AGT CAT CAA ATA GAG ACC GAT ATT 5719 Asp Leu Thr Arg Pro Arg Leu Gly Ser His Gin He Glu Thr Asp He

360 365 370

AGC ACA TCT TCC GAG TCG CCT ACC ACG GAG ACA CCA CGA AAC ATA CAT 5767 Ser Thr Ser Ser Glu Ser Pro Thr Thr Glu Thr Pro Arg Asn He His

375 380 385

ATA ACG TGG GCG AGA CGT TAT CTA AAG GTT ATC ATA GGA ATA ATT TGC 5815 He Thr Trp Ala Arg Arg Tyr Leu Lys Val He He Gly He He Cys 390 395 400 405

GTA GCT GGT ATC CTT TTG ATT GTA ATC TCT ATC ACA TGT TAT ATT CGA 5863 Val Ala Gly He Leu Leu He Val He Ser He Thr Cys Tyr He Arg

410 415 420

TTT CGT CAT ATG CGA TAT AAA CCA TAT GAA GTG ATC AAC CCA TTC CCT 5911 Phe Arg His Met Arg Tyr Lys Pro Tyr Glu Val He Asn Pro Phe Pro

425 430 435

GCG GTA TAT ACC AGC ATT CCT AGT AAC GAT CCC GAC GAA CTC TAC TTT 5959 Ala Val Tyr Thr Ser He Pro Ser Asn Asp Pro Asp Glu Leu Tyr Phe

440 445 450

GAA CGT ATC GCA TCG AAC GAC GAA GAA TCG GCA GAT GAT TCT TTT GAT 6007 Glu Arg He Ala Ser Asn Asp Glu Glu Ser Ala Asp Asp Ser Phe Asp

455 460 465

GAA TCA GAT GAG GAG GAG CCA TTG AAT AAT CAT CAT ATT TCA ACA ACC 6055 Glu Ser Asp Glu Glu Glu Pro Leu Asn Asn His His He Ser Thr Thr 470 475 480 485

CAA CAT ACT GAT ATT AAT CCA GAA AAA TCC GGA TCT GGG TAC AGT GTA 6103 Gin His Thr Asp He Asn Pro Glu Lys Ser Gly Ser Gly Tyr Ser Val

490 495 500

TGG TTT CGT GAT ACA GAA GAT ACA TCA CCT CAG CCC CTA CAC GCT CCT 6151 Trp Phe Arg Asp Thr Glu Asp Thr Ser Pro Gin Pro Leu His Ala Pro

505 510 515 CCA GAT TAC AGT CGC GTA GTT AAA AGA TTA AAG TCT ATT TTA AAA TGA 6199 Pro Asp Tyr Ser Arg Val Val Lys Arg Leu Lys Ser He Leu Lys

520 525 530

CCCGTCGAC 6208

Claims

WE CLAIM:

-1- A recombinant, deletion feline herpesvirus type 1 (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding a polypeptide precursor of gl and gE, wherein the foreign DNA serves as an identification marker for the recombinant, deletion FHV-1.

-2- The recombinant FHV-1 of Claim 1 wherein the foreign DNA encodes beta galactosidase as a marker for the recombinant FHV-1.

-3- The recombinant FHV-1 of any one of Claims 1 or 2 wherein a 1.3 Kb Xbal-BamHI restriction fragment encoding a 5' region of the polypeptide precursor of gl is operably linked to the foreign DNA at one end and wherein at an opposite end of the foreign DNA a 0.95 Kb EcoRV-Sall restriction fragment encoding a 3' region of the polypeptide precursor of gE is operably linked to the foreign DNA.

-4- A method for vaccination against feline viral rhinotracheitis (FVR) which comprises: vaccinating the feline with a recombinant, deletion feline herpesvirus (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding a polypeptide precursor of gl and gE to vaccinate against FVR, wherein the foreign DNA serves as an identification marker for the recombinant, deletion FHV-1.

-5- The method of Claim 4 wherein the foreign DNA encodes beta galactosidase as a marker for the recombinant, deletion FHV-1.

-6- The method of Claims 4 or 5 wherein a 1.3 Kb Xbal- BamHI restriction fragment encoding a 5 • region of the precursor polypeptide of gl is operably linked to the foreign DNA at one end and wherein at an opposite end of the foreign DNA a 0.95 Kb EcoRV-Sall restriction fragment encoding a 3 ' region of the precursor polypeptide of gE is operably linked to the foreign DNA.

-7- The method of Claim 4 wherein vaccination is intramuscularly. -8- The method of Claim 7 wherein the vaccine contains 10⁵ to 10⁶ units of the recombinant, deletion FHV.

-9- A vaccine for feline viral rhinotracheitis (FVR) which comprises: a recombinant, deletion feline herpesvirus (FHV-1) containing foreign DNA encoding a protein foreign to the FHV-1 in place of a portion of the DNA encoding precursor polypeptides of gl and gE in a dosage for treatment of FVR, wherein the foreign DNA serves as an identification marker for the recombinant, deletion FHV-1.

-10- The vaccine of Claim 9 containing between about 10⁵ and 10⁷ units of the recombinant, deletion FHV-1.

-11- A recombinant DNA fragment free of other fragments containing a first DNA segment encoding a 5* portion of a polypeptide precursor of gl of feline herpesvirus (FHV-1) operably linked at one end of a foreign gene and a second DNA segment encoding a 3' portion of a polypeptide precursor of gE of the FHV-1 operably linked at an opposite end to the foreign DNA, wherein the foreign DNA can be operably linked in the FHV-1 in place of DNA encoding a portion of polypeptide precursors of gl and gE to produce a vaccine, and wherein the foreign DNA serves as an identification marker for the recombinant FHV-1. -12- The recombinant DNA fragment of Claim 11 wherein the foreign DNA encodes beta galactosidase as a marker for the recombinant DNA fragment.

-13- The recombinant DNA fragment of Claims 11 or 12 wherein a 1.3 Kb Xbal-BamHI restriction fragment encoding a 5' region of a polypeptide precursor of gl is operably linked to the foreign DNA at one end and wherein at an opposite end of the foreign DNA a 0.95 Kb EcoRV-Sall restriction fragment encoding a 3 ' region of a polypeptide precursor of gE is operably linked to the foreign DNA.

-14-

The recombinant DNA of Claim 13 wherein the first DNA segment is encoded by oligonucleotides 2915 to 4235 of SEQ ID NO:l.

-15- The recombinant DNA of Claim 13 wherein the second DNA sequence is encoded by oligonucleotides 5252 to 6207 of SEQ ID NO:l.

-16- A gene free of other genes encoding a gl precursor polypeptide which comprises ORF 4 of SEQ ID NO: 1.

-17- A gene free of other genes encoding a gE precursor polypeptide which comprises ORF 5 of SEQ ID N0:l. -18- A gl precursor polypeptide which comprises amino acid sequences 3310 to 4419 of SEQ ID NO:l.

-19- A gE precursor polypeptide which comprises amino acid sequences 4601 to 6196 of SEQ ID NO:l.

-20- A purified and isolated polynucleotide having a DNA sequence comprising a 2.1 Kb EcoRV-EcoRV restriction fragment of feline herpesvirus encoding a precursor polypeptide of gl.

-21- A purified and isolated polynucleotide having a DNA sequence comprising a 1.9 Kb EcoRI-Sall restriction fragment of feline herpesvirus encoding a precursor polypeptide of gE.