Technical Field The present invention relates to novel bovine adenovirus (BAV) expression vector systemic in which one or both of the early region 1 (El) and the early region 3 (E3) gene deletions are replaced by a foreign gene and novel recombinant mammalian cell lines stably transformed with BAV El sequences, and therefore, expresses El gene products, to allow a bovine adenovirus with an El gene deletion replaced by a foreign gene to replicate therein. These materials are used in production of recombinant BAV expressing heterologous (antigenic) polypeptides or fragments for the purpose of live recombinant virus or subunit vaccines or for other therapies.

The present invention also relates to novel bovine adenovirus (BAV) expression vector systems comprising BAV genome sequences disclosed herein. The BAV genome sequences can be replaced by one or more foreign genes to generate recombinant BAV expressing heterologous (antigenic) polypeptides or fragments for the purpose of producing live recombinant virus or subunit vaccines or for other therapies. Further, various BAV transcriptional and translational regulatory signals can be used to modulate the expression of foreign genes that have been inserted into the vector systems of the invention. Additionally, the novel sequences of the present invention can be used for diagnostic purposes, to determine the presence of BAV in a subject or biological sample.

Background of the Invention The adenoviruses cause enteric or respiratory infection in humans as well as in domestic and laboratory animals.

The bovine adenoviruses (BAVs) comprise at least nine serotypes divided into two subgroups. These subgroups have been characterized based on enzyme-linked

immunoassays (ELISA). serologic studies with immunofluorescence assays virus- neutralization tests, immunoelectron microscopy, by their host specificity and clinical syndromes. Subgroup 1 viruses include BAV 1, 2, 3 and 9 and grow relatively well in established bovine cells compared to subgroup 2 which includes BAV 4, 5, 6, 7 and 8.

BAV3 was first isolated in 1965 and is the best characterized of the BAV genotypes, containing a genome of approximately 35 kb (Kurokawa et al (1978) J. Virol.

28:212-218). BAV3, a representative of subgroup 1 of BAVs (Bartha (1969)Acta Vet.

Acad. Sci. Hung. 19:319-321), is a common pathogen of cattle usually resulting in subclinical infection (Darbyshire et al. (1965). J. Comp. Patrol. 75:327-330). though occasionally associated with a more serious respiratory tract infection (Darbyshire et al., 1966 Res. Vet Sci 7:81-93; Mattson et al., 1988 J. Vet Res 49:67-69). Like other adenoviruses, BAV3 is a non-enveloped icosahedral particle of 75 nm in diameter (Niiyama ci al. (1975) j Virol. 16:621-633) containing a linear double-stranded DNA molecule. BAV3 can produce tumors when injected into hamsters (Darbyshire, 1966 Nature 211:102) and viral DNA can efficiently effect morphological transformation of mouse, hamster or rat cells in culture (Tsukamoto and Sugino, 1972 J. Virol. 9:465-473; Motoi et al., 1972 Gann 63:415-418; M. Hitt, personal communication). Cross hybridization was observed between BAV3 and human adenovirus type 2 (HAd2) (Hu et al., 1984 J. Virol. 49:604-608) in most regions of the genome including some regions near but not at the left end of the genome.

In the human adenovirus (HAd) genome there are two important regions: El and E3 in which foreign genes can be inserted to generate recombinant adenoviruses (Berkner and Sharp (1984) Nuc. Acid Res., 12:1925-1941 and Haj-Ahmad and Graham (1986) J.

Virol.. 57:267-274). El proteins are essential for virus replication in tissue culture, however, conditional-helper adenovirus recombinants containing foreign DNA in the El region, can be generated in a cell line which constitutively expresses El (Graham et al., (1977) J. Gen. Virol., 36:59-72). In contrast, E3 gene products of HAd 2 and HAd 5 are not required for in vitro or in vivo infectious virion production, but have an important role in host immune responses to virus infection (Andersson et al (1985) Cell 43:215-222; Burgert et al (1987) EMBO J. 6:2019-2026; Carlin et al (1989) Cell 57:135-144; Ginsberg et al (1989) PNAS, USA 86:3823-3827; Gooding et al (1988) Cell 53:341-346; Tollefson et al (1991) J. Virol. 65:3095-3105; Wold and Gooding (1989) Mol. Biol. Med. 6:433-452 and Wold and Gooding (1991) Virology 184:1-8). The E3-19 kiloDalton (kDa)

glycoprotein (gp19) of human adenovirus type 2 (HAd2) binds to the heavy chain of a number of class 1 major histocompatibility complex (MHC) antigens in the endoplasmic reticulum thus inhibiting their transport to the plasma membrane (Andersson et al. (1985) Cell 43:215-922; Burgert and Kvist, (1985) Cell 41:987-997; Burgert and Kvist, (1987) EMBO J. 6:2019-2026). The E3-14.7 kDa protein of HAd2 or HAd5 prevents lysis of virus-infected mouse cells by tumor necrosis factor (TNF) (Gooding et al. (1988) Cell 53:341-346). In addition, the E3-10.4 kDa and E3-14.5 kDa proteins form a complex to induce endosomal-mediated internalization and degradation of the epidermal growth factor receptor (EGF-R) in virus-infected cells (Carlin et al. Cell 57:135-144; Tollefson et al.

(1991) J. Virol. 65:3095-3105). The helper-independent recombinant adenoviruses having foreign genes in the E3 region replicate and express very well in every permissive cell line (Chanda et al (1990) Virology 175:535-547; Dewar et al (1989) J. Virol. 63:129-136; Johnson et al (1988) Virology 164:1-14; Lubeck et al (1989) PNAS USA 86:6763-6767: McDermott et al (1989) Virology 169:244-247; Mittal et al (1993) Virus Res. 28:67-90; Morin et al (1987) PNAS, USA 84:4626-4630; Prevec et al (1990) J. Inf. Dis. 161:27-30; Prevec et al (1989) J. Gen. Virol. 70:429-434; Schneider et al (1989) J. Gen. Virol. 70:417- 427 and Yuasa et al (1991) J. Gen. Virol. 72:1927-1934). Based on the above studies and the suggestion that adenoviruses can package approximately 105% of the wild-type (wt) adenovirus genome (Bett et al (1993) J. Virol. 67:5911-5921 and Ghosh-Choudhury et al (1987) EMBO. J. 6:1733-1739), an insertion of up to 1.8 kb foreign DNA can be packaged into adenovirus particles for use as an expression vector for foreign proteins without any compensating deletion.

The Eel A gene products of the group C human adenoviruses have been very extensively studied and shown to mediate transactivation of both viral and cellular genes (Berk et al., 1979 Cell 17:935-944; Jones and Shenk, 1979 Cell 16:683-689; Nevins, 1981 Cell 26:213-220; Nevins, 1982 Cell 29:913-919; reviewed in Berk, 1986 Ann. Res. Genet 20:45-79), to effect transformation of cells in culture (reviewed in Graham, F.L. (1984) "Transformation by and oncogenicity of human adenoviruses. In: The Adenoviruses." H.S. Ginsberg, Editor. Plenum Press, New York; Branton et al., 1985 Biochim. Biophys.

Acta 780:67-94) and induce cell DNA synthesis and mitosis (Zerler et al., 1987 Mol. Cell Biol. 7:821-929; Bellet et al., 1989 J. Virol. 63:303-310; Howe et al., 1990 PNAS, USA 87:5883-5887; Howe and Bayley, 1992 Virology 186:15-24). The E1A transcription unit comprises two coding sequences separated by an intron region which is deleted from all

processed El A transcripts. In the two largest mRNA species produced from the Eel A transcription unit, the first coding region is further subdivided into exon 1. a sequence found in both the 12s and 13s mRNA species. and the unique region, which is found only in the 13s mRNA species. By comparisons between E1A proteins of human and simian adenoviruses three regions of somewhat conserved protein sequence (CR) have been defined (Kimelman et al., 1985 J. Virol. 53:399-409). CR1 and CR2 are encoded in exon 1. while CR3 is encoded in the unique sequence and a small portion of exon 2.

Binding sites for a number of cellular proteins including the retinoblastoma protein Rb, cyclin A and an associated protein kinase p33Cdk2, and other, as yet unassigned, proteins have been defined in exon 1-encoded regions ofElA proteins (Yee and Branton, 1985 Virology 147:142-153; Harlow et al., 1986 Mol. Cell Biol. 6:1579-1589; Barbeau et al., 1992 Biochem. Cell Biol. 70:1123-1134). Interaction of E 1 A with these cellular proteins has been implicated as the mechanism through which E1A participates in immortalization and oncogenic transformation (Egan et al, 1989 Oncogene 4:383-388; Whyte et al., 1988 Nature 334:124-129; Whyte et al, 1988 J. Virol. 62:257-265). While E1A alone may transform or immortalize cells in culture, the coexpression of both E1A and either the ElB-19k protein or the El B-55k protein separately or together is usually required for high frequency transformation of rodent cells in culture (reviewed in Graham, 1984 supra; Branton et al., 1985 supra; McLorie et al., 1991 J. Gen Virol. 72:1467-1471).

Transactivation of other viral early genes in permissive infection of human cells is principally mediated by the amino acid sequence encoded in the CR3 region ofE1A (Lillie et al. 1986 Cell 46:1043-1051). Conserved cysteine residues in a CysX2CysX13CysX2Cys sequence motif in the unique region are associated with metal ion binding activity (Berg, 1986 supra) and are essential for transactivation activity (Jelsma et al., 1988 Virology 163:494-502; Culp et al., 1988 PNAS, USA 85:6450-6454). As well, the amino acids in CR3 which are immediately amino (N)-terminal to the metal binding domain have been shown to be important in transcription activation, while those immediately carboxy (C)-terminal to the metal binding domain are important in forming associations with the promoter region (Lillie and Green, 1989 Nature 338:39-44; see Fig. 3).

The application of genetic engineering has resulted in several attempts to prepare adenovirus expression systems for obtaining vaccines. Examples of such research include the disclosures in U.S. Patent 4,510,245 on an adenovirus major late promoter for expression in a yeast host; U.S. Patent 4,920,209 on a live recombinant adenovirus type 7

with a gene coding for hepatitis-B surface antigen located at a deleted early region 3; European Patent 389 286 on a non-defective human adenovirus 5 recombinant expression system in human cells for HCMV major envelope glycoprotein: WO 91/11525 on live non-pathogenic immunogenic viable canine adenovirus in a cell expressing E1A proteins; and French Patent 2 642 767 on vectors containing a leader and/or promoter from the E3 region of adenovirus 2.

It is assumed that an indigenous adenovirus vector would be better suited for use as a live recombinant virus vaccine in non-human animal species. as compared to an adenovirus of human origin. This requires that regions suitable for insertion of heterologous sequences be identified in the indigenous adenoviral vector, and that compositions and methods for insertion of heterologous sequence, isolation of recombinants and propagation of recombinants be devised. Regions suitable for insertion could include non-essential regions of a viral genome or essential regions, if an appropriate helper function is provided. For example, if, by analogy to HAds, the E3 regions in other adenoviruses are not essential for virus replication in cultured cells, adenovirus recombinants containing foreign gene inserts in the E3 region could be generated.

The selection of a suitable virus to act as a vector for foreign gene expression, the identification of suitable regions as sites for gene insertion, and the construction, isolation and propagation of recombinant virus pose significant challenges to the development of recombinant viral vaccine vectors. In particular, preferred insertion sites will be non-essential for the viable replication of the virus and its effective operation in tissue culture and also in vivo. Moreover. the insertion sites must be capable of accepting new genetic material, whilst ensuring that the virus continues to replicate. An essential region of a virus genome can also be utilized for foreign gene insertion if the recombinant virus is grown in a cell line which complements the function of that particular essential region in trans.

An efficient method for determining suitable insertion sites in a viral genome is to obtain the complete nucleotide sequence of that genome. This allows the various coding regions to be defined, facilitating their possible use as insertion sites. Definition of nonessential noncoding regions would also be revealed by sequence analysis, and these could also be used as potential insertion sites. The nucleotide sequence of certain regions of the BAV-3 genome has been determined. The sequence of the extreme left end of the genome, including the inverted terminal repeat (ITR), packaging signals, El and pIX, has

been determined by several groups: nucleotides 1-195 (ITR) by Shinagawa et al., 1987.

Gene 55:85-93. nucleotides 1-4060 (ITR, packaging signals. El and pIX) by Zheng et al., 1994. Virus Research 31:163-186; nucleotides 1-4091 (ITR, packaging signals, El and pIX) by Elgadi et al., 1993, Intervirology 36:113-120. (Nucleotide 1 designates the left- most nucleotide of the linear, 34.4 kb BAV-3 genome.) Additional sequences of the BAV- 3 genome that have been determined include: nucleotides 5,235-5,891 (major late promoter, Song et al., 1996. Virology 220:390-401); nucleotides 17,736-20,584 (hexon gene. Hu et al.. 1984, J. Virology 49:604-608); nucleotides 20,408-21,197 (proteinase gene. Cai et al., 1990, Nucleic Acids Res. 18:5568; and nucleotides 26,034-31,132 (E3 region, pVIII and fibre genes, Mittal et al., 1992, J. Gen. Virol. 73:3295-3300).

One of the many uses to which recombinant viruses and viral genomes could be applied, if they were available, is in the development of recombinant subunit vaccines.

Vaccination has proven to be the most effective means for controlling respiratory and enteric viral diseases, especially when live attenuated viral vaccines have been employed.

These vaccines, when administered orally or intranasally, induce strong mucosal immunity, which is required to block the initial infection and to reduce the development of disease caused by these viruses. This approach has been extended by using genetically engineered virus genomes (virulence gene-deleted) as vectors to deliver and express genes of other pathogens in vivo. Ertl et al. (1996) J. Immunol. 156:3579-3582.

A recombinant viral vector system based on human adenoviruses (HAVs) has recently been developed. Graham et al. (1992) in "Vaccines: New approaches to immunological problems" (R.W. Ellis ed.), Butterworth-Heineman, Stoneham, pp. 363- 390. Both replication-defective and replication-competent HAV vectors have been engineered to express various foreign antigens. For review see Grunhaus et al. (1992) Seminar in Virol. 3:237-252; Imler (1995) Vaccine 13:1143-1151. In addition to providing stable foreign gene expression, engineered adenoviruses have been shown to induce humoral, cellular and mucosal immune responses. Buge et al. (1997) J. Virol.

71:8531-8541.

The use of human adenoviruses as vectors for gene therapy has been hampered because of the presence, in the host, of preexisting neutralizing antibodies against HAVs, which may interfere with entry and replication of recombinant virus, and because of the possibility of recombination and/or complementation between recombinant virus and preexisting wild-type HAV in the host. Therefore, animal adenoviruses other than HAV,

which are highly species-specific. are being considered as vectors for gene therapy and recombinant vaccines.

Molecular characterization of bovine adenovirus-3 (BAV3) would aid in the development of bovine adenoviruses as live viral vectors for vaccines and gene therapy, in humans and other mammalian species. Recently, the complete DNA sequence and transcriptional map of the BAV3 genome has been reported. This sequence has been disclosed in the parent U.S. Patent application U.S.S.N. 08/880,234, filed June 23 1997, and in several publications. Baxi et al. (1998) Virus Genes 16:1-4; Lee et al. (1998).

Virus Genes 17:99-100; and Reddy eft awl (1998) J. Virol. 72:1394-1402.

Disclosure of the Invention The present inventors have now completed the sequence of the entire BAV-3 genome comprising 34,446 nucleotides. thereby identifying regions suitable both for insertion of foreign genes and for use as diagnostic probes. The present inventors have also inserted foreign genes into these regions to generate BAV recombinants, and propagated the recombinants. Such recombinants will be useful, for example, as recombinant subunit vaccines for a variety of pathogens, for overexpression of recombinant polypeptides, and for gene therapy purposes.

In one embodiment, the present invention relates to novel bovine adenovirus expression vector systems in which part or all of one or both of the El and E3 gene regions are deleted. It also relates to recombinant mammalian cell lines of bovine origin transformed with El sequences, preferably those of BAV, which constitutively express one or more El gene products to allow bovine adenovirus, having a deletion of part or all of the El gene region replaced by a heterologous nucleotide sequence encoding a foreign gene or fragment thereof, to replicate therein. It further relates to use of these materials in production of heterologous (antigenic) polypeptides or fragments thereof.

In another embodiment, the present invention relates to novel bovine adenovirus expression vector systems that utilize the following regions of the bovine adenovirus genome or fragments thereof: nucleotides 4,092-5,234; nucleotides 5,892-17,735; nucleotides 21,198-26,033 and nucleotides 31,133-34,445. These regions (and fragments thereof) can be used, among other things, for insertion of foreign sequences, for provision of DNA control sequences including transcriptional and translational regulatory sequences, or for diagnostic purposes to detect the presence of viral nucleic acids or proteins encoded by these regions, in a subject or biological sample.

The invention also relates to a method of preparing live recombinant viruses or subunit vaccines, for producing antibodies, cell-mediated and/or mucosal immunity to an infectious organism in a mammal. including bovine, humans and other mammals. The method comprises inserting into the bovine adenovirus genome a gene or gene fragment coding for the antigen which corresponds to said antibodies or which induces said cell-mediated and/or mucosal immunity, together with or without an effective promoter therefor, to produce BAV recombinants.

In another aspect, the invention includes the use of recombinant viruses and recombinant viral vectors for the expression of a DNA sequence or amino acid sequence of interest in a cell system.

Generally, the foreign gene construct is cloned into a nucleotide sequence which represents only a part of the entire viral genome having one or more appropriate deletions.

This chimeric DNA sequence is usually present in a plasmid which allows successful cloning to produce many copies of the sequence. The cloned foreign gene construct can then be included in the complete viral genome, for example, by in vivo recombination following a DNA-mediated cotransfection technique. Incorporation of the cloned foreign gene construct into the viral genome places the foreign gene into a DNA molecule containing replication and packaging signals, allowing generation of multiple copies of the recombinant adenovirus genome that can be packaged into infectious viral particles.

Multiple copies of a coding sequence or more than one coding sequence can be inserted so that the recombinant vector can express more than one foreign protein. The foreign gene can have additions, deletions or substitutions to enhance expression and/or immunological effects of the expressed protein.

The invention also includes an expression system comprising a bovine adenovirus expression vector wherein heterologous nucleotide sequences with or without any exogenous regulatory elements replace the El gene region and/or part or all of the E3 gene region. In another embodiment, the invention includes an expression system wherein one or more regions of the BAV genome are replaced by heterologous sequences, or wherein heterologous nucleotide sequences are introduced into the BAV genome without removal of any BAV sequences. Intergenic regions of the BAV genome comprising DNA regulatory sequences are useful for the expression of homologous and heterologous (i. e., foreign) genes in the practice of the invention.

The invention also includes (A) a recombinant vector system comprising the entire BAV DNA and a plasmid or two plasmids capable of generating a recombinant virus by in vivo homologous recombination following cotransfection of a suitable cell line, comprising BAV DNA representing the entire wild-type BAV genome and a plasmid comprising bovine adenovirus left or right end sequences containing the El or E3 gene regions or bovine adenovirus E2, E4, L1, L2, L3, L4, L5, L6 or L7 sequences, with a heterologous nucleotide sequence encoding a foreign gene or fragment thereof substituted for part or all ofthe El, E2, E3, E4, Ll, L2, L3, L4, L5, L6 or L7 gene regions (i.e., an insertion cassette); (B) a live recombinant bovine adenovirus vector (BAV) system selected from the group consisting of: (a) a system wherein part or all of the El gene region is replaced by a heterologous nucleotide sequence encoding a foreign gene or fragment thereof; (b) a system wherein a part or all of the E3 gene region is replaced by a heterologous nucleotide sequence encoding a foreign gene or fragment thereof; and c) a system wherein part or all of the El gene region and part or all of the E3 gene region are deleted and a heterologous nucleotide sequence encoding a foreign gene or fragment thereof is inserted into at least one of the deletions; C) a recombinant bovine adenovirus (BAV) comprising a deletion of part or all of El gene region, a deletion of part or all of E3 gene region or deletion of both, and inserted into at least one deletion a heterologous nucleotide sequence coding for an antigenic determinant of a disease causing organism; (D) a recombinant bovine adenovirus expression system comprising a deletion of part or all of El, a deletion of part or all of E3, or both deletions, and inserted into at least one deletion a heterologous nucleotide sequence coding for a foreign gene or fragment thereof under control of an expression promoter: or (E) a recombinant bovine adenovirus (BAV) for producing an immune response in a mammalian host comprising: (1) BAV recombinant containing a heterologous nucleotide sequence coding for an antigenic determinant needed to obtain the desired immune response in association with or without (2) an effective promoter to provide expression of said antigenic determinant in immunogenic quantities for use as a live recombinant virus or recombinant protein or subunit vaccine; (F) a mutant bovine adenovirus (BAV) comprising a deletion of part or all of El and/or a deletion of part or all of E3 and/ or a deletion of part or all of at least one of the following regions of the BAV genome: E2, E4, L1, L2, L3, L4, L5, L6 or L7.

In addition to the El and E3 regions, other sites within the BAV genome are also useful for insertion of foreign nucleotide sequences. These include, but are not limited to,

the E2 region. the E4 region, the region between the E4 promoter and the right end of the genome, the late regions (L1-L7), the 33 kD, 52 kD, 100 kD. DBP, pol, pTP and penton genes. and genes IIIA, pV, pVI, pVII, pVIII and pX.

The invention also provides methods and compositions for obtaining, at high efficienecy, viruses whose genomes contain deletions in the E3 region, as well as viruses whose genomes contain insertions of heterologous sequences into a deleted E3 region. In one embodiment, E3-deleted viral genomes (with or without insertion of heterologous sequences) are transfected into a suitable cell line, for example, MDBK cells expressing adenovirus El function or equivalent cells, and recombinant viruses are recovered from the transfected cells. In another embodiment, a segment of the BAV genome containing a deleted E3 region (with or without insertion of heterologous sequences) is allowed to undergo recombination. in a procaryotic cell, with a BAV genome to generate a recombinant BAV genome. For the purposes of The present invention, a BAV genome can be a full-length BAV genome, or it can contain one or more deletions, provided that it comprises one or more BAV replication and/or packaging sequences. The BAV genome segment containing a deleted E3 region can also contain one or more BAV replication and/or packaging sequences. A recombinant BAV genome includes an otherwise full length BAV genome with one or more deletions in particular genomic region(s), as well as BAV genomes, either deleted or undeleted, in which heterologous sequences have been inserted. The recombinant BAV genome is then transfected into a suitable cell line such as, for example, primary fetal bovine retina (PFBR) cells, and recombinant virus is recovered from the transfected cells.

In another aspect, the invention provides recombinant mammalian cell lines stably transformed with BAV El gene region sequences, said recombinant cell lines thereby capable of allowing replication therein of a bovine adenovirus comprising a deletion of part or all of the El or E3 gene regions replaced by a heterologous or homologous nucleotide sequence encoding a foreign gene or fragment thereof. The invention also includes production, isolation and purification of polypeptides or fragments thereof, such as growth factors, receptors and other cellular proteins from recombinant bovine cell lines expressing BAV El gene products.

The invention also includes a method for providing gene therapy to a mammal in need thereof. Gene therapy can be used, for example, to control a gene deficiency, to introduce a therapeutic gene into a host cell, to change the sequence of a mutant gene to

restore wild-tvpe function. to change the sequence of a gene to inactivate its function, to provide exogenous gene function. etc. Gene therapy will be used, for example. in the treatment of cancer, AIDS, other virally-induced pathologies. infectious diseases, hereditary diseases, etc. The process of gene therapy comprises administering to said mammal a live recombinant bovine adenovirus containing a heterologous nucleotide sequence under conditions wherein the recombinant virus vector genome is incorporated into said mammalian genome or is maintained independently and extrachromosomally, to provide expression of the heterologous sequence in a target organ or tissue.

Another aspect of the invention provides a pharmaceutical composition which comprises a therapeutically effective amount of a recombinant virus, recombinant viral vector or recombinant protein in association with or without a pharmaceutically acceptable carrier. One example of such a pharmaceutical composition is a recombinant virus vaccine. The recombinant virus vaccine can be formulated for administration by an oral dosage (e.g. as an enteric coated tablet), by injection or otherwise. More specifically, these include a vaccine for protecting a mammalian host against infection comprising a live recombinant adenovirus or recombinant protein produced by the recombinant adenovirus of the invention wherein the foreign gene or fragment encodes an antigen and formulated with or without a pharmaceutically acceptable carrier. These compositions are capable of expressing antigenic polypeptides or protective antigens, thereby eliciting an immune response to a polypeptide or antigen of interest and providing protection from infection.

Pharmaceutical compositions useful in the practice of the invention may also comprise cells harboring a recombinant BAV vector expressing a polypeptide or antigen of interest, or cells harboring a vector expressing BAV polypeptides or antigens.

The invention also includes methods of producing antibodies, cell-mediated and/or mucosal immunity in a mammal including (1) a method for eliciting an immune response in a mammalian host against an infection comprising: administering a vaccine comprising a live BAV recombinant of the invention wherein the foreign gene or fragment encodes an antigen with or without a pharmaceutically acceptable carrier, and (2) a method for eliciting an immune response in a mammalian host against an infection comprising: administering a vaccine comprising a recombinant antigen prepared by culturing a BAV recombinant wherein the foreign gene or fragment encodes the desired antigen with or without a pharmaceutically acceptable carrier.

The invention additionally provides compositions and methods useful in the practice of diagnostic procedures to detect the presence of BAV DNA and/or BAV- encoded proteins and antigens in a biological sample such as an infected cell or a mammalian subject, or a nucleic acid preparation from these sources. These include but are not limited to BAV genes and coding sequences and fragments thereof, as well as amino acid sequences encoded by the BAV genome and fragments thereof.

The following disclosure will render these and other embodiments of the present invention readily apparent to those of skill in the art. While the disclosure often refers to bovine adenovirus type 3 (BAV3), it should be understood that this is for the purpose of illustration and that the same features apply to bovine adenovirus of other types, e.g., 1, 2, 4, 5, 6, 7 8, and 9; and that the invention described and claimed herein is intended to cover all of these bovine adenovirus types.

Brief Description of the Drawings Figure 1. Complete nucleotide sequence of the bovine adenovirus type-3 (BAV-3) genome.

Figure 2. Transcriptional map of BAV-3. The genome is represented by a solid line numbered from 0 to 100 (map units). Transcripts are represented, with respect to length and direction of transcription, by arrows. The locations ofthe E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, L5, L6 and L7 regions are indicated. Packaging and replication sequences are located in the 195 bp ITR sequences and in a region between the left ITR and the El region.

Figure 3. Construction of a plasmid containing E3-deleted BAV3 genomic DNA.

The plasmid pBAV302 was constructed from different genomic clones as described in Example 3, supra. The origin of the DNA sequences is as follows: plasmid DNA, thin line; BAV3 genomic DNA sequences, thick line. Hollow arrows represent the PCR primers, as follows: a: 5'-ACGCGTCGACTCCTCCTCA (SEQ ID NO 2); b: 5'-TTGACAGCTAGCTTGTTC (SEQ ID NO 3); c: 5'-CCAAGCTTGCATGCCTG (SEQ ID NO 4); and d: 5'-GGCGATATCTCAGCTATAACCGCTC (SEQ ID NO 5).

Figure 4. Restriction enzyme analysis of recombinant BAV3 genomes. The DNAs were obtained from MDBK cells infected with BAV3 (lane b), BAV3.E3d (lanes a and e), BAV3.E3gD (lanes c and f) or BAV3.gDt (lanes d and g). DNA was extracted by

Hirt s method (Hirt (1967).J Mol. Biol. 26:365-369). and digested with BamHI (lanes a and b), NheI (lanes c and d) or NdeI (lanes e, f and g). The 1 kb plus DNA ladder (Gibco/BRL) was used for sizing the viral DNA fragments.

Figure 5. Expression of gD proteins in MDBK cells infected with recombinant BAV3 viruses. (A) Proteins from lysates of radiolabelled MDBK cells uninfected (lane h) or infected with BHV-I (lane a), BAV3.E3d (lanes e, fand g), or BAV3.E3gD (lanes b, c and d) were immunoprecipitated with a pool of gD-specific MAbs and analyzed by SDS- PAGE under reducing conditions. Proteins were labelled from 6 to 16 h (lanes a and h), 36 to 48 h (lanes b and e), 48 to 50 h (lanes c and f), or 60 to 62 h (lanes d and g) post-infection. (B) Proteins from culture medium of radiolabelled MDBK cells infected with BHV-1 (lane a), BAV3.E3d (lanes e, fand g), or BAV3.E3gDt (lanes b, c and d) were immunoprecipitated with a pool of gD-specific MAbs and analyzed by SDS-PAGE under reducing conditions. Proteins were labelled from 6 to 16 h (lane a), 12 to 14 h (lanes b and e), 16 to 18 h (lanes c and f), or 22 to 26 h (lanes d and g), post-infection. Molecular size markers (MW) in kDa.

Figure 6. Antigenic analysis of recombinant gD and gDt proteins. Proteins from lysates (lanes a-e) and culture medium (lanes f-j) of radiolabelled MDBK cells infected with BAV3.E3gD (lane a-e) or BAV3.E3gDt (lanes f-j) recombinant viruses were immunoprecipitated with MAb 136 (lanes a and f), MAb 3E7 (lanes b and g), MAb 4C1 (lanes c and h), MAb 2C8 (lanes d and i), or MAb 3C1 (lanes e and j), and analyzed by SDS-PAGE under reducing conditions. Molecular size marker (MW) in kDa.

Figure 7. Effect of AraC on gD expression in MDBK cells. Proteins from lysates of MDBK cells infected with BAV3.E3gD (lanes a-d) in the presence (lanes a, b) or absence (lanes c, d) of 100 Rg/ml AraC and radiolabelled for 2 h at 22 h (lanes a and c) or 34 h (lanes b and d) post-infection were immunoprecipitated with a pool of gD-specific MAbs and analyzed by SDS-PAGE. Molecular size markers (MW) in kDa.

Figure 8. Antibody responses in cotton rats. Glycoprotein gD (A, B) or BAV3 specific (C, D) IgA (A, C) or IgG (B, D) ELISA titers in sera, lung washes (l.w) and nasal washes (n.w.) 12 days after secondary immunization with recombinant BAV's. Open bars represent BAV3.E3gD, filled bars represent BAV3.E3gDt, and stippled bars represent BAV3.E3d. Error bars represent the standard error of the mean of four animals per group.

Figure 9: Anti-gD IgG titers in calf sera (measured by gD-specific ELISA) at different time points post-immunization with different BAV recombinants. Stippled bars:

animals immunized with BAV3.E3d; open bars: animals immunized with BAV3.E3gD; solid bars: animals vaccinated with BAV3.E3gDt.

Figure 10: Anti-gD IgA titers in calf nasal swabs (measured by gD specific ELISA) at different time points post immunization with different BAV recombinants.

Stippled bars: animals immunized with BAV3.E3d; open bars: animals immunized with BAV3.E3gD; solid bars: animals vaccinated with BAV3.E3gDt.

Figure 11: Temperatures of vaccinated calves observed after BHV-1 challenge.

Filled squares: animals immunized with BAV3.E3d; open triangles: animals immunized with BAV3.E3gD; open circles: animals vaccinated with BAV3.E3gDt.

Figure 12: Observations on the appearance and extent of nasal lesions after BHV- 1 challenge in vaccinated calves. Filled squares: animals immunized with BAV3.E3d; open triangles: animals immunized with BAV3.E3gD; open circles: animals vaccinated with BAV3.E3gDt.

Figure 13: Extent of depression observed in the test and control groups of vaccinated calves. Filled squares: animals immunized with BAV3.E3d; open triangles: animals immunized with BAV3.E3gD.

Figure 14: Isolation of BHV-1 after BHV-1 challenge of test and control groups of vaccinated calves. Filled squares: animals immunized with BAV3.E3d; open triangles: animals immunized with BAV3.E3gD; open circles: animals vaccinated with BAV3.E3gDt.

Modes of Carrying Out the Invention The practice of the present invention will employ, unless otherwise indicated, conventional microbiology, immunology, virology, molecular biology, and recombinant DNA techniques which are within the skill of the art. These techniques are fully explained in the literature. See, g , Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed. (1984)); Nucleic Acid Hvbridization (B. Hames & S. Higgins, eds. (1985)): Transcription and Translation (B. Hames & S. Higgins, eds.

(1984)); Animal Cell Culture (R. Freshney, ed. (1986)); Perbal. A Practical Guide to Molecular Cloning (1984). Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition); vols. I, II & III (1989).

A. Definitions In describing the present invention, the following terminology. as defined below. will be used.

A "replicon" is any genetic element (e.g., plasmid, chromosome. virus) that functions as an autonomous unit of DNA replication in vivo; i.e.. is capable of replication under its own control.

A "vector" is a replicon, such as a plasmid, phage, cosmid or virus, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

By "live virus" is meant, in contradistinction to "killed" virus, a virus which is capable of producing identical progeny in tissue culture and inoculated animals.

A "helper-free virus vector" is a vector that does not require a second virus or a cell line to supply something defective in the vector.

A "double-stranded DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its normal, double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments of DNA from viruses, plasmids, and chromosomes). In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).

A DNA "coding sequence" is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, viral DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

A "transcriptional promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3'

direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at the 3' terminus by the translation start codon (ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often. but not always, contain "TATA" boxes and "CAAT" boxes. Procaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.

DNA "control sequences" refer collectively to promoter sequences, ribosome binding sites, splicing signals, polyadenylation signals. transcription termination sequences, upstream regulatory domains. enhancers. translational termination sequences and the like. which collectively provide for the transcription and translation of a coding sequence in a host cell.

A coding sequence or sequence encoding is "operably linked to" or "under the control of' control sequences in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.

A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. A stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. For mammalian cells, this stability is demonstrated by the ability of the cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.

A "clone" is a population of daughter cells derived from a single cell or common ancestor. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%. and most preferably at least about 95%) of the amino acids match over a defined length of the molecule.

Two DNA sequences are "substantially homologous" when they are identical to or not differing in more that 40% of the nucleotides, preferably not more than about 30% of the nucleotides (i.e. at least about 70% homologous) more preferably about 20% of the nucleotides, and most preferably about 10% of the nucleotides.

DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under. for example, stringent conditions. as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e... Maniatis et al., supra. DNA Cloning, vols. I & II. supra; Nucleic Acid Hybridization. supra.

A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a viral gene, the gene will usually be flanked by DNA that does not flank the viral gene in the genome of the source virus or virus-infected cells. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA. as used herein.

"Bovine host" refers to cattle of any breed, adult or infant.

The term 'protein" is used herein to designate a polypeptide or glycosylated polypeptide, respectively, unless otherwise noted. The term "polypeptide" is used in its broadest sense, i.e., any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term "polypeptide" includes proteins, oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.

"Fusion protein" is usually defined as the expression product of a gene comprising a first region encoding a leader sequence or a stabilizing polypeptide. and a second region encoding a heterologous protein. It involves a polypeptide comprising an antigenic protein fragment or a full length BAV protein sequence as well as (a) heterologous sequence(s), typically a leader sequence functional for secretion in a recombinant host for intracellularly expressed polypeptide, or an N-terminal sequence that protects the protein from host cell

proteases. such as SOD. An antigenic protein fragment is usually about 5-7 amino acids in length.

"Native" proteins or polypeptides refer to proteins or polypeptides recovered from BAV or BAV-infected cells. Thus, the term "native BAV polypeptide" would include naturally occurring BAV proteins and fragments thereof. "Non-native" polypeptides refer to polypeptides that have been produced by recombinant DNA methods or by direct synthesis. "Recombinant" polypeptides refers to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.

A "substantially pure" protein will be free of other proteins, preferably at least 10% homogeneous, more preferably 60% homogeneous, and most preferably 95% homogeneous.

An "antigen" refers to a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is also used interchangeably with "immunogen." A "hapten" is a molecule containing one or more epitopes that does not stimulate a host's immune system to make a humoral or cellular response unless linked to a carrier.

The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds or is recognized by T cells. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

The terms "immunogenic polypeptide" and "immunogenic amino acid sequence" refer to a polypeptide or amino acid sequence, respectively, which elicit antibodies that neutralize viral infectivity, and/or mediate antibody-complement or antibody-dependent cell cytotoxicity to provide protection of an immunized host. An "immunogenic polypeptide" as used herein, includes the full length (or near full length) sequence of the desired protein or an immunogenic fragment thereof.

By "immunogenic fragment" is meant a fragment of a polypeptide which includes one or more epitopes and thus elicits antibodies that neutralize viral infectivity, and/or

mediates antibody-complement or antibody-dependent cell cytotoxicity to provide protection of an immunized host. Such fragments will usually be at least about 5 amino acids in length. and preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence. or even a fusion protein comprising fragments of two or more of the antigens. The term "treatment" as used herein refers to treatment of a mammal, such as bovine or human or other mammal, either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of an infection. The vaccine comprises the recombinant BAV itself or recombinant antigen produced by recombinant BAV.

By "infectious" is meant having the capacity to deliver the viral genome into cells.

B. General Method The present invention discloses the complete nucleotide sequence of the BAV3 genome. See Figure 1. A transcriptional map of the BAV3 genome, derived from transcriptional mapping of mRNAs and sequencing of cDNA clones, is presented in Figure 2. Although the size (34,446 bp) and the overall organization of the BAV3 genome appear to be similar to that of HAVs, there are certain differences. Reddy et al. (1998) supra.

One of the distinctive features of the BAV3 genome is the relatively small size of the E3 coding region (1517 bp). Mittal et al. (1992) J. Gen. Virol. 73:3295-3300; Mittal et al.

(1993).J. Gen. Virol. 74:2825; and Reddy et al. (1998) supra. Analysis of the sequence of the BAV3 E3 region and its RNA transcripts suggests that BAV3 E3 may encode at least four proteins, one of which (121R) exhibits limited homology with the 14.7 kDa protein of HAV5. Idamakanti (1998) "Molecular characterization of E3 region of bovine adenovirus-3," M.Sc. thesis, University of Saskatchewan, Saskatoon, Saskatchewan.

In one embodiment, the present invention identifies and provides a means of deleting part or all of the nucleotide sequence of bovine adenovirus El and/or E3 regions to provide sites into which heterologous or homologous nucleotide sequences encoding foreign genes or fragments thereof can be inserted to generate bovine adenovirus recombinants. By "deleting part of' the nucleotide sequence is meant using conventional genetic engineering techniques for deleting the nucleotide sequence of part of the El and/or E3 region.

In another embodiment, the invention provides compositions and methods for constructing, isolating and propagating E3-deleted recombinant BAV3 (with or without

insertion of heterologous sequences) at high efficiency. These include isolation of recombinant virus in suitable cell lines, such as MDBK cells expressing adenovirus El function or equivalent cell lines. and methods wherein recombinant BAV genomes are constructed via homologous recombination in procaryotic cells, the recombinant genomes obtained thereby are transfected into suitable cell lines such as. for example, primary fetal bovine retina cells or their equivalent, and recombinant virus is isolated from the transfected cells.

In addition, the construction of recombinant BAV3 expressing different forms of BHV- 1 glycoprotein gD are provided. and it is shown that intranasal immunization of cotton rats with gD-expressing recombinant BAV viruses leads to the induction of gD-specific mucosal and systemic immune responses. See Example 3. Intranasal immunization of bovine hosts with BAV recombinants containing BHV- 1 gD genes provides protection against BHV-l challenge in calves. reducing the occurrence of clinical signs, facilitating more rapid clearance of virus, and providing increased titers of both IgG and IgA. See Example 4. In similar fashion, any genes encoding protective determinants of a mammalian pathogen can be inserted into E3-deleted BAV, and the resulting recombinant BAV can be used as a vaccine.

In one embodiment of the invention, a recombinant BAV expression cassette can be obtained by cleaving a wild-type BAV genome with one or more appropriate restriction enzyme(s) to produce a BAV restriction fragment comprising El or E3 region sequences, respectively. The BAV restriction fragment can be inserted into a cloning vehicle, such as a plasmid. and thereafter at least one heterologous sequence (which may or may not encode a foreign protein) can be inserted into the El or E3 region with or without an operatively-linked eukaryotic transcriptional regulatory sequence. The recombinant expression cassette is contacted with a BAV genome and. through homologous recombination or other conventional genetic engineering methods, the desired recombinant is obtained. In the case wherein the expression cassette comprises the El region or some other essential region, recombination between the expression cassette and a BAV genome can occur within an appropriate helper cell line such as, for example, an El-transformed cell line. Restriction fragments of the BAV genome other than those comprising the El or E3 regions are also useful in the practice of the invention and can be inserted into a cloning vehicle such that heterologous sequences can be inserted into the BAV sequences. These

DNA constructs can then undergo recombination in vitro or in vivo. with a BAV genome either before or after transformation or transfection of an appropriate host cell.

Suitable host cells include any cell that will support recombination between a BAV genome and a plasmid containing BAV sequences, or between two or more plasmids, each containing BAV sequences. Recombination is generally performed in procaryotic cells, such as E. coli, while transfection of a plasmid containing a viral genome, to generate virus particles, is conducted in eukaryotic cells, preferably mammalian cells, more preferably bovine cell cultures, most preferably MDBK or PFBR cells, and their equivalents. The growth of bacterial cell cultures, as well as culture and maintenance of eukaryotic cells and mammalian cell lines are procedures which are well-known to those of skill in the art.

One or more heterologous sequences can be inserted into one or more regions of the BAV genome to generate a recombinant BAV vector, limited only by the insertion capacity of the BAV genome and ability of the recombinant BAV vector to express the inserted heterologous sequences. In general, adenovirus genomes can accept inserts of approximately 5% of genome length and remain capable of being packaged into virus particles. The insertion capacity can be increased by deletion of non-essential regions and/or deletion of essential regions whose function is provided by a helper cell line.

In one embodiment of the invention, insertion can be achieved by constructing a plasmid containing the region of the BAV genome into which insertion is desired. The plasmid is then digested with a restriction enzyme having a recognition sequence in the BAV portion of the plasmid, and a heterologous sequence is inserted at the site of restriction digestion. The plasmid, containing a portion of the BAV genome with an inserted heterologous sequence, is co-transformed, along with a BAV genome or a linearized plasmid containing a BAV genome, into a bacterial cell (such as, for example, E. coli), wherein the BAV genome can be a full-length genome or can contain one or more deletions. Homologous recombination between the plasmids generates a recombinant BAV genome containing inserted heterologous sequences.

Deletion of BAV sequences, to provide a site for insertion of heterologous sequences or to provide additional capacity for insertion at a different site, can be accomplished by methods well-known to those of skill in the art. For example, for BAV sequences cloned in a plasmid, digestion with one or more restriction enzymes (with at least one recognition sequence in the BAV insert) followed by ligation will, in some cases, result in deletion of sequences between the restriction enzyme recognition sites.

Alternatively. digestion at a single restriction enzyme recognition site within the BAV insert. followed by exonuclease treatment, followed by ligation will result in deletion of BAV sequences adjacent to the restriction site. A plasmid containing one or more portions of the BAV genome with one or more deletions, constructed as described above, can be co-transfected into a bacterial cell along with a BAV genome (full-length or deleted) or a plasmid containing either a full-length or a deleted BAV genome to generate, by homologous recombination, a plasmid containing a recombinant BAV genome with a deletion at one or more specific sites. BAV virions containing the deletion can then be obtained by transfection of mammalian cells (including. but not limited to, MDBK or PFBR cells and their equivalents) with the plasmid containing the recombinant BAV genome.

In one embodiment of the invention insertion sites are adjacent to and downstream (in the transcriptional sense) of BAV promoters. Locations of BAV promoters, and restriction enzyme recognition sequences downstream of PAV promoters, for use as insertion sites, can be easily determined by one of skill in the art from the BAV nucleotide sequence provided herein. Alternatively, various in vitro techniques can be used for insertion of a restriction enzyme recognition sequence at a particular site, or for insertion of heterologous sequences at a site that does not contain a restriction enzyme recognition sequence. Such methods include, but are not limited to, oligonucleotide-mediated heteroduplex formation for insertion of one or more restriction enzyme recognition sequences (see, for example, Zoller et al. (1982) NucleicAcids Res. 10:6487-6500; <BR> <BR> <BR> Brennan et al. (1990) Roux Arch. Dev. Biol. 199:89-96; and Kunkel et al. (1987) Meth.

Enzynlology 154:367-382) and PCR-mediated methods for insertion of longer sequences.

See, for example, Zheng et al. (1994) Virus Research 31:163-186.

It is also possible to obtain expression of a heterologous sequence inserted at a site that is not downstream from a BAV promoter, if the heterologous sequence additionally comprises transcriptional regulatory sequences that are active in eukaryotic cells. Such transcriptional regulatory sequences can include cellular promoters such as, for example, the bovine hsp70 promoter and viral promoters such as, for example, herpesvirus, adenovirus and papovavirus promoters and DNA copies of retroviral long terminal repeat (LTR) sequences.

In another embodiment. homologous recombination in a procaryotic cell can be used to generate a cloned BAV genome; and the cloned BAV genome can be propagated

as a plasmid. Infectious virus can be obtained by transfection of mammalian cells with the cloned BAV genome rescued from plasmid-containing cells.

The invention also provides BAV regulatory sequences which can be used to regulate the expression of heterologous genes. A regulatory sequence can be, for example, a transcriptional regulatory sequence, a promoter, an enhancer. an upstream regulatory domain, a splicing signal, a polyadenylation signal, a transcriptional termination sequence, a translational regulatory sequence, a ribosome binding site and a translational termination sequence.

In another embodiment, the invention identifies and provides additional regions of the BAV genome (and fragments thereof) suitable for insertion of heterologous or homologous nucleotide sequences encoding foreign genes or fragments thereof to generate BAV recombinants. These regions include nucleotides 4,092-5.234; nucleotides 5,892- 17,735; nucleotides 21,198-26.033 and the region extending from nucleotide 31,133 to the right end of the BAV genome and comprise the E2 region, the E4 region. the late region, the 33 kD, 52 kD, 100 kD, DBP, pol, pTP and penton genes, and genes IIIA, pV, pVI, pVII, pVIII and pX. These regions of the BAV genome can be used, among other things, for insertion of foreign sequences, for provision of DNA control sequences including transcriptional and translational regulatory sequences, or for diagnostic purposes to detect the presence of viral nucleic acids or proteins encoded by these regions, in a biological sample.

In another embodiment, the cloned BAV-3 genome can be propagated as a plasmid and infectious virus can be rescued from plasmid-containing cells.

The presence of viral nucleic acids can be detected by techniques known to one of skill in the art including, but not limited to, hybridization assays, polymerase chain reaction, and other types of amplification reactions. Similarly, methods for detection of proteins are well-known to those of skill in the art and include, but are not limited to, various types of immunoassay, ELISA, Western blotting, enzymatic assay, immunohistochemistry, etc. Diagnostic kits comprising the nucleotide sequences of the invention may also contain reagents for cell disruption and nucleic acid purification, as well as buffers and solvents for the formation, selection and detection of hybrids.

Diagnostic kits comprising the polypeptides or amino acid sequences of the invention may also comprise reagents for protein isolation and for the formation, isolation, purification and/or detection of immune complexes.

Various foreign genes or nucleotide sequences or coding sequences (prokaryotic, and eukaryotic) can be inserted in the bovine adenovirus nucleotide sequence, e.g.. DNA, in accordance with the present invention, particularly to provide protection against a wide range of diseases and many such genes are already known in the art. The problem heretofore has been to provide a safe, convenient and effective vaccine vector for the genes or sequences, as well as safe, effective means for gene transfer to be used in various gene therapy applications.

An exogenous (i.e., foreign) nucleotide sequence can consist of one or more gene(s) of interest, and preferably of therapeutic interest. In the context of the present invention, a gene of interest can code either for an antisense RNA, a ribozyme or for an mRNA which will then be translated into a protein of interest. A gene of interest can be of genomic type. of complementary DNA (cDNA) type or of mixed type (minigene, in which at least one intron is deleted). It can code for a mature protein. a precursor of a mature protein, in particular a precursor intended to he secreted and accordingly comprising a signal peptide, a chimeric protein originating from the fusion of sequences of diverse origins, or a mutant of a natural protein displaying improved or modified biological properties. Such a mutant may be obtained by, deletion, substitution and/or addition of one or more nucleotide(s) of the gene coding for the natural protein, or any other type of change in the sequence encoding the natural protein, such as, for example, transposition or inversion.

A gene of interest may be placed under the control of elements (DNA control sequences) suitable for its expression in a host cell. Suitable DNA control sequences are understood to mean the set of elements needed for transcription of a gene into RNA (antisense RNA or mRNA) and for the translation of an mRNA into protein. Among the elements needed for transcription, the promoter assumes special importance. It can be a constitutive promoter or a regulatable promoter, and can he isolated from any gene of eukaryotic, prokaryotic or viral origin, and even adenoviral origin. Alternatively, it can be the natural promoter of the gene of interest. Generally speaking, a promoter used in the present invention may be modified so as to contain regulatory sequences. As examples, a gene of interest in use in the present invention is placed under the control of the promoter of the immunoglobulin genes when it is desired to target its transfer to lymphocytic host cells. There may also be mentioned the HSV-1 TK (herpesvirus type 1 thymidine kinase) gene promoter, the adenoviral MLP (major late promoter), in particular of human

adenovirus type 2. the RSV (Rous Sarcoma Virus) LTR (long terminal repeat). the CMV (Cytomegalovirus) early promoter, and the PGK (phosphoglycerate kinase) gene promoter. for example. permitting expression in a large number of cell types.

Alternatively, targeting of a recombinant BAV vector to a particular cell type can be achieved by constructing recombinant hexon and/or fiber genes. The protein products of these genes are involved in host cell recognition; therefore. the genes can be modified to contain peptide sequences that will allow the virus to recognize alternative host cells.

Among genes of interest which are useable in the context of the present invention, there may be mentioned: - genes coding for cytokines such as interferons and interleukins; - genes encoding lymphokines; - genes coding for membrane receptors such as the receptors recognized by pathogenic organisms (viruses. bacteria or parasites), preferably by the HIV virus (human immunodeficiency virus); - genes coding for coagulation factors such as factor VIII and factor IX; - genes coding for dystrophins; - genes coding for insulin; - genes coding for proteins participating directly or indirectly in cellular ion channels, such as the CFTR (cystic fibrosis transmembrane conductance regulator) protein; - genes coding for antisense RNAs, or proteins capable of inhibiting the activity of a protein produced by a pathogenic gene which is present in the genome of a pathogenic organism, or proteins (or genes encoding them) capable of inhibiting the activity of a cellular gene whose expression is deregulated, for example an oncogene; - genes coding for a protein inhibiting an enzyme activity, such as ai-antitrypsin or a viral protease inhibitor, for example; - genes coding for variants of pathogenic proteins which have been mutated so as to impair their biological function, such as, for example, trans-dominant variants of the tat protein of the HIV virus which are capable of competing with the natural protein for binding to the target sequence, Thereby preventing the activation of HIV; - genes coding for antigenic epitopes in order to increase the host cell's immunity; - genes coding for major histocompatibility complex classes I and II proteins, as well as the genes coding for the proteins which are inducers of these genes; - genes coding for antibodies;

- genes coding for immunotoxins; - genes encoding toxins; - genes encoding growth factors or growth hormones; - genes encoding cell receptors and their ligands; - genes encoding tumor suppressors; - genes involved in cardiovascular disease including. but not limited to, oncogenes; genes encoding growth factors including, but not limited to. fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF); e-nos, tumor suppressor genes including, but not limited to, the Rb (retinoblastoma) gene; lipoprotein lipase; superoxide dismutase (SOD); catalyse: oxygen and free radical scavengers, apolipoproteins; and pai-l (plasminogen activator inhibitor-1); - genes coding for cellular enzymes or those produced by pathogenic organisms; and - suicide genes. The HSV-l TK suicide gene may be mentioned as an example.

This viral TK enzyme displays markedly greater affinity compared to the cellular TK enzyme for certain nucleoside analogues (such as acyclovir or gancyclovir). It converts them to monophosphorylated molecules, which can themselves be converted by cellular enzymes to nucleotide precursors, which are toxic. These nucleotide analogues can be incorporated into replicating DNA molecules, hence incorporation occurs chiefly in the DNA of dividing cells. This incorporation can result in specific destruction of dividing cells such as cancer cells.

This list is not restrictive, and other genes of interest may be used in the context of the present invention.

It is also possible that only fragments of nucleotide sequences of genes can be used (where these are sufficient to generate a protective immune response or a specific biological effect) rather than the complete sequence as found in the wild-type organism.

Where available, synthetic genes or fragments thereof can also be used. However, the present invention can be used with a wide variety of genes, fragments and the like, and is not limited to those set out above.

In some cases the gene for a particular antigen can contain a large number of introns or can be from an RNA virus, in these cases a complementary DNA copy (cDNA) can be used.

In order for successful expression of the gene to occur. it can be inserted into an expression vector together with a suitable promoter including enhancer elements and polyadenylation sequences. A number of eucaryotic promoter and polyadenylation sequences which provide successful expression of foreign genes in mammalian cells and how to construct expression cassettes are known in the art. for example in U.S. Patent 5,151,267, the disclosures of which are incorporated herein by reference. The promoter is selected to give optimal expression of immunogenic protein which in turn satisfactorily leads to humoral, cell mediated and mucosal immune responses according to known criteria.

The foreign protein produced by expression in vivo in a recombinant virus-infected cell may be itself immunogenic. More than one foreign gene can be inserted into the viral genome to obtain successful production of more than one effective protein.

Thus with the recombinant viruses of the present invention, it is possible to provide protection against a wide variety of diseases affecting cattle, humans and other mammals.

Any of the recombinant antigenic determinants or recombinant live viruses of the invention can be formulated and used in substantially the same manner as described for the antigenic determinant vaccines or live vaccine vectors.

The present invention also includes pharmaceutical compositions comprising a therapeutically effective amount of a recombinant vector, recombinant virus or recombinant protein, prepared according to the methods of the invention, in combination with a pharmaceutically acceptable vehicle and/or an adjuvant. Such a pharmaceutical composition can be prepared and dosages determined according to techniques that are well-known in the art. The pharmaceutical compositions of the invention can be administered by any known administration route including, but not limited to, systemically (for example, intravenously, intratracheally, intravascularly, intrapulmonarilly, intraperitoneally, intranasally, parenterally, enterically, intramuscularly, subcutaneously, intratumorally or intracranially) or by aerosolization or intrapulmonary instillation.

Administration can take place in a single dose or in doses repeated one or more times after certain time intervals. The appropriate administration route and dosage will vary in accordance with the situation (for example, the individual being treated, the disorder to be treated or the gene or polypeptide of interest), but can be determined by one of skill in the art.

The invention also encompasses a method of treatment. according to which a therapeutically effective amount of a BAV vector, recombinant BAV, or host cell of the invention is administered to a mammalian subject requiring treatment.

The antigens used in the present invention can be either native or recombinant antigenic polypeptides or fragments. They can be partial sequences, full-length sequences, or even fusions (e.g., having appropriate leader sequences for the recombinant host, or with an additional antigen sequence for another pathogen). The preferred antigenic polypeptide to be expressed by the virus systems of the present invention contain full- length (or near full-length) sequences encoding antigens. Alternatively, shorter sequences that are antigenic (i.e., encode one or more epitopes) can be used. The shorter sequence can encode a "neutralizing epitope," which is defined as an epitope capable of eliciting antibodies that neutralize virus infectivity in an in vitro assay. Preferably the peptide should encode a "protective epitope" that is capable of raising in the host a "protective immune response;" i.e., an antibody- and/or a cell-mediated immune response that protects an immunized host from infection.

The antigens used in the present invention, particularly when comprised of short oligopeptides, can be conjugated to a vaccine carrier. Vaccine carriers are well known in the art: for example, bovine serum albumin (BSA), human serum albumin (HSA) and keyhole limpet hemocyanin (KLH). A preferred carrier protein, rotavirus VP6, is disclosed in EPO Pub. No. 0259149, the disclosure of which is incorporated by reference herein.

Genes for desired antigens or coding sequences thereof which can be inserted include those of organisms which cause disease in mammals, particularly bovine pathogens such as bovine rotavirus, bovine coronavirus, bovine herpes virus type 1, bovine respiratory syncytial virus, bovine parainfluenza virus type 3 (BPI-3), bovine diarrhea virus, Pasteurella haemolytica, Haemophilus somnus and the like. Genes encoding antigens of human pathogens also useful in the practice of the invention. The vaccines of the invention carrying foreign genes or fragments can also be orally administered in a suitable oral carrier, such as in an enteric-coated dosage form. Oral formulations include such normally-employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like. Oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, containing

from about 10% to about 95% of the active ingredient. preferably about 25% to about 70%. Oral and/or intranasal vaccination may be preferable to raise mucosal immunity (which plays an important role in protection against pathogens infecting the respiratory and gastrointestinal tracts) in combination with systemic immunity.

In addition, the vaccine can be formulated into a suppository. For suppositories, the vaccine composition will include traditional binders and carriers, such as polyalkaline glycols or triglycendes. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.

Protocols for administering to animals the vaccine composition(s) of the present invention are within the skill of The art in view of the present disclosure. Those skilled in the art will select a concentration of the vaccine composition in a dose effective to elicit an antibody and or T-cell mediated immune response to the antigenic fragment. Within wide limits, the dosage is not believed to be critical. Typically, the vaccine composition is administered in a manner which will deliver between about 1 to about 1,000 micrograms of the subunit antigen in a convenient volume of vehicle, e.g., about 1-10 cc. Preferably, the dosage in a single immunization will deliver from about 1 to about 500 micrograms of subunit antigen, more preferably about 5-10 to about 100-200 micrograms (e.g., 5-200 micrograms).

The timing of administration may also be important. For example, a primary inoculation preferably may be followed by subsequent booster inoculations if needed. It may also be preferred, although optional, to administer a second booster immunization to the animal several weeks to several months after the initial immunization. To insure sustained high levels of protection against disease, it may be helpful to readminister a booster immunization to the animals at regular intervals, for example once every several years. Alternatively, an initial dose may be administered orally followed by later inoculations, or vice versa. Preferred vaccination protocols can be established through routine vaccination protocol experiments.

The dosage for all routes of administration of in vivo recombinant virus vaccine depends on various factors including, the size of patient, nature of infection against which protection is needed, carrier and the like and can readily be determined by those of skill in the art. By way of non-limiting example, a dosage of between 103 pfu and 1 10'j pfu, preferably between 105 and 1013 pfu, more preferably between 106 to 1011 pfu and the like

can be used. As with in vitro subunit vaccines. additional dosages can be given as determined bv the clinical factors involved.

In one embodiment of the invention, a number of recombinant cell lines are produced according to the present invention by constructing an expression cassette comprising the BAV El region and transforming host cells therewith to provide complementing cell lines or cultures expressing the El proteins. These recombinant complementing cell lines are capable of allowing a defective recombinant BAV with deleted El sequences to replicate and express a desired foreign gene or fragment thereof which is optionally encoded within the recombinant BAV. These cell lines are also extremely useful in generating recombinant BAV, having an E3 gene deletion replaced by heterologous nucleotide sequence encoding for a foreign gene or fragment, by in vivo recombination following DNA-mediated cotransfection. More generally. defective recombinant BAV vectors lacking one or more essential functions encoded by the BAV genome, can be propagated in appropriate complementing cell lines wherein a particular complementing cell line provides a function or functions that is (are) lacking in a particular defective recombinant BAV vector. Complementing cell lines can provide viral functions through, for example, co-infection with a helper virus, or by integrating or otherwise maintaining in stable form a fragment of a viral genome encoding a particular viral function.

In one embodiment of the invention, the recombinant expression cassette can be obtained by cleaving a BAV genome with an appropriate restriction enzyme to produce a DNA fragment representing the left end or the right end of the genome comprising El or E3 gene region sequences, respectively and inserting the left or right end fragment into a cloning vehicle, such as a plasmid, and thereafter inserting at least one heterologous DNA sequence into the El or E3 deletion with or without the control of an exogenous promoter.

The recombinant expression cassette is contacted with a BAV genome within an appropriate cell and, through homologous recombination or other conventional genetic engineering method, a recombinant BAV genome is obtained. Appropriate cells include both prokaryotic cells, such as, for example, E. coli, and eukaryotic cells. Examples of suitable eukaryotic cells include but are not limited to, MDBK cells, MDBK cells expressing adenovirus El function, primary fetal bovine retina cells, and cells expressing functions that are equivalent to those of the previously-recited cells. Restriction fragments of the BAV genome other than those comprising the El or E3 regions are also useful in the

practice of the invention and can be inserted into a cloning vehicle such that heterologous sequences may be inserted into non-El and E3 BAV sequences. These DNA constructs can then undergo recombination in vitro or in vivo, with a BAV genome. either before or after transformation or transfection of a suitable host cell as described above. For the purposes of the present invention, a BAV genome can be either a full-length genome or a genome containing a deletion in a region other than that deleted in the fragment with which it recombines, as long as the resulting recombinant BAV genome contains BAV sequences required for replication and packaging. Methods for transfection cell culture and recombination in procaryotic and eukaryotic cells such as those described above are well-known to those of skill in the art.

In another embodiment of the invention, El function (or the function of any other viral region which may be mutated or deleted in any particular viral vector) can be supplied (to provide a complementing cell line) by co-infection of cells with a virus which expresses the function that the vector lacks.

The invention also includes an expression system comprising a bovine adenovirus expression vector wherein a heterologous nucleotide sequence, e.g. DNA, replaces part or all of the E3 region, part or all of the El region, part or all of the E2 region, part or all of the E4 region, part or all of the region between E4 and the right end of the genome, part or all of the late regions (L1-L7) and/or part or all of the regions occupied by the 33 kD, 52 kD, 100 kD, DBP, pol, pTP and penton genes, and genes IIIA, pV, pVI, pVII, pVIII and pX. The expression system can be used wherein the foreign nucleotide sequences, e.g.

DNA. is with or without the control of any other heterologous promoter. BAV expression vectors can also comprise inverted terminal repeat (ITR) sequences and packaging sequences.

The BAV 33 kD, 52 kD, 100 kD, DBP, pTP, penton (III), pIIIA, pIVa2, pV, pVI, pVII, pVIII and pX genes are essential for viral replication. Therefore, BAV vectors comprising deletions in any of these genes, or which lack functions encoded by any of these genes, must be grown in an appropriate complementing cell line (i. e., a helper cell line). In human adenoviruses, certain open reading frames in the E4 region (ORF 3 and ORF 6/7) are essential for viral replication. Deletions in analogous open reading frames in the E4 region of BAV-3 could necessitate the use of a helper cell line for growth of the viral vector.

The BAV El gene products of the adenovirus of the invention transactivate most of the cellular genes, and therefore. cell lines which constitutively express El proteins can express cellular polypeptides at a higher level than normal cell lines. The recombinant mammalian, particularly bovine, cell lines of the invention can be used to prepare and isolate polypeptides, including those such as (a) proteins associated with adenovirus E1A proteins: e.g. p300, retinoblastoma (Rb) protein, cyclins, kinases and the like; (b) proteins associated with adenovirus E1B protein: e.g. p53 and the like; growth factors, such as epidermal growth factor (EGF), transforming growth factor (TGF) and the like; (d) receptors such as epidermal growth factor receptor (EGF-R). fibroblast growth factor receptor (FGF-R), tumor necrosis factor receptor (TNF-R), insulin-like growth factor receptor (IGF-R). major histocompatibility complex class I receptor and the like; (e) proteins encoded by proto-oncogenes such as protein kinases (tyrosine-specific protein kinases and protein kinases specific for serine or threonine), p2 1 proteins (guanine nucleotide-binding proteins with GTPase activity) and the like; (f) other cellular proteins such as actins, collagens, fibronectins, integrins, phosphoproteins, proteoglycans, histones and the like, and (g) proteins involved in regulation of transcription such as TATA-box- binding protein (TBP), TBP-associated factors (TAFs), Sp 1 binding protein and the like.

The invention also includes a method for providing gene therapy to a mammal, such as a bovine or a human or other mammal in need thereof, to control a gene deficiency, to provide a therapeutic gene or nucleotide sequence and/or to induce or correct a gene mutation. The method can be used, for example, in the treatment of conditions including, but not limited to hereditary disease, infectious disease, cardiovascular disease, and viral infection. The method comprises administering to said mammal a live recombinant bovine adenovirus containing a foreign nucleotide sequence encoding a non- defective form of said gene under conditions wherein the recombinant virus vector genome is incorporated into said mammalian genome or is maintained independently and extrachromosomally to provide expression of the required gene in the target organ or tissue. These kinds of techniques are currently being used by those of skill in the art for the treatment of a variety of disease conditions, non-limiting examples of which are provided above. Examples of foreign genes, nucleotide sequences or portions thereof that can be incorporated for use in a conventional gene therapy include, cystic fibrosis transmembrane conductance regulator gene, human minidystrophin gene, alpha- 1- antitrypsin gene, genes involved in cardiovascular disease, and the like.

In particular. the practice of the present invention in regard to gene therapy in humans is intended for the prevention or treatment of diseases including, but not limited to, genetic diseases (for example. hemophilia, thalassemias, emphysema. Gaucher's disease, cystic fibrosis, Duchenne muscular dystrophy, Duchenne s or Becker's myopathy, etc.), cancers. viral diseases (for example, AIDS, herpesvirus infection, cytomegalovirus infection and papillomavirus infection), cardiovascular diseases, and the like. For the purposes of the present invention, the vectors, cells and viral particles prepared by the methods of the invention may be introduced into a subject either ex vivo, (i. e., in a cell or cells removed from the patient) or directly in vivo into the body to be treated. Preferably, the host cell is a human cell and. more preferably, is a lung, fibroblast, muscle, liver or lymphocytic cell or a cell of the hematopoietic lineage.

Examples Described below are examples of the present invention. These examples are provided only for illustrative purposes and are not intended to limit the scope of the present invention in any way. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art. The contents of the references cited in the specification are incorporated by reference herein.

Additional methods and techniques can be found in co-owned application PCT/CA94/00678, published as WO 95/16048.

Example 1: Determination of the nucleotide sequence of nucleotides 4,092-5,234; nucleotides 5,892-17,735; nucleotides 21,198-26,033 and nucleotides 31,133-34,445.

To complete the nucleotide sequence of the bovine adenovirus type 3 genome, the following BAV-3 restriction fragments were cloned into bacterial plasmids and their nucleotide sequences were determined by methods known to those of skill in the art.

Hind III B 11.7 - 26.3 m.u.

Hind III K 26.3 - 30.8 m.u.

Hind III A 30.8 - 53.2 m.u.

Eco RI - Hind III 62.5 - 64.5 m.u.

Hind III D 64.3 - 73.7 m.u.

Hind III - Bam HI 73.7 - 76.6 m.u.

Bam HI - C 84.9- 100 m.u.

Example 2: Insertions in the regions of the BAV-3 genome defined by nucleotides 4,092-5,234; nucleotides 5,892-17,735; nucleotides 21,198-26,033 and nucleotides 31,133-34,445.

Insertions are made by art-recognized techniques including, but not limited to, restriction digestion, nuclease digestion, ligation, kinase and phosphatase treatment, DNA polymerase treatment, reverse transcriptase treatment, and chemical oligonucleotide synthesis. Foreign nucleic acid sequences of interest are cloned into plasmid vectors such that the foreign sequences are flanked by sequences having substantial homology to a region of The BAV genome into which insertion is to be directed. These constructs are then introduced into host cells that are coinfected with BAV-3. During infection, homologous recombination between these constructs and BAV genomes will occur to generate recombinant BAV vectors. If the insertion occurs in an essential region of the BAV genome. the recombinant BAV vector is propagated in a helper cell line which supplies the viral function that was lost due to the insertion. For insertions in the E4 region, which is non-essential for viral replication, propagation of BAV vectors in a helper cell line is not necessary.

Example 3: Construction and characterization of E3-deleted BAV3 expressing full length and truncated forms of bovine herpesvirus 1 glycoprotein gD This example demonstrates the construction of a 1.245 kb deletion in the E3 region of BAV3, using the homologous recombination machinery of E. coli. First, a 1.245 kb deletion was introduced in the E3 region of bovine adenovirus-3 (BAV3) genomic DNA cloned in a plasmid. Transfection of linear, restriction enzyme-excised, E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3.E3d) suggesting that all BAV E3-specific open reading frames are non-essential for virus replication in vitro. Using a similar approach, replication-competent BAV3 recombinants expressing full length (BAV3.E3gD) or truncated (BAV3.E3gDt) glycoprotein D of bovine herpesvirus-l (BHV- 1) were constructed. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in BHV- 1 -infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results exemplify the use of replication-competent

bovine adenovirus-3-based vectors for the delivery of vaccine antigens to the mucosal surfaces of animals.

MATERIALS AND METHODS Cells and viruses. Madin Darby bovine kidney (MDBK) cells and primary fetal bovine retina (PFBR) cells were grown in Eagles minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). The wild type (WBR-1 strain) and recombinant BAV3 viruses were propagated in MDBK cells as described previously.

Mittal et al (1995) J. Gen. Virol. 76:93-102. The P8-2 strain of BHV-I was propagated and quantitated as described. Rouse et al. (1974) Jlmmunol. 113:1391-1398.

Animals. An inbred colony of cotton rats (Sigmodon hispidus) maintained at the Veterinary Infectious Disease Organization (Saskatoon) was the source of animals for this study.

Construction of recombinant plasmids. a) Construction of plasmid pBAV302. See Figure 3. A T4 polymerase-treated 587 bp fragment isolated by PCR amplification, using oligonucleotides E3C5' ACGCGTCGACTCCTCCTCA (SEQ ID NO. 2) and E3C3' TTGACAGCTAGCTTGTTG (SEQ ID NO. 3) and plasmid pSM14 (Mittal et al. (1995) supra) as a template, was digested with SacII and ligated to Eco47III-SacII-digested plasmid pSL30l, creating plasmid pE3A. A 164 bp fragment isolated by PCR amplification. using oligonucleotides E3N5' CCAAGCTTGCATGCCTG (SEQ ID NO. 4) and E3N3' GGCGATATCTCAGCTATAACCGCTC (SEQ ID NO. 5) and plasmid pSM14 (Mittal eft awl (1995) supra), was digested with SphI and EcoRV, then ligated to a 531 bp EcoRV-HindIII fragment of pE3A and to SphI-HindIII-digested plasmid pSL301, creating plasmid pE3B. The 614 bp SphI-SacII fragment was isolated from plasmid pE3B and ligated to SphI-SacII-digested pSM14, to create plasmid pE3C.

The plasmid pE3C was digested with EcoRV and ligated to a Srfl linker (TTGCCCGGGCTT, SEQ ID NO 6), creating plasmid pE3CI. A 1.755 kb BamHI fragment of pE3CI was isolated and ligated to BamHI-digested pSMl7 (Mittal et al.

(1995) supra). creating plasmid pE3D. Finally, an 8783 bp KpnI-XbaI fragment of plasmid pE3D was isolated and ligated to KpnI/XbaI-digested plasmid pTG5435 (which contains full-length BAV3 genomic DNA) to create plasmid pE3E. The plasmid pE3E contained end fragments of the BAV3 genome (0-19.7 m.u. and 76.6-100 m.u.), with a

1.245 kb deletion in the E3 region and a unique KpnI site located within the vector sequences.

A plasmid (pBAV302) containing a BAV3 genome with a 1.245 kb deletion in the E3 region was generated by homologous DNA recombination. in E. coli BJ5183. between KpnI-digested pE3E and deproteinized BAV3 genomic DNA. b) Construction of plasmids pBAV302gD and pBAV302gDt. The transfer plasmid for generation of recombinant BAV3 expressing foreign genes in the E3 region was constructed by ligating an 8783 bp KpnI-XbaI fragment of pBAV302 to KpnI/XbaI-digested plasmid pGEM3zf(-), creating plasmid pBAV300. A 1.3 kb Bgl II fragment of plasmid pRSVl.3 (Tikoo et al. (1993) j Virol. 67:2103-2109), containing a full-length BHV-I gD gene, was treated with T4 DNA polymerase and ligated to Srfl-digested pBAV300. creating plasmid pBAV300.gD. Similarly, a 1.3 kb Bgl II fragment of plasmid pRSVl.3XN (Tikoo et al. (1993) supra). containing a truncated BHV-1 gD gene, was treated with T4 DNA polymerase and ligated to Srfl-digested pBAV300, creating plasmid pBAV300.gDt.

Recombinant BAV3 genomes, containing a gene encoding either a full-length or a truncated gD protein, were generated by homologous DNA recombination in E. coli BJ5183 between Srfl-linearized pBAV302 and a 10 kb KpnI/XbaI fragment of pBAV300.gD (creating plasmid pBAV302.gD), or between Srfl-linearized pBAV302 and a 10 kb KpnI/XbaI fragment of pBAV300.gDt (creating plasmid pBAV302.gDt).

Construction of recombinant BAV3. PFBR cell monolayers in 60 mm dishes were transfected with 10 ug of PacI-digested pBAV302, pBAV302.gD or pBAV302.gDt recombinant plasmid DNAs using the calcium phosphate method. Graham et al. (1973) Virology 52:456-467. After 15-20 days of incubation at 370C, the transfected cells showing 50% cytopathic effects were collected, freeze-thawed two times and the recombinant virus was plaque-purified on MDBK cells. Mittal et al. (1995) supra.

Radiolabelling and immunoprecipitation of proteins. About 70-80% confluent MDBK cell monolayers in 28 cm2 wells were infected with 10 PFU of recombinant or wild-type BAV3 per cell. After virus adsorption for 60 min, the cells were incubated in MEM containing 5% FBS. At different times post-infection, the cells were incubated in methionine- and cysteine-free Dulbecco's MEM for 60 min, before labelling with [35S] methionine-cysteine (100 uCi per well). After 2 or 12 h of labelling, cells or medium were harvested. Proteins were immunoprecipitated from the medium, and cells were lysed with

modified RIPA buffer. prior to analysis of proteins by SDS-PAGE as described. Tikoo et al. (1993) supra.

Animal Inoculations. A total of twenty-five 4-6 week old cotton rats of either sex were divided into three groups. Following anesthesia with halotane animals were inoculated twice. at day 1 and day 21, by the intranasal route with 100 Cil of inoculum <BR> <BR> <BR> <BR> containing 107 PFU of individual recombinant virus. Blood samples were collected 0, 21 and 28 days after primary inoculation, to examine the development of antibody responses to BHV-1 gD and BAV3, by enzyme linked immunosorbent assay (ELISA) and virus neutralization (VN) assay. Four animals in each group were euthanized, by an overdose of halothane, at 21 and 28 days after primary inoculation. Lung and nasal secretions were collected separately to monitor the development of BHV-1 gD-specific and BAV3-specific mucosal IgG and IgA antibody responses by ELISA. Papp et (1997) J. Gen. Virol.

78:2933-2943. In addition lungs were collected and the frequency of BHV-1 gD-specific and BAV3-specific, IgA antibody-secreting cells was determined by enzyme linked immunospot (ELISPOT). Papp et al., supra.

Preparation of lung lymphocytes. Aseptically removed lung tissue was cut into small pieces and incubated for one hour in complete medium: MEM supplemented with 10% FBS, 2 mM L-arginine, 1 mM sodium pyruvate, 100 pM non-essential amino acids, 10 mM HEPES buffer, 50 pM 2-mercaptoethanol, 100 U/ml penicillin G, 100 llg/ml streptomycin solution, 150 U/ml collagenase A and 50 U/ml DNaseI. The tissue was then pushed through a plastic mesh. The lung cell suspension was centrifuged through a discontinuous Percoll gradient and washed with MEM. The cells were resuspended in complete medium and incubated for 1 hour in a flask, to allow adherent cells to attach.

The non-adherent cell population was then resuspended and used in the antigen-specific ELM SPOT assay as described earlier. Papp et al., supra.

ELISA. Antibodies specific for BHV-1 and BAV3 in sera, lung secretions, and nasal secretions were determined by ELISA as described earlier. Papp et al., supra.

Briefly, 96-well Immunol-2 microtiter plates were coated with either purified truncated gD (0.01 llg/well) or BAV3 (0.5 llg/well) and incubated with different dilutions of each sample. Antigen-specific IgG was detected using biotinylated rabbit anti-rat IgG.

Antigen-specific IgA was measured using rabbit anti-rat IgA and horseradish peroxidase-conjugated goat anti-rabbit IgG.

Virus neutralization. Two-fold serial dilutions of heat-inactivated serum samples were incubated with 100 PFU of BHV-1 for 1 hour at 370C. The virus-sample mixture was then plated on confluent MDBK cells in 12-well tissue culture plates and incubated for 2 days. Titers were expressed as reciprocals of the highest antibody dilution that caused 50% reduction in the number of plaques relative to the control.

RESULTS Construction of E3-deleted recombinant BAV3. Initially, it was assumed that the role of BAV3 E3 in virus replication would be similar to that of the E3 region of HAV.

Wold et al. (1991) Virology 184:1-8. Accordingly, BAV E3-based vectors were constructed by making deletions of the E3 sequences. However, attempts to isolate an E3-deleted BAV3 recombinant in different bovine cell lines, including MDBK, were unsuccessful. A partially deleted BAV3 recombinant (BAV3-Luc), expressing a luciferase gene. could only be isolated when BAV3 El-transformed MDBK cells were used for transfection. Mittal et al. (1995) supra. However, once isolated, BAV3-Luc could be propagated on normal bovine cells, suggesting that the E3 region of BAV3 is not essential for virus replication in vitro. Mittal et al. (1995) supra. In order to increase the efficiency of isolating a BAV3 recombinant, PBFR cells were used. along with a novel procedure for generating BAV3 recombinants. Degryse (1996) Gene 170:45-50. Using this method, targeted modifications were introduced into plasmid-borne viral sequences, using the highly efficient homologous recombination machinery of E. coli, and infectious virions were isolated after transfection of the modified genome, excised from the plasmid vector, into appropriate host cells. Chartier et al. (1996) J. Virol. 70:4805-4610.

Taking advantage of the homologous recombination machinery of E. coli, a plasmid (pBAV302) was constructed, which contained a 1.245 kb deletion (from nt 26456 to 27701) and a Srfl restriction enzyme site (Fig. 21). PacI-digested pBAV302 DNA, when transfected into PFBR cells, produced cytopathic effects in 14 days. Virus was plaque-purified and expanded in MDBK cells, and named BAV3.E3d. Viral DNA was extracted and analyzed by agarose gel electrophoresis after digestion with BamHI restriction enzyme. As compared to wild-type (Fig. 4, lane b), the 3.019 kb BamHI "D" fragment of the recombinant BAV3.E3d genome was 1.245 kb smaller (Fig. 4, lane a), confirming that an E3-deleted recombinant BAV3 had been isolated. Comparison of the growth characteristics of this recombinant virus with those of wild-type BAV3 revealed no

significant differences in the plaque size or replication. as the E3-deleted recombinant replicated with similar kinetics to wild-type BAV3.

Construction ot recombinant BAV3 expressing BHV-1 glycoprotein D. In order to determine the usefulness of E3 -deleted, replication competent BAV3 recombinants as delivery vehicles for live recombinant vaccine antigens, recombinant BAV3 viruses expressing different forms of BHV-1 glycoprotein gD (Tikoo ci al. (1993) supra) were constructed. Full-length and truncated forms of gD genes (devoid of any exogenous promoter) were inserted individually into the E3 region of the BAV3.E3d genome in a parallel orientation. using the homologous recombination machinery of E. coli. Degryse, (1996) supra. PacI-digested pBAV302.gD or pBAV302.gDt plasmid DNA, when transfected into primary bovine retina cells, produced cytopathic effects in 14 days. Infected cell monolayers showing 50% cytopathic effects were collected. freeze Thawed and recombinant viruses were plaque-purified and propagated in MDBK cells.

The recombinant BAV3s were named BAV3.E3gD (insertion of full-length gD gene in E3 region) and BAV3.E3gDt (insertion of truncated gD gene in E3 region). Viral DNA was extracted and analyzed by agarose gel electrophoresis after digestion with different restriction enzymes. Since the gD gene contains a unique NdeI site, the recombinant viral DNA was cut with NdeI. As seen in Figure 4, compared to the BAV3.E3d (Fig. 4, lane e), the BAV3.E3gD (Fig. 4, lane f) and BAV3.E3gDt (Fig. 4, lane g) genomes contain an additional expected band of 4.6 kb, suggesting that recombinant BAV3.E3gD and BAV3.gDt contained gD or gDt genes in the E3 region. To differentiate between the gD and gDt gene the recombinant viral DNAs were digested with NheI, as the gDt but not the gD gene contains a unique NheI restriction enzyme site. Tikoo et al. (1993) supra. As expected, the 5.4 kb BAV3.E3gD DNA fragment (Fig. 4, lane c) was replaced with a 5.0 kb fragment in BAV3.E3gDt (Fig. 4, lane d). This suggested that recombinant BAV3.E3gD and BAV3.E3gDt contained gD and gDt genes, respectively. A comparison of the growth characteristics of these recombinants with wild type or E3-deleted BAV3 showed no significant differences in either the kinetics of replication or the titer of virus produced.

Analysis of expression of gD by BAV3.E3gD and BAV3.E3gDt. To examine the product(s) expressed by recombinant BAV3 viruses containing the BHV-1 gD or gDt gene, MDBK cells were infected with recombinant BAV3.E3gD, BAV3.E3gDt or BAV3.E3d and metabolically labelled with [35S] methionine-cysteine for different time

periods. For comparison with authentic gD. MDBK cells were infected with BHV-1 and labelled similarly with [35S] methionine-cysteine. The radiolabelled proteins were immunoprecipitated with a pool of gD-specific monoclonal antibodies (MAbs; Hughes et al. (1988) Arch. Virol. 103:47-60), and analyzed by SDS-PAGE under reducing conditions. The immunoprecipitation of recombinant BAV3 . E3 gD-infected cells revealed a major band of approximately 71 kDa (Fig. 5A, lanes b-d). which comigrated with the gD protein produced in BHV-l-infected cells (Fig. 5A, lane a), suggesting that the recombinant gD contained post-translational modifications similar to authentic gD. No similar band was observed in uninfected cells (Fig. SA, lane h), or in cells infected with recombinant BAV3.E3d (Fig. 5A, lanes e-g). Radioimmunoprecipitation of recombinant BAV3.E3gDt-infected cell supernatants revealed a major band of 61 kDa (Fig. 5B, lanes b-d). Both recombinant proteins were expressed throughout the infection of MDBK cells (Fig. 5).

In order to test the antigenicity of recombinant gD proteins, radiolabelled proteins were immunoprecipitated from recombinant BAV3-infected cell lysate (BAV3.E3gD) or supernatant (BAV3.E3gDt) with gD-specific MAbs (Hughes et al. (1988) supra), and analyzed by SDS-PAGE under reducing conditions. As shown in Fig. 6, both gD and gDt proteins were recognized by MAbs directed against discontinuous epitopes Ib (MAb 136; lanes a and f), II (MAb 3E7; lanes b and g), IIIb (MAb 4C 1; lanes c and h) IIIC (MAb 2C8; lanes d and i) and IIId (MAb 3C1; lanes e and j). These results suggest that the antigenic structure of recombinant proteins gD and gDt is similar to that of authentic gD produced in MDBK cells. Tikoo et al. (1993) supra.

To determine whether gD expression occurred in the absence of DNA synthesis. the amount of gD produced in BAV3.E3gD-infected MDBK cells was compared in the presence (Fig. 7, lanes a and b) and absence (Fig. 7, lanes c and d) of an inhibitor of DNA synthesis, 1 -P-D-arabinofuranosyIcytosine (AraC). The results suggest that gD expression was reduced in the presence of AraC.

Antibody responses in animals: To determine the ability of BAV3 recombinants to induce gD-specific immune responses, cotton rats were inoculated twice intranasally, three weeks apart, with 107 PFU of BAV3.E3gD, BAV3.E3gDt or BAV3.E3d recombinants. Serum, lung washes and nasal washes were collected for the analysis of IgG and IgA antibodies, while lungs were collected for analyzing the number of IgA antibody-secreting cells (ASC). Both BAV3.E3gD and BAV3.E3gDt induced gD-specific

IgG antibody response (Fig. 8B) in the serum and lung washes. which was significantly higher (P< 0.05) than the response induced in BAV3.E3d-immunized animals (control).

However, gD-specific IgG response induced by BAV3.E3gDt was higher than the response induced by BAV3.E3gD (P< 0.05).

Immunization with BAV3.E3gD and BAV3.E3gDt induced significantly higher (p<0.05) IgA antibody responses to gD in the serum and lung washes than immunization with BAV3.E3d (Fig. 8A). However, there was no significant difference in the IgA antibody response between the BAV3.E3gD- and the BAV3.E3gDt-immunized groups.

These recombinants also induced a BAV3-specific IgG antibody response (Fig. 8D) in the serum and lung washes and IgA antibody response (Fig. 8C) in serum, lung washes, and nasal washes. which was not significantly different among the groups.

Interestingly, nasal washes contained only IgA antibodies specific for gD (Figs. 26A and 26B) or BAV3 (Figs. 26C and 26D). In addition, IgA antibody-secreting cells specific for both gD and BAV3 could be detected in the lungs of immunized animals, the number of which increased significantly after booster immunization.

To measure the biological activity of the gD-specific serum antibody, anti-BHV-l titers were determined. Immunization with BAV3.E3gDt induced a BHV-1 log2 titer of 4.3 + 0.5, which was significantly higher (P<0.05) than the titers of 3.0 + 0.6 and 0.8 + 0.3 induced by BAV3.E3gD and BAV3.E3d respectively.

DISCUSSION The use of human adenoviruses as a vaccine delivery system in domestic animals is limited. Since non-human adenoviruses are species-specific, development of animal-specific adenovirus as vaccine delivery systems would be a logical choice. Herein is described the development of a replication-competent (E3-deleted) recombinant BAV3 for use in the delivery of vaccine antigens to the mucosal surfaces of animals. In addition, replication-competent BAV3 expressing BHV-1 gD or gDt glycoprotein were constructed and tested for their ability to induce mucosal and systemic immune responses in cotton rats.

Initial attempts to isolate an E3-deleted BAV3 recombinant in different bovine cell lines, which support the formation of infectious progeny following BAV3 DNA-mediated transfection were unsuccessful. However, a partially E3-deleted BAV3 recombinant expressing a luciferase gene was isolated when a BAV3 El -transformed MDBK cell line

was used for transfection. Mittal et al. (1995) supra. This suggested that some function related to the presence of El sequences may be required for the isolation of E3-deleted BAV3. However. subsequent to their isolation, recombinant BAV3 viruses expressing a luciferase gene in their E3 region replicated efficiently in normal MDBK cells. Mittal el al. (1995) supra. Thus, an alternative possibility is that normal MDBK cells may have efficiencies of transfection and/or growth rates that are too low to allow isolation of BAV recombinants.

In order to develop a reliable and efficient method for isolating replication-competent BAV3 recombinants without using BAV3 El-transformed MDBK cells. the homologous recombination machinery of E. coli (Chartier et al. (1996) supra; and Degryse (1996) supra) was first used to introduce a 1.245 kb deletion into the E3 region of a full-length BAV3 genome cloned in a plasmid. Secondly, for isolating the recombinant virus, PFBR cells, instead of MDBK cells, were used for transfection of the modified BAV3 genome, excised from the plasmid.

It has been reported that up to 105% of the wild-type genome can be packaged into the HAV5 virion without causing any rearrangements or deletion of the foreign genes in subsequent rounds of replication. Bett et al. (1993) J. Virol. 67:5911-5921. Recently, it was reported that the insertion capacity of the ovine adenovirus (OAV) vector is 114% of the wild type genome. Xu eft awl (1997) Virology 230:62-71. Since the size of the BAV3 genome is similar to that of the HAV5 genome, the insertion capacity of the BAV3 genome may be similar to that of HAV5. Therefore, as the BAV3.E3d vector described herein has a 1.245 kb E3 deletion, its insertion capacity is at least 3.0 kb.

Earlier, it was reported that recombinant BAV3.luc, containing a 0.696 kb deletion in the E3 region, replicated less efficiently than the wild-type BAV3 in cell culture. Mittal et al. (1995) supra. In contrast, the recombinant BAV3.E3d containing a 1.245 kb deletion in the E3 region replicated as efficiently as the wild-type BAV3 in cell culture. This difference may be due to the insertion of the luciferase gene in recombinant BAV3.luc, which may affect the expression of genes downstream of the E3 region. Mittal et al.

(1995) supra. However, the recombinant BAV3.E3gD or BAV3.E3gDt also replicated as efficiently as wild-type BAV3, suggesting that different foreign gene products may differentially affect the replication of recombinant BAV3 viruses in cell culture.

To confirm the utility of the BAV3 vector system for the expression of potential vaccine antigens, recombinant BAV3.E3gD and BAV3.E3gDt were constructed, which

express BHV-1 gD or gDt glycoprotein. respectively. As expected, gD expressed by BAV3.E3gD had the expected molecular weight and comigrated with the authentic gD expressed in BHV-1-infected cells. Similarly. the gDt expressed by BAV3.E3gDt had the expected molecular weight. Tikoo et al. (1993) supra. In addition, gD and gDt expressed by BAV3 recombinants were recognized by MAbs directed particularly against conformational epitopes. Hughes et al. (1988) supra. These results suggest that recombinant gD and gDt had post-translational modifications and antigenic profiles that were indistinguishable from that of the authentic gD synthesized after viral infection.

Hughes et al. (1988) supra.

Foreign genes without any flanking regulatory sequences, inserted in the left-to-right orientation in the E3 region of human adenoviruses are expressed efficiently, either from the upstream MLP or from the E3 promoter of the viral genome. Dronin et al.

(1993) Gene 126:247-250.; Morin et al. (1987) Proc. Natl. Acad. Sci. USA 84:4626-4630: andXuetal. (1995)J. Gen. Virol. 76:1971-1980. In the present experiments, glycoprotein gD expression was partially reduced in the presence of AraC, a replication inhibitor, suggesting that foreign gene expression was driven by both the MLP (a replication-dependent promoter) and the E3 promoter (an early, replication-independent promoter). Similar results have been reported earlier. Mittal et al. (1995) supra.

Intranasal immunization of cotton rats with BAV3.E3gD or BAV3.E3gDt induced gD-specific mucosal and systemic immune responses. gDt induced a stronger IgG antibody response than gD. In contrast, intradermal immunization of cotton rats with recombinant HAV5 induced lower immune response to gDt than to gD. Mittal et al.

(1996) Virology 222:299-309. In addition, a purified preparation of recombinant gDt incorporated with an adjuvant, when injected into mice, produced a secondary immune response similar to that of authentic gD. Baca-Estrada et al. (1996) Viral Immunol. 9:11- 22. These results strongly suggest that the route of immunization and vaccine formulation may affect the ability of an immunogen to induce an effective immune response to a greater extent than whether the immunogen is anchored in the membrane (gD) or secreted as a soluble protein (gDt).

Surprisingly, gD and gDt elicited similar IgA but significantly different IgG responses in the nasal passage and in the serum. Mucosal and systemic immune responses have been shown to be elicited and regulated with a considerable degree of independence.

Alley et al. (1988) The mucosal immune system. In "B lymphocytes in human disease"

(G. Bird and J.E. Calvert. Eds.). Oxford Universitv Press. Oxford, pp. 222-254: and Conley et al. (1987) Ann. Intern. Mcd 106:892-899. Induction of an immune response in one of these systems does not necessarily lead to a response in the other. Thus, it is possible that the two different forms of gD (full-length and truncated)are recognized differently in the mucosal and systemic compartments.

Induction of mucosal immunity is thought to be crucial in protecting the host from a respiratory or enteric infection. Moreover, the presence of secretory IgA has usually been found to correlate with resistance to such infections. Murphy (1994) Mucosal immunity to viruses. In "Handbook of mucosal immunity" (P.L. Ogra, J. Mestecky, M.E.

Lamm, W. Strober, J.R. McGhee and J. Bienenstock ed.), Academic Press, San Diego, pp.

333-339. Interestingly, intranasal immunization induced a gD-specific IgA antibody response, not only in the serum and lung but also in nasal washes of cotton rats. High levels of IgA and the presence of ASC in the lung suggests that the antibody was produced locally.

In conclusion, the present invention describes an efficient and reliable system for the production of BAV3 recombinants. In addition, intranasal immunization of cotton rats with replication-competent recombinant BAV3 induces vaccine antigen-specific mucosal and systemic immune responses.

Example 4: Immunization of cattle with recombinant BAV3 expressing BHV- 1 glycoprotein D In this example, replication-competent recombinant BAV3 viruses expressing BHV-1 gD (full-length and truncated) were assessed for their ability to provide protection against BHV-1 challenge. Groups of three 3-4 month old calves were immunized intranasally according to the following protocol. Group 1 was immunized with BAV3.E3gD, a virus expressing full-length BHV-1 gD. See Example 3. Group 2 was immunized with BAV3.E3gDt, a virus expressing a truncated BHV-1 gD. See Example 3 and Tikoo et al. (1993) supra. Group 3 was immunized with BAV3.E3d, a virus with a deleted E3 region, but no inserted heterologous sequences. See Example 3.

Animals were vaccinated on days 1 and 28, and challenged with aerosolized BHV- 1 on day 42. On days 1, 28, 40 and 52, blood was taken for serology, and lymphocyte proliferation was measured in the day 28 blood sample. Clinical signs, including temperature, nasal lesions and depression, were assessed daily for ten days after challenge

(days 42 through 52). Between days 1 and 15, nasal swabs for virus isolation were taken every third day. After challenge, viral titers were measured every two days. for ten days, by nasal swab and nasartampon. On days 22 and 40, and from days 42-52. antibody titers were determined. using nasal tampons.

The results indicate that, in animals immunized with gD- or gDt-expressing BAV recombinants, IgG titers increased after both initial and booster vaccinations, and were increased further after challenge (Figure 9). By comparison. animals vaccinated with E3-deleted BAV3 lacking an inserted gD gene showed increased IgG titers only after challenge, and the titers were at least one log lower than those obtained in vaccinated animals. IgA titers were measured and the results are shown in Figure 10. As seen, there was no detectable increase in the IgA titers after first immunization. However, gD-specific IgA titers increased after second immunization and after BHV-I challenge. Once again, control animals vaccinated with E3-deleted BAV3 lacking an inserted gD gene exhibited increased IgA titers only after challenge, and to a lower extent than animals vaccinated with gD- or gDt-expressing BAV.

Clinical symptoms were reduced in animals vaccinated with gD- and gDt-expressing BAV, compared to animals that had been vaccinated with E3-deleted BAV. These included fever (Figure 11), appearance and extent of nasal lesions (Figure 12) and depression (Figure 13). Finally, viral titers in nasal fluids were reduced more rapidly after challenge in the BAV.E3gD- and BAV.E3gDt-vaccinated animals, compared to controls (Figure 14).

These results indicate that recombinant BAV viruses expressing a BHV-1 glycoprotein provide protection against BHV-1 challenge in calves, reducing the occurrence of clinical signs, facilitating more rapid clearance of virus, and providing increased titers of both IgG and IgA, indicating induction of both humoral and mucosal immunity in a bovine host.

Deposit of Biological Materials The following materials were deposited and are maintained with the Veterinary Infectious Disease Organization (VIDO), 120 Veterinary Road, Saskatoon Saskatchewan, S7N 5l Canada.

The nucleotide sequences of The deposited materials are incorporated by reference herein. as well as the sequences of the polypeptides encoded thereby. In the event of any discrepancy between a sequence expresslv disclosed herein and a deposited sequence, the deposited sequence is controlling.

Material Internal Accession No. Deposit Date Recombinant plasmids pSM5l pSMSl Dec 6, 1993 pSM71 pSM71 Dec 6, 1993 Recombinant cell lines MDBK cells transformed with BAV3 EI sequences (MDBK-BAVE 1) Dec 6, 1993 Fetal bovine kidney cells transfonned with BAV3 El sequences (FBK-BAV-El) Dec 6, 1993 While the present invention has been illustrated above by certain specific embodiments, the specific examples are not intended to limit the scope of the invention as described in the appended claims.

SEQUENCE LISTING @@ GENERAL INFORMATION: (1) APPLICANT: TIKOO, SURESH K.

REDDY, POLICE SESHIDAR BABIUK, LORNE A.

(11) TITLE OF INVENTION: BOVINE ADENOVIRUS 3 GENOME (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SCOTT & AYLEN (E) STREET: 60 QUEEN STREET (C) CITY: OTTAWA (D; COUNTRY: CANADA (E) ZIP: K1P 5Y7 (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 #1.30 (vi) CURRENT APPLICATION DATA: (j APPLICATION NUMBER: (E) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: FRITZ, JOACHIM T.

(B) REGISTRATION NUMBER: 4173 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (613)237-5160 (B) TELEFAX: (613) 230-8842 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34446 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CATCATCAAT AATCTACAGT ACACTGATGG CAGCGGTCCA ACTGCCAATC ATTTTTGCCA 60 CGTCATTTAT GACGCAACGA CGGCGAGCGT GGCGTGCTGA CGTAACTGTG GGGCGGAGCG 120 CGTCGCGGAG GCGGCGGCGC TGGGCGGGGC TGAGGGCGGC GGGGGCGGCG CGCGGGGCGG 180 CGCGCGGGGC GGGGCGAGGG GCGGAGTTCC GCACCCGCTA CGTCATTTTC AGACATTTTT 240 TAGCAAATTT GCGCCTTTTG CAAGCATTTT TCTCACATTT CAGGTATTTA GAGGGCGGAT 300 TTTTGGTGTT CGTACTTCCG TGTCACATAG TTCACTGTCA ATCTTCATTA CGGCTTAGAC 360 AAATTTTCGG CGTCTTTTCC GGGTTTATGT CCCCGGTCAC CTTTATGACT GTGTGAAACA 420 CACCTGCCCA TTGTTTACCC TTGGTCAGTT TTTTCGTCTC CTAGGGTGGG AACATCAAGA 480

ACAPATTTC- CGAGTAATTG TGCACCTTTT TCCGCGTTAG GACTGCGTTT CACACGTAGA 540 CAGACTTTTT CTCATTTTCT CACACTCCGT CGTCCGCTTC AGAGCTCTGC GTCTTCGCTG 600 CCACCATGAA GTACCTGGTC CTCGTTCTCA ACGACGGCAT GAGTCGAATT GAAAAAGCTC 660 TCCTGTGCAG CGATGGTGAG GTGGATTTAG AGTGTCATGA GGTACTTCCC CCTTCTCCCG 720 CGCCTGTCCC CGCTTCTGTG TCACCCGTGA GGAGTCCTCC TCCTCTGTCT CCGGTGTTTC 780 CTCCGTCTCC GCCAGCCCCG CTTGTGAATC CAGAGGCGAG TTCGCTGCTG CAGCAGTATC 840 GGAGAGAGCT GTTAGAGAGG AGCCTGCTCC GAACGGCCGA AGGTCAGCAG CGTGCAGTGT 900 GTCCATGTGA GCGGTTGCCC GTGGAAGAGG ATGAGTGTCT GAATGCCGTA AATTTGCTGT 960 TTCCTGATCC CTGGCTAAAT GCAGCTGAAA ATGGGGGTGA TATTTTTAAG TCTCCGGCTA 1020 TGTCTCCAGA ACCGTGGATA GATTTGTCTA GCTACGATAG CGATGTAGAA GAGGTGACTP. 1080 GTCACTTTTT TCTGGATTGC CCTGAAGACC CCAGTCGGGA GTGTTCATCT TGTGGGTTTC 1140 ATCAGGCTCA AAGCGGAATT CCAGGCATTA TGTGCAGTTT GTGCTACATG CGCCAAACCT 1200 ACCATTGCAT CTATAGTAAG TACATTCTGT AAAAGAACAT CTTGGTGATT TCTAGGTATT 1260 GTTTAGGGAT TAACTGGGTG GAGTGATCTT AATCCGGCAT AACCAAATAC ATGTTTTCAC 1320 AGGTCCAGTT TCTGAAGAGG AAATGTGAGT CATGTTGACT TTGGCGCGCA AGAGGAAATG 1380 TGAGTCATGT TGACTTTGGC GCGCCCTACG GTGACTTTAA AGCAATTTGA GGATCACTTT 1440 TTTGTTAGTC GCTATAAAGT AGTCACGGAG TCTTCATGGA TCACTTAAGC GTTCTTTTGG 1500 ATTTGAAGCT GCTTCGCTCT ATCGTAGCGG GGGCTTCAAA TCGCACTGGA GTGTGGAAGA 1560 GGCGGCTGTG GCTGGGACGC CTGACTCAAC TGGTCCATGA TACCTGCGTA GAGAACGAGA 1620 GCATATTTCT CAATTCTCTG CCAGGGAATG AAGCTTTTTT AAGGTTGCTT CGGAGCGGCT 1680 ATTTTGAAGT GTTTGACGTG TTTGTGGTGC CTGAGCTGCA TCTGGACACT CCGGGTCGAG 1740 TGGTCGCCGC TCTTGCTCTG CTGGTGTTCA TCCTCAACGA TTTAGACGCT AATTCTGCTT 1800 CTTCAGGCTT TGATTCAGGT TTTCTCGTGG ACCGTCTCTG CGTGCCGCTA TGGCTGAAGG 1860 CCAGGGCGTT CAAGATCACC CAGAGCTCCA GGAGCACTTC GCAGCCTTCC TCGTCGCCCG 1920 ACAAGACGAC CCAGACTACC AGCCAGTAGA CGGGGACAGC CCACCCCGGG CTAGCCTGGA 1980 GGAGGCTGAA CAGAGCAGCA CTCGTTTCGA GCACATCAGT TACCGAGACG TGGTGGATGA 2040 CTTCAATAGA TGCCATGATG TTTTTTATGA GAGGTACAGT TTTGAGGACA TAAAGAGCTA 2100 CGAGGCTTTG CCTGAGGACA ATTTGGAGCA GCTCATAGCT ATGCATGCTA AAATCAAGCT 2160 GCTGCCCGGT CGGGAGTATG AGTTGACTCA ACCTTTGAAC ATAACATCTT GCGCCTATGT 2220 GCTCGGAAAT GGGGCTACTA TTAGGGTAAC AGGGGAAGCC TCCCCGGCTA TTAGAGTGGG 2280

GGCCATGGCC CTCGGTCCGT GTGTAACAGG AATGACTGGG GTGACTTTTC TGAATTGTAG 2340 GTTTGAGAGA GACTCAACAA TTAGGGGGTC CCTGATACGA GCTTCAACTC ACGTGCTGTT 2400 TCATGGCTGT TATTTTATGG GAATTATGGG CACTTGTATT GAGGTGGGGG CGGGAGCTTA 2460 CATTCGGGGT TGTGAGTTTG TGGGCTGTTA CCGGGGAATC TGTTCTACTT CTAACAGAGA 2520 TATTAAGGTG AGGCAGTGCA ACTTTGACAA ATGCTTACTG GGTATTACTT GTAAGGGGGA 2580 CTATCGTCTT TCGGGAAATG TGTGTTCTGA GACTTTCTGC TTTGCTCATT TAGAGGGAGA 2640 GGGTTTGGTT AAAAACAACA CAGTCAAGTC CCCTAGTCGC TGGACCAGCG AGTCTGGCTT 2700 TTCCATGATA ACTTGTGCAG ACGGCAGGGT TACGCCTTTG GGTTCCCTCC ACATTGTGGG 2760 CAACCGTTGT AGGCGTTGGC CAACCATGCA GGGGAATGTG TTTATCATGT CTAAACTGTA 2820 TCTGGGCAAC AGAATAGGGA CTGTAGCCCT GCCCCAGTGT GCTTTCTACA AGTCCAGCAT 2880 TTGTTTGGAG GAGAGGGCGA CAAACAAGCT GGTCTTGGCT TGTGCTTTTG AGAATAATGT 2940 ACTGGTGTAC AAAGTGCTGA GACGGGAGAG TCCCTCAACC GTGAAAATGT GTGTTTGTGG 3000 GACTTCTCAT TATGCAAAGC CTTTGACACT GGCAATTATT TCTTCAGATA TTCGGGCTAA 3060 TCGATACATG TACACTGTGG ACTCAACAGA GTTCACTTCT GACGAGGATT AAAAGTGGGC 3120 GGGGCCAAGA GGGGTATAAA TAGGTGGGGA GGTTGAGGGG AGCCGTAGTT TCTGTTTTTC 3180 CCAGACTGGG GGGGACAACA TGGCCGAGGA AGGGCGCATT TATGTGCCTT ATGTAACTGC 3240 CCGCCTGCCC AAGTGGTCGG GTTCGGTGCA GGATAAGACG GGCTCGAACA TGTTGGGGGG 3300 TGTGGTACTC CCTCCTAATT CACAGGCGCA CCGGACGGAG ACCGTGGGCA CTGAGGCCAC 3360 CAGAGACAAC CTGCACGCCG AGGGAGCGCG TCGTCCTGAG GATCAGACGC CCTACATGAT 3420 CTTGGTGGAG GACTCTCTGG GAGGTTTGAA GAGGCGAATG GACTTGCTGG AAGAATCTAA 3480 TCAGCAGCTG CTGGCAACTC TCAACCGTCT CCGTACAGGA CTCGCTGCCT ATGTGCAGGC 3540 TAACCTTGTG GGCGGCCAAG TTAACCCCTT TGTTTAAATA AAAATACACT CATACAGTTT 3600 ATTATGCTGT CAATAAAATT CTTTATTTTT CCTGTGATAA TACCGTGTCC AGCGTGCTCT 3660 GTCAATAAGG GTCCTATGCA TCCTGAGAAG GGCCTCATAT ACCATGGCAT GAATATTAAG 3720 ATACATGGGC ATAAGGCCCT CAGAAGGGTT GAGGTAGAGC CACTGCAGAC TTTCGTGGGG 3780 AGGTAAGGTG TTGTAAATAA TCCAGTCATA CTGACTGTGC TGGGCGTGGA AGGAAAAGAT 3840 GTCTTTTAGA AGAAGGGTGA TTGGCAAAGG GAGGCTCTTA GTGTAGGTAT TGATAAATCT 3900 GTTCAGTTGG GAGGGATGCA TTCGGGGGCT AATAAGGTGG AGTTTAGCCT GAATCTTAAG 3960 GTTGGCAATG TTGCCCCCTA GGTCTTTGCG AGGATTCATG TTGTGCAGTA CCACAAAAAC 4020 AGAGTAGCCT GTGCATTTGG GGAATTTATC ATGAAGCTTG GAGGGGAAGG CATGAAAAAA 4080 TTTTGAGATG GCTTTATGGC GCCCCAGGTC TTCCATGCAT TCGTCCATAA TAATAGCAAT 4140

AGGCCCGGTT TTGGCTGCCT GGGCAAACAC GTTCTGAGGG TGGGCGACAT CATAGTTGTA 4200 GTCCATGGTC AGGTCTTCAT AGGACATGAT CTTAAAGGCA GGTTTTAGGG TGCTGCTTTG 4260 AGGAACCAGA GTTCCTGTGG GGCCGGGGT GTAGTTCCCT TCACAGATTT GGGTCTCCCA 4320 AGCAAGCAGT TCTTGCGGGG GTATCATGTC AACTTGGGGG ACTATAAAAA AAACAGTTTC 4380 GGGAGGTGGT TGAATGAGGC CCGTAGACAT AAGGTTTCTG AGGAGCTGGG ATTTTCCACA 4440 ACCGGTTGGT CCGTAGACCA CCCCAATAAC GGGTTGCATG GTAAAGTTTA AAGATTTGCA 4500 TGAACCGTCA GGGCGCAGAT ATGGCATGGT GGCATTCATG GCATCTCTTA TCGCCTGATT 4560 ATAGTCTGAG AGGGCATTGA GTAGGGTGGC GCCCCCCATA GCCAGTAGCT CGTCCAAGGA 4620 AGAAAAGTGT CTAAGAGGTT TGAGGCCTTC AGCCATGGGC ATGGACTCTA AGCACTGTTG 4680 CATGAGAGCA CATTTGTCCC AAAGCTCAGA GACGTGGTCT AGTACATCTC CATCCAGCAT 4740 AGCTCTTTGT TTCTTGGGTT GGGGTGGCTG TTGCTGTAGG GGGCGAGACG GTGACGGTCG 4800 ATGGCCCGCA GGGTGCGGTC TTTCCAGGGC CTGAGCGTCC TCGCCAGGGT CGTCTCGGTG 4860 ACCGTGAAGG GCTGCTGATG CGTCTGTCTG CTGACCAGCG AGCGCCTCAG GCTGAGCCTG 4920 CTGGTGCCGA ACTTTTCGTC GCCTAGCTGT TCAGTGGAAT AATAACAAGT CACCAGAAGG 4980 TCGTAGGAGA GTTGTGAGGT GGCATGGCCT TTGCTCGAAG TTTGCCAGAA CTCTCGGCGG 5040 CGGCAGCTTG GGCAGTAGAT GTTTTTAAGG GCATATAGTT TGGGGGCTAA GAAGACAGAT 5100 TCCTGGCTGT GGGCGTCTCC GTGGCAGCGG GGGCACTGGG TCTCGCATTC CACAAGCCAA 5160 GTCAGCTGAG GGTTGGTGGG ATCAAAGACC AGAGGACGGT TATTACCTTT CAGGCGGTGC 5220 TTGCCTCGGG TGTCCATGAG TTCCTTTCCC CTTTGGGTGA GAAACATGCT GTCCGTGTCT 5280 CCGTAGACAA ATTTGAGAAT CCGGTCTTCT AGGGGAGTGC CTCTGTCTTC TAAATAGAGG 5340 ATGTCTGCCC ATTCAGAGAC AAAGGCTCTA GTCCACGCGA GGACAAATGA AGCTATGTGT 5400 GAGGGGTATC TGTTATTAAA TATGAGAGAG GATTTTTTTT GCAAAGTATG CAGGCACAGG 5460 GCTGAGTCAT CAGCTTCCAG AAAGGTGATT GGTTTGTAAG TGTATGTCAC GTGATGGTTC 5520 TGGGGGTCTC CCAGGGTATA AAAGGGGGCG TCTTCGTCTG AGGAGCTATT GCTAGTGGGT 5580 GTGCACTGAC GGTGCTTCCG CGTGGCATCC GTTTGCTGCT TGACGGGTGA GTAGGTGATT 5640 TTTAGCTCTG CCATGACAGA GGAGCTCAGG TTGTCAGTTT CCACGAAGGC GGTGCTTTTG 5700 ATGTCGTAGG TGCCGTCTGA AATGCCTCTA ACATATTTGT CTTCCATTTG GTCAGAAAAG 5760 ACAGTGACTC TGTTGTCTAG CTTAGTGGCA AAGCTGCCAT ACAGGGCATT GGACAGCAGT 5820 TTGGCAATGC TTCTGAGAGT TTGGTTTTTC TCTTTATCCG CCCTTTCCTT GGGCGCAATG 5880 TTAAGTTGCA CGTAGTCTCT AGCCAGACAC TCCCACTGGG GAAATACTGT GGTGCGGGGG 5940

TCGTTGAGAA TTTGGACTCT CCAGCCGCGG TTATGAAGCG TGATGGCATC CAAACAAGTT 6000 ACCACTTCCC CCCGTAGTGT CTCGTTGGTC CAGCAGAGGC GACCTCCTTT TCTGGAGCAG 6060 AAGGGCGGTA TAACGTCCAA GAATGCTTCT GGGGGTGGGT CTGCATCAAT GGTGAATATC 6120 GCGGGCAGTA GGGTGCGATC AAAATAGTCA ATGGGTCTGT GCAACTGGGT TAGGCGGTCT 6180 TGCCAGTTTT TAATTGCAAG CGCTCGATCA AAGGGGTTCA AAGGTTTTCC CGCTGGGAAA 6240 GGATGGGTGA GGGCGCTGGC ATACATGCCG CAGATGTCAT ACACATAGAT GGCTTCTGTT 6300 AGGACGCCTA TGTAGGTAGG ATAGCATCGG CCGCCCCGAA TACTTTCTCT AACGTAATCA 6360 TACATTTCAT TGGAAGGGGC TAGTAGAAAG TTGCCCAGAG AGCTCCTGTT GGGACGCTGG 6420 GATCGGTAGA CTACCTGTCT GAAGATGGCA TGGGAATTGG AGCTGATGGT GGGCCTTTGG 6480 AGGACATTGA AATTGCAGTG GGGCAGCCCC ACTGACGTGT GAACAAAGTC CAAATAAGAT 6540 GCTTGGAGTT TTTTAACCAA TTCGGCCGTA ACCAGCACGT CCATAGCACA GTAGTCCAAG 6600 GTGCGTTGCA CAATATCATA GGCACCTGAA TTCTCTTGCA GCCAGAGACT CTTATTGAGA 6660 AGGTACTCCT CGTCGCTGGA CCAGTAGTCC CTCTGAGGAA AAGAATCTGC GTCGGTTCGG 6720 TAGGTACCTA ACATGTAAAA TTCATTTACA GCTTTGTAAG GGCAGCAGCC TTTTTCCACG 6780 GGTAAAGCGT AAGCGGCAGC TGCGTTCCTG AGACTCGTGT GCGTGAGAGC AAAGGTATCT 6840 CGGACCATGA ACTTCACAAA CTGAAATTTA TAGTCTGCTG AGGTGGGAGT GCCTTCCTCC 6900 CAGTCTTTGA AGTCTTTTCG AGCAGCATGT GTGGGGTTAG GCAGAGCAAA AGTTAAGTCA 6960 TTGAAAAGAA TCTTGCCACA ACGAGGCATG AAATTTCTAC TGACTTTAAA AGCAGCTGGA 7020 ATACCTTGTT TGTTGTTAAT GACTTGTGCG GCTAGAACAA TCTCATCAAA GCCGTTTATG 7080 TTGTGCCCTA CGACATAGAC TTCCAAGAAA GTCGGTTGCC CTTTGAGTTC AAGCGTACAC 7140 AGTTCCTCGA AAGGAATGTC GCTGGCATGG ACATAGCCCA GTTTGAGGCA GAGGTTTTCT 7200 AAGCACGGAT TATCTGCCAG GAACTGGCGC CAAAGCAAAG TGCTGGCAGC TTCTTGAAGG 7260 GCATCCCGAT ACTGTTTAAA CAAGCTGCCT ACTTTGTTTC TTTGCGGGTT GAGGTAGTAG 7320 AAGGTATTTG CTTGCTTTGG CCAGCTTGAC CACTTTTGCT TTTTAGCTAT GTTAACAGCC 7380 TGTTCGCATA GCTGCGCGTC ACCAAACAAA GTAAACACGA GCATAAAAGG CATGAGTTGC 7440 TTGCCAAAGC TACCGTGCCA AGTGTATGTT TCCACATCAT AGACGACAAA GAGGCGCCGG 7500 GTGTCGGGGT GAGCGGCCCA GGGGAAAAAC TTTATTTCTT CCCACCAGTC CGAAGATTGG 7560 GTGTTTATGT GGTGAAAGTA AAAGTCCCGG CGGCGAGTGC TGCAGGTGTG CGTCTGCTTA 7620 AAATACGAAC CGCAGTCGGC ACATCGCTGG ACCTCTGCGA TGGTGTCTAT GAGATAGAGC 7680 TTTCTCTTGT GAATAAGAAA GTTGAGGGGG AAGGGAAGGC GCGGCCTGTC AGCGCGGGCC 7740 GGGATGCTTG TAATTTTCAG CTTCCCCTTG TATGTTTTGT AAACGCACAT ATTTGCGTTG 7800

CAGAACCGGA CGAGCGTGTC TTGGAATGrS. AGGATATTTT CTGGTTTTAA ATCrrATGGG 7860 CAGTGCTCCA AGTGCAGTTC AAAAAGGTTT CGGAGACTGC TGGAAACGTC TGCGTGATAC 7920 TTGACTTCCA GGGTGGTCCC GTCTTCAGTC TGACCGTGCA GCCGTAGGGT ACTGCGTTTG 7980 GCGACCAGGG GCCCCCTTGG GGCTTTCTTT AAAGGGGACG TCGAGGGCCG AGGGGCGGCC 8040 TTTGCCTTTC GGGCCTGAGG GGCGGTAGCT GGACCGGATC GTTGAGTTCG GGCATGGGTT 8100 GCAGCTGTTG GCGCAGGTCT GATGCGTGCT GCACGACTCT GCGGTTGATT CTCTGAATCT 8160 CCGGGTGTTG GGTGAATGCT ACTGGCCCCG TCACTTTGAA CCTGAAAGAG AGGTCGACAG 8220 AGTTAATAGA TGCATCGTTA AGCTCCGCCT GTCTAATAAT TTCTTCCACG TCACCGCTGT 8280 GGTCTCGGTA AGCAATGTCT GTCATAAACC GTTCGATCTC TTCCTCGTCC AGTTCTCCGC 8340 GACCAGCTCG GTGGACCGTG GCTGCCAAGT CCGTGCTAAT GCGTCGCATG AGCTGGGAAA 8400 AGGCATTGGT TCCCGGTTCA TTCCACACTC TGCTGTATAT AACAGCGCCA TCTTCGTCTC 8460 GGGCTCGCAT GACCACCTGG CCCAAGTTTA GCTCCACGTG GCGAGCAAAG ACGGGGCTGA 8520 GGCGGAGGTG GTGGTGCAGA TAATTGAGAG TGGTGGCTAT GTGCTCCACG ATGAAGAAGT 8580 AGATGACCCA TCTGCGGATG GTCGACTCGT TAATGTTGCC CTCTCGCTCC AGCATGTTTA 8640 TGGCTTCGTA AAAGTCCACA GCGAAGTTAA AAAACTGCTC GTTGCGGGCG GAGACTGTCA 8700 GCTCTTCTTG CAGGAGACGA ATGACTTCGG CTACGGCGGC GCGGACTTCT TCGGCAAAGG 8760 AGCGCGGCGG CACGTCCTCC TCCTCCTCTT CTTCCCCCTC CAGCGGGGGC ATCTCCAGCT 8820 CTACCGGTTC CGGGCTGGGG GACAGGGAAG GCGGTGCGGG CCGAACGACC CGTCGGCGTC 8880 GGGTGGGCAA GGGGAGACTC TCTATGAATC GCTGCACCAT CTCGCCCCGG CGTATCCGCA 8940 TCTCCTGGGT AACGGCACGC CCGTGTTCTC GGGGTCGGAG CTCAAAAGCT CCGCCCCGCA 9000 GTTCGGTCAG AGGCCGCGCC GCGGGCTGGG GCAGGCTGAG TGCGTCAATA ACATGCGCCA 9060 CCACTCTCTC CGTAGAGGCG GCTGTTTCGA ACCGAAGAGA CTGAGCATCC ACGGGATCGC 9120 TGAAGCGTTG CACAAAAGCT TCTAACCAGT CGCAGTCACA AGGTAGGCTG AGCATAGGTG 9180 AGGCTCGCTC GGTGTTGTTT CTGTTTGGCG GCGGGTGGCT GAGGAGAAAA TTAAAGTACG 9240 CGCACCGCAG GCGCCGGATG GTTGTCAGTA TGATGAGATC CCTGCGACCC GCTTGTTGGA 9300 TTCTGATGCG GTTTGCAAAG CCCCAGGCTT GGTCTTGGCA TCGCCCAGGT TCATGCACTG 9360 TTCTTGGAGG AATCTCTCTA CGGGCACGTT GCGGCGCTGC GGGGGCAGGG TCAGCCATTT 9420 CGGTGCGTCC AAACCCACGC AATGGTTGGA TGAGAGCCAA GTCCGCTACT ACGCGCTCTG 9480 CTAGGACGGC TTGCTGGATC TGCCGCAGCG TTTCATCAAA GTTTTCCAAG TCAATGAAGC 9540 GGTCGTAGGG GCCCGCGTTT ATGGTGTAGG AGCAGTTTGC CATGGTGGAC CAGTCCACAA 9600

TCTGCTGATC TACCCGCACC GTTTCTCCCT ACACCAGTCG CCTATAGC?T CGCCTSTCGA 9660 AAACATAGTC GTTGCAAACG CGCACCACGT ATTGGTAGCC GATTAGGAAG TGCGGCGGCG 9720 GGTATAAGTA GAGCGGCCAG TTTTGCGTGG CCGGCTGTCT GGCGCCCAGA TTCCGTAGCA 9780 TGAGTGTGGG GTATCGGTAC ACGTGACGCG ACATCCAGGA GATGCCCGCG GCCGAAATGG 9840 CGGCCCTGGC GTACTCCCGG GCCCGGTTCC ATATATTCCT GAGAGGACGA AAGATTCCAT 9900 GGTGTGCAGG GTCTGCCCCG TAAGACGCGC GCAATCTCTC GCGCTCTGCA AAAAACATAC 9960 AGATGAAACA TTTTTGGGGC TTTTCAGATG ATGCATCCCG CTTTACGGCA AATGAAGCCC 10020 AGATCCGCGG CAGTGGCGGG GGTTCCTGCT GCGGCCGCCG GCGCGAGCGT TGACTCAGGC 10080 GGTACTACCG CGCCCCCTGG TGTCGAGTGC GGCGAGGGGG AAGGGTTAGC TCGGCTGTAC 10140 GCGCACCCGG ACACACACCC GCGCGTGTGC GTGAAGCGCG ATGCGGCGGA GGCGTACGTT 10200 CCCCGGGAGA ACTTATTCCG CGACCGCAGC GGGGAGGAAC CCGAAGGGAG CCGAGACCTA 10260 AAGTACAAGG CCGGTCGGCA GTTGCGCGCC GGCATGCCCC GAAAGCGGGT GCTGACCGAA 10320 GGGGACTTTG AGGTGGATGA GCGCACTGGC ATCAGCTCAG CCAAAGCCCA CATGGAGGCG 10380 GCCGATCTAG TGCGGGCTTA CGAGCAAACG GTGAAGCAAG AGGCTAATTT TCAAAAGTCA 10440 TTTAATAACC ACGTGCGGAC ACTGATCTCC CGCGAGGAGA CCACCCTGGG TTTGATGCAC 10500 TTGTGGGACT TTGCGGAGGC ATACGCGCAG AACCCCGGCA GCAAGACCCT TACGGCCCAA 10560 GTCTTTCTCA TCGTGCAGCA CTTGCAAGAT GAGGGCATTT TTGGGGAAGC TTTCTTAAGC 10620 ATAGCAGAGC CCGAGGGACG ATGGATGCTA GATCTGCTAA ACATATTGCA GTCCATTGTG 10680 GTGCAAGAGC GCCAGCTTTC GCTATCTGAA AAGGTAGCCG CGGTGAACTA CTCCGTAGTT 10740 ACCCTGGGCA AACATTATGC CCGCAAGATC TTTAAGAGCC CCTTTGTGCC GCTTGACAAG 10800 GAGGTGAAGA TCAGTACATT TTATATGCGC GCGGTGCTTA AGGTCCTGGG TCTAAGTCAC 10860 GACCTGGGCA TGTACAGAAA CGAAAAGGTG GAGAAGCTAG CTAGCATAGG CAGGCGTTCG 10920 GGAGATGAGC GACGCGGAGC TGCTGTTCAA CCTCCGCCGC GCACTAACCA CTGGCGATTC 10980 TGAAGCATTC GATGAAGGCG GGGACTTTAC CTGGGCTCCG CCAACTCGCG CGACCGCGGC 11040 GGCCGCTTTG CCGGGGCCCG AGTTTGAGAG TGAAGAGACG GACGATGAAG TCGACGAATG 11100 AGTGATGCGG ACCCCCGTAT CTTTCAGCTG GTCAGTCGGC AAGAGACCGT AGCCATGGCC 11160 GAAGCGCCCC GAAGCCTGGG CCCCGCCCCT TCCAATCCTA GTTTGCAGGC TTTATTCCAA 11220 AGCCAGCCCA GCGCCGAGCA GGAGTGGCAC GGCGTGCTGG AGAGAGTCAT GGCCCTTAAC 11280 AAAAATGGAG ACTTTGGCTC GCAGCCCCAG GCGAACCGGT TTGGAGCCAT CCTCGAAGCC 11340 GTGGTGCCCC CGCGCTCCGA TCCCACCCAT GAAAAAGTGC TAGCTATTGT GAATGCGCTC 11400 TTGGAGACTC AGGCCATCCG TCGCGATGAG GCCGGACAGA TGTACACCGC GCTGTTGCAG 11460

CGGGTGGCCA CATACAACAG TGTGAATGTG CAGGGCAATT TGGACAGGCT GATTCAGGAC 11520 GTGRAGGAGG CTCTGGCGCA GCGCGAGCGC ACCGGGCCGG GGGCCGGCCT AGGGTCTGTG 11580 GTAGCCTTGA ATGCCTTCCT GAGCACACAG CCAGCGGTGG TGGAGAGGGG CCAGGAGAAC 11640 TATGTGGCCT TTGTGAGCGC CTTAAAACTC ATGGTGACCG AGGCGCCGCA GTCTGAGGTT 11700 TACCAGGCCG GACCTAGTTT CTTTTTTCAA ACCAGCCGGC ACGGTTCGCA GACGGTAAAC 11760 CTCAGTCAGG CCTTTGATAA CTTGCGACCC CTCTGGGGCG TGCGCGCGCC AGTACACGAG 11820 CGTACTACCA TCTCCTCTCT GCTCACACCA AACACCCGCT TGCTCTTGCT CCTCATTGCG 11880 CCCTTTACGG ACAGCGTGGG CATATCCCGG GACAGTTACC TGGGGCATCT GCTGACCCTT 11940 TACCGGGAGA CCATAGGTAA CACTCGAGTT GATGAGACCA CGTACAACGA GATCACGGAA 12000 GTGAGTCGGG CCCTGGGCGC CGAAGACGCG TCTAACTTGC AAGCCACTCT CAACTACTTA 12060 CTCACAAATA AGCAGAGCAA GTTGCCACAG GAGTTTTCTC TGAGTCCCGA AGAGGAGCGG 12120 GTGCTGCGCT ACGTGCAGCA ATCTGTCAGT TTATTTTTAA TGCAGGATGG ACACACGGCC 12180 ACCACTGCTC TAGATCAGGC TGCGGCCAAC ATAGCGCCCT CGTTTTACGC GTCCCACCGC 12240 GACTTTATAA ACCGACTGAT GGACTATTTC CAGCGAGCTG CGGCTATGGC CCCTGACTAC 12300 TTTTTACAGG CTGTTATGAA TCCCCACTGG CTCCCGCCGC CGGGTTTCTT TACTCAGGAG 12360 TTTGACTTTC CGGAGCCCAA CGGAGGCTTC CTGTGGGATG ATTTGGACAG CGCGCTCCTA 12420 CGCGCGCACG TAAAAGAAGA GGAGGATCAA GGAGCTGTGG GCGGCACGCC GGCGGCTTCG 12480 GCGCCCGCGT CTCGCGCGCA CACACCACCG CCGCCGCCCG GTGCCGCGGA CCTCTTTGCT 12540 CCTAACGCCT TCCGCAATGT GCAAAATAAC GGCGTGGATG AACTTATTGA CGGCTTAAGC 12600 AGATGGAAGA CTTACGCCCA GGAGAGGCAG GAAGTCGTTG AGCGGCACAG GCGCAGAGAG 12660 GCGCGTCGCC GGGCGCGCGA GGCGCGTCTA GAGTCGAGCG ATGATGACGA CAGCGACCTA 12720 GGGCCGTTTC TACGGGGCAC GGGGCACCTC GTTCACAACC AGTTTATGCA TCTGAAGCCC 12780 CGGGGTCCCC GCCAGTTTTG GTAACCGCAC TGTATTAAGC TGTAAGTCCT CTCATTTGAC 12840 ACTTACCAAA GCCATGGTCT TGCTTCGCCT CTGACACTTT CTCTCCCCCC ACACGCGGCA 12900 CCCTACAGCC TAGGGGCGAT GCTCCAGCCC GAACTGCAGC CAATTCCGCT GTCCCGCCGC 12960 CGGCTTATGA GGCGGTGGTG GCTGGGGCCT TCCAGACGCT TTCTCTTCGA CGAGATCCAC 13020 GTCCCGCCGC GATATGCTGC CGCGTCTGCG GGGAGAAACA GTATCCGTTA TTCCATGCTG 13080 CCCCCGTTGT ATGACACCAC GAAGATATAC CTTATCGACA ACAAATCTTC AGACATCCAA 13140 ACTCTGAATT ACCAAAACGA CCACTCAGAT TACCTCACTA CCATCGTGCA GAACAGCGAC 13200 TTCACGCCCC TGGAGGCTAG CAACCACAGC ATCGAGCTAG ACGAGCGGTC CCGCTGGGGC 13260

GGA?A.CCTTA AAACCATCCT TTATAC?-3 CTGCCTAATA TCACCSAGCA CATGTTTTCT 13320 AACTCTTTTC GGGTAAAGAT GATGGCCTCA AAAAAAGACG GCGTGCCCCA GTACGAGTGG 13380 TTCCCCCTAA SGCTGCCCGA GGGTAACTTT TCTGAGACTA TGGTCATTGA CCTCATGAAC 13440 AATGCCATCG TAGAGCTGTA CTTGGCTTTG GGGCGCCAGG AGGGCGTGAA GGAAGAGGAC 13500 ATCGGGGTA AGATCGATAC GCGCAACTTT AGTCTGGGCT ATGACCCGCA GACCCAGTTA 13560 GTGACGCCCG GCGTATACAC CAATGAAGCT ATGCATGCGG ACATCGTGTT GCTGCCGGGC 13620 TGTGCTATAG ACTTTACGCA CTCCCGATTA AACAACCTCT TGGGCATACG CAAGCGTTTT 13680 CCGTACCAAG AGGGCTTCGT CATCTCCTAT GAGGACCTTA AGGGGGGTAA CATCCCCGCT 13740 TTGATGGACG TGGAGGAGTT TAACAAGAGC AAGACGGTTC GAGCTTTGCG GGAGGACCCC 13800 AAGGGGCGCA GTTATCACGT GGGCGAAGAC CCAGAAGCCA GAGAAAACGA AACCGCCTAC 13860 CGCAGCTGGT ACCTGGCTTA CAATTACGGG GACCCAGAAA AAGGGGTGCG GGCCACCACA 13920 CTGCTGACTA CCGGCGACGT GACCTGCGGG GTGGAACAGA TCTACTGGAG CTTGCCGGAC 13980 ATGGCACTGG ACCCAGTCAC TTTCAAGGCT TCGCTGAAAA CTAGCPATTA CCCCGTGGTG 14040 GGCACAGAAC TTTTGCCACT GGTGCCGCGT AGCTTTTATA ACGCTCAGGC TGTGTACTCA 14100 CAGTGGATAC AAGAAAAAAC TAACCAGACC CACGTTTTCA ATCGCTTTCC CGAAAATCAG 14160 ATCTTGGTGC GGCCCCCTGC GCCTACCATC ACGTCCATAA GTGAAAATAA GCCCAGCTTG 14220 ACAGATCACG GAATCGTGCC GCTCCGGAAC CGCTTGGGGG GCGTGCAACG TGTGACTTTG 14280 ACTGACGCGC GGCGAAGATC CTGCCCCTAC GTCTACAAGA GCTTAGGCAT TGTGACGCCG 14340 CAAGTGCTAT CTAGCCGCAC GTTTTAAGCA GACAGGGGCA CAGCAGCCGT TTTTTTTTTT 14400 TTTTTTTCGC TCCACCAGGG ACTGTCAGGA ACATGGCCAT TCTAATCTCT CCTAGCAATA 14460 ACACGGGCTG GGGCCTGGGA TGCAATAAGA TGTACGGGGG CGCTCGCATA CGTTCAGACT 14520 TGCATCCAGT GAAGGTGCGG TCGCATTATC GGGCCGCCTG GGGCAGCCGC ACCGGTCGGG 14580 TGGGTCGCCG CGCAACCGCA GCTTTAGCCG ATGCCGTCGC GGCCACCGGT GATCCGGTGG 14640 CCGACACAAT CGAGGCGGTG GTGGCTGACG CCCGCCAGTA CCGGCGCCGC AGACGGCGAG 14700 GGGTGCGCCG AGTCAGAAGG TTGCGTCGGA GCCCCCGCAC TGCCCTGCAG CGACGGGTTC 14760 GTAGCGTACG CCGACAAGTG GCGAGGGCCC GCAGGGTGGG CCGGCGCGCG GCCGCTATCG 14820 CAGCAGACGC GGCCATGGCC ATGGCGGCGC CAGCTCGGCG ACGCCGTAAC ATCTACTGGG 14880 TACGCGATGC GGCAACCGGA GCCCGCGTTC CGGTGACAAC CCGGCCTACG GTCAGCAACA 14940 CCGTTTGAAA TGTCTGCTAC TTTTTTTTGC TTCAATAAAA GCCCGCCGAC TGATCAGCCA 15000 CACCTTGTCA CGCAGAATTC TTTCAAACCA TTGCGCTCTC AGCGCGCGCG CCGATAAACC 15060 CACTGTGATG GCCTCCTCTC GGTTGATTAA AGAAGAAATG TTAGACATCG TGGCGCCTGA 15120

GATTTACAAG SGCAAACGGC CCAGGCGAGA ACGCGCAGCA CCGTATGCTG TGAAGCAGGA 15180 GGAGAAGCCT TTAGTAAAGG CGGAGCGCAA AATTAAGCGC GGCTCCAGAA AGCGGGCCTT 15240 GTCAGGCGTT GACGTTCCTC TGCCCGATGA CGGCTTTGAG GACGACGAGC CCCACATAGA 15300 ATTTGTGTCT GCGCCGCGTC GGCCCTACCA GTGGAAGGGC AGGCGGGTGC GCCGGGTTTT 15360 GCGTCCCGGC GTGGCCGTTA GTTTCACGCC CGGCGCGCGC TCCCTCCGTC CGAGTTCCAA 15420 GCGGGTGTAT GACGAGGTGT ACGCAGACGA CGACTTCTTA GAAGCGGCCG CGGCCCGTGA 15480 GGGGGAGTTT GCTTACGGAA AGCGGGGACG CGAGGCGGCC CAGGCCCAGC TGCTACCGGC 15540 TGTGGCCGTG CCGGAACCGA CTTACGTAGT TTTGGATGAG AGCAACCCCA CCCCGAGCTA 15600 CAAGCCTGTA ACCGAGCAGA AAGTTATTCT TTCCCGCAAG CGGGGTGTGG GGAAGGTAGA 15660 GCCTACCATC CAGGTTTTAG CTAGCAAGAA GCGGCGCATG GCCGAGAATG AGGATGACCG 15720 CGGGGCCGGC TCCGTGGCCG AAGTGCAGAT GCGAGAAGTT AAACCGGTAA CCGCTGCCTT 15780 GGGTATTCAG ACCGTGGATG TTAGCGTGCC CGACCACAGC ACTCCCATGG AGGTCGTGCA 15840 GAGTCTCAGT CGGGCGGCTC AAGTAGCTCA ACGCCTGACC CAACAACAGG TGCGGCCTTC 15900 GGCTAAGATT AAAGTGGAGG CCATGGATCT TTCTGCTCCC GTAGACGCAA AGCCTCTTGA 15960 CTTAAAACCC GTGGACGTAA AGCCGACCCC GACCTTCGTG CTTCCCAGCT TTCGTTCACT 16020 CAGCACCCAA ACTGACTCTT TGCCCGCGGC AGTGGTCGTG CCGCGCAAGC CCCGCGTGCA 16080 CCGTGCTACT AGGCGTACTG CGCGCGGCTT GCTGCCCTAT TACCGCCTGC ATCCTAGCAT 16140 CACGCCGACA CCGGGTTACC GAGGATCTGT CTACACGAGC TCGGGTGTGC GCCTGCCCGC 16200 CGTCCGGGCG CCGCCGTCGC CGCCGTACCC GCAGGGCGAC TCCCCGCCTC AGCGCTGCCG 16260 CGGCCGCGGC GCTGCTGCCC GGCGTGCGCT ATCACCCTAG CATCCGCCAA GCGGCCACAG 16320 TAACCCGGCT CCGCCGTTAA GCGCTGTGAA ACTGCAACAA CAACAACAAA AATAAAAAAA 16380 AGTCTCCGCT CCACTGTGCA CCGTTGTCCA TCGGCTAATA AAGTCCCGCT TTGTGCGCCG 16440 CAGGAACCAC TATCCGTAAC CTGCGAAAAT GAGTCCCCGC GGAAATCTGA CTTACAGACT 16500 GAGAATACCG GTCGCCCTCA GTGGCCGGCG CCGGCGCCGA ACAGGCTTGC GAGGAGGGTC 16560 TGCGTACCTG CTCGGCCGCC GCAGAAGGCG CGCGGGCGGC GGCCGCCTGC GCGGGGGCTT 16620 CCTTCCCCTC CTGGCTCCCA TCATTGCAGC CGCCATCGGC GCAATCCCCG GCATCGCATC 16680 AGTGGCCATT CAGGCGGCCC ACAACAAATA GGGACAGTGT AAAGAAAGCT CAATCTCAAT 16740 AAAACAAACC GCTCGATGTG CATAACGCTC TCGGCCTGCA ACTTCTGCTG CTTACGTCTT 16800 TGACCAAAGT CACTACTGTT TTCCTTTTAC CCAGAGCCGG CGCCAGCCCC ACACAGCTTG 16860 TTAACACGCC ATGGACGAAT ACAATTACGC GGCTCTTGCT CCCCGGCAAG GCTCCCGACC 16920

CATGCTGAGC CAGTGGTCCG GCATCGGCAO GCACGAAATG CACGGCGGAC GTTTTAATCT 16980 GGGCAGTTTG TGGAGCGGGA TCAGGAATGT GGGCAGCGCG TTAAGAACTG GGGCTCTCGG 17040 GCCTGGCACA GCAATGCGGG CAAGCGTTGC GCGCCCAGCT GAAAAAGACG GGCTTGCAAG 17100 AAAAGATATT GAGGGCGTTA GCGCCGGTAT CCACGGAGCC GTGGATCTGG GCCGTCAGCA 17160 GCTAGAGAAA GCTATTGAGC AGCGCCTAGA GCGTCGCCCC ACCGCTGCCG GTGTGGAAGA 17220 CCTTCCGCTT CCCCCGGGAA CAGTCTTAGA AGCTGATCGT TTACCGCCCT CCTACGCCGA 17280 AGCGGTGGCT GAGCGCCCGC CGCCGGCTGA CGTTCTCCTG CCCGCATCCT CAAAGCCGCC 17340 GGTGGCGGTG GTGACCTTGC CCCCGAAAAA GAGAGTGTCT GAAGAGCCTG TGGAGGAAGT 17400 TGTGATTCGT TCCTCCGCAC CGCCGTCGTA CGACGAGGTT ATGGCACCGC AGCCGACTCT 17460 GGTAGCCGAG CAGGGCGCCA TGAAAGCAGT GCCCGTGATT AAGCCGGCTC AACCTTTTAC 17520 CCCAGCTGTG CACGAAACGC AACGCATAGT GACCAACTTG CCAATCACCA CAGCTGTGAC 17580 ACGGCGACGC GGGTGGCAGG GCACTCTGAA TGACATCGTG GGCCTCGGCG TTCGTACCGT 17640 GAAGCGCCGG CGGTGCTATT GAGGGGGCGC GCAGCGGTAA TAAAGAGAAC ATAAAAAAGC 17700 AGGATTGTGT TTTTTGTTTA GCGGCCACTG ACTCTCCCTC TGTGTGACAC GTCCTCCGCC 17760 AGAGCGTGAT TGATTGACCG AGATGGCTAC CCCGTCGATG CTGCCGCAAT GGTCCTACTG 17820 CACATCGCCG GTCAGGACGC GTCCGAGTAC CTGTCCCCCG GCTTGGTGCA ATTCGCACAA 17880 GCCACCGAAT CCTACTTTAA CATTGGGAAC AAGTTTAGAA ACCCCACCGT CGCCCCGACG 17940 CACGATGTCA CCACGGAGCG TTCGCAGCGT CTGCAGCTCC GCTTCGTGCC CGTAGACCGG 18000 GAGGACACAC AGTACTCCTA CAAAACCCGC TTCCAGCTAG CCGTGGGCGA CAACCGGGTG 18060 CTGGACATGG CCAGCACGTA TTTTGACATC CGCGGTACGC TGGAGAGGGG CGCCAGTTTC 18120 AAGCCTTACA GCGGCACGGC CTACAACTCC TTTGCCCCCA ACAGTGCCCC TAACAATACG 18180 CAGTTTAGGC AGGCCAACAA CGGTCATCCT GCTCAGACCA TAGCTCAAGC TTCTTACGTG 18240 GCTACCATCG GCGGTGCCAA CAATGACTTG CAAATGGGTG TGGACGAGCG TCAGCAGCCG 18300 GTGTATGCGA ACACTACGTA CCAGCCGGAA CCTCAGCTCG GCATTGAAGG TTGGACAGCT 18360 GGATCCATGG CGGTCATCGA TCAAGCAGGC GGGCGGGTTC TCAGGAACCC TACTCAAACT 18420 CCCTGCTACG GGTCCTATGC TAAGCCGACT AACGAGCACG GGGGCATTAC TAAAGCAAAC 18480 ACTCAGGTGG AGAAAAAGTA CTACAGAACA GGGGACAACG GTAACCCGGA AACAGTGTTT 18540 TATACTGAAG AGGCTGACGT GCTAACGCCC GACACCCACC TTGTTCACGC GGTACCGGCC 18600 GCGGATCGGG CAAAGGTGGA GGGGCTATCT CAGCACGCAG CTCCCAACAG GCCGAACTTT 18660 ATCGGCTTTC GGGACTGCTT TGTAGGCTTG ATGTATTATA ACAGCGGGGG CAACCTGGGC 18720 GTCTTAGCGG GTCAATCCTC TCAGCTGAAT GCCGTGGTAG ACCTGCAAGA CCGCAACACT 18780

GAGCTTTCCT ATCAGATGCT@TCTTGCAAAC ACGACGGACA GATCCCGCTA TTTTAGCATG 18840 TGGAACCAAG CCATGGACTC GTACGACCCG GAGGTCAGGG TGATAGATAA CGTGGGCGTA 1890C GAGGACGAGA TGCCTAATTA CTGCTTTCCG TTGTCGGGGG TTCAGATTGG AAACCGTAGC 18960 CACGAGGTTC AAAGAAACCA ACAACAGTGG CAAAATGTAG CTAATAGTGA CAACAATTAC 19020 ATAGGCAAGG GGAACCTACC GGCCATGGAG ATAAATCTAG CGGCCAATCT CTGGCGTTCC 19080 TTTTTGTACA GTAATGTGGC GTTGTACTTG CCAGACAACC TTAAATTCAC CCCTCACAAC 1914C ATTCAACTCC CGCCTAACAC GAACACCTAC GAGTACATGA ACGGGCGAAT CCCCGTTAGC 1920C GGCCTTATTG ATACGTACGT .-AATATAGGC ACGCGGTGGT CGCCCGATGT GATGGACAAC 19260 GTGAATCCCT TTAACCACCA CCGCAACTCG GGCCTGCGTT ACCGCTCCCA GCTGCTGGGC 19320 AACGGCCGCT TCTGCGACTT TCACATTCAG GTGCCACAAA AGTTTTTTGC TATTCGAAAC 19380 CTGCTTCTCC TGCCCGGCAC GTACACTTAC GAGTGGTCCT TTAGAAAGGA CGTAAACATG 19440 ATCCTTCAGA GCACTCTGGG CAATGATCTG CGGGTCGATG GGGCCACTGT TAATATTACC 19500 AGCGTCAACC TCTACGCCAG CTTCTTTCCC ATGTCACATA ACACCGCTTC CACTTTGGAA 19560 GCTATGCTCC GCAACGACAC TAATGACCAG TCTTTTAATG ACTATCTCTC GGCGGCTAAC 19620 ATGTTGTATC CCATTCCGCC CAATGCCACC CAACTGCCCA TCCCCTCACG CAACTGGGCA 19680 GCGTTCCGTG GCTGGAGTCT CACCCGGCTA AAACAGAGGG AGACACCGGC GCTGGGGTCC 19740 CCGTTCGATC CCTATTTCAC CTATTCGGGC ACCATCCCGT ACCTGGACGG CACTTTTTAC 19800 CTCAGCCACA CCTTTCGCAA GGTGGCCATC CAGTTTGACT CTTCTGTGAC CTGGCCCGGC 19860 AATGACAGGC TTTTAACCCC TAACGAGTTC GAAATAAAAA TAAGTGTGGA CGGTGAAGGC 19920 TACAACGTGG CTCAGAGCAA TATGACTAAG GACTGGTTCC TGGTGCAGAT GCTAGCGAAT 19980 TACAACATAG GCTACCAGGG ATATCACCTG CCCCCGGACT ACAAGGACAG GACATTTTCC 2004C TTCCTGCATA ACTTCATACC CATGTGCCGA CAGGTTCCCA ACCCAGCAAC CGAGGGCTAC 20100 TTTGGACTAG GCATAGTGAA CCATAGAACA ACTCCGGCTT ATTGGTTTCG ATTCTGCCGC 20160 GCTCCGCGCG AGGGCCACCC CTACCCCCAA CTGGCCTTAC CCCCTCATTS GGACCCACGC 2022C CATGCCCTCC GTGACCCAGA GAGAAAGTTT CTCTGCGACC GCACCCTCTG GCGAATCCCC 20280 TTCTCCTCGA ACTTCATGTC CATGGGGTCC CTCACAGATC TCGGACAGAA CCTACTGTAT 20340 GCCAATGCCG CGCATGCCCT AGACATGACT TTTGAGATGG ATCCCATCAA TGAGCCCACT 20400 CTGCTGTACG TTCTGTTTGA SGTGTTTGAC GTGGCCCGCG TTCACCAGCC CCACAGAGGC 20460 GTGATCGAAG TGGTGTACTT GAGAACGCCA TTCTCAGCCG GCAACGCTAC CACATAAGTG 20520 CCGGCTTCCC TCTCAGGCCC CGCGATGGGT TCTCGGGAAG AGGAGCTGAG ATTCATCCTT 20580

CACGATCTCG GTGTGGGGCC ATACTTCCTC GGCACTTTCG ATAAACACTT TCCGGGGTTC 20640 ATCTCCAAAG ACCGAATGAG CTGTGCCATA GTCAACACTG CCGGACGCGA AACCGGGGGC 20700 GTGCATTGGC TGGCCATGGC TTGGCACCCA GCCTCGCAGA CCTTTTACAT GTTTGACCCT 20760 TTCGGTTTCT CGGATCAAAA GCTAAAGCAA ATTTACAACT TTGAGTATCA GGGCCTCCTA 20820 AAGCGCAGCG CCCTGACTTC CACTGCTGAC CGCTGCCTGA CCCTTATTCA AAGCACTCAA 20880 TCTGTCCAGG GACCCAACAG CGCCGCCTGC GGTCTGTTCT GCTGCATGTT CCTCCACGCC 20940 TTTGTCCGCT GGCCGCTTAG GGCCATGGAC AACAATCCCA CCATGAACCT CATCCACGGA 21000 GTTCCCAACA ACATGTTGGA GAGCCCCAGC TCCCAAAATG TGTTTTTGAG AAACCAGCAA 21060 AATCTGTACC GTTTCCTAAG ACGCCACTCC CCCCATTTTG TTAAGCATGC GGCTCAAATT 21120 GAGGCTGACA CCGCCTTTGA TAAAATGTTA ACAAATTAGA CCGTGAGCCA TGATTGCAGA 21180 AGCATGTCAT TTTTTTTTTA TTGTTTAAAA TAAAAACAAC ACATAACATC TGCCGCCTGT 21240 CCTCCCGTGA TTTCTTCTGC TTTATTTGCA AATGGGGGGC ACCTTAAAAC AAAGAGTCAT 21300 CTGCATCGTA CTGATCGATG GGCAGAATAA CATTCTGATG CTGGTACTGC GGGTCCCAGC 21360 GGAATTCGGG AATGGTAATG GGGGGGCTCT GTTTAACCAG CGCGGACCAC ATCTGCTTAA 21420 CCAGCTGCAA GGCTGAAATC ATATCTGGAG CCGAAATCTT GAAATCGCAG TTTCGCTGGG 21480 CATTAGCCCG CGTCTGCCGG TACACAGGGT TACAGCACTG AAATACTAAC ACCGATGGGT 21540 GTTCTACGCT GGCCAGGAGT TTGGGATCTT CTACGAGGCT CTTATCTACC GCAGAGCCCG 21600 CGTTGATATT AAAGGGCGTT ATCTTGCATA CCTGACGGCC TAGGAGGGGC AATTGGGAGT 21660 GACCCCAGTT ACAATCACAC TTTAAAGGCA TAAGCAGATG AGTTCCGGCA CTTTGCATCC 21720 TGGGGTAACA GGCTTTCTGA AAGGTCATGA TCTGCCAGAA AGCCTGCAAA GCCTTGGGCC 21780 CCTCGCTGAA AAACATACCA CAAGACTTTG AGGTAAAGCT GCCGGCCGGC AAAGCGGCGT 21840 CAAAGTGACA GCAAGCCGCG TCTTCATTCT TTAGCTGCAC TACGTTCATA TTCCACCGGT 21900 TGGTGGTGAT CTTTGTCTTA TGCGGGGTCT CTTTTAAAGC CCGCTGCCCA TTTTCGCTGT 21960 TCACATCCAT CTCTATCACT TGGTCTTTGG TAAGCATAGG CAGGCCATGC AGGCAGTGAA 22020 GGGCCCCGTC TCCCCCCTCG GTACACTGGT GGCGCCAGAC CACACAGCCC GTGGGGCTCC 22080 ACGAGGTCGT CCCCAGGCCT GCGACTTTTA ACACAAAATC ATACAAGAAG CGGCCCATAA 22140 TAGTTAGCAC GGTTTTCTGA GTACTGAAAG TAAGAGGCAG GTACACTTTA GACTCATTAA 22200 GCCAAGCTTG TGCAACCTTC CTAAAACACT CGAGCGTGCC AGTGTCGGGC AGCAAGGTTA 22260 AGTTTTTAAT ATCCACTTTC AAAGGCACAC ACAGCCCCAC TGCTAATTCC ATGGCCCGCT 22320 GCCAAGCAAC TTCGTCGGCT TCCAGCAAGG CCCGGCTGGC CGCCGGCAGG GCGGGAGCGG 22380 CGGCCTCAGC GGCTGGGGCT GAAGGTTTGA AAATCTTGGC GCGCTTAACG GCTGTGACAT 22440

CTTCGGCGGG GGGCTCAGCG ATCGGCGCGC GCCGTTTGCG GCTGACTTTT TTCCGGGGCG 22500 TCTCATCTAT CACTAAGGGG TTCTCGTCCC CGCTGCTGTC AGCCGAACTC GTGGCTCGCG 22560 TTAAGTCACC GCTGCGATTC ATTATTCTCT CCTAGATAAC GACAACAAAT GGCAGAGAAA 22620 GGCAGTGAAA ATCAGCGGCC AGAGAACGAC ACTGAGCTAG CAGCGGTTTC AGAAGCCCTA 22680 GGCGCGGCCG CTTCGGCCCC CTCACGTAAC TCCCCGACTG ACACGGATTC AGGGGTGGAA 22740 ATGACGCCCA CCAGCAGCCC CGAGCCGCCC GCCGCTCCCC CAAGTTCGCC TGCCGCAGCA 22800 CCTGCCCCTC AGAAGAACCA GGAGGAGCTC TCTTCCCCCG AGCCCGCGGT AGCAGCAGCG 22860 GAGCCAGAAG CCGCTTCGCG GCCCAGACCA CCCACACCCA CCGTTCAGGT CCCGCGGGAG 22920 CCGAGCGAGG ATCAACCTGA CGGACCCGCG ACGAGGCCTT CGTACGTGAG CGAGGATTGC 22980 CTCATCCGCC ATATCTCTCG CCAGGCTAAC ATTGTTAGAG ACAGCCTGGC AGACCGCTGG 23040 GAGTTAGAGC CCACCGTGTC GGCTCTCTCC GAGGCTTACG AAAAGCTCCT CTTTTGTCCC 23100 AAGGTACCAC CCAAGAAGCA AGAGAATGGC ACTTGCGAAC CTGAACCTCG CGTTAATTTT 23160 TTCCCCACCT TTGTAGTGCC CGAAACTTTA GCCACGTACC ACATCTTTTT CCAAAACCAA 23220 AAAATCCCCC TGTCTTGTCG CGCCAACCGC ACCCACACAG ACACCATCAT GCACCTCTAC 23280 TCGGGGGACT CCTTACCGTG CTTCCCCACG CTGCAGCTGG TCAACAAAAT CTTTGAAGGC 23340 TTGGGCTCAG AGGAGCGGCG CGCAGCCAAC TCGCTGAAAG ATCAAGAGGA TAACAGCGCG 23400 TTAGTTGAGC TCGAAGGGGA CAGTCCCCGA CTGGCTGTGG TTAAGCGCAC ACTGTCTTTG 23460 ACACATTTCG CCTACCCTGC CATAACACTA CCGCCTAAGG TGATGGCAGC TGTCACTGGC 23520 AGCCTCATTC ATGAATCAGC AGCGACCGCC GAACCGGAAG CTGAGGCGCT GCCAGAAGCC 23580 GAGGAGCCCG TGGTTAGTGA CCCTGAACTT GCTCGCTGGT TGGGGCTCAA CTTACAACAG 23640 GAGCCCGAGG CCACGGCCCA GGCTTTGGAA GAAAGACGCA AGATTATGTT GGCAGTATGC 23700 TTAGTCACAC TTCAGCTCGA GTGCCTGCAC AAGTTTTTTT CTTCAGAGGA TGTCATCAAA 23760 AAGCTGGGAG AGAGCCTCCA CTACGCCTTT CGCCACGGCT ACGTGCGCCA AGCCTGCTCC 23820 ATTTCTAACG TGGAACTAAC GAACATCGTC TCATACCTGG GTATCTTGCA CGAAAACCGC 23880 TTGGGACAGA GTACCCTACA CGCCACCCTT AAAGACGAGA ACCGCAGAGA CTACATCAGA 23940 GACACAGTCT TTCTCTTTCT GGTTTATACT TGGCAGACTG CCATGGGCAT TTGGCAGCAG 24000 TGCCTCGAGA CTGAGAACGT AAAAGAACTT GAAAAGCTCT TGCAAAAAAG CAAGAGGGCT 24060 CTCTGGACGG GCTTCGACGA GCTCACCATA GCTCAAGACC TAGCTGACAT AGTGTTCCCC 24120 CCCAAATTCT TGCACACCTT GCAAGCCGGC CTGCCAGACC TTACATCCCA GAGTCTCCTT 24180 CACAACTTTC GCTCCTTCAT TTTCGAACGC TCGGGCATTC TACCCGCCAT GTGCAATGCA 24240

CTGCCCACCG ACTTCATCCC TATCAGCTAC CGGGAGTGCC CTCCAACTTT CTGGGCCTAC 24300 ACCTACCTCT TTAAACTGGC CAATTACCTC ATGTTTCACT CCGACATCGC TTACGATCGG 24360 AGCGGCCCCG GTCTCATGGA ATGCTACTGT CGCTGCAACC TGTGCAGTCC TCACCGCTGC 24420 TTGGCGACCA ACCCCGCCCT GCTCAGCGAG ACCCAAGTTA TCGGTACCTT CGAGATTCAG 24480 GGCCCTCCTG CTCAAGACGG ACAGCCGACC AAACCGCCCC TCAGGCTGAC TGCAGGTCTC 24540 TGGACTTCCG CCTACCTGCG CAAATTTGTA CCGCAAGACT TCAACGCCCA CAAAATAGCC 24600 TTCTACGAAG ACCAATCCAA AAAGCCGAAA GTGACCCCCA GCGCTTGTGT CATCACTGAA 24660 GAAAAAGTTT TAGCCCAATT GCATGAAATT AAAAAAGCGC GGGAAGACTT TCCTCTTAAA 24720 AAGGGGCACG GAGTCTATCT GGACCCTCAG ACCGGCGAGG AGCTGAACGG ACCCGCACCC 24780 TCCGCAGCTA GGAATGAAAC CCCGGAGCAT GTCGGCAGCC GGGCCTTCCG CGGCTCAGGC 24840 TTCGGAGGGC CAACAGCTGC CGCCACAGAC AGCGGGGCTG CAGCCGAGCA AGAGGGCTGT 24900 GAGGAAGGTA GTAGCTTCTC TGAATCCCAC CGCCGCCCTG GAAGACATAT CCGAGGGGGA 24960 GGAAGGCTTC CCCCTGACGG ACGAGGAAGA CGGGGACACC CTGGAGAGCG ATTTCAGCGA 25020 CTTCACGGAC GAAGACGTCG AGGAGGAGGA TATGATTTCG ATACCCCGCG ACCAGGGGCA 25080 CTCCGGCGAG CTCGAGGAGG GCGAAATTCC CGCAACGGTA GCGGCGACGG CGGTCAAGAA 25140 GGGCCAGGGC AAGAAGAGTA GGTGGGACCA GCAGGTCCGC TCCACAGCGC CTCTAAAGGG 25200 CGCTAGAGGT AAGAGGAGCT ACAGCTCCTG GAAACCCCTC AAGCCCACTA TCCTTTCATG 25260 CTTACTGCAG AGCTCCGGCA GCACTGCCTT CACTCGCCGC TATCTGCTTT TTCGCCATGG 25320 CGTGTCCGTT CCCTCCAGGG TAATTCATTA CTATAATTCT TACTGCAGAC CCGAAGCTGA 25380 CCAAAACCGC CACTCAGAGC AAAAAGAGCC GCCGGAGTGC CAGCGCGGCG CGCCCTCGCC 25440 CTCCTCCTCT TCCTCCCAAG CGTGCTCGGG CGCCCCGCCG CCCCAAAGGC CAGCGCCATC 25500 AGGCCGACGA CGCAAGCACC GAGGGCCGCG ACAAGCTTCG GGAGCTGATC TTTCCCACTC 25560 TCTATGCCAT ATTCCAACAA AGTCGCGCTC AGCGGTGTCA CCTCAAAGTG AAAAATAGAT 25620 CCTTACGTTC ACTGACGCGC AGCTGCCTCT ACCACAACAA GGAGGAACAG CTCCAGCGAA 25680 CCCTAGCAGA CTCCGAGGCG CTTCTCAGTA AATACTGCTC TGCAGCTCCG ACACGATTCT 25740 CGCCGCCCTC TTATACCGAG TCTCCCGCCA AGGACGAATC CGGACCCGCC TAAACTCTCA 25800 GCATGAGCAA AGAAATTCCC ACACCTTATG TTTGGACCTT TCAACCTCAG ATGGGAGCGG 25860 CCGCAGGTGC CAGTCAAGAT TACTCGACCC GCATGAATTG GTTCAGCGCG GGACCTGATA 25920 TGATCCACGA CGTTAACAAC ATTCGTGACG CCCAAAACCG CATCCTTATG ACTCAGTCGG 25980 CCATTACCGC CACTCCCAGG AATCTGATTG ATCCCAGACA GTGGGCCGCC CACCTCATCA 26040 AACAACCCGT GGTGGGCACC ACCCACGTGG AAATGCCTCG CAACGAAGTC CTAGAACAAC 26100

ATCTGACCTC ACATGGCGCT CAAATCGCGG GCGGAGGCGC TGCGGGCGAT TACTTTAAAA 2616C GCCCCACTTC AGCTCGAACC CTTATCCCGC TCACCGCCTC CTGCTTAAGA CCAGATGGAG 26220 TCTTTCAACT AGGAGGAGGC TCGCGTTCAT CTTTCAACCC CCTGCAAACA GATTTTGCCT 26280 TCCACGCCCT GCCCTCCAGA CCGCGCCACG GGGGCATAGG ATCCAGGCAG TTTGTAGAGG 26340 AATTTGTGCC CGCCGTCTAC CTCAACCCCT ACTCGGGACC GCCGGACTCT TATCCGGACC 26400 AGTTTATACG CCACTACAAC GTGTACAGCA ACTCTGTGAG CGGTTATAGC TGAGATTGTA 26460 AGACTCTCCT ATCTGTCTCT GTGCTGCTTT TCCGCTTCAA GCCCCACAAG CATGAAGGGG 26520 TTTCTGCTCA TCTTCAGCCT GCTTGTGCAT TGTCCCCTAA TTCATGTTGG GACCATTAGC 26580 TTCTATGCTG CAAGGCCCGG GTCTGAGCCT AACGCGACTT ATGTTTCTGA CTATGGAAGC 26640 GAGTCAGATT ACAACCCCAC CACGGTTCTG TGGTTGGCTC GAGAGACCGA TGGCTCCTGG 26700 ATCTCTSTTC TTTTCCGTCA CAACGGCTCC TCAACTGCAG CCCCCGGGGT CGTCGCGCAC 26760 TTTACTGACC ACAACAGCAG CATTGTGGTG CCCCAGTATT ACCTCCTCAA CAACTCACTC 26820 TCTAAGCTCT GCTGCTCATA CCGGCACAAC GAGCGTTCTC AGTTTACCTG CAAACAAGCT 26880 GACGTCCCTA CCTGTCACGA GCCCGGCAAG CCGCTCACCC TCCGCGTCTC CCCCGCGCTG 26940 GGAACTGCCC ACCAAGCAGT CACTTGGTTT TTTCAAAATG TACCCATAGC TACTGTTTAC 27000 CGACCTTGGG GCAATGTAAC TTGGTTTTGT CCTCCCTTCA TGTGTACCTT TAATGTCAGC 27060 CTGAACTCCC TACTTATTTA CAACTTTTCT GACAAAACCG GGGGGCAATA CACAGCTCTC 27120 ATGCACTCCG GACCTGCTTC CCTCTTTCAG CTZTTTAAGC CAACGACTTG TGTCACCAAG 27180 GTGGAGGACC CGCCGTATGC CAACGACCCG GCCTCGCCTG TGTGSCGCCC ACTGCTTTTT 27240 GCCTTCGTCC TCTGCACCGG CTGCGCGGTG TTGTTAACCG CCTTCGGTCC ATCGATTCTA 27300 TCCGGTACCC GAAAGCTTAT CTCAGCCCGC TTTTGGAGTC CCGAGCCCTA TACCACCCTC 27360 CACTAACAGT CCCCCCATGG AGCCAGACGG AGTTCATGCC GAGCAGCAGT TTATCCTCAA 27420 TCAGATTTCC TGCGCCAACA CTGCCCTCCA GCGTCAAAGG GAGGAACTAG CTTCCCTTGT 27480 CATGTTGCAT GCCTGTAAGC GTGGCCTCTT TTGTCCAGTC AAAACTTACA AGCTCAGCCT 27540 CAACGCCTCG GCCAGCGAGC ACAGCCTGCA CTTTGAAAAA AGTCCCTCCC GATTCACCCT 27600 GGTCAACACT CACGCCGGAG CTTCTGTGCG AGTGGCCCTA CACCACCAGS GAGCTTCCGG 27660 CAGCATCCGC TGTTCCTGTT CCCACGCCGA GTGCCTCCCC GTCCT=CTCA AGACCCTCTG 27720 TGCCTTTAAC TTTTTAGATT AGCTGAAAGC AAATATAAAA TGGTGTGCTT ACCGTAATTC 27780 TGTTTTGACT TGTGTGCTTG ATTTCTCCCC CTGCGCCGTA ATCCAGTGCC CCTCTTCAAA 27840 ACTCTCGTAC CCTATGCGAT TCGCATAGGC ATATTTTCTA AAAGCTCTGA AGTCAACATC 27900

ACTCTCAAAC ACTTCTCCGT TGTAGGTTAC TTTCATCTAC AGATAAAGTC ATCCACCGGT 27960 TAACATCATG AAGAGAAGTG TGCCCCAGGA CTTTAATCTT GTGTATCCGT ACAAGGCTAA 28020 GAGGCCCAAC ATCATGCCGC CCTTTTTTGA CCGCAATGGC TTTGTTGAAA ACCAAGAAGC 28080 CACGCTAGCC ATGCTTGTGG AAAAGCCGCT CACGTTCGAC AAGGAAGGTG CGCTGACCCT 28140 GGGCGTCGGA CGCGGCATCC GCATTAACCC CGCGGGGCTT CTGGAGACAA ACGACCTCGC 28200 GTCCGCTGTC TTCCCACCGC TGGCCTCCGA TGAGGCCGGC AACGTCACGC TCAACATGTC 28260 TGACGGGCTA TATACTAAGG ACAACAAGCT AGCTGTCAAA GTAGGTCCCG GGCTGTCCCT 28320 CGACTCCAAT AATGCTCTCC AGGTCCACAC AGGCGACGGG CTCACGGTAA CCGATGACAA 28380 GGTGTCTCTA AATACCCAAG CTCCCCTCTC GACCACCAGC GCGGGCCTCT CCCTACTTCT 28440 GGGTCCCAGC CTCCACTTAG GTGAGGAGGA ACGACTAACA GTAAACACCG GAGCGGGCCT 28500 CCAAATTAGC AATAACGCTC TGGCCGTAAA AGTAGGTTCA GGTATCACCG TAGATGCTCA 28560 AAACCAGCTC GCTGCATCCC TGGGGGACGG TCTAGAAAGC AGAGATAATA AAACTGTCGT 28620 TAAGGCTGGG CCCGGACTTA CAATAACTAA TCAAGCTCTT ACTGTTGCTA CCGGGAACGG 28680 CCTTCAGGTC AACCCGGAAG GGCAACTGCA GCTAAACATT ACTGCCGGTC AGGGCCTCAA 28740 CTTTGCAAAC AACAGCCTCG CCGTGGAGCT GGGCTCGGGC CTGCATTTTC CCCCTGGCCA 28800 AAACCAAGTA AGCCTTTATC CCGGAGATGG AATAGACATC CGAGATAATA GGGTGACTGT 28860 GCCCGCTGGG CCAGGCCTGA GAATGCTCAA CCACCAACTT GCCGTAGCTT CCGGAGACGG 28920 TTTAGAAGTC CACAGCGACA CCCTCCGGTT AAAGCTCTCC CACGGCCTGA CATTTGAAAA 28980 TGGCGCCGTA CGAGCAAAAC TAGGACCAGG ACTTGGCACA GACGACTCTG GTCGGTCCGT 29040 GGTTCGCACA GGTCGAGGAC TTAGAGTTGC AAACGGCCAA GTCCAGATCT TCAGCGGAAG 29100 AGGCACCGCC ATCGGCACTG ATAGCAGCCT CACTCTCAAC ATCCGGGCGC CCCTACAATT 29160 TTCTGGACCC GCCTTGACTG CTAGTTTGCA AGGCAGTGGT CCGATTACTT ACAACAGCAA 29220 CAATGGCACT TTCGGTCTCT CTATAGGCCC CGGAATGTGG GTAGACCAAA ACAGACTTCA 29280 GGTAAACCCA GGCGCTGGTT TAGTCTTCCA AGGAAACAAC CTTGTCCCAA ACCTTGCGGA 29340 TCCGCTGGCT ATTTCCGACA GCAAAATTAG TCTCAGTCTC GGTCCCGGCC TGACCCAAGC 29400 TTCCAACGCC CTGACTTTAA GTTTAGGAAA CGGGCTTGAA TTCTCCAATC AAGCCGTTGC 29460 TATAAAAGCG GGCCGGGGCT TACGCTTTGA GTCTTCCTCA CAAGCTTTAG AGAGCAGCCT 29520 CACAGTCGGA AATGGCTTAA CGCTTACCGA TACTGTGATC CGCCCCAACC TAGGGGACGG 29580 CCTAGAGGTC AGAGACAATA AAATCATTGT TAAGCTGGGC GCGAATCTTC GTTTTGAAAA 29640 CGGAGCCGTA ACCGCCGGCA CCGTTAACCC TTCTGCGCCC GAGGCACCAC CAACTCTCAC 29700 TGCAGAACCA CCCCTCCGAG CCTCCAACTC CCATCTTCAA CTGTCCCTAT CGGAGGGCTT 29760

GGTTGTGCAT AACAACGCCC TTGCTCTCCA ACTGGGAGAC GGCATGGAAG TAAATCAGCA 29820 CGGACTTACT TTAAGAGTAG GCTCGGGTTT GCAAATGCGT GACGGCATTT TAACAGTTAC 29880 ACCCAGCGGC ACTCCTATTG AGCCCAGACT GACTGCCCCA CTGACTCAGA CAGAGAATGG 29940 AATCGGGCTC GCTCTCGGCG CCGGCTTGGA ATTAGACGAG AGCGCGCTCC AAGTAAAAGT 30000 TGGGCCCGGC ATGCGCCTGA ACCCTGTAGA AAAGTATGTA ACCCTGCTCC TGGGTCCTGG 30060 CCTTAGTTTT GGGCAGCCGG CCAACAGGAC AAATTATGAT GTGCGCGTTT CTGTGGAGCC 30120 CCCCATGGTT TTCGGACAGC GTGGTCAGCT CACATTTTTA GTGGGTCACG GACTACACAT 30180 TCAAAATTCC AAACTTCAGC TCAATTTGGG ACAAGGCCTC AGAACTGACC CCGTCACCAA 30240 CCAGCTGGAA GTGCCCCTCG GTCAAGGTTT GGAAATTGCA GACGAATCCC AGGTTAGGGT 30300 TAAATTGGGC GATGGCCTGC AGTTTGATTC ACAAGCTCGC ATCACTACCG CTCCTAACAT 30360 GGTCACTGAA ACTCTGTGGA CCGGAACAGG CAGTAATGCT AATGTTACAT GGCGGGGCTA 30420 CACTGCCCCC GGCAGCAAAC TTTTTTTGAG TCTCACTCGG TTCAGCACTG GTCTAGTTTT 30480 AGGAAACATG ACTATTGACA GCAATGCATC CTTTGGGCAA TACATTAACG CGGGACACGA 30540 ACAGATCGAA TGCTTTATAT TGTTGGACAA TCAGGGTAAC CTAAAAGAAG GATCTAACTT 30600 GCAAGGCACT TGGGAAGTGA AGAACAACCC CTCTGCTTCC AAAGCTGCTT TTTTGCCTTC 30660 CACCGCCCTA TACCCCATCC TCAACGAAAG CCGAGGGAGT CTTCCTGGAA AAAATCTTGT 30720 GGGCATGCAA GCCATACTGG GAGGCGGGGG CACTTGCACT GTGATAGCCA CCCTCAATGG 30780 CAGACGCAGC AACAACTATC CCGCGGGCCA GTCCATAATT TTCGTGTGGC AAGAATTCAA 30840 CACCATAGCC CGCCAACCTC TGAACCACTC TACACTTACT TTTTCTTACT GGACTTAAAT 30900 AAGTTGGAAA TAAAGAGTTA AACTGAATGT TTAAGTGCAA CAGACTTTTA TTGGTTTTGG 30960 CTCACAACAA ATTACAACAG CATAGACAAG TCATACCGGT CAAACAACAC AGGCTCTCGA 31020 AAACGGGCTA ACCGCTCCAA GAATCTGTCA CGCAGACGAG CAAGTCCTAA ATGTTTTTTC 31080 ACTCTCTTCG GGGCCAAGTT CAGCATGTAT CGGATTTTCT GCTTACACCT TTTTAGACAG 31140 CAGTTTACAC TCATTTCCGT TAAAGGATTA CAACTGCGGC ATATGAGAAT TAAGTATATA 31200 CAACTATTGC CCTTTACCCA CAAACACTCC CCCCACGGGG TGCACCTGAT GTAGCTGCCC 31260 TCCTCAATCA TGAAAGTGCT ATTAAAGTAA ATTAAATGAA CATTATTCAC ATACACGCTT 31320 CCCACATAGG CCAAAAAAAC AGAGGACAAC TTTGACAGCT CCCGCCTGAA ATACCAATAC 31380 ACTCTATCAA ACTGCGCACC GTGCACGCAC TGCTTTACCA GGCCTTGAAA GTAAACAGCG 31440 GCGGACCGAC ACTGCAAGCT TCTAGGCTTT GGGCAGTGGC AGTGAATATA TAGCCACTCC 31500 TCCCCATGCA CGTAGTAGGA ACGCCGCTTC CCGGGAATCA CAAATGACAA GCAGTAGTCA 31560

CAGAGGCAAC TAGTCAAGTG AGCGTCCTCC TGAGGCATGA TTACCTTCCA TGGAATGGGC 31620 CAGTGAATCA TAGTGGCAAA GCCAGCTGCA TCTGGAGCGC TGCGAACCTT GGCTACATGT 31680 GGTGATTGGC GACGCAGATG GAGACAGGAC CTTGCATTCT GAAGACCACT GCAACAGCTT 31740 CTGCGTACGC TTGTATTTAC AGTACATAAA AAAGCACTTT TGCCACAGAG CGGTCTTACT 31800 CAACCGACAG CTTTTTTCTT TCTGACGCTG CCTTCTGCTA CTCAGGTAGT ACAAGTCCAA 31860 AAGAGCCAAA CGGACACTCA AATCCGGGTT ATCTCGATGC TGAAGCCAGA GTCCAAAAGT 31920 AACCACGCTA AAAGCCTGCA TCCATATTTT GTAACTGCTG TAACTCCATC CCAGAGCCGG 31980 GCACCGCACT TGGTCCACCA TAGCTGCAAA CAAACGGGAC AATTAAGGAA AGTAAAATGA 32040 GCGCTGGGGG CGGACTCTTC TCCCGTTCGT AGGAAACAGC CACGTATCAA ACACCCTTTT 32100 CAACACTGGC TCTCCAGCCG CTACTCGTTG AATTAATTTG TCCCTGTGCT CAAACAACCC 32160 ACACTGGTAA CGGTGGTCGC TAGGCAAACA TGTCAAATAG CACATAATCA TTTCCTTCAC 32220 TTTAAGCAAA CATCGACTAG CAGACACTTC ACTTAATTCA GCACAGTCAT AGCAAGGAAT 32280 GATTATACAC TTGTCATCTA ATCCACTGCC CATGTACACA TTGCCCCAGG CAAAAGTGGG 32340 CAGGGACTTT AAGAGCTGAT TGCTCGCCCC GACATAGTTG GTAAAATACA GCAAATGCAC 32400 CTTGTTAACA TACACACTCC CCACATAGTA AATATACCGA GTAGACAGCT TAGAAAGCTC 32460 CCTCCGAAAA AATGGGAACA TGGTATCAAA GGCAGTGCCC GCAACACACA TCTTGAACAG 32520 ATCCATCAGG ATAGTAGCTC GACACAGCCC CTGCAGACTT TGGTCAGCTT GCTTGCTGCA 32580 GCAGTACACT CTCCACGTAG CATCTCCGCT GATGAAGTAT TCGCTATCGC AGCGACCAAA 32640 AATACAGCAA TCACAAGGCA GACGCAACAG TCTTTCATCC AGACTGTTCA TGAGAGGCTT 32700 TAGAGGTATG GGAAAAAATC CAAAGTGCTC AAAATAAGCA GCGCTGGGCT CATTCTGACA 32760 TTCCCCCAAC ATGCTGAGTC GAACCATAGC ACAGTCATAC AAACTCAGCT GTCGGAATTG 32820 ATCTTCCATG ATTGAGTTTC TACTGAGATA TTATCTCAAA CTTAAAACTG TTGCTCACCA 32880 ACTCTATGCG AACTTGCTCA AGAAGCTCTT GGTTTAGGGC GACCTCTTCT GGTCGTCGGA 32940 AGTTACTGAT GGAACAACAA GCGCCGCCCA ACTTCAAATT TCCAGCCGAC CCAATCCAGT 33000 GGTCTCTCAA CTCACGCGCA CAAGCTACTA TGCAGTCCTC ACTTTCGTCA AAGTCAGCAG 33060 CGCCTATAGA AATCAACACA CTGAGTCCAC CATCTTCAGC TTTTAAGGGA TAACAGCTGA 33120 TAGCAAACTG GTTCTGAGAC CACGGCAAAG CACGTAGGAA TTGCTGTTAA GTTAATTTCC 33180 AAACACCGCT GAAGCAGCTC TATGGTTGCT GGACATATGT CCTCTGCATA GAAGCTTTGA 33240 ACATAACTTA AGACAGGGCC GGGCACATGA AACACAAACA GAGAACTATA CACAATCTGG 33300 GCCATGATCA CTCACATTTA AATAGCAGCT GAAAAGTGGC TTTCTTCACT TGGGAGCAAA 33360 ATTAGCGAAG ACTGTGCCAG AATGCTCACG TCGAAAGGCG GTGGGTCTCG CAGAGGCAGG 33420

TTCGGAGCTC TAATTAAACA CAGGTGGGTA ATCCAGTCAA CGATGAGGAC CAGCTGAAAA 33480 GTGGCTTTCT TCACTTGGGA GCAAAATTAG CGAAGACTGT GCCAGAATGC TCACGTCGAA 33540 AGGCGGTGGG TCTCGCAGAG GCAGGTTCGG AGCTCTAATT AAACACAGGT GGGTAATCCA 33600 GTCAACGATG AGGACTTTTA AAAAACTGTC TAAAACTGAA GCAGTTAAGT TAGAGGCAGA 33660 CACAGAAAAA ACTACAGTTA AACTATCAGT TGCTGAAATT GAAAAGCACC CAATAATTAT 33720 GCGCGAGGGC ACAGGCAATA AAAGTGTTAG CCCCTCGGCT AACGCGTCAG CTAAAAAATC 33780 TTTAGCTAAA GTATCTACTG GCCGCGTGGT AAAAGTTTGA ATATAATTTA CGACAGGAGC 33840 TGGCAAGTGA AACTCCACAA AAAAAGTAAA TGGCTGCACA CACGCCATTA TTTTGAAAAT 33900 AAGAAGTACT CACAAAATCA GCTGGAGCTG CCGCAAGTGA AAAAGACCAG CTGAAGTCTT 33960 ATTTTAAACT GTAAAATATA AAAAAAAAAA TAGGGCGTGA ACAAAAATGA GAAAATAATA 34020 CCGGATATGA CTATTAAGGG CGTACACTGA AACTGGGTAA TATTTGAGAA AAAGATTAAG 34080 ATAATAGCTG AACAAATGAA GTGTGCAGAA CACGGAACAA TGGTGGCGAA AAAAAAAAAC 34140 AGTGTAAGCA CATGGCGCGC ACGTACTTCC GTGAGAAAAA TTAAAAAAAT TTACCCAGTA 34200 TAAGGTGCGT CATTAGACCC GCCTTGTGGC GCGGTTGTAG CCCTGCCCTT TGCCCCGCCC 34260 CGCGCGCCGC CCCGCGCGCC GCCCCCGCCG CCCTCAGCCC CGCCCAGCGC CGCCGCCTCC 34320 GCGACGCGCT CCGCCCCACA GTTACGTCAG CACGCCACGC TCGCCGTCGT TGCGTCATAA 34380 ATGACGTGGC AAAAATGATT GGCAGTTGGA CCGCTGCCAT CAGTGTACTG TAGATTATTG 34440 ATGATG 34446 (3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACGCGTCGAC TCCTCCTCA (4) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TTGACAGCTA C-OTTGTTG (5) INFORMATION FOR@ SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCAAGCTTGC ATGCCTG (6) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGCGATATCT CAGCTATAAC CGCTC (7) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTGCCCGGGC TT