WO1999007852A1 - Vaccine against fowlpox virus - Google Patents

Vaccine against fowlpox virus Download PDF

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WO1999007852A1
WO1999007852A1 PCT/AU1998/000628 AU9800628W WO9907852A1 WO 1999007852 A1 WO1999007852 A1 WO 1999007852A1 AU 9800628 W AU9800628 W AU 9800628W WO 9907852 A1 WO9907852 A1 WO 9907852A1
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fpv
rev
stranded dna
dna molecule
plasmid
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PCT/AU1998/000628
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French (fr)
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Christian Hertig
Barbara Elizabeth Howieson Coupar
Allan Robert Gould
David Bernard Boyle
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Commonwealth Scientific And Industrial Research Organisation
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Priority to AU86193/98A priority Critical patent/AU8619398A/en
Publication of WO1999007852A1 publication Critical patent/WO1999007852A1/en

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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a vaccine for the control of fowlpox and, more particularly, to a vaccine for the control of fowlpox which is free of contamination from the reticuloendotheliosis virus (REV) .
  • REV reticuloendotheliosis virus
  • Fowlpox virus is the type species of the avipox viruses ( Poxviridae, Chordopoxvirinae, Avipoxvirus) Infection of chickens or birds is characterised by the development of lesions on various parts of the skin, particularly unfeathered areas. With the most severe, diphtheritic, forms lesions occur on mucous membranes of respiratory and gastrointestinal tracts. Production losses in chickens are due to a decrease in egg production in layers and to reduced growth rates in broilers. Mortalities can occur with the severe, generalised or diphtheritic forms .
  • Vaccination and insect-proof screening to prevent mechanical transmission by mosquitoes are the methods generally used for controlling fowlpox disease in production flocks.
  • the vaccines are based on low virulence strains of FPV, and are usually delivered by wing web stab or feather follicle inoculation. Depending upon the residual virulence of the vaccine, vaccination may be carried out on day-old chickens or during the first four to five weeks of age, or older birds.
  • FPV M Cyanamid-Websters Pty Ltd Mild vaccine strain
  • Reticuloendotheliosis virus has been shown to have integrated into the genome of an avian herpesvirus
  • Marek's disease virus (MDV), during mixed infections of cell cultures used for attenuation of the MDV by continuous passage (Isfor, et al., 1991). Although insertion sites appeared clustered, insertions did occur in several different regions of the genome (Jones et al . , 1993). Integrated proviruses were unstable in undergoing recombination deletion, leaving fragments of the long terminal repeats in the MDV genome.
  • Retrovirus integration into the genome of herpesviruses has the potential to generate all the changes, e.g., gene activation, and mutation, that are usually associated with retrovirus integration into cellular genomes, as well as providing a novel means of retrovirus transmission via the infectious cycle of the herpesvirus.
  • Baculoviruses have also been identified as being able to spontaneously accommodate host cell-derived transposons. For both baculoviruses and herpesviruses, DNA replication occurs within the nucleus of an infected cell.
  • provirus DNA Integration of the provirus into the cellular DNA occurs, providing an opportunity for viral genomes to be an alternative target for integration.
  • poxvirus DNA replication occurs within the cytoplasm of the infected cell.
  • the present invention is based on the discovery that vaccine and field strains of FPV carry integrated REV sequences. Some of these FPV strains carry a near-full-length provirus of REV, and can give rise to infectious REV when transfected into cell cultures and when chickens are infected. Thus, having identified the nature of the apparent contamination of the FPV vaccines, the present invention provides the means by which a FPV vaccine free from contamination by REV may be produced.
  • FPV fowlpox virus
  • REV active reticuloendotheliosis virus
  • the present inventors have found that the REV sequence is inserted into the region of approximately right-hand one-third (or 3' end) of the FPV genome in a previously uncharacterised region of the FPV genome. PCR analysis suggests that there is a near-full-length provirus present .
  • FPV fowlpox virus
  • FPV fowlpox virus
  • said REV sequence has been excised from the one third of the FPV genome adjacent its 3' end.
  • said REV sequence is excised from a location which maps to a 17kb EcoRI restriction endonuclease fragment.
  • a method of preparing a recombinant fowlpox virus (FPV) whose nucleic acid does not encode active reticuloendotheliosis (REV) comprising the steps of:
  • said isolated DNA fragment is inserted into a plasmid.
  • the amplification in step (2) is performed using the polymerase chain reaction with primers based on the FPV sequences which flank the REV sequence.
  • the person skilled in the art will be aware of other suitable methods of DNA amplification, such as ligase chain reaction.
  • said isolated DNA fragment is an EcoRI- BamHl fragment.
  • said isolated DNA fragment is amplified in plasmid pCH21, preferably by a method wherein plasmid pCH21 is digested with Bel and the digested DNA ligated to itself to produce plasmic pCH29. Said isolated DNA fragment is then recombined into the nucleic acid of a fowlpox virus strain.
  • a preferred method for modifying a strain of FPV to excise an REV sequence comprises the following steps : (1) Inserting an EcoRI-BamHI fragment of the FPV mild vaccine strain into plasmid pUC19 so as to create plasmid pCH21, which plasmid includes the FPV sequences which flank said REV sequence and said REV sequence;
  • the FPV sequence from pCH29 is recombined into a fowlpox virus by a method comprising the steps of:
  • the FPV sequence from pCH29 is recombined into a fowlpox virus by a method comprising the steps of: (1) isolating a DNA fragment carrying the P.L promoter- ⁇ -galactosidase gene and the wP.7.5 promoter ECOGPT gene from plasmid pAF09;
  • the present invention also envisages novel nucleic acid molecules and plasmids including these novel nucleic acid molecules are also envisaged.
  • a plasmid selected from the group comprising:
  • first single-stranded DNA molecule which has a molecular size of 17kb and is derived from FPV S
  • second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single-stranded DNA hybridises with the product of amplification of an REV LTR sequence, and is an EcoRI restriction endonuclease fragment.
  • a first single-stranded DNA molecule which has a molecular size of 9.8kb and is derived from FPV S, and
  • an isolated DNA molecule comprising an REV sequence and flanking FPV sequences and selected from the group consisting of the DNA sequences having the DNA sequence shown in Fig 5 including the FPV S 5' LTR (SEQ ID NO: 2), the FPV S 3' LTR (SEQ ID NO: 3) or FPV M3 LTR (SEQ ID NO : 4), or the DNA sequences shown in Fig 6 including FPV S 5' LTR (SEQ ID NO: 5), FPV S 3' LTR (SEQ ID NO: 7) and FPV M3 LTR (SEQ ID NO: 6).
  • nucleic acid molecule having the sequence shown in Fig 14 (SEQ ID NO: 9) .
  • nucleic acid molecule having the sequence shown in Figure 16 (SEQ ID NO: 10) .
  • nucleic acid molecule having the sequence shown in Fig 18 (SE ID NO: 11)
  • a vaccine against fowlpox virus which does not give rise to reticuloendotheliosis virus (REV) infection when administered to chickens, comprising an attenuated strain of FPV and a pharmaceutically acceptable carrier, and including means for preventing expression of active REV.
  • FPV fowlpox virus
  • REV reticuloendotheliosis virus
  • said means for preventing expression of active REV comprises using a recombinant FPV as described above, but any other means for preventing expression of active REV could be used.
  • any other means for preventing expression of active REV could be used.
  • the means for preventing expression of active REV could be used.
  • REV sequence can be mutated so that inactive REV is expressed. Typically, said mutation is by insertion, deletion or non-conservative substitution.
  • said means for preventing expression of active REV comprises an expression cassette for expressing antisense RNA corresponding to the REV sequence.
  • the expression cassette may be incorporated into the FPV or may comprise a further expression vector.
  • said means for preventing expression of active REV comprises a catalytic ribozyme for cleaving REV mRNA, or an expression cassette for expression of such a ribozyme.
  • said means for preventing expression of active REV comprises means for downregulating the LTR promoter in the REV sequence.
  • a method for preventing the occurrence of fowlpox virus (FPV) in a chicken without giving rise to reticuloendotheliosis virus (REV) infection comprising the step of administering a vaccine as described above to said chicken.
  • FMV fowlpox virus
  • REV reticuloendotheliosis virus
  • said chicken is vaccinated at older than one day of age, optionally subsequently to vaccination at one day of age with FPV M.
  • REV sequence means a DNA sequence, which may or may not encode active reticuloendotheliosis virus, derived from the incorporation of the reticuloendotheliosis virus into the genome of a fowlpox virus .
  • Plaque purified derivatives of this strain are designated FPV M3 and FPV M A-F and have been described in Boyle, D.B. Pye, A.D. and Coupar, B.E.H. (1997) "Comparison of Field and Vaccine Strains of Australian Fowlpox Viruses" Arch.
  • Figure 1 shows evidence for REV sequences in field and vaccine strains of FPV.
  • FPV DNAs were digested with Pstl restriction and endonucleases and transferred to nylon membranes by Southern blotting.
  • A Ethidium bromide stained
  • B 32 P-labeled 291-bp LTR PCR product hybridized to 9.8 pb fragment from FPV S .
  • Lanes 1 to 5 contain DNA isolated from FPV M-F, M3, S, AWPL 1136 and 1137, respectively. Lambda Hindi! markers are shown as size markers at the left of the Figure;
  • Figure 2 shows evidence for complete REV provirus incorporation into FPV S and possible remnants in FPV M3, FPV DNAs were digested with EcoRI restriction endonuclease and transferred to nylon membranes by Southern blotting.
  • A Ethidium bromide stained.
  • B 32 P-labeled 291-bp LTR PCR product hybridized to the DNA fragments and autoradiographed. Lanes 1 and 2 contain DNA isolated from FPV M3 and S, respectively. Lambda Hindlll markers are shown as size markers at the left of the Figure;
  • Figure 3 shows a restriction endonuclease map of the FPV genome showing region of inserted REV sequences .
  • P Pstl
  • E EcoRI
  • B BamHl (BamHl and £ ⁇ oRI sites marked are incomplete)
  • LTR long terminal repeat of REV.
  • LTR truncated LTR present in FPV M3 and the 3 * end of the REV provirus inserted into FPV S.
  • REV genes gag and env are marked.
  • Open reading frames from the FPV genomic region flanking the provirus insertion are marked 1, 2 and 3.
  • Ps ll fragments F, J, P, A' and D' are at the right-hand end of the FPV genome, respectively (as reported by Coupar et al . , 1990). Regions from which partial or complete nucleotide sequence has been determined are marked by dashed lines;
  • Figure 4 shows long-range (XL) PCR analysis of the REV provirus insert in FPV S strain.
  • Primer pairs used for LX PCR were derived from lane 1 flanking FPV sequences (primer pair 1/2), from lanes 2 and 3 flanking FPV sequences and internal REV sequences (lane 2 primers 1/4 and lane 3 primers 2/3) .
  • the locations of the primers are marked in Figure 3.
  • ⁇ DNA digested with Hindlll was used as size markers, lO ⁇ l from a 100- ⁇ l XL PCR was analyzed on a 0.6% agarose gel;
  • FIG. 5 shows the alignment of REV LTR and flanking FPV sequences present in FPV strains.
  • the REV LTR present in chicken syncytial virus provirus (ACRLTRl) Swift et al., 1987) (SEQ ID NO: 1) was aligned with REV LTR sequences present at the 5' end (FPV M5 LTR) (SEQ ID NO: 2) and the 3' end (FPV S3') (SEQ ID NO: 3) and the FPV M3 LTR (SEQ ID NO: 4) .
  • the U3, R and U5 regions are those identified by Swift et al . , (1987). conserveed nucleotides are indicated by dots; deletions are indicated by a dash.
  • Flanking FPV sequences are shown in lower case letters .
  • the duplicated U3 terminus present in FPV S 3 ' LTR is in boldface and underlined.
  • flanking REV sequences present on the 5 ' end of FPV S 3 ' and the 3 ' end of the FPV S 5' LTR have been omitted;
  • Figure 6 shows the alignment of REV sequences, including the 5' LTR, 3' LTR and some of the intervening REV sequence, and flanking FPV sequences in FPV S AND FPV M3. Sequences containing the FPV S 5' LTR (SEQ ID NO : 5 ) , the FPV M3 LTR ( SEQ ID NO : 6 ) , the FPV S 3 ' LTR (SEQ ID NO 7) and the FPV M3 3' flanking sequence (SEQ ID NO: 8) are shown but the REV insert is not shown in full for clarity.
  • Figure 7 is a restriction endonuclease cleavage map of plasmid pCH21;
  • Figure 8 is a restriction endonuclease cleavage map of plasmid pCH2 ;
  • Figure 9 is a restriction endonuclease cleavage map of plasmid pCH30a
  • Figure 10 is a restriction endonuclease cleavage map of plasmid pCH30b
  • Figure 11 is a restriction endonuclease cleavage map of plasmid pCH31a
  • Figure 12 is a restriction endonuclease cleavage map of plasmid pCH31b.
  • Figure 13 is a restriction endonuclease map of the FPV S REV neg beta-gal positive construct constructed from FPV S using plasmid pCH30a;
  • Figure 14 is the DNA sequence of the construct shown in Figure 13 (SEQ ID NO: 9);
  • Figure 15 is a restriction endonuclease map of the FPV S REV neg beta-gal positive construct constructed from FPV S using pCH30b;
  • Figure 16 is the DNA sequence of the construct shown in Figure 15 (SEQ ID NO: 10);
  • Figure 17 is a restriction endonuclease map of the FPV S REV negative construct constructed from FPV S using plasmids pCH31a and b;
  • Figure 18 is the DNA sequence of the construct shown in Figure 17 (SEQ ID NO: 11) .
  • CES Primary chicken embryo skin cell cultures
  • SPF pathogen free
  • CEF Chicken embryo fibroblast
  • the primers and protocols described by Aly et al . , (1993) were used for amplification of REV LTR sequences present in FPV S, the AWPL field strains and CEF cells infected with REV virus.
  • 32 P-labelled REV LTR PCR products were prepared by including 32 P-labelled nucleotide in the PCR reaction mix with a corresponding reduction in the equivalent unlabelled nucleotide to 1/10 the normal coneentration.
  • XL PCR (Perkin Elmer GenAmp XL PCR) was used to characterize the REV sequences in FPV S strain.
  • Primer 1 (sense) (5' -CCATCGAATTCACGTATTAC-3 ' ) (SEQ ID NO: 12) located at the EcoRI end of the sequenced region of FPV M3;
  • primer 2 (antisense) ( 5 ' -CGGAATTCGGATCCGCGTGAATAGCTTCTACGGG-3 ' ) (SEQ ID NO: 13) located at the BamHl end of the FPV M3 sequenced region (EcoRI site added to aid cloning) ;
  • primer 3 (sense) (5' -TTTCTGCATCCCTCTGGC-3 ' ) (SEQ ID NO: 14) derived from the polymerase region of REV (sequence determined from the EcoRI-Pstl fragment of FPV S) ; and primer 4
  • PCR conditions included the use of a hot start, 93° C for 1 min, 55° C for 2 min, 68° C for 5 min with a 10 sec extension per cycle using a total of 30 cycles. Less than 100 ng of FPV DNA was used as template in each 100 ⁇ l reaction.
  • FPV M3 and S genomic DNA fragments carrying REV sequences were separated by agarose gel electrophoresis after restriction endonuclease digestion. Selected DNA bands were excised from the gel, purified and cloned into the pUC19 plasmid vector. Desired plasmids were identified by hybridization and restriction endonuclease digestion analysis. Initial nucleotide sequence was determined from these plasmids using universal forward and reverse sequencing primers. Additional sequence was obtained by a variety of strategies including cloning of subfragments into pUC19 and M13 vectors and primer walking strategies, followed by manual and automated sequencing methods .
  • the location of the REV LTR insertion within the FPV genome was mapped by hybridization of selected fragments to restriction endonuclease digested genomic DNA.
  • the previously constructed Pstl and partial BamH maps of the FPV genome (Coupar et al., (1990) "Restriction endonuclease mapping of the fowlpox virus genome” Virology 179, 159- 167), the contents of which are incorporated herein by reference, were used to locate the REV LTR insertion within the FPV genomic map.
  • RT lysis buffer (30 mM Tris-HCl pH 8.0, 80 mM KC1, 1.0 mM EDTA, 0.1 % v/v Triton X-100, 10 % v/v glycerol and 2 mM DTT) and dilution series were tested for reverse transcriptase activity using RT-detect (NEN Du Pont) according to the manufacturer's instructions.
  • the 4 x RT buffer used contained 130 mM Tris-HCl pH 8.0, 120 mM KCl and 33.6 mM MgCl 2 .
  • Cellular genomic DNA was prepared from uninfected and REV infected CES and CEF cell cultures .
  • DNA was prepared from 5 x 10 s cells using the Qiagen genomic DNA purification procedure (Qiagen Pty. Ltd.). Cultures infected with REV were inoculated as 50-80% confluent monolayers and cells harvested for DNA extraction 10 to 14 days later .
  • FPV DNA was purified from FPV S and M3 infected CES cell cultures.
  • CES cell cultures were infected at a multiplicity of 0.05 to 0.1 pfu per cell.
  • the CPE had reached 80 to 90% (5 to 8 days after infection) the cells were harvested, resuspended in 4°C lOmM Tris-HCl pH 7.6 (20ml per 5 x 10 7 cells) and dounce homogenized. Nuclei were removed by centrifugation at 5,000 g for 5 min and RNAase (20 ⁇ g/ml) and DNAase (25 ⁇ g/ml) added.
  • DNA was extracted from the cores using the Qiagen genomic DNA purification procedure.
  • FPV DNA, FPV M3 infected CES cells were mixed with an equal number of REV infected CES cells prior to commencing the protocol for purification of FPV DNA.
  • REV IDEXX, USA
  • IDEXX a detergent and heat inactivated antigen preparation from the Cook strain of REV.
  • REV was isolated from heparinized blood by direct inoculation of CEF cell cultures. After an additional passage in CEF cells, the cultures were stained by immunofluorescence with the gag (P29) monoclonal antibody to detect REV.
  • the 9.8 kb fragment is absent from Pstl digests of FPV M and its plaque purified derivatives, but is present in all of the AWPL series field isolates and the FPV S vaccine strain. (Fig.lA) . This data suggested that part of the REV genome was present within the genome of the FPV S and AWPL isolates.
  • the 9.8 kb Pstl fragment of FPV S shown to hybridize with the REV LTR PCR product, was cloned into pUC. This cloned fragment hybridized to the 9.8 kb fragment present in the field isolates (Fig. IC) . Confirmation that part of this fragment was derived from FPV was shown by its hybridization to the largest Pstl fragments of FPV M-F and M3 (Fig. IC) . The location of the REV LTR sequences within this cloned fragment was determined by restriction endonuclease digestion and Southern hybridization. An EcoRI-PstI subfragment (4.3 kb) was identified as containing the REV LTR hybridizing region.
  • Nucleotide sequence determination of this fragment revealed 903 bp of apparent FPV genome sequence (adjacent to the EcoRI site), a complete REV LTR and sequence of the REV gag region extending to the Pstl site - a total of 3388 bp of REV genomic sequence.
  • the presence of REV genomic sequences up to the Pstl site suggested that the REV integrated sequences extended into an adjoining Pstl fragment .
  • the FPV M3 EcoRI genomic fragment (9.0 kb) shown to weakly hybridize to REV LTR product was cloned into pUC19. This fragment hybridized only to the largest Pstl fragment of FPV M-F and M3 and to the largest fragment of FPV S and the field strains, and to the 9.8 kb fragment of these strains shown to contain the REV LTR sequences (data not shown) . Although weakly hybridizing with the REV LTR probe (Fig. 2), a specific PCR product could not be generated from FPV M3 DNA suggesting that an incomplete REV LTR may be present in the genome of this virus.
  • nucleotide sequence determination revealed a truncated REV LTR (248 bp) remnant in the FPV M3 genome at the same location as the FPV S REV insertion.
  • XL PCR analysis suggested that there was a near full length provirus present in the FPV strain.
  • XL PCR analysis of FPV M3 and FPV S using primers 1 and 2 yielded products of 2.8 kb from both virus DNA's (Fig. 4).
  • the absence of a larger product from FPV S DNA was indicative of heterogeneity present in the genomes of the viruses carrying near full length REV provirus inserts. Genome heterogeneity in these viruses would be expected since the REV LTR direct repeats present would make the genome inherently unstable in the region of the insert. This heterogeneity was not apparent in the hybridization analysis of the FPV DNAs.
  • the protein (285 amino acids) encoded by ORF 2 is related to the hypothetical 33.6K protein (a member of the protein kinase family - 287 amino acids) of shope fibroma virus (Genbank entry JQ1743) .
  • the deduced amino acid sequence from the incompletely sequenced ORF 3 is related to the serpin from ectromelia virus (Genbank entry S24676).
  • the sequences of the integrated retroviral provirus are closely related to REV and spleen necrosis virus (SNV) (Fig.5).
  • Alignment of the FPV S 5' LTR with REV LTR (Genbank entry ACRLTRl) revealed a single base deletion and two base substitutions in the FPV S 5' LTR over the 517bp LTR region.
  • the FPV S 3' LTR and the remnant LTR in FPV M 3 were identical to each other except for one deletion, one base substitution and the 23bp duplication of the U3 5' terminus present in FPV S 3' LTR.
  • both the FPV S 3' LTR and the FPV M3 remnant LTR had a large deletion (262bp) spanning part of the U3, all of the R and part of the U5 regions.
  • significant changes had occurred in the remnant (51bp) of the U5 region with three base deletions, five base substitutions and a three base insertion (Fig.5).
  • the truncated and rearranged 3 ' LTR and the absence of the classical direct repeats at the integration site suggests that the integration of the REV provirus into the genome of FPV occurred in an unusual manner or has undergone rearrangement after integration.
  • Example 6 Testing biological activity of REV provirus in FPV S in vitro and in vivo
  • Plasmid pCH21 carries an EcoRI-BamHI fragment of the FPV Mild vaccine strain genomic DNA. This plasmid is based upon the widely-used cloning vector pUCl9.
  • the FPV genomic fragment carries the FPV sequences which flank the site of insertion of the REV genome in the FPV Standard strain genome (these same sequences surround the site of integration of a near full-length REV genome in the FPV Standard vaccine strain) . It also carries the REV long terminal repeat remnant, which we have shown to be present in FPV Mild vaccine strain.
  • Polymerase chain reaction primers have been specifically designed to amplify the complete pCH21 plasmid, deleting the REV LTR remnant and inserting specific restriction endonuclease sites in its place between the FPV genes identified as M-l and M-l 3' as per the map of pCH21.
  • Plasmid pCH29 was digested with Bell and ligated to the DNA fragment from pAF09. Plasmids pCH30a and pCH30b were recovered and characterised by standard techniques. These plasmids carry the FPV DNA sequences with the DNA fragment from pAF09 inserted at the Bell site.
  • Plasmid pCH29 was digested with BamHl and ligated to the DNA fragment from pAF09. Plasmids pCH31a and pCH31b were recovered and characterised by standard techniques . These plasmids carry the FPV DNA sequences with the DNA fragment from pAF09 flanking the FPV DNA and inserted at the BamHl site.
  • plasmids pCH30 a & b and pCH31a & b were combined with FPV Standard vaccine strain. These same plasmids could be used to recombine with any other isolated fowlpox virus shown to similarly contain the REV genome sequences.
  • re ⁇ ombinants with the REV genome deleted and the ⁇ -galactosidase gene and ECOGPT gene inserted in its place were selected on the basis of growth under selection conditions for ECOGPT and on the basis of expression of the ⁇ -galactosidase gene.
  • the recombinants were plaque purified and virus stocks prepared. This strain was designated FPV Standard REV negative ⁇ - galactosidase positive. (FPV S REV neg ⁇ -gal pos) .
  • pCH31 recombinants with the REV genome deleted were selected on the basis of transient dominant insertion (Davison, A.J. and Elliot, R.M. (1993), Molecular Virology: A Practical Approach. IRL Press. Oxford University Press, Oxford, New York, Tokyo. Chapter 9. Smith, G.L. "Expression of Genes by Vaccinia Virus Vectors" pp 257-283, which is incorporated herein by reference), of the ⁇ -galactosidase and ECOGPT genes inserted in the FPV genome and the REV genome deleted and replaced by the short sequence surrounding the Bell site inserted between the M-l and M-l 3' genes identified in the map of pCH31a & b.
  • FPV standard strain derivatives were selected upon the basis of insertion of the ⁇ -galactosidase and ECOGPT genes (blue plaques) then derivatives were selected upon the basis of the loss of the ⁇ -galactosidase gene (white plaques) .
  • the recombinants with the REV genome deleted and the ⁇ - galactosidase and ECOGPT genes lost after transient dominant selection were plaque purified and virus stocks prepared. This strain was designated FPV Standard REV negative (FPV S REV -) .
  • FPV S REV - ⁇ -gal + and FPV S REV - can be used to vaccinate and revaccinate chickens at ages greater than one day old. These strains generated by recombinant DNA techniques to remove the REV genome integrated in the FPV genome and thus to remove the REV contamination of the
  • FPV S vaccine will overcome the deficiencies present in the FPV Mild Strain and the contamination problem which resulted in the use of the FPV Standard strain being discontinued. Similarly this method can be used to eliminate REV from recently-derived field isolates of FPV, enabling them to be used for the development of fowlpox vaccines .
  • a restriction endonuclease map has been prepared for the FPV S REV neg beta-gal positive strains constructed from FPV S using pCH30a and pCH30b, and DNA sequences obtained for these constructs . These are shown in Figures 11, 12, 13 and 14, respectively.
  • a restriction endonuclease map for the FPV S REV negative strain constructed from FPV S using plasmids pCH31a has been prepared and is shown in Figure 15, and the DNA sequence of this construct is shown in Figure 16.
  • the present invention is useful in the poultry industry for the control of fowlpox in chickens without the introduction of reticulendotheliosis virus to these chickens .
  • Reticuloendotheliosis virus Experimental infection of poultry and immunofluorescent identification of Australian isolates. Aust . Vet . J. 53, 506-508.
  • Herpesviral gD as a common target of integration. Virology 203, 125-133.
  • Retroviral insertions into a herpesvirus are clustered at the junction of the short repeat and short unique sequences. Proc . Natl . Acad . Sci . USA 90, 3855-3859.
  • the baculovirus- integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase. J. Virol . 66, 1590-1601.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Avian reticuloendotheliosis virus
  • CTTTTTCTTC TATATCCTCA GATTGGCAGT GAGAGGAGAT TTTGTTCGTG GTGTTGGCTG 480
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAATACTACG ATATTTTCGA ATTAAAATAT CTAGTTGATA ACTGTAAACT ACCTATGCCT 1080
  • MOLECULE TYPE DNA (genomic)
  • AGGATGTCAT ATCTACGATA ATACTAAGTC TATAATAGCC GAAGCAATGT TTTTTAC AT 1440 GAAACACGAA TCTATATTCG GTTCAACAAG AAAAGATACT ATAACCTTCT ATAAGTACGA 1500
  • CACATGATAA GAGATTGTAT CAGTTTCGTA GTCTTGAGTA TTGGTATTAC TATATAGTAT 1860 ATGTCGGGAA TTCAGATCCA TGCTAGATCC CAAATAGTAC ATAATGGATT TCCTTACGCG 1920
  • AAATACGGGC AGACATGGCC TGCCCGGTTA TTATTATTTT TGACACCAGA CCAACTGGTA 1980
  • CGTATCGCCA AAATCACCGC CGTAAGCCGA CCACGGGTTG CCGTTTTCAT CATATTTAAT 3300
  • CTGTAGCCAG CTTTCATCAA CATTAAATGT GAGCGAGTAA CAACCCGTCG GATTCTCCGT 4680
  • AAAAGCCCTT ATTGGTATAT TCGTGTGATT CTTATAGGTT ATATAACGCT AAATATGACA 5280
  • ATAAAAATTT AACCAGTGAA TCTTTTTACA TGACTAATAA TGGTGTTTTA AAAATAGGTA 5640
  • GGAATTATAT GATTCCGGAG AGCCTTTATT ATTTTCACCT AGAGGGCTAC ATAAAATATT 6120
  • AAAAGTAGAA AATATATTCT AATTTATTGC ACGGTAAGGA AGTAGATCAT AAAGAACAGT 4380
  • TGTTCAAACC CCGCTTTGGG CCATGGGGAG ATCATATTAT TGTATAATAT TATATTTTGT 5100
  • AAAGCCCTTA TTGGTATATT CGTGTGATTC TTATAGGTTA TATAACGCTA AATATGACAA 5280
  • GTACATATTG GATATATCCG TTCCTTTATG TAGCCTGGTG GTTGAAAATA ACTACCTTAC 5460
  • AAATTATACT ATAAAATCCG AAATTTATAG ATTCGGTATT GTTATATGGG AAATTATTAC 1620
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid

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Abstract

A recombinant fowlpox virus (FPV) whose nucleic acid does not encode an active reticuloendotheliosis virus (REV).

Description

TITLE
VACCINE AGAINST FOWLPOX VIRUS
FIELD OF THE INVENTION
The present invention relates to a vaccine for the control of fowlpox and, more particularly, to a vaccine for the control of fowlpox which is free of contamination from the reticuloendotheliosis virus (REV) .
BACKGROUND ART
Fowlpox virus (FPV) is the type species of the avipox viruses ( Poxviridae, Chordopoxvirinae, Avipoxvirus) Infection of chickens or birds is characterised by the development of lesions on various parts of the skin, particularly unfeathered areas. With the most severe, diphtheritic, forms lesions occur on mucous membranes of respiratory and gastrointestinal tracts. Production losses in chickens are due to a decrease in egg production in layers and to reduced growth rates in broilers. Mortalities can occur with the severe, generalised or diphtheritic forms .
Vaccination and insect-proof screening to prevent mechanical transmission by mosquitoes are the methods generally used for controlling fowlpox disease in production flocks. The vaccines are based on low virulence strains of FPV, and are usually delivered by wing web stab or feather follicle inoculation. Depending upon the residual virulence of the vaccine, vaccination may be carried out on day-old chickens or during the first four to five weeks of age, or older birds.
In Australia, the Cyanamid-Websters Pty Ltd Mild vaccine strain (FPV M) is the only vaccine widely available to the poultry industry for vaccination to control fowlpox. This vaccine is a very mild vaccine suitable for application to chickens at around one day of age. Because it is a very mild vaccine and is not generally considered suitable for application to chickens of older age or for revaccination of chickens previously vaccinated at one day old, and provides a low level of protection against FPV.
Previously a more virulent fowlpox vaccine, Cyanamid Websters Pty Ltd Standard strain (FPV S) , was available to the industry. It was routinely used to vaccinate chickens older than one day of age, and was suitable for the revaccination of older chickens previously vaccinated at one day of age. This vaccine was withdrawn from use in Australia because it was considered to be contaminated with REV. Attempts to remove the REV contamination were unsuccessful, and no other FPV isolate has been found from the field to provide an adequate replacement free of REV.
In the present invention it has been shown that this apparent contamination of the FPV Standard vaccine with REV is not due to free REV, but rather is due to the integration of REV into the genome of FPV. In addition we have now found that many, if not all, field strains of FPV in Australia carry similarly integrated REV sequences in their genome.
Reticuloendotheliosis virus (REV) has been shown to have integrated into the genome of an avian herpesvirus,
Marek's disease virus (MDV), during mixed infections of cell cultures used for attenuation of the MDV by continuous passage (Isfor, et al., 1991). Although insertion sites appeared clustered, insertions did occur in several different regions of the genome (Jones et al . , 1993). Integrated proviruses were unstable in undergoing recombination deletion, leaving fragments of the long terminal repeats in the MDV genome.
Co-infection studies with two different avian retroviruses, REV and avian leukosis virus (ALV) , and two different avian herpes viruses, MDV and the herpesvirus of turkeys (HVT) , showed that integration of retrovirus into the herpesvirus genome could occur as early as the fourth to sixth in vitro passage (Isfort et al., 1994). Integration occurred at a number of sites including the HVT gD gene which was disrupted by the insertion. In one case the integrated REV provirus in the HVT genome appeared to be full length, as it was infectious when transfected into chicken embryo fibroblast cells (Isfort et al . , 1994). Short regions of nucleotide sequence homology within the R and U3 regions of REV LTR observed in other isolates of MDV type I suggest that REV insertion into the MDV genome has occurred frequently in the past (Isfort et al., 1992). To date no field isolates of MDV have been described containing full-length REV provirus. Retrovirus integration into the genome of herpesviruses has the potential to generate all the changes, e.g., gene activation, and mutation, that are usually associated with retrovirus integration into cellular genomes, as well as providing a novel means of retrovirus transmission via the infectious cycle of the herpesvirus.
Baculoviruses have also been identified as being able to spontaneously accommodate host cell-derived transposons. For both baculoviruses and herpesviruses, DNA replication occurs within the nucleus of an infected cell.
Integration of the provirus into the cellular DNA occurs, providing an opportunity for viral genomes to be an alternative target for integration. In contrast, poxvirus DNA replication occurs within the cytoplasm of the infected cell.
Surprisingly, despite this difference REV appears to be able to integrate into the genome of FPV, and the present invention is based on the discovery that vaccine and field strains of FPV carry integrated REV sequences. Some of these FPV strains carry a near-full-length provirus of REV, and can give rise to infectious REV when transfected into cell cultures and when chickens are infected. Thus, having identified the nature of the apparent contamination of the FPV vaccines, the present invention provides the means by which a FPV vaccine free from contamination by REV may be produced.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a recombinant fowlpox virus (FPV) whose nucleic acid does not encode an active reticuloendotheliosis virus (REV) .
The present inventors have found that the REV sequence is inserted into the region of approximately right-hand one-third (or 3' end) of the FPV genome in a previously uncharacterised region of the FPV genome. PCR analysis suggests that there is a near-full-length provirus present .
Accordingly, in a preferred embodiment of the present invention there is provided a recombinant fowlpox virus (FPV) in which an REV sequence is substantially absent from the FPV genome.
According to a still more preferred embodiment of the present invention there is provided a recombinant fowlpox virus (FPV) from which an REV sequence previously present in the FPV genome has been excised.
Preferably said REV sequence has been excised from the one third of the FPV genome adjacent its 3' end. In FPV S said REV sequence is excised from a location which maps to a 17kb EcoRI restriction endonuclease fragment.
According to another aspect of the invention, there is provided a method of preparing a recombinant fowlpox virus (FPV) whose nucleic acid does not encode active reticuloendotheliosis (REV) , comprising the steps of:
(1) providing an isolated DNA fragment comprising a REV sequence flanked by FPV sequences;
(2) amplifying said FPV sequences but not said REV sequence; and
(3) inserting said FPV sequences into a fowlpox virus.
Preferably, said isolated DNA fragment is inserted into a plasmid. Typically, the amplification in step (2) is performed using the polymerase chain reaction with primers based on the FPV sequences which flank the REV sequence. The person skilled in the art will be aware of other suitable methods of DNA amplification, such as ligase chain reaction.
Typically said isolated DNA fragment is an EcoRI- BamHl fragment. Typically said isolated DNA fragment is amplified in plasmid pCH21, preferably by a method wherein plasmid pCH21 is digested with Bel and the digested DNA ligated to itself to produce plasmic pCH29. Said isolated DNA fragment is then recombined into the nucleic acid of a fowlpox virus strain.
A preferred method for modifying a strain of FPV to excise an REV sequence comprises the following steps : (1) Inserting an EcoRI-BamHI fragment of the FPV mild vaccine strain into plasmid pUC19 so as to create plasmid pCH21, which plasmid includes the FPV sequences which flank said REV sequence and said REV sequence;
(2) Amplifying plasmid pCH21 using the polymerase chain reaction with primers based on the FPV sequences which flank said REV sequence, so as to generate a DNA product encompassing the entire plasmid but without the REV sequence;
(3) Digesting the DNA product with Bell and ligating the digested DNA product to itself so as to recover plasmid pCH29; and
(4) Inserting the FPV sequence from plasmid pCH29 into a FPV construct so as to create a recombinant FPV free of the REV sequence.
Preferably, the FPV sequence from pCH29 is recombined into a fowlpox virus by a method comprising the steps of:
(1) isolating a DNA fragment carrying the P.L promoter-β-galactosidase gene and the wP.7.5 promoter
ECOGPT gene from plasmid pAF09;
(2) digesting plasmid pCH29 with Bell and ligating to the DNA fragment from pAF09 to produce plasmids pCH30a and pCH30b; and
(3) combining plasmids pCH30a and/or pCH30b with the fowlpox virus .
Alternatively, the FPV sequence from pCH29 is recombined into a fowlpox virus by a method comprising the steps of: (1) isolating a DNA fragment carrying the P.L promoter-β-galactosidase gene and the wP.7.5 promoter ECOGPT gene from plasmid pAF09;
(2) digesting plasmid pCH29 with BamHl and ligating to the DNA fragment from pAF09 to produce plasmids pCH31a and pCH31b; and
(3) combining plasmids pCH31a and/or pCH31b into the fowlpox .
The present invention also envisages novel nucleic acid molecules and plasmids including these novel nucleic acid molecules are also envisaged. In a preferred embodiment of the present invention there is provided a plasmid selected from the group comprising:
plasmid pCH21 as herein defined;
plasmid pCH29 as herein defined;
plasmid pCH30a as herein defined;
plasmid pCH30b as herein defined;
plasmid pCH31a as herein defined; and
plasmid pCH31b as herein defined.
In a further embodiment of the invention there is provided an isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 17kb and is derived from FPV S, and (2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single-stranded DNA hybridises with the product of amplification of an REV LTR sequence, and is an EcoRI restriction endonuclease fragment.
In another embodiment of the present invention there is provided an isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 9.0kb and is derived from FPV M3, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single-stranded DNA hybridises to a product of amplification of an REV LTR sequence, and is an EcoRI restriction endonuclease fragment.
In still another embodiment of the present invention there is provided an isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 9.8kb and is derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single-stranded DNA hybridises with a product of amplification of an REV LTR sequence, and is a PstI restriction endonuclease fragment.
In yet another embodiment of the present invention there is provided an isolated DNA molecule comprising an REV sequence and flanking FPV sequences and selected from the group consisting of the DNA sequences having the DNA sequence shown in Fig 5 including the FPV S 5' LTR (SEQ ID NO: 2), the FPV S 3' LTR (SEQ ID NO: 3) or FPV M3 LTR (SEQ ID NO : 4), or the DNA sequences shown in Fig 6 including FPV S 5' LTR (SEQ ID NO: 5), FPV S 3' LTR (SEQ ID NO: 7) and FPV M3 LTR (SEQ ID NO: 6).
In yet another embodiment of the present invention there is provided an isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 7.
In still another embodiment of the present invention there is provided an isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 8.
In still another embodiment of the present invention there is provided an isolated double-stranded DNA comprising:
( 1 ) a first single-stranded DNA molecule derived from FPV S, and (2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 9.
In still another embodiment of the present invention there is provided a nucleic acid molecule having the sequence shown in Fig 14 (SEQ ID NO: 9) .
In still another embodiment of the present invention there is provided a nucleic acid molecule having the sequence shown in Figure 16 (SEQ ID NO: 10) .
In still another embodiment of the present invention there is provided a nucleic acid molecule having the sequence shown in Fig 18 (SE ID NO: 11)
According to a further aspect of the invention there is provided a vaccine against fowlpox virus (FPV) which does not give rise to reticuloendotheliosis virus (REV) infection when administered to chickens, comprising an attenuated strain of FPV and a pharmaceutically acceptable carrier, and including means for preventing expression of active REV.
Preferably, said means for preventing expression of active REV comprises using a recombinant FPV as described above, but any other means for preventing expression of active REV could be used. For example, the
REV sequence can be mutated so that inactive REV is expressed. Typically, said mutation is by insertion, deletion or non-conservative substitution. Alternatively, said means for preventing expression of active REV comprises an expression cassette for expressing antisense RNA corresponding to the REV sequence. The expression cassette may be incorporated into the FPV or may comprise a further expression vector. As a further alternative, said means for preventing expression of active REV comprises a catalytic ribozyme for cleaving REV mRNA, or an expression cassette for expression of such a ribozyme. In a still further alternative, said means for preventing expression of active REV comprises means for downregulating the LTR promoter in the REV sequence.
According to a still further aspect of the present invention there is provided the use of a recombinant FPV as described above in the preparation of a vaccine against fowlpox virus (FPV) .
According to yet another aspect of the invention there is provided a method for preventing the occurrence of fowlpox virus (FPV) in a chicken without giving rise to reticuloendotheliosis virus (REV) infection, comprising the step of administering a vaccine as described above to said chicken.
Preferably said chicken is vaccinated at older than one day of age, optionally subsequently to vaccination at one day of age with FPV M.
DEFINITIONS
Throughout the specification and claims the following definitions are used:
"REV sequence" means a DNA sequence, which may or may not encode active reticuloendotheliosis virus, derived from the incorporation of the reticuloendotheliosis virus into the genome of a fowlpox virus .
FPV S or the words "Standard strain" means the
Cyanamid Websters Pty Ltd Standard strain fowlpox vaccine, a relatively virulent strain withdrawn from sale in Australia due to apparent contamination with REV but shown by the present inventors to include an REV sequence encoding active reticuloendotheliosis virus.
FPV M or the words "Mild strain" means the
Cyanamid Websters Pty Ltd mild strain fowlpox vaccine. Plaque purified derivatives of this strain are designated FPV M3 and FPV M A-F and have been described in Boyle, D.B. Pye, A.D. and Coupar, B.E.H. (1997) "Comparison of Field and Vaccine Strains of Australian Fowlpox Viruses" Arch.
Virol. 142, 737-748, the contents of which are incorporated herein by reference.
Throughout this speci ication and the claims the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, ie, the word "comprising' means "including but not limited to" and the words "comprises" and "comprise" have corresponding meanings .
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows evidence for REV sequences in field and vaccine strains of FPV. FPV DNAs were digested with Pstl restriction and endonucleases and transferred to nylon membranes by Southern blotting. (A) Ethidium bromide stained, (B) 32P-labeled 291-bp LTR PCR product hybridized to 9.8 pb fragment from FPV S . Lanes 1 to 5 contain DNA isolated from FPV M-F, M3, S, AWPL 1136 and 1137, respectively. Lambda Hindi! markers are shown as size markers at the left of the Figure;
Figure 2 shows evidence for complete REV provirus incorporation into FPV S and possible remnants in FPV M3, FPV DNAs were digested with EcoRI restriction endonuclease and transferred to nylon membranes by Southern blotting. (A) Ethidium bromide stained. (B) 32P-labeled 291-bp LTR PCR product hybridized to the DNA fragments and autoradiographed. Lanes 1 and 2 contain DNA isolated from FPV M3 and S, respectively. Lambda Hindlll markers are shown as size markers at the left of the Figure;
Figure 3 shows a restriction endonuclease map of the FPV genome showing region of inserted REV sequences . P, Pstl; E, EcoRI ; B, BamHl (BamHl and £σoRI sites marked are incomplete), LTR, long terminal repeat of REV. "LTR", truncated LTR present in FPV M3 and the 3 * end of the REV provirus inserted into FPV S. REV genes gag and env are marked. Open reading frames from the FPV genomic region flanking the provirus insertion are marked 1, 2 and 3. Ps ll fragments F, J, P, A' and D' (terminal fragment) are at the right-hand end of the FPV genome, respectively (as reported by Coupar et al . , 1990). Regions from which partial or complete nucleotide sequence has been determined are marked by dashed lines;
Figure 4 shows long-range (XL) PCR analysis of the REV provirus insert in FPV S strain. Primer pairs used for LX PCR were derived from lane 1 flanking FPV sequences (primer pair 1/2), from lanes 2 and 3 flanking FPV sequences and internal REV sequences (lane 2 primers 1/4 and lane 3 primers 2/3) . The locations of the primers are marked in Figure 3. λ DNA digested with Hindlll was used as size markers, lOμl from a 100-μl XL PCR was analyzed on a 0.6% agarose gel;
Figure 5 shows the alignment of REV LTR and flanking FPV sequences present in FPV strains. The REV LTR present in chicken syncytial virus provirus (ACRLTRl) ( Swift et al., 1987) (SEQ ID NO: 1) was aligned with REV LTR sequences present at the 5' end (FPV M5 LTR) (SEQ ID NO: 2) and the 3' end (FPV S3') (SEQ ID NO: 3) and the FPV M3 LTR (SEQ ID NO: 4) . The U3, R and U5 regions are those identified by Swift et al . , (1987). Conserved nucleotides are indicated by dots; deletions are indicated by a dash. Flanking FPV sequences are shown in lower case letters . The duplicated U3 terminus present in FPV S 3 ' LTR is in boldface and underlined. For clarity, flanking REV sequences present on the 5 ' end of FPV S 3 ' and the 3 ' end of the FPV S 5' LTR have been omitted;
Figure 6 shows the alignment of REV sequences, including the 5' LTR, 3' LTR and some of the intervening REV sequence, and flanking FPV sequences in FPV S AND FPV M3. Sequences containing the FPV S 5' LTR (SEQ ID NO : 5 ) , the FPV M3 LTR ( SEQ ID NO : 6 ) , the FPV S 3 ' LTR (SEQ ID NO 7) and the FPV M3 3' flanking sequence (SEQ ID NO: 8) are shown but the REV insert is not shown in full for clarity.
Figure 7 is a restriction endonuclease cleavage map of plasmid pCH21;
Figure 8 is a restriction endonuclease cleavage map of plasmid pCH2 ;
Figure 9 is a restriction endonuclease cleavage map of plasmid pCH30a;
Figure 10 is a restriction endonuclease cleavage map of plasmid pCH30b;
Figure 11 is a restriction endonuclease cleavage map of plasmid pCH31a;
Figure 12 is a restriction endonuclease cleavage map of plasmid pCH31b; and
Figure 13 is a restriction endonuclease map of the FPV S REV neg beta-gal positive construct constructed from FPV S using plasmid pCH30a; Figure 14 is the DNA sequence of the construct shown in Figure 13 (SEQ ID NO: 9);
Figure 15 is a restriction endonuclease map of the FPV S REV neg beta-gal positive construct constructed from FPV S using pCH30b;
Figure 16 is the DNA sequence of the construct shown in Figure 15 (SEQ ID NO: 10);
Figure 17 is a restriction endonuclease map of the FPV S REV negative construct constructed from FPV S using plasmids pCH31a and b; and
Figure 18 is the DNA sequence of the construct shown in Figure 17 (SEQ ID NO: 11) .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preferred embodiments of the present invention will now be described, by way of example only, with reference to the following examples, and to the figures.
Cells, Viruses and Virus DNA
Australian FPV vaccines, FPV M (mild vaccine strain) and FPV S (standard vaccine strain) were obtained from Cyanamid-Websters Pty. Ltd., Castle Hill, Australia. The FPV S vaccine is not currently used in Australia - its use was discontinued because of suspected REV contamination. The FPV M has been widely used in Australia, and is known to be free of REV. Plaque purified derivatives of this strain (designated FPV M3 and FPV M - A to F) have been described elsewhere (Boyle et al., 1997). Field isolates of FPV, designated AWPL 1136, 1137, 1138, 1139 and 1140, were obtained from scab material collected from poultry infections occurring in New South Wales, Australia during 1988 and 1989. A comparison of these field isolates with the vaccine strains has been described elsewhere (Boyle et al . , 1997). An Australian isolate of REV, designated REV/Vic/1/76, was obtained from Dr . . Ignatovic, CSIRO, Parkville, Australia and cultivated in chicken embryo fibroblast (CEF) cell cultures (Bagust and Dennett, 1977).
Primary chicken embryo skin cell cultures (CES) were prepared from 13 day old specific pathogen free (SPF) embryos (Silim et al . , 1982). Chicken embryo fibroblast (CEF) cell cultures were prepared from 10 day old SPF embryos. FPV DNA was extracted from partially purified FPV grown in CES cells and DNA restriction endonuclease fragments were separated by horizontal agarose gel electrophoresis, transferred to hybridization membranes and hybridized with radio-isotope labelled probes (Coupar et al . , 1990) .
Example 1 - REV LTR PCR
The primers and protocols described by Aly et al . , (1993) were used for amplification of REV LTR sequences present in FPV S, the AWPL field strains and CEF cells infected with REV virus. 32P-labelled REV LTR PCR products were prepared by including 32P-labelled nucleotide in the PCR reaction mix with a corresponding reduction in the equivalent unlabelled nucleotide to 1/10 the normal coneentration.
XL PCR analysis of REV sequences in FPV
XL PCR (Perkin Elmer GenAmp XL PCR) was used to characterize the REV sequences in FPV S strain. Primer 1 (sense) (5' -CCATCGAATTCACGTATTAC-3 ' ) (SEQ ID NO: 12) located at the EcoRI end of the sequenced region of FPV M3; primer 2 (antisense) ( 5 ' -CGGAATTCGGATCCGCGTGAATAGCTTCTACGGG-3 ' ) (SEQ ID NO: 13) located at the BamHl end of the FPV M3 sequenced region (EcoRI site added to aid cloning) ; primer 3 (sense) (5' -TTTCTGCATCCCTCTGGC-3 ' ) (SEQ ID NO: 14) derived from the polymerase region of REV (sequence determined from the EcoRI-Pstl fragment of FPV S) ; and primer 4
(antisense) (5' -CGAGCCAGAGACCTAGTAGC-3 ' ) (SEQ ID NO: 15) derived from the end of the polymerase region of REV (Ref ; ACRPOLENV, #K02537 - Genbank) . PCR conditions included the use of a hot start, 93° C for 1 min, 55° C for 2 min, 68° C for 5 min with a 10 sec extension per cycle using a total of 30 cycles. Less than 100 ng of FPV DNA was used as template in each 100 μl reaction.
Restriction enzyme mapping, cloning and nucleotide sequence determination of REV insertion site
FPV M3 and S genomic DNA fragments carrying REV sequences were separated by agarose gel electrophoresis after restriction endonuclease digestion. Selected DNA bands were excised from the gel, purified and cloned into the pUC19 plasmid vector. Desired plasmids were identified by hybridization and restriction endonuclease digestion analysis. Initial nucleotide sequence was determined from these plasmids using universal forward and reverse sequencing primers. Additional sequence was obtained by a variety of strategies including cloning of subfragments into pUC19 and M13 vectors and primer walking strategies, followed by manual and automated sequencing methods . The location of the REV LTR insertion within the FPV genome was mapped by hybridization of selected fragments to restriction endonuclease digested genomic DNA. The previously constructed Pstl and partial BamH maps of the FPV genome (Coupar et al., (1990) "Restriction endonuclease mapping of the fowlpox virus genome" Virology 179, 159- 167), the contents of which are incorporated herein by reference, were used to locate the REV LTR insertion within the FPV genomic map. Reverse transcriptase assay and Western Blotting
Supernatants from FPV S and FPV M3 infected CES cell cultures and from REV infected CEF cell cultures were clarified by centrifugation at 2500 rpm for 10 min. Half of the resulting supernatants were directly centrifuged at 28,000 rpm in a Beckman SW28 rotor for 3 h at 4 °C and the second half similarly centrifuged after ultrafiltration through 0.2 μm filters to remove remaining cell debris and FPVs . A greater than 100 fold concentration of the culture supernatants was achieved by the ultracentrifugation. Pellets were resuspended in RT lysis buffer (30 mM Tris-HCl pH 8.0, 80 mM KC1, 1.0 mM EDTA, 0.1 % v/v Triton X-100, 10 % v/v glycerol and 2 mM DTT) and dilution series were tested for reverse transcriptase activity using RT-detect (NEN Du Pont) according to the manufacturer's instructions. The 4 x RT buffer used contained 130 mM Tris-HCl pH 8.0, 120 mM KCl and 33.6 mM MgCl2. For immunoblot analysis equal amounts of 2 x SDS gel-loading buffer (100 mM Tris- HCl, pH 6.8, 20 % v/v glycerol, 4 % w/v SDS, 0.2 % w/v bromophenol blue, 200 mM DTT) and pelleted material resuspended in RT lysis buffer were mixed. The samples were resolved by SDS-PAGE on 12 % gels and transferred onto nitrocellulose membranes by electroblotting. After blocking the membranes with 5 % non-fat powdered milk in 0.02 % Tween 20 and PBS, they were incubated with a monoclonal antibody to the gag (P29) product of REV for 1 h, washed three times, incubated with sheep anti-mouse antibody conjugated with horseradish peroxidase (Silenus), washed three times and reacted with 4-chloro-l-naphthol and hydrogen peroxide.
Example 2 - "In vitro" recovery of REV from FPV S DNA
Cellular genomic DNA was prepared from uninfected and REV infected CES and CEF cell cultures . DNA was prepared from 5 x 10s cells using the Qiagen genomic DNA purification procedure (Qiagen Pty. Ltd.). Cultures infected with REV were inoculated as 50-80% confluent monolayers and cells harvested for DNA extraction 10 to 14 days later .
FPV DNA was purified from FPV S and M3 infected CES cell cultures. CES cell cultures were infected at a multiplicity of 0.05 to 0.1 pfu per cell. When the CPE had reached 80 to 90% (5 to 8 days after infection) the cells were harvested, resuspended in 4°C lOmM Tris-HCl pH 7.6 (20ml per 5 x 107cells) and dounce homogenized. Nuclei were removed by centrifugation at 5,000 g for 5 min and RNAase (20μg/ml) and DNAase (25μg/ml) added. After incubation for 15 min at 37°C, trypsin was added to a final concentration of 250μg/ml followed by a further incubation for 15 min at 37°C. 20 ml of extract was layer onto a 16 ml cushion consisting of 36% sucrose (8ml) overlain with 10% Dextran T10 (8ml) in lOmM Tris-HCl pH 7.6. Virus was pelleted by centrifugation at 20,000 rpm for 80 min in a
SW28 Beckman rotor then resuspended in 18 ml lOmM Tris-HCl pH7.6, l%Triton X-100 and 35mM mercaptoethanol . Following a further 10 min incubation on ice with occasional gentle mixing, viral cores were pelleted by another round of ultracentrifugation through the Dextran/Sucrose cushions.
DNA was extracted from the cores using the Qiagen genomic DNA purification procedure.
As a control to demonstrate the removal of REV cellular provirus DNA and free REV during purification of
FPV DNA, FPV M3 infected CES cells were mixed with an equal number of REV infected CES cells prior to commencing the protocol for purification of FPV DNA.
Purified cellular and FPV DNAs (0.5-lμg) were digested to completion with EcoRI {the REV provirus does not contain any EcoRI sites (Chen et al . , 1981)}. The DNA was then transfected into 50-80% confluent monolayers of CEF cells using LipofectAMINE (Gibco BRL) . 10 to 14 days later the culture supernatants were harvested and passaged onto fresh 50-80% confluent monolayers of CEF cells. Supernatants from the second passage were harvested 10 days later. CEF cells were infected in chamber well slide cultures with both the first and second passage culture supernatants from the transfections . 10 days later the monolayers were fixed with methanol and stained by immunofluorescenee with the monoclonal antibody to the REV gag (p29) gene product.
Example 3 - "In vivo" recovery of REV from FPV S
15 chickens at 3 weeks of age (SPF hybrid white leghorn strain) were inoculated by wing web stab and subcutaneous injection into the wing web (0.05 ml/chicken) . To ensure isolation from possible sources of REV infection the chickens were held in a PC3 animal containment facility isolated from all other poultry and totally protected from insect vectors. Each chicken received 2.5 x 106 pfu of FPV S. Prior to infection and 32 days after infection heparinized biood was collected from the wing vein. Plasma was collected for antibody assays . Antibody responses to REV were determined using a commercially available test kit
(IDEXX, USA) which is based upon a detergent and heat inactivated antigen preparation from the Cook strain of REV. REV was isolated from heparinized blood by direct inoculation of CEF cell cultures. After an additional passage in CEF cells, the cultures were stained by immunofluorescence with the gag (P29) monoclonal antibody to detect REV.
Example 4 - Evidence for REV sequences in FPV S and field isolates
In checking a number of field isolates by PCR for REV contamination we observed that a specific, 291 bp, product from the LTR was obtained when partially purified FPV DNA was used as the template for the PCR reaction. Contamination by REV proviral DNA from the infected cells could not be excluded, however the FPV S and field isolates had been cultivated on CES cells derived from SPF embryonated eggs known to be free from REV. The FPV M strain had also been cultivated on these cells and this virus was negative by PCR for REV LTR sequences.
Southern hybridization with 32P labelled 291 bp LTR fragment generated by PCR on FPV S DNA or on DNA extracted from CEF cells infected with REV, demonstrated specific hybridization to a 9.8 kb Pstl fragment of FPV S DNA and to a fragment of the same size in field isolates AWPL 1136 to 1140 (Fig. IB) . Hybridization to Pstl fragments of the FPV M, M3 or A-F DNA was not obvious, however very weak hybridization to the largest Pstl fragments of these viruses was sometimes observed. The 9.8 kb fragment is absent from Pstl digests of FPV M and its plaque purified derivatives, but is present in all of the AWPL series field isolates and the FPV S vaccine strain. (Fig.lA) . This data suggested that part of the REV genome was present within the genome of the FPV S and AWPL isolates.
When the 32P labelled REV 291 bp PCR product was hybridized to EcoRI digested DNA from FPV S and M3, strong hybridization was detected with a 17 kb fragment from FPV S and weak hybridization with a 9 kb fragment from FPV M3.
(Fig 2) . Since REV provirus is reported not to contain any EcoRI sites (Chen et al., 1981), the difference in size of the EcoRI fragments from FPV S and M3 might be attributable to the insertion of a complete REV provirus (=8.3 kb) (Chen et al., 1981). The lack of a specific LTR PCR product and weak hybridization to FPV M3 was suggestive of a remnant of the LTR being present . Example 5 - Cloning and mapping of REV sequences in the FPV genomes
The 9.8 kb Pstl fragment of FPV S, shown to hybridize with the REV LTR PCR product, was cloned into pUC. This cloned fragment hybridized to the 9.8 kb fragment present in the field isolates (Fig. IC) . Confirmation that part of this fragment was derived from FPV was shown by its hybridization to the largest Pstl fragments of FPV M-F and M3 (Fig. IC) . The location of the REV LTR sequences within this cloned fragment was determined by restriction endonuclease digestion and Southern hybridization. An EcoRI-PstI subfragment (4.3 kb) was identified as containing the REV LTR hybridizing region. Nucleotide sequence determination of this fragment revealed 903 bp of apparent FPV genome sequence (adjacent to the EcoRI site), a complete REV LTR and sequence of the REV gag region extending to the Pstl site - a total of 3388 bp of REV genomic sequence. The presence of REV genomic sequences up to the Pstl site suggested that the REV integrated sequences extended into an adjoining Pstl fragment .
The FPV M3 EcoRI genomic fragment (9.0 kb) shown to weakly hybridize to REV LTR product was cloned into pUC19. This fragment hybridized only to the largest Pstl fragment of FPV M-F and M3 and to the largest fragment of FPV S and the field strains, and to the 9.8 kb fragment of these strains shown to contain the REV LTR sequences (data not shown) . Although weakly hybridizing with the REV LTR probe (Fig. 2), a specific PCR product could not be generated from FPV M3 DNA suggesting that an incomplete REV LTR may be present in the genome of this virus. Additional restriction endonuclease mapping and nucleotide sequence determination showed that an EcoRI-BamHI sub-fragment (2.8 kb) was identical with the region identified in the FPV S cloned fragment (except for the extent of REV sequences present) and appeared to span the site of insertion of the REV provirus sequences. In addition, nucleotide sequence determination revealed a truncated REV LTR (248 bp) remnant in the FPV M3 genome at the same location as the FPV S REV insertion. From this data it was possible to conclude that the REV sequences were inserted into the right hand 1/3 of the FPV genome and that the REV sequences in FPV M3 and S appeared to be located at the same place within a previously uncharacterized region of the FPV genome (Fig. 3).
XL PCR analysis suggested that there was a near full length provirus present in the FPV strain. XL PCR analysis of FPV M3 and FPV S using primers 1 and 2 (Fig. 3) yielded products of 2.8 kb from both virus DNA's (Fig. 4). The absence of a larger product from FPV S DNA was indicative of heterogeneity present in the genomes of the viruses carrying near full length REV provirus inserts. Genome heterogeneity in these viruses would be expected since the REV LTR direct repeats present would make the genome inherently unstable in the region of the insert. This heterogeneity was not apparent in the hybridization analysis of the FPV DNAs. However, given the large size difference (2.5kb vs ~10kb) between the shorter PCR product and that anticipated from the near full length REV provirus insert, a low level of contamination with the short rearranged genome would ensure that the shorter PCR product predominated when the flanking FPV primers were used in the XL PCR. When the FPV primers were used in combination with primers derived from REV (Primer pair 1 and 4) and (Primer pair 2 and 3) no products were generated with FPV M3 DNA (Fig. 4) . In comparison FPV S DNA generated PCR products of 5.9 and 6.3 kb indicative of a complete REV provirus present in the FPV S DNA. (Fig. 4). The 3' end of the REV provirus inserted into FPV S was sequenced from the XL PCR product derived using primers 2 and 3. This revealed a truncated and rearranged 3 ' LTR almost identical to the remnant in FPV M3 and downstream of the env gene and flanked by the same FPV genomic sequences present in FPV M3.
The FPV genome region in which the REV near full length provirus integration has occurred has not previously been characterized. Comparisons of the deduced proteins encoded by open reading frames (ORF) 1, 2 and 3 (Fig.4) with available data base sequences using BLAST WWW Server (National Center for Biotechnology Information) identified distant but definite relationships with characterized genes of other poxviruses . Regions of the deduced protein (283 amino acids) encoded by ORF 1 have identifiable but very limited relationships to the molluscum contagiosum virus subtype 1 MC14 14R hypothetical protein (Genbank entry U60315) and to the A49L protein of variola virus. The protein (285 amino acids) encoded by ORF 2 is related to the hypothetical 33.6K protein (a member of the protein kinase family - 287 amino acids) of shope fibroma virus (Genbank entry JQ1743) . The deduced amino acid sequence from the incompletely sequenced ORF 3 is related to the serpin from ectromelia virus (Genbank entry S24676).
The sequences of the integrated retroviral provirus are closely related to REV and spleen necrosis virus (SNV) (Fig.5). Alignment of the FPV S 5' LTR with REV LTR (Genbank entry ACRLTRl) revealed a single base deletion and two base substitutions in the FPV S 5' LTR over the 517bp LTR region. The FPV S 3' LTR and the remnant LTR in FPV M 3 were identical to each other except for one deletion, one base substitution and the 23bp duplication of the U3 5' terminus present in FPV S 3' LTR. However, in comparison with the FPV S 5' LTR and the REV LTR both the FPV S 3' LTR and the FPV M3 remnant LTR had a large deletion (262bp) spanning part of the U3, all of the R and part of the U5 regions. In addition, significant changes had occurred in the remnant (51bp) of the U5 region with three base deletions, five base substitutions and a three base insertion (Fig.5). The truncated and rearranged 3 ' LTR and the absence of the classical direct repeats at the integration site suggests that the integration of the REV provirus into the genome of FPV occurred in an unusual manner or has undergone rearrangement after integration. The close relationship of the FPV S near full length REV insert to REV was further apparent upon comparison of the nucleotide and deduced amino acid sequences from the regions of REV sequence determined. Homologies ranging from 85 to 99% were observed when compared with available REV and SNV Genbank sequences at both the nucleotide and protein level. The sequence of FPV S and FPV M3 in the region of the REV insert (but omitting some of the REV sequence) is shown in Fig 6.
Example 6 - Testing biological activity of REV provirus in FPV S in vitro and in vivo
The presence of a near full length REV provirus integrated into FPV S posed a number of questions regarding biological function i.e. is the apparent contamination of the FPV S due to free REV or due to REV arising from the integrated provirus. We tested for the presence of free REV virus in FPV S stocks by harvesting CES culture supernatants, concentrating any possible REV by ultracentrifugation and testing the pellet for reverse transcriptase activity and for REV gag antigens by Western blotting using a monoclonal antibody for antigen detection. Culture supernatants from FPV S and M (concentrated by a factor of 100 fold) were negative for RT activity and antigen by Western blotting whilst controls from REV infected CEF cultures were positive. Additionally, immunofluorescence staining of CEF and CES cultures infected with FPV M, M3, S and AWPL1136 to 1140 for REV gag gene products using the monoclonal antibody to gag (p29) was negative.
When purified and EcoRI digested FPV S DNA was transfected into CEF cell cultures REV virus was recovered (Table 1) . Cellular DNA purified from CEF and CES cells infected with REV yielded REV when transfected into CEF cells. DNA purified from FPV M3, uninfected CEF and CES cells failed to yield REV upon transfection into CEF cell cultures. When DNA was purified from FPV M3 deliberately contaminated with REV infected cells, purification of the FPV M3 DNA removed REV cellular proviral DNA and REV since REV was not detected when the DNA was transfected into CEF cell cultures (Table 1) .
That FPV S could give rise to REV upon replication in chickens was determined by infecting 15 3- week old SPF chickens with FPV S via the wing web. Serum samples were collected prior to infection and 32 days after infection. All fifteen inoculated chickens developed high titered antibodies to REV (10,000 to 30,000). None were positive prior to inoculation. Peripheral blood collected 32 days after infection with FPV S yielded REV from eight of fourteen chickens . This confirms the experience of using FPV S vaccine in commercial poultry where its use was associated with the apparent spread of REV. The widely used Australian FPV M vaccine and its plaque purified derivatives are free of REV contamination when similarly tested by poultry inoculation.
Example 7 - Construction of Recombinant FPV
Deletion of REV sequences from the FPV standard strain has been carried out in the following manner.
1. Plasmid pCH21 carries an EcoRI-BamHI fragment of the FPV Mild vaccine strain genomic DNA. This plasmid is based upon the widely-used cloning vector pUCl9. The FPV genomic fragment carries the FPV sequences which flank the site of insertion of the REV genome in the FPV Standard strain genome (these same sequences surround the site of integration of a near full-length REV genome in the FPV Standard vaccine strain) . It also carries the REV long terminal repeat remnant, which we have shown to be present in FPV Mild vaccine strain.
2. Polymerase chain reaction primers have been specifically designed to amplify the complete pCH21 plasmid, deleting the REV LTR remnant and inserting specific restriction endonuclease sites in its place between the FPV genes identified as M-l and M-l 3' as per the map of pCH21.
Primer 1. 5 ' GCATGATCATATTATTGTATAATATTATATTTTG3 ' (SEQ ID NO: 16) Immediately 3' of REV insert LTR in FPV-M3. To be used to delete REV LTR from pCH21 by XL-PCR and religation.
Primer 2. 5 ' GCGTGATCACCCATGGTTTTTATATTGTTACTGTTCCG3 ' (SEQ
ID NO: 17) Positionl230-1260 FPV-M3 immediately 5' adjacent to REV insert. To be used to. delete REV LTR from pCH21 PCR.
Using long-range PCR, these primers were used to generate a DNA product from pCH21 encompassing the entire plasmid without the LTR remnant. Following digestion with Bell restriction endonuclease the DNA was ligated to itself and plasmid pCH29 recovered. PCH29 carries the FPV sequences without the REV LTR remnant and an inserted Bell site immediately downstream of the FPV gene identified as M-l.
3. Using established techniques (Heine, H-G and Boyle, D.B. (1993) Virol 131: 277-292 and Heine, H-G,
Foord, A.J., Young, P.L., Hooper, P.T., Lehrbach, P.R. and Boyle, D.B. (1996) Virus Research 50: 23-33, the contents of which are incorporated herein by reference, a DNA fragment (BamHl to Bglll) carrying the P.L promoter-β- galactosidase gene and the w P.7.5 promoter ECOGPT gene was isolated from plasmid pAF09.
4. Plasmid pCH29 was digested with Bell and ligated to the DNA fragment from pAF09. Plasmids pCH30a and pCH30b were recovered and characterised by standard techniques. These plasmids carry the FPV DNA sequences with the DNA fragment from pAF09 inserted at the Bell site.
5. Plasmid pCH29 was digested with BamHl and ligated to the DNA fragment from pAF09. Plasmids pCH31a and pCH31b were recovered and characterised by standard techniques . These plasmids carry the FPV DNA sequences with the DNA fragment from pAF09 flanking the FPV DNA and inserted at the BamHl site.
6. Using established techniques (Boyle, D.B. and Coupar, B.E.H.(1988) Virus Research 10: 343-356; Davison, A.J. and Elliot, R.M. (1993) Molecular Virology: A Practical Approach - Chapter 9, pages 257-283; Heine, H- G. and Boyle D.B. (1993) Arch. Virol. 131: 277-292 and Heine, H-G et al (1997) Virus Research 50: 23-33, the contents of which are incorporated herein by reference) for the construction of recombinant fowlpox viruses, the plasmids pCH30 a & b and pCH31a & b were combined with FPV Standard vaccine strain. These same plasmids could be used to recombine with any other isolated fowlpox virus shown to similarly contain the REV genome sequences.
7. For pCH30, reσombinants with the REV genome deleted and the β-galactosidase gene and ECOGPT gene inserted in its place were selected on the basis of growth under selection conditions for ECOGPT and on the basis of expression of the β-galactosidase gene. The recombinants were plaque purified and virus stocks prepared. This strain was designated FPV Standard REV negative β- galactosidase positive. (FPV S REV neg β-gal pos) .
8. For pCH31, recombinants with the REV genome deleted were selected on the basis of transient dominant insertion (Davison, A.J. and Elliot, R.M. (1993), Molecular Virology: A Practical Approach. IRL Press. Oxford University Press, Oxford, New York, Tokyo. Chapter 9. Smith, G.L. "Expression of Genes by Vaccinia Virus Vectors" pp 257-283, which is incorporated herein by reference), of the β-galactosidase and ECOGPT genes inserted in the FPV genome and the REV genome deleted and replaced by the short sequence surrounding the Bell site inserted between the M-l and M-l 3' genes identified in the map of pCH31a & b. FPV standard strain derivatives were selected upon the basis of insertion of the β-galactosidase and ECOGPT genes (blue plaques) then derivatives were selected upon the basis of the loss of the β-galactosidase gene (white plaques) . The recombinants with the REV genome deleted and the β- galactosidase and ECOGPT genes lost after transient dominant selection were plaque purified and virus stocks prepared. This strain was designated FPV Standard REV negative (FPV S REV -) .
9. FPV S REV - β-gal + and FPV S REV - can be used to vaccinate and revaccinate chickens at ages greater than one day old. These strains generated by recombinant DNA techniques to remove the REV genome integrated in the FPV genome and thus to remove the REV contamination of the
FPV S vaccine will overcome the deficiencies present in the FPV Mild Strain and the contamination problem which resulted in the use of the FPV Standard strain being discontinued. Similarly this method can be used to eliminate REV from recently-derived field isolates of FPV, enabling them to be used for the development of fowlpox vaccines . A restriction endonuclease map has been prepared for the FPV S REV neg beta-gal positive strains constructed from FPV S using pCH30a and pCH30b, and DNA sequences obtained for these constructs . These are shown in Figures 11, 12, 13 and 14, respectively. A restriction endonuclease map for the FPV S REV negative strain constructed from FPV S using plasmids pCH31a has been prepared and is shown in Figure 15, and the DNA sequence of this construct is shown in Figure 16.
INDUSTRIAL APPLICABILITY
The present invention is useful in the poultry industry for the control of fowlpox in chickens without the introduction of reticulendotheliosis virus to these chickens .
REFERENCES
The contents of those of the following references not specifically incorporated by reference elsewhere in the specification are now incorporated herein by reference.
Aly, M. M., Smith, E. J., and Fadly, A. M. (1993). Detection of reticuloendotheliosis virus infection using the polymerase chain reaction. Avian Pathology 22, 543-554.
Ball, L. A. (1987). High-frequency homologous recombination in vaccinia virus DNA. J.Virol. 61, 1788- 1795.
Bagust, T. J., and Dennett, D. P. (1977).
Reticuloendotheliosis virus : Experimental infection of poultry and immunofluorescent identification of Australian isolates. Aust . Vet . J. 53, 506-508.
Binns, M. M., Boursnell, M. E. G., and Skinner, M. A.
(1992) . Gene translocations in poxviruses : the fowlpox virus thymidine kinase gene is flanked by 15bp direct repeats and occupies the locus which in vaccinia virus is occupied by the ribonucleotide reductase large subunit gene. Virus Res . 24, 161-172.
Blissard, G. W., and Rohrmann, G. F. (1990). Baculovirus diversity and molecular biology. Annu. Rev. Entomol . 35, 127-155.
Boyle, D. B., Pye, A. D., and Coupar, B. E. H. (1997). Comparison of field and vaccine strains of Australian fowlpox viruses. Arch . Virol . 142, 737-748.
Chen, I. S., Mak, T. W., O'Rear, J.J., and Temin, H. M. (1981) . Characterisation of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning. J. Virol . , 40, 800-811.
Isfort, R. J., Jones, D., Kost, R., Witter, R., and Kung, H-J. (1992) . Retrovirus insertion into herpesvirus in vitro and in vivo. Proc . Natl . Acad . Sci . USA 89, 991-995.
Isfort, R. j., Qian, Z., Jones, D., Silva, R. F., Witter, R., and Kung, H. J. (1994). Integration of multiple chicken retroviruses into multiple chicken herpesviruses :
Herpesviral gD as a common target of integration. Virology 203, 125-133.
Jones, D., Isofort, R., Witter, R., Kost, R., and Kung, H- J. (1993). Retroviral insertions into a herpesvirus are clustered at the junction of the short repeat and short unique sequences. Proc . Natl . Acad . Sci . USA 90, 3855-3859.
Jones, D., Brunovskis, P., Witter, R., and Kung, H-J. (1996) . Retroviral insertional activation in a herpesvirus
: transcriptional activation of Uβ genes by an integrated long terminal repeat in a Marek's disease virus clone. J. Virol . 70, 2460-2467.
Lerch, R. A., and Friesen, P. D. (1992). The baculovirus- integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase. J. Virol . 66, 1590-1601.
Silim, A., Masy, E. A., and Roy, R. S. (1982). A simple technique for preparation of chicken-embryo-skin cell cultures. Avian Dis . 26, 182-185.
Slabaugh, M. B., and Roseman, N. A. (1989). Retroviral protease-like gene in the vaccinia virus genome. Proc .
Natl . Acad . Sci . USA 86, 4152-5155. Smith, G. L. (1993) . Vaccinia virus glycoproteins and immune evasion. J. Gen . Virol . 74, 1725-1740.
Swift, R. S., Boerkoel, C, Ridgway, A., Fujita, D. J., Dodgson, J. B. and Kung, H. -J. (1987). B-Lymphoma induction by reticuloendotheliosis virus : Characterization of a mutated chicken syncytial virus provirus involved in c-myc activation. J. Virol. 61, 2084-2090.
Coupar, B.E.H., Teo, T., Boyle, D.B. (1990) Restriction endonuclease mapping of the fowlpox virus genome. Virology 179: 159-167.
Davison, A.J. and Elliott, R.M. (1993) . Molecular Virology : A Practical Approach. IRL Press. Oxford University Press, Oxford, New York, Tokyo. Chapter 9. Smith, G.L. "Expression of genes by vaccinia virus vectors." Pages 257-283.
Heine, H-G. and Boyle, D.B. (1993). Infectious bursal disease virus structural protein VP2 expressed by a fowlpox virus recombinant confers protection against disease in chickens. Arch. Virol. 131 : 277-292.
Heine, H.G., Foord, A.J., Young, P.L., Hooper, P.T.,
Lehrbach, P.R. and Boyle, D.B. (1996) Recombinant fowlpox virus vaccines against Australian virulent Marek's disease virus : Gene sequence analysis and comparison of vaccine efficacy in specific pathogen free and production chickens. Virus Research 50 : 23-33 (July 1997).
Rural Industries Research and Development Corporation. Chicken Meat Research and Development Committee. "Call for applications to conduct research to develop a new fowlpox vaccine strain" 1995. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL
RESEARCH ORGANISATION
(B) STREET: Limestone Avenue
(C) CITY: Campbell (D) STATE: ACT
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP) : 2612
[ii) TITLE OF INVENTION: VACCINE AGAINST FOWLPOX VIRUS
iii) NUMBER OF SEQUENCES: 17
(iv) 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 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: AU PO 8454
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Avian reticuloendotheliosis virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TGTGGGAGGG AGCTCCGGGG GAAATAGCGC TGGCTCGCTA ACTGCCATAT TAGCTTCTGT 60
AATCATGCTT GCTTGCCTTA GCCGCCATTG TACTTGATAT ATTTCGCTGA TATCATTTCT 120
CGGAATCGGC ATCAAGAGCA GGCTCATAAA CCATAAAAGG AAATGTTTGT TGAAGGCAAG 180
CATCAGACCA CTTGCACCAT CCAATCACGA ACAAACACGA GATCGAACTA TCATACTGAG 240
CCAATGGTTG TAAAGGGCAG ATGCTATCCT CCAATGAGGG AAAATGTCAT GCAACATCCT 300
GTAAGCGGCT ATATAAGCCA GGTGCATCTC TTGCTCGGGG TCGCCGTCCT ACACATTGTT 360
GTGACGTGCG GCCCCGATTC GAATCTGTAA TAAAAGCTTT TTCTTCTATA TCCTCAGATT 420
GGCAGTGAGA GGAGATTTTG TTCGTGGTGT TGGCTGGCCT ACTGGGTGGG GTAGGGATCC 480
GGACTGAATC CGTAGTATTT CGGTACAACA TT 512 ( 2 ) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 536 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic!
iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Fowlpox virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AACGGAACAG TAACAATATA AAAAGTGTGG AGGGAGCTCC GGGGGGAATA
GCGCTGGCTC 60
GCTAACTGCC ATATTAGCTT CTGTAATCAT GCTTGCTTGC CTTAGCCGCC ATTGTACTTG 120
ATATATTTCG CTGATATCAT TTCTCGGAAT CGGCATCAAG AGCAGGCTCA TAAACCATAA 180
AAGGAAATGT TTGTTGAAGG CAAGCATCAG ACCACTTGCA CCATCCAATC ACGAACAAAC 240
ACGAGATCGA ACTATCATAC TGAGCCAATG GTTGTAAAGG GCAGATGCTA TCCTCCAATG 300
AGGGAAAATG TCATGCAACA TCCTGTAAGC GGCTATATAA GCCAGGTGCA TCTCTTGCTC 360 GGGGTCGCCG TCCTACACAT TGTTGTGACG TGCGGCCCAG ATTCGAATCT GTAATAAAAG 420
CTTTTTCTTC TATATCCTCA GATTGGCAGT GAGAGGAGAT TTTGTTCGTG GTGTTGGCTG 480
GCCTACTGGG TGGGGTAGGG ATCCGGACTG AATCCGTAGT ATTTCGGTAC AACATT 536
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 296 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Fowlpox virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TGTGGGAGGG AGCTCCGGGG AATGTGGGAG GGAGCTCCGG GGGGAATAGC GCTGGCTCGC 60
TAACTGCCAT ATTAGCTTCT GTAATCATGC TTGCTTGCCT TAGCCGCCAT
TGTACTTGAT 120
ATATTTCGCT GATATCATTT CTCGGAATCG GCATCAAGAG CAGGCTCATA AACCATAAAA 180
GAAAATGTTT GTTGAAGGCA AGCATCAGAC CACTTGCACA CTAGGTGGGG CAGCAGGGGT 240 CCGGACTGAA TCGTCGTAGT TCGGTACAAC AGTATTATTG TATAATATTA TATTTT 296
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Fowlpox virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
AACGGAACAG TAACAATATA AAAAGTGTGG AGGGAGCTCC GGGGGGAATA GCGCTGGCTC 60
GCTAACTGCC ATATTAGCTT CTGTAATCAT GCTTGCTTGC CTTAGCCGCC ATTGTACTTG 120
ATATATTTCG CTGATATCAT TTCTCGGAAT CGGCATCAAG AGCAGGCTCA
TAAACCATAA 180
AAGGAAATGT TTGTTGAAGG CAAGCATCAG ACCACTTGCA CACTAGGTGG GGCAGCAGGG 240
GTCCGGACTG AATCGTCCGT GTTCGGTACA ACAGTATTAT TGTATAATAT TATATTTT 2 98 (2) INFORMATION FOR SEQ ID NO : 5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4643 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Fowlpox virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AGGTATACTA GATTCAGTGT ACCTTTCGTG GATATCTTTC ATGTATACTA CTAGGGTAAG 60
ATGTTATATA GTATCTGCAA AAAAAGATTT GAGCATTAAC TGGATGGTAA ACGGTGTTAT 120
TACTGAAGGG ATGTCATTTA ATGAAATTAG TTATGACGAT AGTTATTTGT
CCGGGTGTTT 180
TATGATAGAG TTAAAGATAA TTAATAGCTT TAGTGATAAG GTAATTTCTC CTATATGTAG 240
GGTTAATTTC GGCTCTAAAG GATACAAAGA ATATAGAATA AATCTAACAA ATACACTTCC 300
TGATACCTTA TACGATAAAT ATTCTAATCC TATAAGATAT TATAATCCAT CGAATTCACG 360
TATTACAAAA ATGAAATAAT AAAACTAGTT TGTAATGTGA TGGCAACGGT TGCTAGAATG 420
TATAAAACTA TAAATACCAC CGGTATATCA TGCGTCTTGA AAAGTTTGAT ACCAGATAGC 480
TATAATGAAG AATACAACAT AGATGACCTA GATCTATTAA AGATAAAAGA GTTTATAGAG 540
ATATCCATGC AAAGATGCTT TTCTATAAAA TCCGTCACAG ATTCCACAGT ATTATACATA 600
GAGAACAGGA CTAACAGATA TTCTATATCT ACTAGTCACG ATAAGAATGA ACCGTATGAA 660
GAAAATGGTA TTATAATGAA CAATATAGAG TGTTATTTTG TTGCGTGTCT AGAAGGATCG 720
TGTACAGTAA ATGTAAATCT TGGAGACAGA CAAATATCAG ACAATATATC TGAATCATCA 780
GGATTCCTAA TGGATGTAAA CACCGATCAC GTTATAGATA CAAAATATGT AGGATTATTT 840
ATTACAAAAA TCAAAGTAGA TGCGCATGTA TTTTACGGGC AAAATGTGAT AATGTTTCCA 900
GAAAAAAACT TGTTTTCTCA AACTAATGGT CCTAATTTCA TTTTATATGA TATAACAGTT 960
CAAGATCGTA ATGTACTTTT GCTTATAACG AGCAAGTATA TTTACAATTT
GTGCGACGAT 1020
AAATACTACG ATATTTTCGA ATTAAAATAT CTAGTTGATA ACTGTAAACT ACCTATGCCT 1080
CTTATTCCAC TATCGAAGTA CGATTTTACA TTTACTGATT TGAGTGTTAT CAAATCAGAG 1140 AATGTTAAAA CGGTACTCTC TAAAGTTCAT ACGAGTATGA AATCGTACTA CAACAATGAT 1200
ACGTCTCTTC CTGTCGCCGT TAAGGTGATT TACGGAACAG TAACAATATA AAAAGTGTGG 1260
AGGGAGCTCC GGGGGGAATA GCGCTGGCTC GCTAACTGCC ATATTAGCTT CTGTAATCAT 1320
GCTTGCTTGC CTTAGCCGCC ATTGTACTTG ATATATTTCG CTGATATCAT TTCTCGGAAT 1380
CGGCATCAAG AGCAGGCTCA TAAACCATAA AAGGAAATGT TTGTTGAAGG CAAGCATCAG 1440
ACCACTTGCA CCATCCAATC ACGAACAAAC ACGAGATCGA ACTATCATAC TGAGCCAATG 1500
GTTGTAAAGG GCAGATGCTA TCCTCCAATG AGGGAAAATG TCATGCAACA
TCCTGTAAGC 1560
GGCTATATAA GCCAGGTGCA TCTCTTGCTC GGGGTCGCCG TCCTACACAT TGTTGTGACG 1620
TGCGGCCCAG ATTCGAATCT GTAATAAAAG CTTTTTCTTC TATATCCTCA GATTGGCAGT 1680
GAGAGGAGAT TTTGTTCGTG GTGTTGGCTG GCCTACTGGG TGGGGTAGGG ATCCGGACTG 1740
AATCCGTAGT ATTTCGGTAC AACATTTGGG GGCTCGTCCG GGATTCCTCC CCATCGGCAG 1800
AGGTGCCTAC TGTTTCTTCG AACTCCGGCG CCGGTAAGTA AGTACTTGAT TTTGGTACCT 1860 CGCGAGGGTT TGGGAGGTTC GGAGTGGCGG GACGCTGCCG GGAAGCTCCA CCTCCGCTCA 1920
GCAGGGGACG CCCTGGTCTG AGCTCTGTGG TATCTGATTG TTGTTGAACC GTCTCTAAGA 1980
CGGTGATACT ATAAGTCGTG GTTTGTGTGT TTGTTTGTTA CCTTGTGTTT GTTCGTCACT 2040
TGTCGACAGC GCCCTGCGAA TTGGTGTACC CACACCGCGC GGCTTGCGAA TAATACTTTG 2100
GAGAGTCTTT TGCCTCCAGT GTCTTCCGTT TGTACTCGTC CTCCTCTCCC TCTCCGGCCG 2160
GGATGGGACA GGCAGGATCG AAGGGGCTTT TAACCCCTCT AGAGTGCATT CTGAAGAACT 2220
TCTCTGACTT TAAGAAGAGG GCGGGAGACT ATGGGGAGGA TGTGGATTCG TTTACCCTGC 2280
GCAAGTTATG TGAGTTGGAA TGGCCCACGT TTGGCGTGGG GTGGCCGAAG GAAGGGACTC 2340
TAGACTTTAG GGTGGTAGCC GCGGTCAGGA ATATAGTTTT TGGGAATCCA GGGCATCCAG 2400
ACCAGGTAAT ATATATAACC GTCTGGATAG ATATAACCAT AGAAAGGCCT AAATACTTG 2460
AAGATTGCGG GTGTAAACCC ACAGGACCCT CTAAAGTTCT GTTAGCTAGC CAAAAAGTTA 2520
ATCCCAGGCG GCCCGTGCTC CCCTCAGCCC CAGAAAGCCC CCCTCGGATG AGGAGGGCTC 2580
AATTCCTGGA TGAGAGACCT CTCTCTCCGG CCCCAGCCCC TCCACCTCCA TATCCTGAGG 2640
TACCTGCCAT TGCAGAGGAA GGTGAGGAGG GGCAACAACC AGACTCTACT GTAATGGCGA 2700
GCCCTCCCCA CACCCGAAGT GGGTTAGAGT TCGGAGCACA AGGGCCGTCA GGGATGTACC 2760
CCCTTAGGGA AACTGGGGAA CGGGATATGG GTGGTCGCCC CATGAGAACA TATGTCCCAT 2820
TCACCACCTC GGATCTGTAT AATTGGGAAA ACCAAAACCC ATCATTCTCC CAGGCTCCAG 2880
ATGAAGTAAT TAGCCTATTA GAATCCGTTT TCTATACACA CCAGCCTACC TGGGATGATT 2940
GCCAGCAACT CCTCCGTACC CTGTTTACGA CGGAGGAGAG GGAGAGGGTG AGGACAGAAA 3000
GTAGGCGGGA GGTCAGGAAT GATCAGGGAG TACAGGTCAC TGACGAGCGA GAAATAGAAG 3060
CCCAGTTCCC AGCTACTCGG CCCGACTGGG ATCCTAACAC AGGGAGAGGA AACGACAATC 3120
TAGAACGATA TCGCCAGATC TTGCTGAGGG GGCTCCGGGC AGCGGCACGG AAACCCACCA 3180
ATCTTTCTAA AATAACTGAG GTTCGGCAGG GGGCGGACGA AAGTCCTACA
GCTTACCTGG 3240
AGAGACTCTA TCAGGCTTAT CGGACCTGGT CCCCCATAGA TCCAAGGGCA CCCGAGAATC 3300
AGGCTGCCAT AGTCATTCAA TTTGTATCTC AATCCGCGCC CGATATTAGG AAGAAAATCC 3360 AAAAGATAGA TGGGTTCCAG GGGAAATCTC TCTCCGAGTT AGTAGCTATT GCCCAGAAGG 3420
TTTTCGACCA ACGGGAAGAC CCAGCCAAGG CAACTCACGA ACTCACTCAG AAGATGGCCA 3480
AGGTACTACT CGCCCAGGAG AGTAGAGCGG AGAGAGGAAG TAAGAAGACG CCTCCGGGTA 3540
AGGGACGCCC GCCTTTGGGG AAAAACCAAT GCGCGTACTG TAAGGAGGAG GGACATTGGA 3600
AGAAGAACTG TCCAAAACTC GTAAGCGGGG CAACCCCAGT GTTGGTAGAA GAATTACAAT 3660
AGGGCCGTCA GGGTTCTTCC GCCCTCCGTG AACCCAGGCT AAAAGTTAAA GTAGGGGGGC 3720
AAATAATAGA TTTTCTAGTA GATACGGGAG CGACCCATTC TGTAGTGCAG
AAACCTGTGG 3780
GGCCTATGTC TAAAGAATCT GTAGCAATTA TCGGGGCTAC TGGGAACATA CGGAATTACC 3840
CTAAGTCTGA AGGGCGTCTT GTGGACCTAG GGAGGGGACT AGTGACTCAT TCATTTC AG 3900
TGATTCCTGA GTGCCCAGAC CCACTATTGG GGAGGGATCT ATTACAGAAG CTAAGGGCTA 3960
CCATCTCGTT CACCGGGGAA GGGCCCCCCG AAATACGGAC AGAAGGGAAA CTATTGGTAA 4020
CGGCTCCCCT GGAAGAGGAA TACCGTTTGT TTTTAGAGGC GCCGATACAA AATGTTACGC 4080 TGCTAGAGCA GTGGAAACGG GAAATCCCGA AAGTCTGGGC CGAGATAAAT CCCCCGGGGT 4140
TGGCATCCAC ACAAGCCCCC ATTCATGTCC AGCTATTAAG TACCGCCCTA CCTGTGAGGG 4200
TAAGACAGTA TCCTATTACC CTGGAGGCAA AACGAAGCCT GCGCGAAACT ATTCGCAAAT 4260
TCAGAGCAGC GGGCATCCTG AGACCCGTCC ACTCCCCCTG GAACACTCCC CTCCTCCCTG 4320
TGCGAAAGTC CGGCACGTCC GAGTATCGGA TGGTTCAAGA TTTAAGGGAA GTAAACAAGA 4380
GAGTAGAGAC TATTCACCCA ACTGTCCCTA ACCCATACAC CCTCCTCAGC CTATTACCCC 4440
CTGACCGAAT ATGGTATTCT GTTTTGGACC TGAAAGACGC CTTTTTCTGC ATCCCTCTGG 4500
CCCCTGAGTC GCAATTGATC TTCGCATTCG AGTGGGCAGA TGCGGAGGAA GGAGAATCAG 4560
GGCAACTAAC CTGGACTAGG CTACCTCAGG GTTTTAAGAA ATCACCCACC CTTTTTGATG 4620
AAGCCCTTAA CAGGGATCTG CAG 4643
\ 2 ) INFORMATION FOR SEQ ID NO : 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1100 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAATTCACGT ATTACAAAAA TGAAATAATA AAACTAGTTT GTAATGTGAT GGCAACGGTT 60
GCTAGAATGT ATAAAACTAT AAATACCACC GGTATATCAT GCGTCTTGAA AAGTTTGATA 120
CCAGATAGCT ATAATGAAGA ATACAACATA GATGACCTAG ATCTATTAAA GATAAAAGAG 180
TTTATAGAGA TATCCATGCA AAGATGCTTT TCTATAAAAT CCGTCACAGA TTCCACAGTA 240
TTATACATAG AGAACAGGAC TAACAGATAT TCTATATCTA CTAGTCACGA
TAAGAATGAA 300
CCGTATGAAG AAAATGGTAT TATAATGAAC AATATAGAGT GTTATTTTGT TGCGTGTCTA 360
GAAGGATCGT GTACAGTAAA TGTAAATCTT GGAGACAGAC AAATATCAGA CAATATATCT 420
GAATCATCAG GATTCCTAAT GGATGTAAAC ACCGATCACG TTATAGATAC AAAATATGTA 480
GGATTATTTA TTACAAAAAT CAAAGTAGAT GCGCATGTAT TTTACGGGCA AAATGTGATA 540
ATGTTTCCAG AAAAAAACTT GTTTTCTCAA ACTAATGGTC CTAATTTCAT TTTATATGAT 600 ATAACAGTTC AAGATCGTAA TGTACTTTTG CTTATAACGA GCAAGTATAT TTACAATTTG 660
TGCGACGATA AATACTACGA TATTTTCGAA TTAAAATATC TAGTTGATAA CTGTAAACTA 720
CCTATGCCTC TTATTCCACT ATCGAAGTAC GATTTTACAT TTACTGATTT GAGTGTTATC 780
AAATCAGAGA ATGTTAAAAC GGTACTCTCT AAAGTTCATA CGAGTATGAA ATCGTACTAC 840
AACAATGATA CGTCTCTTCC TGTCGCCGTT AAGGTGATTT ACGGAACAGT AACAATATAA 900
AAAGTGTGGA GGGAGCTCCG GGGGGAATAG CGCTGGCTCG CTAACTGCCA TATTAGCTTC 960
TGTAATCATG CTTGCTTGCC TTAGCCGCCA TTGTACTTGA TATATTTCGC TGATATCATT 1020
TCTCGGAATC GGCATCAAGA GCAGGCTCAT AAACCATAAA AGGAAATGTT TGTTGAAGGC 1080
AAGCATCAGA CCACTTGCAC 1100
( 2 ) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1022 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE : DNA ( genomi c ; (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Fowlpox virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
AAATAGAAGG GGGTTAGACC TATGGACTGC CGAACAAGGA GGAATATGTC TCGCACTCCA 60
GGAGAAGTGC TGTTTTTACG CCAACAAGTC GGGTATCGTA CGTGACAAGA TCCGGAAACT 120
CCAAGAGGAC CTTATCGCGA GGAAACGTGC ACTGTACGAC AACCCCCTGT GGAACGGCTT 180
GAACGGCTTC CTTCCATATT TGCTACCCTT GTTGGGCCCC CTGTTTGGGC TCATATTGTT 240
CCTGACCCTC GGCCCGTGCA TCAGTAAGAC CCTGACTCGC ATTATCCATG CACAAAAATC 300
AGGCAGTAAA AATCCTAGCA TTCAGTCACC CGCAGTACAA GCCACTCCCA ACAGAGATGG 360
ATACCCTAGG TCAATGATTT GACCAGAATA TACAAGAGCA GTGGGGAATG TGGGAGGGAG 420
CTCCGGGGAA TGTGGGAGGG AGCTCCGGGG GGAATAGCGC TGGCTCGCTA ACTGCCATAT 480
TAGCTTCTGT AATCATGCTT GCTTGCCTTA GCCGCCATTG TACTTGATAT ATTTCGCTGA 540
TATCATTTCT CGGAATCGGC ATCAAGAGCA GGCTCATAAA CCATAAAAGA AAATGTTTGT 600 TGAAGGCAAG CATCAGACCA CTTGCACACT AGGTGGGGCA GCAGGGGTCC GGACTGAATC 660
GTCGTAGTTC GGTACAACAG TATTATTGTA TAATATTATA TTTTGTAATA TATAAAAAAA 720
TAGAAAATAA ATAATATATT ATTTTTATAA TGGATATTAT AACTAATACA ACTATGTTTG 780
ATATACAATT TAACGATATA CCGAATATAC CCTATGTAGA TATAGAAAAG CCCTTATTGG 840
TATATTCGTG TGATTCTTAT AGGTTATATA ACGCTAAATA TGACAACAAT CCCGTCAGTT 900
TGAAGACTTT TACATGCCCA TCTAAAAATA GTATAAGACA GTTCATAAAA GAAC AGATC 960
TGTTACGTTC TCTACAATCT TCTGAACACG TTATTAAACT TTACGGGTAC ATATTGGATA 1020
TA 1022
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1504 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Fowlpox virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
ACTAGGTGGG GCAGCAGGGG TCCGGACTGA ATCGTCGTAG TTCGGTACAA CAGTATTATT 60
GTATAATATT ATATTTTGTA ATATATAAAA AAATAGAAAA TAAATAATAT ATTATTTTTA 120
TAATGGATAT TATAACTAAT ACAACTATGT TTGATATACA ATTTAACGAT ATACCGAATA 180
TACCCTATGT AGATATAGAA AAGCCCTTAT TGGTATATTC GTGTGATTCT TATAGGTTAT 240
ATAACGCTAA ATATGACAAC AATCCCGTCA GTTTGAAGAC TTTTACATGC CCATCTAAAA 300
ATAGTATAAG ACAGTTCATA AAAGAACTAG ATCTGTTACG TTCTCTACAA TCTTCTGAAC 360
ACGTTATTAA ACTTTACGGG TACATATTGG ATATATCCGT TCCTTTATGT AGCCTGGTGG 420
TTGAAAATAA CTACCTTACG TTAAGAAACT TTTTAGATAT GGAAAAAGAT ATAGATTACG 480
CCAAGAAAAC AAGAATTATC ATAGATGCCG CAAAAGGTCT AAATGCTATG CATACTAGCT 540
ACTCGACTCC CATACTACAT AAAAATTTAA CCAGTGAATC TTTTTACATG
AC AATAATG 600
GTGTTTTAAA AATAGGTAGC GGGGCATATT ATAATATATA CAAAAGAGTA AATTTTATGG 660
CATATTTTGA TTATGACATG TTAAAAGATA TCTTTTCAAA TTATACTATA AAATCCGAAA 720 TTTATAGATT CGGTATTGTT ATATGGGAAA TTATTACCCG TAAAATACCT TTTGAAAATA 780
TGGACTACCA AGGAATATAC AATATGCTAA TAAAGGAAAA TAAAGGCGAA TATATGCCTC 840
TAGACTGTCC TCTGGAATTA CAGTGTATTG TTATCGCGTG TAGAAATACA AATTCTATAT 900
TTAGACCTTC TATAAGTGCA ATAATTGATT TTCTGGAAAC TTTTTATTCT AATATAATTA 960
AAAACAGAAA CTTAAAATAG ACTAAGTAGA GTATATACAC ATATTACACG GTAACATGTT 1020
TGCTATTTCA GTGTTAAAGG AATTATATGA TTCCGGAGAG CCTTTATTAT TTTCACCTAG 1080
AGGGCTACAT AAAATATTAT GTAATATCAG GCACGGGTGC AACGGAAATA
CTAAAAATCA 1140
ATTAGATAAT TTATTAGAAG AAACCATATA TGATTACGGA GAAGACCAGG CGCTTAAACA 1200
TATAATAACT AACATTTCTG TTCTACTAAT AAAAAGATGT TATAATATAA ACGAGAAATT 1260
TATTAAAGAT AGTAATACGA TATATAATAC CGATGTATTA GAATTTTATA ATGTAAGACA 1320
AATACCTAGA ATAATGAATA AATGGATTAG ATCAAGATCT AATAATAAAA TAACAGATAT 1380
AGGATGTCAT ATCTACGATA ATACTAAGTC TATAATAGCC GAAGCAATGT TTTTTAC AT 1440 GAAACACGAA TCTATATTCG GTTCAACAAG AAAAGATACT ATAACCTTCT ATAAGTACGA 1500
TGGA 1504
( 2 ) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6561 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "plasmid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9:
GAATTCACGT ATTACAAAAA TGAAATAATA AAACTAGTTT GTAATGTGAT
GGCAACGGTT 60
GCTAGAATGT ATAAAACTAT AAATACCACC GGTATATCAT GCGTCTTGAA AAGTTTGATA 120
CCAGATAGCT ATAATGAAGA ATACAACATA GATGACCTAG ATCTATTAAA GATAAAAGAG 180
TTTATAGAGA TATCCATGCA AAGATGCTTT TCTATAAAAT CCGTCACAGA TTCCACAGTA 240
TTATACATAG AGAACAGGAC TAACAGATAT TCTATATCTA CTAGTCACGA TAAGAATGAA 300
CCGTATGAAG AAAATGGTAT TATAATGAAC AATATAGAGT GTTATTTTGT TGCGTGTCTA 360 GAAGGATCGT GTACAGTAAA TGTAAATCTT GGAGACAGAC AAATATCAGA CAATATATCT 420
GAATCATCAG GATTCCTAAT GGATGTAAAC ACCGATCACG TTATAGATAC AAAATATGTA 480
GGATTATTTA TTACAAAAAT CAAAGTAGAT GCGCATGTAT TTTACGGGCA AAATGTGATA 540
ATGTTTCCAG AAAAAAACTT GTTTTCTCAA ACTAATGGTC CTAATTTCAT TTTATATGAT 600
ATAACAGTTC AAGATCGTAA TGTACTTTTG CTTATAACGA GCAAGTATAT TTACAATTTG 660
TGCGACGATA AATACTACGA TATTTTCGAA TTAAAATATC TAGTTGATAA CTGTAAACTA 720
CCTATGCCTC TTATTCCACT ATCGAAGTAC GATTTTACAT TTACTGATTT GAGTGTTATC 780
AAATCAGAGA ATGTTAAAAC GGTACTCTCT AAAGTTCATA CGAGTATGAA ATCGTACTAC 840
AACAATGATA CGTCTCTTCC TGTCGCCGTT AAGGTGATTT ACGGAACAGT AACAATATAA 900
AAACCATTGG TGATCTCCCC ATGGCCCAAA GCGGGGTTTG AACAGGGTTT CGCTCAGGTT 960
TGCCTGTGTC ATGGATGCAG CCTCCAGAAT ACTTACTGGA AACTATTGTA ACCCGCCTGA 1020
AGTTAAAAAG AACAACGCCC GGCAGTGCCA GGCGTTGAAA AGATTAGCGA CCGGAGATTG 1080
GCGGGACGAA TACGACGCCC ATATCCCACG GCTGTTCAAT CCAGGTATCT TGCGGGATAT 1140
CAACAACATA GTCATCAACC AGCGGACGAC CAGCCGGTTT TGCGAAGATG GTGACAAAGT 1200
GCGCTTTTGG ATACATTTCA CGAATCGCAA CCGCAGTACC ACCGGTATCC ACCAGGTCAT 1260
CAATAACGAT GAAGCCTTCG CCATCGCCTT CTGCGCGTTT CAGCACTTTA AGCTCGCGCT 1320
GGTTGTCGTG ATCGTAGCTG GAAATACAAA CGGTATCGAC ATGACGAATA CCCAGTTCAC 1380
GCGCCAGTAA CGCACCCGGT ACCAGACCGC CACGGCTTAC GGCAATAATG CCTTTCCATT 1440
GTTCAGAAGG CATCAGTCGG CTTGCGAGTT TACGTGCATG GATCTGCAAC ATGTCCCAGG 1500
TGACGATGTA TTTTTCGCTC ATGTGAAGTG TCCCAGCCTG TTTATCTACG GCTTAAAAAG 1560
TGTTCGAGGG GAAAATAGGT TGCGCGAGAT TATAGAGATC CGTCACTGTT CTTTATGATC 1620
TACTTCCTTA CCGTGCAATA AATTAGAATA TATTTTCTAC TTTTACGAGA AATTAATTAT 1680
TGTATTTATT ATTTATGGGT GAAAAACTTA CTATAAAAAG CGGGTGGGTT TGGAATTAGT 1740
GATCAGTTTA TGTATATCGC AACTACCGGC ATATGGCTAT TCGACATCGA GAACATTACC 1800
CACATGATAA GAGATTGTAT CAGTTTCGTA GTCTTGAGTA TTGGTATTAC TATATAGTAT 1860 ATGTCGGGAA TTCAGATCCA TGCTAGATCC CAAATAGTAC ATAATGGATT TCCTTACGCG 1920
AAATACGGGC AGACATGGCC TGCCCGGTTA TTATTATTTT TGACACCAGA CCAACTGGTA 1980
ATGGTAGCGA CCGGCGCTCA GCTGGAATTC CGCCGATACT GACGGGCTCC AGGAGTCGTC 2040
GCCACCAATC CCCATATGGA AACCGTCGAT ATTCAGCCAT GTGCCTTCTT CCGCGTGCAG 2100
CAGATGGCGA TGGCTGGTTT CCATCAGTTG CTGTTGACTG TAGCGGCTGA TGTTGAACTG 2160
GAAGTCGCCG CGCCACTGGT GTGGGCCATA ATTCAATTCG CGCGTCCCGC AGCGCAGACC 2220
GTTTTCGCTC GGGAAGACGT ACGGGGTATA CATGTCTGAC AATGGCAGAT
CCCAGCGGTC 2280
AAAACAGGCG GCAGTAAGGC GGTCGGGATA GTTTTCTTGC GGCCCTAATC CGAGCCAGTT 2340
TACCCGCTCT GCTACCTGCG CCAGCTGGCA GTTCAGGCCA ATCCGCGCCG GATGCGGTGT 2400
ATCGCTCGCC ACTTCAACAT CAACGGTAAT CGCCATTTGA CCACTACCAT CAATCCGGTA 2460
GGTTTTCCGG CTGATAAATA AGGTTTTCCC CTGATGCTGC CACGCGTGAG CGGTCGTAAT 2520
CAGCACCGCA TCAGCAAGTG TATCTGCCGT GCACTGCAAC AACGCTGCTT CGGCCTGGTA 2580 ATGGCCCGCC GCCTTCCAGC GTTCGACCCA GGCGTTAGGG TCAATGCGGG TCGCTTCACT 2640
TACGCCAATG TCGTTATCCA GCGGTGCACG GGTGAACTGA TCGCGCAGCG GCGTCAGCAG 2700
TTGTTTTTTA TCGCCAATCC ACATCTGTGA AAGAAAGCCT GACTGGCGGT TAAATTGCCA 2760
ACGCTTATTA CCCAGCTCGA TGCAAAAATC CATTTCGCTG GTGGTCAGAT GCGGGATGGC 2820
GTGGGACGCG GCGGGGAGCG TCACACTGAG GTTTTCCGCC AGACGCCACT GCTGCCAGGC 2880
GCTGATGTGC CCGGCTTCTG ACCATGCGGT CGCGTTCGGT TGCACTACGC GTACTGTGAG 2940
CCAGAGTTGC CCGGCGCTCT CCGGCTGCGG TAGTTCAGGC AGTTCAATCA ACTGTTTACC 3000
TTGTGGAGCG ACATCCAGAG GCACTTCACC GCTTGCCAGC GGCTTACCAT CCAGCGCCAC 3060
CATCCAGTGC AGGAGCTCGT TATCGCTATG ACGGAACAGG TATTCGCTGG
TCACTTCGAT 3120
GGTTTGCCCG GATAAACGGA ACTGGAAAAA CTGCTGCTGG TGTTTTGCTT CCGTCAGCGC 3180
TGGATGCGGC GTGCGGTCGG CAAAGACCAG ACCGTTCATA CAGAACTGGC GATCGTTCGG 3240
CGTATCGCCA AAATCACCGC CGTAAGCCGA CCACGGGTTG CCGTTTTCAT CATATTTAAT 3300
CAGCGACTGA TCCACCCAGT CCCAGACGAA GCCGCCCTGT AAACGGGGAT ACTGACGAAA 3360
CGCCTGCCAG TATTTAGCGA AACCGCCAAG ACTGTTACCC ATCGCGTGGG CGTATTCGCA 3420
AAGGATCAGC GGGCGCGTCT CTCCAGGTAG CGAAAGCCAT TTTTTGATGG ACCATTTCGG 3480
CACAGCCGGG AAGGGCTGGT CTTCATCCAC GCGCGCGTAC ATCGGGCAAA TAATATCGGT 3540
GGCCGTGGTG TCGGCTCCGC CGCCTTCATA CTGCACCGGG CGGGAAGGAT CGACAGATTT 3600
GATCCAGCGA TACAGCGCGT CGTGATTAGC GCCGTGGCCT GATTCATTCC CCAGCGACCA 3660
GATGATCACA CTCGGGTGAT TACGATCGCG CTGCACCATT CGCGTTACGC GTTCGCTCAT 3720
CGCCGGTAGC CAGCGCGGAT CATCGGTCAG ACGATTCATT GGCACCATGC CGTGGGTTTC 3780
AATATTGGCT TCATCCACCA CATACAGGCC GTAGCGGTCG CACAGCGTGT ACCACAGCGG 3840
ATGGTTCGGA TAATGCGAAC AGCGCACGGC GTTAAAGTTG TTCTGCTTCA TCAGCAGGAT 3900
ATCCTGCACC ATCGTCTGCT CATCCATGAC CTGACCATGC AGAGGATGAT GCTCGTGACG 3960
GTTAACGCCT CGAATCAGCA ACGGCTTGCC GTTCAGCAGC AGCAGACCAT TTTCAATCCG 4020
CACCTCGCGG AAACCGACAT CGCAGGCTTC TGCTTCAATC AGCGTGCCGT CGGCGGTGTG 4080 CAGTTCAACC ACCGCACGAT AGAGATTCGG GATTTCGGCG CTCCACAGTT TCGGGTTTTC 4140
GACGTTCAGA CGTAGTGTGA CGCGATCGGC ATAACCACCA CGCTCATCGA TAATTTCACC 4200
GCCGAAAGGC GCGGTGCCGC TGGCGACCTG CGTTTCACCC TGCCATAAAG AAACTGTTAC 4260
CCGTAGGTAG TCACGCAACT CGCCGCACAT CTGAACTTCA GCCTCCAGTA CAGCGCGGCT 4320
GAAATCATCA TTAAAGCGAG TGGCAACATG GAAATCGCTG ATTTGTGTAG TCGGTTTATG 4380
CAGCAACGAG ACGTCACGGA AAATGCCGCT CATCCGCCAC ATATCCTGAT CTTCCAGATA 4440
ACTGCCGTCA CTCCAACGCA GCACCATCAC CGCGAGGCGG TTTTCTCCGG
CGCGTAAAAA 4500
TGCGCTCAGG TCAAATTCAG ACGGCAAACG ACTGTCCTGG CCGTAACCGA CCCAGCGCCC 4560
GTTGCACCAC AGATGAAACG CCGAGTTAAC GCCATCAAAA ATAATTCGCG TCTGGCCTTC 4620
CTGTAGCCAG CTTTCATCAA CATTAAATGT GAGCGAGTAA CAACCCGTCG GATTCTCCGT 4680
GGGAACAAAC GGCGGATTGA CCGTAATGGG ATAGGTTACG TTGGTGTAGA TGGGCGCATC 4740
GTAACCGTGC ATCTGCCAGT TTGAGGGGAC GACGACAGTA TCGGCCTCAG GAAGATCGCA 4800 CTCCAGCCAG CTTTCCGGCA CCGCTTCTGG TGCCGGAAAC CAGGCAAAGC GCCATTCGCC 4860
ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA 4920
GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG GGTTTTCCCA 4980
GTCACGACGT TGTAAAACGA CGGGATCTAG CATGGATCTA GCCATTTAGT ATCCTAAAAT 5040
TGAATTGTAA TTATCGATAA TAAATGGACG GATCATATTA TTGTATAATA TTATATTTTG 5100
TAATATATAA AAAAATAGAA AATAAATAAT ATATTATTTT TATAATGGAT ATTATAACTA 5160
ATACAACTAT GTTTGATATA CAATTTAACG ATATACCGAA TATACCCTAT GTAGATATAG 5220
AAAAGCCCTT ATTGGTATAT TCGTGTGATT CTTATAGGTT ATATAACGCT AAATATGACA 5280
ACAATCCCGT CAGTTTGAAG ACTTTTACAT GCCCATCTAA AAATAGTATA AGACAGTTCA 5340
TAAAAGAACT AGATCTGTTA CGTTCTCTAC AATCTTCTGA ACACGTTATT AAACTTTACG 5400
GGTACATATT GGATATATCC GTTCCTTTAT GTAGCCTGGT GGTTGAAAAT AACTACCTTA 5460
CGTTAAGAAA CTTTTTAGAT ATGGAAAAAG ATATAGATTA CGCCAAGAAA ACAAGAATTA 5520
TCATAGATGC CGCAAAAGGT CTAAATGCTA TGCATACTAG CTACTCGACT CCCATACTAC 5580
ATAAAAATTT AACCAGTGAA TCTTTTTACA TGACTAATAA TGGTGTTTTA AAAATAGGTA 5640
GCGGGGCATA TTATAATATA TACAAAAGAG TAAATTTTAT GGCATATTTT GATTATGACA 5700
TGTTAAAAGA TATCTTTTCA AATTATACTA TAAAATCCGA AATTTATAGA TTCGGTATTG 5760
TTATATGGGA AATTATTACC CGTAAAATAC CTTTTGAAAA TATGGACTAC CAAGGAATAT 5820
ACAATATGCT AATAAAGGAA AATAAAGGCG AATATATGCC TCTAGACTGT CCTCTGGAAT 5880
TACAGTGTAT TGTTATCGCG TGTAGAAATA CAAATTCTAT ATTTAGACCT TCTATAAGTG 5940
CAATAATTGA TTTTCTGGAA ACTTTTTATT CTAATATAAT TAAAAACAGA AACTTAAAAT 6000
AGACTAAGTA GAGTATATAC ACATATTACA CGGTAACATG TTTGCTATTT CAGTGTTAAA 6060
GGAATTATAT GATTCCGGAG AGCCTTTATT ATTTTCACCT AGAGGGCTAC ATAAAATATT 6120
ATGTAATATC AGGCACGGGT GCAACGGAAA TACTAAAAAT CAATTAGATA
ATTTATTAGA 6180
AGAAACCATA TATGATTACG GAGAAGACCA GGCGCTTAAA CATATAATAA CTAACATTTC 6240
TGTTCTACTA ATAAAAAGAT GTTATAATAT AAACGAGAAA TTTATTAAAG ATAGTAATAC 6300 GATATATAAT ACCGATGTAT TAGAATTTTA TAATGTAAGA CAAATACCTA GAATAATGAA 6360
TAAATGGATT AGATCAAGAT CTAATAATAA AATAACAGAT ATAGGATGTC ATATCTACGA 6420
TAATACTAAG TCTATAATAG CCGAAGCAAT GTTTTTTACT ATGAAACACG AATCTATATT 6480
CGGTTCAACA AGAAAAGATA CTATAACCTT CTATAAGTAC GATGGAACTT CGTTACCCGT 6540
AGAAGCTATT CACGCGGATC C 6561
(2) INFORMATION FOR SEQ ID NO : 10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6560 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "plasmid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 10:
GAATTCACGT ATTACAAAAA TGAAATAATA AAACTAGTTT GTAATGTGAT
GGCAACGGTT 60
GCTAGAATGT ATAAAACTAT AAATACCACC GGTATATCAT GCGTCTTGAA AAGTTTGATA 120
CCAGATAGCT ATAATGAAGA ATACAACATA GATGACCTAG ATCTATTAAA GATAAAAGAG 180 TTTATAGAGA TATCCATGCA AAGATGCTTT TCTATAAAAT CCGTCACAGA TTCCACAGTA 240
TTATACATAG AGAACAGGAC TAACAGATAT TCTATATCTA CTAGTCACGA TAAGAATGAA 300
CCGTATGAAG AAAATGGTAT TATAATGAAC AATATAGAGT GTTATTTTGT TGCGTGTCTA 360
GAAGGATCGT GTACAGTAAA TGTAAATCTT GGAGACAGAC AAATATCAGA CAATATATCT 420
GAATCATCAG GATTCCTAAT GGATGTAAAC ACCGATCACG TTATAGATAC AAAATATGTA 480
GGATTATTTA TTACAAAAAT CAAAGTAGAT GCGCATGTAT TTTACGGGCA AAATGTGATA 540
ATGTTTCCAG AAAAAAACTT GTTTTCTCAA ACTAATGGTC CTAATTTCAT
TTTATATGAT 600
ATAACAGTTC AAGATCGTAA TGTACTTTTG CTTATAACGA GCAAGTATAT TTACAATTTG 660
TGCGACGATA AATACTACGA TATTTTCGAA TTAAAATATC TAGTTGATAA CTGTAAACTA 720
CCTATGCCTC TTATTCCACT ATCGAAGTAC GATTTTACAT TTACTGATTT GAGTGTTATC 780
AAATCAGAGA ATGTTAAAAC GGTACTCTCT AAAGTTCATA CGAGTATGAA ATCGTACTAC 840
AACAATGATA CGTCTCTTCC TGTCGCCGTT AAGGTGATTT ACGGAACAGT AACAATATAA 900 AAACCATGGT GATCCGTCCA TTTATTATCG ATAATTACAA TTCAATTTTA GGATACTAAA 960
TGGCTAGATC CATGCTAGAT CCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG 1020
TTACCCAACT TAATCGCCTT GCAGCACATC CCCCTTTCGC CAGCTGGCGT AATAGCGAAG 1080
AGGCCCGCAC CGATCGCCCT TCCCAACAGT TGCGCAGCCT GAATGGCGAA TGGCGCTTTG 1140
CCTGGTTTCC GGCACCAGAA GCGGTGCCGG AAAGCTGGCT GGAGTGCGAT CTTCCTGAGG 1200
CCGATACTGT CGTCGTCCCC TCAAACTGGC AGATGCACGG TTACGATGCG CCCATCTACA 1260
CCAACGTAAC CTATCCCATT ACGGTCAATC CGCCGTTTGT TCCCACGGAG AATCCGACGG 1320
GTTGTTACTC GCTCACATTT AATGTTGATG AAAGCTGGCT ACAGGAAGGC CAGACGCGAA 1380
TTATTTTTGA TGGCGTTAAC TCGGCGTTTC ATCTGTGGTG CAACGGGCGC TGGGTCGGTT 1440
ACGGCCAGGA CAGTCGTTTG CCGTCTGAAT TTGACCTGAG CGCATTTTTA CGCGCCGGAG 1500
AAAACCGCCT CGCGGTGATG GTGCTGCGTT GGAGTGACGG CAGTTATCTG GAAGATCAGG 1560
ATATGTGGCG GATGAGCGGC ATTTTCCGTG ACGTCTCGTT GCTGCATAAA CCGACTACAC 1620
AAATCAGCGA TTTCCATGTT GCCACTCGCT TTAATGATGA TTTCAGCCGC GCTGTACTGG 1680
AGGCTGAAGT TCAGATGTGC GGCGAGTTGC GTGACTACCT ACGGGTAACA GTTTCTTTAT 1740
GGCAGGGTGA AACGCAGGTC GCCAGCGGCA CCGCGCCTTT CGGCGGTGAA ATTATCGATG 1800
AGCGTGGTGG TTATGCCGAT CGCGTCACAC TACGTCTGAA CGTCGAAAAC CCGAAACTGT 1860
GGAGCGCCGA AATCCCGAAT CTCTATCGTG CGGTGGTTGA ACTGCACACC GCCGACGGCA 1920
CGCTGATTGA AGCAGAAGCC TGCGATGTCG GTTTCCGCGA GGTGCGGATT GAAAATGGTC 1980
TGCTGCTGCT GAACGGCAAG CCGTTGCTGA TTCGAGGCGT TAACCGTCAC GAGCATCATC 2040
CTCTGCATGG TCAGGTCATG GATGAGCAGA CGATGGTGCA GGATATCCTG CTGATGAAGC 2100
AGAACAACTT TAACGCCGTG CGCTGTTCGC ATTATCCGAA CCATCCGCTG TGGTACACGC 2160
TGTGCGACCG CTACGGCCTG TATGTGGTGG ATGAAGCCAA TATTGAAACC CACGGC TGG 2220
TGCCAATGAA TCGTCTGACC GATGATCCGC GCTGGCTACC GGCGATGAGC
GAACGCGTAA 2280
CGCGAATGGT GCAGCGCGAT CGTAATCACC CGAGTGTGAT CATCTGGTCG GTGGGGAATG 2340
AATCAGGCCA CGGCGCTAAT CACGACGCGC TGTATCGCTG GATCAAATCT GTCGATCCTT 2400 CCCGCCCGGT GCAGTATGAA GGCGGCGGAG CCGACACCAC GGCCACCGAT ATTATTTGCC 2460
CGATGTACGC GCGCGTGGAT GAAGACCAGC CCTTCCCGGC TGTGCCGAAA TGGTCCATCA 2520
AAAAATGGCT TTCGCTACCT GGAGAGACGC GCCCGCTGAT CCTTTGCGAA TACGCCCACG 2580
CGATGGGTAA CAGTCTTGGC GGTTTCGCTA AATACTGGCA GGCGTTTCGT CAGTATCCCC 2640
GTTTACAGGG CGGCTTCGTC TGGGACTGGG TGGATCAGTC GCTGATTAAA TATGATGAAA 2700
ACGGCAACCC GTGGTCGGCT TACGGCGGTG ATTTTGGCGA TACGCCGAAC GATCGCCAGT 2760
TCTGTATGAA CGGTCTGGTC TTTGCCGACC GCACGCCGCA TCCAGCGCTG
ACGGAAGCAA 2820
AACACCAGCA GCAGTTTTTC CAGTTCCGTT TATCCGGGCA AACCATCGAA GTGACCAGCG 2880
AATACCTGTT CCGTCATAGC GATAACGAGC TCCTGCACTG GATGGTGGCG CTGGATGGTA 2940
AGCCGCTGGC AAGCGGTGAA GTGCCTCTGG ATGTCGCTCC ACAAGGTAAA CAGTTGATTG 3000
AACTGCCTGA ACTACCGCAG CCGGAGAGCG CCGGGCAACT CTGGCTCACA GTACGCGTAG 3060
TGCAACCGAA CGCGACCGCA TGGTCAGAAG CCGGGCACAT CAGCGCCTGG CAGCAGTGGC 3120 GTCTGGCGGA AAACCTCAGT GTGACGCTCC CCGCCGCGTC CCACGCCATC CCGCATCTGA 3180
CCACCAGCGA AATGGATTTT TGCATCGAGC TGGGTAATAA GCGTTGGCAA TTTAACCGCC 3240
AGTCAGGCTT TCTTTCACAG ATGTGGATTG GCGATAAAAA ACAACTGCTG ACGCCGCTGC 3300
GCGATCAGTT CACCCGTGCA CCGCTGGATA ACGACATTGG CGTAAGTGAA GCGACCCGCA 3360
TTGACCCTAA CGCCTGGGTC GAACGCTGGA AGGCGGCGGG CCATTACCAG GCCGAAGCAG 3420
CGTTGTTGCA GTGCACGGCA GATACACTTG CTGATGCGGT GCTGATTACG ACCGCTCACG 3480
CGTGGCAGCA TCAGGGGAAA ACCTTATTTA TCAGCCGGAA AACCTACCGG ATTGATGGTA 3540
GTGGTCAAAT GGCGATTACC GTTGATGTTG AAGTGGCGAG CGATACACCG CATCCGGCGC 3600
GGATTGGCCT GAACTGCCAG CTGGCGCAGG TAGCAGAGCG GGTAAACTGG CTCGGATTAG 3660
GGCCGCAAGA AAACTATCCC GACCGCCTTA CTGCCGCCTG TTTTGACCGC TGGGATCTGC 3720
CATTGTCAGA CATGTATACC CCGTACGTCT TCCCGAGCGA AAACGGTCTG CGCTGCGGGA 3780
CGCGCGAATT GAATTATGGC CCACACCAGT GGCGCGGCGA CTTCCAGTTC AACATCAGCC 3840
GCTACAGTCA ACAGCAACTG ATGGAAACCA GCCATCGCCA TCTGCTGCAC GCGGAAGAAG 3900
GCACATGGCT GAATATCGAC GGTTTCCATA TGGGGATTGG TGGCGACGAC TCCTGGAGCC 3960
CGTCAGTATC GGCGGAATTC CAGCTGAGCG CCGGTCGCTA CCATTACCAG TTGGTCTGGT 4020
GTCAAAAATA ATAATAACCG GGCAGGCCAT GTCTGCCCGT ATTTCGCGTA AGGAAATCCA 4080
TTATGTACTA TTTGGGATCT AGCATGGATC TGAATTCCCG ACATATACTA TATAGTAATA 4140
CCAATACTCA AGACTACGAA ACTGATACAA TCTCTTATCA TGTGGGTAAT GTTCTCGATG 4200
TCGAATAGCC ATATGCCGGT AGTTGCGATA TACATAAACT GATCACTAAT TCCAAACCCA 4260
CCCGCTTTTT ATAGTAAGTT TTTCACCCAT AAATAATAAA TACAATAATT AATTTCTCGT 4320
AAAAGTAGAA AATATATTCT AATTTATTGC ACGGTAAGGA AGTAGATCAT AAAGAACAGT 4380
GACGGATCTC TATAATCTCG CGCAACCTAT TTTCCCCTCG AACACTTTTT AAGCCGTAGA 4440
TAAACAGGCT GGGACACTTC ACATGAGCGA AAAATACATC GTCACCTGGG
ACATGTTGCA 4500
GATCCATGCA CGTAAACTCG CAAGCCGACT GATGCCTTCT GAACAATGGA AAGGCATTAT 4560
TGCCGTAAGC CGTGGCGGTC TGGTACCGGG TGCGTTACTG GCGCGTGAAC TGGGTATTCG 4620
SUBSΗTUTE SHEET (Rule 26) TCATGTCGAT ACCGTTTGTA TTTCCAGCTA CGATCACGAC AACCAGCGCG AGCTTAAAGT 4680
GCTGAAACGC GCAGAAGGCG ATGGCGAAGG CTTCATCGTT ATTGATGACC TGGTGGATAC 4740
CGGTGGTACT GCGGTTGCGA TTCGTGAAAT GTATCCAAAA GCGCACTTTG TCACCATCTT 4800
CGCAAAACCG GCTGGTCGTC CGCTGGTTGA TGACTATGTT GTTGATATCC CGCAAGATAC 4860
CTGGATTGAA CAGCCGTGGG ATATGGGCGT CGTATTCGTC CCGCCAATCT CCGGTCGCTA 4920
ATCTTTTCAA CGCCTGGCAC TGCCGGGCGT TGTTCTTTTT AACTTCAGGC GGGTTACAAT 4980
AGTTTCCAGT AAGTATTCTG GAGGCTGCAT CCATGACACA GGCAAACCTG
AGCGAAACCC 5040
TGTTCAAACC CCGCTTTGGG CCATGGGGAG ATCATATTAT TGTATAATAT TATATTTTGT 5100
AATATATAAA AAAATAGAAA ATAAATAATA TATTATTTTT ATAATGGATA TTATAACTAA 5160
TACAACTATG TTTGATATAC AATTTAACGA TATACCGAAT ATACCCTATG TAGATATAGA 5220
AAAGCCCTTA TTGGTATATT CGTGTGATTC TTATAGGTTA TATAACGCTA AATATGACAA 5280
CAATCCCGTC AGTTTGAAGA CTTTTACATG CCCATCTAAA AATAGTATAA GACAGTTCAT 5340
SUBSΗTUTE SHEET (Rule 26) AAAAGAACTA GATCTGTTAC GTTCTCTACA ATCTTCTGAA CACGTTATTA AACTTTACGG 5400
GTACATATTG GATATATCCG TTCCTTTATG TAGCCTGGTG GTTGAAAATA ACTACCTTAC 5460
GTTAAGAAAC TTTTTAGATA TGGAAAAAGA TATAGATTAC GCCAAGAAAA CAAGAATTAT 5520
CATAGATGCC GCAAAAGGTC TAAATGCTAT GCATACTAGC TACTCGACTC CCATACTACA 5580
TAAAAATTTA ACCAGTGAAT CTTTTTACAT GACTAATAAT GGTGTTTTAA AAATAGGTAG 5640
CGGGGCATAT TATAATATAT ACAAAAGAGT AAATTTTATG GCATATTTTG ATTATGACAT 5700
GTTAAAAGAT ATCTTTTCAA ATTATACTAT AAAATCCGAA ATTTATAGAT TCGGTATTGT 5760
TATATGGGAA ATTATTACCC GTAAAATACC TTTTGAAAAT ATGGACTACC AAGGAATATA 5820
CAATATGCTA ATAAAGGAAA ATAAAGGCGA ATATATGCCT CTAGACTGTC CTCTGGAATT 5880
ACAGTGTATT GTTATCGCGT GTAGAAATAC AAATTCTATA TTTAGACCTT CTATAAGTGC 5940
AATAATTGAT TTTCTGGAAA CTTTTTATTC TAATATAATT AAAAACAGAA ACTTAAAATA 6000
GACTAAGTAG AGTATATACA CATATTACAC GGTAACATGT TTGCTATTTC AGTGTTAAAG 6060
GAATTATATG ATTCCGGAGA GCCTTTATTA TTTTCACCTA GAGGGCTACA TAAAATATTA 6120
TGTAATATCA GGCACGGGTG CAACGGAAAT ACTAAAAATC AATTAGATAA TTTATTAGAA 6180
GAAACCATAT ATGATTACGG AGAAGACCAG GCGCTTAAAC ATATAATAAC TAACATTTCT 6240
GTTCTACTAA TAAAAAGATG TTATAATATA AACGAGAAAT TTATTAAAGA TAGTAATACG 6300
ATATATAATA CCGATGTATT AGAATTTTAT AATGTAAGAC AAATACCTAG AATAATGAAT 6360
AAATGGATTA GATCAAGATC TAATAATAAA ATAACAGATA TAGGATGTCA TATCTACGAT 6420
AATACTAAGT CTATAATAGC CGAAGCAATG TTTTTTACTA TGAAACACGA ATCTATATTC 6480
GGTTCAACAA GAAAAGATAC TATAACCTTC TATAAGTACG ATGGAACTTC GTTACCCGTA 6540
GAAGCTATTC ACGCGGATCC 6560
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2402 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
( ii ) MOLECULE TYPE : other nucleic acid
(A) DESCRIPTION : /desc = " plasmid " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GAATTCACGT ATTACAAAAA TGAAATAATA AAACTAGTTT GTAATGTGAT GGCAACGGTT 60
GCTAGAATGT ATAAAACTAT AAATACCACC GGTATATCAT GCGTCTTGAA AAGTTTGATA 120
CCAGATAGCT ATAATGAAGA ATACAACATA GATGACCTAG ATCTATTAAA GATAAAAGAG 180
TTTATAGAGA TATCCATGCA AAGATGCTTT TCTATAAAAT CCGTCACAGA TTCCACAGTA 240
TTATACATAG AGAACAGGAC TAACAGATAT TCTATATCTA CTAGTCACGA TAAGAATGAA 300
CCGTATGAAG AAAATGGTAT TATAATGAAC AATATAGAGT GTTATTTTGT TGCGTGTCTA 360
GAAGGATCGT GTACAGTAAA TGTAAATCTT GGAGACAGAC AAATATCAGA CAATATATCT 420
GAATCATCAG GATTCCTAAT GGATGTAAAC ACCGATCACG TTATAGATAC AAAATATGTA 480
GGATTATTTA TTACAAAAAT CAAAGTAGAT GCGCATGTAT TTTACGGGCA AAATGTGATA 540
ATGTTTCCAG AAAAAAACTT GTTTTCTCAA ACTAATGGTC CTAATTTCAT
TTTATATGAT 600
ATAACAGTTC AAGATCGTAA TGTACTTTTG CTTATAACGA GCAAGTATAT TTACAATTTG 660
TGCGACGATA AATACTACGA TATTTTCGAA TTAAAATATC TAGTTGAT A CTGTAAACTA 72 0 CCTATGCCTC TTATTCCACT ATCGAAGTAC GATTTTACAT TTACTGATTT GAGTGTTATC 780
AAATCAGAGA ATGTTAAAAC GGTACTCTCT AAAGTTCATA CGAGTATGAA ATCGTACTAC 840
AACAATGATA CGTCTCTTCC TGTCGCCGTT AAGGTGATTT ACGGAACAGT AACAATATAA 900
AAACCATTGG TGATCATATT ATTGTATAAT ATTATATTTT GTAATATATA AAAAAATAGA 960
AAATAAATAA TATATTATTT TTATAATGGA TATTATAACT AATACAACTA TGTTTGATAT 1020
ACAATTTAAC GATATACCGA ATATACCCTA TGTAGATATA GAAAAGCCCT TATTGGTATA 1080
TTCGTGTGAT TCTTATAGGT TATATAACGC TAAATATGAC AACAATCCCG
TCAGTTTGAA 1140
GACTTTTACA TGCCCATCTA AAAATAGTAT AAGACAGTTC ATAAAAGAAC TAGATCTGTT 1200
ACGTTCTCTA CAATCTTCTG AACACGTTAT TAAACTTTAC GGGTACATAT TGGATATATC 1260
CGTTCCTTTA TGTAGCCTGG TGGTTGAAAA TAACTACCTT ACGTTAAGAA ACTTTTTAGA 1320
TATGGAAAAA GATATAGATT ACGCCAAGAA AACAAGAATT ATCATAGATG CCGCAAAAGG 1380
TCTAAATGCT ATGCATACTA GCTACTCGAC TCCCATACTA CATAAAAATT TAACCAGTGA 1440 ATCTTTTTAC ATGACTAATA ATGGTGTTTT AAAAATAGGT AGCGGGGCAT ATTATAATAT 1500
ATACAAAAGA GTAAATTTTA TGGCATATTT TGATTATGAC ATGTTAAAAG ATATCTTTTC 1560
AAATTATACT ATAAAATCCG AAATTTATAG ATTCGGTATT GTTATATGGG AAATTATTAC 1620
CCGTAAAATA CCTTTTGAAA ATATGGACTA CCAAGGAATA TACAATATGC TAATAAAGGA 1680
AAATAAAGGC GAATATATGC CTCTAGACTG TCCTCTGGAA TTACAGTGTA TTGTTATCGC 1740
GTGTAGAAAT ACAAATTCTA TATTTAGACC TTCTATAAGT GCAATAATTG ATTTTCTGGA 1800
AACTTTTTAT TCTAATATAA TTAAAAACAG AAACTTAAAA TAGACTAAGT AGAGTATATA 1860
CACATATTAC ACGGTAACAT GTTTGCTATT TCAGTGTTAA AGGAATTATA TGATTCCGGA 1920
GAGCCTTTAT TATTTTCACC TAGAGGGCTA CATAAAATAT TATGTAATAT
CAGGCACGGG 1980
TGCAACGGAA ATACTAAAAA TCAATTAGAT AATTTATTAG AAGAAACCAT ATATGATTAC 2040
GGAGAAGACC AGGCGCTTAA ACATATAATA ACTAACATTT CTGTTCTACT AATAAAAAGA 2100
TGTTATAATA TAAACGAGAA ATTTATTAAA GATAGTAATA CGATATATAA TACCGATGTA 2160
TTAGAATTTT ATAATGTAAG ACAAATACCT AGAATAATGA ATAAATGGAT TAGATCAAGA 2220
TCTAATAATA AAATAACAGA TATAGGATGT CATATCTACG ATAATACTAA GTCTATAATA 2280
GCCGAAGCAA TGTTTTTTAC TATGAAACAC GAATCTATAT TCGGTTCAAC AAGAAAAGAT 2340
ACTATAACCT TCTATAAGTA CGATGGAACT TCGTTACCCG TAGAAGCTAT TCACGCGGAT 2400
CC 2402
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
CCATCGAATT CACGTATTAC 20
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid
( C ) STRANDEDNESS : s ingl e
( D ) TOPOLOGY : l inear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CGGAATTCGG ATCCGCGTGA ATAGCTTCTA CGGG 34
(2) INFORMATION FOR SEQ ID NO : 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
TTTCTGCATC CCTCTGGC 18
(2) INFORMATION FOR SEQ ID NO : 15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGAGCCAGAG ACCTAGTAGC 20
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : If
GCATGATCAT ATTATTGTAT AATATTATAT TTTG 34
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GCGTGATCAC CCATGGTTTT TATATTGTTA CTGTTCCG 38
SUBSΗTUTE SHEET (Rule 26)

Claims

1. A recombinant fowlpox virus (FPV) whose nucleic acid does not encode an active reticuloendotheliosis virus (REV) .
2. A recombinant fowlpox virus (FPV) according to claim 1 in which an REV sequence is substantially absent from the FPV genome.
3. A recombinant fowlpox virus (FPV) according to claim 1 from which an REV sequence previously present in the FPV genome has been excised.
4. A recombinant fowlpox virus (FPV) as claimed in claim 3, wherein said REV sequence has been excised from the one third of the FPV genome adjacent its 3' end.
5. A recombinant fowlpox virus (FPV) as claimed in claim 4 wherein said REV sequence has been excised from a location in FPV S which maps to a 17kb EcoRI restriction endonuclease fragment .
6. A method of preparing a recombinant fowlpox virus (FPV) whose nucleic acid does not encode active reticuloendotheliosis (REV), comprising the steps of:
(1) providing an isolated DNA fragment comprising a REV sequence flanked by FPV sequences;
(2) amplifying said FPV sequences but not said REV sequence; and
(3) inserting said FPV sequences into a fowlpox virus.
7. A method according to claim 6, wherein said isolated DNA fragment is inserted into a plasmid.
8. A method according to claim 7, wherein said plasmid is amplified by the polymerase chain reaction with primers based on the FPV sequences which flank the REV sequence .
9. A method according to any one of claims 6 to 8 wherein said isolated DNA fragment is an EcoRI-BamHI fragment of FPV M.
10. A method according to claim 9, wherein said isolated DNA fragment is amplified in plasmid pCH21.
11. A method according to claim 10, wherein plasmid pCH21 is digested with Bell, and the digested DNA product is ligated to itself to produce plasmid pCH29.
12. A method according to claim 11, wherein plasmid pCH29 is recombined with a fowlpox virus in a process comprising the steps of:
(1) isolating a DNA fragment carrying the P.L promoter-╬▓-galactosidase gene and the wP.7.5 promoter ECOGPT gene from plasmid pAF09;
(2) digesting plasmid pCH29 with Bell and ligating to the DNA fragment from pAF09 to produce plasmids pCH30a and pCH30b; and
(3) combining plasmids pCH30a and/or pCH30b with the fowlpox virus .
13. A method according to claim 11, wherein the FPV sequence from pCH29 is recombined with a fowlpox virus in a process comprising the steps of: (1) isolating a DNA fragment carrying the P.L promoter-╬▓-galactosidase gene and the wP.7.5 promoter ECOGPT gene from plasmid pAF09;
(2) digesting plasmid pCH29 with BamHl and ligating to the DNA fragment from pAF09 to produce plasmids pCH31a and pCH31b; and
(3) combining plasmids pCH31a and/or pCH31b with the fowlpox virus.
14. A plasmid selected from the group consisting of:
plasmid pCH21 as herein defined;
plasmid pCH29 as herein defined;
plasmid pCH30a as herein defined;
plasmid pCH30b as herein defined;
plasmid pCH31a as herein defined; and
plasmid pCH31b as herein defined.
15. An isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 17kb and is derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single stranded DNA hybridises with the product of amplification of an REV LTR sequence, and is an EcoRI restriction endonuclease fragment.
16. An isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 9.0kb and is derived from FPV M3, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said first single-stranded DNA molecule hybridises to a product of amplification of an REV LTR sequence, and is an EcoRI restriction endonuclease fragment.
17. An isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule which has a molecular size of 9.8kb and is derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, wherein said single-stranded DNA hybridises with a product of amplification of an REV LTR sequence, and is a Pstl restriction endonuclease fragment.
18. An isolated DNA molecule comprising an REV sequence and flanking FPV sequences and selected from the group consisting of the DNA sequences having the DNA sequence shown in Fig 5 including the FPV S 5' LTR (SEQ ID NO: 2 ) , the FPV S 3 ' LTR (SEQ ID NO : 3 ) or FPV M3 LTR (SEQ ID NO: 4), or the DNA sequence shown in Fig 6 including the FPV S 5 ' LTR (SEQ ID NO : 5 ) , FPV S 3 ' LTR (SEQ ID NO : 7 ) and FPV M3 LTR (SEQ ID NO: 6) .
19 . An isolated double- stranded DNA molecule comprising : (1) a first single-stranded DNA molecule derived from FPV S, and
(2) a second single-stranded DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 7.
20. An isolated double-stranded DNA comprising:
(1) a first single-stranded DNA molecule derived from FPV S, and
(2) a second DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 8.
21. An isolated double-stranded DNA molecule comprising:
(1) a first single-stranded DNA molecule derived from FPV S, and
(2) a second DNA molecule complementary to said first single-stranded DNA molecule, which has the restriction endonuclease cleavage map shown in Fig 9.
22. A nucleic acid molecule having the sequence shown in Fig 14 (SEQ ID NO: 9).
23. A nucleic acid molecule having the sequence shown in Fig 16 (SEQ ID NO: 10).
24. A nucleic acid molecule having the sequence shown in Fig 18 (SEQ ID NO: 11).
25. A vaccine against fowlpox virus (FPV) which does not give rise to reticuloendotheliosis virus (REV) infection when administered to chickens, comprising an attenuated strain of FPV and a pharmaceutically acceptable carrier, and including means for preventing expression of active REV.
26. A vaccine according to claim 30, comprising the a recombinant fowlpox virus (FPV) as claimed in any one of claims 1 to 5.
27. Use of a recombinant fowlpox virus (FPV) as claimed in any one of claims 1 to 5 in the preparation of a vaccine against fowlpox virus (FPV) .
28. A method of preventing the occurrence of fowlpox virus (FPV) in a chicken without giving rise to reticuloendotheliosis virus (REV) infection, comprising the step of administering a vaccine as claimed in claim 25 or claim 26 to said chicken.
29. A method according to claim 28, wherein said chicken is vaccinated at older than one day of age.
30. A method according to claim 29, wherein a chicken older than one day of age is vaccinated with said vaccine subsequent to vaccination with FPV M at one day of age.
PCT/AU1998/000628 1997-08-08 1998-08-07 Vaccine against fowlpox virus WO1999007852A1 (en)

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US5403582A (en) * 1993-01-21 1995-04-04 Nippon Zeon Co., Ltd. Vaccine comprising fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus

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CN112143693A (en) * 2019-06-28 2020-12-29 杭州康万达医药科技有限公司 Method for producing virus and harvesting solution composition

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