EP0904394A1 - Chicken embryo lethal orphan (celo) virus - Google Patents

Abstract

The invention relates to a chicken embryo lethal orphan (CELO) virus obtained by in vitro manipulation of plasmid-cloned CELO virus DNA. Said virus is suitable for production of vectors for gene therapy and as a vaccine against infectious diseases in humans and animals, in particular birds.

Description

CELO virus

The invention relates to adenoviruses. The large family of adenoviruses is divided into adenoviruses that infect mammals (the mastadenoviridae) and adenoviruses that infect birds (the aviadenoviridae) according to their host. The CELO virus ("Chicken Embryo Lethal Orphan"; review by McFerran, et al., 1977; McCracken and Adair, 1993) was identified as an infectious agent in 1957 (Yates and Fry, 1957). The CELO virus is classified as poultry adenovirus type 1 (FAV-1) and initially aroused interest due to its property of being tumorigenic in baby hamsters. However, since infection with the CELO virus has no serious health and economic consequences, interest in this virus has waned in recent years. The FAV-1 adenoviruses can be isolated from healthy chickens and do not cause disease when experimentally reintroduced into chickens (Cowen, et al., 1978). Isolation from diseased birds is likely the result of adenovirus replication in a host that has a weakened immune system due to other factors.

The general structural organization of CELO virus is with an icosahedral capsid of 70-80 nm, made up of hexon and penton structures, similar to that of mammalian adenoviruses (Laver, et al., 1971). The CELO virus genome is a linear, double-stranded DNA molecule, the DNA being condensed within the virion by virus-encoded core proteins (Laver, et al., 1971; Li, et al., 1984b). The CELO virus genome has covalently bound terminal proteins (Li, et al., 1983), and the genome has inverted terminal repeats (ITRs) when also shorter than the mammalian ITRs (Aleström, et al., 1982b; Sheppard and Trist, 1992). The CELO virus encodes a protease with 61-69% homology to the mammalian adenovirus proteases (Cai and Weber, 1993).

There are clear differences between the CELO virus and the mastadenovirus. CELO virus has a larger genome, with a sequence homology to Ad5 (Aleström, et al., 1982a) that can only be determined in two short regions of the CELO virus genome (by hybridization). The CELO virion has been reported to have two fibers of different lengths at each vertex. The CELO virus cannot complement the E1A functions of Ad5, and the replication of CELO virus is not facilitated by the activity of Ad5El (Li, et al., 1984c).

In the context of the present invention, a complete sequence analysis of the CELO virus was carried out; on the one hand because it is useful for understanding the biology of adenoviruses to elucidate the genomic organization of an adenovirus that is far from the generally studied mammalian adenoviruses. Since probably the transmission and

Survival conditions for a virus that infects an avian species are different than for mammalian viruses, it is possible that the avian adenoviruses have acquired new viral functions or show a greater degree of variability than the mastadenoviridae. The complete CELO virus sequence also enables changes in the CELO virus genome with a view to fiction analysis.

Since adenovirus vectors have proven to be very powerful vectors for gene transfer {review article by Graham, 1990; Kozarsky and Wilson, 1993; Trapnell and Gorziglia, 1994), on the other hand, is the complete CELO virus sequence of particular interest as a basis for the production of new recombinant vectors for gene transfer.

Sequence analysis has shown that the CELO virus genome has 43.8 kb, which is more than 8 kb longer than the human subtypes Ad2 and Ad5. The genes for the main structural proteins (hexon, penton base, purple, fiber, pVI, pVII, pVIII) are both present on the one hand, and on the other hand they are also located at the corresponding locations in the genome. The Early Region 2 genes (E2; DNA binding protein, DNA polymerase and terminal protein) are also present. However, the CELO virus lacks sequences which are homologous to the regions E1, E3 and E4 of the mammalian adenoviruses. There is approximately 5 kb at the left end and 15 kb at the right end of the CELO virus genome, where there is little or no homology with the mastadenovirus genomes. These new sequences contain a number of open reading frames and are believed to code for functions that replace the missing E1, E3 and possibly E4 regions.

Parts of the CELO virus sequence have already been published; they are listed in Table 1, as are the differences between the sequence known from the database and the sequence determined in the context of the present invention. Studies focusing on certain viral genes have made a homologue of the VA-RNA gene of mastadenovirus known (Larsson, et al., 1986) and part of the genome sequence carrying the endoprotease has been described (Cai and Weber , 1993). Fragments of the CELO virus genome have also been published (Akopian, et al., 1990; Akopian, et al., 1992; Hess, et al., 1995). The sequence of the penton base from the related virus FAV-10 has also been reported (Sheppard and Trist, 1992). Some other sequence fragments were stored in the database and are also shown in Table 1. In total, about 50% of the CELO virus

Genome available in the form of fragments (approx. 24 kb in total). The sequence obtained in the context of the present invention is complete and has the advantage that it was obtained from a single isolate.

The complete sequence of the CELO virus is shown in the sequence listing (in the sequence listing "complementary" means that the respective open reading frames are in reverse order). It shows a large number of striking differences between Ad2 and the CELO virus. The organization of the recognizable open reading frames (ORFs) of the CELO virus genome on the basis of the sequence analysis, in comparison with Ad2, is shown in FIG. 1A: The figure shows an overview of the genomic organization of Ad2 / 5 and CELO virus. The arrows indicate the location of the coding regions, but not the exact cleavage pattern of the gene products. The CELO virus pattern also indicates (in the first 6,000 bp and in the last 13,000 bp) all unassigned open reading frames that start with a methionine and that code for more than 99 amino acid residues. The central region of the two genomes, which show homology based on the dot matrix analysis (cf. FIG. 3), and the regions at the ends of the CELO virus genome which have no homology with other adenoviruses ("Unique to CELO") are given. The abbreviations in the figure, which also correspond to those in the tables, mean: PB, penton base; EP, endoproteinase; DBP, DNA binding protein; bTP, pre-terminal protein; pol, DNA polymerase.

The sequenced CELO virus genome is 43,804 bp in length and has a G + C content of 54.3%. It previously it was suspected that the CELO virus genome is much larger than the 34-36 kb mastadenovirus genome; it was found that the CELO virus DNA

Has a weight of 30 x 10 ⁶ daltons, determined according to its sedimentation coefficient (Laver, et al., 1971), compared to 24 x 10 ⁶ daltons for Ad2 (Green, et al., 1967). The size of the CELO virus genome, determined by adding the restriction fragments, is approximately 43 kb (Cai and Weber, 1993; Denisova, et al., 1979). A pulsed field gel analysis of the CELO virus genome isolated from purified virions is shown in FIG. 2A and is carried out with the DNA isolated from Ad5 dll014 (34,600 bp; Bridge and Ketner, 1989) or wild-type Ad5 virions (35,935 bp; vt300) ; Chroboczek, et al., 1992; Jones and Shenk, 1978); a mixture of uncleaved bacteriophage λ-DNA and λ-DNA, digested with five different restriction enzymes (Biorad), was used as the size marker (lanes 1 and 7 show the molecular weight markers, lane 2 the DNA of

Ad5 dll014, lane 3 the DNA from Ad5 wt300, lane 4 the CELO virus DNA, lane 5 the DNA from OTE, lane 6 the DNA from Indiana C). Figure 2A shows that the CELO virus genome is 44 kb in length. From this analysis it can be seen that the CELO virus genome is actually significantly larger than the genome of the mammalian virus. Calculations based on the migration of fragments of the lambda bacteriophage result in a size of 43 kb for the CELO virus genome. The DNA extracted from two other FAV-1 isolates, Indiana C and OTE, co-migrates with the CELO virus species, which is further evidence of the size of the CELO virus genome. Fig. 2B shows that the CELO virus sequence contained on the bacterial plasmid pBR327 has the same size. There is no identifiable El region. There was no significant homology between the CELO virus

Genome and the first 4,000 bp of Ad2. There are some small open reading frames in the first 5,000 bp of CELO virus that may accomplish some of the El tasks. An open reading frame at the right end of the virus genome (GAM-1) can replace E1B 19K in functional assays without significant homology between GAM-1 and E1B 19K. To confirm that the left end is from the wild-type CELO virus genome and does not represent the sequence of a cloned variant, various tests were carried out: comparison of the direct sequence analysis of CELO virions at three different sites with the corresponding sites of the cloned sequences; Southern analyzes with DNA from different virus isolates, which gave the same restriction fragments; Pulsed field gel electrophoresis of various virus genomes that showed no heterogeneity.

There is no identifiable E3 region; the two small open reading frames in the corresponding region of CELO virus have no significant homology with the E3 functions described.

There is a group of small open reading frames between 36,000 and 31,000, the location of which indicates the mammalian virus E4 region, but with an additional 8 kb sequence at the right end of the CELO virus.

No sequence similar to Protein IX was identified either (Protein IX is essential for the hexon-hexon interactions and the stability of the mammalian adenovirus virions). A protein V gene was also not identified.

The following regions are conserved between the CELO virus and Ad2: The central part of the CELO virus genome, from the IVa2 gene (approx. From nucleotide (nt) 5,000) on the left strand to the fiber genes on the right strand (approx nt 33,000) is organized like mastadenoviruses, and most of the important viral genes can be identified both by position and by sequence homology. Previous studies of homology between CELO and Ad2 (Aleström, et al., 1982a) showed two regions of the CELO virus that cross-hybridize with the Ad2 sequence. These two fragments are nt 5,626 to 8,877 (coding for IVa2 and the carboxy terminus of DNA polymerase) and nt 17,881 to 21,607 (coding for the hexon). The dot matrix analysis shown in FIG. 3 (carried out using the UWGCG program Compare with a window of 30 and a stringency of 20; summarized in FIG. 1A) shows that the entire DNA sequence homology between CELO virus and Ad5 mapped in the central region of the CELO virus genome. This can be expected because the capsid proteins are encoded in this central region and the coarse structure of the CELO virion is comparable to the capsid of the mammalian adenovirus (Laver, et al., 1971; Li, et al., 1984a). The genes coding for proteins corresponding to the human adenovirus proteins hexon, purple, penton base, protein VI and protein VIII are present in the expected order and location (Fig. 1A and Table 2A; Table 2B shows unassigned open reading frames which code for gene products with more than 99 amino acid residues). Each vertex of the mastadenovirus virion contains a pentamer of the penton base protein in association with a single fiber consisting of three copies of the fiber polypeptide. Ad2, like most mastadenoviruses, has a single fiber gene, some types of adenovirus have two fiber genes. The CELO virus genome codes for two fiber polypeptides of different lengths and sequences.

DNA binding proteins were identified in region E2 (Li, et al., 1984c); Four proteins with similar peptide maps have been described, suggesting a single precursor that is then cleaved or degraded. The left open reading frame of the CELO virus genome, starting at nt 23,224, is in the expected DNA binding protein region. The genes coding for DNA polymerase and pTP (pre-terminal protein) are present and in the expected positions (Fig. 1A, Table 2A).

With regard to the production of vectors based on the CELO virus, it is of interest to identify the mechanisms that the CELO virus uses to package nearly 44 kb of DNA into a virion that is similar in size to human Adenoviruses that are severely restricted in their packaging capacity (Bett, et al., 1983; Caravokyri and Leppard, 1995; Ghosh-Choudhury, 1987). One possibility is that the CELO virion, although almost identical in size to Ad2 and Ad5, has enough expanded structure to accommodate the larger genome. An alternative hypothesis is that CELO has a different mechanism of DNA condensation and therefore has differences in the level of core proteins that are responsible for DNA packaging. Laver, et al. , 1971 identified two proteins in the core of CELO virus and noted the lack of a protein V-like molecule. Li, et al. , 1984b, used higher resolution electrophoresis and reported a core structure with three polypeptides (20 kD, 12 kD and 9.5 kD). It can be concluded from both findings that the CELO virus may lack the larger basic nuclear protein V (41 kD), which is found in mammalian adenoviruses. Perhaps the lack of Protein V and / or the presence of smaller, basic proteins is responsible for the additional packaging capacity of the CELO virion. The smallest of those described by Li, et al. , 1984b, identified CELO virus core proteins (9.5 kD) most closely associated with virus DNA, similar to protein VII of human adenovirus. An open reading frame, which suggests a protein with 8,597 D with 72 amino acids, is nt 16,679; the encoded protein is rich in arginine (32.9 mol%) and contains two cleavage sites for protease (pVII of Ad2 has only one cleavage site). An open reading frame, which suggests a protein with 19,777 D with 188 amino acid residues, is nt 16,929. The protein has protease cleavage sites after residues 22, 128 and 145, and the carboxy-terminal residues have homology with pX from mastadenovirus. 4 shows the amino acid sequences of protein VII and pX of various mastadenoviruses in comparison to the CELO virus and the core proteins core 2 and core 1 of FAV-10. The sequences were arranged using the UWGCG Bestfit program with a gap weight of 3.0 and a weight and a gap length of 0.1. The protease cleavage sites of adenovirus are underlined. In this context, it is of interest that the 19 residue mastadenovirus DNA binding protein called "rough" is formed by two protease cleavages of the pX precursor (Hosokava and Sung, 1976; Weber and Anderson, 1988; Anderson, et al ., 1989;). Cleavage of the 188 residue protein after residues 128 and 145 would be 17 residues mu-like basic protein (41% arginine, 12% lysine). The uncleaved form of the protein is also strongly basic; the uncleaved copies of this protein might correspond to the 20 kD core protein observed by Li, et al., 1984b; a third 12 kD core protein identified by these authors could not yet be assigned.

Some new or unassigned open reading frames were also found in the CELO virus genome. A summary of these open reading frames is shown in Table 2A; these open reading frames are also shown in Fig. 1A. This compilation was limited to the sequences from nt 0 - 6,000 and 31,000 - 43,804 and only ORFs which contain a methionine residue and which code for a protein of> 99 amino acid residues are given. As already mentioned, there is an ORF at nt 1999 which codes for a protein with homologies to Parvovirus REP, and an ORF at nt 794 with homology to dUTPase and Ad2 E4 ORF1. The object of the present invention was to provide a new CELO virus.

Based on the complete CELO virus genome sequence, the present invention thus relates to a CELO virus which was obtained by in vitro manipulation of a plasmid-cloned CELO virus DNA.

In a preferred embodiment, the CELO virus according to the invention derived from the genomic DNA contains the left and right terminal repeat and the packaging signal and has modifications in the regions of the CELO virus DNA in the form of insertions and / or deletions and / or mutations on. The left or right terminal repeat ("Inverted Terminal Repeat", ITR) extends from nucleotides 1 - 68 and from nucleotides 43734 -

43804, the packaging signal (also referred to as "Psi") extends from nucleotides 70-200. Modifications in DNA sections other than these have the effect that the genes affected by the modification are non-functional or deleted.

Modifications of the CELO virus genome are preferably carried out, which lie on a section of the CELO virus DNA which comprises the nucleotides from approx. 201 to approx. 5,000 (following the left terminal repeat, the section on the left end, in the hereinafter referred to as "Section A") and / or on a section which comprises the nucleotides of approximately 31,800 - approximately 43,734 (the section located in front of the right terminal repeat at the right end, hereinafter referred to as "Section B") and / or on a section comprising the nucleotides of approximately 28,114-30,495 (the region of the fiber 1 gene, hereinafter referred to as "section C").

A CELO virus that has certain genes non-functional or deleted, e.g. Genes that affect the host's immune response, such as antagonists of genes from the E3 region of mammalian adenoviruses, can be used as the vaccine.

In one embodiment of the invention, the CELO virus contains one or more foreign DNA molecules, in particular a foreign DNA to be expressed in a host organism. In this embodiment, the CELO virus acts as a vector which can transport and express the foreign DNA into higher eukaryotic cells, tissues or organisms, in particular mammals and birds. Suitable insertion sites for the foreign DNA are sections A and / or B and / or C.

The foreign DNA preferably replaces one or more sequences from these sections.

The CELO virus according to the invention is contained on a plasmid which is replicable in bacteria or yeast and which, after being introduced into suitable cells, delivers virus particles. Examples of suitable cells are bird embryonic kidney or liver cells.

With regard to the use of a recombinant CELO virus vector for gene therapy, the foreign DNA can consist of one or more therapeutically active genes. Examples are genes coding for immunomodulators or modulators of

Inflammatory processes (cytokines such as IL-2, GM-CSF, IL-1, IL-6, IL-12; interferons, tumor antigens, IκB and derivatives of IκB that lack serine phosphorylation sites (Traenckner, et al., 1995) or that lack lysine ubiquitination sites ; Glucocorticoid receptors; enzymes such as catalase,

Manganese superoxide dismutase, glutathione peroxidase, LIP members of the C / EBP family such as LIP or LAP (Descombes and Schibier, 1991), ADF (Tagaya, et al., 1989)), genes which influence apoptosis (members of Bcl-2- Family such as Bcl-2, adenovirus E1B19K, Mcl-2; BAX; IRF-2; members of the ICE protease family; variants of cJun such as TAM-67 (Brown, et al., 1991); adenovirus E1A; p53) and Genes coding for other therapeutic proteins (e.g. coagulation factors such as factor VIII or IX; growth factors such as erythropoietin; the cystic fibrous transmembrane regulator gene (CFTR); dystrophin and its derivatives; globin; the LDL receptor; genes that lysosomal storage diseases such as β-glucuronidase are absent; Etc.) .

With regard to the production of cellular tumor vaccines or for pharmaceutical

Compositions with which the immune response to tumors is to be strengthened are encoded by the foreign DNA for immunostimulating proteins or tumor antigens or fragments thereof.

The therapeutically effective DNA can also code for antisense molecules which prevent the expression of genes or the transcription of certain RNA sequences in the target cell.

With regard to the use of the recombinant CELO virus vector as a vaccine, the foreign DNA codes for one or more antigens which elicit an immune response in the treated individual.

In one embodiment of the invention, the foreign DNA codes for an antigen which is derived from a human pathogen, in particular an infectious agent.

Epitopes that can be expressed by recombinant CELO viruses include any human viral pathogen such as HIV, hepatitis A, B, C, hantavirus, poliovirus, influenza virus, respiratory syncytiavirus, measles, mumps, rubella, papilloma and epitopes derived from many other viruses. Non-viral pathogens include trypanosomes (causes of sleeping sickness and Chagas' disease), Leishmania, Plasmodium Falciparum

(Malaria), various bacterial pathogens, such as the causative agents of tuberculosis, leprosy, Pseudomonas aeruginosa

(Complications in cystic fibrosis) and many others. An overview of vaccines based on mastadenoviruses is given in Table 3; the epitopes exemplified there can also be used to insert into a vector based on the CELO virus.

With regard to the use of the recombinant CELO virus vector as a veterinary vaccine, e.g. for avian, in particular poultry, vaccine, in a further preferred embodiment the foreign DNA codes for an antigen derived from a protein of a pathogen of animal diseases, in particular infectious avian diseases.

Avian Infectious Bronchitis Virus (IBV, a coronavirus; Jia, et al., 1995; Ignjatovic and McWaters, 1991; Küsters, et al., 1990; Lenstra, et al., 1989; Cavanagh, et al ., 1988; Cunningham, 1975), Avian Influenza Virus (Orthomyxovirus Type A; Kodihalli, et al., 1994; Treanor, et al., 1991; Tripathy and Schnitzlein, 1991), Fowlpox virus (McMillen, et al., 1994), Avian Infectious Laryngotracheitis Virus (Guo, et al., 1994; Scholz, et al., 1993; Keeler, et al., 1991), Mycoplasma Gallisepticum (Nascimento, et al., 1993), Avian Pasteurella Multocida (Wilson , et al., 1993; Lee, et al., 1991; Hertman, et al., 1980; Hertman, et al., 1979), Avian Reovirus (Ni and Kemp, 1992; Huang, et al., 1987), Marek's Disease Virus (MDV; Malkinson, et al., 1992; Scott, et al., 1989), Turkey herpes virus (HVT, Herpesvirus of Turkeys), Newcastle Disease Virus (NDV; Cosset, et al., 1991; Morrison, et al., 1990), Avia n Paramyxovirus type 1 (Jestin, et al. , 1989), Avipoxvirus Isolate (Schnitzlein, et al., 1988) such as Juncopox, Pigeon Pox, and Feld (Field) and vaccine strains of bird poxviruses, Avian Encephalomyelitis Virus (Shafren and Tannock, 1991; Nicholas, et al. , 1987; Deshmukh, et al. , 1974), Avian Sarcoma Virus, Rotavirus (Estes and Graham, 1985), Avian Reovirus (Haffer, 1984; Gouvea, et al., 1983; Gouvea and Schnitzer, 1982), H7 Influenza Virus (Fynan, et al., 1993).

In addition to DNA sequences which code for therapeutically effective gene products or for antigens, the foreign DNA can code for proteins or protein fragments which determine the behavior of the CELO virus, in particular its ability to bind to the cells, with a view to use on mammals especially on mammalian cells. Examples of such proteins are fiber or penton-based proteins from mammalian and other adenoviruses, surface proteins from other viruses, and ligands which have the ability to bind to mammalian cells and to transport the CELO virus into the cells. Suitable ligands include transferrin from various mammalian species, lectins, antibodies or antibody fragments, etc. Such ligands are known to the person skilled in the art, further examples can be found in WO 93/07283.

The recombinant CELO virus can contain one or more foreign DNA molecules, these can either be inserted in tandem or, at a distance from one another, into different sections of the CELO virus sequence.

The foreign DNA is under the control of regulatory sequences; suitable promoters are e.g. the CMV immediate early promoter / enhancer, the Rous Sarcoma Virus LTR, the adenovirus major late promoter, the CELO virus major late promoter.

The suitability of the CELO virus for the production of vectors and the advantages of these vectors and their Applications are based in particular on the following properties of the CELO virus:

i) Safety: The CELO virus does not naturally replicate in mammalian cells. Therefore, vectors based on this virus can be used in humans without the risk that subsequent infection with a wild-type human adenovirus could complement the vector and allow replication. This is an advantage over the Ad2 and Ad5 vectors currently used.

ii) Increased packaging capacity: The CELO virus genome is approximately 44 kb long compared to the 36 kb of the Ad5 genome. Both viruses have comparable virion dimensions, so that with a CELO virus vector there is the possibility of expanding the strict packaging limit of 35 kb available with Ad5. On the basis of the sequencing carried out in the context of the present invention, DNA-packaging core proteins of the CELO virus were identified and striking ones

Differences to Ad2 found that could be responsible for the increased packaging capacity. There are approx. 13 kb at both ends of the CELO virus, which should not code for structural proteins (eg capsid components) or for proteins that are required directly for virus replication (eg DNA polymerase). For the production of vectors, these sequences on the CELO virus genome can be removed and, if necessary, replaced by complementing cell lines. These sequences can be assumed to encode the immune functions or the apoptotic functions (eg GAM-1) of the host cell or that they are involved in the activation of the host cell for virus replication (opponents to the El-, E3- and E4- Regions of Ad2). These are the gene types that are either unnecessary for virus growth in cell culture, such as the E3 genes from Ad2 (Wold and Gooding, 1991; Gooding, 1992), or that are easily removed from the virus and from a complementing cell line can be expressed, such as the El region in 293 cells (Graham, et al, 1977) and the E4 region in W162 cells (Weinberg and Ketner, 1983).

iii) Stability: The CELO virion is remarkably stable. The infectivity and the ability to transport DNA survive treatment at 60 ° C for 30 min. In comparison, Ad5 loses two orders of magnitude of infectivity at 48 ° C and is completely inactivated at 52 ° C. Presumably the CELO virus did not develop its heat stability naturally, rather the heat stability may indicate a response to another type of selective pressure on the virion. The natural route of the CELO virus infection is a fecal-oral one, which requires that the virion can survive contact with a chemically harsh environment with extreme pH values as well as proteases. An even more resistant virus would be desirable for special applications in gene therapy, e.g. would survive in the digestive tract or lungs of a patient with cystic fibrosis.

iv) Targeted use: The CELO virus binds only weakly to mammalian cells and requires the addition of a ligand for efficient entry into the cell (transferrin or lectin; Cotten, et al., 1993). Therefore, recombinant CELO virions cannot penetrate human cells, resulting in the following possible uses: As indicated above, the virus can be genetically modified in order to express ligands on its surface which enable targeted transport, such as specific peptides, or fibers and / or penton bases of human adenoviruses.

Another possibility is the chemical modification of the virus in order to couple specific ligands such as transferrin, e.g. propose in WO 94/24299, furthermore the virus can be biotinylated

(WO 93/07283) and via streptavidin to biotinylated ligands such as wheat germ agglutinin or other lectins

(WO 93/07283) are bound. CELO virus vectors therefore do not have the disadvantages of human adenoviruses which, although they have a good ability to bind to human cells, have to be masked for a specific, targeted application mediated by the ligand.

v) Applicability for vaccines: The CELO virus is rarely associated with diseases in birds, which indicates that it causes a strong protective immune response in bird hosts. The CELO virus vector can be easily adapted for the expression of new vaccine epitopes.

With regard to the removal of regions of the CELO virus DNA, it was concluded from the knowledge gained from the mastadenovirus that if the central sections of the CELO genome were removed, these sections must be made available in trans, for example from a packaging cell line. This limitation is due to the large amount of virion components necessary to assemble the virus and the need to make a cell line that can can produce appropriate amounts of these proteins without toxicity.

The approaches to mastadenoviruses that have so far been more successful in this regard have been the production of cell lines that express regulatory proteins (E1, E4 regions) or enzymatic proteins (DNA polymerase, DNA binding protein) because these proteins do not become large during a productive virus infection Quantities are needed.

Based on the analysis of the known CELO genes, the sections of the genome from nt approximately 12,000 to approximately 33,000 which code for structural components of the virus are preferably not interrupted. The region from approximately nt 5,000 to approximately 12,000 codes for the E2 genes IVa2 (a viral transcription factor), the viral DNA polymerase (POL) and the terminal viral protein (Viral Terminal Protein; pTP). These genes are essential for the function of mastadenoviruses; they should therefore also be essential for the CELO virus. In principle, however, deletions of such essential genes are also possible, provided that they are in trans, e.g. B. from a packaging line can be produced. For example, produced a packaging cell line that produces Ad5 DNA polymerase, which made it possible to delete this gene from the virus genome. The construction of CELO virus vectors can be carried out in a similar manner by removing sections or the entire region from nt 5,000 to 12,000 from the CELO virus and contributing the corresponding functions in trans from a packaging cell line.

Another possible limitation is the presumed major late promoter, which was provisionally assigned to the region at around nt 7,000 (TATA box at nt 7,488). In the mastadenovirus, this promoter is essential to drive late gene expression. Therefore, any change in the region at nt 7,000 of the CELO genome must be made in a manner that maintains that region's promoter function.

Table 4 lists the sequence elements of the CELO virus genome and, with regard to their deletion and / or mutation in the production of CELO virus vectors, is divided into different categories (in Table 4, L1, L2, etc. mean late Message 1, 2, etc., according to the designation common for mastadenoviruses):

Category 1 includes sequence elements that are required in ice and therefore cannot be provided in trans by a complementing cell line or by a complementing plasmid. These sections are required and are therefore present on the CELO virus according to the invention; it is the left and right terminal repeat as well as the packaging signal.

Category 2 sequences code for proteins that are required for virion production in large quantities. These proteins can optionally be produced from a gene contained in a complementing cell line or on a complementing plasmid. Other sequences of category 2 are the major late promoter, the tripartite leader sequence, also the splice acceptor sites (SA) or the

Polyadenylation sites (poly A sites) of genes that are essential and cannot be provided in trans. In principle, it must be ensured that any modifications to the CELO virus DNA that are carried out at the borders of genes do not interrupt or otherwise impair control signals that may be present, for example polyA sites.

The genes deleted or non-functional on the CELO virus can be trans, e.g. of complementing cell lines.

Complementing cell lines ("helper cells") can be produced in a manner known from the literature, analogously to helper cells which complement the functions of mammalian adenoviruses. For this purpose, the relevant CELO virus gene is introduced on a plasmid, preferably in combination with a selectable marker, into cells which allow the replication of CELO virus, preferably into immortalized cell lines such as LMH (Kawaguchi, et al., 1987) or immortalized quail cell lines, such as by Guilhot et al. 1993, described. The defective CELO viruses can replicate in helper cells that express the relevant CELO virus genes, possibly stably integrated.

Instead of making the regions of the CELO virus deleted in the vector available by means of a cell line, the deletions can also be complemented by a copy of the gene in question contained on a plasmid. For this purpose, for example, that described in WO 96/03517 by Cotten et al. 1994a and 1994b) or that of Wagner et al. , 1992, method used, wherein a deletion-containing CELO virus vector as part of a transfection complex, containing a conjugate of polylysine and a UV / psoralen-inactivated adenovirus (human or CELO) and optionally transferrin-polylysine, in embryonic Chicken kidney cells or liver cells, embryonic or immortalized quail cells, for example liver or kidney cells, are introduced and the transfection complex also contains a plasmid which carries a copy of the gene or genes which the CELO virus vector is missing. The combination of genes contained on the vector and genes carried by the plasmid results in a normal virus replication cycle. (Similar approaches have been used in mastadenovirus systems to complement El-deficient (Goldsmith, et al., 1994) or E4-deficient (Scaria, et al., 1995) adenoviruses.) The subsequent amplification of the virus can be performed by the defective virus is used as a carrier to which the complementing plasmid is attached according to the methods given above.

Another way to replace the genes missing from the CELO virus is to use helper viruses.

A CELO virus (wild-type or partially defective) can be used as the helper virus. In this embodiment, the mutation-carrying CELO plasmid (for example a derivative of pCEL07) is used, for example according to the method described in WO 96/03517 or by Cotten et al. , 1994, method described, introduced into chicken cells, the carrier for the derivative being, for example, psoralen / UV-inactivated adenovirus (human or CELO) together with an adenovirus (human or CELO) as a carrier for the plasmid (s) with the genes which complement the defect is used. Alternatively, a wild-type CELO virus can be used both as a carrier and as a source for complementary gene functions. The subsequent amplification of the defective CELO viruses obtained is by means of co-infection of the defective CELO virus with a complementing adenovirus (eg wild-type CELO or a CELO that has mutations at other parts of the genome).

Category 3 CELO virus genes include the sequences on sections A, B and C. These are sequences which code for a protein or an RNA molecule which interacts with the host cell machinery or with the host immune system is required. These proteins should be required in lower concentrations or not necessary for the cultivation of the virus in tissue culture.

The genes of category 3 are thus preferably replaced by the gene of interest in the CELO virus vectors according to the invention; if necessary, complementing cell lines or plasmids or helper viruses can be produced which produce the corresponding gene products.

In one embodiment of the invention, the vectors according to the invention contain the gene of interest instead of one of the fiber genes. The CELO virus has two fiber proteins (Laver, et al., 1971; Gelderblom and Maichle-Lauppe, 1982; Li, et al., 1984a). It is believed that one of the fibers of the CELO virus is not required for virion assembly and infectivity. This assumption is supported by electron microscopic observations that the longer fiber (fiber 1) may associate with the penton base along the side of the complex, while the shorter fiber (fiber 2) protrudes from the center of the penton base, similar to the penton / fiber complexes in the Mastadenoviruses (Hess, et al., 1995). In adenoviruses with only one fiber, the fiber molecule is for the Assembly of the virus required; in the absence of fiber, no stable, mature viruses are formed. The CELO virion should therefore require fiber 2 for stability and as a ligand, while fiber 1 only has a ligand function. As part of the present

Invention, the correctness of the assumption that of the two fiber genes of the CELO virus, the fiber gene 1 located in region C is superfluous and can be replaced by the gene of interest, was confirmed by removing the fiber 1 gene and by a luciferase expression unit has been replaced.

Other examples are insertions in region A and / or B.

In the context of the present invention, it was found that destruction of the reading frame at nt 794 (region A) which codes for dUTPase provides viable viruses. The dUTPase gene is thus a gene that is not required for growth in cell culture.

In one embodiment of the invention, the recombinant CELO virus thus contains a foreign gene which is inserted in the region of the reading frame coding for dUTPase.

In a further aspect, the present invention relates to a method for producing recombinant CELO virus.

The method is characterized in that the CELO virus genome, or sections thereof, contained on a plasmid is genetically manipulated.

In one aspect of the invention, the genetic manipulation consists of an insertion and / or deletion. Insertions and / or deletions can be made by naturally occurring in these sections in the CELO virus DNA

Restriction enzyme interfaces are used, e.g. the Fsel interface occurring in section B at position 35.693; the insertion into this interface can be made directly, over this interface or in the vicinity of this interface, or this interface is used to facilitate recombination in the neighborhood.

In a preferred embodiment, the manipulation consists in making insertions and / or deletions using standard molecular biological methods (Maniatis, 1989). The naturally occurring restriction interfaces can be used for this, e.g. Locations located in regions of the genome which are unnecessary for culturing the virus in the host cell, e.g. the Fsel interface occurring in section B at position 35.693. The insertion can be made in this interface directly, over this interface or in the vicinity of this interface, or this interface is used to facilitate recombination in the neighborhood. Foreign DNA sequences, e.g. Marker genes or genes coding for therapeutically active proteins can be inserted.

An alternative possibility is to remove CELO sequences flanked by two restriction sites and to replace them with new sequences. (An example of this is the dUTPase mutation, as was carried out in Example 7.) In these cases, the manipulation is carried out with the entire CELO virus genome. In the event that restriction sites are present, the Deletion / insertion can be carried out on a subfragment, which is then reinstalled into the entire genome by means of ligation and possibly recloning in bacteria.

Another possibility is to insert the foreign gene into artificial restriction enzyme interfaces produced using conventional methods of recombinant DNA technology (Maniatis, 1989).

In one embodiment, the method is characterized in that manipulations are carried out in a plasmid DNA which contains the CELO virus genome, in CELO DNA sequences, except in the left and right inverted terminal repeat and in the packaging signal.

In a further preferred method, the manipulation of the CELO genome is carried out by means of recombination. For this purpose, a subfragment of the CELO genome is manipulated in order to introduce mutations and / or new sequences. Subfragments can be produced in various ways, by means of PCR (polymerase chain reaction), by ligation between PCR products or between restriction fragments or by subcloning in bacteria (as described in the examples of the previous invention; see also Chartier et al. 1996 ). Examples of suitable bacterial strains for recombination are BJ 5183 (Hanahan, 1983) or JC 8679 (Gillen et al., 1974) or JC 5176 (Capado-Kimball and Barbour, 1971).

For the recombination with the aid of PCR products, the sequence to be inserted into the CELO genome is made using primers which contain the sequence plus approximately 15 nucleotides of the insertion site in the CELO genome flank complementary sequence, prepared by PCR (Oliner et al., 1993). In a second round of PCR, a further 15 nucleotides of the sequence complementary to CELO are added, which leads to a PCR product which consists of the sequence to be inserted with 30 nucleotides of the CELO sequence at both ends. This fragment is mixed with a plasmid which contains the CELO-DNA (for example the plasmid pCEL07 produced in the context of the present invention) and which has been linearized with a restriction enzyme which only cuts between the two flanking sequences which are attached to the sequence by means of PCR were.

In principle, the recombination using ligation reaction products (produced using conventional techniques, as described, for example, by Maniatis et al. 1989) is carried out in exactly the same way as in the case of recombination with cloned fragments, with the difference that the intermediate cloning step is omitted.

In all cases, the manipulated product obtained is characterized and used to produce the virus by transfecting avian cells (for example using the method described by Wagner et al., 1992; Cotten et al., 1994; or Cotten et al., 1993) and then cultivated, whereupon the virus is harvested.

For the production of recombinant CELO virus by means of cloned fragments, the procedure is preferably that of subcloning a small fragment from the relevant region of CELO virus into which the foreign gene is to be inserted on a bacterial plasmid in order to achieve that restriction sites that occur multiple times on the CELO virus genome, occur only once on the plasmid. These restriction sites will be used to remove a region from the small fragment. This region is replaced by foreign DNA for the production of the CELO virus vector. The foreign DNA can consist only of a linker with a unique restriction site, or of a sequence coding for a protein or for an antigen. The sequence can also code for reporter gene with a unique restriction site. This facilitates further manipulation of the CELO virus by inserting the foreign DNA coding for a therapeutically active gene product or for an antigen into this restriction site, and at the same time the reporter gene enables rapid information about the efficiency of the vector by the plasmid is introduced into cells and the expression of the reporter gene is monitored.

Figure overview

Fig. 1A: Comparison of the genomic organization of Ad2 / 5 with the CELO virus

Figure 1B: Restriction map of the CELO virus genome

2A and B: Pulsed field gel electrophoretic analysis of the genome size of adenoviruses

2C: Characterization of plasmid-cloned copies of the CELO virus genome by means of restriction endonucleases

Fig. 3: Dot matrix analysis of the DNA sequence homology between CELO virus and Ad2 4: Amino acid sequences of protein VII and pX from different mastadenoviruses in comparison with CELO virus and the core proteins core 2 and core 1

Fig. 5: Construction of a plasmid containing the entire length of the CELO genome

6: Production of a CELO vector from a copy of the CELO virus genome contained on a plasmid

Figure 7: Identification of bacterial clones containing a deletion in the dUTPase gene

Figure 8: Comparison of wild-type CELO and CELO containing a deletion in the dUTPase gene Western blot analysis

Unless otherwise stated, the following materials and methods were used in the examples:

a) Virus and virus DNA

A plaque-purified isolate of CELO virus (FAV-1,

Phelps strain), which was used as the starting material for the DNA for both direct sequencing and for the formation of bacterial plasmid clones, was grown in 9 day old pathogen-free chicken embryos as described by Cotten, et al. , 1993. The FAV-1 isolates OTE (Kawamura, et al., 1963) and Indiana C (Calnek and Cowen, 1975; Cowen, et al., 1978) were grown in chicken embryonic kidney cells. The virus was purified from allantoic fluid or from infected embryonic kidney cells by separation in CsCl gradients, as described by Laver, et al. , 1971, and Cotten, et al. , 1993. Virus DNA was obtained by treating the purified virions with Proteinase K (0.1 mg / ml) and SDS (0.2%) at 56 ° C. for 45 min, and subsequent equilibrium centrifugation of the DNA in a CsCl gradient in the presence of ethidium bromide. After the second gradient, the ethidium bromide was removed by extraction with CsCl-saturated isopropanol and the virus DNA was dialyzed extensively against 10 mM Tris, 0.1 mM EDTA, pH 8.

b) Chicken embryonic kidney cells

The kidneys from 14 day old chicken embryos were collected, washed in PBS and digested with pancreatic trypsin (2.5 mg / ml in PBS) at 37 ° C. The dispersed cells were mixed with an equal volume of fetal calf serum, the cells obtained by centrifugation, washed once with FCK medium and taken up again in the same medium. (The FCK medium is Medium 199 with Earle's salts (Sigma M2154), supplemented with 10% tryptose phosphate (Sigma T8159), with 10% fetal calf serum, 2 mM glutamine, 100 μg / ml streptomycin, 100 IU / ml penicillin.) Die Cells were plated in 175 cm ² tissue culture bottles (2 embryos per bottle), stored at 37 ° C / 5% CO ₂ and infected 24 to 48 hours later. The cells were infected with approximately 1,000 virus particles per cell and harvested 3-4 days after infection when the cytopathic effect was complete.

c) Pulsed field electrophoresis

Aliquots of purified adenovirus DNA (10-20 ng) were loaded onto a 1% agarose gel (BioRad, PFC-agarose) and using a BioRad CHEF Mapper Pulsed Field electrophoresis system (FIGE mode) in 0.5 x TBE for 24 h , cooled to 14 C, separated. The switching time, both in forward and Reverse direction, was changed logarithmically from 0.22 see to 0.92 see with a ramp factor of 0.357 (21%). The forward voltage gradient was 9 V / cm (300 V), the reverse voltage gradient was 6 V / cm (200 V). After the run, the gel was stained in water for 0.5 min in 0.5 μg / ml ethidium bromide solution and then decolorized for 1 h before the DNA pattern was made visible by UV illumination.

d) sequencing methods, data analysis

For the sequencing, EcoRI and HindIII restriction fragments of CELO virus DNA were cloned into pBlueScript SK ( ^" ). Three of the EcoRI clones containing the EcoRI fragments C, D and E (see FIG. 1B) and five the HindiII clones containing HindiII fragments F, A, G, B and E (see FIG. 1B) were selected for the creation of unidirectional deletions using exonuclease III (in FIG. 1B the cleavage sites are for the restriction enzymes EcoRI, HindiII, BamHI and BglII; the alphabetical designation of the EcoRI and HindiII fragments (because of their relative sizes) is also given 373 sequenced according to the manufacturer's instructions The sequence analysis of the terminal 2,000 bp on the left and the 1,000 bp on the right end of the CELO virus genome, the

Sequencing to close the gaps between the fragments EcoRI C / HindIII G and the fragments HindIII B / EcoRI D as well as sequencing to confirm the sequence at different sites in the genome were carried out by direct sequencing of the viral DNA. All sequence data are the result of at least three sequence reactions. The sequence data were merged using the SeqEd (ABI) and SeqMan (Lasergene) programs. Sequence analysis was performed using the University of Wisconsin program GCG.

example 1

Production of a recombinant bacterial plasmid clone of the CELO virus genome

a) Preparation of a plasmid vector with a low copy number for the cloning of the CELO virus

The bacterial vector pBR327 (ATCC No. 37516) was chosen because it is retained in bacterial host strains with a somewhat low copy number (instead of this plasmid, any other plasmid with a low copy number, such as pBR322, could equally be used). It was essential to create a unique restriction site on the vector that does not appear in the CELO virus sequence. As described below, the virus sequence must be excised from the plasmid vector sequences to inject a productive infection, therefore restriction sites flanking the CELO sequence (but which are not within the CELO sequence) must be incorporated into the vector . In the experiments carried out, the restriction enzyme Spei was chosen; however, other enzymes that have no recognition sites in the CELO sequence, such as Ascl, Pacl and Sfil, can be used instead.

The plasmid p327SpeI was prepared by ligating a Spel linker (New England Biolabs) into the Klenow-treated EcoRI site of pBR327, whereby the EcoRl site was destroyed and a unique Spel site was created.

b) Cloning the ends of CELO

The two terminal HindII fragments were cloned. For this purpose, CsCl-purified genomic CELO-DNA was digested with HindIII and separated on a low-melting agarose gel (0.7% low-melting agarose in TAE). The 1601 bp left end fragment and the 959 bp right end fragment were cut from the gel, and each gel fragment was suspended in 300 µl 10 mM Tris, ImM EDTA pH 7.4 and heated at 70 ° C for 10 min to melt the agarose. The terminal peptides were removed by adding NaOH to 0.3 N and heating to 37 ° C for 90 min (Hay, et al., 1984). The solutions were then cooled to room temperature, then Tris pH 7.4 (to 0.1 M) and HC1 (to 0.3 M) were added to neutralize the NaOH. The fragments were heated to 56 ° C for 20 min and slowly (1 h) cooled to room temperature to facilitate reannealing. The DNA was then purified on a Qiaquick column and ligated to a Spel linker (New England Biolabs) at 16 ° C for 4 hours using a Pharmacia T4 ligase reaction (New England Biolabs). The ligase was inactivated by heating to 70 ° C for 10 minutes, excess linker was removed (and an overhang complementary to Spei was formed) by digesting with Spei restriction endonuclease for two hours. The DNA fragments were again purified by Qiaquick column chromatography and ligated to p327SpeI treated with Spei / HindiII / calf alkaline phosphatase. The ligation product was transformed into the DH5alpha bacterial strain and plasmid clones were identified which either contained the 1601 bp left end fragment or the 959 bp right end fragment (both released by Spel / Hindlll digestion). A DNA sequence analysis was carried out to confirm the terminal 300 bp of both fragments.

c) Cloning both CELO ends on the same plasmid

The 1601 bp left end fragment and the 959 bp right end fragment were cut out of their vectors by Hindi / SpeI digestion, separated by gel electrophoresis and purified by Qiaquick chromatography. The two fragments were mixed in approximately equimolar amounts and ligated for 30 minutes using the Pharmacia T4 ligase reaction. An aliquot of Spel / CIP treated p327SpeI was added and the ligation was continued for 4 hours. The ligation mixture was transformed into DH5alpha and plasmid clones were identified which carried the correct double insert (pWü # 1 and pWü # 3).

The second Hindlll site was removed by cleaving pWü # 3 with Clal and BamHI, treating with Klenow enzyme, religating, transforming DH5alpha and selecting a clone that contained the Clal / BamHI (which had contained a Hindlll site). The resulting plasmid, designated pWü-H35, now contained a single HindIII site between the left and right CELO end fragments.

d) Cloning of the entire CELO genome

The plasmid pWü-H35 obtained in c) was treated with HindIII and CIP and purified on a low-melting agarose gel, followed by Qiaquick chromatography. The linearized vector pWü-H35 was mixed with 0.3 ug purified CELO virus DNA, then 30 μl of electrocompetent bacterial strain JC8679 (Gillen, et al., 1974; Oliner, et al., 1993) were added to the DNA mixture on ice. Ten minutes later, the mixture was pulsed into a BioRad electroporation chamber and with an electrical charge of 2.4 kV (BioRad Gene Pulser; Oliner, et al., 1993). The bacteria were then plated on LB ampicillin plates and the ampicillin-resistant colonies examined for their plasmid content. The recombination between the terminal CELO sequences on pWü-H35 and the ends of the genomic CELO DNA restores the circularity of the linearized plasmid and allows growth on ampicillin. A plasmid containing the full length of the CELO genome was identified and this plasmid, designated pCEL07, was used for subsequent studies. The characterization of plasmid-cloned copies of the CELO virus genome is shown in FIG. 2C. Plasmid DNA from clones of the designation pCEL07, 8, 9 and 13 or DNA isolated from purified CELO virus was digested with either BglII (lanes 2-6) or Hindlll (lanes 7-11) and separated on a 0.6% agarose gel, and the DNA was visualized using ethidium bromide staining. The molecular weight marker (lanes 1 and 12) was bacteriophage λ-DNA cut with HindIII and EcoRI. The sizes of some molecular weight fragments (in base pairs) are shown on the right in the figure. For each enzyme, the two final CELO fragments that are bound to the bacterial plasmid during cloning (and therefore are not released after restriction digestion) are shown on the left in the figure (in base pairs). These are the fragments with 5832 and 5102 bp with Bglll, and 1601 and 959 with Hindlll.

The construction of pCEL07 is shown in Fig. 5. e) Initiation of a CELO virus infection from a cloned CELO genome

pCEL07 was digested with SpeI (which cleaves at the sites flanking the adenovirus termini), extracted with phenol / chloroform and passed through an HBS-equilibrated gel filtration column (Pharmacia Nick column) to remove contaminants. The digested DNA was then in streptavidin-polylysine / transferrin-polylysine / biotin adenovirus

(UV / Psoralen inactivated) incorporated, as described in WO 93/07283. Complexes containing 0.5 μg SpeI-cleaved pCEL07 plus 5.5 μg carrier DNA (pSP65; Boehringer Mannheim) were used to transfect primary embryonic chicken kidney cells (the complexes contained 4 mg DNA per 180 cm ² bottle, containing approx. 3 x 10 Cells), and the cells were examined for the cytopathic effect caused by virus replication. Five days after the transfection, when most of the transfected cells had rounded and detached from the plate surface, the cells were collected by centrifugation and the CELO virus was purified as described by Cotten, et al. , 1993. The virus yield from the plasmid-cloned CELO virus is comparable to the yields when using pure CELO virus DNA

(purified from virions) can be obtained.

Example 2

Production of a CELO mutant, which lacks the sequences from nt 35,870 to 42,373 at the right end

There are no identifiable viral structural genes beyond the two fiber genes with which L5 polyadenylation site at position 31771. (There is a cryptic VA gene at position 39.841 to 39.751.) It was therefore examined whether the sequences between approximately 32,000 and the right ITR are necessary for the virus to grow in cell culture. For this, a cluster of seven Asel positions at positions 35,870, 36,173, 38,685. 38.692, 39.015, 42.348 and 42.373, which does not occur anywhere else in the CELO virus genome. pCEL07 was digested with Asel, religated, and a plasmid lacking the inner Asel fragments was identified and named pALMCELO_35870-42373. In this connection it should be noted that the plasmid vector also has an Asel site; however, this is in the ampicillin resistance gene, and selection for ampicillin resistance requires that all positive colonies have at least the two fragments that carry the right and left halves of the amp gene.

In order to simplify further manipulations of the virus with the missing left end of the genome, pALMCELO_35870-42373 was digested with Asel (which cuts once in the ampicillin resistance gene of the plasmid and once at position 35.870) and ligated to a linker oligonucleotide TACCCTTAATTAAGGG, which is used for an interface encoded for the restriction endonuclease Pacl and for ends that are complementary to those formed during the Asel digest. Religion followed by selection for ampicillin resistance identified plasmids that did not integrate the oligonucleotide at the Asel site of the ampicillin resistance gene. Restriction digestion identified a plasmid that carried a PacI site at the former Asel site of CELO at position 35,870. This plasmid was named pALMCELO_35870-42373P.

Example 3 Production of a CELO virus vector in which a fiber gene is missing, which is replaced by a gene of interest

The CELO fiber genes are contained on a Hindi I I fragment which extends from nt 27,060 to 33,920 (the HindIII B fragment, cf. the restriction map in FIG. 1B). On this fragment, the sequence coding for fiber 1 extends from nt 1,054 to 3,435. The 5H3 fragment was digested with Bglll (which cuts at nt 1,168) and Hpal (which cuts at 3,440), the Bglll end was filled in with Klenow enzyme and attached to a blunt CMV / luciferase / β-globin

Cleavage / polyadenylation signal fragment from plasmid pCMVL (Plank et al., 1992) ligated to form plasmid p5H_28227-30502 (luc) which lacks almost all of the fiber 1 sequence which is replaced by a luciferase expression unit.

The relevant restriction interfaces in CELO are as follows:

Bglll A'GATCJT

Cuts at: 0 5102 15979 23472 28227 37972 43804

Size: 5102 10877 7493 4755 9745 5832

Hindlll A'AGCTJT

Cuts at: 0 1601 5626 17881 23327 27060 33920 38738 42845

42845 43804

Size: 1601 4025 12255 5446 3733 6860 4818 4107

959

Hpal GTT'AAC

Cuts at: 0 5503 20673 23355 30502 43804

Large: 5503 15170 2682 7147 13302 Notl GC'GGCCJGC

Cuts at: 0 17389 43804

Size: 17389 26415

Xbal T'CTAG_A

Cuts at: 0 1659 1988 28608 39268 41746 43804

Gbxße: 1659 329 26620 10660 2478 2058

The modifications made to p5H_28227-30502 (luc) were introduced into the entire CELO genome in the following manner: The CELO / Luciferase / CELO fragment was excised from p5H_28227-30502 (luc) as a Hindi II fragment. This fragment was generated with the 26kbXbaI fragment (CELO nucleotides 1988-28608) and the terminal Hpal fragments derived from pCEL07 (obtained by cutting with hpal containing the left end of the CELO virus and pBR327 sequences defined by the Hpal Positions) recombined. The three DNA fragments (approximately 50 ng each) were mixed in water and electroporated into JC8679 cells as described above.

Example 4

Introduction of a reporter gene (luciferase) into the CELO genome

i) Preparation of a left end fragment containing a CMV luciferase construct

The EcoRI fragment of the designation 7R1 (nucleotides from positions 79 to 8877) was cloned into a pSP65 derivative of the designation pAAALM (described in WO 95/33062). The plasmid was in the DAM methylase negative bacterial strain JM110 was transformed to allow clal sites in the fragment to be cleaved. The plasmid was purified, cut with Clal (at position 1083) and Ncol (at position 4334), treated with Klenow enzyme to fill in the overhanging ends, and attached to a blunt CMV / luciferase / β-globin cleft / polyadenylation signal (Plank et al., 1992). The resulting plasmid was named p7Rl__1083- 4334Luc.

ii) Recombination of the luciferase link end fragment into a complete (full length) CELO sequence

The plasmid p7Rl_1083-4334Luc was cleaved with Eco47 III, which cleaves at the CELO nucleotides 937, 1292, 2300 and 8406 (the sites at nucleotide 1292 and 2300 are missing in p7Rl_1083-4334Luc) to a large fragment containing the sequence

CELOnt937-1083 / CMVLucPA / CELOnt4334-8406. This fragment was recombined in pCEL07. pCEL07 was cleaved at the only Pmel site at CELO nt7433 and extensively dephosphorylated with calf intestinal phosphatase. The linearized pCEL07 was mixed with an approx. 3 to stache molar excess of CELOnt937-1083 / CMVLucPA / CELOnt4334-8406. The mixture was electroporated into the JC8679 bacterial strain and ampicillin resistant colonies were examined for plasmids containing the desired recombinant DNA. The correct plasmid was identified, characterized by restriction enzyme analysis and designated pCELOLucI.

iii) A luciferase expressing CELO virus was made by transfecting pCELOLucI into primary embryonic chicken kidney cells as described above. Example 5

Preparation of a CELO vector from a copy of the CELO virus genome contained on a plasmid

The region between the Dralll site (originally contained at nt 34,426 in the CELO virus genome) and the Xhol site (originally contained at nt 36,648 in the CELO virus genome) was derived from the plasmid pAALMH3, which contains the HindiII fragment from nt 33,920 to nt 38,738, cloned in pAALM, contains, removed. It was then treated with T4 DNA polymerase to produce blunt ends and ligated with the CMV / luciferase / β-globin fragment (see Example 4). The plasmid p7H3Δ 34426-36648 Luc was thus obtained. The CELO / Luciferase / CELO fragment was cut out on a Hind3 fragment and inserted into the CELO genome of pCEL07 by recombination via the unique Fsel site at position 35,694. This resulted in the plasmid pCELOΔ 34426-36648Luc. Digest with Spei and transfection into embryonic chicken kidney cells revealed a virus CELOΔ 34426-36648Luc. Subsequently, further insertions were made to replace the luciferase sequence with other genes of interest, using the unique Pacl site introduced with the luciferase sequence.

Figure 6 shows the cloning strategy used in this example in general form: a small CELO fragment is subcloned into a plasmid (containing restriction site C); the

Restriction sites A and B, which now occur only once in this plasmid, are used to replace the sequence with foreign DNA. The next step is the entire fragment, containing the foreign DNA between CELO sequences cut out of the plasmid and mixed with the plasmid which contains all of the CELO DNA and which was cut with a restriction enzyme (D) which cleaves the CELO DNA only once. This mixture is used to transform bacteria (for example from strain JC8679; Oliner et al., 1993; or another strain of Bakery with a similar ability to recombine); Recombination provides the desired plasmid, containing the foreign DNA as an insert in the CELO virus genome.

Example 6

Preparation of a quail cell line that complements the 7R1 deletions and / or the 9R1 deletions in CELO

The plasmids pX7Rl and pX9Rl (described in WO 95/33062) were introduced into primary embryonic quail kidney or liver cells by means of transfer infection, as described in WO 93/07283. Four days after the transfection, the cells were trypsinized and seeded at 1/5 of the original density. The cells were replenished with FCK medium twice a week. Clonal lines were expanded and clones carrying either the 7R1, 9R1 plasmid, or both plasmids were identified by PCR analysis. RNA expression from the integrated plasmids was determined by Northern analysis.

Example 7

a) Generation of a CELO virus genome with a mutation in the ORF794 dUTPase gene A plasmid called pWüΔdut was prepared by removing a 540 bp AfIIII-SacI fragment from the ORF794 in pWü-H35 (see Example 1 c). To prepare pCELOΔdut, pWüΔdut was linearized with HindIII and dephosphorylated using alkaline shrimp phosphatase. After gel purification, the DNA was mixed with purified CELO DNA and used to transform E.coli BJ5183 (Degryse, 1996) to ampicillin resistance. The DNA was extracted from the ampicilli-resistant bacterial colonies obtained and thus E.coli DH5a was transformed. DNA extracted from these bacteria was analyzed by restriction mapping to identify recombinant virus plasmids. The identity of the clones was determined by means of restriction mapping (FIG. 7; pWü-H35 is labeled "pWü" in the figure). The digestion of the wild type plasmid pWü-H35 with Hindlll and Spei gives fragments of 2944 bp, 1607 bp and 961 bp

(Lane 2). The deletion that changes the dUTPase converts the 1607 bp fragment into a 1071 bp fragment

(Lane 3; the modified fragments are marked with an asterisk). The plasmids containing the complete CELO coding sequence or the complete CELO sequence plus the dUTPase mutation were analyzed by Spel / HindIII digestion and showed the same change in the 1607 bp fragment to a 961 bp fragment (lanes 4 and 5) .

b) Production of recombinant CELO clones from chicken cells

Either 6 μg pCEL07 (see Example 1 d) or 6 μg pCELOΔdut (see above; digested with SpeI) were used to isolate primary embryonic chicken kidney cells (approx. 500,000 cells in a 2.5 cm well) using polyethyleneamine (PEI) / adenovirus Complex to transfect. The Qiagen-purified DNA was used for this Triton X-114 extracted to remove lipopolysaccharide as described by Cotten et al. 1994. Transfection complexes were prepared by diluting 6 μg of digested DNA in 250 μl 20 mM HEPES, pH 7.4. 20 ul 10 mM PEI (molecular weight 2,000, pH 7) were diluted in 250 ul 20 mM HEPES, pH 7.4. The PEI solution was added dropwise to the DNA solution

Allow to incubate for 20 min at room temperature and then mix with 1.5 μl of an adenovirus preparation (psoralen / UV-inactivated adenovirus type 5, cf. WO 1719, 1.5 × 10 ¹² particles / ml). After a further 20 min, the complex was applied to the cells in DMEM without serum (250 μl complex on 1.25 ml medium). The medium was changed to normal growth medium (with serum), and

4 to 5 days later, the cells were harvested, resuspended in 100 μl HBS and sonicated for 2 minutes. A 10 ul aliquot of this sonicate (virus in passage 1) was used to infect the same number of primary embryonic chicken kidney cells in a 2.5 cm well of a cell culture plate. After another 4 to

The cells were counted for 5 days to determine the cytophatic effect (CPE endpoint assay or plaque assay, Precious and Rüssel, 1985). The cells were harvested (virus in passage 2) as in the first step and used to infect fresh chicken cells; harvesting these cells resulted in 3rd passage viruses, the analysis of which is shown in FIG. 8.

c) Western blots

The virus-infected cells were harvested, resuspended in HBS and sonicated. Aliquots were mixed with 5x loading buffer (250 mM Tris-Cl, pH 6.8, 500 mM DTT, 10% SDS, 0.5% bromophenol blue, 50% glycerin),

Heated to 95 ° C for 3 minutes and then run over a 10% polyacrylamide gel. The proteins were transferred to nitrocellulose, blocked overnight in 5% defatted milk / TBST (10mM Tris-Cl, pH 7.4, 150mM NaCl, 0.05% Tween-20). Viral proteins were detected using anti-CELO antisera from rabbits (1: 1000) and anti-rabbit horseradish peroxidase (DAKO; 1: 20000) and visualized using ECL (Amersham). CELO virus (2.5 x 10 ¹² virus particles / ml) was used as a control. The rabbit serum was prepared using CsCl purified CELO virus and heat inactivated at 60 ° C for 30 minutes.

d) extraction of virus DNA

Virus infected cells were harvested and in 100 ul HBS / 0. 1% SDS / lg / ml proteinase K was added, incubated at 56 ° C. for 1 hour and extracted with phenol / chloroform. The DNA was precipitated with ethanol.

The analyzes performed showed that the recombination between the linearized plasmid pWüΔdut and the CELO DNA gave two types of plasmids. Recombination at the left end of the dUTPase mutation resulted in a wild-type CELO genome; recombination on the right side of the dUTPase mutation resulted in a CELO genome that carried the dUTPase mutation.

Infection of primary embryonic chicken cells with both CELO-DNA and CELO-Δdut-DNA showed cytopathic effects, which manifested themselves in the form of swollen, detached cells after 36 h, while control cells (treated with lysates from cells with an empty vector (Bluescript pBS, Stratagene) had been transfected) remained healthy in their morphology. Western blot analysis showed that CELO virus and CELO-Δdut virus produced by plasmid DNA are indistinguishable from wild-type virus grown in fertilized 9-day-old chicken eggs.

Overall, the experiments carried out in this example show that pCEL07 encodes a viable CELO virus genome. This DNA, after excision with Spei and transfection into primary embryonic chicken kidney cells, delivers infectious, passable virus. Lysates from 1st and 2nd passage viruses produce a cytopathic effect on primary embryonic chicken kidney cells. It should be noted that these lysates were produced by sonication, which excludes the possibility that the CPE in the secondary and tertiary infections can be attributed to the expression of viral genes which originate from residual plasmid DNA in the lysates; plasmid DNA is not expected to endure the method used to prepare the lysates. It was also found that with each round of infection there is a 100-fold amplification of the CPE-causing agent, which is in accordance with the amplification of a virus, but not with the simple passage of residual plasmid DNA from the first transfection.

The deletion of 540 bp in the CELO genome, with which sequences between an AfUli site at bp 609 and a SacI site at bp 1145 were removed and the open reading frame coding for dUTPase was destroyed, yielded a virus genome which was also found in primary embryonic chicken cells is viable. A virus gene was therefore identified with the UTPase gene, which is not required for growth in cell culture. Table 1

CELO virus sequences, published or from databases

REPLACEMENT BLAπ (RULE 26)

Table 2B Unassigned open reading frames, greater than 99 amino acid residues

Table 3 Recombinant adenovirus vaccines

REPLACEMENT BLAπ ₍ RULE 26)

REPLACEMENT SHEET (RULE 28) Bibliography

Akopian, T.A., et al. , 1990, Nucleic Acids Res. 18:

2825. (Corrigendum: Nucleic Acids Res 19: 424). Akopian, T.A. , et al., 1992, Mol. Gen. Microbiol.

Virol. 11: 19-23. Aleström, P., et al. , 1982a, J. Virology 42:

306-310. Aleström, et al. , 1982b, Gene 18: 193-197. Anderson, J., et al. , 1969a, J. Natl. Cancer Inst. 42

1-7. Anderson, J., et al. , 1969b, J. Natl. Cancer Inst. 43

575-580. Anderson, C.W., et al. , 1989, Virology 172: 506-512. Bailey, A. and Mautner, V., 1994. Virology 205:

438-452. Bennett, D.D. and Wrigth, S.E., 1987, Virus Res. 8:

73-7. Bett, A.J., Prevec, L. and Graham, F.L., 1983,

J. Virol. 67: 5911-5921. Both et al., 1993, Virology 193: 940-950.

Boulanger, P., et al. , 1979, J. Gen. Virol., 44:

783-800. Bridge, E. and Ketner, G., 1989, J. Virol. 63:

631-638. Brown, P.H., et al. , 1993, Oncogene 9: 791-799. Cai, F. and Weber, J., 1993, Virology 196: 358-362. Calnek, B.W. and Cowen, B.S., 1975, Avian Dis. 19:

91-103. Capado-Kimball and Barbour, 1997, J. Bacteriol. 106:

204-212 Caravokyri, C. and Leppard, K.N., 1995, J. Virol. 69

6627-6633. Cavanagh, D., et al. , 1988, Virus Res. 11: 141-50. Charlton et al. , 1992, Arch. Virol. 123: 169-179. Chartier, C, et al. , 1996, J. Virol. 70: 4805-4810 Cheng et al. , 1992, J. Virol. 66: 6721-6727. Chengalvalva, et al. , 1994, J. Gen. Virol. 75:

125-131. Chiocca, S., et al. , 1996, J. Virol. 70: 2939-2949 Chroboczek, J., Bieber, F. and Jacrot, B., 1992,

Virology 186: 280-285. Colby, W.W. and Shenk, T., 1981, J. Virol. 39:

977-980. Cosset, FL, et al. , 1991, Virology 185: 862-6 Cotten, M., et al. , 1993, J.Virol 67: 3777-3785. Cotten, M., et al. , 1994, Virology 205: 254-261 Cotten, M., et al. , 1994, Gene Therapy 1: 239-246 Cowen, B., et al. , 1978, Avian Diseases 22: 459-470. Cunningham, CH, 1975, Dev. Biol. Stand. 28: 546-62. Davison, AJ, et al. , 1993, J. Mol. Biol. 234:

1308-1316. Degryse, E., 1996, Gene 170: 45-50 Denisova, T.S., Sitnikov, B.S. and Ghibadulin, R.A.,

1979, Mol. Biol. (USSR) 13: 1021-1034. Descombes, P., and Schibier, U., 1991, Cell 67:

569-579 Deshmukh, D.R., et al. , 1974, at. J. Vet. Res. 35:

1463-4. Eloit et al., 1990, J. Gen. Virol. 71: 2425-2431. Estes, M.K. and Graham, D.Y., 1985, Adv. Exp. Med.

Biol. 185: 201-14. Everitt, E., et al. , 1973, Virology 52: 130-147. Fooks et al., 1995, Virology 210: 456-465.

Furcinitti, P.S., van Oostrum, J. and Burnett, R.M.,

1989, EMBO J. 8: 3563-3570. Fynan, E.F., et al. , 1993, DNA Cell Biol. 12: 785-9. Gallichan et al. , 1993, J. Infect. Dis. 168: 622-629. Gelderblom, H. and Maichle-Lauppe, I., 1982, Arch.

Virol. 72: 289-298. Ghosh-Choudhury, G., Haj-Ahmad, Y. and Graham, F.L.,

1987, EMBO J. 6: 1733-1739. Gillen, J.R., et al. , 1974, Mechanisms in Genetic

Recombination (R.F. Grell, ed.) Plenum, New York, pp. 123-135 Gooding, L.R., 1992, Cell 71: 5-7. Gouvea, V. and Schnitzer, T.J., 1982, Infect.

Immune. 38: 731-8. Gouvea, V., et al. , 1983, Virology 126: 240-7. Grable, M. and Hearing, P., 1990, J. Virol. 64:

2047-2056. Grable, M. and Hearing, P., 1992, J. Virol. 66:

723-731. Graham, F.L., 1990, Trends in Biotechnology 8: 85-87. Graham, F.L., et al. , 1975, Cold Spring Harbor Symp.

Quant. Biol. 39: 637-650. Graham, F.L., et al. , 1977, J. Gen. Virol. 36: 59-72. Green, M., et al. , 1967, Proc. Natl. Acad. Be. USA 57

1302-1309. Green, NM, et al. , 1983, EMBO J. 2: 1357-1365. Guilhot, C, et al., 1993, Oncogene 8: 619-624. Haffer, K., 1984, Avian Dis. 28: 669-76. Hanahan, D., 1983, J. Mol. Biol. 166: 557-580 Hertmann, I., et al. , 1979, Avian Dis. 23: 863-9. Hertmann, I., et al. , 1980, Prog. Clin. Biol. Res. 47:

125-32. Hess, M., et al., 1995, J. Mol. Biol. 252: 379-385. Hosokawa, K. and Sung, M.T., 1976, J. Virol. 17:

924-934. Hsu, et al., 1994, Vaccine 12: 607-612.

Huang, D.D., et al. , 1987, Avian Dis. 31: 446-54. Ignjatovic, J. and McWaters, P.G., 1991, J. Gen.

Virol. 72: 2915-22. Javier, R., Raska, K.J. and Shenk, T., 1992, Science

257: 1267-1271. Javier, R.T., 1994, J. Virol. 68: 3917-3924. Jia, W., et al., 1995, Arch. Virol. 140: 259-271. Johnson, D.C., et al. , 1988, Virology 164: 1-14. Jones, N., and Shenk, T., 1978, Cell 13: 181-188. Jones, R.F., Asch, B.B. and Yohn, D.S., 1970, Cancer

Res. 30: 1580-1585. Kalicharran et al. , 1992, Can J. Vet Res. 56: 28-33. Kawamura, H., Sato, T., Tsubahara, H. and Isogai, S.,

1963, Jap. Nat. Inst. Anim. Health quart. 3:

1-10. Keeler, C.L., et al. , 1991, Avian Dis. 35: 920-9. Kidd, A.H., et al., 1993 Virology 192: 73-84. Kodihalli, S., et al. , 1994, Vaccine 12: 1467-1472. Kozarsky, K.F. and Wilson, J.M., 1993, Current Opinion in Genetics and Development 3: 499-503. Küsters, J.G., et al. , 1990, Vaccine 8: 605-8. Larsson, S., Bellett, A.J. and Akusjarvi, G., 1986,

J. Virol. 58: 600-609. Laver, W.G., Younghusband, H.B. and Wrigley, N.G.,

1971, J.A., et al. , 1989, Mol. Immunol. 26: 7-15. Li, P., Bellett, A.J.D. and Parish, C.R., 1983,

J. Gen. Virol. 64: 1375-1379. Li, P., Bellett, A.J.D. and Parish, C.R., 1984a,

J. Gen. Virol. 65: 1803-1815. Li, P., Bellett, A.J.D. and Parish, C.R., 1984b,

J. Virol. 52: 638-649. Li, P., Bellett, A.J.D. and Parish, C.R., 1984c,

J. Virol. 65: 1817-1825. Maizel, J.V., et al. , 1968, Virology 36: 126-136. Malkinson, M., et al. , 1992, Arch. Virol. 127: 169-84. Mancini, L.O., et al. , 1970a, arch. For entire

Virus Research 30: 257-260. Mancini, L.O., et all, 1970b, Arch. For Entire

Virus Research 30: 261-262. Mangel, W., et al. , 1993, Nature 361: 274-275. Maniatis, T., et al. , 1989, Molecular cloning, A laboratory Manual, Second Edition. Cold Spring

Habor University Press, Cold Spring Habor, NY. Marshall, et al. , 1990, J. Infect. Dis. 162:

1177-1181. Mautner, V., 1989, Adenoviridae. in: Porterfield ed.

Andrewes' Viruses of Vertebrates. pp. 249-282.

London: Baillierre Tindall. McFerran, J.B. and Adair, B.M., 1977, Avian Pathology

6: 189-217. McCracken, R.M. and Adair, B.M., 1993, Avian adenoviruses. in "Viral Infections of Vertebrates"

(volume 3. Viral Infections of Birds) Eds. J.B.

McFerran and M.S. McNulty, Elsevior Scientific

Publishers. Amsterdam. Mittal, S.K., et al. , 1995, J. Gen. Virol. 76:

93-102. Moran, E., 1993, the FASEB J. 7: 880-885. Moran, E., 1994, Seminars in Virology 5: 327-340. Morrison, T, et al., 1990, Microb. Pathog. 9: 387-96 Moscovici, C, et al. , 1977, Cell 11: 95-103. Natuk, et al. , 1993, AIDS Res. Hum Retroviruses. 9:

395-404 Ni, Y., and Kemp, M.C., 1992, J. Gen. Virol. 73:

3107-13. Nicholas, R.A. , et al. , 1987, Arch. Virol. 96: 283-7. Oliner, J.D., et al. , 1993, Nucleic Acids Research 21:

5192-5197 Pieniazek, N.J., et al. , 1990, Nucl. Acids Res. 18:

1901. Plank, C., et al. , 1992, Bioconjugate Chemistry 3:

533-539 Precious, B. and Russell, W.C., 1985, Virology, ed.

Mahy, B.W.J., IRL Press, Oxford, Washington, DC,

193-205. Rao, L., et al. , 1992, Proc. Natl. Acad. Be. USA 89:

7742-7746. Raviprakash, K.S., et al. , 1989, J. Virol. 63:

5455-5458. Roberts, R.J. , et al. , 1986, A consensus sequence for the adenovirus-2 genome. (in) Doerfler, W. (Ed.);

Adenovirus DNA: 1-51; Martinus Nijhoff Publishing,

Boston. Ruley, H.E., 1983, Nature 304: 602-606. Sarma, P.S., Huebner, R.J. and Lane, W.T., 1965,

Science 149: 1108. Schnitzlein, W.M., et al. , 1988, Virus Res. 10: 65-75. Scott, S.D., et al. , 1989, J. Gen. Virol. 70: 3055-65. Shafren, D.R. and Tannock, G.A. , 1991, J. Gen.

Virol. 72: 2713-9. Sheppard, M. and Trist, H., 1992, Virology 188:

881-886. Shinagawa, M., et al. , 1983, Virology 125: 491-495. Stouten, PFW, et al. , 1992, J. Mol. Biol. 226:

1073-1084. Tagaya, Y., et al. , 1989, Embo J. 8: 757-764. Taylor, J., et al. , 1995, Vaccine 13: 539-549. Traenckner, E., B-M. , et al. , 1995, Embo J. 14:

2876-2883. Trapnell, B.C. and Gorziglia, M., 1994, Current

Opinion in Biotechnology 5: 617-625. Treanor, J.J., et al. , 1991, Vaccine 9: 459-501. Tripathy, D.N. and Schnitzlein, W.M. , 1991, Avian

Dis. 35: 186-91. Van den Ende, M., Don, P. and Kipps, A., 1949,

J. Gen. Microbiol. 3: 174-183. Van der Eb, A.J., et al. , 1980, Cold Spring Harbor

Symp. Quant. Biol. 44: 383-399. Vrati, S., et al. , 1995, Virology 209: 400-408. Wagner, E., et al. , 1992, Proc. Natl. Acad. Be.

USA 89: 6099-6103. Weber, J.M., 1995, Current Topics in Microbiology and

Immunology 199/1. eds. W. Doerfler and P. Böhm.

Springer publishing house, Heidelberg-Berlin. Weber, J. and Anderson, C.W., 1988, J. Virol. 62:

1741-1745. Webster, A., Hay, R. and Kemp, G., 1993, Cell 72:

97-104. Webster, A., Leith, I.R. and Hay, R.T., 1994,

J. Virol. 68: 7292-7300. Weinberg, D.H. and Ketner, G., 1983, Proc. Natl. Acad.

Be. USA 80: 5383-5386. White, E., 1994, Seminars in Virology 5: 341-348. Wold, W.S.M. and Gooding, L.R., 1991, Virology 184,

1-8. Yates, V.J., and Fry, D.E., 1957, Amer. J. Vet.

Res. 18: 657-660.

Zheng, et al. , 1993, Vaccine 11: 1191-1198.

SEQUENCE LOG

(1) AU-GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Ingelheim International GmbH

(B) STREET: Binger Strasse 173

(C) LOCATION: Ingelheim am Rhein

(E) COUNTRY: Germany

(F) POSTAL NUMBER: 55216

(G) TELEPHONE: 06132/772282 (H) TELEFAX: 06132/774377

(ii) NAME OF THE INVENTION: CELO Virus (iii) NUMBER OF SEQUENCES: 39

(iv) COMPUTER READABLE VERSION:

(A) DA'l'i-NlKAGER: Flqppy disk

(B) COMPUTER: IBM PC compatible

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

LOCUS AAU46933 43804 bp DNA

GENBANK ACCESSION No: U46933

SOURCE Chickens Adenovirus CELO. ORGANISM chickens adenovirus CELO virus; dsDNA virus;

Adenoviridae; Aviadenovirus.

JOURNAL J. Virol. 70 (5), 2939-2949 (1996) TITLE The complete DNA sequence and genomic organization of the avian adenovirus CELO

AUTHORS Chiocca, S., Kurzbauer, R. , Conductor, G. .

Baker, A. , Mautner, V. and Cotten, M.

CHARACTERISTICS Location / Qualifiers

Source 1..43804

Organism chickens adenovirus CELO strain "Phelps (ATCC VR-432)" CDS 794. , 1330

Note: ORF1

Translation: MDPFGSSSVPPCSTSDLPEPKLYFVRLSPHA

VPPVRATHGAAGYDLFSAYDIKVPARGRALVPTDLVFQFPPGC

YGRIAPRSGLAAKFFIDVGAGVIDPDYRGNVSWLFNFSESSF

NIRRGDRVAQLILERIMVPELSELTQLGETDRGASGFGSTGMG

AVDRNQRSVLEWLTPGSR

CDS Complementary (1191..1514)

Note: ORF15

Translation: MVASCHTLTIIPKEARSNCYRAYSRASCWCC LRTDNVRMCRRPPQNLLASVQRSRLRRKGPINGNQGSAIPTQS ADCGLQHPYLWTRNPTPRGLSRLAASVPTAPEP

CDS 1999..2829

Note: ORF2

Translation: MSRESERYWTLVHALIDRGWSREQWQMVDP

AQYQFYHRSKQRGFKVRHILRDVIRHMCWSRTLLDYMSSASTP

SPDDVLRNPLYQLLLCNGYNPAWGTALIRWAGHQSNRNTVWI

RGTPMSGAPYLAQAIAYCSPLVGSVDWRNKSNPFEGCPDSLVF

WWDGGYVYDCCVGLVKQVFRGEHVILPPEGLRGPNPCSELFRT

PVLMYSQADICMTRLRSGELSAEHAVGLRDCMYLIRLTEDFDC

AGGISCADVKQFVAWSREHPGEVRETHELK

CDS Complementary (2892..3374)

Note: ORF14

Translation: MYPFKHSPHCITDEECDLQLRSFCSWIRVIE MRCTDWTIQYICSCETPRSLFCLSLIRVLTAHWAKTWNFVAQ HDHQPQLPLNLILYTYATHCRLCNLNPALEQIYTAVTVARRQG AYTRLEGQTLYVVRLACPFYHLVCLPRPIVNH

CDS Complementary (3549..4568)

Note: ORF13

Translation: MTTTPCALSYARTKECSVPARGPPHAMLVTH HMTYNSLPQCTKRRRESQSSLSSEEEQIASCIPDTPSPCLFPS TSPMDQLVERLFVEGVAHEVQWNFPSKNLIPTYERERVLEALK ERFGPGQSLINQLPSEEPDTLKAAFYNVCDNWFHQMMEAEGYE GKVAANAILRWLRGELNTLVLCGGRLSNAKSLFNALCACFPLA ISDSRINSILSLGEIAPHASLYCLPFVDEKPDPLMLHFMEGNA ATCRLNKKTFHIPSTPMLIHCADLSLANEFTARNTWFFLTGD HTKTPPCYHPRKELRDFVANAAACACLMTLHCKRDNKLCNPCI RTPLQNQ CDS 3781. , 4095

Note: ORF3

Translation: MGVEGMWNVFLFSLQVAALPSIKCSINGSGF

SSTKGRQYREAWGAISPSDSMELIRLSEIASGKHAHKALKRLL

ALESLPPQSTRVFSSPRSHRRMALAATFPS

CDS Complementary (4462..5094)

Note: ORF12

Translation: MLEAEGYNAPVAIYAIYLWMSAMSISRLCHY TNTLYWGEPSSAADIFTASILRLFQFVLTANINAFDFGQYAR QQDLVKMLYFPCTAHCNTFKDPVANQLLKGRSFTTMTRDGLVD ISEKKCLVRLYQLPHFFFPTGDHLIFI

CDS Complementary (5366..6685)

Product: IVa2

Translation: MSTQIPARQETYDPSQSSGTKTPSHPYDGNP TRSYPKRNAGKFTTYSSQMIAPRKRKAWEYEEEEYEASRDFYQ RVTSWYDGAVDLAPQLFREQHFPSYDEFYSLGGVNEKFLEAHE EVKAQEQMDSRYLQHGQLPSINMGKQPIIGVIYGPTGSGKSHL LRALISCNMLDPIPETVIFITPEKNMIPPIEQTSWNLQLVEAN FDCREDGTIAPKTSTFRPEFMEMTYEEATAPEHLNIDHPDNIY VKVSKRGPVAIIMDECMDKLCSGSSVSVLFHALPSKLFARSAH CTAFYIFWLHNMAPRTAIGNVPTLKVNAKMHILSCHIPQFQF ARFLYAFAHNISKDLWLLKAYFSFLQQNQRFSWVMYTPDPVS ESFRWCSIDQQYSIIPLNVNIQERFLKTAKSIIKFSETHRKQL ERNPKLTDLEKLSPPGTFQET

CDS 5963.-6373

Note: ORF4

Translation: MVDVEMFGCGGLLVSHLHKFGTERACLRGDG

AVFPAVEIGLDQLQVPGRLFDGWNHVLFRSDEDDRFGDRVQHV

ARDERPQQMRLAGSGGSVDDPDDGLLAHVDGRQLSVLEVATVH

LFLGFNFFVGFEKLLINAP CDS Complementary (6501..9866)

Product: E2b pol

Translation: MLIAKNVTGEWVWITSRTPVQQCPTCGRHWV RRHSCNERRSAFYYHAVQGSGSDLWQHVHFSCPAQHPHIRQLY ITYDIETYTVFEKKGKRMHPFMLCFMLSGDPQLVSRAERLARQ DDRLKALDEGFYWLDSHPGEVARRFRNFRSRLQIEFAQNLVDR YAAANRDYCDQLVKDGKYGSVHKIPYELFEKPTSPLSLPDNFY SVDIWLGHNICKFDELLLATELVERRDLFPEACKCDRSFMPR VGRLLFNDIIFRMPNPNYVKKDASRVERWSRGIVSHQDARSVF VRFMVRDTLQLTSGAKLSKAAAAYALDLCKGHCPYEAINEFIS TGRFHADADGFPVERYWEDPSVIAEQKNLWQKEHPGQQYDIVQ ACLEYCMQDVRVTQKLAHTLHDSYDAYFQRELGMEGHFNIFVR PTIPSNTHAFWKQLTFSNYVREQRATCPPSVPEPPKKKGRTKK KKQPSPDYVAEVYAPHRPMFKYIRQALRGGRCYPNVLGPYLKP VYVFDICGMYASALTHPMPHGMPLDPKFTAQHVEELNRLLTNE SHLSYFDARIKPSILKIEAYPPPPEMLDPLPPICSRRGGRLVW TNEALYDEWTVIDILTLHNRGWRVQVLHDEMNIVFPEWKTLC ADYVTKNILAKEKADREKNEVIRSISKMLSNALYGAFATNMDT TRIIFEQDLSEADKKNIYEGTEIVKHVTLLNDDSFNGTEVTLE NAPNPFSEESLRQQFRYADDPEQEEPEAEEDGEEEGDDSDRES ARKPKNALTEDDPLVAVDLEVEATLATGPYIPEGELSSAHYAR ANETRFKPMRLLEATPEALTVLHLESLDKQVANKRYATQIACF VLGWSRAFFSEWCDILYGPDRGVHILRREEPRSLYGDTDSLFV TETGYHRMKSRGAHRIKTESTRLTFDPENPGLYWACDCDIKCK ACGSDTYSSETIFLAPKLYGLKNSICVNEQCRTV GPGKIRSKG HRQSELIYDTLLRCWRRHEDVQFGAQSNIPELHTRRTIFKTTL LNKVSRYDPFTIHNEQLTRVLRPWKDLTLYEHGDYLYPYDNEH PNPRTTGDVRPVPIVGHEDPLAPHDLEWFGVVQQ

REPLACEMENT BLAπ (RULE 26) CDS Complementary (10269..11996)

Product: E2b pTP

Translation: MQLRDLAPRSPNVAAPPYNGLPPPHLLLGYQ AMHRALNDYLFDNRVFMQIGYDSPPQRPRRLFWTCLTDCSYAV NVGQYMRFLDLDNFHGTFTQMHNAVLMDRVAADMGRAHLRGRG IDVGRHGQVLPQLDAEHHSLLSGNGAGGLQEGVLMRTASAADA ELLAAIRQLRVALCHYLFCYAYDLFQTEERYRFLPGSDVFLEP NWLSYFAEAFAELDTQQLVRDAERKFRGRRDVEEPTETMARCF MSTLASDAVSLAGTGLSGGAITLCSRRVTDRTGLRPRDRHGRA ITASEARRIRPRAVRAFVDRLPRVTRRRRRPPSPAPPPEEIEE AAMEVEEPEEEEEELLDEVIRTALEAIGALQDELSGAARRHEL FRFANDFYRMLLTARDAGLMGESFLRKWVLYFFLAEHIASTLY YLYSHFIANREFRRYVDVLTLQVLWGWDVNAQQVFKRIWSEQ SNPATIFETLWERILRDFLMMVERTGQFEGMDDADQQLFLSDI QYRDRSGDIEEVLKQLNLSEELIDSIDISFRIKFKGIVAIATN EEIKANLRRVLRHRREDIEAAARRGQPL

CDS 12193..13329

Gen: Ll

Product: Ll 52K

Translation: MHPVLQSVRNASVSAGGPHQQQPQQQQHGVS SVRRPPSPPRYPAQHAYPGAGATPTAGRGDFDGALDPDEGPVA CGLAAGAGVDEVRMRERDAARRATVPEINLFKARRDWPNGDY ERDLMYHSGQAIDIDRQRVLTPEDFKGSEPAFTPAVNHMRAAE LKRAAEQTAFGEELRNTCHQTRIRTALLRPEIGAGIYYLYDFV QTYLEHPDGRVKLNPQLVLVAQHAGNTMLAQRLWAIAEEKNAW LRDLIEMAYMIVNDPYLNTEQQLSAICTTWELSMKYAKLAAK NGYPSMAQMAKAQEFFYRVMQAVLDLGVQVGVYNNRPARYRQK RMSEIPQMTDAEYMFGLTQALESRPPQGESFADEGPSESDDED DFI Gen 12193..15043

Gen: Ll

CDS 133 16. . 15043

Gen: Ll

Product: Ll purple

Translation: MTSSDTFLALAPYGRQEVADALSSLPDGKDA RSLRHAPYANRLIKLQSAMVPPKVDGTSERVAEIVKGLAEQGA IYPDQMGAIHSDLLNRAYTWNSMGVQESIQALVNDVIHGQNRT LQDELARTKEIANASLLTQFFDSLYKTVDRGQRNFEGFKKLLR LFVNNVPNAEVYGSSGSFSVQINLGGSSQNINLTNAFENLKPI WGARWDAVNNPRIGALLTPNTRALLFFVSSFYDYGAMEPGSYL DNIMRLYKEAIRADVDAEGDAIMELGEAGANLNLRFNDYKDTL NYLLQNREWPDTAPLELSAEQEMLLKYLMRQLRQALKDGVPA DISISTMTQYLDPRLYQTNKVFVEKLQNYLLAAQARNPVYYRL LVLDPNWRPPAGLYTGNYVIPDRYDFEDVQSELEYAGPSRDEY FDDSLFAPGPQRRLNSAEEAQLERDIESLTGHIDEELGVQSQA GWLADHRLPVAFDGALΞLTERNAYNTPLPPDSHMRSRSSSVAS DLGLLNLSGTGGPGFFASLRPSIGSRQPTGTAVGLRPTTPYSG SGCMRGTGLARKVLNPAASRRGRKLRFY

Special feature 15080

Note: L2 region penton base splice acceptor site

CDS 15110..16657

Gen: L2

Product: Penton base

Translation: MYRSLRPPTSIPPPPPSGPSPYPAMINGYPP DVPVGSPANGDAELFVPLQRVMPPTGGRNSIRYRNYAPCQNTT KFFYVDNKLSDLDTYNEDANHSNFRTTVIHNQDLDPSTAATET IQLDNRSCWGGELKTAVKTNCPNISSFFQSDTVRVRLMSKRDP GGTDPDAGVNNPPGAEYKWYDLRIPEGNYALNEIIDLLNEGIV QLYLQEGRQNNVLKSDIGVKFDTRYLDLLKDPVTGLVTPGTYV YKGYHPDIILLPGCAVDFTFSRLSLLLGIAKREPYSKGFTITY EDLQGGNVPALLDLSSVQVDDQDEDVIWADARPLLKDSKGVS YNVITTGVTQPQTAYRSWLLAYHTLDSPARNKTLLTVPDMAGG IGAMYTSMPDTFTAPAGFKEDNTTNLCPWAMNLFPSFNKVFY QGASAYVQRLENATQSATAAFNRFPENEILKQAPPMNVSSVCD NQPAWQQGVLPLKNSLSGLQRVLITDDRRRPIPYVYKTIATV QPRVLSSSTLQ

Gen 15110..17495

Gen: L2

REPLACEMENT SHEET (RttaüL 26) CDS 16679..16897 Gen: L2

Product: L2pVII

Translation: MSILISPSDNRGWGANMRYRRRASMRGVGRR RLTLRQLLGLGSRRRRRSRPTTVSNRLVWSTRRRSSRRRRR

CDS 16929..17495

Gen: L2

Product: L2 mu (pX, 11K)

Translation: MCAVAIHRSDWMPSVLLTGGRTAKGKKRAS RRRVKVPKLPKGARRKRASVTPVPTVATATASERAALTNLARR LQRGDYAAWRPADYTSPAVSEAARAAASSGTPATARDLATGTL ARAVPMTKTGATIFGKI

CDS 17559..18230

Gen: L3

Product: L3 pVI

Translation: MDYAALSPHLGGWALRDHHIGDSSLRGGAIN WGNLGSRITSALNSTGRWLYNTGNRFVHSNTFNQIKQGIQDSG VIRNVANLAGETLGALTDIGRLKLQQDLEKLRRKALGEEGPAT QAELQALIQALQAQVAAGEPPAAPAAPAPAPPLVPTTRPIPEM VTEVKPPVTSSAPAVPVDVPTTLEMRPPPPKRRRKRARPGQWR ARLDSLSGTGVATATRRMCY gene 17559..21754

Gen: L3 special trait 18261

Gen: L3 Remark: hexon splice acceptor site

ERSATZBLRI i (KLÜLL 2Ö) CDS 18289..21117

Gen: L3

Product: L3 hexon

Translation: MTALTPDLTTATPRLQYFHIAGPGTREYLSE DLQQFISATGSYFDLKNKFRQTWAPTRNVTTEKAQRLQIRFY PIQTDDTPNSYRVRYSVNVGDSWVLDMGATYFDIKGVLDRGPS FKPYGGTAYNPLAPREAIFNTWVESTGPQTNWGQMTNVYTNQ TRNDKTATLQQVNSISGWPNVNLGPGLSQLASRADVDNIGW GRFAKVDSAGVKQAYGAYVKPVKDDGSQSLNQTAYWLMDNGGT NYLGALAVEDYTQTLSYPDTVLVTPPTAYQQVNSGTMRACRPN YIGFRDNFINLLYHDSGVCSGTLNSERSGMNVWELQDRNTEL SYQYMLADMMSRHHYFALWNQAVDQYDHDVRVFNNDGYEEGVP TYAFLPDGHGAGEDNGPDLSNVKIYTNGQQDKGNWAGTVSTQ LNFGTIPSYEIDIAAATRRNFIMSNIADYLPDKYKFSIRGFDP VTDNIDPTTYFYMNRRVPLTNWDLFTNIGARWSVDQMDNVNP FNHHRNWGLKYRSQLLGNSRYCRFHIQVPQKYFAIKNLLLLPG TYTYEWVLRKDPNMILQSSLGNDLRADGAQIVYTEVNLMANFM PMDHNTSNQLELMLRNATNDQTFADYLGAKNALYNVPAGSTLL TINIPARTWEGMRGWSFTRLKASETPQLGAQYDVGFKYSGSIP YSDGTFYLSHTFRSMSVLFDTSINWPGNDRLLTPNLFEIKRPV ATDSEGFTMSQCDMTKDWFLVQMATNYNYVYNGYRFWPDRHYF HYDFLRNFDPMSRQGPNFLDTTLYDLVSSTPWNDTGSQPSQD NVRNNSGFIAPRSWPVWTAQQGEAWPANWPYPLIGNDAISSNQ TVNYKKFLCDNYLWTVPFSSDFMYMGELTDLGQNPMYTNNSHS MVINFELDPMDENTYVYMLYGVFDTVRVNQPERNVLAMAYFRT PFATGNAV Special feature 21102

Gen: L3

Remark: protease splice acceptor site Special feature 21123

Gen: L3

Note: protease splice acceptor site

CDS 21134. . 21754

Gen: L3

Product: L3 protease

Translation: MSGTTETQLRDLLSSMHLRHRFLGVFDKSFP GFLDPHVPASAIVNTGSRASGGMHWIGFAFDPAAGRCYMFDPF GWSDQKLWELYRVKYNAFMRRTGLRQPDRCFTLVRSTEAVQCP CSAACGLFSALFIVSFDRYRSKPMDGNPVIDTWGVKHENMNS PPYRDILHRNQERTYYWWTKNSAYFRAHQEELRRETALNALPE NHV polyA_site 21767 21824 polyA_site polyA_site 21836 21882 polyA_site

CDS Complementary (21899..23224)

Product: E2a DBP

Translation: MERTPKRAHGFRSTKPVKRTAEVMMEEEEEE VEWAPGRGATRKKVSRREESPSPVRRVTRRRETWDDEENAS DEESPEAPLSDPWYGAQRAMATVASICEALDLQWQGASVRPD DSIWTKMGGTYVRKKHPEFRLTFSSYDSFNAQVGRFLAAVIYS RAGLEPKFVPGGAHVWRHGWFPALQEPFPKCMHGVDMVTKPRT VELNPSSEAGKRALAEQNGVIEKNRFGRQVWLRFDANAVCYK DQEHSGFPHPHAHGSCAMVFSDAAKAVSAMRHDIDWTKALYPN ADKRRAEECVLISTNCNCNYASDRAISGRQFCKMTPYKLNGTD DITRDMVESRPDMKAHKKNPHTMVFTCCNPQAASGGAGRGLKK TEKTCAWRLSAMDLRYAYVFATELFTAVMGSSEPTHVPEFRWN ESYAFKTEVLAPVSPIASDDPFA

Special feature 23608

Note: 100K splice acceptor site

Special feature 23649

Note: 100K splice acceptor site

CDS 23680..26634

Gen: L4

Product: L4 10OK

Translation: MADKITREEKTIATLDLVLRVWDAGNWDVF SKRLVRYTREQYGIELPEDIGDLPDTSEVSKVLLSHLGEDKAV LSAYRIAELTQPSEMDRAKVTEGGLAVLNASRDESEAQNPSNP EPESIESDAVEDLGVAAESDPSDDEPDPEPEYDHREADHDSDA DSGYYSADGGRPGTPVDEEPQDDSPSSEETASTVIEEAQTSAS NDSHDDDTHRDDGSASEEDLERDALVAPADPFPNLRKCFERQA MMLTGALKDAADTADPPETLSVDSVQRQLERFVFNPDRRVPAE HLEVRYNFYPPFLTPKAIASYHIFAVTASIPLSCKANRSGSDL LAKAKESTFFKRLPKWRLGIEIDDGLGTEVTAVTELEEAKMVP LKDDVSRLQWAKMRGEHIRFFSYPSLHMPPKISRMLMETLLQP FADENQKAEEALPCLSDEEVLAIVDPTGRLHGEDALKAVEKRR AAVTMAVRYTATLELMERVFREPSMVKKMQEVLHHTFHHGFVA LVRETAKVNLSNYATFHGLTYNNPLNNCIMSKLLEGADKEDYV VDSIYLFLVLTWQTAMGMWQQAIDDMTIQMYTEVFTKNKYRLY SLPNPTAIGKAIVDILMDYDRLTEEMRKALPNFTCQSQITAFR HFLLERSNIPAVAAPFMPSDFVPLAYKQSPPLLWDQVYLLQLA FYLTKHGGYLWEAPEEEANNPSNRTYCPCNLCSPHRMPGHNAA LHNEILAIGTFEIRSPDGKTFKLTPELWTNAYLDKFDAEDFHP FTVFHYPENASRFASTLKACVTQSPEILSLIRQIQESREEFLL TKGKGVYKDPNTGETISRQPRDTARAQHAGDGQALPAPGAYTT GGNRAETAPAGAVRLAPDYQDGQFPIAKVGPHYHGPKNVRRED QGYRGGPGGVRGEREWLSRRAGGRRFGRRNTRQSGYNERANR YFGRGGGGSVRGQQGEHPTTSPSASEPPAPSRIL ARGTPPSPE RRDRQEE

Gen 23680.27886

Gen: L4

CDS 27149..27886

Gen: L4

Product: L4 pVIII

Translation: MNLMNATPTEYVWKYNPVSGIPAGAQQNYGA TIDWVLPGGTGFAIATNDIRRQTLNPAVTRAITARFEAESDQQ PYASPHETNVIAANVLDSGYPKSGLYPLELSGNQRVQLAGGLM VGRTEGRMQLAGGLTEGRVQLSGGFHGRPLVRGRSRRPPRWCG AELTGNGLPEQAEVTSDTYKYFLRTQGPSQWEEPGVFSQRQF MTTFLPSWPHPFDSTNPGDFPAQYSAIYKGRTAFEDTFWDW polyA_site 27920

Special feature 28315

Note: fiber splice acceptor site

Special feature 28341

Note: fiber splice acceptor site

Gen 28363..31768

Gen: L5

CDS 28363..30495

Gen: L5

Product: L5 fiber 1

Translation: MTSPLTLSQRALALKTDSTLTLNTQGQLGVS LTPGDGLVLNTNGLSINADPQTLAFNNSGALEVNLDPDGPWSK TATGIDLRLDPTTLEVDNWELGVKLDPDEAIDSGPDGLCLNLD ETLLLATNSTSGKTELGVHLNTSGPITADDQGIDLDVDPNTMQ VNTGPSGGMLAVKLKSGGGLTADPDGISVTATVAPPSISATAP LTYTSGTIALTTDTQTMQVNSNQLAVKLKTGGGLTADADGISV SVAPTPTISASPPLTYTNGQIGLSIGDQSLQVSSGQLQVKLKS QGGIQQSTQGLGVAVDQTLKIVSNTLEVNTDPSGPLTSGNNGL SLAAVTPLAVSSAGVTLNYQSPLTVTSNSLGLSIAAPLQAGAQ GLTVNTMEPLSASAQGIQLHYGQGFQWAGTLQLLTNPPIWS SRGFTLLYTPAFTVSNNMLGLNVDGTDCVAISSAGLQIRKEAP LYVTSGSTPALALKYSSDFTITNGALALANSGGGGSSTPEVAT YHCGDNLLESYDIFASLPNTNAAKVAAYCRLAAAGGWSGTIQ VTSYAGRWPKVGNSVTDGIKFAIWSPPMDKDPRSNLSQWLGA TVFPAGATTALFSPNPYGSLNTITTLPSIASDWYVPESNLVTY TKIHFKPTGSQQLQLASGELWAAAKSPVQTTKYELIYLGFTL KQNSSGTNFFDPNASSDLSFLTPPIPFTYLGYYQ Special feature 30511

Gen: L5

Note: fiber splice acceptor site

REPLACEMENT BLAπ (RULE 26) CDS 30536. , 31768

Gen: L5

Product: L5 fiber 2

Translation: MADQKRKLADPDAEAPTGKMARAGPGELDLV YPFWYQVAAPTEITPPFLDPNGPLYSTDGLLNVRLTAPLVIIR QSNGNAIGVKTDGSITVNADGALQIGISTAGPLTTTANGIDLN IDPKTLWDGSSGKNVLGVLLKGQGALQSSAQGIGVAVDESLQ IVDNTLEVKVDAAGPLAVTAAGVGLQYDNTQFKVTNGTLQLYQ APTSSVAAFTSGTIGLSSPTGNFVSSSNNPFNGSYFLQQINTM GMLTTSLYVKVDTTTMGTRPTGAVNENARYFTVWVSSFLTQCN PSNIGQGTLEPSNISMTSFEPARNPISPPVFNMNQNIPYYASR FGVLESYRPIFTGSLNTGSIDVRMQVTPVLATNNTTYNLIAFT FQCASAGLFNPTVNGTVAIGPWHTCPAARAPVTV polyA_site 31770

CDS Complementary (31812..32429)

Note: ORF22

Translation: MNDEQILEMVLQHQQRRQQEAEREEEVGDDM EDDEDDDGLQMPTPLHAYQLLCYDSFELHFGGCACHGLPLHRM GLSACHLAPSDLATYVWARLEDDLNVAGVYFVAMWASPGFSDF SPVFMQRPIGNVCGMLIHVDLHSRLPFLIAVSRLGEAGGSPCL YMRKIDVDLDTQRVHFYTEDWFSEFANLLYYWQMSEWKHLAER MQ

CDS Complementary (32735..33058)

Note: ORF21

Translation: MCTGSTGSLIPSVSDSANSVRRGNLSLCSVL LSWLICAMCLWNDARESLVNVRIANYVFDFAVLWTLLARVLGP PGRPVLQQHHPVQLPVPTEPSVFVKLCNQRVRL

CDS Complementary (32892..33707)

Note: ORF20

Translation: MERLNEYRINRAVASLRCFDNDLMRRLHSSV TVLVTVRSAKFVCFKRRDYVLMNCIVRIVSALHLNRAEKTALL HYLSRRLLFITPGMKYDLEPWMLARRKTDFKFFTTGFLIAEKI SVKMALRSMSFEVSFSQVPSSVPFVRSPWLMNACRVTVTATI MVETISRSSAVTQPVCLRSMLRVMVSPELWPIVSQGLCYFPGY RRLSYANVEEWVFHVHGKYGESHPECFGQCKQCSTRQPLΞLFC SAQLAYLRNVFMERRARVAGERPYS

REPLACEMENT BUTT (RULE 26) CDS 33030..33476

Note: ORF5

Translation: MRLPVLPVHMKYPLFDVSVGQPTVTGEVTQS LRHDRPQFRRHHHAEHASQADGLGHGAAARNSFHHDGGRHGHA TRIHENNRRPHKRNRRRHLRKGHLKAHRAESHLYGYLLRNQLKPGEKLHQQKPKKKLPRQK

CDS 33169..33483

Note: ORF6

Translation: MLLRQTGWVTALLREIVSTMMVAVTVTRHAF

MRTTGDRTKGTDEGTCEKDTSKLIERRAIFTDIFSAIRKPWK

NLKSVFLRASIHGSKSYFIPGVIKSNLRER

CDS Complementary (34238..35599)

Note: ORF19

Translation.-MRGFVPPTSSPDRGSKKVGRIVALDPPLESF QGYRMDLHTKGLNLLLSSGGHWSANRDADSVISRDDADYVWI ASSIGSYGFDRPIGDEYIRSDLTGQKHEACESRAWWKGQICAW SYSGRRHCEDVHIPFDFLRSDGLCYHIMAPLTFMKALDTHQAD QLLSMHGSVPSAWSAYVTGRDYSQPTQYYTEEVADWRMLLRED DMASSYLLLWTEGNAAELWTYDPYYTKTIGMEHGYSVRWYFI RDRNVGEAPIVLYARGGGVLKFIRLYKGRGTLTSLGARAMTTQ EVTEFTCFRTHTYYFTGTKKYDCHPGGHRFDVPRWRSHINVSA HHLPVPPKCGCLKFPKLFKDYVIFDHPNWGRAGEYVSLGPWS TGLQAWTFKPQPRRHRWLATYWDACSNTKRRVGIDVRTDRK NHMVWLKADKPVSREMWFVSEVDWRVYVTWLSPE

CDS Complementary (35536..36144)

Note: ORF18

Translation: MSALSSCFNGSDSRWDPPYPKADVRRLMGTY SPDFPSWPKLIVWWNETFLTFSDGPWWSQMRRLGVLDGKDSG ELIILVQDMYPDVCPLINRARYDGTYKWTSEMMRKILRMHTIM TPESPVILLDWDVALLTVGTQKV

CDS 35629..36024

Note: ORF7

Translation: MNSMVLELRKKMSSGPDCVIGRPPHILPPQK

GVYLLTNISQLIGPVQQNDRGLWRHNGMHTQNLSHHFTGPFIC

AVIARPINKRTHIGIHVLNQNNELPAIFTIQYPEPPHLTDNPG

AVRKSQKSLIPPYN

ERSATZBUπ (RULE 26) CDS 37391..38239

Note: ORF8

Translation: MARNPFRMFPGDLPYYMGTISFTSWPVDPS

QRNPTTSLREMVTTGLIFNPNLTGEQLREYSFSPLVSMGRKAI

FADYEGPQRIIHVTIRGRSAEPKTPSEALIMMEKAVRGAFAVP

DWVAREYSDPLPHGITHVGDLGFPIGSVHALKMALDTLKIHVP

RGVGVPGYEGLCGTTTIKAPRQYRLLTTGVFTKKDLKRTLPEP

FFSRFFNQTPEVCAIKTGKNPFSTEIWCMTLGGDSPAPERNEP

RNPHSLQDWARLGVMETCLRMSRRGLGSRHHPYHSL

CDS Complementary (38717..39256)

Note: ORF17

Translation: MPLYLCFGAAAPVSILWREELFWGFVAAVKR RWHTVYARTNVDIQYPMAYCVGIQSLSPCKCHVTVWCLTFLD LRMSAINEATKIMRAFFKTFFYHHGKVPRGRWFKLYRNDWCKD PNLTVGNYIVASSTALPLRSWARDRDEGLLVRGSKL

CDS Complementary (39286..39705)

Note: ORF16

Translation: MYYFHLRVTLMEPNLAVFHDLKLTVINAWES LTVEMLSHYSVDYLFRLEEFAGVYSASIFLPTHKVDWTFLKRA VALLRECIWRRFECTQVPRGVASIYAVRNTWTPSANRVARHFV KRGALVGMQPCLH

CDS 40037..41002

Note: ORF9

Translation: MEPPHNSPVPFSIAKMGNPTLLLLSGLLSLT

QAISIGEHENKTRHVIVWRHSSSHQCSDWRTVTEWFPPQKGNP

VRPPYTQRVSLDTANNTLTVKPFETNNGCWETTSQGINHPPTT

IQYRVWNITTTPTIQTINITKITVREGEDFTLYGPVSETMSII

EWEFIKDVTPQFILQYYLSINSTIVYASYQGRVTFNPGKNTLT

LKGAKTTDSGTYKSTVNLDQVSVHNFRVGVTPIEKKEEATAET

PASKPTPIPRVRADARSTALWVGLALCILTVIPALIGWYFRDR

LCVPDPIIELEIPGQPHVTIHILKGPDDDCET

REPLACEMENT BLAπ (RULE 26) CDS 41002..41853

Note: ORF10

Translation: MIDKRNKKAVTHISTCLCHSSIPIYGDSPFL NTHRAAMDPRPLVLLLLLASHISTFRQMYFEGETIHFPMGIYG NETTLYMNDIILEGTRANTTTRTISLTTTKKNAGTNLYTVISE TGHNATYLITVQPLGQSIHHAYTWAGNTFTLQGQVFEHGNYTR WVRLENAEPKLIISWALSNRTINKGPAYTANMDFDPGNNTLTL HPVLITDAGIFQCVIDQQTNLTLTINFTVSENPPIVAHLDIHK TISRTIAICSCLLIAVIAVLCCLRQLNVNGRGNSEMI

CDS 41958..42365

Comment: ORF 11

Translation: MLLLTWLLVGVTLAADHPTLYAPKGGSIEL GVGAKQKGQYKFEWRFGNLKIVIAEMSSTNQLEIKFPDNGFQN RSEFNPTKHNLTIHNASYEDSGTYSLHQEENDGTEHTDNFKTIVQQMSLY

REPLACEMENT BLAπ (RULE 26) 1 gatgatgtat aataacctca aaaactaacg cagtcataac cggccataac cgcacggtgt

61 cactcgggta caaattatga attcgatctt tggacttttc gacgcgccca gtgactgtac

121 tttattgcgc caattcacca cgcccgggag atttcgaaat tgctatttcc gtgcagttcc

181 gcattccgaa gtacaattta accggtttta tgggtgttcg gtgtttttct agcttaatca

241 ttgtttttag acgacacagt gggtatctgt tttcgcttgg acttggctcc gctttgtgaa

301 aattcaactc gatccaacat tttccttatt gatggaaggc ttttattatt tgcacaacag

361 acatcgcgct atttacacag aacgcaaagt gctgtctttt ttattccttg ttccgggtac

421 atcttttatt gctagtgcct cgcctatttt tagtcacgta tcttccttgt tctatagcta

481 tatattcacg cggttttcgg tctctcctca ctcggcagat gacttcggaa gagaagctgc

541 agagttcgtc tccggagacc ggcctcgccg ctgtcgtcct gcaaagcccc cttgaggtac

601 gtgtgcctgc cgttcttcct cctccagtgc gaattgacat ctaccgctac ccaggctttc

661 cgccaacgga gaccatctgg cacggtctca tcacgcagac tgagttaaac caggctttgg

721 agagcatcgt tgagcaattg daytaagtgt cagtccctat ttttctgttt tttcttgtat

781 ttcctcttag acgatggacc cgttcggttc ttcttcagtc cctccgtgct ctacatcaga

841 ccttcccgaa cccaagctct atttcgtccg cttgtcaccc catgcagtgc ctccagttag

901 ggctacgcac ggagctgcag gatacgattt gtttagcgct tacgacatta aagtgcctgc

961 tcgcggtcgg gcgctagttc ccacagattt agtttttcaa tttccgcccg gctgttacgg

1021 tcggattgct cctcgctcgg gcttggccgc caaatttttc attgacgtcg gagcgggtgt

1081 tatcgatccc gattaccgcg ggaacgttag cgtggttttg ttcaatttct ccgagagctc

1141 gttcaacatc aggcgaggcg atagggtagc acagcttatt ctggagcgta ttatggttcc

1201 ggagctgtcg gaactgacgc agctaggcga gacagaccgc ggggcgtcgg gtttcgggtc 1261 cacaggtatg ggtgctgtag accgcaatca gcgctctgtg ttggaatggc tgacccctgg 1321 ttccccttgactgacgcttggggggctctg

1381 tgagggggac gacgacacat acggacattg tctgtgcgaa ggcagcacca gcaagaggcc 1441 ctgctgtacg ctctgtaaca gttactgcgt gcttctttag gtatgattgt gagcgtgtgg 1501 cagctcgcga ccatcgcgat tgagttttgc gataagtggg tggggaaata ctacagattc 1561 cgaccgtatc atgagagact tttgcttatg cagagtcggc aagctttgga aaggagcttg 1621 cgccgctgcg taagtgaagt tggacctccg ccagagcctc tagaatagcg atggagtccg 1681 gcgcacgtgg tgcttcctga ctttctgcac cattaccgtg gtgtttctgg ccttctttct 1741 gcagaaactt ctcaactaca tagatttcag agatagcgac tgcacagaat gtttttttgt 1801 gtagttgaca ggactatgga ccagacagca acccatacag ctttgattct tctgatcgtc 1861 ttgacggtgt tcacgggcgc ggtggtagct ttgatgttgt atattgcgat aactggactt 1921 ccttgctcta tgctttgctc tcaataaaga ttttcagaat ggtgattcta tggtattttg 1981 tcttttttct agacagtcat gtcgcgtgag tctgaacgtt actggacttt ggtgcacgct 2041 ctgattgatc ggggtgtagt cagccgtgaa cagtggcaaa tggttgaccc tgcgcaatac 2101 cagttctacc accgctccaa acagaggggt tttaaggtcc gtcacattct tcgtgatgtg 2161 attcgccaca tgtgttggtc tcggactctg ttagattata tgtcctcggc ctcgacacct 2221 a gtccggacg atgtattacg caatcccctg tatcagttgc tgttatgtaa tggatataac 2281 cccgctgttg tggggacagc gctgatccgg tgggcgggcc atcagagcaa ccgtaacact 2341 gtttggattc gaggcacccc tatgtccgga gctccgtact tggcacaggc tatcgcgtac 2401 tgctctcccc tcgtagggag cgttgattgg cgcaacaagt ctaacccatt cgaagggtgt 2461 ccagatagtc tggtgttttg gtgggacggc ggttatgttt atgattgttg tgtgggtctg 2521 gtgaagcagg tgttccgggg agaacatgtt attttgcctc ctgagggctt gcgtggcccc 2581 aacccgtgct ctgaactctt caggacccca gtcttgatgt acagccaggc ggatatttgt 2641 atgactaggc tgagatcagg ggaactaagt gcagagcatg cggtgggcct cagggattgt 2701 atgtacctga tccgtttgac agaagatttt gactgcgcgg gtggtatatc gtgtgcagat 2761 gtcaaacagt ttgtggcgtg gagccgcgaa caccctgggg aggttcgcga gacccacgaa 2821 ctcaaataaa aattcgggac ttctgtgtac gttccttttc atgtttatta aacactgttc 2881 tttcgagtga gtcatatcac gtggaagtta attgcgactg ggagccgcag aagcaggtgg 2941 taaaagcaag ctatgcaggg atagtttacg atgtcccttg gaagacagac atagagtgtt 3001 tgtccttcca gtcgcgtgta ggcgccttgg cgccgcgcaa cggttactgc tgtatatatt 3061 tgttcga ggg cagggttcaa gttgcataac ctgcagtgag tagcatatgt gtataagatg

REPLACEMENT BLAπ (RULE 28) 3121 agattaagag ggagttgggg ctggtggtcg tgttgagcaa cgaaattgac gaccgttttg 3181 gcccagtgag ctgtaagcac tcggatgagg gataaacaaa agagggaacg gggtgtctcg 3241 cagctgcaga tgtactggat agtccagtcg gtacatcgca tctcaataac tcttatccag 3301 ctgcagaatg acctgagctg gaggtcacac tcttcgtccg taatgcagtg gggcgagtgc 3361 ttgaaggggt acatctgtct tttaaggaga aagagtagga aatcgggatc tgtgattagg 3421 gtaatgccca cgcgcgtgaa caggggctcg atgtagatat gccaactgtg ttggggctcg 3481 tcctcatcgt tgtcactatc ccaaacaagg tcgcacgact cgaatgtctg taaaacatca 3541 aaagtttatt actgattttg aagaggggta cgtatacagg ggttacagag tttattatcg 3601 cgtttgcaat gcagtgtcat taagcaagca caagcggcag cattagcaac aaagtcgcgt 3661 agctctttgc gcgggtggta gcatggaggg gtcttggtgt ggtctcctgt gaggaagaag 3721 acgaccgtgt tccgcgccgt gaactcgttg gcgagcgaga ggtccgcgca gtggattagc 3781 atgggggtcg aggggatgtg gaacgttttc ttattcagcc tgcaggtggc agcattgcct 3841 tccataaagt gcagcatcaa cgggtccggc ttctcgtcta cgaagggcag acagtataga

3901 gaggcgtggg gtgcgatttc gcccagtgat agtatggagt tgattcggct gtcggagatc 3961 gcgagcggga aacacgcgca taaggcatta aagagactct tggcgttgga aagtcttcct 4021 ccgcagagca cgagggtgtt tagttctcct cggagccatc ggaggatggc gttagctgcc

4081 acttttccct cgtagccttc ggcttccatc atctgatgga accagttgtc gcagacgttg 4141 tagaacgcag ccttgagggt gtcgggctct tcggagggta actggttaat gaggctctgt 4201 ccgggtccga accgttcctt gagggcttcg agtacacgct ctcgttcgta ggtgggtatg 4261 aggttcttgg acgggaagtt ccactggact tcgtgtgcta caccttcgac aaacaaccgt 4321 tcaaccaact gatccatggg ggacgtggac ggaaataagc agggtgaagg ggtgtctgga 4381 atgcaggatg ctatttgctc ctcttcgcta cttaaagacg actgagactc gcgtcgcctc 4441 ttggtgcact gtgggagaga attatatgtc atgtgatgag tgacaagcat tgcatgggga 4501 ggtccacgag cgggaacaga gcattccttt gttctcgcat aggagagcgc gcaaggcgtc 4561 gttgtcatta tctgcctgta attggtgtat gcgatgaatg tagcgggaga gctctcccag 4621 aaagaacccg cagccgttgg cgggttcgta gaacctaata atgatatgtt cgtcgggagc 4681 agtgggcaga tgttcgggat gggggagctg atataagcgg acgaggcatt ttttctcact 4741 gatgtccacg agaccgtcgc gggtcattgt ggtgaatgac ctgcctttca gcagctggtt 4801 agcaacggga tctttgaacg tgttacaatg agctgtgcag gggaaataaa gcatcttgac 4861 taaatcttgc tgtctggcgt actggccaaa gtcgaacgcg ttaatgttgg cagtgaggac 4921 a aattggaat aatctgagga tggatgcagt gaatatatct gcggcagagg aaggttctcc 4981 tacgacatag agcgtgttag tataatggca caggcgacta atgctcatgg cagacatcca 5041 cagataaatg gcgtagatgg ctaccggagc attgtaacct tcggcttcta gcatggctcg 5101 gagatctctt tgtgaggaca gctcgaagcc ttgcagattg aaattgatcg tttgactgag 5161 cccgtagctg cagtataatt tgcgcgcttc ttccagaagc gcgggggcga ccgattcgaa 5221 tagttcagca tcttggggat accgagtgag ccaatctttg taggtaaaga tgttgtttcg 5281 ctggagatcg aatattagcc tgtgtgtgac taaatcgtcg tcgtcttctc ccgtgtggcg gttcctgagt 5341 ctcttaacgc tatccttaag tttcctgaaa cgttcctggg ggagaaagtt 5401 tttcgagatc ggttagtttg gggtttctct ctaactgctt tctatgtgtt tcgctaaatt 5461 tgatgataga tttggctgtt ttcaggaatc tctcctgaat gttaacattg agagggatga 5521 tcgagtactg ctgatctata ctgcaccacc taaaggactc ggatactggg tccggagtgt 5581 acatgaccca gctgaaccgc tggttctgct gcaggaagga aaagtaagct ttgagaagga 5641 caacgaggtc cttcgagatg ttgtgtgcga acgcatagag gaacctagcg aactggaatt 5701 ggggaatatg acaggatagg atgtgcattt tcgcgttcac tttgagggtg ggaacgtttc 5761 ctatcgc ggt gcgcggtgcc atgttgtgca agactacgaa aatgtagaag gctgtacagt 5821 gggcagagcg agcaaagagc ttagaaggaa gggcgtgaaa gaggacagag acgctggagc 5881 ctgaacagag cttatccatg cactcgtcca tgataatggc gacgggtccc cgcttggaga 5941 ctttcacgta aatgttgtct ggatggtcga tgttgagatg ttcgggtgcg gtggcctcct 6001 cgtaagtcat ctccataaat tcgggacgga acgtgcttgt cttaggggcg atggtgccgt 6061 cttccctgca gtcgaaattg gcctcgacca gctgcaggtt ccaggacgtc tgttcgatgg 6121 gtggaatcat gttcttttcc ggagtgatga agatgaccgt ttcggggatc gggtccaaca 6181 tgttgcacga gatgagcgcc cgcagcagat gcgacttgcc ggatccggtg ggtccgtaga 6241 tgaccccgat gatgggctgc ttgcccatgt tgatggacgg cagctgtccg tgttggaggt 6301 agcgactgtc catctgttcc tggctctctggtcctt

REPLACEMENT BLAπ (RULE 26) 6361 taacgccccc taggctgtag aactcgtcgt aggaggggaa gtgttgctcg cggaagagct 6421 gcggtgctag gtcgacagct ccgtcgtacc agctggtgac gcgctggtag aagtcccgcg 6481 aggcttcgta ctcttcttcc tcatactccc aggctttccg cttcctggga gctatcatct 6541 gcgaagagta ggtcgtgaac ttgcccgcat tcctcttcgg ataggaacgc gtagggttcc 6601 catcgtaggg gtgcgagggg gtcttcgtgc ccgacgattg ggacgggtcg tacgtctcct 6661 gtcgtgcggg gatttgggtg ctcattgtcg taggggtaca ggtagtcccc gtgctcgtat 6721 agggtgaggt ccttccacgg acgcagcact cgcgtgagct gctcgttgtg aatggtgaaa 6781 gggtcgtagc gactgacctt gttcagaagc gtggttttaa agatggttct ccgcgtgtgt 6841 agctctggga tgttgctctg cgctccgaat tgcacgtcct cgtgtctacg ccaacagcgc 6901 agcagcgtgt cgtagatgag ttcggactgc ctgtgtccct tcgatctgat tttcccgggt 6961 cctaccgtgc ggcactgttc gttgacgcag attgagtttt tcagtccgta cagttttggc 7021 gctaggaaga tggtttccga gctgtacgtg tcacttccgc aggctttgca cttgatgtcg 7081 caatcgcagg cccagtagag gccgggattt tctggatcga aagtcagtcg agtggattct 7141 gttttgattc ggtgcgcgcc gcggcttttc atgcgatgat agcctgtttc tgtgacgaac 7201 a ggctgtcgg tatcgccata gaggctgcgc ggctcctccc ttcgcaggat gtgcactcct 7261 ctgtccggtc cgtacaggat gtcacaccac tcgctgaaga aggccctcga ccagcccagc 7321 acgaagcagg cgatttgcgt ggcgtatctt ttgtttgcca cctgcttgtc caggctttcc 7381 agatggagca cggttagggc ttctggtgtg gcttcgagga gacgcatagg tttaaaccgg 7441 gtctcgttag cgcgagcgta gtgggcggag cttagctccc cctcgggtat ataagggccc 7501 gtcgcgaggg tcgcctcgac ttccaggtct acggcgacga gaggatcgtc ttcggtaagt 7561 gcgtttttcg gcttacgggc actctcgcga tcgctgtcgt ctccttcttc ttccccatcc 7621 tcttctgctt cgggctcttc ctgttcgggg tcgtctgcgt agcggaactg ttgtcgtaga 7681 ctctcctcac tgaaggggtt aggcgcgttt tcgagggtga cttccgttcc gttgaacgag 7741 tcgtcattga gcagcgtgac gtgtttgacg atttcagtgc cttcgtagat gtttttctta 7801 tctgcttccg agaggtcctg ttcaaagatg atgcgcgtgg tgtccatgtt ggtggcaaac 7861 gcaccgtaca gcgcgttgct cagcattttg gagatggatc gaatcacttc gttcttctcg 7921 cgatcggctt tttctttggc gaggatgttt ttcgtgacgt agtcggcaca tagcgttttc 7981 cattccggaa aaacaatgtt catctcgtca tggaggacct ggactcgcca tccccggttg 8041 tgcagcg tga ggatatctat gacggtgacc acctcgtcgt agagagcctc gttggtccag 8101 accagtctgc ctcccctccg ggagcagatg ggagggagtg ggtctaacat ttcgggcggg 8161 ggagggtagg cttctatttt caggatggaa ggcttgatac gcgcatcgaa gtagctcaga 8221 tgcgattcgt tggtcagcag ccggttgagc tcctccacgt gctgcgcggt aaattttgga 8281 tctaggggca ttccgtgggg catggggtgg gtgagggcgg aagcgtacat gccgcagatg 8341 tcaaagacgt agacgggttt caggtaaggt ccgagcacgt tggggtagca tcgtccgccg 8401 cggagcgctt ggcgtatgta tttgaacatg gggcggtggg gggcgtagac ttcggccacg 8461 tagtcggggg agggttgttt tttctttttg gttcgacctt tctttttggg gggttcgggg 8521 acggagggag ggcatgtcgc acgctgttcg cggacgtaat tggaaaaggt aagttgcttc 8581 caaaaggcat gagtgttgct ggggatggtg ggccgcacga agatgttaaa atggccttcc 8641 atccctagtt ctcgttggaa ataggcgtcg tagctgtcgt gtaacgtgtg ggccagcttt 8701 tgggtgacgc ggacgtcctg catgcagtat tcgaggcacg cttggacgat gtcgtactgc 8761 tggcccgggt gttctttctg ccatagattc ttctgttcag cgatgacgga tgggtcttcc 8821 cagtaccttt cgacaggaaa gccgtcggcg tccgcgtgaa agcgccccgt ggaaatgaat 8881 tcgttgatgg cc tcgtatgg gcaatgtccc ttgcagaggt ctagcgcgta ggctgccgcg 8941 gctttggaga gtttggcccc gctggtgagc tgtagagtgt ctcgcaccat gaaccgcaca 9001 aataccgagc gcgcatcctg atgggacacg atcccgcgag accagcgttc tacgcgggag 9061 gcgtctttct tcacgtagtt ggggtttggc atgcggaaaa tgatatcatt gaacagaagg 9121 cgaccgacgc gaggcatgaa ggatcgatca catttgcacg cttccgggaa taggtccctg 9181 cgctcgacga gttccgtggc taagaggagt tcatcgaact tacatatgtt gtgacctagc 9241 actacgatgt ctacggaata aaagttatcc gggagggaga ggggggaggt gggtttctcg

9301 aagagctcgt acggtatttt gtgaacggag ccgtactttc cgtccttgac tagctggtcg 9361 caataatctc ggttggcagc cgcgtagcgg tcgactagat tttgggcaaa ttctatttgc

9421 agacgggacc tgaagttgcg aaaccttctg gcaacctcgc ccggatggct gtctagccaa 9481 tagaagcctt cgtcgagggc tttgagacgg tcgtcctgcc gtgctaagcgttcggcccgggcggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg

ERS _A TZBLAπ (RULE 20) 9601 cctttctttt cgaacacggt atacgtctcg atatcgtagg tgatgtacaa ctgacgtatg 9661 tgggggtgtt gggctggaca ggagaagtgg acgtgctgcc acaaatcgct gcccgatccc 9721 tggacggcgt ggtagtagaa ggcagagcgg cgttcgttgc acgagtgtcg tctgacccag 9781 tgtcggccgc aggtgggaca ctgttgaacc ggagtgcggc tggtgatcca gacccattct 9841 ccggtgacgt ttttggcgat gagcatgggg ggaagggcgg gatgttcgga gatgtgcacg 9901 gtccgcaggc gcgctgtttt gcctttaaag ttgatgacgg tgacctgggc gggcttaaag 9961 tcggggaggg agcgttcggg gtctcgggca tagtgcagat agtctattcg gtcgtagcct 10021 ctggctctct tagacagcag gaagcggtgg gtgcgtagga atttcttgag acctatgggg 10081 aatacagtgg gttggaagcg gaagggctgt tcctcgatgt aatagacgtt tgtgcgtagg 10141 gggttgcccc ggaagctctt gaagtacttt cgggtctctt ccagccgttt agcggaaatg 10201 gttcggatga cgtgcgaatc ggggcccagc acgctgtccg tgaggggagg ccccgcgtct 10261 gcttccattt acagaggctg acctcgtcgc gccgccgctt cgatgtcttc gcggcggtgg 10321 cggagcacgc gtctgaggtt ggctttgatc tcctcgttgg tagcgatggc tacgatgcct 10381 ttaaatttga tgcggaaact gatgtcgatg ctatcgatca gctcttcgct gaggttgagc 10441 tgcttcagca cttcttcgat gtcaccggag cggtctctgt attgaatatc agagagaaac 10501 agttgttggt ccgcatcgtc catgccttcg aattgacccg tccgttcgac catcataaga 10561 aaatcgcgta atatacgttc ccacagggtt tcgaatatgg tggcggggtt ggattgctcg 10621 ctccatatgc gtttaaaaac ctgctgcgcg ttgacgtccc atcccacgac gagtacttgt 10681 aaggtcagga cgtcgacgta ccggcggaac tcgcggttgg cgatgaagtg gctgtacagg 10741 tagtagagtg tagaggcgat atgttcggct aagaagaagt acagcaccca cttgcgcagg 10801 aacgactctc ccatgagtcc cgcgtcgcgc gcggtcagga gcatgcggta gaagtcgttg 10861 gcaaacctga agagttcgtg tctccgggcg gccccgctga gctcgtcttg cagtgccccg 10921 atggcttcga gcgctgtgcg aatcacctcg tctaacagct cttcttcctc ctcttctggt 10981 tcttctactt ccatggcggc ttcttctatt tcttcgggag ggggcgcggg ggaggggggt 11041 ctccgtcgcc gccgcgtgac gcggggcagg cggtctacga aggcccgcac ggcacggggc 11101 ctaatgcggc gcgcttcgga cgcggtgatg gctctgccgt ggcggtctct agggcgcagg 11161 ccggtgcggt cggttacccg ccggctgcag agggtgatgg cgcctcctga cagacccgtt 11221 cctgctaagg aaacggcgtc gctcgctaga gtgctcatga aacatctcgc ca ttgtttcc 11281 gtaggttcct ctacgtcccg tcttcctcga aacttgcgct cggcatcccg caccagttgc 11341 tgggtgtcta gctccgcgaa ggcttccgcg aagtaggaga gccagtttgg ttcaaggaac 11401 acatcggatc caggtaagaa ccgatatctt tcttccgttt gaaatagatc atatgcgtag 11461 cagaatagat agtggcagag ggcgactctt agttggcgga tggcggcgag cagttcggcg 11521 tcggcggcag aggccgttcg catgaggacg ccttcttgca agccacccgc tccgttgccc 11581 gacagtaggc tgtggtgttc ggcgtcgagc tgcggcaaca cttgtccgtg acggcctacg 11641 tcgattcccc tacctcgcag atgcgcccgg cccatgtccg cggccacgcg gtccatgagt 11701 acggcgttgt gcatctgcgt gaacgtaccg tgaaagttgt cgagatcgag aaatcgcatg 11761 tactgcccta cattgacggc gtaggagcag tcggtcagac aggtccaaaa gaggcgtctg 11821 ggtctttggg gtgggctatc gtaacctatc tgcataaaaa cgcggttgtc gaaaaggtaa 11881 tcgttgagcg cgcggtgcat agcttggtac ccgaggagaa ggtgcggcgg cggcaatccg 11941 ttgtagggcg gcgcggcgac gttcggcgat cgcggcgcga ggtctcggag ttgcattaaa 12001 cggtagtcgt acacccggct gacgagaaac acgcttcggg ggtggaccag ggctggttct 12061 tcccgaacga ggacatagtc agcggtgatg acgggttcgc agaag cggac ggtggctaga 12121 ctctgtccgg tgagatccgc gaagactctg taagcctgaa attgagcccc tgacgttttt 12181 agaccgctcg taatgcaccc cgtcctgcaa agcgttcgaa acgcgagcgt gagcgccgga 12241 ggaccccatc aacagcaacc gcagcagcaa cagcacggtg tgtcgtcggt ccgtcgtcct 12301 ccttcaccac cccgatatcc cgcacaacat gcctatcccg gcgcgggcgc gacacccacg 12361 gcaggacgag gcgatttcga cggcgcgctt gatcccgatg aaggaccggt cgcgtgcggg 12421 ctggcggccg gggccggtgt ggacgaagtt agaatgaggg agcgggacgc cgcgcggcga 12481 gccacggtgc ccgagatcaa tctttttaag gctcgacgtg acgtggtgcc caatggggat 12541 tacgagaggg atctgatgta ccactcggga caggcaatcg atatcgatcg gcaacgtgtg 12601 ctcactccgg aagactttaa ggggtccgag ccggctttca cgccggctgt caaccatatg 12661 cgcgcggccg agttgaagag ggcggctgag cagacggcat ttggggagga attgaggaat 12721 acctgccatc agacccgcat ccgcacggct ctgttaaggc ccgagatcgg agcgggaatc 12781 tactatctgt acgatttcgt ccagacttat ctggagcatc cggacggtcg ggtgaagctc

REPLACEMENT BLAπ (RULE 26) 12841 aatcctcagc tggtgttggt ggctcagcac gcgggcaata ctatgctggc gcagcgcttg 12901 tgggccatcg cagaggagaa gaatgcgtgg ttgagagatt tgatagagat ggcgtacatg 12961 atcgtgaacg atccgtacct caatacggag cagcagctgt cggccatctg cacgacggtg 13021 gtcgagttga gcatgaaata cgccaagttg gccgccaaga acggttaccc gtccatggcg 13081 cagatggcta aggcgcagga atttttctac cgggtcatgc aagcggtgct cgatttaggt 13141 gtccaagtgg gggtgtataa caaccgacca gctcggtacc gtcagaagcg catgagcgag 13201 attccgcaga tgactgacgc cgagtacatg ttcggtttga cccaggcgct ggagagcagg 13261 cctccgcagg gcgaatcttt tgccgacgag gggccgtcag aatcggacga cgaggatgac 13321 ttcatctgat acgtttctgg ctcttgcgcc ctacgggcgt caggaggtgg cggacgccct 13381 cagttcgctc ccagatggca aggacgcgcg gtcgctacgt catgcaccct acgcaaatcg 13441 cctcatcaaa ctccagagcg ccatggtgcc tccaaaagtg gacggtactt ccgagcgggt 13501 ggccgaaatc gtgaaagggc tagccgagca aggcgccatc taccccgatc agatgggcgc 13561 gatccactca gatttgctta atcgagctta cacgtggaat tccatggggg tgcaggagag 13621 catccaggcg ctggtcaacg acgtgatcca cggacagaac cggacattgc aag acgagct 13681 tgcgcggacg aaagaaatag cgaatgcttc gctcttgacc caatttttcg attccctgta 13741 caaaacggtg gatcgtgggc agcgaaattt tgagggcttt aagaaacttt tgcgtctttt 13801 cgtgaataac gtgccgaatg ccgaagtgta cgggtcttcg gggtccttta gcgtgcagat 13861 aaatcttggc ggatctagtc aaaacatcaa tctgaccaat gcgtttgaga atttgaagcc

13921 gatatggggc gcacggtggg acgcggtgaa taatcctcgc atcggggcgc ttctgacacc 13981 caacactcga gcgttgttgt ttttcgtgag ctctttttac gactacgggg ctatggagcc 14041 cggtagttac ttggacaata tcatgaggct gtacaaggag gctatcagag ccgatgtgga 14101 cgcggagggt gatgccatta tggagctcgg ggaggcgggc gcaaatctca acttgcggtt 14161 caacgattac aaggacacac taaactacct cctgcaaaat cgagaggttg tacccgacac 14221 ggctccgctg gagctgagcg cggagcagga aatgctcttg aagtacctga tgaggcaact 14281 acgacaggct cttaaggacg gggtcccggc ggacatttct atcagtacca tgactcagta 14341 cctagatcct aggctgtatc agacgaacaa ggtgttcgtg gagaaattgc aaaactacct 14401 gttggcggct caggcgcgca atcctgtgta ttaccgactg ttggtgctgg accccaactg 14461 gcggcctccg gcaggcctat atacgggtaa ttacgtgata cccgaccgct acgactttga 14521 ggacgtgcag agcgagcttg aatacgcggg tccctccaga gacgagtatt tcgatgattc 14581 tttgttcgca ccaggtcctc agcgccgctt aaattcggcc gaggaggctc aattggagcg

14641 tgacatcgaa tctttgaccg gccacattga cgaagagctg ggcgtccaat ctcaggctgg 14701 ctggctcgcc gatcaccgcc tgcctgtcgc gttcgatggc gctctcagcc ttaccgaacg 14761 caacgcctac aacacgccgt tgccccccga ttcccacatg cgtagccgtt ctagctccgt 14B21 cgctagcgat cttgggctat tgaacctatc tgggacgggg ggaccgggct ttttcgctag 14881 tctgcggcct tccatcggca gccgtcaacc gaccggcacg gccgtgggcc tccgcccgac 14941 gacaccgtac agcggttcgg ggtgtatgag gggcaccggt ctggcgcgca aagttttaaa 15001 cccggccgcg tcgcgccggg ggcgcaagct acggttctac tgaaccctag actctgacga 15061 agaaacttaa aaacgcttac cgccatttcg ccgcgcagaa gttggaagga tgtaccggag 15121 cctgcgaccg ccgacgtcga ttcctcctcc gcctccctct ggtccctcgc cttatccggc 15181 gatgatcaac ggatatcccc cggatgtgcc ggtggggtca cctgccaacg gagatgcgga 15241 gctgttcgtg ccgctccaga gggtgatgcc gcctacgggt ggacggaaca gcattagata 15301 ccggaattat gcgccgtgcc aaaacaccac caagtttttt tacgtagaca ataagctgag 15361 cgacttagac acctacaacg aggacgcgaa tcacagcaat tttaggacga cagtcattca 15421 taatcaggac ttagacccgt caacggccgc cacagagacc attcagctcg aca ataggtc 15481 gtgttggggc ggagagctaa aaacagcggt gaaaaccaat tgcccgaaca tcagctcgtt 15541 tttccaaagt gatacagtgc gcgtgcgtct gatgagcaag cgcgatccgg ggggtaccga 15601 cccagacgcg ggggtgaaca acccacccgg ggccgagtac aagtggtatg atctgaggat 15661 tcccgaaggt aactacgcgt tgaacgagat cattgacctt ttgaacgaag gcatcgtcca 15721 gctgtacctg caggaggggc gccaaaacaa tgtgctcaag agcgatatcg gggttaagtt 15781 cgatacgcgg tatctggatt tgctgaagga ccccgtgacg gggctggtga cgcccggcac 15841 ctacgtttac aaaggatacc atcccgacat catcctcctc cccggctgcg cggtcgactt 15901 tacgttcagc aggcttagtc ttctgctcgg tatcgcgaag cgcgagccct actcgaaggg 15961 gtttacgatt acttacgaag atcttcaagg agggaacgtg cccgcgctgc tcgatctgtc 16021 ctccgtgccag gtagacgagggtggagacgagggt

REPLACEMENT SHEET (REGELä§) 16081 tttaaaagac tccaagggcg tttcctataa cgtgatcacc actggcgtga ctcaaccgca 16141 aaccgcttat cggtcttggc tccttgccta ccacaccctg gactcccccg cgcgcaataa 16201 aacgttattg actgttccgg atatggcagg tggtatcggc gctatgtaca catcgatgcc 16261 ggacacgttt accgcacctg ccggatttaa ggaagacaat acgaccaacc tttgtcctgt 16321 ggtggccatg aacctgttcc cgagtttcaa taaggtattt taccagggcg cgtccgccta 16381 cgtgcagcgc ttagaaaatg ccacgcaatc cgcaacggcc gctttcaacc ggtttcccga 16441 aaacgaaatt ctaaagcagg ccccacccat gaatgtttcc tcggtgtgtg ataaccaacc 16501 cgccgtcgtt cagcagggtg tgctaccgct gaagaattct ctgtctggcc tacagcgcgt 16561 gttgatcacc gacgaccggc gccgtcccat tccatacgtg tacaaaacca tcgccaccgt 16621 gcaaccgcgc gttttgagca gttcaaccct gcagtgagga gcggaaggat tttcaaacat 16681 gtccattctg atttcaccca gtgataacag aggttgggga gcaaacatgc gttaccgccg 16741 tagagcatcc atgcgcgggg tcggtcgccg tcgtctcacc ctgaggcagc tattgggtct 16801 ggggtctcgc cggagacggc gatccaggcc cacgaccgtc agtaaccgtt tggtggttgt 16861 gagcacccgc cgccgctctt cccgaagacg ccgatgaagc aagcagctga tga gatgttc 16921 ttctgactat gtgtgccgtc gctatacaca ggagcgacgt cgttatgcct tccgttcttt 16981 tgaccggcgg gcggaccgcc aagggcaaga agagagcctc tcgtcgtcga gtgaaagtgc 17041 ctaagttgcc taagggagcg cgccgaaagc gtgcgtcggt gacgccggtc cctaccgtag 17101 ctaccgcgac cgcttccgag cgcgcggctc tgacgaacct agccagacgg ctccagcgcg 17161 gcgactacgc cgcttggagg cccgccgact acacgtcacc ggccgtttcc gaggcggctc 17221 gcgcagccgc ctcgtccggc acccccgcga ccgcgaggga tctcgcgacg ggaaccctcg 17281 ctcgcgccgt gcccatgacg ggtaccggcg gaaggcggcg caagcgcacc gctacccgcc 17341 gccgatctct gaaggggggc ttcctgccgg ctctgatacc tatcattgcg gccgctatcg 17401 gcgccattcc gggcatcgca ggcaccgccg tgggcatcgc caatctgaag gagcagcaga 17461 gacagtttaa taagatttac ggggacaaaa agtgatgctg actggacgca ctaaaaggcc 17521 ctttcaataa acgcgttttt gtagaaccgg ctcgcgtcat ggactacgct gcgctatcac 17581 cgcatctcgg tgggtgggcc ctgagagacc accacatcgg cgactctagc ttgagagggg 17641 gagccatcaa ctggggcaac ctcgggtcgc gcataaccag cgcgctgaac tccaccggtc 17701 gctggctgta taacaccggc aaccgcttcg tgcattcgaa cacttt CAAC cagattaaac 17761 aaggcataca agacagcggg gtcatacgca acgtggctaa tttggccgga gagacgctgg 17821 gggccctgac cgacatcggc cggttgaagt tgcaacagga tctggagaag ctgcggcgta 17881 aagctttggg ggaggaaggt ccagcgaccc aggccgaact gcaggctctc attcaggccc 17941 tgcaggcgca agtggctgcc ggagagccgc ccgccgcacc cgcggcgccg gcgccggccc 18001 cgccgctcgt gcccaccact cgtcctattc ccgaaatggt aacggaggtt aagcctcccg 18061 ttacgtcttc ggcgccagcc gtccccgtag acgtgccgac cacgctggaa atgcgacctc 18121 cgccgcccaa gcgcaggcgc aagagggcac gaccgggaca atggagggca cgcttggaca 18181 gcctctcggg taccggagta gcgaccgcca ctagacgtat gtgttactaa aattccgtcg 18241 ttccgctatg tctaattttt agctcaccgg ttgtctcccg aaggcgtcat gactgcgctt 18301 actcccgacc tgaccacggc gacgccgcgg ctgcagtact ttcatatcgc gggccctggc 18361 acccgagagt atctatccga ggatctccag cagtttatct cggccacggg gagctacttt 18421 gacttgaaaa acaaattcag gcagacggtc gtagctccca ctcgcaatgt caccaccgaa 18481 aaggcacaac gtctgcagat cagattctac ccgatccaga cggatgacac gccaaacagc 18541 tatcgcgtgc gctacagcgt caacgttggg gacagctgg g tgttggacat gggggcgacc 18601 tacttcgaca taaagggtgt gctggaccgc ggaccttcct tcaagccgta cggcggaacg 18661 gcttataatc cccttgcgcc aagagaagct attttcaaca cctgggtgga gagcactggt 18721 cctcagacca atgtggtggg acagatgacc aacgtgtaca caaatcagac caggaacgac 18781 aagacggcca cgcttcagca ggtcaatagc atctccgggg tggttcccaa cgtcaacctg 18841 ggacccggcc tcagtcaact agcatcccgg gccgacgtgg ataatattgg cgtggtggga 18901 cgtttcgcca aggtagactc agcgggcgtg aagcaggcgt acggagccta tgtcaagccc 18961 gtgaaggacg acgggtctca gtctctgaac cagaccgcgt actggctgat ggacaacgga 19021 ggtaccaact atctgggtgc cctggctgtg gaagactaca ctcagaccct gagttacccc 19081 gataccgtgc tcgtgacccc tcccaccgct taccagcaag tcaactccgg caccatgcgg 19141 gcatgcaggc ccaactacat cggcttccga gacaacttta tcaacctact gtaccacgac 19201 tcgggcgtct gcagcggaac gctcaactcc gagcgctccg gcatgaacgt ggtcgtggaa 19261 ctccaggaca gaaacacaga actgagttac cagtacatgc tggcggacat gatgtcccgt

REPLACEMENT SHEET (REG & g§) 19321 catcactact tcgcgctgtg gaaccaggcc gtcgaccagt acgaccacga cgtgcgcgtc 19381 ttcaacaacg acggctacga agagggcgtg cctacttacg ccttcctgcc cgacgggcac 19441 ggggcgggcg aagacaacgg tcccgacctc agcaatgtca aaatttacac caacggacag 19501 caagataagg gcaacgtggt ggccggaacg gtttccacac agctcaattt cggtaccatt 19561 ccctcctacg agatcgacat tgctgctgcc accaggcgca acttcatcat gagcaacatt 19621 gccgactacc tgcccgacaa atacaagttt agcattcgcg gtttcgaccc tgttacagac 19681 aacatcgacc ctaccaccta cttttacatg aatcgcaggg ttcccttgac caacgtggta 19741 gacctgttta ccaacattgg tgccagatgg tccgtggacc agatggacaa cgtcaatccc 19801 ttcaaccacc accgtaactg ggggttgaag tacaggtctc agctgctcgg aaacagcaga 19861 tactgccgtt tccatattca ggtgccgcag aaatactttg ccatcaagaa tctgctcctg 19921 ttgcccggca cctacactta cgagtgggtc ctcagaaagg atcccaacat gattctgcag 19981 tccagccttg gcaacgactt gcgcgcggac ggcgcgcaga tcgtgtatac cgaggtgaac 20041 cttatggcca atttcatgcc catggaccac aataccagca accagctgga gctgatgttg 20101 cgcaacgcta ccaacgacca gaccttcgcg gactacttgg gcgccaagaa cgc tctctac 20161 aacgttccgg ccggctccac gctgctgacc atcaatattc ccgccagaac atgggagggt 20221 atgcggggct ggtcttttac ccgcctcaag gcctcggaga cgccccagct gggcgctcag 20281 tacgacgtcg gtttcaagta ttcaggctcc attccctatt cggatggcac cttttacctg 20341 tcccacacgt tccgcagtat gagcgtgttg tttgatacct ctatcaactg gcctggcaac 20401 gaccgtctgc tcacacctaa cctgttcgag atcaagaggc cagtggccac cgacagcgaa 20461 ggcttcacta tgtcgcagtg cgacatgacc aaggactggt tcctcgtgca gatggccacc 20521 aactacaact acgtgtacaa cggttatagg ttctggcctg acagacacta cttccactat 20581 gacttcctac gcaacttcga ccccatgtcg cgtcagggcc ccaacttcct ggacaccacg 20641 ctgtacgacc tggtgtccag cactcccgtt gttaacgaca ccggctcaca gccgtctcag 20701 gacaacgtgc gtaacaactc cggctttatc gcccctcgca gctggcccgt atggaccgca 20761 cagcagggcg aagcctggcc cgctaactgg ccgtacccgc tgatcgggaa cgacgccatc 20821 agttccaacc aaaccgtcaa ctacaagaag ttcctgtgcg ataactacct ctggaccgtg 20881 ccgttcagct cggactttat gtatatggga gagctgaccg atctgggtca gaaccccatg 20941 tacacaaaca actcccatag catggttatc aactttgagt tggacc ccat ggatgagaat 21001 acttacgtgt acatgctgta cggggtattt gataccgttc gcgtgaacca gcccgagcgt 21061 aacgtgctag ccatggctta cttccgtacg cctttcgcca caggcaacgc tgtgtaaaaa 21121 aaagacggct gggatgtcgg gaaccaccga gacccaactg cgggacctgc tgtcctctat 21181 gcacctgcgg caccgcttcc tgggtgtttt tgacaaaagt ttcccaggat ttctcgatcc 21241 gcacgtgccc gcctcagcta tcgtcaacac cggctcccgg gcctccggag gtatgcactg 21301 gatcgggttc gcgttcgacc ctgccgcagg acgatgttac atgtttgacc ctttcgggtg 21361 gtcagaccag aagctgtggg agttatacag agtcaagtac aacgctttca tgcgtcggac 21421 cggcttacgg cagcccgatc gctgttttac cctggtccgt tctaccgagg ccgtgcagtg 21481 cccctgctcg gccgcttgtg ggctttttag tgcccttttt atcgtctctt tcgaccgtta 21541 ccggtcgaag cccatggatg gcaatcccgt gatcgacacc gtagtcggtg tgaagcacga 21601 aaatatgaat tctccgccct accgcgacat cctgcaccgt aaccaagagc gcacctatta 21661 ctggtggacc aagaatagcg cctattttcg tgctcatcaa gaggaactcc gacgagaaac 21721 ggcccttaac gccctacctg aaaatcacgt ttaatgaccg actgtaaata aagaacgacg 21781 cacacacgta ctgtacatat ttgtgaatag agcaaccgt t tattagataa acgtcaataa 21841 atgccgaccg atagaccgac aaggctcttc actggcttta tttaaagaaa caaaaggatt 21901 aagcgaacgg gtcgtcactg gcgatgggcg agactggcgc caacacctcc gttttaaagg 21961 cgtacgactc gttccaacgg aactcaggca catgtgtggg ctctgaagaa cccatcacgg 22021 cagtaaacag ctccgtagca aagacgtacg cgtagcgcag atccatggcc gacagacgcc 22081 aggcgcaggt tttttcggtc ttcttcagac cgcggcctgc tccgcccgac gccgcctgcg 22141 ggttgcagca ggtgaacacc atggtatgcg ggtttttctt gtgagccttc atatcgggcc 22201 tgctctcgac catgtcgcga gtaatgtcgt ctgtgccgtt gagcttataa ggagtcattt 22261 tacagaactg tctccctgaa atggctcgat cggaggcgta gttgcagttg caattggttg 22321 agatgaggac acattcctct gcccggcgct tgtccgcgtt ggggtaaagc gccttcgtcc 22381 agtctatgtc gtgacgcatc gcgctgaccg ccttggcggc gtcggaaaag accatggcgc 22441 aactgccgtg cgcgtgggga tgagggaagc cgctgtgctc ctgatccttg tagcacaccg 22501 cgttcgcgtc gaacctgagc accaccacct gtcgtccaaa ccggttcttc tcgattacgc

ERS _A TZBLAπ (RULE 26) 22561 cgttctgttc ggccagagcc ctctttcccg cctcgctaga cgggttcaac tccacggtac 22621 gaggtttcgt caccatgtcc acaccgtgca tgcatttcgg gaagggctcc tggagcgctg 22681 ggaaccagcc atggcgccaa acgtgcgccc ctccgggcac gaacttgggc tccagacccg 22741 cgcggctgta gataacggct gccaggaacc gccctacctg agcgttgaaa gagtcgtagc 22801 tagaaaaggt caggcgaaat tcgggatgct ttttgcgaac gtatgtaccc cccattttgg 22861 tccaaatgga gtcgtcgggg cgcacgctgg ctccctgcca ctgtaggtcg agagcttcgc

22921 agatgctggc gacggtggcc atggcgcgtt gcgcgccgta gaccacgggg tctgacagag 22981 gggcctccgg ggattcctcg tcgctggcgt tttcttcgtc atcgacaacg gtttcccgcc

23041 ggcgggtaac tcgccttacg ggggatgggg actcctcgcg gcggctgacc ttcttgcgag 23101 tcgcgcctcg gcccggggcg accacttcca cttcttcctc ctcctcttcc atcatgactt 23161 ctgccgttct cttgacaggc ttggtgctgc gaaagccatg agctcttttc ggggttcttt 23221 ccatgacttc tgcttcggtg acgggatctc gcgtttcaaa aagttcttgc tctccctcct 23281 cttcagagtc agggactact gccggagagg gtggaagcgt cttttgaagc ttcctgggac 23341 ctatagggta aagttaacgc ccatcgtcag cgagaccacg cctcgctggc cgatgggatc 23401 acgagacacg ataaaagacc gcgaccaaaa cactcttggg gctagtatcc ctacccgggt 23461 gcgagcgtgg cagatcttcg ctcttctgct tctccagtgg attctcgggg tctttcggcc 23521 ccgtcggtct ctggggtggg agaggcctgc tcctccctct gtttgacttg attaccgtcg 23581 acggcccggg ctcttcgagg tccacgaagt ccgccacgtc ttcgtcgctg ctgatcgtct 23641 ctgggtgaag cgtttctgcc atcgtggctg tcatcgaaaa tggcagacaa gattacccga 23701 gaggaaaaaa ccatagcgac gctggacctc gtgttacgcg tggtcgtcga tgctggtaac 23761 tgggacgtgt tctcgaaacg tttggttcgc tacacacgcg aacagtacgg aatcgagctg 23821 cccgaagata tcggggactt accggacaca tctgaggtct cgaaagtgct gtt gagtcat 23881 ttgggggaag acaaggcggt actgtccgcg taccgaatcg cggaactgac gcaaccttcc

23941 gaaatggacc gcgctaaggt cacagaggga ggcctggccg tacttaacgc gagtcgcgat 24001 gaaagcgaag ctcagaaccc ctcgaacccc gaacccgaga gcatcgagag cgacgccgta 24061 gaggatctcg gcgttgcagc agagagcgac cctagcgatg acgaacccga cccagaaccc 24121 gagtatgacc atcgagaggc ggatcatgac tctgatgcgg atagcggata ctattcggca 24181 gatgggggac gacctggaac accagtggac gaggagcccc aggacgattc tccctcttcc 24241 gaggagaccg catccactgt catcgaagaa gcgcagacta gcgctagcaa cgattctcat 24301 gacgacgaca ctcaccgcga cgacggcagt gcttctgaag aggatctcga gcgggacgcc 24361 ctcgtggccc cggccgatcc ttttcccaac ttgcggaagt gtttcgagcg ccaagccatg 24421 atgctgaccg gggcgttaaa agacgccgcg gacacggctg atccgccaga aacgctctcc 24481 gtcgacagcg tgcaaaggca gctcgaacgc ttcgtcttta accccgaccg ccgcgtgccc 24541 gccgaacact tggaggtacg ctacaatttc taccctcctt tcctcacccc caaggccatc 24601 gcgagctatc acatctttgc cgtcaccgct tccatccctc taagctgcaa agccaaccgc 24661 agcggcagcg accttctagc caaagcaaaa gagagcactt tcttcaaacg cttacctaaa 24721 tggcgtctcg ggatagagat cgacgacggg ttgggaacgg aagtcacggc ggt aacagag 24781 ctggaagagg approx

24961 gaggcacttc cctgtctgtc ggacgaggaa gtgctggcca tcgtggaccc gacagggcgc 25021 ctccacggcg aggacgcgct caaggccgtg gaaaagcgga gggccgcggt cactatggcg 25081 gtacgctaca ccgcgaccct cgaactcatg gaacgcgtgt tccgcgaacc gtctatggtc 25141 aaaaagatgc aggaggtcct ccaccatacc ttccaccacg gcttcgtcgc cctggtacgc 25201 gaaaccgcaa aagtcaacct gagcaactat gcgaccttcc atgggcttac ctacaacaac 25261 cccctgaaca actgcatcat gtccaagctc ctagaaggag cagacaagga ggactatgtg 25321 gtggactcga tctacctttt cttggtcctg acgtggcaaa cggctatggg tatgtggcag 25381 caggccatag acgatatgac tatccagatg tacaccgagg tctttaccaa gaataagtac 25441 aggctgtact cgctgcccaa cccgaccgcc atcggcaagg ccatcgtgga catcctcatg 25501 gactacgacc ggctcaccga ggaaatgcgg aaagcgctgc ccaacttcac ctgtcagagc 25561 cagattactg ccttccgcca ttttctactg gaacggtcca acatcccagc ggtcgccgcg 25621 cctttcatgc caagcgactt tgtgcctctg gcttacaagc agagccctcc cctcctctgg 25681 gaccaggtct atctgctgca gctggccttc tatctcacta agcacggagg ctacctgtgg 25741 gaagccccgg aggaagaggc caacaacccg tccaaccgga cttactgtcc ttg caatctc 25801 tgcagtccgc accggatgcc aggtcacaat gcggcattgc acaacgagat tctggctatc 25861 ggaacgttcg agatccgcag tccggacggg aagaccttca agctcacgcc tgagctgtgg 25921 accaacgcat acctcgacaa atttgacgcc gaggacttcc acccgttcac ggtgttccac 25981 tatcccgaga acgcatcgcg gttcgcatcc actctaaaag catgcgtcac gcagagcccc 26041 gaaatcttga gcctgattcg ccagattcag gaatcgaggg aggagtttct gctcaccaag 26101 ggcaaggggg tgtacaaaga cccgaacacc ggagaaacca tctccagaca gccccgggac 26161 actgcccgcg cgcagcacgc tggagacggt caagctctac cagcccctgg agcctatacc 26221 accggaggaa atagagcgga gacagcgcct gctggagctg tacggcttgc cccggactac 26281 caagacgggc agtttcctat cgcgaaagtc ggcccgcact accatggccc aaagaatgtt 26341 agacgagaag accagggtta cagaggcggg cccggaggtg tacggggaga gcgcgaggtc 26401 gtcctttcac gaagagcagg aggaagacgt ttcggacgga gaaacactag gcagtcagga 26461 tacaacgaac gggctaaccg atatttcgga agaggaggag gaggatctgt tcgagggcaa 26521 caaggagaac atcccaccac ctcgccgtcc gcctcggaac cgccggctcc gagccgcata 26581 ctcgctcgag gaacccctcc ttcccccgag cgccgcgacc gacaagaaga gta agaaagt 26641 cccaaaaagg cgaggtaaat atcgcagctg ggctaagcac cgcgtggcga tatgccaggc 26701 acttcgcgat gcggtctttg accgcaaaaa ggcgggcgaa atcctcaagc ggggtcaccg 26761 gctcttcgtg cccgctactg tcataggcta ctatgctcgc aaactctctc cctcatttct 26821 cgctcctctc tccagccaca ccgcacccct cctcccacca aaaaaacacc ggcgctaagg 26881 ctgtgcgtct gcgccaagat ccggtgccgc agcacatcgc ggacctcaga ggggaaatac 26941 tcgacatcct gttggaaatc gagtcgtacg cccgccgccg tcccgaccgc cacgtgtcca 27001 ttcgcaacag aacgcgcgaa agcatcaccc gaaaactgca ttacgagaaa aatgaagata 27061 agcttacccg tatgaagagc gatgctatca agttgctcgc tctctggcag accgtttaac 27121 tcgtgttcct ttatagccct tcggaaccat gaacctgatg aacgccacac ccaccgaata 27181 cgtatggaag tacaacccag tctccggcat tcccgccggc gcgcaacaga attacggcgc 27241 cactatagac tgggtgttgc caggaggaac cggtttcgca atagcaacca acgacattcg 27301 aagacaaacc cttaacccgg ccgtgacccg tgcaattacc gcgcgttttg aagctgagtc 27361 agaccagcaa ccgtacgcta gccctcacga gaccaatgtt atcgcggcca atgtcctcga 27421 ctcgggttat cccaaatccg gtctctaccc attagagctc agcggc AATC agcgcgtaca 27481 gctggcaggc ggcctaatgg taggtcgcac tgagggcagg atgcaattag cgggcggttt 27541 aacagaagga agagtgcaac tttctggagg tttccacgga cggccgttgg ttagagggcg 27601 gagcagaaga ccgcccagat ggtgcggcgc cgaactgact gggaacggac tgcccgagca 27661 agccgaagtc acttctgaca cttacaagta cttcctgaga acacagggtc ccagccaagt 27721 ggttgaagag cccggcgtct tttcgcaaag acaatttatg actaccttcc tcccctccgt 27781 tgtccctcat cccttcgaca gcaccaaccc cggcgatttc cccgcgcagt acagtgccat 27841 ctacaaaggc cgcacggcct tcgaagacac cttttgggac tggtgaagcg caccttttgt 27901 tggcgatgct ccgtttcgca ataaatttct tccaattctc tgtcgttaaa cggctcccgt 27961 ctggtcactg tcacgcgctc gccgccctcg ctcgtcaccc gcgcgcggta ccgtcgcctc 28021 agccagaata caaaaccggg gttcaggggt tcgtcgaacc gtaccacagc ctggtcgttt 28081 aatctcaacc aatattttct agggttcgac atcatgaacg aggaggttcc cctaaagcgt 28141 gtcagccctg acgaaaccga gacggttccc aaaaaaccgc gaaccgacgt tcgcgacacc 28201 gtcagggccg gcactgacga cacggtagat ctcgtgtacc ctttttggtg gaatctcgga 28261 acgggagggg gcggaggagg aggaggcggg ggcggcggc a gtggaacctc tctccagccc 28321 aatgacccgc tttacgccgc cagcgggacc atcaacctac gcatgacatc cccgctaacg 28381 ttgtcacaac gagccttggc tctcaaaacg gacagcaccc tcaccctcaa cacgcaaggc 28441 cagctggocg tcagcctcac ccccggagac gggctcgtcc tcaacaccaa cgggctcagc 28501 atcaacgcag acccgcaaac cctcgcattc aacaacagcg gggcgctcga agtcaaccta 28561 gaccccgacg gaccctggtc taaaaccgcc acggggatcg atctgcgtct agatccgacg 28621 acgctcgaag tagacaattg ggaactagga gtcaagctcg atcccgacga agccatcgat 28681 tccgggcccg acggtctctg cctcaacctg gacgagactc tgctgctcgc caccaacagc 28741 acatccggca aaacggagct cggggtacac ctcaacacca gcggtcccat tactgcggac 28801 gaccagggca tcgacctgga cgtcgatccc aacaccatgc aggtgaacac aggaccttcc 28861 ggaggcatgc tggccgtcaa actcaaatct ggcggcgggc tcaccgctga ccccgacggt 28921 atctcggtca cggccaccgt cgcgcctccg tccatcagtg cgacagctcc tctcacctac 28981 accagcggca ccattgcact cactacggat acgcaaacga tgcaagtcaa cagcaaccaa

ERS _A TZBLAπ (RULE 28) 29041 ctggccgtga agctcaaaac gggaggcggt ctgacggctg acgcggacgg aatctccgtt 29101 tcggttgcac cgaccccgac gatcagcgct tctcccccgc taacctacac caacgggcaa 29161 atagggctct ctatcggaga ccaaagcctc caagtcagct ctggacagct ccaagtcaaa 29221 ctgaaaagcc agggcggtat tcaacagagc acgcaggggc tgggagtggc ggttgatcaa 29281 acccttaaga ttgtgtctaa cacgctcgag gtcaacacgg acccgagcgg acccctcacc 29341 agcggcaaca acggtctcag cttagcggcc gtcacacccc tagcagtgtc ttccgccggc 29401 gtcaccctga actatcagtc ccctcttaca gtcacgagta actctctcgg gctctccata 29461 gccgcgccac tccaggcggg tgcgcaaggc ttgacggtaa acacgatgga acccttgagc 29521 gcctcggcgc agggcatcca gctgcactac ggacagggat ttcaggtcgt cgcgggcacg 29581 ctgcagctgc tcactaatcc ccccatcgtt gtctcatccc gcgggttcac cttactctac 29641 actcccgcct tcacggtgag caacaatatg ttggggttga atgtagacgg cactgactgc 29701 gtggctatca gttcagccgg cctacagatc cgtaaggaag ccccgctgta cgtgacctcg 29761 ggaagcactc cagcattagc ccttaagtac agctccgact ttaccattac caatggtgcg 29821 ctcgcgttag cgaacagcgg cggaggagga agttccacac ccgaggtggc cac ctatcac 29881 tgcggggata acctactcga gtcctacgac atcttcgcct ctctacccaa caccaacgcg

29941 gctaaggtgg cggcttactg ccgtttagct gctgcaggtg gcgtggtcag cgggaccatt

30001 caagtgacaa gctatgccgg acgatggcct aaagtgggca acagcgttac ggacggcatc 30061 aaatttgcca tcgtcgtgtc tccccccatg gacaaagacc cacgatcgaa cctcagtcag 30121 tggctgggtg ccaccgtatt ccctgcgggc gcgactactg ctctcttctc acccaacccg 30181 tacggctccc tcaacaccat caccacactg ccatccatcg cctcggactg gtacgtgccc 30241 gagtccaacc tggtcacgta taccaagatc cattttaaac caacggggtc gcagcagctg 30301 cagctcgcga gcggagaact cgttgttgca gcggcgaaat cgcccgtgca gacgacgaaa 30361 tacgaattga tctatctggg atttacgctt aagcagaact cctcgggtac caacttcttc 30421 gatcccaatg cctcctccga tctatccttt ctgacaccac cgattccgtt tacttatctg 30481 gggtactatc aatgaacttg ttaactcctg cagcagcagc agcagcagca gcagcatggc 30541 tgaccagaaa aggaagctgg cggatccgga tgccgaggct ccgacgggca agatggcccg 30601 cgcgggtccg ggagaactgg acctcgtcta ccctttctgg taccaagtag ccgctcccac 30661 ggaaatcaca cctccgttct tggacccgaa cggtcccctg tactccacgg acggcttgtt 30721 gaacgtcagg ctcacggcac ccctcgttat catccgtcaa tctaacggca acgcgatcgg 30781 ggtcaagacc gacggaagca ttaccgtcaa tgcggacggc gcgctgcaga tcg gaatcag 30841 cacggccgga cctctcacca ctaccgccaa cggcatcgat cttaatatcg atcccaaaac 30901 cctggtcgtt gacggtagca gcggcaagaa cgtcttggga gtgcttctga aaggacaggg 30961 ggcgctacag agcagcgcgc aaggcatagg cgttgccgtc gacgagtctc tacaaatcgt 31021 cgataacacc ttggaagtga aggtagatgc tgcaggtccg ctcgccgtca cagcagccgg 31081 cgtagggttg cagtacgaca acacccaatt taaagtcacg aatgggactt tgcaactgta 31141 ccaagcgccc actagcagcg tggccgcatt tacatccggg acgatcggct tgtcctcccc

31201 tacgggcaat tttgtgagct ctagcaacaa cccgtttaac gggagctact tcctgcagca 31261 gatcaatacc atgggcatgc tgactacctc gctctacgtc aaagtcgaca caaccaccat 31321 gggtacgcgt cccacgtaggcggggtc

31381 ctccttcctc acgcagtgca acccctcgaa catcggtcaa gggaccctag agccaagcaa 31441 catcagtatg acctcttttg aacccgccag aaaccccatc tcacctcccg tgttcaatat 31501 gaaccaaaac ataccctact acgcttcccg attcggggta ctggagtctt accggcctat 31561 cttcaccggc tcgctcaaca cgggaagtat cgacgtacgg atgcaagtga cgcccgtcct 31621 cgccaccaac aacacgacct acaatctcat cgcctttacc ttccaatgcg ccagtgccgg 31681 actgttcaat cccaccgtga acggcaccgt ggccatcgga ccggtggtgc atacctgtcc 31741 cgctgcccgc gcccccgtta cggtctgaac aataaagaca aggtgaacca tttatacagt 31801 ctcacgtctc tttattgcat acgctccgct aaatgtttcc attcgctcat ttgccagtaa 31861 tacagcagat tcgcaaactc actgaaccaa tcttctgtat aaaaatgtac gcgctgcgtg 31921 tccaaatcaa catcaatttt cctcatatac agacaggggc tgccacccgc ctcccccaag 31981 cgcgacaccg caattaggaa tggtagcctg ctgtgcaggt ccacgtgaat taacatcccg 32041 cacacgttcc cgatcggtcg ctgcataaat actggagaga aatcgctaaa ccccggtgac 32101 gcccacatag ccacgaagta cacccctgcc acattcaagt catcctccaa cctggcccaa 32161 acataagtgg ccaaatcgga aggagccagg tggcaagccg ataaccccat acg atgcaaa 32221 ggtaacccgt ggcaagcgca tcccccgaaa tgaagttcga aagaatcgta acacagtagc 32281 tgataggcat gaagcggcgt cggcatctga agaccgtcat catcttcgtc gtcttccatg 32341 tcatccccaa cttcctcctc gcgctccgct tcctgttggc ggcgctgctg gtgctgcagc 32401 accatctcca ggatctgctc gtcgttcatc ttaatccgga attatcgcgt acggatgttc 32461 ctcgtcgtcc gaactgacaa cagaaggcgg aggagctgtc agtggtgctg tagaggctaa 32521 cgatgctgca gcaccggtct cttgcaattc gaaataccaa gggttgctac tgacggtcca 32581 gttcccgccc cgtgaaccag gccagcggga aatcggtgca ggtaggggat ccggtgaagg 32641 agaccgggaa tggagggaag gaactgcgag atccttatcc actcgataca aaccgtataa 32701 cagggagccc aacgccaggt acaccaggaa cgtactacaa acgaacacgc tgattacaaa 32761 gtttaacgaa gacagatggt tctgtaggaa caggaagctg cacaggatga tgctgctgta 32821 ggacagggcg accaggaggg ccaagaactc gcgccaatag cgtccacaac actgcaaaat 32881 caaacacgta attagctata cggacgttca ccagcgactc tcgcgcgtcg ttccataaac 32941 acattgcgca gataagccaa ctgagcagaa cagaacagag agagaggttg ccgcgtcgaa 33001 cactgtttgc actgtccgaa acactcggga tgagactccc cgtacttccc gtgcacatga 33061 aatacccact cttcgacgtt agcgtaggac aaccgacggt aaccggggaa gta acacagt 33121 ccctgcgaca cgatcggcca cagttccggc gacaccatca cgcggagcat gcttctcagg 33181 cagacgggtt gggtcacggc gctgctgcga gaaatagttt ccaccatgat ggtggccgtc 33241 acggtcacgc gacacgcatt catgagaaca accggagacc gcacaaaagg aacagacgaa 33301 ggcacttgcg aaaaggacac ctcaaagctc atcgagcgga gagccatctt tacggatatc 33361 ttctccgcaa tcagaaagcc tgtggtgaaa aacttaaaat ctgtcttcct gcgagcaagc 33421 atccacggtt ccaagtcgta cttcatccca ggtgtaataa aaagcaacct acgtgagagg 33481 tagtgcagca gggcggtctt ctctgcccgg ttcaggtgaa gggcactcac aatccggact 33541 atgcaattca tgagtacgta gtctcgccgt ttgaaacaca caaactttgc gctgcgtacc 33601 gtcactagca ccgtgacaga actatgcaat cgcctcatca gatcattatc gaaacaccgc 33661 aagctagcca cggctctatt tatgcggtac tcgttcagtc tctccatttc ctcctgtcga 33721 caagttggat cgtgacgtag gagaaaacta aaccatatta ctcctacttc gttatgaaag 33781 ccacaagctc tgctgacggt taaagactcc ttaagaaaga aaaggtagta cagtcaagct 33841 gacccataca ggtgaacccg ccccaagtca cgtaagtcaa ctcaccgaaa gaggacacag 33901 agccatatcc gctgcttcaa agctttattg acgggtctaa aggcgt AAAG aaaagaagaa 33961 tttaccgttc tgcatctcaa accccaccac cacgcgaaaa agtccgaacg atgctgcagc 34021 accgttcacg caaaagtccc ggacgcgcac aaataaaccc ttaatcccga taacggtgct 34081 gcagcaccgt cacgctgctg cagcatcgtc agacgtttat gacatgcagc cgattccgtg 34141 cggatttatg gtcccataac ggttccaggt cctttcgttg ctgcagcacc gtttcacgta 34201 aagaagctgt acaggtcaaa ctggtccgga ttactgatta ttcgggggag agccacgtga 34261 cgtagactcg aacgacgtcc acttccgata caaaccacat ctctctggac acaggcttgt 34321 ccgccttgag ccaaaccatg tgattcttcc ggtccgttct gacgtcaatg ccgacacgcc 34381 tcttggtgtt tgaacaggca tcccagtacg tagccaggac cactcggtga cgtcgaggtt 34441 gaggtttaaa ggtcactacg gcctgtaacc cggtactcca gggccctaaa ctaacatatt 34501 ctccggccct gcccactaca ttcgggtgat caaatatgac ataatcctta aacaatttgg 34561 ggaacttcaa acagccacat ttgggcggga caggaagatg gtgcgcagaa acattgatat 34621 gagagcgcca tctagggaca tcaaagcggt gcccgcctgg atggcaatcg tacttcttag 34681 ttccggtaaa gtaataggtg tgagtccgga aacacgtaaa ctccgtcact tcctgtgtcg 34741 tcattgccct cgcccctagt gacgtcagag tgccacgcc c cttgtacagt ctaataaatt 34801 ttaatacacc cccgccccta gcatataaaa caatgggagc ctcgcccaca ttcctatccc 34861 taataaaata ccatctgacc gaatacccgt gttccatccc tattgtttta gtataataag 34921 ggtcataagt ccacaactcc gcggcattgc cctctgtcac caccaacagc aaataggaag 34981 atgccatgtc atcctctcgt aaaagcatcc tccaatcagc tacctcttcc gtgtagtact 35041 gagttggctg actgtaatca cgccccgtga cgtaggctga ccacgcggaa ggaacacttc 35101 cgtgcatgct cagtagctgg tcagcctggt gagtatccag ggccttcata aaggtcaaag 35161 gcgccataat gtgataacac agtccatccg atcggaggaa atcaaaggga atatgtacat 35221 cttcacaatg gcgcctccca gaataagacc atgcacagat ttgacctttc caccaagcac 35281 gtgactcgca agcctcatgt ttttgaccag tcagatcgct ccgtatatac tcgtctccta 35341 ttggtcgatc aaaaccgtac gaaccaatac tagacgcaat caccactacg taatccgcgt 35401 catccctaga gataacacta tccgcatccc tattggctga ccaatgacca ccggaagaca 35461 gcaaaaggtt taaccctttt gtgtgtaagt ccatcctata accctggaaa ctttccaatg

JRSATZBLAπ (RULE2β) 35521 ggggatctag cgccactatg cggccaacct tttttgaccc tctgtccgga ctagaagttg 35581 gcgggacaaa gccgcgcata cagtgccccc tagcgacatc cctatgcaat gaattcgatg 35641 gtccttgaac tccgtaaaaa aatgagcagt ggtcctgact gcgtaatagg ccggccccct 35701 cacatcctgc ccccacaaaa gggcgtctac cttcttacaa atatctctca gctgattggt 35761 ccagtccaac agaatgaccg gggactctgg cgtcataatg gtatgcatac gcaaaatctt 35821 tctcatcatt tcactggtcc atttatatgt gccgtcatag cgcgccctat taataagcgg 35881 acacacatcg ggatacatgt cctgaaccag aataatgagc tccccgctat ctttaccatc 35941 caataccccg agccgccgca tttgactgac aacccagggg ccgtccgaaa aagtcaaaaa 36001 agtctcattc caccatacaa ttaacttggg ccacgaagga aaatccggcg aataggtgcc 36061 catcaagcgc ctgacgtcag ccttaggata tggcggatcc catctggaat ccgacccatt 36121 aaagcacgaa gatagggcag acatcggcca atggccagga gaatgggtag aattaataag 36181 gactccgcct ccatttccga gcttttaaaa aaaagagaaa atggaaatca gccaagagac 36241 caccaccccg tgattggatg attggtcatc agaagatcga taagggaatt tattttctgg 36301 gagccccccc cccccctact cctatttaaa aaataaccct ttcctcacca agc tcagaag 36361 acagaggagg agagtagagc gccgctcaga ggtcatccgc cgagagaaaa tcccgcgcag 36421 agaacagagc tctcaggtag gggtctggag ctctctggaa aaatcgcggg ctcttataca 36481 cttactctcc gcccattcga aagccgcgcc tgactagagt acacactata aattccattc 36541 cggtgactta ctactaggcg ctggccactt atcaaaagaa acagttctaa gaataggaca 36601 aagtccaacc gcaataaaac acccttgtca aacatgataa gagtgttctc gagaaggtac 36661 tggaaaagca aacagtccaa ctcccaagtt aaatattacg caaagaggcg taacgagaaa 36721 agactagaaa gtgtaaacac acctctccta gttatatata aacccagcgg ggcagtccct 36781 agaagaacac tacctcaatc cagttacaca ttaacccggg aacctattat tgattaacta 36841 gacagtactt cctcattttc tactggaact ttccactgcc ctccggggat tttccattgg 36901 caatcattaa cttgactttg tactttatgt ttactctcca tagcaacgca ccttatatgg 36961 aaaatatgct cctccccgga ccgcccatcg taccacctga gcaggtaggc tgtacctttt 37021 cctattggcc cattatgagc tcacctggtt aatcatatac ccgctccgcc tatataggta 37081 gcataccggg acaggttccc tcacagtcta ttgcagactg ccgaagagag aggagctccg 37141 cataggactg ggaccagaac cccgagactc tgccggtaat atttta ATTT catttaatcg 37201 aatcaaataa atcaaaaatc aactcaaacc catgattctc aatggaaatt tcttgtgatt 37261 ttctttcgcg cgcgaccacc ccctatggca cccccctgta cacccccctg tacacccccc 37321 tgtacaaggg aacctacccc cctgtacagc gaccaccccc catggacacc cccctgtaca 37381 ttctacaggt atggcccgca acccattccg catgttccct ggggaccttc catactacat 37441 ggggaccatt tcctttactt cggtggtccc tgtggaccct agccagcgga atcccaccac 37501 tagccttaga gaaatggtga ccaccggcct gatttttaac cctaacctga ccggcgagca 37561 actgcgggaa tactcattca gccccctagt gtccatgggg agaaaggcaa tcttcgcaga 37621 ctacgagggt ccccagcgca ttatccacgt taccattagg gggcgctccg cggaacccaa 37681 gacccccagt gaggccctca ttatgatgga gaaggcggtc cgtggcgcgt tcgcggttcc 37741 tgattgggtg gccagggaat actcggatcc cctcccccac ggcataaccc acgtggggga 37801 cctgggcttc cccattggtt ccgtgcatgc cctgaagatg gcgctagaca cactgaagat 37861 ccatgtccct cgcggagtgg gggtccctgg ctatgagggt ctctgtggga ccaccaccat 37921 caaagccccc cgacaatatc ggctcctgac cactggagtt ttcaccaaaa aagatctgaa 37981 aagaacactt ccagaaccat tcttcagccg attttttaa c caaactcccg aagtttgtgc 38041 catcaagact ggcaaaaatc cgttttctac agaaatttgg tgtatgactc tcggcgggga 38101 tagccccgcc cccgagagaa atgaacccag aaatccccat tctctccaag attgggcaag 38161 actgggtgtc atggaaacct gcctacgtat gagtaggcgg ggactcgggt ctcggcacca 38221 cccctaccat tctctgtaac caatccctga ataaagattt gcataacaga actttgactc 38281 ctccttttat gtgggtgggg taatgggcgg cacttggggg taatggcggt tcctattgga 38341 tgggtaacac cgactccgcc ctacaaagtt aatgattgat ttttcggact tagaaaaatt 38401 tcgactgtca cctggatgtt tttccccact taacctctag ggggagatag atcgcgtcca 38461 aggggaggag ctcaataccg gaccgcctat taggtgtggc ttcgggctcc gcctagtggg 38521 aggagacagg aaaaccacgc ctagtgacgc tgggtcaaag tccaagggga gtggtttatg 38581 cgcaccgcct tggggcgtgg tttgggcggc gcaaggtaac ccttggactg ggaggagact 38641 tctgtccctt gggcgtgtca aacaggtaaa ccccacccgc gcgattaatg attaattttt 38701 cggacttaga aaattttcaa cctgatactt tattttcaag cttttcccgc cgacgggcaa 38761 gcctcctatc tctccgtcta tgactccaca gagcctcatc tgaatatgta aatgtgctga 38821 accgcaaccc cgtagaccgc gcccacccca gcatcaaagg taacgccccc gatgccacaa 38881 tgtaattacc cactgttaaa ttaggatcct tacaccaatc atttctgtac aatttaaacc 38941 accgcccacg cgggactttc ccgtggtggt agaaaaaggt tttgaaaaac gcgcgcatta 39001 ttttcgtggc ttcattaata gcggacatgc gcagatccag aaaggtcaga cacaccacca 39061 cggtcacatg acatttgcat ggagacaggg attggatacc gacacaatac gccatcggat 39121 actgaatgtc cacattggtc cgtgcatata cagtgtgcca cctcctcttg accgcagcca 39181 cgaatcccca gaagagttct tcccgccaca aaatcgaaac cggggcggcg gccccaaagc 39241 acaaatacaa aggcatcctc ctgaggctct agaaaaaaca actcattaac aggcatcccg 39301 ctcataggta cattcgtgta aacagggctg catgccaacc aatgccccgc gttttacaaa 39361 gtgacgggcc accctattgg cggagggggt ccacgtattg cgcaccgcgt aaatagaagc 39421 cacccctcgc ggaacctgtg tacattcaaa tctcctccaa atacattcgc gcagtaaagc 39481 caccgccctt ttcaagaaag tccaatcaac cttatgcgtg ggcaaaaaaa tagaagctga 39541 atataccccc gcaaactcct ccaatcggaa caggtaatct acactatagt ggg acagcat 39601 ctcaacagtt aaactttccc aggcatttat caccgtcaat ttcagatcat ggaatacggc 39661 caagttaggc tccatcaagg tcacgcggag gtggaagtaa tacatcccga aataccctgt 39721 taaaaaaaat agaaaaatga actaaccgac aataagatcg gcagtaccca gtttcgatct 39781 ggggacctcc ggagtgcaag tccgacgctc ttacggctga gctacactgt cgatcttgat 39841 ccgctagggt acgcagtccg gagaagaaat atactaagtg agacccggtc ctatatatac

39901 aggttggttc aaaggaacct ttgtacccat taaaacaggt gcgtgactgt agaagccaca 39961 cccctacctg taccgataag gcacaccctg agcaaacaaa ccataaaggt atacttcctt 40021 attcagacag gtataaatgg aacctccgca caacagtccg gtaccatttt ccatcgcgaa 40081 aatgggcaac cctactctgc tccttctttc aggtctcctt tctctgaccc aggccatttc 40141 catcggagaa cacgaaaaca aaacccggca tgtgattgta tggcggcact cctcctccca 40201 ccaatgctct gattggagaa cagtcacgga atggttcccg ccccaaaaag gcaacccggt 40261 gagaccaccc tacacccagc gggtttccct ggatacggca aacaataccc tcacggtaaa

40321 acccttcgag acaaacaacg ggtgttggga aactacgtca caaggcatta accatccacc 40381 aaccaccatt cagtaccggg tatggaacat caccaccacg cccaccatac agacaatcaa 40441 cattaccaaa ataactgttc gggaggggga ggactttacc ttatacggac ctgtgtccga 40501 aaccatgagt attatcgaat gggaattcat caaggatgtc acgccccagt tcatcctcca 40561 atactatctc tccattaact ctactattgt gtacgcaagc taccaaggga gagttacctt 40621 taaccccggt aaaaacacac taaccttaaa aggcgcgaag accaccgaca gcggcaccta 40681 caagtccacg gtgaacctcg accaggtatc cgtccacaac ttccgagtag gagtcacgcc 40741 catcgagaaa aaagaagaag ctaccgcaga gacacctgcc agcaagccca cgcccatacc 40801 acgtgtccga gcggatgctc gaagtactgc cctatgggtt ggacttgccc tttgcatcct 40861 gactgttata cccgccctta ttgggtggta cttcagagat aggctctgtg ttcccgatcc 40921 aatcattgaa ctggaaatcc ccggacaacc ccatgtaaca atacacatat tgaaaggtcc 40981 cgatgatgat tgcgaaactt aatgattgac aaacgtaata aaaaagctgt gacgcacata 41041 agtacgtgtc tgtgtcattc atccatacct atatatggtg atagcccctt cctcaataca 41101 caccgagccg cgatggaccc cagaccactt gttctgctcc tcctcctagc gtc ccatata 41161 agtacattcc ggcaaatgta ctttgaaggg gaaaccatcc atttccctat gggcatatat 41221 ggaaatgaga ccaccctcta tatgaatgac atcatcctgg aaggaacacg cgccaatacg 41281 accacccgta caatcagcct cacgaccacc aagaagaatg cgggaactaa cctgtacact 41341 gtgatctccg aaacgggaca caacgccacc tatctgataa ctgtacaacc gctgggacaa 43'01 tcgatacacc acgcctacac ttgggctgga aatactttta ccttacaagg acaggtattt 4. -51 gaacacggta attatacacg atgggtgcgg ctggagaatg cggaaccgaa actcattatc 41st ..1 agctgggcat tgtccaacag aacaataaac aaaggaccgg cctatactgc aaacatggac 41581 tttgatcccg gaaacaacac cctcactctc caccctgtgc tgataacaga tgccgggatt 41641 ttccaatgcg tcattgatca gcaaacaaac ctaaccctca ccataaactt tacagtctcc 41701 gagaatccac caatcgtagc acacctggat atccataaaa ctatttctag aacaattgcc 41761 atttgtagct gtttgcttat cgcggtaatt gcggtcttgt gttgcctacg tcagctcaat 41821 gtaaacgggc ggggaaattc cgaaatgata taaaacaata aagcagtgtg cgtcatggaa 41881 acttttctca ggtgcttcct cattcacaca ggtatatata gggaatggaa aattagacag 41941 atacccacac cggaacaatg ctacttctca cagtagttct gttg gtgggg gtcaccctcg

_k REPLACEMENT BLAπ (RULE 26) 42001 ctgcggacca tcctactcta tacgctccga aagggggcag tatagaattg ggtgtggggg 42061 ctaaacagaa agggcaatac aaatttgaat ggcggtttgg aaatctaaaa attgtgatag 42121 ccgaaatgtc atccactaac caattagaaa tcaaatttcc cgataacggt ttccaaaatc 42181 gatccgagtt taaccccacc aaacataact taaccattca taatgccagc tacgaggaca 42241 gcggaaccta ctcactccac caggaagaaa atgatggcac ggaacacacg gacaacttca 42301 aagtgattgt tcaaggtatg tcattatata catatttaca atatgcatta atatcaccta 42361 tctaatagag cattaattat ccagacccga ttccacgccc tgaagtcaag ggaaccacta 42421 tgcaaatcaa cgggaaaact ttcaccaata tatcctgcca tctaccggcc ggttcctacg 42481 gcaatgtctc ctggcattgg aattataccg acccaatcat agtcgggtac gaaaatcaga 42541 gcatgctcgt tggaccttta ggggtaatgt attcatgtac ggcatccaat caagtctcaa 42601 aaaactccag tgcaataagt atggacaccg ccgaaccatc agagagtaag tagcgccctc 42661 tatagacatt atatagaata taactgaaca cattaagaaa cctctgtaat tatttatagg 42721 agcggagtgc gcatatacag gatacattgc gggcattata atcttaggag tgctttgcat 42781 attgttcatt tacctatatg caaatacccc tgaagtgcgg cagagaataa ccg accaatt 42841 agaaaagctt ctcggaacat tctgtgacgt cagtatagaa gacggaatac cggaacgcac 42901 cagaagaaac aaaaaaagaa tcatcttaaa ggagccctcc cataggtgga tctggattca 42961 gtcatttgca taatatgctg tgtcataacc gccaccatca ccatagcaat cattgggcgt 43021 aagtattgtg acgtaagaaa aggcatgtct aaaaaaacgg tcactcacca taatgctgcc 43081 cccgataggt tgcgaccgcc ttccggagtt tgacgtagta tgcccgcgag actggatcgg 43141 atttcaaagc aagtgctact acttttcgga gtcagagtcc aattggagtg aagccgaaaa 43201 attttgtaga cagcaagagg cggagctagc agttcggcgt tccgaggagg aaaaggtaaa 43261 aagttaaatt ccaggaaact cctaattccc cgaaaaatta caaaaattaa cggagaccct 43321 ttacaggagt tccttctgcg ccaatgcgga acaggaacta actggctggg cgtaaccagg 43381 aagtcgaagg acggagctga ttgggtggat gcatcgtacg atgattacgt accatggtga 43441 gtcatgttat acgtcacatc cgggatgtga cgtatgcgga agttgatccg ggagtgaaaa 43501 cccggaagta acctgttaat ttgcatacag gtatgaaatt cggggaggcg gagactgcgt 43561 gtatttaaat ggagaccgag tgacgtcagc ctactgtgat acccagaagc tatttgtctg 43621 ttcctgtcaa gattcgtatt cgtattggtt agaaaacaaa taaatc aata aactaattta 43681 tgatatcatt catatttatg ggtgtggttt tattatgcgt cataaaacta ttttgcgtat 43741 agcgacacgc tgcggttatg gccggttatg actgcgttag tttttgaggt tattatacat 43801 catc

REPLACEMENT BLAπ (RULE 26)

Claims

claims

1. CELO virus obtained by in vitro manipulation of a plasmid cloned CELO virus DNA.

2. CELO virus according to claim 1, characterized in that it contains the left and right inverted terminal repeat and the packaging signal and in different regions thereof the CELO virus DNA has modifications in the form of insertions and / or deletions and / or mutations .

3. CELO virus according to claim 2, characterized in that it has modifications which are located on a section of the CELO virus DNA, which comprises the nucleotides from about 201 - about 5,000 and / or on a section which the Nucleotides of approx. 31,800 - approx. 43,734 and / or on a section which contains the nucleotides of approx. 28,114 -

30,495 comprises.

4. CELO virus DNA, contained on a plasmid which is replicable in bacteria or yeast and, after introduction into cells, optionally together with a plasmid which contains a gene which is possibly missing from the CELO virus DNA and is required for the development of mature virus particles complemented, delivers virus particles.

5. CELO virus according to any one of claims 2 to 4 or derived therefrom CELO virus DNA containing foreign DNA.

6. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a coding for a therapeutically active protein DNA.

7. CELO virus or CELO virus DNA according to claim 6, containing as foreign DNA a coding for an immunostimulating protein DNA.

8. CELO virus or CELO virus DNA according to claim 7, containing a DNA coding for a cytokine.

9. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA coding for a tumor antigen or a fragment thereof.

10. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA coding for an antigen derived from a human pathogen.

11. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA coding for an antigen derived from an animal pathogen.

12. CELO virus or CELO virus DNA according to claim 11, containing as foreign DNA a DNA coding for an antigen derived from a bird pathogen.

13. CELO virus or CELO virus DNA according to claim 5, containing as foreign DNA a DNA coding for a ligand for mammalian cells.

14. CELO virus or CELO virus DNA according to any one of claims 5 to 13, characterized in that it contains the foreign DNA in the Fsel interface at position 35.693 or over this interface or in the vicinity of this interface.

15. CELO virus or CELO virus DNA according to one of claims 5 to 13, characterized in that it contains the foreign DNA on a section which comprises the nucleotides of approximately 28.114-30.495 which contains the fiber 1 gene .

16. CELO virus or CELO virus DNA according to one of claims 5 to 13, characterized in that it contains the foreign DNA in the region of the reading frame at nucleotide 794, which codes for dUTPase.

17. A process for the production of recombinant CELO virus or recombinant CELO virus DNA according to any one of claims 2 to 16, characterized in that the CELO virus genome or sections thereof contained on a plasmid is genetically manipulated.

18. The method according to claim 17, characterized in that the CELO virus genome contained on a plasmid or sections thereof are manipulated in an area which is different from the left and right inverted terminal repeat and the packaging signal.

19. The method according to claim 17 or 18, characterized in that one carries out insertions and / or deletions.

20. The method according to claim 19, characterized in that a foreign gene is inserted into a naturally occurring or artificially introduced restriction site on a section of the CELO virus DNA which contains a sequence which is not required for the replication of the virus in the host cell or can be complemented.

21. The method according to claim 20, characterized in that, in addition to the insertion, a deletion is carried out in another region of the CELO virus DNA which contains a sequence which is not required or can be complemented for the replication of the virus in the host cell .

22. The method according to claim 17 or 18, characterized in that the manipulation is carried out by recombination.

23. The method according to claim 22, characterized in that recombining DNA molecules obtained by means of polymerase chain reaction.

24. The method according to claim 22, characterized in that recombining DNA molecules obtained by ligation.

25. The method according to claim 22, characterized in that recombining DNA molecules obtained by cloning in bacteria.

26. A process for the production of recombinant CELO virus DNA according to claim 25, characterized in that a CELO virus DNA fragment, containing two restriction sites, is cloned into a bacterial plasmid that occurs only once between them on the plasmid Restriction sites inserted the foreign DNA by cutting out the fragment containing the foreign DNA from the plasmid and mixing it with a plasmid which contains the entire CELO virus DNA and which was cut at a unique restriction site, and that with this mixture of DNA molecules bacteria transformed and the bacteria grown, a plasmid is obtained by recombination of the DNA molecules, which contains the entire CELO virus DNA with the foreign DNA as an insert.

27. The method according to any one of claims 20 to 26, characterized in that a reporter gene is inserted as foreign DNA.

28. The method according to claim 27, characterized in that the reporter gene has a restriction site, in which, in an additional step, a foreign DNA defined in one of claims 6 to 13 is inserted.

29. A method for producing CELO virus, characterized in that bird cells are transformed with a plasmid containing CELO virus or DNA derived therefrom, defined in one of claims 1 to 16, the cells are cultivated and the CELO virus particles are harvested.

30. The method according to claim 29, characterized in that the CELO virus DNA sequences are missing, which are necessary for the formation of mature virus particles and that the missing DNA sequences are complemented.

31. The method according to claim 30, characterized in that helper cells which complement the missing gene or genes are used as avian cells.

32. Helper cells containing, integrated in their genome, CELO virus genes.

33. helper cells according to claim 32, that the cells are avian cells.

34. The method according to claim 30, characterized in that the cells are also transformed with a plasmid which complements the missing gene or the CELO virus DNA, necessary for the formation of mature virus particles.

35. The method according to claim 30, characterized in that the cells are also infected with a helper virus which complements the missing gene or the CELO virus DNA, necessary for the formation of mature virus particles.

36. Medicament for use in gene therapy, containing a CELO virus according to claim 6.

37. Vaccine against human infectious diseases, containing a CELO virus according to claim 10.

38. vaccine against infectious diseases of animals, containing a CELO virus according to claim 11.

39. vaccine against infectious diseases of birds, containing a CELO virus according to claim 12.

40. CELO virus according to claim 7, 8 or 9 for the manufacture of cancer vaccines.