cDNA CLONE FOR SOUTH AFRICAN ARBOVIRUS NO. 86

Abstract: The present invention provides a recombinant DNA comprising a cDNA coding for an infectious South African Arbovirus No. 86 (S.A.AR86) virus RNA transcript and a heterologous promoter positioned upstream from the cDNA and operatively associated therewith. The present invention also provides an infectious RNA transcript encoded by the cDNA, and infectious attenuated viral particles produced from cells transfected with the RNA transcript encoded by the cDNA.

Description: cDNA CLONE FOR SOUTH AFRICAN ARBOVIRUS NO. 86 Field of the Invention The present invention relates to live attenuated vaccines in general, and particularly relates to attenuated vaccines produced from South African Arbovims No. 86 (S.A.AR86) virus. Background of the Invention This invention was made with government support under Grant No. 2-ROIA122186 (09-13) awarded by the National Institutes of Health. The government has certain rights in the invention. South African Arbovims No. 86 (S. A. AR86) is an isolate of Sindbis vims. S.A.AR 86 was originally isolated from mosquitoes. The vims is antigenically related to Sindbis vims and to two other Sindbis vims isolates, Girdwood S.A. and Ockelbo82. See, Malherbe et al. , South African Medical Journal, 37:547 (1963) and Niklasson et al., Am. J. Trop. Med. Hyg. 33:1212 (1984), respectively. The latter is associated with a human disease, also known as Ockelbo. Sindbis vims is the prototype member of the alphavims genus of the family Togaviridae. The Sindbis vims includes various strains, including S.A.AR86 and Sindbis AR339. The genome of Sindbis vimses consists of a single strand of RNA which contains the information for the viral genes, and which is infectious when introduced into the cytoplasm of cells. Full-length cDNA clones of positive-strand RNA vimses are important tools for the study of the biology of vimses including Sindbis vimses. It is known with respect to several viral systems that in vitro transcripts of cDNA clones, and in some cases the cDNA itself, can initiate a complete and productive infectious cycle upon introduction into susceptible cells. See Racaniello et al., Science 214:916 (1981); Ahlquist et al. , Proc. Natl. Acad. Sci. USA 81:7066 (1984); Kaplan et al. , Proc. Natl. Acad. Sci. USA 82:8424 (1985); Mizutani et al., J. Virol. 56:628 (1985); van der Werf, Proc. Natl. Acad. Sci. USA 83:2330 (1986); Rice et al., J. Virol. 61:3809 (1987); and Nos et al. , Virology 165:33 (1988). This has made it possible to test progeny vims for phenotypic manifestations of directed mutations and recombinations which have been introduced into the cDNA clone. Pathogenesis studies with several positive-strand vimses, including the picornaviruses and the alphavirases have been advanced significantly by the use of full-length cDNA clones. As another useful application, live attenuated viral vaccines may be produced using full-length cDN A clones. Live attenuated viral vaccines are among the most successful means of controlling viral disease. However for some vims pathogens, immunization with a live vims strain may be either impractical or unsafe. Sindbis vims is subclinical in humans, but is closely related to other vimses which do induce clinical diseases in humans, such as Ockelbo, an epidemic polyarthritis syndrome common in areas of Scandinavia and Northern Europe. Accordingly, Sindbis vims vaccines are desirable for producing an immunogenic response to such clinical diseases. Sindbis vims vaccines are also desirable as viral carriers in vims constmcts which express genes encoding immunizing antigens for other vimses. See U.S. Patent No. 5,217,879 to Huang et al. Huang et al. describes Sindbis infectious viral vectors. However, the reference does not describe the cDNA sequence of S.A.AR86 vims, or clones or viral vectors produced therefrom. Accordingly, there remains a need in the art for full-length cDNA clones of positive-strand RNA vimses, such as the S.A.AR86 strain of Sindbis. In addition, there is a need in the art for full-length cDNA clones of S.A.AR86 encoding infectious RNA transcripts. Further, there remains a need in the art for cDNA clones of S.A.AR86 which encode RNA transcripts which may be used to produce infectious attenuated viral particles, and methods of producing such viral particles. Summary of the Invention As a first aspect, the present invention provides a recombinant DNA comprising a cDNA coding for an infectious South African Arbovims No. 86 (S.A.AR86) vims RNA transcript and a heterologous promoter positioned upstream from the cDNA and operatively associated therewith. The cDNA is selected from the group consisting of (i) cDNA having the sequence given herein as SEQ ID NO. : 1, (ii) cDNA having the same RNA coding sequence as the cDNA given herein as SEQ ID NO. : 1, and (Hi) cDNA according to (0 or (ii) above and further containing at least one attenuating mutation. Preferably at least one attenuating mutation is included in the cDNA, and more preferably at least two attenuating mutations are included in the cDNA. Attenuating mutations may, for example, be provided in any of the nsPl, E2, and nsP2 coding regions of the cDNA. Preferably at least one silent mutation is included in the cDNA in addition to the attenuating mutation(s). As a second aspect, the present invention provides an infectious RNA transcript encoded by the cDNA. As a third aspect, the present invention provides infectious attenuated viral particles containing the RNA transcript encoded by the cDNA. The foregoing and other aspects of the present invention are explained in detail in the detailed description set forth below. Brief Description of the Drawings Figure 1 shows the relationship of the 3' half of the nsP3 gene among various Sindbis-like isolates. Figure 2 shows the replacement of of the AR339-derived cDNAs into the plasmid pTotollOl background. Detailed Description of the Invention The South African Arbovims No. 86 (S.A.AR86) viral clones employed in practicing the present invention are genomic clones which code for an RNA transcript, which RNA trasncript is capable of producing live encapsidated S.A.AR86 vims when used to transfect a S.A.AR86 vims-permissive cell. S.A.AR86 vims-permissive cells are cells which, upon transfection with the viral RNA transcript, are capable of producing viral particles. The S.A.AR86 vims has a broad host cell range. Examples of suitable host cells include, but are not limited to Nero cells, baby hamster kidney (BHK-21) cells, and chicken embryo fibroblast cells. Uptake of the RΝA into the cells can be achieved by any suitable means, such as for example, by treating the cells with DEAE-dextran, treating the cells with "LIPOFECTIN™", and by electrophoresis, with electrophoresis being the currently preferred means of achieving RNA uptake into the host cells. The phrases "attenuating mutation" and "attenuating amino acid, " as used herein, mean a nucleotide mutation or an amino acid coded for in view of such a mutation which result in a decreased probability of causing disease in its host (i.e., a loss of vimlence), in accordance with standard terminology in the art. See, e.g., B. Davis, et al., Microbiology 132 (3d ed. 1980), whether the mutation be a substitution mutation or an in-frame deletion mutation. The phrase "attenuating mutation" excludes mutations which would be lethal to the vims. The phrase "silent mutation" as used herein refers to mutations in the cDNA coding sequence which do not produce mutations in the corresponding protein sequence translated therefrom. The cDNA clone has a sequence as given herein as SEQ ID NO.: 1. Alternatively, the cDNA clone may have a sequence which differs from the cDNA of SEQ ID NO. : 1, but which has the same RNA coding sequence as the cDNA given herein as SEQ ID NO.: 1. Thus, the cDNA clone may include one or more silent mutations. For example, the clone sequence may differ from the wild-type S.A.AR86 sequence given herein as SEQ ID NO.: 1, by the inclusion of silent mutations at any or all of nucleotides 215, 3863, 5984, and 9113. The silent mutations at the foregoing nucleotides may be substitution or in-frame deletion mutations, such as for example, the substitution of guanine for adenine at nucleotide 215 of the cDNA sequence given herein as SEQ ID NO.: 1; or the substitution of guanine for cytosine at nucleotide 3863 of the cDNA sequence given herein as SEQ ID NO. : 1; or the substitution of guanine for adenine at nucleotide 5984 of the cDNA sequence given herein as SEQ ID NO.: 1; or the substitution of cytosine for thymine at nucleotide 9113 of the cDNA sequence given herein as SEQ ID NO. : 1. In yet another emobidment, the cDNA clone has a sequence according to either of the foregoing described sequences, but which also includes attenuating mutations. The attenuating mutations being described more fully hereinafter. Promoter sequences and S.A.AR86 vims cDNA clones are operatively associated in the present invention such that the promoter causes the cDNA clone to be transcribed in the presence of an RNA polymerase which binds to the promoter. The promoter is positioned on the 5' end (with respect to the virion RNA sequence), or "upstream" from, the cDNA clone. An excessive number of nucleotides between the promoter sequence and the cDNA clone will result in the inoperability of the constmct. Hence, the number of nucleotides between the promoter seuqence and the cDNA clone is preferably not more than eight, more preferably not more than five, still more preferably not more than three, and most preferably not more than one. Examples of promoters which are useful in the cDNA sequences of the present invention include, but are not limited to T3 promoters, T7 promoters, and SP6 promoters. The DNA sequence of the present invention may reside in any suitable transcription vector. The DNA sequence preferably has a complementary DNA sequence bonded thereto so that the double-stranded sequence will serve as an active template for RNA polymerase. The transcription vector preferably comprises a plasmid. When the DNA sequence comprises a plasmid, it is preferred that a unique restriction site be provided 3' (with respect to the virion RNA sequence) to (i.e., "downstream" from) the cDNA clone. This provides a means for linearizing the DNA sequence to allow the transcription of genome-length RNA in vitro. The cDNA clone can be generated by any of a variety of suitable methods known to those skilled in the art. A preferred method is the method set forth in U.S. Patent No. 5,185,440 to Davis et al., and Gubler et al. , Gene 25:263 (1983), the disclosures of which are incorporated herein by reference in their entirety. Attenuating mutations of S.A.AR86 are identified by sequencing attenuated strains of the S.A.AR86 vims and comparing the sequence of the attenuated strain with the sequence of the corresponding wild-type vims. Serial passage techniques for the generation of attenuated strains may be carried out in accordance with known procedures. Preferably, the atenuated strains are generated by selecting strains at each passage during serial passage in cell culture which either grow rapidly or penetrate the cell more rapidly. This selection process, which reduces the number of serial passages required to obtain attenuated strains, is known. See, e.g., Olmstead et al., Science 225:424 (1984); and Johnston et al. , Virology 162:437 (1988). the disclosures of which are incorporated herein by reference in their entirety. cDNA clones may be modified to incorporate attenuating mutations by site-directed mutagenesis in accordance with known procedures. An exemplary technique is that of Kunkel Proc. Natl. Acad. Sci. (USA) 82:488 (1985). These same techniques may be used to join the heterologous promoter to the cDNA clone. RNA is preferably synthesized from the DNA sequence in vitro using purified RNA polymerase in the presence of ribonucleotide triphosphates in accordance with conventional techniques. Pharmaceutical compositions, such as vaccines, containing the S.A.AR86 clone of the present invention comprise an immunogenic amount of a live attenutated vims as disclosed herein in combination with a pharmaceutically acceptable carrier. An "effective immunogenic amount" is an amount of the attenuated vims sufficient to evoke an immune response in the subject to which the vaccine is administered. An amount of about 10 1 to about 10 5 plaque forming units per dose is believed to be suitable, depending upon the age and species of the subject being treated. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution. Subjects which may be administered immunogenic amounts of the live attenuated vimses of the present invention include both human and animal (e.g., horse, donkey, mouse, hamster, or monkey) subjects. Administration may be by a suitable means, such as intraperitoneoal, intracerebral or intramuscular injection. Complimentary DNA clones of the S.A.AR86 vims are made in accordance with the procedures described herein, as supplemented with procedures known in the art. We employed as a starting material, the S.A.AR86 vims. A first exemplary attenuating substitution mutation in a S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably isoleucine, at nsPl amino acid residue 538. A second exemplary attenuating subsitution mutation in a S. A. AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably threonine, at E2 amino acid residue 304. A third exemplary attenuating subsitution mutation in a S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably lysine, at E2 amino acid residue 314. A fourth exemplary attenuating subsitution mutation in a S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably valine, at E2 amino acid residue 372. A fifth exemplary attenuating substitution mutation in a S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably alanine, at E2 amino acid residue 376. One embodiment of the present invention contains, in combination, said attenuating substitution mutations at E2 amino acid residues 304, 314, 372, and 376. A sixth exemplary attenuating subsitution mutation in a S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably glycine, at nsP2 amino acid residue 96. A seventh exemplary attenuating subsitution mutation in a S. A. AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably valine, at nsP2 amino acid residue 372. One embodiment of the present invention contains, in combination, said attenuating substitution mutations at nsP2 amino acid residues 96 and 372. An eighth exemplary attenuating substitution mutation in an S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably leucine, at nsP2 amino acid residue 529. A nineth exemplary attenuating substitution mutation in an S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably asparagine, at nsP2 amino acid residue 571. A tenth exemplary attenuating substitution mutation in an S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably arginine, at nsP2 amino acid residue 682. An eleventh exemplary attenuating substitution mutation in an S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably arginine, at nsP2 amino acid residue 804. A twelveth exemplary attenuating substitution mutation in an S.A.AR86 clone useful in practicing the present invention is a substitution mutation which codes for an attenuating amino acid, preferably arginine, at nsP3 amino acid residue 22. One embodiment of the present invention contains, in combination, said attenuating substitution mutations at nsP2 amino acid residues 529, 571, 682, and 804, and at nsP3 amino acid residue 22. The cDNA clones according to the present invention are useful for the preparation of pharmaceutical formulations, such as vaccines, as described above. In addition, the cDNA clones of the present invention are useful for administration to animals for the purpose of producing antibodies to the S . A. AR86 vims, which antibodies may be collected and used in known diagnostic techniques for the detection of S.A.AR86 vims. The following examples are provided to illustrate the present invention, and should not be constmed as limiting thereof. In these examples, nt means nucleotide. EXAMPLE 1 Relationship of S.A.AR86 Clone to Other Sindbis Strains At the nucleotide level, S.A.AR86 differs from the consensus sequence of Sindbis strain AR339 by 685 nucleotides (111 amino acids). From the published Sindbis HR sequence (See, Strauss et al., Virol. 133:92 (1984)), S.A.AR86 differs by 704 nucleotides and 119 amino acids. S.A.AR86 differs from the sequence of Ockelbo82 (See, Shirako et al. , Virol. 182:753 (1991)) by 430 nucleotides (67 amino acids). Included in these differences are several insertions and deletions present in S.A. AR86 relative to the Sindbis sequences The relationship of the 3' half of the nsP3 gene among various Sindbis-like isolates is shown in Figure 1. EXAMPLE 2 Observed Mortality in Mice Infected with S.A.AR86 When adult mice are inoculated intracerebrally (i.e.) with low doses of S.A.AR86, 100% motality is observed. The methods employed for evaluating the instance mortality in mice are set forth in D. Russell, et al., J. Virol. 63(4): 1619 (1989), the disclosure of which is incorporated herein by reference in its entirety. This high mortality rate is unique among "Sindbis-like" vimses which are characteristically avimlent in adult mice. Also unique in the S. A.AR86 strain is the cys substitution for the opal stop codon normally found between the nsP3 and nsP4 genes. EXAMPLE 3 Construction of S.A.AR86 Clone The S.A.AR86 clone is constmcted by substituting partial cDNA clones of S.A.AR86 genomic RNA into pTR5000, one of a series of Sindbis AR339 cDNA clones. Constmction of pTR5000 (a full-length cDNA clone of Sindbis following the SP6 phage promoter and containing mostly Sindbis AR339 sequences) is accomplished by sequential replacement of AR339-derived cDNAs into the plasmid pTotollOl background, according to the technique described in Rice et al., J. Virol. 61:3809 (1987). The replacement of of the AR339-derived cDNAs into the plasmid pTotollOl background is shown in Figure 2. Production of the cDNAs used in constmcting pTR5000 has been described previously in Polo et al., J. Virol. 62:2124 (1988), as has the construction of pTR2000, see Polo et al. , J. Virol. 62:2124 (1988) and Polo et al. , J. Virol. 64:4438 (1990). Nucleotide numbering follows that of Strauss et al. Virol. 133:92 (1984). The StuI (nt8571) to SacII (ntll484) fragment of pTotollOl is removed and replaced with the analogous fragment from clone pSB4 to form pTR2000, using the loss of the pTotollOl StuI site at ntl0770 (a site not present in AR339) as a screen, as shown in Figure 2. The sequences of AR339 and pTotollOl are identical from ntl l485 to the 3'-end (ntll703). Therefore, as shown in Figure 2, these 3' sequences are of AR339 origin. Constmction of pTR3000 is accomplished by replacement of the BssHII (nt9804) to StuI (nt8571) fragment of pSB3 into pTR2000 from which the analogous fragment had been removed. The Aflll site found at nt8835 in pTotollOl but absent in AR339 is used to screen the recombinants. An AR339 fragment from pSBl, Spel (nt5262) to BssHII, is used to replace the Spel-BssHII fragment from pTR2000, using the Aflll screen and forming pTR4000. To constmct pTR5000, ρSB5 is subcloned into pUC119, and the PstI site at nt3953 is ablated using site-directed mutagenesis, as described in Kunkel, Methods Enzymol. 154:367 (1987), to change nt3950 from U to C. The Clal (nt2713) to Spel fragment is removed from the mutagenized subclone and for pTR5000, is used to replace the analogous fragment of pTR4000 using the ablated PstI site as a screen. Partial cDNA clones of S.A.AR86 were obtained using classical reverse transcriptase (RT) procedures according to Polo et al., J. Virol. 62:2124 (1988), as well as RT-PCR protocols according to Heidner et al. , J. Virol. 68:2683 (1994). These cDNA clones are used to replace analogous portions of the clone pTR5000, culminating in the constmction of a full-length cDNA of the S.A.AR86 genomic sequence downstream of an SP6 promoter and followed by a poly (A) tract and a unique Xbal site. During the course of replacing S.A.AR86 sequences into pTR5000, it was observed that the pTR5000 nonstructural proteins are incompatible with those of S.A.AR86, so that the chimeric clones yielded transcripts which are not infectious for baby hamster kidney (BHK-21) cells. In addition, the restriction sites in the nonstractural region of S.A.AR86 are very different from the Sindbis AR339-based clones. The first complete S.A.AR86 clone, pSIO, also failed to yield infectious transcripts. The constmction is repeated, beginning with pTR5000, using sequences derived from the same partial cDNA clones of S.A.AR86 as are used for the constmction of pSIO except for nucleotides 3171 to 6410 (numbering from nucleotide 1 of the S.A.AR86 sequence), which are derived by RT-PCR of the genomic RNA. The resulting constmct, pS22, gives infectious transcripts, but the vims derived therefrom is temperature-sensitive. Replacement of nucleotides 3171 to 6410 with an alternative RT-PCR derived cDNA corrected the temperature- sensitive defect, yielding clone pS24. EXAMPLE 4 Observed Mortality in Mice Infected with S.A.AR86 Upon i.e. inoculation vims derived from pS24 is avirulent, whereas S.A.AR86 caused 100% mortality. Clearly, pS24 contained one or more mutations which were strongly attenuating. The complete sequence of pS24 was determined directly from pS24 and related clones. Comparison with the S.A.AR86 genomic RNA sequence reveals 5 mutations or clusters of mutations which are potentially associated with the avimlent phenotype of vims from pS24. These included the mutations or clusters of mutations indicated in clones pS56, pS51, and pS57, a mutation at ntl278 A-C (nsPl 407 K→Q), and a mutation at nt5972 T→G (nsP3 228 N →S). While said nsPl 407 substitution alone is a lethal mutation, the vimses are viable when the mutation co-exists with a serine at the nsP3 amino acid residue 228. Clone pS24 is corrected by by a combination of site- directed mutagenesis and replacement of specific pS24 sequences with cDNA fragments which do not contain the subject mutations. The resulting cDNA is pS55 which contains 5' and 3' untranslated sequences identical to S.A.AR86 genomic RNA, no coding differences with S.A.AR86 genomic RNA, and the 4 non-coding changes at nt 215 A-G, nt 3863 C-G, nt 5984 A-G, and nt 9113 T-C. Vims derived from pS55 is indistinguishable from native S.A.AR86 in tests of vimlence, and growth in adult mice and by histopathological analysis of tissues from infected animals. These results, which are reported in Table 1 below, indicate that vims derived from clone pS55 accurately reflects native S.A.AR86 both in terms of coding sequence and in vivo phenotype. Table 1 VIRULENCE IN MICE AGE OF MICE S.A.AR86 S55 S56 2 S51 3 4 to 6 Weeks Mortality 100% 100% 80% 20% AST 1 6.36 ± 1.39 6.37 ± 1.4 9.37 + 1.77 8.5 ± 0.7 'Average Survival Time in days. 2 Virus isogenic with S55 except at nucleotide 6 (C -*A). 3 Virus isogenic with S55 except at nucleotide 1648 (C →T). This is nsPl amino acid 538 (T →l). The isoleucine is the amino acid found in all other Sindbis isolates sequenced to date. The 3 mutations or clusters of mutations in clones pS56, pS51, and pS57 are placed independently into the pS55 background. Vims derived from each of these clones is highly attenuated in adult mice inoculated i.e. In clone pS53, the mutations at nucleotides 6 and 1672 are combined, and the resulting vims is avimlent in the adult mouse model. The mutations in pS61 are present in pS48, an intermediate clone constructed during the repair of pS24. The vims from pS48 produced small plaques on BHK-21 cells. When these mutations are placed in the pS55 background, they also gave a highly attenuated phenotype. (S61 , vims derived from pS61, gave 33.3% mortality (6±4.2)). The foregoing is illustrative of the present invention and is not to be constmed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Johnston, Robert E. Simpson, Dennis Davis. Nancy L. (ii) TITLE OF INVENTION: cDNA Clone for South African Arbov rus No. 86 (ii ) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Kenneth D. Si ley (B) STREET: Post Office Drawer 34009 (C) CITY: Charlotte (D) STATE: NC (E) COUNTRY: USA (F) ZIP: 28234 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0. Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Sibley. Kenneth D. (B) REGISTRATION NUMBER: 31,665 (C) REFERENCE/DOCKET NUMBER: 5470-118 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (919) 881-3140 (B) TELEFAX: (919) 881-3175 (C) TELEX: 575102 (2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11663 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: ATTGGCGGCG TAGTACACAC TAπGAATCA AACAGCCGAC CAAπGCACT ACCATCACAA 60 TGGAGAAGCC AGTAGπAAC GTAGACGTAG ACCCTCAGAG TCCGTTTGTC GTGCAACTGC 120 AAAAGAGCπ CCCGCAAπT GAGGTAGTAG CACAGCAGGT CACTCCAAAT GACCATGCTA 180 ATGCCAGAGC ATTTTCGCAT CTGGCCAGTA AACTAATCGA GCTGGAGGπ CCTACCACAG 240 CGACGATTπ GGACATAGGC AGCGCACCGG CTCGTAGAAT GT CCGAG CACCAGTACC 300 AπGCGπTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAA TATGCCAGCA 360 AACTGGCGGA AAAAGCATGT AAGAπACAA ACAAGAACTT GCATGAGAAG ATCAAGGACC 420 TCCGGACCGT ACπGATACA CCGGATGCTG AAACGCCATC ACTCTGCTTC CACAACGATG 480 πACCTGCAA CACGCGTGCC GAGTACTCCG TCATGCAGGA CGTGTACATC AACGCTCCCG 540 GAACTATTTA CCACCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATT GGCTTCGACA 600 CCACCCAGπ CATGπCTCG GCTATGGCAG GπCGTACCC TGCATACAAC ACCAACTGGG 660 CCGACGAAAA AGTCCTTGAA GCGCGTAACA TCGGACTCTG CAGCACAAAG CTGAGTGAAG 720 GCAGGACAGG AAAGπGTCG ATAATGAGGA AGAAGGAGπ GAAGCCCGGG TCACGGGTπ 780 ATFTCTCCGT TGGATCGACA CTΓTACCCAG AACACAGAGC CAGCπGCAG AGCTGGCATC 840 πCCATCGGT GπCCACπG AAAGGAAAGC AGTCGTACAC πGCCGCTGT GATACAGTGG 900 TGAGCTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATC ACGGGAGAAA 960 CCGTGGGATA CGCGGπACA AACAATAGCG AGGGCπcπ GCTATGCAAA GπACCGATA 1020 CAGTAAAAGG AGAACGGGTA TCGπCCCCG TGTGCACGTA TATCCCGGCC ACCATATGCG 1080 ATCAGATGAC CGGCATAATG GCCACGGATA TCTCACCTGA CGATGCACAA AAACπCTGG 1140 πGGGCTCAA CCAGCGAATC GTCAπAACG GTAAGACTAA CAGGAACACC AATACCATGC 1200 AAAAπACCT TCTGCCAATC AπGCACAAG GGπCAGCAA ATGGGCCAAG GAGCGCAAAG 1260 AAGATCTTGA CAATGAAAAA ATGCTGGGCA CCAGAGAGCG CAAGCπACA TATGGCTGCT 1320 TGTGGGCGπ TCGCACTAAG AAAGTGCACT CGπCTATCG CCCACCTGGA ACGCAGACCA 1380 TCGTAAAAGT CCCAGCCTCT TTTAGCGCπ TCCCCATGTC ATCCGTATGG ACTACCTCπ 1440 TGCCCATGTC GCTGAGGCAG AAGATGAAAT TGGCAπACA ACCAAAGAAG GAGGAAAAAC 1500 TGCTGCAAGT CCCGGAGGAA πAGπATGG AGGCCAAGGC TGCTTTCGAG GATGCTCAGG 1560 AGGAATCCAG AGCGGAGAAG CTCCGAGAAG CACTCCCACC AπAGTGGCA GACAAAGGTA 1620 TCGAGGCAGC TGCGGAAGπ GTCTGCGAAG TGGAGGGGCT CCAGGCGGAC ACCGGAGCAG 1680 CACTCGTCGA AACCCCGCGC GGTCATGTAA GGATAATACC TCAAGCAAAT GACCGTATGA 1740 TCGGACAGTA TATCGπGTC TCGCCGATCT CTGTGCTGAA GAACGCTAAA CTCGCACCAG 1800 CACACCCGCT AGCAGACCAG GπAAGATCA TAACGCACTC CGGAAGATCA GGAAGGTATG 1860 CAGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG AAGTGCCGTA CCATGGCCAG 1920 AAπcπAGC ACTGAGTGAG AGCGCCACGC πGTGTACAA CGAAAGAGAG TTTGTGAACC 1980 GCAAGCTGTA CCATAπGCC ATGCACGGTC CCGCTAAGAA TACAGAAGAG GAGCAGTACA 2040 AGGπACAAA GGCAGAGCTC GCAGAAACAG AGTACGTGπ TGACGTGGAC AAGAAGCGAT 2100 GCGπAAGAA GGAAGAAGCC TCAGGACTTG TCCTTTCGGG AGAACTGACC AACCCGCCCT 2160 ATCACGAACT AGCTCπGAG GGACTGAAGA CTCGACCCGC GGTCCCGTAC AAGGπGAAA 2220 CAATAGGAGT GATAGGCACA CCAGGATCGG GCAAGTCAGC TATCATCAAG TCAACTGTCA 2280 CGGCACGTGA TCπGπACC AGCGGAAAGA AAGAAAACTG CCGCGAAAπ GAGGCCGACG 2340 TGCTACGGCT GAGGGGCATG CAGATCACGT CGAAGACAGT GGAπCGGπ ATGCTCAACG 2400 GATGCCACAA AGCCGTAGAA GTGCTGTATG πGACGAAGC GπCCGGTGC CACGCAGGAG 2460 CACTACπGC CπGAπGCA ATCGTCAGAC CCCGTAAGAA GGTAGTACTA TGCGGAGACC 2520 CTAAGCAATG CGGAπCTTC AACATGATGC AACTAAAGGT ACAπTCAAC CACCCTGAAA 2580 AAGACATATG TACCAAGACA πCTACAAGT TTATCTCCCG ACGπGCACA CAGCCAGTCA 2640 CGGCTAπGT ATCGACACTG CAπACGATG GAAAAATGAA AACCACAAAC CCGTGCAAGA 2700 AGAACATCGA AATCGACAπ ACAGGGGCCA CGAAGCCGAA GCCAGGGGAC ATCATCCTGA 2760 CATGTπCCG CGGGTGGGπ AAGCAACTGC AAATCGACTA TCCCGGACAT GAGGTAATGA 2820 CAGCCGCGGC CTCACAAGGG CTAACCAGAA AAGGAGTATA TGCCGTCCGG CAAAAAGTCA 2880 ATGAAAACCC GCTGTACGCG ATCACATCAG AGCATGTGAA CGTGπGCTC ACCCGCACTG 2940 AGGACAGGCT AGTATGGAAA ACTTTACAGG GCGACCCATG GAπAAGCAG CTCACTAACG 3000 TACCTAAAGG AAATITTCAG GCCACCATCG AGGACTGGGA AGCTGAACAC AAGGGAATAA 3060 πGCTGCGAT AAACAGTCCC GCTCCCCGTA CCAATCCGπ CAGCTGCAAG ACTAACGπT 3120 GCTGGGCGAA AGCACTGGAA CCGATACTGG CCACGGCCGG TATCGTACTT ACCGGπGCC 3180 AGTGGAGCGA GCTGTTCCCA CAGTTTGCGG ATGACAAACC ACACTCGGCC ATCTACGCCT 3240 TAGACGTAAT πGCAπAAG πTTTCGGCA TGGACπGAC AAGCGGGCTG TTTTCCAAAC 3300 AGAGCATCCC GπAACGTAC CATCCTGCCG ACTCAGCGAG GCCAGTAGCT CATTGGGACA 3360 ACAGCCCAGG AACACGCAAG TATGGGTACG ATCACGCCGT TGCCGCCGAA CTCTCCCGTA 3420 GATTTCCGGT GπCCAGCTA GCTGGGAAAG GCACACAGCT TGATTTGCAG ACGGGCAGAA 3480 CTAGAGπAT CTCTGCACAG CATAACπGG TCCCAGTGAA CCGCAATCTC CCTCACGCCT 3540 TAGTCCCCGA GCACAAGGAG AAACAACCCG GCCCGGTCGA AAAAπcπG AGCCAGπCA 3600 AACACCACTC CGTACπGTG ATCTCAGAGA AAAAAAπGA AGCTCCCCAC AAGAGAATCG 3660 AATGGATCGC CCCGAπGGC ATAGCCGGCG CAGATAAGAA CTACAACCTG GCTπCGGGT 3720 πCCGCCGCA GGCACGGTAC GACCTGGTGT TCATCAATAT TGGAACTAAA TACAGAAACC 3780 ATCACTTTCA ACAGTGCGAA GACCACGCGG CGACCπGAA AACCCπTCG CGπCGGCCC 3840 TGAACTGCCT TAACCCCGGA GGCACCCTCG TGGTGAAGTC CTACGGπAC GCCGACCGCA 3900 ATAGTGAGGA CGTAGTCACC GCTCπGCCA GAAAAπTGT CAGAGTGTCT GCAGCGAGGC 3960 CAGAGTGCGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CCGACAACTA GACAACAGCC 4020 GCACACGACA AπCACCCCG CATCATTTGA AπGTGTGAT πCGTCCGTG TACGAGGGTA 4080 CAAGAGACGG AGπGGAGCC GCACCGTCGT ACCGTACTAA AAGGGAGAAC AπGCTGATT 4140 GTCAAGAGGA AGCAGπGTC AATGCAGCCA ATCCACTGGG CAGACCAGGA GAAGGAGTCT 4200 GCCGTGCCAT CTATAAACGT TGGCCGAACA GTTTCACCGA πCAGCCACA GAGACAGGTA 4260 CCGCAAAACT GACTGTGTGC CAAGGAAAGA AAGTGATCCA CGCGGπGGC CCTGATTTCC 4320 GGAAACACCC AGAGGCAGAA GCCCTGAAAT TGCTGCAAAA CGCCTACCAT GCAGTGGCAG 4380 ACπAGTAAA TGAACATAAT ATCAAGTCTG TCGCCATCCC ACTGCTATCT ACAGGCATTT 4440 ACGCAGCCGG AAAAGACCGC CTTGAGGTAT CACTTAACTG CπGACAACC GCGCTAGACA 4500 GAACTGATGC GGACGTAACC ATCTACTGCC TGGATAAGAA GTGGAAGGAA AGAATCGACG 4560 CGGTGCTCCA AOTAAGGAG TCTGTAACTG AGCTGAAGGA TGAGGATATG GAGATCGACG 4620 ACGAGπAGT ATGGATCCAT CCGGACAGπ GCCTGAAGGG AAGAAAGGGA πCAGTACTA 4680 CAAAAGGAAA GπGTAπCG TACTTTGAAG GCACCAAAπ CCATCAAGCA GCAAAAGATA 4740 TGGCGGAGAT AAAGGTCCTG πCCCAAATG ACCAGGAAAG CAACGAACAA CTGTGTGCCT 4800 ACATAπGGG GGAGACCATG GAAGCAATCC GCGAAAAATG CCCGGTCGAC CACAACCCGT 4860 CGTCTAGCCC GCCAAAAACG CTGCCGTGCC TCTGTATGTA TGCCATGACG CCAGAAAGGG 4920 TCCACAGACT CAGAAGCAAT AACGTCAAAG AAGπACAGT ATGCTCCTCC ACCCCCCTTC 4980 CAAAGTACAA AATCAAGAAT GπCAGAAGG πCAGTGCAC AAAAGTAGTC CTGTTTAACC 5040 CGCATACCCC CGCAπCGπ CCCGCCCGTA AGTACATAGA AGCACCAGAA CAGCCTGCAG 5100 CTCCGCCTGC ACAGGCCGAG GAGGCCCCCG GAGπGTAGC GACACCAACA CCACCTGCAG 5160 CTGATAACAC CTCGCTTGAT GTCACGGACA TCTCACTGGA CATGGAAGAC AGTAGCGAAG 5220 GCTCACTCπ πCGAGCπT AGCGGATCGG ACAACTACCG AAGGCAGGTG GTGGTGGCTG 5280 ACGTCCATGC CGTCCAAGAG CCTGCCCCTG πCCACCGCC AAGGCTAAAG AAGATGGCCC 5340 GCCTGGCAGC GGCAAGAATG CAGGAAGAGC CAACTCCACC GGCAAGCACC AGCTCTGCGG 5400 ACGAGTCCCT TCACCTTTCT TTTGATGGGG TATCTATATC CTTCGGATCC CππCGACG 5460 GAGAGATGGC CCGCπGGCA GCGGCACAAC CCCCGGCAAG TACATGCCCT ACGGATGTGC 5520 CTATGTCΗT CGGATCGTTT TCCGACGGAG AGAπGAGGA GπGAGCCGC AGAGTAACCG 5580 AGTCGGAGCC CGTCCTGTTT GGGTCATπG AACCGGGCGA AGTGAACTCA AπATATCGT 5640 CCCGATCAGC CGTATCTΠT CCACCACGCA AGCAGAGACG TAGACGCAGG AGCAGGAGGA 5700 CCGAATACTG TCTAACCGGG GTAGGTGGGT ACATATπTC GACGGACACA GGCCCTGGGC 5760 ACπGCAAAA GAAGTCCGπ CTGCAGAACC AGCπACAGA ACCGACCπG GAGCGCAATG 5820 πCTGGAAAG AATCTACGCC CCGGTGCTCG ACACGTCGAA AGAGGAACAG CTCAAACTCA 5880 GGTACCAGAT GATGCCCACC GAAGCCAACA AAAGCAGGTA CCAGTCTCGA AAAGTAGAAA 5940 ACCAGAAAGC CATAACCACT GAGCGACTGC πTCAGGGCT ACGACTGTAT AACTCTGCCA 6000 CAGATCAGCC AGAATGCTAT AAGATCACCT ACCCGAAACC ATCGTAπCC AGCAGTGTAC 6060 CAGCGAACTA CTCTGACCCA AAGTfTGCTG TAGCTGTTTG TAACAACTAT CTGCATGAGA 6120 AπACCCGAC GGTAGCATCT TATCAGATCA CCGACGAGTA CGATGCTTAC πGGATATGG 6180 TAGACGGGAC AGTCGOTGC CTAGATACTG CAACTΠTTG CCCCGCCAAG CπAGAAGπ 6240 ACCCGAAAAG ACACGAGTAT AGAGCCCCAA ACATCCGCAG TGCGGπCCA TCAGCGATGC 6300 AGAACACGπ GCAAAACGTG CTCAπGCCG CGACTAAAAG AAACTGCAAC GTCACACAAA 6360 TGCGTGAACT GCCAACACTG GACTCAGCGA CAπCAACGT TGAATGCTTT CGAAAATATG 6420 CATGCAATGA CGAGTAπGG GAGGAGπTG CCCGAAAGCC AAπAGGATC ACTACTGAGT 6480 TCGπACCGC ATACGTGGCC AGACTGAAAG GCCCTAAGGC CGCCGCACTG πCGCAAAGA 6540 CGCATAATTT GGTCCCAπG CAAGAAGTGC CTATGGATAG ATTCGTCATG GACATGAAAA 6600 GAGACGTGAA AGπACACCT GGCACGAAAC ACACAGAAGA AAGACCGAAA GTACAAGTGA 6660 TACAAGCCGC AGAACCCCTG GCGACCGCπ ACCTATGCGG GATCCACCGG GAGπAGTGC 6720 GCAGGCπAC AGCCGππG CTACCCAACA πCACACGCT CTTTGACATG TCGGCGGAGG 6780 ACTTTGATGC AATCATAGCA GAACACπCA AGCAAGGTGA CCCGGTACTG GAGACGGATA 6840 TCGCCTCGπ CGACAAAAGC CAAGACGACG CTATGGCGπ AACCGGCCTG ATGATCπGG 6900 AAGACCTGGG TGTGGACCAA CCACTACTCG ACπGATCGA GTGCGCCTTT GGAGAAATAT 6960 CATCCACCCA TCTGCCCACG GGTACCCGπ TCAAAπCGG GGCGATGATG AAATCCGGAA 7020 TGπCCTCAC GCTCTTTGTC AACACAGπC TGAATGTCGT TATCGCCAGC AGAGTATTGG 7080 AGGAGCGGCT TAAAACGTCC AAATGTGCAG CATTTATCGG CGACGACAAC AπATACACG 7140 GAGTAGTATC TGACAAAGAA ATGGCTGAGA GGTGTGCCAC CTGGCTCAAC ATGGAGGπA 7200 AGATCAπGA CGCAGTCATC GGCGAGAGAC CACCTTACπ CTGCGGTGGA πCATCπGC 7260 AAGAπCGGT TACCTCCACA GCGTGTCGCG TGGCGGACCC CTTGAAAAGG CTGTπAAGT 7320 TGGGTAAACC GCTCCCAGCC GACGATGAGC AAGACGAAGA CAGAAGACGC GCTCTGCTAG 7380 ATGAAACAAA GGCGTGGπT AGAGTAGGTA TAACAGACAC CTTAGCAGTG GCCGTGGCAA 7440 CTCGGTATGA GGTAGACAAC ATCACACCTG TCCTGCTGGC AπGAGAACT πTGCCCAGA 7500 GCAAAAGAGC ATTTCAAGCC ATCAGAGGGG AAATAAAGCA TCTCTACGGT GGTCCTAAAT 7560 . AGTCAGCATA GTACATTTCA TCTGACTAAT ACCACAACAC CACCACCATG AATAGAGGAT 7620 TCTTTAACAT GCTCGGCCGC CGCCCCTTCC CAGCCCCCAC TGCCATGTGG AGGCCGCGGA 7680 GAAGGAGGCA GGCGGCCCCG ATGCCTGCCC GCAATGGGCT GGCTTCCCAA ATCCAGCAAC 7740 TGACCACAGC CGTCAGTGCC CTAGTCAπG GACAGGCAAC TAGACCTCAA ACCCCACGCC 7800 CACGCCCGCC GCCGCGCCAG AAGAAGCAGG CGCCAAAGCA ACCACCGAAG CCGAAGAAAC 7860 CAAAAACACA GGAGAAGAAG AAGAAGCAAC CTGCAAAACC CAAACCCGGA AAGAGACAGC 7920 GTATGGCACT TAAGπGGAG GCCGACAGAC TGπCGACGT CAAAAATGAG GACGGAGATG 7980 TCATCGGGCA CGCACTGGCC ATGGAAGGAA AGGTAATGAA ACCACTCCAC GTGAAAGGAA 8040 CTAπGACCA CCCTGTGCTA TCAAAGCTCA AAπCACCAA GTCGTCAGCA TACGACATGG 8100 AGπCGCACA GπGCCGGTC AACATGAGAA GTGAGGCGπ CACCTACACC AGTGAACACC 8160 CTGAAGGGπ CTACAACTGG CACCACGGAG CGGTGCAGTA TAGTGGAGGC AGATTTACCA 8220 TCCCCCGCGG AGTAGGAGGC AGAGGAGACA GTGGTCGTCC GAπATGGAT AACTCAGGCC 8280 GGGπGTCGC GATAGTCCTC GGAGGGGCTG ATGAGGGAAC AAGAACCGCC CTTTCGGTCG 8340 TCACCTGGAA TAGCAAAGGG AAGACAATCA AGACAACCCC GGAAGGGACA GAAGAGTGGT 8400 CTGCTGCACC ACTGGTCACG GCCATGTGCT TGCTTGGAAA CGTGAGCTTC CCATGCAATC 8460 GCCCGCCCAC ATGCTACACC CGCGAACCAT CCAGAGCTCT CGACATCCTC GAAGAGAACG 8520 TGAACCACGA GGCCTACGAC ACCCTGCTCA ACGCCATATT GCGGTGCGGA TCGTCCGGCA 8580 GAAGTAAAAG AAGCGTCACT GACGACπTA CCπGACCAG CCCGTACTTG GGCACATGCT 8640 CGTACTGTCA CCATACTGAA CCGTGCTTTA GCCCGAπAA GATCGAGCAG GTCTGGGATG 8700 AAGCGGACGA CAACACCATA CGCATACAGA CπCCGCCCA GTTTGGATAC GACCAAAGCG 8760 GAGCAGCAAG CTCAAATAAG TACCGCTACA TGTCGCTCGA GCAGGATCAT ACTGTCAAAG 8820 AAGGCACCAT GGATGACATC AAGATCAGCA CCTCAGGACC GTGTAGAAGG CπAGCTACA 8880 AAGGATACπ TCTCCTCGCG AAGTGTCCTC CAGGGGACAG CGTAACGGπ AGCATAGCGA 8940 GTAGCAACTC AGCAACGTCA TGCACAATGG CCCGCAAGAT AAAACCAAAA πCGTGGGAC 9000 GGGAAAAATA TGACCTACCT CCCGπCACG GTAAGAAGAT TCCπGCACA GTGTACGACC 9060 GTCTGAAAGA AACAACCGCC GGCTACATCA CTATGCACAG GCCGGGACCG CATGCCTATA 9120 CATCCTATCT GGAGGAATCA TCAGGGAAAG TTTACGCGAA GCCACCATCC GGGAAGAACA 9180 πACGTACGA GTGCAAGTGC GGCGAπACA AGACCGGAAC CGπACGACC CGTACCGAAA 9240 TCACGGGCTG CACCGCCATC AAGCAGTGCG TCGCCTATAA GAGCGACCAA ACGAAGTGGG 9300 TCπCAACTC GCCGGACTCG ATCAGACACG CCGACCACAC GGCCCAAGGG AAAπGCAπ 9360 TGCCTTTCAA GCTGATCCCG AGTACCTGCA TGGTCCCTGT TGCCCACGCG CCGAACGTAG 9420 TACACGGCπ TAAACACATC AGCCTCCAAT TAGACACAGA CCATCTGACA πGCTCACCA 9480 CCAGGAGACT AGGGGCAAAC CCGGAACCAA CCACTGAATG GATCATCGGA AACACGGπA 9540 GAAACπCAC CGTCGACCGA GATGGCCTGG AATACATATG GGGCAATCAC GAACCAGTAA 9600 GGGTCTATGC CCAAGAGTCT GCACCAGGAG ACCCTCACGG ATGGCCACAC GAAATAGTAC 9660 AGCAπACTA TCATCGCCAT CCTGTGTACA CCATCTTAGC CGTCGCATCA GCTGCTGTGG 9720 CGATGATGAT TGGCGTAACT GπGCAGCAT TATGTGCCTG TAAAGCGCGC CGTGAGTGCC • 9780 TGACGCCATA TGCCCTGGCC CCAAATGCCG TGAπCCAAC πCGCTGGCA CTπTGTGCT 9840 GTGπAGGTC GGCTAATGCT GAAACAπCA CCGAGACCAT GAGπACTTA TGGTCGAACA 9900 GCCAGCCGπ CπCTGGGTC CAGCTGTGTA TACCTCTGGC CGCTGTCGTC GπCTAATGC 9960 GCTGπGCTC ATGCTGCCTG COTΠTTAG TGGπGCCGG CGCCTACCTG GCGAAGGTAG 10020 ACGCCTACGA ACATGCGACC ACTGπCCAA ATGTGCCACA GATACCGTAT AAGGCACTTG 10080 πGAAAGGGC AGGGTACGCC CCGCTCAAπ TGGAGAπAC TGTCATGTCC TCGGAGGTTT 10140 TGCCπCCAC CAACCAAGAG TACAπACCT GCAAAπCAC CACTGTGGTC CCCTCCCCTA 10200 AAGTCAGATG CTGCGGCTCC πGGAATGTC AGCCCGCCGC TCACGCAGAC TATACCTGCA 10260 AGGTCTTTGG AGGGGTGTAC CCCTTCATGT GGGGAGGAGC ACAATGTITT TGCGACAGTG 10320 AGAACAGCCA GATGAGTGAG GCGTACGTCG AAπGTCAGT AGAπGCGCG ACTGACCACG 10380 CGCAGGCGAT TAAGGTGCAT ACTGCCGCGA TGAAAGTAGG ACTGCGTATA GTGTACGGGA 10440 ACACTACCAG TTTCCTAGAT GTGTACGTGA ACGGAGTCAC ACCAGGAACG TCTAAAGACC 10500 TGAAAGTCAT AGCTGGACCA ATTTCAGCAT TGTπACACC AπCGATCAC AAGGTCGπA 10560 TCAATCGCGG CCTGGTGTAC AACTATGACT πCCGGAATA CGGAGCGATG AAACCAGGAG 10620 CGTTTGGAGA CAπCAAGCT ACCTCCπGA CTAGCAAAGA CCTCATCGCC AGCACAGACA 10680 TTAGGCTACT CAAGCCTTCC GCCAAGAACG TGCATGTCCC GTACACGCAG GCCGCATCTG 10740 GAπCGAGAT GTGGAAAAAC AACTCAGGCC GCCCACTGCA GGAAACCGCC CCTπTGGGT 10800 GCAAGAπGC AGTCAATCCG CTTCGAGCGG TGGACTGCTC ATACGGGAAC AπCCCAπT 10860 CTAπGACAT CCCGAACGCT GCCTTTATCA GGACATCAGA TGCACCACTG GTCTCAACAG 10920 TCAAATGTGA TGTCAGTGAG TGCACπAπ CAGCGGACTT CGGAGGGATG GCTACCCTGC 10980 AGTATGTATC CGACCGCGAA GGACAATGCC CTGTACAπC GCATTCGAGC ACAGCAACCC 11040 TCCAAGAGTC GACAGπCAT GTCCTGGAGA AAGGAGCGGT GACAGTACAC πCAGCACCG 11100 CGAGCCCACA GGCGAACπC AπGTATCGC TGTGTGGTAA GAAGACAACA TGCAATGCAG 11160 AATGCAAACC ACCAGCTGAT CATATCGTGA GCACCCCGCA CAAAAATGAC CAAGAAπCC 11220 AAGCCGCCAT CTCAAAAACT TCATGGAGπ GGCTGTTTGC CC I 1 1 ICGGC GGCGCCTCGT 11280 CGCTAπAAT TATAGGACπ ATGAI 1 I I IG CπGCAGCAT GATGCTGACT AGCACACGAA 11340 GATGACCGCT ACGCCCCAAT GACCCGACCA GCAAAACTCG ATGTACTTCC GAGGAACTGA 11400 TGTGCATAAT GCATCAGGCT GGTATAπAG ATCCCCGCTT ACCGCGGGCA ATATAGCAAC 11460 ACCAAAACTC GACGTATTTC CGAGGAAGCG CAGTGCATAA TGCTGCGCAG TGπGCCAAA 11520 TAATCACTAT AπAACCAπ TAπCAGCGG ACGCCAAAAC TCAATGTAπ TCTGAGGAAG 11580 CATGGTGCAT AATGCCATGC AGCGTCTGCA TAACI 1 1 1 IA πAπTCTTT TAπAATCAA 11640 CAAAAI I I I G 1 1 1 1 lAACAT πC 11663

Claims: That Which Is Claimed Is: 1. A recombinant DNA comprising a cDNA coding for an infectious South African Arbovims No. 86 (S.A.AR86) vims RNA transcript, and a heterologous promoter positioned upstream from said cDNA and operatively associated therewith. 2. The recombinant DNA according to Claim 1, wherein said cDNA is selected from the group consisting of (i) cDNA having the sequence given herein as SEQ ID NO.: 1, (ii) cDNA having the same protein coding sequence as the cDNA given herein as SEQ ID NO. : 1, and (Hi) cDNA according to (0 or (ii) above, and further comprising at least one attenuating mutation in said cDNA. 3. The recombinant DNA according to Claim 1, wherein said cDNA has the sequence given herein as SEQ ID NO.: 1. 4. The recombinant DNA according to Claim 1, further comprising at least one attenuating mutation in said cDNA clone. 5. The recombinant DNA according to Claim 1, further comprising at least two attenuating mutations in said cDNA clone. 6. The recombinant DNA according to Claim 5, wherein each of said attenuating mutations are in the region selected from the group consisting of the nsPl coding region, E2 coding region, and nsP2 coding region. 7. The recombinant DNA according to Claim 1, further comprising at least one attenuating mutation selected from the group consisting of codons at nsPl amino acid position 538 which specify an attenuating amino acid, codons at E2 amino acid position 304 which specify an attenuating amino acid, codons at E2 amino acid position 314 which specify an attenuating amino acid, codons at E2 amino acid position 372 which specify an attenuating amino acid, codons at E2 amino acid position 376 which specify an attenuating amino acid, codons at nsP2 amino acid position 96 which specify an attenuating amino acid, codons at nsP2 amino acid position 372 which specify an attenuating amino acid, codons at nsP2 amino acid position 529 which specify an attenuating amino acid, codons at nsP2 amino acid position 571 which specify an attenuating amino acid, codons at nsP2 amino acid position 682 which specify an attenuating amino acid, codons at nsP2 amino acid position 804 which specify an attenuating amino acid, and codons at nsP3 amino acid position 22 which specify an attenuating amino acid. 8. The recombinant DNA according to Claim 7, wherein said attenuating mutation comprises a substitution mutation. 9. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for isoleucine at nsPl amino acid 538. 10. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for threonine at E2 amino acid 304. 11. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for lysine at E2 amino acid 314. 12. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for valine at E2 amino acid 372. 13. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for alanine at E2 amino acid 376. 14. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for glycine at nsP2 amino acid 96. 15. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for valine at nsP2 amino acid 372. 16. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for leucine at nsP2 amino acid 529. 17. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for asparagine at nsP2 amino acid 571. 18. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for arginine at nsP2 amino acid 682. 19. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for arginine at nsP2 amino acid 804. 20. The recombinant DNA according to Claim 8, wherein said substitution mutation codes for arginine at nsP3 amino acid 22. 21. The recombinant DNA according to Claim 1 further comprising at least one silent mutation. 22. The recombinant DNA according to Claim 21, wherein said silent mutation is located at a position selected from the group consisting of nucleotide 215, nucleotide 3863, nucleotide 5984 and nucleotide 9113. 23. The recombinant DNA according to Claim 1, wherein not more than eight nucleotides are positioned between said promoter and said cDNA clone. 24. The recombinant DNA according to Claim 23, wherein said promoter is selected from the group consisting of T3 promoters, T7 promoters, and SP6 promoters. 25. The recombinant DNA according to Claim 1, wherein said recombinant DNA comprises a plasmid, and wherein said recombinant DNA further comprises a unique restriction site positioned downstream from said cDNA clone. 26. An infectious RNA transcript encoded by a cDN A according to Claim 1. 27. Infectious attenuated viral particles containing an RNA transcript of Claim 26. 28. A pharmaceutical composition comprising an effective immunogenic amount of an infectious attenuated vims according to Claim 27 in combination with a pharmaceutically acceptable carrier.

Inventors: DAVIS NANCY L, JOHNSTON ROBERT E, SIMPSON DENNIS A, DAVIS, NANCY, L., JOHNSTON, ROBERT, E., SIMPSON, DENNIS, A.
Applicants: DAVIS NANCY L, JOHNSTON ROBERT E, SIMPSON DENNIS A, UNIV NORTH CAROLINA, DAVIS, NANCY, L., JOHNSTON, ROBERT, E., SIMPSON, DENNIS, A., THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL
Application year: 1996
Main classification: A61K39
IPC codes: A61K39, A61P31, C07K14, C12N7, C12N15