RECOMBINANT DNA-VECTOR COMPRISING GENOMIC EQUINE ARTERITIS VIRUS SEQUENCES

Abstract: The present invention relates to a recombinant DNA vector comprising a cDNA copy of at least a part of the genome of equine arteritis virus. The recombinant DNA vector according to the present invention is stable in a vector host cell and yields, after transcription, infectious RNA. Due to the invention the genetic engineering of the virus is very much simplified and it may be used for, among other things, the preparation of pharmaceutical compositions. Licensing possible.

Description: RECOMBINANT DNA-VECTOR COMPRISING GENOMIC EQUINE ARTER1TIS VTRUS SEQUENCES The present invention relates to a recombinant DNA- vector comprising a DNA sequence (-DNA) complementary to at least a part of the genome (+RNA) of eguine arteritis virus (EA-virus) , said DNA-vector having adjacent to the 3' end of the DNA sequence (-DNA) a non-EA virus-specific promotor for a non-EA virus-specific RNA poly erase and, after linearization of the vector and transcription of the DNA sequence (-DNA) by the non-EA virus-specific RNA polymerase, yields +RNA which is infectious in a virus host cell, the DNA sequence (-DNA) comprising a complementary DNA sequence (+DNA) corresponding to said part of the genome, said complementary DNA sequence (+DNA) consisting of a first sequence and adjacent at the 5' and of the first sequence a second sequence, the first sequence being the sequence represented by SEQ nr. 1 or a genetically engineered, together with the second sequence infectious derivative thereof, and the second sequence being the sequence represented by SEQ nr. 2 or a functional derivative thereof, wherein the DNA-vector comprising the complementary DNA sequence (+DNA) consisting of SEQ nr. 1 and SEQ nr. 2 can be maintained in E. coli K12 PC2495 (Phabagen Collection, University of Utrecht, P.O Box 80.056, 3508 TB Utrecht, the Netherlands) as a vector host cell and, after isolation and an in vitro transcription reaction, with T7 promotor (SEQ nr. 3) as non-EA virus-specific promotor and T7 polymerase as non-EA virus-specific RNA polymerase, yields +RNA which is infectious in baby hamster kidney cells BHK-21 (C13) (ATCC CCL10) as a virus host cell and leads to the formation of virus particles, as well as a genetically engineered derivative of this vector the RNA transcription product of which is capable of replication in an eu aryote . Eguine arteritis virus (EA virus) is a virus that, as far as is known, in nature only occurs in horses and donkeys, but which can replicate in other mammalian cell lines. The genome of the EA virus is an RNA strand. The RNA is a single, positive strand, that is, the RNA is immedi- ately available for translation as soon as it has entered a virus host cell. This means that based on the RNA, the ribosomes of the virus host cell are capable of making proteins. The first protein for which the RNA strand codes is the EAV replicase, an RNA-polymerase capable of making a negatively stranded copy of the positive RNA strand. Just like the positive RNA strand, this negatively stranded RNA copy has at the 3' end a nucleotide sequence functioning as a very selective point of action for the replicase. The positive RNA genome of the EA virus is surrounded by capsids and a membrane. Of other viruses it is known that the ends of the RNA strand are very important during the formation of the virus particle. The nucleotide sequence of the EA virus is by and large known in the state of the art (SEQ nr. 1 disclosed by Den Boon, J.A. et al . , J. Virol. (1991), 6_5: pp 2910-2920). This sequence has been determined by sequencing a large number of clones comprising cDNA obtained using reverse transcriptase. The various fragments of the cDNA overlap, allowing the almost complete determination of the sequence. Reverse transcriptase makes relatively many errors during the synthesis of the complementary DNA, as a result of which the sequence determined does not necessarily correspond to the RNA which was used as starting material. Reverse transcriptase tends to terminate before the 5' end of the positive EA virus RNA is reached. Thus a cDNA (-DNA) is formed being incomplete at the 3' end. The determination of this nucleotide sequence is very difficult. As indicated above, the end of the RNA strand is very important. EAV replicase makes errors also during the duplication of positive and negative RNA strands also. This may result in non-infectious virus mutants. If these are used when making cDNA, which is used to transform a vector host cell in a manner known it itself, a vector is obtained of which the +RNA transcript is not infectious. In principle it is possible, but very laborious, to make a clone through genetic engineering wherein all cDNA fragments are placed in the correct order, in correspon- dance to the +RNA sequence of the virus . Until now that has not been realized. Firstly, there is a lack of information on possibly missing nucleotides. Secondly, there is the risk described above that, when the clone obtained is used to make +RNA, the +RNA obtained is no longer infectious. Thirdly, there is the problem that there is absolutely no certainty that the clone will be stable in the vector host cell chosen. For example, protein products of the cDNA may be toxic for the vector host cell. For example, the genome of the EA virus codes for three proteases which may disrupt the metabolism of the vector host cell. Stepwise combining cDNA fragments does not necessarily offer a solution, as instances are described in the literature wherein in the presence of a first gene the absence of a second gene was toxic. In case of toxicity, it is highly likely that a clone is selected in which the gene causing toxicity is no longer active and accordingly there is a chance that a derived RNA is no longer infectious. In summary, combining cDNA guarantees a lot of work, while there is no reasonable expectation of success. Applicant has found that a recombinant DNA-vector comprising a nucleotide sequence with SEQ nr. 2 immediately adjacent to and contiguous with SEQ nr. 1 in E. coli K12 PC2495 (Phabagen Collection, University of Utrecht, P.O Box 80.056, 3508 TB Utrecht, the Netherlands) together with a non-EA virus-specific promotor can stably be maintained in a vector host cell and the +RNA product thereof is infectious in baby hamster kidney cells BHK-21 (C13) (ATCC CCL10) . Thus applicant provides for the possibility of simple genetic engineering of the DNA by a method known in itself, by which recombinant RNA can be formed. Instead of the sequence used by SEQ nr. 2, a functional derivative thereof may be used. A functional derivative is to be understood as a sequence in which one or more nucleotides are deleted, inserted or replaced by one or more other nucleotides while the capability of forming virus particles is maintained. The first sequence may be adapted, for example, by introducing a non-EA virus-specific gene to express a new protein product in a virus host cell. It is also possible to change the virulence of the virus or to modify the host specificity. Though the present invention makes it possible to obtain recombinant DNA-vectors and recombinant RNA products in a simple way, the expert is well aware of the fact that when introducing a new gene there is a good chance that the ability to replicate and/or form infectious virus particles is eliminated by knocking out essential viral genes or other functional parts of the viral genome, such as sequences essential for RNA replication. Fig. 1 shows the known EAV genes. From this it appears that a gene may be introduced between 0RF1 and ORF2. However, applicant has found that here a gene unknown to this day, which herein will be referred to as ORF2a, (from nucleotide 9734 to 9937; see SEQ nr. 1) overlapping with ORF2. In other words, this site is not suitable for the insertion either, unless a complete sequence of ORF2a is provided on the vector according to the invention or another vector for complementation purposes, which is brought to expression. It has been found that the nucleotides 211 to 987 (SEQ nr. 1) , which code for a non-structural protein 1 (NSPl; Snijder E.J. et al., J. Virol. 6_6_, pp. 7040-7048 (1992)), may be removed yielding a recombinant DNA-vector coding for a recombinant RNA product capable of replication but not capable of forming infectious virus particles. Instead of the indicated nucleotide sequence a new gene may be inserted. If desired an intact NSPl gene may be intro- duced adjacent to the new gene. According to an advantageous embodiment, the new gene is inserted closely behind nucleotide 990, as NSPl, a protease, cuts the protein product between the two glycine residues for which the nucleotides 985 to 990 code (see SEQ nr . l) . Preferably a nucleotide sequence is provided after the nucleotide sequence for the new gene where the resulting protein products can be cut, advantageously by the serin protease for which the viral cDNA of the vector already codes. A suit- able nucleotide sequence is, for example, that of 5209 to 5268 (see SEQ nr . 1) . According to the present invention the overlapping open reading frames of the first gene and the second gene may be unbraided by the insertion of a nucleotide sequence i) wherein the overlapping part is duplicated; ii) in the first gene at least one nucleotide of the ATG start codon for the second gene is replaced by another nucleotide as a result of which a) the ATG triplet in the first gene no longer functions as start codon for the second gene; and b) the other nucleotide is chosen such that the gene product of the first gene still functions. To this end the nucleotide is preferably chosen such that using the degeneracy of the genetic code the change does not lead to a change in the amino acid sequence. By unbraiding the first and the second gene, a sequence comprising a new gene may be inserted. To prevent that the new gene and the second gene result in a gene fusion product, a stop codon may be inserted between the new gene and the second gene, or a Internal Ribosomal Entry Site (IRES) , as described in the state of the art. If necessary, such a promotor sequence or IRES may also be inserted between the first gene and the new gene. Elimination of the overlap between two genes is pre- ferably accomplished between those genes showing little overlap, causing a relatively small increase of the total length of the vector. In addition, applicant has found that a vector in which ORF1 and 0RF2a are intact, and nucleotide 10,035 in ORF2 tot nucleotide 12,545 in ORF7 are removed, results in an RNA product capable of replication. The sequence removed may be partially or completely be replaced by a new gene capable of expression from a sub-geno ic RNA. In all cases where a gene is knocked out, an intact copy of this gene may be provided on the vector according to the present invention or a further vector to be provided in the virus host cell, in order to take over the function of the knocked out gene . The present invention also comprises a genetically engineered recombinant DNA-vector according to the invention, in particular one which is modified by the insertion of a non-EA virus-specific gene. The invention also relates to a vector host cell containing a recombinant DNA-vector according to claim 1, and in particular to such a vector host cell which is E. coli K12 PC2495 and contains the recombinant DNA-vector pEAV030, deposited on 11 July 1996 under accession nr. 760- 96 with the Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, as well as to recombinant DNA-vector pEAV030. In addition, the invention relates to the DNA sequence according to SEQ nr. 2 or a derivative thereof which, when included in a DNA-vector comprising a DNA sequence (+DNA) consisting of SEQ nr. 1 and contiguous therewith at the 5' end of SEQ nr. 2 or the derivative thereof, can be maintained in a vector host cell and, with a non-EA virus-specific promotor for a non-EA virus-speci- fie RNA polymerase and the non-EA virus-specific RNA polymerase, results in RNA which is infectious in a virus host cell and yields virus particles. By modifying this oligonucleotide which is at least for replication in the virus host cell important, it may be possible, for example, to impede the encapsidation of the +RNA in virus particles. The invention also relates to the use of a oligonucleotide chosen from the group consisting of the DNA sequence with SEQ nr. 2, the complement thereof, and frag- ments thereof, as a primer for a chain extension reaction. Such primers may be utilized in the detection of the virus with the Polymerase Chain Reaction technique, and in particular for making a full DNA copy of the viral genome also. RNA may be obtained by transcription of the DNA sequence (-DNA) of the recombinant DNA-vector according to the invention. The transcription may proceed in vivo, although, according to an advantageous embodiment, it may be carried out in vitro. Here use is made of a suitable non-EA virus-specific promotor. RNA is, amongst others, useful for the preparation of virus particles and a pharmaceutical composition. Thus the present invention also relates to two advantageous methods for obtaining virus particles, wherein according to a first embodiment RNA according to the invention is introduced in virus host cells, the virus host cells are cultured and virus particles are isolated in a manner known in itself . According to a second advantageous embodiment, a recombinant DNA-vector according to the invention is introduced in virus host cells, the virus host cells are cultured and virus particles are isolated in a manner known in itself. Preferably the recombinant DNA-vector is provided with a non-EAV-specific promotor which is recognized by a non-EAV-specific polymerase of the virus host cells, which may or may not occur in the virus host cells by nature. The present invention also relates to a virus host cell transfected with RNA according to the invention or with virus particles obtained by one of the methods described above. Finally, the invention relates to two methods for the preparation of a pharmaceutical preparation. According to a first method for the preparation of a recombinant RNA-containing pharmaceutical composition, the recombinant DNA-vector according to the invention is multiplied in a vector host cell, the DNA-vector is transcribed yielding recombinant RNA, if desired the recombinant RNA is introduced into a virus host cell, yielding recombinant RNA-containing viruses which, if desired, are isolated, and the thus obtained recombinant RNA is processed with a pharmaceutically acceptable carrier or excipient to a pharmaceutical composition. According to a second method for the preparation of a recombinant DNA-vector containing pharmaceutical composition, the recombinant DNA-vector according to the invention is multiplied in a vector host cell and isolated in a manner known in itself, and the thus obtained recombinant DNA is processed with a pharmaceutically acceptable carrier or excipient into a pharmaceutical composition. In such a case the recombinant DNA-vector is preferably provided with a non-EAV-specific promotor which may be recognized by a non- EAV-specific polymerase of the virus host cells, which may or may not occur in the virus host cell by nature. The invention will now be elucidated with reference to the drawing in which the only Figure shows a schematic overview of the EAV-specific part of a recombinant DNA- vector according to the invention and the way in which it is reconstructed. The Figure shows that twelve cDNA clones, obtained using Avian Myeloblastosis Virus reverse transcriptase (Pharmacia) , are used for reconstructing the almost complete sequence of a cDNA copy of the RNA genome of EA virus. The clones and genes (open reading frames) shown in the top of the Figure are described in detail by Den Boon, J.A. et al (J. Virol. (1991), 6_5: pp. 2910-2920). Above the clones a restriction map is shown indicating the restriction enzymes used for removing the overlapping cDNA parts of the clones. Clone 108, to the right in the figure, comprised a poly-A-tail with only 25 of the 140 nucleotides from SEQ nr. 1 and this was extended in a manner known in itself to about 140 nucleotides. At the 5' end an unknown number of nucleotides was missing due to premature termination of reverse transcriptase. By direct RNA sequence determination 16 nucleotides could be determined. A method described by Dumas J.B. et al. (Nucl. Acids Res. (1991) 19: pp. 5227-5232) gave nucleotide 17. This sequence was, together with a part of the 5' sequence, synthesized and added to the clone in a manner known in itself. The thus obtained cDNA of EAV (SEQ nr. 2+1) and the T7 promotor sequence (SEQ nr. 3) were connected by means of the polymerase chain reaction in the order 5' SEQ nr. 3 -SEQ nr. 2-SEQ nr. 1-3'. During this reaction the 5' end was extended with the nucleotide sequence 15,491 - 15,511 (see SEQ nr. 4) , comprising unique restriction sites for Ndel and Notl. At the 3' end the nucleotide sequence 12,845 - 12,851 was added, coding for a unique Xhol-restriction site. The construct was introduced in the vector pUC18 (EMBL database accession L08752) between the unique EcoRI and Hindlll restriction sites. After cutting these sites were made blunt to allow ligation of the construct. This yielded the (circular) vector pEAV030 (SEQ nr. 4) . The fact that a single point mutation, C instead of T at place 7491 of SEQ nr. 1, resulted in non-infectious RNA, shows how great the danger is that indeed a stable recombinant DNA-vector is obtained, but that this does not yield infectious RNA. The present invention will now be elucidated by the following two, non-limiting Examples. All standard molecu- lar biology products and cell culture products were obtained from Life Technologies (Breda, the Netherlands) . Example l Transfection of several cell lines with +RNA from pEAV030. a) Linearization of pEAV030 plasmid DNA To make an RNA strand with a 3' terminal polyA tail, the plasmid is linearized using the unique Xhol restriction site. pEAV030 plasmid DNA was isolated from Escherichia coli strain PC2495 according to the invention (Phabagen Collection, University of Utrecht, P.O Box 80.056, 3508 TB Utrecht, the Netherlands) in a manner known in itself (alkaline lysis method; Sambrook, J. , Fritsch, E.F. and Maniatis, T. (1989) . Molecular cloning: a laboratory man- ual. Cold Spring Harbour Laboratory, N.Y., U.S.A.; hereinafter referred to as Maniatis et al.) and the DNA concentration was established spectrophotometrically. Two micrograms pEAV030 plasmid DNA was linearized by digestion with 10 units of the restriction enzyme Xhol (which cuts this vector only once) (digestion volume: 20 microliters; buffer conditions: 10 M Tris-HCl, pH 7.9, 10 mM MgCl 2 , 1 M dithiothreitol (DTT) . 100 micrograms/ml bovine serum albamine (BSA) ; incubation: 2 hours at 37°C) . One microliter of this digestion solution was checked to be sure that the digestion was complete in a manner known in itself (agarose gel electrophoresis of DNA; see Maniatis et al . ) . To the remaining 19 microliters 80 microliters aqua bidest was added and l microliter of a 20 mg/ml Proteinase K solution (incubation: l hour at 37°C) . The linearized plasmid DNA was extracted twice with 100 microliter phenol/chloroform/isoamylalcohol and concentrated through precipitation with methanol in a manner known in itself (see Maniatis et al.). After centrifugation (15 minutes, 15,000 x g) and washing the DNA pellet with 500 microliters 70% ethanol, the plasmid DNA was taken up in 15 microliters aqua bidest . b) In vitro transcription To make an RNA transcript of the linearized DNA in vitro using T7 RNA polymerase, the following components were added together: - 15 microliters Xhol-linearized pEAV030 DNA (see above) 5 microliters 1 mg/ml BSA (Life Technologies) 5 microliters 50 mM DTT 5 microliters of a ribonucleotide mixture (of ATP, CTP GTP and UTP, each 10 mM) (Life Technologies) 5 microliters 10 mM capping analogon (m7G(5' )ppp(5' )G) (Life Technologies) - 10 microliters 5 x concentrated T7 transcription buffer (200 mM Tris-HCl, pH 8.0 , 40 mM MgCl 2 , 10 mM spermidine, 125 mM NaCl) (Life Technologies) - 2.5 microliters 8 units/microliter RNAse inhibitor (RNA- guard; Pharmacia, Bergen op Zoom, the Netherlands) - 2.5 microliters 8 units/microliter T7-RNA-polymerase (Life Technologies) Transcription volume: 50 microliters. Incubation was carried out for 1 hour at 37°C. One microliter of the transcription reaction was checked to be sure that the transcription reaction had succeeded in a manner known in itself (agarose gel electrophoresis of DNA; see Maniatis et al.) The estimated yield of the above reaction was 10 micrograms RNA. c) Cell culture The cell lines BHK-21 (hamster; ATCC CCL10) , HeLa (human; ATCC CCL-2.1) and L (mouse; ATCC CCL10) , were cultured in a manner known in itself at 37°C in Dulbecco's Modification of Eagle's Medium (DMEM) , supplemented with 10 foetal calf serum, and the usual amounts of the antibiotics vancomycin and gentamycin. Care was taken that the cell density at the moment of use for transfection was 60-80%. d) Electroporation and virus harvest For the transfection of eukaryotic cells with RNA use was made of an electroporator of the type EquiBio Easy- Ject Plus (Eurogentec, Seraing, Belgium) and the accompanying cuvettes (diameter 0.4 cm). Cells were washed in a manner known in itself in PBS (137 mM NaCl, 27 mM KL1, 8 M Na 2 HP0 4 , 1.5 mM KH 2 P0 4 , pH 7.2) and incubated in a trypsin solution (Life Technologies; 1 ml per 75 cm 2 culture dish surface area) during about 3-5 minutes at 37°C to release the cells from the culture dish. Per ml cell suspension 9 ml culture medium (DMEM with supplements; see above) were added and the cells were resuspended thoroughly. The cells were pelleted by centrifugation (10 minutes, 3000 x g) en resuspended in 10 ml PBS per ml of the original cell suspension. The cells were again pelleted by centrifugation, taken up in PBS at a concentration of 10 7 cells/ml, and kept on ice from this moment on. A mixture of 0.6 ml of the above cell suspension and 49 microliters transcription reaction (see above) was introduced into an electroporation cuvette. Using the above-mentioned electroporator, two successive pulses were given with the following set values: 850 V, 2310 Ohm and 25 μF . Hereafter the cell suspension was mixed with culture medium (DMEM), plated in culture dishes, incubated at 39.5°C and used in a manner known in itself for various virological analyses. Infectious virus could be obtained for each of the above cell lines by, in a manner known in itself, harvesting and storing the medium of the transfected cells 20-30 hours after electroporation. Finally, it has been shown that the transcription product of the vector according to the invention is capable of replication in strains which do not act as a virus host cell line by nature, yielding viruses infectious for natural virus host cell lines. Example 2 Construction of a recombinant expression vector The gene for the Green Fluorescent Protein (GFP) was inserted in the Hindlll restriction site on nucleotide 12303-12308 in SEQ nr . 4, being a part of pEAV030, in the proper orientation (that is, the ÷strand of the GFP gene resides in the +DNA of the EAV part of the DNA vector) . This restriction site resides just before ORF7 (see Fig. 1) . The insertion disrupts 0RF6 (the reading frame for a membrane protein of EAV) , and prevents the translation of 0RF7 (coding for the nucleocapsid protein of EAV) . The genetically modified vector (pEAVGFP7) is consequently capable of replication and transcription (including GFP expression) but not capable of virus production and propagation, as two essential structural proteins are not made. a) Construction of the intermediate construct pM115128. As there are several Hindlll restriction sites in pEAV030, it was not possible to insert the GFP gene directly into the Hindlll site at position 12303-12308 of SEQ nr. 4. Thus an intermediate construct (plasmid) was made in which said Hindlll site is unique. This construct (pM115128) was made by cutting pEAV030 using EcoRI (position 11488-11493 of SEQ nr . 4) and Xhol (nucleotides 12,845-12,851 in SEQ. nr. 4). These are both unique restriction sites. The resulting DNA fragment corresponds to the 3' and of the viral genome and contains said Hindlll site at position 12303-12308 of sequence 4. The EcoRI-XhoI DNA fragment was obtained by digesting 1 microgram pEAV030 plasmid DNA with 10 units of each of the restriction enzymes EcoRI and Xhol (digestion volume: 20 microliters; buffer conditions: 10 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , l mM dithiothreitol (DTT) , 100 micrograms/ml bovine serum albumin (BSA) ,- incubation: 2 hours at 37°C) . The digestion was analyzed in a manner known in itself (agarose gel electrophoresis of DNA; see Maniatis et al.) and the required DNA fragment was purified from the agarose gel in a manner known in itself and taken up in 10 microliters aqua bidest. As basic vector for pM115128 plasmid pBluescript SK- (pSK; Stratagene, La Jolla, US) was used, of which 1 microgram was cut with EcoRI and Xhol (as described above) , purified from agarose gel and taken up in 10 microliters aqua bidest. The EcoRI-Xhol DNA fragment from pEAV030 and the vector pSK cut with EcoRI-XhoI were ligated to pM115128 using T4 DNA ligase in the following system: 1 microliter purified pSK vector cut with EcoRI-Xhol 6 microliters purified EcoRI-Xhol DNA fragment from pEAV030 2 microliters 5x concentrated ligate buffer (250 mM Tris-HCl, pH 7.8, 50 mM MgCl 2 , 50 mM DTT, 5 mM ATP, 125 micrograms/ml BSA) 1 microliter T4 DNA ligase (1 unit/microliter; Life Technologies) . After one hour incubation at 20°C the ligation mix- ture was transformed to E_-_ coli strain PC2495 in a manner known in itself . The transformed bacteria were cultured in the presence of the antibiotic ampicillin to which the vector contains a resistance gene in a manner known in itself. The plasmid was subsequently isolated from the bacteria in a manner known in itself and checked using restriction enzymes in a manner known in itself on the presence of the EcoRI-XhoI DNA fragment from pEAV030. b) Insertion of the GFP gene in intermediate construct pM115128. To insert the GFP gene into the now unique Hindlll restriction site (position 12303-12308 of SEQ. nr. 4) of pM115128, a DNA fragment with the GFP gene was isolated by digesting 1 microgram of the plasmid pGFP (CLontech, Palo Alto, US) in a manner known in itself (and analogous to the procedure described above) using the restriction enzymes Asp718 and EcoRI. Before the appropriate DNA fragment was purified from gel, the sticky ends were made blunt in a manner known in itself by treatment with the large (or "Klenow") fragment of E ^ coli DNA polymerase I. After puri- fication from agarose gel the fragment was again taken up in 10 microliters aqua bidest. Analogous tot the above procedure, l microgram PM115128 plasmid DNA was digested with the enzyme Hindlll, treated with the large (or "Klenow") fragment of E. coli DNA polymerase I to make the sticky ends blunt, purified from agarose gel, and taken up in 10 microliters aqua bidest . The Asp718-EcoRI DNA fragment from pGFP and the vector pM115128 with Hindlll were ligated in a manner known in itself and using the above method using T4 DNA ligase to pMll5128/GFP. The ligation mixture was transformed to E. coli strain PC2495. The transformed bacteria were cultured in the presence of the antibiotic ampicillin, to which the vector contains a resistance gene. The plasmid was isolated from the bacteria and using restriction enzymes the presence and proper orientation of the GFP gene was confirmed, c) Back-cloning of the GFP gene to pEAV030. To put the gene of pM115128/GFP back to the full- length pEAV030, use was made of the earlier used unique restriction sites EcoRI and Xhol. Using these enzymes a DNA fragment of the GFP gene plus the flanking EAV sequences was obtained from pM115128/GFP and this fragment was back- ligated between the unique EcoRI and Xhol restriction sites of pEAV030. Using the method described in Example l under d) , the RNA transcript of the DNA vector was introduced into BHK-21 cells, where replication of the RNA transcript occurred and GFP was expressed. Because ORF6 and ORF7 were knocked out, no virus particles could be formed. SEQ nr. 1 Source organism information: Molecule type RNA (genomicl Strandedness single Topology linear Anti-sense no Scientific name: Equine arteritis virus 1 TGCCATATAC GGCTCACCAC CATATACACT GCAAGAATTA CTATTCTTGT GGGCCCCTCT 61 CGGTAAATCC TAGAGGGCTT TCCTCTCGTT ATTGCGAGAT TCGTCGTTAG ATAACGGCAA 121 GTTCCCTTTC TTACTATCCT ATTTTCATCT TG GGCTTGA CGGGTCAtrTG CCATCGTCGT 1S1 CGATCTCTAT CAACTACCCT TGCGACTATG GCAACCTTCT CCGCTACTGG ATTTGGAGGG 241 AGTTΓTGTΓA GGGACTGGTC CCΓGGACΓTA CCCGACGCTT GTGAGCATGG CGCGGGATTG 301 TGCTGCGAAG TGGACGGCTC CACCTTATGC GCCGAGTGTT TTCGCGGTTG CGAAGGAATG 361 GAGCAATGTC CTGGCTTGTT CATGGGACTG TTAAAACTGG CTTCGCCAGT TCCAGTGGGA 421 CATAAGTTCC TGATTGGTTG GTATCGAGCT GCCAAAGTCA CCGGGCGTTA CAATTTCCTT 481 GAGCTGTTGC AACACCCTGC TTTCGCCCAG CTGCGTGTGG TTGATGCTAG GTTAGCCATT 541 GAAGAGGCAA GTGTGTTTAT TTCCACTGAC CACGCGTCTG CTAAGCGTTT CCCTGGCGC 601 AGATTTGCGC TGACACCGGT GTATGCTAAC GCTTGGGT G TGAGCCCGGC TGCTAACAGT 661 TTGATAGTGA CCACTGACCA GGAACAAGAT GGGTTCTGCT GGTTAAAACT TTTGCCACCT 721 GACCGCCGTG AGGCTGGTTT GCGGTTGTAT TACAACCATT ACCGCGAACA AAGGACCGGG 781 TGGCTGTCTA AAACAGGACT TCGCTrATGG CTTGGAGACC TGGGTTTGGG CATCAATGCG 841 AGCTCTGGAG GGCTGAAATT CCACATTATG AGGGGTTCGC CTCAGCGAGC TTGGCATATC 901 ACAACACGCA GCTGCAAGCT GAAGAGCTAC TACGTTTGTG ACATCTCTGA AGCAGACTGG 961 TCCTGTTTGC CTGCTGGCAA CTACGGCGGC TACAATCCAC CAGGGGACGG AGCTTGCGGT 1021 TACAGGTGC TGGCCTTCAT GAATGGCGCC ACTGTTGTGT CGGCTGGTTG CAGTTCTGAC 1081 TTGTGGTGTG ATGATGAGTT GGCTTATCGA GTCTTTCAAT TGTCACCCAC GTTCACGGTT 1141 ACCATCCCAG GTGGGCGAGT TTGTCCGAAT GCCAAGTACG CAATGATTTG TGACAAGCAG 1201 CACTGGCGCG TCAAACGTGC AAAGGGCGTC GGCCTG GTC TCGATGAAAG CTGTTTCAGG 1261 GGCATCTGCA ATTGCCAACG CATGAGTGGA CCACCACCTG CACCCG GTC AGCCGCCG G 1321 TTAGATCACA TACTGGAGGC GGCGACGTTT GGCAACGTTC GCGTGGTTAC ACCTGAAGGG 1381 CAGCCACGCC CCGTACCAGC GCCGCGAGTT CGTCCCAGCG CCAACTCTTC TGGAGATGTC 1441 AAAGATCCGG CGCCCGTTCC GCCAGTACCA AAACCAAGGA CCAAGCTTGC CACACCGAAC 1501 CCAACTCAGG CGCCCATCCC AGCACCGCGC ACGCGACTTC AAGGGGCCTC AACACAGGAG 1561 CCACTGGCGA GTGCAGGAGT TGCTTCTGAC TCGGCACCTA AATGGCGTGT GGCCAAAACT 1621 GTOTACAGCT CCGCGGAGCG CTTTCGGACC GAACTGGTAC AACGTGC CG GTCCGTTGGG 1681 GACGTTCTTG TTCAAGCGCT ACCGCTCAAA ACCCCAGCAG TGCAGCGGTA TACCATGACT 1741 CTGAAGATGA TGCGTTCACG CTTCAGTTGG CACTGCGACG TGTGGTACCC TTTGGCTGTA 1801 ATCGCTTGTT TGCTCCCTAT ATGGCCATCT CTTGCTTTGC TCCTTAGCTT TGCCATTGGG 1861 TTGATACCCA GTGTGGGCAA TAATGTTGTT CTGACAGCGC TTCTGGTTTC ATCAGCTAAT 1921 TATGTTGCGT CAATGGACCA TCAATGTGAA GGTGCGGCTT GCTTAGCCTT GCTGGAAC5AA 1981 GAACACTATT ATAGAGCGGT CCGTTGGCGC CCGATTACAG GCGCGCTGTC GCTTGTGCTC 20 1 AATTTACTGβ GGCAGGTAGG CTATGTAGCT CGTTCCACCT TTGATGCAGC TTATGTTCCT 2101 TGCAC-TGTGT TCGATCTTTG CAGCTTTGCT ATTCTGTACC TCTGCCGCAA TCGTTGCTGG 2161 AGATGCTTCG GACGCTGTGT GCGAGTTGGG CCTGCCACGC ATGTTTTGGG CTCCACCGGG 2221 CAACGAGTTT CCAAACTGGC GCTCATTGAT TTGTGTGACC ACTTTTCAAA GCCCACCATC 2281 GATGTTGTGG GCATGGCAAC TGGTTGGAGC GGATGTTACA CAGGAACCGC CGCAATGGAG 23 1 CGTCAGTGTG CCTCTACGGT GGACCCTCAC TCGTTCGACC AGAAGAAGGC AGGAGCGACT 240 GTTTACCTCA CCCCCCCTGT CAACAGCGGG TCAGCGCTGC AGTGCCTCAA TGTCATGTGG 2461 AAGCGACCAA TTGGGTCCAC TGTCCTTGGG GAACAAACAG GAGCTGTTGT GACGGCGGTC 2521 AAGAGTATCT CTTTCTCACC TCCCTGCTGC GTCTCTACCA CTTTGCCCAC CCGACCCGGT 2581 GTGACCGTTG TCGACCATGC TCTTTACAAC CGGTTGACTG CTTCAGGGGT CGATCCCGCT 2641 TTATTGCGTG TTGGGCAAGG TGATTTTCTA AAACTTAATC CGGGGTTCCG GCTGATAGGT 2701 GGATGGATTT ATGGGATATG CTATTTTGTG TTGGTGGTTG TGTCAACTTT TACCTGCTTA 2 61 CCTATCAAAT GTGGCATTGG CACCCGCGAC CCTTTCTGCC GCAGAGTGTT TTCTGTACCC 2821 GTCACCAAGA CCCAAGAGCA CTGCCATGCT GGAATGTGTG CTAGCGCTGA AGGCATCTCT 2881 CTGGACTCTC TGGGGTTAAC TCAGTTACAA AGTTACTGGA TCGCAGCCGT CACTAGCGGA 29 1 TTAGTGATCT TGTTGGTCTG CCACCGCCTG GCCATCAGCG CCTTGGACTT GTTGACTCTA 3001 GCTTCCCCTT TAGTGTTGCT TGTGTTCCCT TGGGCATCTG TGGGGCTTTT ACTTGCTTGC 3061 AGTCTCGCTG GTGCTGCTGT GAAAATACAG TTGTTGGCGA CGCTTTTTGT GAATCTGTTC 3121 TTTCCCCAAG CTACCCTTGT CΛCTATGGGA TACTGGGCGT GCGTGGCGGC TTTGGCCGTT 3181 TACAGTTTGA TGGGCTTGCG AGTGAAAGTG AATGTGCCCA TGTGTGTGAC ACCTGCCCAT 324 TTTCTGCTGC TGGCGAGGTC AGCTGGACAG TCAAGAGAGC AGATGCTCCG GGTCAGCGCT 3 01 GCTGCCCCCA CCAATTCACT GCTTGGAGTG GCTCGTGATT GTTATGTCAC AGGCACAACT 3361 CGGCTGTACA TACCCAAGGA AGGCGGGATG GTGTTTGAAG GGCTATTCAG GTCACCGAAG 3421 GCGCGCGGCA ACGTCGGCTT CGTGGCTGGT AGCAGCTACG GCACAGGGTC AGTGTGGACC 3481 AGGAACAACG AGGTCGTCGT ACTGACAGCG TCACACGTGG TTGGCCGCGC TAACATGGCC 3541 ACTCTGAAGA TCGGTGACGC AATGCTGACT CTGACTTTCA AAAAGAATGG CGACTTCGCC 3601 GAGGCAGTGA CGACACAGTC CGAGCTCCCA GGCAATTGGC CACAGTTGCA TTTCGCCCAA 3661 CCAACAACCG GGCCCGCTTC ATGGTGCACT GCCACAGGAG ATGAAGAAGG CTTGCTCAGT 3721 GGCGAGGTTT GTCTGGCGTG GACTACTAGT GGCGACTCTG GATCTGCAGT GGTTCAGGGT 3781 GACGCTGTGG TAGGGGTCCA CACCGGTTCG AACACAAGTG GTGTTGCCTA CGTGACCACC 3841 CCAAGCGGAA AACTCCTTGG CGCCGACACC GTGACTTTGT CATCACTGTC AAAGCATTTC 3901 ACAGGCCCTT TGACATCAAT CCCGAAGGAC ATCCCTGACA ACATTATTGC CGATGTTGAT 3961 GCTGTTCCTC GTTCTCTGGC CATGCTGATT GATGGCTTAT CCAATAGAGA GAGCAGCCTT 4021 TCTGGACCTC AGTTGTTGTT AATTGCTTGT TTTATGTGGT CTTATCTTAA CCAACCTGCT 4081 TACTTGCCTT ATGTGCTGGG CTTCTTTGCC GCTAACTTCT TCCTGCCAAA AAGTGTTGGC 4141 CGCCCTGTGG TCACTGGGCT TCTATGGTTG TGCTGCCTCT TCACACCGCT TTCCATGCGC 4201 TTGTGCTTGT TCCATCTGGT CTGTGCTACC GTCACGGGAA ACGTGATATC TTTGTGGTTC 4261 TACATCACTG CCGCTGGCAC GTCTTACCTT TCTGAGATGT GGTTCGGAGG CTATCCCACC 4321 ATGTTGTTTG TGCCACGGTT CCTAGTGTAC CAGTTCCCCG GCTGGGCTAT TGGCACAGTA 4381 CTAGCGGTAT GCAGCATCAC CATGCTGGCT GCTGCCCTCG GTCACACCCT GTTACTGGAT 4441 GTGTTCTCCG CCTCAGGTCG CTTTGACAGG ACTTTCATGA TGAAATACTT CCTGGAGGGA 4501 GGAGTGAAAG AGAGTGTCAC CGCCTCAGTC ACCCGCGCTT ATGGCAAACC AATTACCCAG 4561 GAGAGTCTCA CTGCAACATT AGCTGCCCTC ACTGATGATG ACTTCCAATT CCTCTCTGAT 4621 GTGCTTGACT GTCGGGCCGT CCGATCGGCA ATGAATCTGC GTGCCGCTCT CACAAGTTTT 4681 CAAGTGGCGC AGTATCGTAA CATCCTTAAT GCATCCTTGC AAGTCGATCG TGACGCTGCT 47 CGTAGTCGCA GACTAATGGC AAAACTGGCT GATTTTGCGG TTGAACAAGA AGTAACAGCT 4801 GGAGACCGTG TTGTGGTTAT CGACGGTCTG GACCGCATGG CTCACTTCAA AGACGATTTG 4861 GTGCTGGTTC CTTTGACCAC CAAAGTAGTA GGCGGTTCTA GGTGCACCAT TTGTGACGTC 4921 GTTAAGGAAG AAGCCAATGA CACCCCAGTT AAGCCAATGC CCAGCAGGAG ACGCCGCAAG 4981 GGCCTGCCTA AAGGTGCTCA GTTGGAGTGG GACCGTCACC AGGAAGAGAA GAGGAACGCC 50 1 GGTGATGATG ATTTTGCGGT CTCGAATGAT TATGTCAAGA GAGTGCCAAA GTACTGGOAT 5101 CCCAGCGACA CCCGAGGCAC GACAGTGAAA ATCGCCGGCA CTACCTATCA GAAAGTGGTT 5 61 GACTATTCAG GCAATGTGCA TTACGTGGAG CATCAGGAAG ATCTGCTAGA CTACGTGCTG 5221 GGCAAGGGGA GCTATGAAGG CCTAGATCAG GACJ^AAGTGT TGGACCTCAC AAACATGCTT 5281 AAAGTGGACC CCACGGAGCT CTCCTCCAAA GACAAAGCCA AGGCGCGTCA CGTTGCTCAT 5341 CTGCTGTTGG ATCTGGCTAA CCCAGTTGAG GCAGTGAATC AGTTAAACTG AGAGCGCCCC 5401 ACATCTTTCC CGGCGATGTG GGGCGTCGGA CCTTTGCTGA CTCTAAAGAC AAGGGTTTCG 5461 TGGCTCTACA CAGTCGCACA ATGTTTTTAG CTGCCCGGGA CTTTTTATTT AACATCAAAT 5S21 TTGTGTGCGA CGAAGAGTTC ACAAAGACCC CAAAAGACAC ACTGCTTGGG TACGTACGCG 5581 CCTGCCCTGG TTACTGGTTT ATTTTCCGTC GTACGCACCG GTCGCTGATT GATGCATACT 5641 GGGACAGTAT GGAGTGCGTT TACGCGCTTC CCACCATATC TGATTTTGAT GTGAGCCCAG 5701 GTGACGTCGC AGTGACGGGC GAGCGATGGG ATTTTGAATC TCCCGGAGGA GGCCGTGCAA 5761 AACGTCTCAC AGCTGATCTG GTGCACGCTT TTCAAGGGTT CCACGGAGCC TCTTATTCCT 5821 ATGATGACAA GGTGGCAGCT GCTGTCAGTG GTGACCCGTA TCGGTCGGAC GGCGTCTTGT 5881 ATAACACCCG TTGGGGCAAC ATTCCATATT CTGTCCCAAC CAATGCTTTG GAAGCCACAG 5941 CTTGCTACCG TGCTGGATGT GAGGCCGTTA CCGACGGGAC CAACGTCATC GCAACAATTG 6001 GGCCCTTCCC GGAGCAACAA CCCATACCGG ACATCCCAAA GAGCGTGCTT GACAACTGCG 6061 CTGACATCAG CTGTGACGCT TTCATAGCGC CCGCTGCAGA GACAGCCCTG TGTGGAGATT 6121 TAGAGAAATA CAACCTATCC ACGCAGGGTT TTGTGTTGCC TAGTGTTTTC TCCATGGTGC 6181 GGGCGTACTT AAAAGAGGAG ATTGGAGACG CTCCACCACT CTACTTGCCA TCTACTGTAC 6241 CATCTAAAAA TTCACAAGCC GGAATTAACG GCGCTGAGTT TCCTACAAAG TCTTTACAGA 6301 GCTACTGTTT GATTGATGAC ATGGTGTCAC AGTCCATGAA AAGCAATCTA CAAACCGCCA 6361 CCATGGCGAC TTGTAAACGG CAATACTGTT CCAAATACAA GATTAGGAGC ATTCTGGGCA 6421 CCAACAATTA CATTGGCCTA GGTTTGCGTG CCTGCCTTTC GGGGGTTACG GCCGCATTCC 6481 AAAAAGCTGG AAAGGATGGG TCACCGATTT ATTTGGGCAA GTCAAAATTC GACCCGATAC 6541 CAGCTCCTGA CAAGTACTGC CTTGAAACAG ACCTGGAGAG TTGTGATCGC TCCACCCCGG 6601 CTTTGGTGCG TTGGTTCGCT ACTAATCTTA TTTTTGAGCT AGCTGGCCAG CCCGAGTTGG 6661 TGCACAGCTA CGTGTTGAAT TGCTGTCACG ATCTAGTTGT GGCGGGTAGT GTAGCATTCA 6721 CCAAACGCGG GGGTTTGTCA TCTGGAGACC CTATCACTTC CATTTCCAAT ACCATCTATT 6781 CATTGGTGCT GTACACCCAG CACATGTTGC TATGTGGACT TGAAGGCTAT TTCCCAGAGA 6841 TTGCAGAAAA ATATCTTGAT GGCAGCCTGG AGCTGCGGGA CATGTTCAAG TACGTTCGAG 6901 TGTACATCTA CTCGGACGAT GTGGTTCTAA CCACACCCAA CCAGCATTAC GCGGCCAGCT 6961 TTGACCGCTG GGTCCCCCAC CTGCAGGCGC TGCTAGGTTT CAAGGTTGAC CCAAAGAAAA 7021 CTGTGAACAC CAGCTCCCCT TC ΓTTTTGG GCTGCCGGTT CAAGCAAGTG GACGGCAAGT 708 1 GTTATCTAGC CAGTCTTCAG GACCGCGTTA CACGCTCTCT GTTATACCAC ATTGGTGCAA 71 41 AGAATCCCTC AGAGTACTAT GAAGCTGCTG TTTCCATCTT TAAGGACTCC ATTATCTGCT 7201 GTGATGAAGA CTGGTGGACG GACCTCCATC GACGTATCAG TCW3CGCTGCG CGTACCGACG 7261 GAGTTGAGTT CCCCACCATT GAAATGTTAA CATCCTTCCG CACCAAGCAG TATGAGAGTG 732 1 CCGTGTGCAC AGTTTGTGGG GCCGCCCCCG TGGCCAAGTC TGCTTGTGGA GGGTGGTTCT 7381 GTGGCAATTG TGTCCCGTAC CACGCGGGTC ATTGTCACAC AACCTCGCTC TTCGCCAACT 74 4 1 GCGGGCACGA CATCATGTAC CGCTCCACTT ACTGCACAAT GTGTGAGGGT TCCCCAAAAC 7501 AGATGGTACC AAAAGTGCCT CACCCGATCC TGGATCATTT GCTGTGCCAC ATTGATTACG 7561 GCAGTAAAGA GGAACTAACT CTGGTAGTGG CGGATGGTCG AACAACATCA CCGCCCGGGC 7621 GCTACAAAGT GGGTCACAAG GTAGTCGCCG TGGTTGCAGA TGTGGGAGGC AACATTGTGT 7681 TTGGGTGCGG TCCTGGATCA CACATCGCAG TACCACTTCA GGATACGCTC AAGGGCGTGG 7741 TGGTGAATAA AGCTCTGAAG AACGCCGCCG CCTCTGAGTA CGTGGAAGGA CCCCCTGGGA 7801 GTGGGAAGAC TTTTCACCTG GTCAAAGATG TGCTAGCCGT GGTCGGTAGC GCGACCTTGG 7861 TTGTGCCCAC CCACGCGTCC ATGCTGGACT GCATCAACAA GCTCAAACAA GCGGGCGCCG 7921 ATCCATACTT TGTGGTGCCC AAGTATACAG TTCTTGACTT TCCCCGGCCT GGCAGTOGAA 7981 ACATCACAGT GCGACTGCCA CAGGTCGGAA CCAGTGAGGG AGAAACCTTT GTGGATGAGG 80 1 TGGCCTACTT CTCACCAGTG GATCTGGCGC GCATTTTAAC CCAGGGTCGA GTCAAGGOTT 8101 ACGGTGATTT AAATCAGCTC GGGTGCGTCG GACCCGCGAG CGTGCCACGT AACCTTTGGC 8161 TCCGACATTT TGTCAGCCTG GAGCCCTTGC GAGTGTGCCA TCGATTCGGC GCTGCTGTGT 8221 GTGATTTGAT CAAGGGCATT TATCCTTATT ATGAGCCAGC TCCACATACC ACTAAAGTGG 8281 TGTTTGTGCC AAATCCAGAC TTTGAGAAAG GTGTAGTCAT CACCGCCTAC CACAAAGATC 83 1 GCGGTCTTGG TCACCGCACA ATTGATTCAA TTCAAGGCTG TACATTCCCT GTTGTGACTC 8 01 TTCGACTGCC CACACCCCAA TCACTGACGC GCCCGCGCGC AGTTGTGGCG GTTACTAGGG 8461 CGTCTCAGGA ATTATACATC TACGACCCCT TTGATCAGCT TAGCGGGTTG TTGAAGTTCA 8521 CCAAGGAAGC AGAGGCGCAG GACTTGATCC ATGGCCCACC TACAGCATGC CACCTGGOCC 8581 AAGAAATTGA CCTTTGGTCC AATGAGGGCC TCGAATATTA CAAGGAAGTC AACCTGCTGT 8641 ACACACACGT CCCCATCAAG GATGGTGTAA TACACAGTTA CCCTAATTGT GGCCCTGCCT 8701 GTGGCTGGGA AAAGCAATCC AACAAAATTT CGTGCCTCCC GAGAGTGGCA CAAAATTTGG 8 61 GCTACCACTA TTCCCCAGAC TTACCAGGAT TTTGCCCCAT ACCAAAAGAA CTCGCTGAGC 8821 ATTGGCCCGT AGTGTCCAAT GATAGATACC CGAATTGCTT GCAAATTACC TTACAGCAAG 8881 TATGTGAACT CAGTAAACCG TGCTCAGCGG GCTATATGGT TGGACAATCT GTTTTCG GC 89 1 AGACGCCTGG TGTGACATCT TACTGGCTTA CTGAATGGGT CGACGGCAAA GCGCGTGCTC 9001 TACCAGATTC CTTATTCTCG TCCGGTAGGT TCGAGACTAA CAGCCGCGCT TTCCTCGATG 9061 AAGCCGAGGA AAAGTTTGCC GCCGCTCACC CTCATGCCTG TTTGGGAGAA ATTAATAAGT 9121 CCACCGTGGG AGGATCCCAC TTCATCTTTT CCCAATATTT ACCACCATTG CTACCCGCAG 9181 ACGCTGTTGC CCTGGTAGGT GCTTCATTGG CTGGGAAAGC TGCTAAAGCT GCTTGCAGCG 9241 TTGTTGATGT CTATGCTCCA TCATTTGAAC CTTATCTACA CCCTCAGACA CTGAGTCGCG 9301 TGTACAAGAT TATGATCGAT TTCAAGCCGT GTAGGCTTAT GGTGTGGAGA AACGCGACCT 9361 TTTATGTCCA AGAGGGTGTT GATGCAGTTA CATCAGCACT AGCAGCTGTG TCCAAACTCA 9421 TCAAAGTGCC GGCCAATGAG CCTGTTTCAT TCCATGTGGC ATCAGGGTAC AGAACCAACG 9481 CGCTGGTAGC GCCCCAGGCT AAAATTTCAA TTGGAGCCTA CGCCGCCGAG TGGGCACTGT 9541 CAACTGAACC GCCACCTGCT GGTTATGCGA TCGTGCGGCG ATATATTGTA AAGAGGCTCC 9601 TCAGCTCAAC A(»AGT<?rTC TTGTGCCGCA GGGGTGTTGT GTCTTCCACC TCAGTGCAGA 9661 CCATTTGTGC ACTAGAGGGA TGTAAACCTC TGTTCAACTT CTTACAAATT GGTTCAGTCA 9721 TTGGGCCCGT GTGATGGGCT TAGTGTGGTC ACTGATTTCA AATTCTATTC AGACTATTAT 9781 TGCTGATTTT GCTATTTCTG TGATTGATGC AGCGCTTTTC TTTCTCATGC TACTTGCATT 98 1 GGCTGTTGTT ACTGTGTTTC TTTTCTGGCT CATTGTTGCC ATCGGCCGCA GCTTGGTGGC 9901 GCGGTGTTCA CGAGGTGCGC GTTACAGACC TGTTTAAGGA TTTGCAGTGC GACAACCTGC 9961 GCGCGAAAGA TGCCTTCCCG AGTCTGGGAT ATGCTCTGTC GATTGGCCAG TCGAGGCTAT 10021 CGTATATGCT GCAGGATTGG TTGCTTGCTG CGCACCGCAA GGAAGTTATG CCTTCCAATA 10081 TCATGCCTAT GCCCGGTCTT ACTCCTGATT GCTTTCΪACCA TCTGGAGTCT TCTAGCTATG 10141 CTCCATTTAT CAATGCCTAT CGGCAGGCAA TTTTGAGTCA ATACCCACAA GAGCTCCAGC 10201 TCGAAGCCAT CAACTGTAAA TTGCTTGCTG TGGTTGCACC GGCATTGTAT CATAATTACC 10261 ATCTAGCCAA TTTGACCGGA CCGGCCACAT GGGTCGTGCC TACAGTGGGC CAGTTGCACT 10321 ATTATGCTTC TTCCTCTATT TTTGCTTCAT CTGTGGAAGT GTTGGCAGCA ATAATACTAC 10381 TATTTGCATG CATACCACTA GTGACACGAG TGTACATCTC TTTTACGCGG CTAATGTCAC 104 1 CTTCCCGTCG CACTTCCAGC GGCACTTTGC CGCGGCGCAA GATTTTGTAG TGCACACGGG 10S01 TTATGAATAT GCCGGGGTCA CTATGTTAGT GCACTTGTTT GCCAACTTGG TTCTGACATT 10561 TCCGAGCTTA GTTAATTGTT CCCGCCCTGT GAATGTCTTT GCTAATGCTT CTTGCGTGCA 10621 AGTGGTTTGT AGTCATACCA ACTCAACTAC TGGCTTGGGT CAACTTTCTT TTTCCTTTGT 10681 AGATGAAGAT CTACGGCTGC ATATCAGGCC TACTCTTATT TGTTGGTTTG CCTTGTTGTT 10741 GGTGCACTTT CTACCCATGC CACGCTGCAG AGGCTCGTAA TTTTACTTAC ATTAGTCATO 10801 GATTGGGCCA CGTGCACGGT CATGAGGGGT GTAGGAATTT TATTAATGTC ACTCATTCTG 10861 CATTTCTTTA TCTTAATCCC ACCACTCCCA CTGCGCCGGC TATAACTCAT TGTTTACTTC 10921 TGGTTCTGGC AGCCAAAATG GAACACCCAA ACGCTACTAT CTGGCTGCAG CTGCAGCCGT 10 81 TTGGGTATCA TGTGGCTGGC GATGTCATTG TCAACTTGGA AGAGGACAAG AGGCATCCTT 110 1 ACΠTAAACT TTTGAGAGCG CCGGCTT AC CGCTTGGTTT TGTGGCTATA GTTTATGTTC 11101 TTTTACGACT GGTACGTTGG GCTCAACGAT GTTATCTATG ATTGTATTGC TMTCTTGCT 11161 TTGGGGTGCG CCATCACATG CTTACTTCTC ATACTACACC GCTCAGCGCT TCACAGACTT 11221 CACCTTGTGT ATGCTGACGG ATCGCGGCGT ATTGCCAAT TTGCTGCGAT ATGATGAGCA 11281 CACTGCTTTG TACAATTGTT CCGCCAGTAA AACCTGTTGG TATTGCACAT TCCTGGACGA 11341 ACAGATTATC ACGTTTGGAA CCGATTGTGA TGACACCTAC GCGGTCCCAG TTGCTGAGGT 11401 CCTGGAACAG GCGCATGGAC CGTACAGTGC GCTGTTTGAT GACATGCCCC CTTTTATTTA 11461 CTATGGCCGT GAATTCGGCA TAGTTGTGTT GGATGTCTTT ATGTTCTATC CCGTTTTλGT 11521 TCTGTTTTTC TTATCAGTAC TACCCTATGC TACGCTTATT CTTGAAATGT GTGTATCTAT 11581 TCTGTTTATA ATCTATGGCA TTTACAGCGG GGCCTACTTG GCCATGGGCA TATTTGCGGC 11641 CACGCTTGCT ATACATTCAA TTGTGGTCCT CCGCCAATTA CTGTGGTTAT GCCTGGCTTG 11701 GCGATACCGC TGTACGCTTC ACGCGTCCTT TATATCAGCT GAGGGGAAAG TGTACCCCGT 11761 AβACCCCGGA CTCCCGGTTG CCGCCGTGGG CAATCGGTTG TTAGTCCCAG GTAGGCCCAC 11821 TATCGATTAT GCAGTGGCCT ACGGCAGCAA AGTCAACCTT GTGAGGTTGG GGGCAGCTGA 11881 GGTATGGGAG CCATAGATTC ATTTTQTGGT GACGGGATTT TAGGTGAGTA TCTAGATTAC 11941 TTTATTCTGT CCGTCCCACT CTTGCTGTTG CTTACTAGGT ATGTAGCATC TGGGTTAGTG 12001 TATGTTTTGA CTGCCTTGTT CTATTCCTTT GTATTAGCAG CTTATATTTG GTTTGTTATA 12061 GTTGGAAGAG CCTTTTCTAC TGCTTATGCT TTTCyTGCTTT TGGCTGCTTT TCTGTTATTA 12121 GTAATGAGGA TGATTGTGGG TATGATGCCT CGTCTTCGGT CCATTTTCAA CCATCGCCAA 12181 CTGGTGGTAG CTGATTTTGT GGACACACCT AGTGGACCTG TTCCCATCCC CCGCTCAACT 122 1 ACTCAGGTAG TGGTTCGCGG CAACGGGTAC ACCGCAGTTG GTAACAAGCT TGTCGATGGC 12301 GTCAAGACGA TCACGTCCGC AGGCCGCCTC TTTTCGAAAC GGACGGCGGC GACAGCCTAC 12361 AAGCTACAAT GACCTACTGC GCATGTTTGG TCAGATGCGG GTCCGCAAAC CGCCCGCGCA 12421 ACCCACTCAG GCTATTATTG CAGAGCCTGG AGACCTTAGG CATGATTTAA ATCAACAGGA 12481 GCGCGCCACC CTTTCGTCGA ACGTACAACG GTTCTTCATG ATTGGGCATG GTTCACTCAC 12541 TGCAGATGCC GGAGGACTCA CGTACACCGT CAGTTGGGTT CCTACCAAAC AAATCCAGCG 12601 CAAAGTTGCG CCTCCAGCAG GGCCGTAAGA CGTGGATATT CTCCTGTGTG GCGTCATGTT 12661 GAAGTAGTTA TTAGCCACCC AGGAACCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 12721 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 12781 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA SEQ nr. 2 Source organism information: Molecule type: RNA (genomic) Strandedness : single Topology : linear Anti-sense : no Scientific name: Equine arteritis virus 1 GCTCGAAGTG TGTATGG SEQ nr. 3 Source organism information: Molecule type: DNA (genomic) Strandedness : double Topology : linear Anti-sense : no Scientific name: Bacteriophage T7 1 TAATACGACT CACTATA Construction of pEAV030 nt 1 to 17 = sequence corresponding with SEQ nr. 2 nt 18 to 12,844 = sequence corresponding with SEQ nr.l nt 12,845 to 12,851 = synthetic nucleotide sequence with unique Xhol restriction site nt 12,852 to 15,490 = pUC18 vector sequence (beginning on the Hindlll made blunt and ending on the EcoRI restriction site made blunt) 2L nt 1 5,4 9 1 to 15,511 = synthetic nucleotide sequence with two unique restriction sites (Ndel and NotI) nt 15 ,512 to 1 5,528 = sequence corresponding to S E Q nr. 3 (T7 promotor) 1 GCTCGAAGTG TGTATGGTGC CATATACGGC TCACCACCAT ATACACTGCA AGAATTACTA 61 TTCTTGTGGG CCCCTCTCGG TAAATCCTAG AGGGCTTTCC TCTCGTTATT GCGAGATTCG 121 TCGTTAGATA ACGGCAAGTT CCCTTTCTTA CTATCCTATT TTCATCTTGT GGCTTGACGG 181 GTCACTGCCA TCGTCGTCGA TCTCTATCAA CTACCCTTGC GACTATGGCA ACCTTCTCCG 241 CTACTGGATT TGGAGGGAGT TTTGTTAGGG ACTGGTCCCT GGACTTACCC GACGCTTGTG 301 AGCATGGCGC GGGATTGTGC TGCGAAGTGG ACGGCTCCAC CTTATGCGCC GAGTGTTTTC 361 GCGGTTGCGA AGGAATGGAG CAATGTCCTG GCTTGTTCAT GGGACTGTTA AAACTGGCTT 421 CGCCAGTTCC AGTGGGACAT AAGTTCCTGA TTGGTTGGTA TCGAGCTGCC AAAGTCACCG 481 GGCGTTACAA TTTCCTTGAG CTGTTGCAAC ACCCTGCTTT CGCCCAGCTG CGTGTGGTTG 541 ATGCTAGGTT AGCCATTGAA GAGGCAAGTG TGTTTATTTC CACTGACCAC GCGTCTGCTA 601 AGCGTTTCCC TGGCGCTAGA TTTGCGCTGA CACCGGTGTA TGCTAACGCT TGGGTTGTGA 661 GCCCGGCTGC TAAC-AGTTTG ATAGTGACCA CTGACCAGGA ACAAGATGGG TTCTGCTGGT 721 TAAAACTTTT GCCACCTGAC CGCCGTGAGG CTGGTTTGCG GTTGTATTAC AACCATTACC 781 GCGAACAAAG GACCGGGTGG CTGTCTAAAA CAGGACTTCG CTTATGGCTT GGAGACCTGG 841 GTTTGGGCAT CAATGCGAGC TCTGGAGGGC TGAAATTCCA CATTATGAGG GGTTCGCCTC 901 AGCGAGCTTG GCATATCACA ACACGCAGCT GCAAGCTGAA GAGCTACTAC GTTTGTGACA 961 TCTCTGAAGC AGACTGGTCC TGTTTGCCTG CTGGCAACTA CGGCGGCTAC AATCCACCAG 1021 GGGACGGAGC TTGCGGTTAC AGGTGCTTGG CCTTCATGAA TGGCGCCACT GTTGTGTCGG 1081 CTGGTTGCAG TTCTGACTTG TGGTGTGATG ATGAGTTGGC TTATCGAGTC TTTCAATTGT 1141 CACCCACGTT CACGGTTACC ATCCCAGGTG GGCGAGTTTG TCCGAATGCC AAGTACGCAA 1201 TGATTTGTGA CAAGCAGCAC TGGCGCGTCA AACGTGCAAA GGGCGTCGGC CTGTGTCTCG 1261 ATGAAAGCTG TTTCAGGGGC ATCTGCAATT GCCAACGCAT GAGTGGACCA CCACCTGCAC 1321 CCGTGTCAGC CGCCGTGTTA GATCACΛTAC TGGAGGCGGC GACGTTTGGC AACGTTCGCG 1381 TGGTTACACC TGAAGGGCAG CCACGCCCCG TACCAGCGCC GCGAGTTCGT CCCAGCGCCA 1441 ACTCTTCTGG AGATGTCAAA GATCCGGCGC CCGTTCCGCC AGTACCAAAA CCAAGGACCA 1501 AGCTTGCCAC ACCGAACCCA ACTCAGGCGC CCATCCCAGC ACCGCGCACG CGACTTCAAG 1561 GGGCCTCAAC ACAGGAGCCA CTGGCGAGTG CAGGAGTTGC TTCTGACTCG GCACCTAAAT 1621 GGCGTGTGGC CAAAACTGTG TACAGCTCCG CGGAGCGCTT TCGGACCGAA CTGGTACAAC 1681 GTGCTCGGTC CGTTGGGGAC GTTCTTGTTC AAGCGCTACC GCTCAAAACC CCAGCAGTGC 1741 AGCGGTATAC CATGACTCTG AAGATGATGC GTTCACGCTT CAGTTGGCAC TGCGACGTGT 1801 GGTΛCCCTTT GGCTGTAATC GCTTGTTTGC TCCCTATATG GCCATCTCTT GCTTTGCTCC 1861 TTAGCTTTGC CATTGGGTTG ATACCCAGTG TGGGCAATAA TGTTGTTCTG ACAGCGCTTC 1921 TGGTTTCATC AGCTAATTAT GTTGCGTCAA TGGACCATCA ATGTGAAGGT GCGGCTTGCT 1981 TAGCCTTGCT GGAAGAAGAA CACTATTATA GAGCGGTCCG TTGGCGCCCG ATTACAGGCG 20 1 CGCTGTCGCT TGTGCTCAAT TTACTGGGGC AGGTAGGCTA TGTAGCTCGT TCCACCTTTG 2101 ATGCAGCTTA TGTTCCTTGC ACTGTGTTCG ATCTTTGCAG CTTTGCTATT CTGTACCTCT 2161 GCCGCAATCG TTGCTGGAGA TGCTTCGGAC GCTGTGTGCG AGTTGGGCCT GCCACGCATG 2221 TTTTGGGCTC CACCGGGCAA CGAGTTTCCA AACTGGCGCT CATTGATTTG TGTGACCACT 2281 TTTCAAAGCC CACCATCGAT GTTGTGGGCA TGGCAACTGG TTGGAGCGGA TGTTACACAG 2341 GAACCGCCGC AATGGAGCGT CAGTGTGCCT CTACGGTGGA CCCTCACTCG TTCGACCAGA 2401 AGAAGGCAGG AGCGACTGTT TACCTCACCC CCCCTGTCAA CAGCGGGTCA GCGCTGCAGT 2461 GCCTCAATGT CATGTGGAAG CGACCAATTG GGTCCACTGT CCTTGGGGAA CAAACAGOAG 2521 CTGTTGTGAC GGCGGTCAAG AGTATCTCTT TCTCACCTCC CTGCTGCGTC TCTACCACTT 2581 TGCCCACCCG ACCCGGTGTG ΛCCGTTGTCG ACCATGCTCT TTACAACCGG TTGACTGCTT 26 1 CAGGGGTCGA TCCCGCTTTA TTGCGTGTTG GGCAAGGTGA TTTTCTAAAA CTTAATCCGG 2701 GGTTCCGGCT GATAGGTGGA TGGATTTATG GGATATGCTA TTTTGTGTTG GTGGTTGTGT 2761 CAACTTTTAC CTGCTTACCT ATCAAATGTG GCATTGGCAC CCGCGACCCT TTCTGCCGCA 2821 GAGTGTTTTC TGTACCCGTC ACCAAGACCC AAGAGCACTG CCATGCTGGA ATGTGTGCTA 2881 GCGCTGAAGG CATCTCTCTG GACTCTCTGG GGTTAACTCA GTTACAAAGT TACTGGATCG 29 1 CAGCCGTCAC TAGCGOATTA GTGATCTTGT TGGTCTGCCA CCGCCTGGCC ATCAGCGCCT 3001 TGGACTTGTT GACTCTAGCT TCCCCTTTAG TGTTGCTTGT GTTCCCTTGG GCATCTGTGG 3061 GGCTTTTACT TGCTTGCAGT CTCGCTGGTG CTGCTGTGAA AATACAGTTG TTGGCGACGC 3121 TTTTTGTGAA TCTGTTCTTT CCCCAAGCTA CCCTTGTCAC TATGGGATAC TGGGCGTGCG 3181 TGGCGGCTTT GGCCGTTTAC AGTTTGATGG GCTTGCGAGT GAAAGTGAAT GTGCCCATGT 3241 GTGTGλCλCC TGCCCATTTT CTGCTGCTGG CGAGGTCAGC TGGACAGTCA AGAGAGCAGA 3301 TGCTCCGGGT CAGCGCTGCT GCCCCCACCA ATTCACTGCT TGGAGTGGCT CGTGATTGTT 3361 ATGTCACAGG CACAACTCGG CTGTACATAC CCAAGGAAGG CGGGATGGTG TTTGAAGGGC 3421 TATTCAGGTC ACCGAAGGCG CGCGGCAACG TCGGCTTCGT GGCTGGTAGC AGCTACGGCA 3481 CAGGGTCAGT GTGGACCAGG AACAACGAGG TCGTCGTACT GACAGCGTCA CACGTGGTTG 3541 GCCGCGCTAA CATGGCCACT CTGAAGATCG GTGACGCAAT GCTGACTCTG ACTTTCAAAA 3601 AGAATGGCGA CTTCGCCGAG GCAGTGACGA CACAGTCCGA GCTCCCAGGC AATTGGCCAC 3661 AGTTGCATTT CGCCCAACCA ACAACCGGGC CCGCTTCATG GTGCACTGCC ACAGGAGATG 3721 AAGAAGGCTT GCTCAGTGGC GAGGTTTGTC TGGCGTGGAC TACTAGTGGC GACTCTGGAT 3781 CTGCAGTGGT TCAGGGTGAC GCTGTGGTAG GGGTCCACAC CGGTTCGAAC ACAAGTGGTG 3841 TTGCCTΛCGT GACCACCCCA AGCGGAAAAC TCCTTGGCGC CGACACCGTG ACTTTGTCAT 3901 CACTGTCAAA GCATTTCACA GGCCCTTTGA CATCAATCCC GAAGGACATC CCTGACAACA 3961 TTATTGCCGA TGTTGATGCT GTTCCTCGTT CTCTGGCCAT GCTGATTGAT GGCTTATCCA 4021 ATAGAGAGAG CAGCCTTTCT GGACCTCAGT TGTTGTTAAT TGCTTGTTTT ATGTGGTCTT 4081 ATCTTAACCA ACCTGCTTAC TTGCCTTATG TGCTGGGCTT CTTTGCCGCT AACTTCTTCC 141 TGCCAAAAAG TGTTGGCCGC CCTGTGGTCA CTGGGCTTCT ATGGTTGTGC TGCCTCTTCA 4201 CACCGCTTTC CATGCGCTTG TGCTTGTTCC ATCTGGTCTG TGCTACCGTC ACGGGAAACG 4261 TGATATCTTT GTGGTTCTAC ATCACTGCCG CTGGCACGTC TTACCTTTCT GAGATGTGGT 4321 TCGGAGGCTA TCCCACCATG TTGTTTGTGC CACGGTTCCT AGTGTACCAG TTCCCCGGCT 4381 GGGCTATTGG CACAGTACTA GCGGTATGCA GCATCACCAT GCTGGCTGCT GCCCTCGGTC 4441 ACACCCTGTT ACTGGATGTG TTCTCCGCCT CAGGTCGCTT TGACAGGACT TTCATGATGA 4501 AATACTTCCT GGAGGGAGGA GTGAAAGAGA GTGTCACCGC CTCAGTCACC CGCGCTTATG 4561 GCAAACCAAT TACCCAGGAG AGTCTCACTG CAACATTAGC TGCCCTCACT GATGATGACT 621 TCCAATTCCT CTCTGATGTG CTTGACTGTC GGGCCGTCCG ATCGGCAATG AATCTGCGTG 4681 CCGCTCTCAC AAGTTTTCAA GTGGCGCAGT ATCGTAACAT CCTTAATGCA TCCTTGCAAG 4741 TCGATCGTGA CGCTGCTCGT AGTCGCAGAC TAATGGCAAA ACTGGCTGAT TTTGCGGTTG 4801 AACAAGAAGT AACAGCTGGA GACCGTGTTG TGGTTATCGA CGGTCTGGAC CGCATGGCTC 4861 ACTTCAAAGA CGATTTGGTG CTGGTTCCTT TGACCACCAA AGTAGTAGGC GGTTCTAGGT 4921 GCACCATTTG TGACGTCGTT AAGGAAGAAG CCAATGACAC CCCAGTTAAG CCAATGCCCA 4981 GCAGGAGACG CCGCAAGGGC CTGCCTAAAG GTGCTCAGTT GGAGTGGGAC CGTCACCAGG 5041 AAGAGAAGAG GAACGCCGGT GATGATGATT TTGCGGTCTC GAATGATTAT GTCAAGAGAG 5101 TGCCAAAGTA CTGGGATCCC AGCGACACCC GAGGCACGAC AGTGAAAATC GCCGGCACTA 5161 CCTATCAGAA AGTGGTTGAC TATTCAGGCA ATGTGCATTA CGTGGAGCAT CAGGAAGATC 5221 TGCTAGACTA CGTGCTGGGC AAGGGGAGCT ATGAAGGCCT AGATCAGGAC AAAGTGTTGG 5281 ACCTCACAAA CATGCTTAAA GTGGACCCCA CGGAGCTCTC CTCCAAAGAC AAAGCCAAGG 5341 CGCGTCACGT TGCTCATCTG CTGTTGGATC TGGCTAACCC AGTTGAGGCA GTGAATCAOT 5401 TAAACTGAGA GCGCCCCACA TCTTTCCCGG CGATGTGGGG CGTCGGACCT TTGCTGACTC 5461 TAAAGACAAG GGTTTCGTGG CTCTACACAG TCGCACAATG TTTTTAGCTG CCCGGGACTT 5521 TTTATTTAAC ATCAAATTTG TGTGCGACGA AGAGTTCACA AAGACCCCAA AAGACACACT 5581 GCTTGGGTAC GTACGCGCCT GCCCTGGTTA CTGGTTTATT TTCCGTCGTA CGCACCGGTC 5641 GCTGATTGAT GCATACTGGG ACAGTATGGA GTGCGTTTAC GCGCTTCCCA CCATATCTGA 5701 TTTTGATGTG AGCCCAGGTG ACGTCGCAGT GACGGGCGAG CGATGGGATT TTGAATCTCC 5761 CGGAGGAGGC CGTGCAAAAC GTCTCACAGC TGATCTGGTG CACGCTTTTC AAGGGTTCCA 5821 CGGAGCCTCT TATTCCTATG ATGACAAGGT GGCAGCTGCT GTCAGTGGTG ACCCGTATCG 5881 GTCGGACGGC GTCTTGTATA ACACCCGTTG GGGCAACATT CCATATTCTG TCCCAACCAA 5941 TGCTTTGGAA GCCACAGCTT GCTACCGTGC TGGATGTGAG GCCGTTACCG ACGGGACCAA 6001 CGTCATCGCA ACAATTGGGC CCTTCCCGGA GCAACAACCC ATACCGGACA TCCCAAAGAG 6061 CGTGCTTGAC AACTGCGCTG ACATCAGCTG TGACGCTTTC ATAGCGCCCG CTGCAGAGAC 6121 AGCCCTGTGT GGAGATTTAG AGAAATACAA CCTATCCACG CAGGGTTTTG TGTTGCCTAG 6181 TGTTTTCTCC ATGGTGCGGG CGTACTTAAA AGAGGAGATT GGAGACGCTC CACCACTCTA 6241 CTTGCCATCT ACTGTACCAT CTAAAAATTC ACAAGCCGGA ATTAACGGCG CTGAGTTTCC 6301 TACAAAGTCT TTACAGAGCT ACTGTTTGAT TGATGACATG GTGTCACAGT CCATGAAAAG 6361 CAATCTACAA ACCGCCACCA TGGCGACTTG TAAACGGCAA TACTGTTCCA AATACAAGAT 6421 TAGGAGCATT CTGGGCACCA ACAATTACAT TGGCCTAGGT TTGCGTGCCT GCCTTTCGGG 6481 GGTTACGGCC GCATTCCAAA AAGCTGGAAA GGATGGGTCA CCGATTTATT TGGGCAAGTC 6541 AAAATTCGAC CCGATACCAG CTCCTGACAA GTACTGCCTT GAAACAGACC TGGAGAGTTG 6601 TGATCGCTCC ACCCCGGCTT TGGTGCGTTG GTTCGCTACT AATCTTATTT TTGAGCEAGC 6661 TGGCCAGCCC GAGTTGGTGC ACAGCTACGT GTTGAATTGC TGTCACGATC TAGTTGTGGC 6721 GGGTAGTGTA GCATTCACCA AACGCGGGGG TTTGTCATCT GGAGACCCTA TCACTTCCAT 6781 TTCCAATACC ATCTATTCAT TGGTGCTGTA CACCCAGCAC ATGTTGCTAT GTGGACTTGA 68 1 AGGCTATTTC CCAGAGATTG CAGAAAAATA TCTTGATGGC AGCCTGGAGC TGCGGGACAT 6901 GTTCAAGTAC GTTCGAGTGT ACATCTACTC GGACGATGTG GTTCTAACCA CACCCAACCA 6961 GCATTACGCG GCCAGCTTTG ACCGCTGGGT CCCCCACCTG CAGGCGCTGC TAGGTTTCAA 7021 GGTTGACCCA AAGAAAACTG TGAACACCAG CTCCCCTTCC TTTTTGGGCT GCCGGTTCAA 7081 GCAAGTGGAC GGCAAGTGTT ATCTAGCCAG TCTTCAGGAC CGCGTTACAC GCTCTCTGTT 7141 ATACCACATT GGTGCAAAGA ATCCCTCAGA GTACTATGAA GCTGCTGTTT CCATCTTTAA 7201 GGACTCCATT ATCTGCTGTG ATGAAGACTG GTGGACGGAC CTCCATCGAC GTATCAGTGG 7261 CGCTGCGCGT ACCGACGGAG TTGAGTTCCC CACCATTGAA ATGTTAACAT CCTTCCGCAC 7321 CAAGCAGTAT GAGAGTGCCG TGTGCACAGT TTGTGGGGCC GCCCCCGTGG CCAAGTCTGC 7381 TTGTGGAGGG TGGTTCTGTG GCAATTGTGT CCCGTACCAC GCGGGTCATT GTCACACAAC 7441 CTCGCTCTTC GCCAACTGCG GGCACGACAT CATGTACCGC TCCACTTACT GCACAATGTG 7501 TGAGGGTTCC CCAAAACAGA TGGTACCAAA AGTGCCTCAC CCGATCCTGG ATCATTTGCT 7561 GTGCCACATT GATTACGGCA GTAAAGAGGA ACTAACTCTG GTAGTGGCGG ATGGTCGAAC 7621 AACATCACCG CCCGGGCGCT ACAAAGTGGG TCACAAGGTA GTCGCCGTGG TTGCAGATGT 7681 GGGAGGCAAC ATTGTGTTTG GGTGCGGTCC TGGATCACAC ATCGCAGTAC CACTTCAGGA 7741 TACGCTCAAG GGCGTGGTGG TGAATAAAGC TCTGAAGAAC GCCGCCGCCT CTGAGTACGT 7801 GGAAGGACCC CCTGGGAGTG GGAAGACTTT TCACCTGGTC AAAGATGTGC TAGCCGTGGT 7861 CGGTAGCGCG ACCTTGGTTG TGCCCACCCA CGCGTCCATG CTGGACTGCA TCAACAAGCT 79 1 CAAACAAGCG GGCGCCGATC CATACTTTGT GGTGCCCAAG TATACAGTTC TTGACTTTCC 7981 CCGGCCTGGC AGTGGAAACA TCACAGTGCG ACTGCCACAG GTCGGAACCA GTGAGGGAGA 80 1 AACCTTTGTG GATGAGGTGG CCTACTTCTC ACCAGTGGAT CTGGCGCGCA TTTTAACCCA 8101 GGGTCGAGTC AAGGGTTACG GTGATTTAAA TCAGCTCGGG TGCGTCGGAC CCGCGAGCGT 8161 GCCACGTAAC CTTTGGCTCC GACATTTTGT CAGCCTGGAG CCCTTGCGAG TGTGCCATCG 8221 ATTCGGCGCT GCTGTGTGTG ATTTGATCAA GGGCATTTAT CCTTATTATG AGCCAGCTCC 8281 ACATACCACT AAAGTGGTGT TTGTGCCAAA TCCAGACTTT GAGAAAGGTG TAGTCATCAC 8341 CGCCTACCAC AAAGATCGCG GTCTTGGTCA CCGCACAATT C^TTCAATTC AAGGCTGTAC 8401 ATTCCCTGTT GTGACTCTTC GACTGCCCAC ACCCCAATCA CTGACGCGCC CGCGCGCAGT 8461 TGTGGCGGTT ACTAGGGCGT CTCAGGAATT ATACATCTAC GACCCCTTTG ATCAGCTTAG 8521 CGGGTTGTTG AAGTTCACCA AGGAAGCAGA GGCGCAGGAC TTGATCCATG GCCCACCTAC 8581 AGCATGCCAC CTGGGCCAAG AAATTGACCT TTGGTCCAAT GAGGGCCTCG AATATTACAA 8641 GGAAGTCAAC CTGCTGTACA CACACGTCCC CATCAAGGAT GGTGTAATAC ACAGTTACCC 8701 TAATTGTGGC CCTGCCTGTG GCTGGGAAAA GCAATCCAAC AAAATTTCGT GCCTCCCOAG 8761 AGTGGCACAA AATTTGGGCT ACCACTATTC CCCAGACTTA CCAGGATTTT GCCCCATACC 8821 AAAAGAACTC GCTGAGCATT GGCCCGTAGT GTCCAATGAT AGATACCCGA ATTGCTTGCA 8881 AATTACCTTA CAGCAAGTAT GTGAACTCAG TAAACCGTGC TCAGCGGGCT ATATGGTTGG 8941 ACAATCTGTT TTCGTGCAGA CGCCTGGTGT GACATCTTAC TGGCTTACTG AATGGGTCGA 9001 CGGCAAAGCG CGTGCTCTAC CAGATTCCTT ATTCTCGTCC (ΪGTAGGTTCG AGACTAACAG 9061 CCGCGCTTTC CTCGATGAAG CCGAGGAAAA GTTTGCCGCC GCTCACCCTC ATGCCTGTTT 9121 GGGAGAAATT AATAAGTCCA CCGTGGGAGG ATCCCACTTC ATCTTTTCCC AATATTTACC 9181 ACCATTGCTA CCCGCAGACG CTGTTGCCCT GGTAGGTGCT TCATTGGCTG GGAAAGCTGC 9241 TAAAGCTGCT TGCAGCGTTG TTGATGTCTA TGCTCCATCA TTTGAACCTT ATCTACACCC 9301 TGAGACACTG AGTCGCGTGT ACAAGATTAT GATCGATTTC AAGCCGTGTA GGCTTATGGT 9361 GTGGAGAAAC GCGACCTTTT ATGTCCAAGA GGGTGTTGAT GCAGTTACAT CAGCACTAGC 9421 AGCTGTGTCC AAACTCATCA AAGTGCCGGC CAATGAGCCT GTTTCATTCC ATGTGGCATC 9481 AGGGTACAGA ACCAACGCGC TGGTAGCGCC CCAGGCTAAA ATTTCAATTG GAGCCTACGC 9541 CGCCGAGTGG GCACTGTCAA CTGAACCGCC ACCTGCTGGT TATGCGATCG TGCGGCGATA 9601 TATTGTAAAG AGGCTCCTCA GCTCAACAGA AGTGTTCTTG TGCCGCAGGG GTGTTGTGTC 9661 TTCCACCTCA GTGCAGACCA TTTGTGCACT AGAGGGATGT AAACCTCTGT TCAACTTCTT 9721 ACAAATTGGT TCAGTCATTG GGCCCGTGTG ATGGGCTTAG TGTGGTCACT GATTTCAAAT 9781 TCTATTCAGA CTATTATTGC TGATTTTGCT ATTTCTGTGA TTGATGCAGC GCTTTTCTTT 9841 CTCATGCTAC TTGCATTGGC TGTTGTTACT GTGTTTCTTT TCTGGCTCAT TGTTGCCATC 9901 GGCCGCAGCT TGGTGGCGCG GTGTTCACGA GGTGCGCGTT ACAGACCTGT TTAAGGATTT 9961 GCAGTGCGAC AACCTGCGCG CGAAAGATGC CTTCCCGAGT CTGGGATATG CTCTGTCGAT 10021 TGGCCAGTCG AGGCTATCGT ATATGCTGCA GGATTGGTTG CTTGCTGCGC ACCGCAAGGA 10081 AGTTATGCCT TCCAATATCA TGCCTATGCC CGGTCTTACT CCTGATTGCT TTGACCATCT 10141 GGAGTCTTCT AGCTATGCTC CATTTATCAA TGCCTATCGG CAGGCAATTT TGAGTCAATA 10201 CCCACAAGAG CTCCAGCTCG AAGCCATCAA CTGTAAATTG CTTGCTGTGG TTGCACCGGC 10261 ATTGTATCAT AATTACCATC TAGCCAATTT GACCGGACCG GCCACATGGG TCGTGCCTAC 10321 AGTGGGCCAG TTGCACTATT ATGCTTCTTC CTCTATTTTT GCTTCATCTG TGGAAGTGTT 10381 GGCAGCAATA ATACTACTAT TTGCATGCAT ACCACTAGTG ACACGAGTGT ACATCTCTTT 10441 TACGCGGCTA ATGTCACCTT CCCGTCGCAC TTCCAGCGGC ACTTTGCCGC GGCGCAAGAT 10501 TTTGTAGTGC ACACGGGTTA TGAATATGCC GGGGTCACTA TGTTAGTGCA CTTGTTTGCC 10561 AACTTGGTTC TGACATTTCC GAGCTTAGTT AATTGTTCCC GCCCTGTGAA TGTCTTTGCT 10621 AATGCTTCTT GCGTGCAAGT GGTTTGTAGT CATACCAACT CAACTACTGG CTTGGGTCAA 10681 Cri'lVl'l'l' T CCTTTGTAGA TGAAGATCTA CGGCTGCATA TCAGGCCTAC TCTTATTTGT 107 1 TGGTTTGCCT TGTTGTTGGT GCACTTTCTA CCCATGCCAC GCTGCAGAGG CTCGTAATTT 10801 TACTTACATT AGTCATGGAT TCX3GCCACGT GCACGGTCAT GAGGGGTGTA GGAATTTTAT 10861 TAATGTCACT CATTCTGCAT TTCTTTATCT TAATCCCACC ACTCCCACTG CGCCGGCTAT 10921 AACTCATTGT TTACTTCTGG TTCTGGCAGC CAAAATGGAA CACCCAAACG CTACTATCTG 10981 GCTGCAGCTG CAGCCGTTTG GGTATCATGT GGCTGGCGAT GTCATTGTCA ACTTGGAAGA 11041 GGACAAGAGG CATCCTTACT TTAAACTTTT GAGAGCGCCG GCTTTACCGC TroGTTTTQT 11101 GGCTATAGTT TATGTTCTTT TACGACTGGT ACGTTGGGCT CAACGATGTT ATCTATGATT 1116 GTATTGCTAT TCTTGCTTTG GGGTGCGCCA TCACATGCTT ACTTCTCATA CTACACCGCT 11221 CAGCGCTTCA CAGACTTCAC CTTGTGTATG CTGACGGATC GCGGCGTTAT TGCCAATTTG 11281 CTGCGATATG ATGAGCACAC TGCTTTGTAC AATTGTTCCG CCAGTAAAAC CTGTTGGTAT 113 1 TGCACATTCC TGGACGAACA GATTATCACG TTTGGAACCG ATTGTGATGA CACCTACGCG 11401 GTCCCAGTTG CTGAGGTCCT GGAACAGGCG CATGGACCGT ACAGTGCGCT GTTTGATGAC 11461 ATGCCCCCTT TTATTTACTA TGGCCGTGAA TTCGGCATAG TTGTGTTGGA TGTGTTTATG 11521 TTCTATCCCG TTTTAGTTCT GTTTTTCTTA TCAGTACTAC CCTATGCTAC GCTTATTCTT 11581 GAAATGTGTG TATCTATTCT GTTTATAATC TATGGCATTT ACAGCGGGGC CTACTTGGCC 11641 ATGGGCATAT TTGCGGCCAC GCTTGCTATA CATTCAATTG TGGTCCTCCG CCAATTACTG 11701 TGGTTATGCC TGGCTTGGCG ATACCGCTGT ACGCTTCACG CGTCCTTTAT ATCAGCTGAG 11761 GGGAAAGTGT ACCCCGTAGA CCCCGGACTC CCGGTTGCCG CCGTGGGCAA TCGGTTGTTA 11821 GTCCCAGGTA GGCCCACTAT CGATTATGCA GTGGCCTACG GCAGCAAAGT CAACCTTGTG 11881 AGGTTGGGGG CAGCTGAGGT ATGGGAGCCA TAGATTCATT TTGTGGTGAC GGGATTTTAG 11941 GTGAGTATCT AGATTACTTT ATTCTGTCCG TCCCACTCTT GCTGTTGCTT ACTAGGTATG 12001 TAGCATCTGG GTTAGTGTAT GTTTTGACTG CCTTGTTCTA TTCCTTTGTA TTAGCAGCTT 12061 ATATTTGGTT TGTTATAGTT GGAAGAGCCT TTTCTACTGC TTATGCTTTT GTGC-TTTTGG 12121 CTGCTTTTCT GTTATTAGTA ATGAGGATGA TTGTGGGTAT GATGCCTCGT CTTCGGTCCA 12181 TTTTCAACCA TCGCCAACTG GTGGTAGCTG ATTTTGTGGA CACACCTAGT GGACCTGTTC 12241 CCATCCCCCG CTCAACTACT CAGGTAGTGG TTCGCGGCAA CGGGTACACC GCAGTTGGTA 12301 ACAAGCTTGT CGATGGCGTC AAGACGATCA CGTCCGCAGG CCGCCTCTTT TCGAAACGGA 12361 CGGCGGCGAC AGCCTACAAG CTACAATGAC CTACTGCGCA TGTTTGGTCA GATGCGGGTC 12421 CGCAAACCGC CCGCGCAACC CACTCAGGCT ATTATTGCAG AGCCTGGAGA CCTTAGGCAT 12481 GATTTAAATC AACAGGAGCG CGCCACCCTT TCGTCGAACG TACAACGGTT CTTCATGATT 12541 GGGCATGGTT CACTCACTGC AC5ATGCCGGA GGACTCACGT ACACCGTCAG TTGGGTTCCT 12601 ACCAAACAAA TCCAGCGCAA AGTTGCGCCT CCAGCAGGGC CGTAAGACGT GGATATTCTC 12661 CTGTGTGGCG TCATGTTGAA GTAGTTATTA GCCACCCAGG AACCAAAAAA AAAAAAAAAA 12721 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 12781 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 12841 AAAACTCGAG GAGCTTGGCA CTGGCCGTCG TTTTACAACG TCGTGACTGG GAAAACCCTG 12901 GCGTTACCCA ACTTAATCGC CTTGCAGCAC ATCCCCCTTT CGCCAGCTGG CGTAATAGCG 12961 AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG CCTGAATGGC GAATGGCGCC 13021 TGATGCGGTA TTTTCTCCTT ACGCATCTGT GCGGTATTTC ACACCGCATA TGGTGCACTC 13081 TCAGTACAAT CTGCTCTGAT GCCGCATAGT TAAGCCAGCC CCGACACCCG CCAACACCCG 13141 CTGACGCGCC CTGACGGGCT TGTCTGCTCC CGGCATCCGC TTACAGACAA GCTGTGACCG 13201 TCTCCGGGAG CTGCATGTGT CAGAGGTTTT CACCGTCATC ACCGAAACGC GCGAGACGAA 13261 AGGGCCTCGT GATACGCCTA TTTTTATAGG TTAATGTCAT GATAATAATG GTTTCTTAGA 13321 CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA 13381 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT 13441 GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC TTTTTTGCGG 13501 CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA GATGCTGAAG 13561 ATCAGTTGGG TGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG 13621 AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTG 13681 GCGCGGTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGC ATACACTATT 13741 CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA 1 801 CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG GCCAACTTAC 1 861 TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGOATC 13921 ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC 13981 GTGACACCλC GATGCCTGTA GCAATGGCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC 14041 TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGOAT GGAGGCGGAT AAAGTTGCAG 14101 GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG 14161 GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC ACΛTGGTAAG CCCTCCCGTA 14221 TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG 14281 CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTOTC AGACCAAGTT TACTCATATA 1 3 TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG AAGATCCTTT 14401 TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGA GCGTCAGACC 14461 CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT 14521 TGCλλλCλλλ AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA 14581 CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTCCTTCTAG 14641 TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC 14701 TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAλ GTCGTGTCTT ACCGGGTTGG 14761 ACTCAAGACG ATλ β TTλCCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCβTGCλ 14821 CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT 14881 GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG 14941 TCGGAACAGG AβλβCGCλCβ AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC 15001 CTGTCGGGTT TCGCCACCTC TGACTTβλGC GTCGλTTTTT GTGλTGCTCG TCAGGGGGGC 15061 GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC 15121 CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG 15181 CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGλ 15241 GCGAGGAAGC GGAAGAGCGC CCAATACGCA AACCGCCTCT CCCCGCGCGT TGGCCGATTC 15301 ATTAATGCAG CTGGCACGAC AGGTTTCCCG ACTGGAAAGC GGGCAGTGAG CGCAACGCAA 15361 TTAATGTGAG TTAGCTCACT CATTAGGCAC CCCAGGCTTT ACACTTTATG CTTCCGGCTC 15421 GTATGTTGTG TGGAATTGTG AGCGGATAAC AATTTCACAC AGGAAACAGC TATGACCATG 15481 ATTACGAATT GATCCCCCAT ATGGCGGCCG CTAATACGAC TCACTATA

Claims: CLAIMS 1. A recombinant DNA-vector comprising a DNA sequence (-DNA) complementary to at least a part of the genome (+RNA) of eguine arteri tis virus (EA-virus) , said DNA-vector having adjacent to the 3' end of the DNA sequence (-DNA) a non-EA virus-specific promotor for a non- EA virus-specific RNA polymerase and, after linearization of the vector and transcription of the DNA sequence (-DNA) by the non-EA virus-specific RNA polymerase, yields +RNA which is infectious in a virus host cell, the DNA sequence (-DNA) comprising a complementary DNA sequence (+DNA) corresponding to said part of the genome, said complementary DNA sequence (+DNA) consisting of a first sequence and adjacent at the 5' and of the first sequence a second sequence, the first sequence being the sequence represented by SEQ nr. 1 or a genetically engineered, together with the second sequence infectious derivative thereof, and the sec- ond sequence being the sequence represented by SEQ nr. 2 or a functional derivative thereof, wherein the DNA-vector comprising the complementary DNA sequence (+DNA) consisting of SEQ nr. 1 and SEQ nr. 2 can be maintained in E. coli K12 PC2495 (Phabagen Collection, University of Utrecht, P.O Box 80.056, 3508 TB Utrecht, the Netherlands) as a vector host cell and, after isolation and an in vitro transcription reaction, with T7 promotor (SEQ nr. 3) as non-EA virus-specific promotor and T7 polymerase as non-EA virus-specific RNA polymerase, yields +RNA which is infectious in baby hamster kidney cells BHK-21 (C13) (ATCC CCL10) as a virus host cell and leads to the formation of virus particles, as well as a genetically engineered derivative of this vector the RNA transcription product of which is capable of replication in an eukaryote. 2. Vector host cell comprising a recombinant DNA vector according to claim 1. 3. Vector host cell according to claim 2, which is E. coli K12 PC2495 and contains the recombinant DNA vector, deposited on 11 July 1996 under accession number 760-96 with the Centraalbureau voor Schimmelcultures, Baarn, the Netherlands . 4. Recombinant DNA vector pEAV030. 5. DNA sequence according to SEQ nr. 2 or a deriva- tive thereof which, when introduced in a DNA vector comprising a DNA sequence (+DNA) consisting of SEQ nr. 1 and contiguous at the 5' end thereof SEQ nr. 2 or the derivative thereof, can be maintained in a vector host cell and results, with a non-EA virus-specific RNA polymerase and the non-EA virus-specific RNA polymerase, in RNA which is infectious in a virus host cell and yields virus particles. 6. Use of an oligonucleotide chosen from the group consisting of the DNA sequence according to claim 5, the complement thereof and fragments thereof, as a primer for a chain extension reaction. 7. +RNA obtained by transcription of the DNA sequence (-DNA) of the recombinant DNA vector according to claim 1. 8. Method of obtaining virus particles, wherein +RNA according to claim 7 is introduced into virus host cells, the virus host cells are cultured and virus particles are isolated in a manner known in itself. 9. Method of obtaining virus particles, wherein a recombinant DNA vector according to claim l is introduced into virus host cells, the virus host cells are cultured and virus particles are isolated in a manner known in itself . 10. Virus host cell transformed using +RNA according to claim 7 or with virus particles obtained using the method according to claim 8 or 9. 11. Method of preparing a recombinant RNA-containing pharmaceutical composition, wherein the recombinant DNA vector according to claim 1 is multiplied in a vector host cell, the DNA-vector is transcribed yielding recombinant RNA, if desired the recombinant RNA is introduced into a virus host cell, yielding recombinant RNA-containing viruses which, if desired, are isolated, and the thus obtained recombinant RNA is processed with a pharmaceuti- cally acceptable carrier or excipient to a pharmaceutical composition. 12. Method for the preparation of a recombinant DNA vector-comprising pharmaceutical composition, wherein the recombinant DNA vector according to claim 1 is multiplied in a vector host cell and is isolated in a manner known in itself, and the thus obtained recombinant DNA is processed with a pharmaceutically acceptable carrier or excipient into a pharmaceutical composition.

Inventors: BREDENBEEK PETRUS JOHANNES, DEN BOON JOHAN ARIE, SNIJDER ERIC JOHN, SPAAN WILHELMUS JOSEPHUS MARIA, VAN DINTEN LEONIE CHRISTINA, WASSENAAR ALFRED LEONARD MARIA, BREDENBEEK, PETRUS, JOHANNES, DEN BOON, JOHAN, ARIE, SNIJDER, ERIC, JOHN, SPAAN, WILHELMUS, JOSEPHUS, MARIA, VAN DINTEN, LEONIE, CHRISTINA, WASSENAAR, ALFRED, LEONARD, MARIA
Applicants: BREDENBEEK PETRUS JOHANNES, DEN BOON JOHAN ARIE, DINTEN LEONIE CHRISTINA VAN, SNIJDER ERIC JOHN, SPAAN WILHELMUS JOSEPHUS MARIA, UNIV LEIDEN, WASSENAAR ALFRED LEONARD MARIA, BREDENBEEK, PETRUS, JOHANNES, DEN BOON, JOHAN, ARIE, RIJKSUNIVERSITEIT LEIDEN, SNIJDER, ERIC, JOHN, SPAAN, WILHELMUS, JOSEPHUS, MARIA, VAN DINTEN, LEONIE, CHRISTINA, WASSENAAR, ALFRED, LEONARD, MARIA
Application year: 1997
Main classification: C12N15
IPC codes: A61K48, C07K14, C12N1, C12N5, C12N7, C12N15, C12Q1, C12R1