DE19709512A1 - Recombinant influenza virus - Google Patents

Abstract

Influenza virus vector for replication and expression of foreign genes comprises one or more segments with foreign or altered gene sequences in addition to or partial substitution for the RNA segments of the normal viral genome. Also claimed is a method for the indirect selection of influenza virus variants as above by FACS separation of infected cells, in which the foreign genes are preceded by the gene for Aequoria victoria green fluorescent protein (GFP) flanked by vRNA splice donor and acceptor signals or duplicate influenza virus replication signal sequences with an activity such that the GFP gene will be lost at a moderate rate during virus replication or passage.

Description

Technical field of the invention

The invention relates to a method for producing recombinant influenza A viruses, the corresponding (fusion) genes in their genome, as well as in the shell their virions complete membrane-anchored foreign glycoproteins or membrane-ver anchored any protein in addition to their normal genes and surface proteins contain. The foreign type I glycoproteins can be viral, animal or human Be of origin and will use their own membrane segment or that of influenza hemagglutinin incorporated into the virion envelope. The same The transmembrane domain can also be coupled to the C-terminus of other proteins be the z. Let T. anchor as artificial membrane proteins in the virus envelope. Technical elements of this invention belonging to the field of genetic engineering are also a Indirect selection process (in two versions) with which recombinant viruses can be strongly enriched, which carry any foreign gene (regardless of its properties), and the use of promoter-enhancing mutants and the Generation of (recombinant) influenza vRNA molecules in vivo by the cellular RNA poly merase I (both the subject of patent application PCT 95/03663, G. Hobom et al. and Bayer AG).

Preparations of recombinant influenza viruses of the described composition (also in the Mixture of several constructs) meet the criteria of live vaccines for influenza Human or animal, or of double vaccines z. B. for influenza viruses and swine fever viruses with simultaneous immunization in pigs etc. Other virus constructs with foreign, especially cellular glycoproteins and possibly another cytotoxic Genes are suitable as vector systems in genetics and somatic gene therapy.

State of the art

1. A number of well-examined viruses have already been able to target the virus vector systems for the transfer of foreign genes are transformed. By deleting some at least under laboratory conditions of unnecessary genes there is room for the inclusion of the Foreign genes created during infection (mostly under the control of virus borrowed signal sequences) are expressed together with the virus's own genes. In addition to the methods for recombinant protein production such. B. by Baculovirus Vek gates (in insects or insect cells) serve different vector systems derived from the vaccinia, herpes, adeno or adeno-associated viruses to the heterologous Gene transfer into the cells of an infected organism, or in the latter case also for  in vivo gene therapy. In all of these cases, however, the foreign gene is only present in the virion Gene sequence is present and does not appear as a protein in the infected cells until after infection Appearance. In addition to the DNA viruses mentioned, this principle is also applicable to retroviruses (especially derived from the murine Moloney leukemia virus MoLV) and on RNA viruses such as the Rabies virus (Schnell et al., 1994) or the measles virus (Sidhu et al., 1995) or Have been transferred. While here too the heterologous gene product is usually only found in the infected cell appears, in some cases the virus-inherent glycoprotein that of another virus has been replaced, especially in the case of MoLV vector systems thereof env protein by the glycoprotein of the vesicular stomatitis virus (VSV; Burns et al., 1993), which then leads to a VSV-specific infection process for the recombinant virus.

This principle of vector modification of basic virus structures is in the present Invention has been applied to influenza A viruses, and particularly expression of foreign glycoproteins that not only enter the genome as foreign genes, but also as heterologous proteins in addition to its own proteins hemagglutinin and neuraminidase in the envelope of these viruses can be integrated. Unlike most viruses is an additional inclusion of these foreign genes and proteins in the Virus association, d. H. associated with an expansion of the genome and a change in its Surface structure. Recombinant viruses of this structure are therefore basically double (or multiple) vaccine suitable - also for nasal / oral application - provided that it can be reliably attenuated (see below). A particular advantage is the use recombinant influenza viruses in that the added foreign genes with a particular strong promoter variant (1104; Neumann and Hobom, 1995) can be linked to the leads to high expression rates of the foreign proteins.

2. The vaccination against influenza A viruses has so far been inactivated or through Detergents disintegrated viruses (i.e. on protein mixtures) turned off. After an injection these preparations stimulate a B cell response with antibody production, however (as good as) no reaction of the CD8-T cells, like that with a normal infection or with a live vaccination would also be the case. It is also unphysiological for this Injection method, the synthesis of specific IgG antibodies in the serum, not however, that of secreted IgA molecules in the respiratory tract increased markedly perform the first protective function on the surface of the mucosa. And finally they can antibodies raised against a "fixed" hemagglutinin (even when used as a mixture) do not anticipate the later modification of the virus structures in their "genetic drift",  they also become their protective effect in the serum against the greater spread of Lose influenza infection in the body again later.

The currently introduced influenza vaccine is in principle other viral vaccines inferred using an attenuated, live virus (e.g. polio vaccine), and in which a (limited) viral replication and protein synthesis in the infected cells in addition to B-cell stimulation in MHC I-mediated contact specific T cell reactions (CD4 and CD8) can be initiated. The usual is also done Influenza immunization in an unphysiological way, namely by injections, with what against the natural infection in the pharynx mucosal cells groups of Lymphocytes in different locations can be reached in the body, which - not completely understood - to a different B cell response (e.g. in the ratio IgG to IgA, see above) leads. In other cases too, an immunization mode has come as close as possible to that proven natural infection, which in the case of influenza viruses as with other aerosol Respiratory tract infections indicate live nasal / oral vaccination. This way is supposed to of the present invention, also as a double immunization stra strategy if e.g. B. in pigs at the same time against the swine influenza virus and against Swine plague virus, both of which are transmitted by an aerogenic route, is immunized. For the first time, foreign viral glycoproteins such as swine fever virus protein E2 in the influenza virion completely and integrated in (largely) authentic conformation, what the formation of the (non-linear) on the surfaces of these glycoproteins antigenic B cell epitopes is important. At the same time, the genetic anchoring in the Influenza genome that these proteins reproduced in the abortively infected cells and appear on their surface in the same conformation, which is the B cell reaction stimulated further, supplemented by stimulation of the CD4 helper cells on the Based on MHC I-presented T cell epitopes. - So far, only genetically-anchored presented short-chain segments of foreign proteins on the surface of influenza virions are integrated into influenza-specific glycoproteins, especially in the stalk of Neuraminidase (Percy et al., 1995). However, that for immunizations was also possible unsuitable form of transient integration of foreign viral glycoproteins after virus duplication infections with the so-called "phenotypic mixing".

3. In the past, attenuated variants of viruses are often under through serial passages marginal growth conditions or in unnatural host cells, d. H. by random mutation, and generated under unclear selection conditions. First with the reverse genetics and now through targeted mutations can attenuate  Virus variants are built up in a controlled manner, but then as before the impact of these mutations must be examined, first in the Cell culture and animal experiments. So far there are attenuated influenza variants for vaccines in particular, various (multiple) temperature-sensitive mutants have been proposed, the In principle, however, nucleotide substitutions are subject to reversion each time they multiply can. Instead, two different principles are used in this invention. First, an over-potent viral promoter in conjunction with the foreign gene is in only one who then uses a total of nine different influenza vRNA molecules to Overexpression and overreplication of this one vRNA compared to the eight remaining ones are from the helper virus and are all required for virus replication. At statistical packaging of all nine vRNA molecules according to their individual Concentration and with a limited capacity of about 15 virions The molecules are then separated by the increased number of copies of foreign RNA eight influenza vRNAs are displaced from the virions that form, resulting in loss of these the ability to reproduce and overall to decrease the rate of plaque formation forming particles, typically in a passage around three to four Powers of ten. Ribozymes are used as the second attenuation principle sequence-specific response against individual of the helper vRNA segments are directed. As a result, the respective gene sequence is destroyed in high yield and cannot be used in the Packaging process in the vaccine viruses that are formed. The failure of the associated gene product is hereby by DNA transfection with the corresponding Constructs or by multiplication in cell lines with chromosonal expression of the missing Protein balanced.

Thus, this invention describes and discloses the construction of recombinant Influenza viruses with high expression of foreign gene products including foreign glycoproteins, through statistical repression, especially through controlled destruction of individuals Genes are attenuated in their virulence and can therefore be used as vaccines.

Construction elements of the invention 1. RNA polymerase I method for the expression of (recombinant) influenza vRNA molecules

The structure of recombinant influenza viruses on the Bals corresponding Plasmid constructs in E.coli K12 uses the method of developed in this laboratory intracellular generation of recombinant vRNA molecules by the enzyme RNA-Poly merase I after plasmid DNA transfection of the corresponding cDNA constructs (Zobel  et al., 1994; Neumann et al., 1994), laid down in patent application PCT 95/03663 (Hobom et al. and Bayer AG). In addition to the method mentioned there, either the murine RNA polymerase I promoter (in mouse cell lines) or the human RNA polymerase I-Pro motor (in human or in monkey cell lines, especially in COS-7) used while the Terminator sequence interchangeable in all cell lines from one or the other rDNA is active.

2. RNA polymerase II method for the expression of (recombinant) influenza vRNA molecules

As an alternative to the above-mentioned standard method, RNA molecules with influenza vRNA-typical end sequences can be generated in two steps instead of one by the normal cellular transcription method with RNA polymerase II, but here in a minus-strand orientation. For this purpose, the 3 'end of the vRNA is formed from the primary transcript by a specifically constructed cis-ribozyme (the 3'-end of the mRNA that is formed, including the hepatitis δ-ribozyme sequence itself, is thus removed by nucleolysis; Menke and Hobom, 1997). The 5 'end of the primary transcript which extends beyond the viral 5'-vRNA promoter sequence (for example 12 nucleotides with a 5'-cap structure) is subsequently lost in the course of the vRNA amplification reaction, which also occurs in the case of a template which has been extended in this way the synthesis of the influenza mRNA and cRNA starts in the normal 5 'position. In two steps, a vRNA molecule that can be reproduced and packaged is also created from an mRNA precursor, such as is formed under the control of a p _CMV promoter.

3. Mutations of the viral promoter sequence at the 5 'and 3' ends of the vRNA

Reinforcing mutations of the viral promoter sequence in different gradations, especially but the clearly effective mutations 1104 and 1102 (Neumann and Hobom 1995; in the already mentioned patent) are consistently used as end sequences in the recombinant vRNA molecules, d. H. used in conjunction with the foreign genes. They are based on the "corkscrew" secondary structure interpretation for the activated form of this Promoter sequences (Flick et al., 1996).

4. Expression of foreign glycoprotein genes and incorporation of the glycoproteins into the Virions

The genes of various foreign glycoproteins of general type I, i.e. H. With A hydrophobic membrane anchor sequence close to the C-terminus are used for expression other genes also used to construct corresponding cDNA constructs, so that  Appropriate artificial vRNA molecules can be formed, in minus-strand orien tion or counter-strand direction. The foreign genes take the place of coding sequence of influenza genes, flanked by authentic or light modified non-coding areas as found in the influenza vRNA molecules available. In addition to its own signal peptide or one derived from hemagglutinin (HA) The glycoprotein sequence then comprises the complete own, ie HA-foreign ecto domain and then either your own or more often the hemagglutinin transmembrane domain including a C-terminal "cytoplasmic" end sequence (for HA: 26 + 11 Amino acids). The same design principle also applies to non-glycoproteins that first through the connection with the two flanking signal and membrane segments of the Hemagglutinins are transformed into artificial surface proteins on the Virion, e.g. B. for the green fluorescent protein from Aequoria victoria (GFP). With the Haemagglutinin Se sequences anchored in the membrane are indeed fusion proteins, whose entire unchanged ectodomain is nevertheless unaffected by this in the Can form normal conformation, recognizable in the case mentioned by the GFP fluorescence.

5. Indirect selection method for recombinant influenza viruses

The by RNA polymerase I (or RNA polymerase II) in the cDNA-transfected cells formed recombinant vRNA molecules need for their conversion into pseudo-viral mRNAs and for the subsequent amplification via cRNAs back to VRNAs the viral RNA polymerase of the helper viruses, for the subsequent packaging together with the Helper virus vRNAs then also all other gene products including the structural proteins. The unchanged helper viruses formed at the same time dominate in number in the mixture of the daughter viruses.

The selection of the recombinantly transfected or recombinantly infected cells (after "Virus passage" of the primarily formed cell supernatant with the daughter viruses therein to fresh ones Cells) compared to the non-infected or non-recombinantly infected cells with the help of the expression of GFP and its fluorescence emission of 590 nm when excited by 483 nm (Chalfie et al., 1994; Cubitt et al. 1995; Siemering et al., 1996). The GFP gene has been introduced into bicistronic vRNA segments for this purpose as first cistron at the 5 'end of these mRNAs, while the one that is actually interesting Foreign gene takes the distal place on the viral mRNAs. On the vRNA counter strand the sequence is therefore inverted. Before and after the inverse GFP sequence are here Splice donor and splice acceptor signals with partial effectiveness arranged in the  infected cells (during the "virus passage") the GFP gene in some of the vRNA molecules remove by a splice reaction. Have the cells infected in the next passage then the GFP fluorescence is lost and now instead express the previously distal one Gene product of the bicistonic RNA segments. In the second (or third) FACS sort the non-fluorescent "dark" cells and their virus over are then sorted out was then used for further propagation. - The procedure describes for the first time a splice reaction at the level of a genomic vRNA, and thus at one Negative strand RNA.

A second indirect selection method for foreign genes is also based on fluorescence of an upstream GFP gene, which, however, here is caused by an alternative one Replication signal is separated from the subsequent, indirectly selectable gene. Bicistronic vRNA segments of this type contribute to their localization at the 3 'end another 3 'promoter sequence in the middle between the two cistrons, so that the The entire promoter structure formed from the 5 'and 3' portions alternatively on the one hand can train across both cistrons, or on the other hand only via the foreign gene away, which leads to replicative loss of the non-included GFP gene. The two-stage Selection by FACS sorting leads here to isolation or Enrichment of influenza viruses with any foreign genes.

6. Targeted mutagenesis of the influenza genes - selection by ribozyme cleavage

The incorporation of foreign genes into recombinant influenza viruses is because of this associated new phenotype easier to detect and isolate than the targeted constructed variants of virus genes are - hidden in a majority of unchanged helper viruses. To select them, a detour must be made, which is in the There is construction of a series of specifically modified helper viruses, all of which have a ribozyme cut place in each of their vRNA segments, in the area of 5 'and / or 3' non-coding edge sequences. You became yourself for this purpose specifically changed and isolated by multiple plaque cleaning. Mixed with "his" specific helper virus, the analog, specifically modified in the coding area, however, in the outer flanks, the untouched wild-type sequence has vRNA segment isolate the RNA polymerase I transcription by exposure to ribozyme. To that For this purpose, the corresponding host cells are previously transient with a cDNA construct transfected, which expresses a specific acting ribozyme (of the hammerhead type), directed against the interface in the one vRNA flank sequence in the selected one  Helper virus. The cells pretreated in this way are then mixed with the virus mixture infected (in a "passage"), with the majority of one of the eight helper virus segments being infected Cleavage is destroyed while the mutant replacement segment survives and even is amplified. This treatment can be repeated in a second pass and delivers the specifically modified virus mutants in a uniform population. For a higher one Reaction yield and also to protect against a possible mutation in the course of genetic drifts in the ribozyme recognition site on the vRNA are just like that associated ribozymes in the form of tandem dimers (Menke and Hobom, 1997).

7. Virus attenuation by vRNA ribozyme cleavage and compensating protein com implementation

The previously described ribozyme cleavage of the vRNA of a helper virus segment performs the same if there is no compensation through a newly formed segment Function to an only abortive infection, there will be no or at most very few Daughter viruses formed. However, the missing gene function can be complemented by a vRNA also by the associated protein, d. H. through protein expression on the basis of an RNA polymerase II expression, which however cannot be packaged RNA molecules are created. In the previously transient double DNA transfected cells - (Ribozyme construct; expression construct for a viral protein) are the result of a subsequent virus infection formed daughter viruses, but in the absence of Gene segment only one-stage (abortive) capable of infection, but not in the newly infected Cells are capable of reproduction. The viruses thus attenuated are therefore suitable as Vaccine viruses; in addition to the partial expression of the own genes in the abortively infected For them, the expression of foreign proteins and glycoproteins is for the purpose of Increasing or expanding the immune response possible.

8. Helper viruses and tissue culture cells

As standard helper viruses are different Chicken influenza viruses (KP or FPV viruses) used (H7-HA): FPV-Bratislava, FPV-Rostock or FPV casting. In addition, several virus reassortants were used for this, formed in double infections from an H7 and an H2 virus (Scholtissek et al., 1979), and finally, for individual experiments, the special helper virus strains mentioned under (6). The later virus passages were routinely carried out on the standard MDCK and cell lines MDBK performed during the first step towards the synthesis of recombinant viruses via the RNA polymerase I expression of cDNA constructs either on mouse cell lines  (e.g. B 82; constructs contain the mouse rDNA promoter) or on human or monkey cell lines (e.g. COS-7; constructs contain the human rDNA-Pro engine).

example 1 Incorporation of CSFV glycoprotein E2 into influenza virions

The swine fever virus (CSFV = contagious swine fever virus) is still a significant threat to pig breeding in Germany and Europe. With increased traffic and trade with the Eastern European countries, the risk of infection could increase further and the need for an effective vaccine e.g. B. to curb an epidemic, - even in the Eastern countries themselves. As a pestivirus, CSFV has an RNA-plus-strand genome with a long reading frame, which is soon converted into a proteolytically broken down polyprotein. Three of the cleavage products are found as envelope proteins on the surface of the virions: glycoproteins Eo (= E ^ms ), E1 and E2, of which the E2 protein contains the receptor binding site (Rümenapf et al., 1993). The morphogenesis of the CSFV virions takes place in the area of the Trans-Golgi network, i.e. not on the plasma membrane of the cell surface, which is also not reached by any of the individual glycoproteins. The intracellular transport processes are therefore completely different from those of the influenza virus and its glycoproteins.

The cDNA segment with the coding sequence for the CSFV glycoprotein E2 ectomain domain including a preceding region corresponding to a 23 amino acid signal peptide (signalase sensitive), covering a total of 364 codons, from nucleotide position (Meyer et al., 1989): 2362 (ATA = isoleucine, but here transformed into ATG = methionine) to 3454 (TTT = phenylalanine, here transformed into TGG = tryptophan) has been cloned in exchange into a vRNA expression construct for hemagglutinin. The flanking 5 'and 3' ends of the sequence of the influenza segment 4 are obtained, however, before the start codon of the fusion protein is changed in accordance with the Kozak sequence (Kozak, 1991), and the entire section in accordance with the HA transmembrane Domain and the C-terminus (nucleotides 1-29 and 1623 to 1768 in the cRNA numbering). The promoter mutant 1104 used in construct pHL1959 ( Fig. 1) also contains nucleotide exchanges at positions 3, 5 and 8 (Neumann and Hobom, 1995). The fusion limit of the protein between the E2 ectodomain and the HA transmembrane domain is marked at the cDNA level by a BamHI site. The described central cDNA section of the construct is framed by the murine rDNA promoter and terminator, ie from segments -254 to -1 (based on the start of transcription of the precursor rRNA) and +, for nucleotide expression in pHL1959 for expression as a vRNA minus strand 571 to +745 (based on the 3 'end of the mature 285 rRNA). The replication in E.coli K12 and the ampicillin selection are carried by the surrounding base vector pBluescript SK (see Fig. 1).

After lipofectamine-mediated DNA transfection of pHL1959 DNA in murine B82 cells, followed after 24 hours (2-30 hours) by the FPV-Bratislava helper virus infection (moi: 2-5) are in the nucleoli of the transfected and infected cells the cellular RNA polymerase I pseudo-vRNA molecules generated, and then converted into viral mRNA molecules by the viral RNA polymerase (according to the "cap snatching"principle; Krug et al., 1989). In the later phase of the virus infection, cPNA molecules are then also formed, and daughter vRNA molecules are finally rewritten. The viral mRNA molecules lead to the synthesis of the E2-HA fusion protein, which appears on the surface of the cells and can be detected there by indirect immunofluorescence ( Fig. 2). There it is incorporated further into the influenza virions that are forming ( Figs. 3 and 4), as the third glycoprotein in the virion envelope ("envelope"), next to the glycoproteins HA and NA of the helper influenza virus. Transport to the cell surface and incorporation into the (foreign) virions is dependent on the HA transmembrane domain ( Fig. 5), and control clones with the E2 transmembrane segment are not transported to or into the surface even under the conditions of an influenza infection Influenza virions incorporated (lane 6 in Fig. 3). The C-terminal sequence of 11 amino acids can be modified without having a decisive effect on the transport behavior in the cell and the incorporation of the fusion protein into the virions. (Lanes 3 to 5 in Fig. 3). The transport of the fusion protein to the cell surface is independent of other viral proteins and is also carried out when expressed by RNA polymerase II in the absence of the influenza infection (not shown).

In addition to incorporating the fusion protein into the virions, the associated vRNA is also used packed into the viruses and can be (multiple) virus passage on the surface of the new infected cells as well as in the virions formed there, because of the Connection with an enhanced promoter in increasing concentration (up to 1: 1 relative to the HA). The infectivity of the viruses is not affected by what is different from each other independent incorporation and oligomerization of the different glycoproteins in the Virions prove that the homo-trimerization of HA as well as the homo-tetramerization of NA is a prerequisite for their function in the course of the infection.  

Example 2 Expression of green fluorescent protein by recombinant Influenza viruses and FACS cell sorting of the GFP influenza-infected cells

The cDNA for the sequence of the green fluorescent protein (GFP) from Aequoria victoria is in pHL1972 ( Fig. 6) or in other such constructs including pHL2251 in an activity-increasing mutated form (Chalfie et al. 1994, Cubitt et al. 1995 Siemering et al., 1996) have been integrated into RNA polymerase I expression vectors. The 246 codons of the GFP sequence plus six codons of a histidine sequence at the C-terminus replace the coding sequence of the NP gene in segment 5; only the flanking non-translated sequences at the 5 'and 3' ends of this segment are then obtained , modified however according to the 1104 promoter variant (Neumann and Hobom, 1995) and for an optimization of the translation start according to Kozak (1991). In this case, the central part of the construct is framed by the RNA polymerase I promoter from the human rDNA (-407 to -1, based on the starting point of the precursor rRNA) and the RNA polymerase I terminator from the murine rDNA (within the same limits as in Example 1), while the E.coli K12 cassette also corresponds to the construct pHL1959 in Example 1.

The cell specificity of the human promoter means that lipofectamine DNA trans fection is carried out in COS-7 cells. Superinfection with FPV _Bratislava as helper viruses (moi: 1-5) after 20-24 hours leads to GFP fluorescence in both transfected and infected cells, typically in one, in pHL1972 after three to four hours, in pHL2251 after 2 to 3 hours Share in the cell population of 15% -25% ( Fig. 7), the yield depending on the success of the lipofectamine treatment. The mixed cell population can be split into fluorescent and non-fluorescent cells (not transfected, or only infected by helper viruses) in a fluorescence-activated cell sorter (FACS; Becton-Dickinson), their extinction greatly differentiated, see. Fig. 8. The fluorescent cells are re-concentrated by centrifugation and taken up in tissue culture medium. The viruses released by them in the further incubation are highly enriched for recombinant viruses, as can be seen in their further passage: the cells infected by them are recombinantly infected in high yield, ie they show the fluorescence caused by the GFP segment. The FACS selection can also be carried out after the first passage, ie on the cells infected with a mixture of recombinant and helper viruses, and then separates the recombinantly infected cells from the other cells which are either only infected by helper viruses or not at all.

Literature quotes

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Claims (10)

1. An influenza virus vector system, suitable for replication, transcription and Translation (expression) of foreign gene products, characterized in that it is one or contains several segments with foreign or modified gene sequence, in addition to RNA segments of the normal virus genome or in their partial substitution. 2. Viruses according to claim 1, in which one or more mutations in a targeted manner into the non-coding and / or coding regions, at one or several segments, which quantitative and / or qualitative changes to the various viral gene products, including complete deletion one of the viral gene segments. 3. Viruses according to claims 1 and 2, in which at least one additional, modified segment added to the eight original influenza vRNA segments coding for one or more foreign (or modified own) genes or Gene segments. 4. Viruses according to claims 1 to 3, in which the or an incorporated Foreign segment for a glycoprotein of foreign viral, animal, human or other Origin coded, with or without raster fusion connection to be coded influenza Se sequences, and at the same time the glycoprotein itself in the envelope of the virions is incorporated. 5. Viruses according to claim 4, in which the foreign, as a vRNA and as a protein incorporated glycoprotein sequence is taken from the genome of swine fever virus (CSFV), Bovine Diarrhea Virus (BVDV), Vesicular Stomatitis Virus (VSV), of the Borna virus (BDV), the Marburg virus, the Ebola virus, the hepatitis C virus, the Early Summer Meningo Encephalitis Virus (TBE) or the West Nile Virus or of the human immunodeficiency virus (HIV). 6. Viruses according to claims 1 to 3, in which one or more of the incorporated Coding foreign genes for a lymphokine of human or animal origin, which is derived from the Influence vector-infected cell is secreted. 7. Viruses according to claims 1 to 3, in which one of the foreign genes for Expression of an apoptosis inducing gene or a toxin gene encoded, effective in the primary infected cell or after secretion in neighboring cells. 8. A method for the indirect selection of influenza virus variants according to the Claims 1 to 7 by the FACS separation of infected cells, in any Foreign genes the gene for the green fluorescent protein (GFP) from Aequoria victoria or an artificial derivative thereof was flanked by vRNA splice donor and acceptor signals or duplicate influenza replication signal sequences such activity that the GFP gene with during virus propagation or passage middle rates are lost. 9. Use of a virus according to claims 1 to 7 for the preparation of active pharmaceutical ingredients. 10. The pharmaceutical preparation of a virus vaccine against influenza, or with double Effect at the same time against influenza and against a second virus via the incorporated one Glycoprotein, or use as a vector for somatic gene therapy, from one the virus according to claims 1 to 7.