JPH08511424A - High-yielding influenza virus - Google Patents

Abstract

  (57) [Summary] The present invention relates to an influenza virus strain capable of reliably growing to a high titer in a host such as hatched chicken eggs. High-yielding virus strains are disclosed, including influenza viruses that display chimeric virus surface molecules such as reshaped influenza virus, chimeric HA and / or NA surface molecules. Such high-yielding viruses can be used for the production of antiviral vaccines as well as the production of chimeric influenza viruses.

Description

Detailed Description of the Invention High-yielding influenza virus 1. Introduction The present invention relates to an influenza virus strain that can be reliably propagated to a high titer in a host such as hatched chicken eggs. High yield strains containing reassortment influenza viruses and influenza viruses displaying chimeric virus surface molecules (such as chimeric HA and / or NA surface molecules) are described. Additionally, methods for producing such high yielding influenza A, B and / or C virus strains are described. Such high-yielding viruses can be used in the production of antiviral vaccines and the production of chimeric influenza viruses. 2. Background of the Invention The manufacture of antiviral vaccines offers many challenges. For example, a constantly changing group of influenza virus subtypes [influenza A (H3N2) and A (H1N1) etc.] and influenza B spread throughout the population. Furthermore, antigenic drift causes large numbers of mutations to rapidly emerge within each influenza A subtype and influenza B. Therefore, there is a need to continually isolate influenza viruses and evaluate their antigenic properties. Such information is used annually in determining the components of influenza vaccines for the following reasons (Robert son, JS et al., 1988, Influenza Vaccines, Development and Perspectives, Giornale di Igiene e Medicina Preventiva 29: 4- 58). However, freshly isolated influenza virus strains from patients usually do not grow to high titers. This is especially true when the virus is propagated in embryonated chicken eggs, which is the preferred host for producing large amounts of virus for use as an inactivated virus vaccine. In order to improve the growth characteristics of freshly isolated influenza virus strains, attempts are often made to reconstruct new strains with laboratory strains. For example, a new influenza A virus strain is crossed with a high-yielding influenza A / PR / 8/34 virus strain in order to generate potentially high-yield reassortant strains. Reassortant viruses with high growth properties usually receive 6 genomic RNA segments from the high-yielding parental virus strain and hemagglutinin (HA) and neuraminidase (NA) segments from the newly isolated parental strain (Kilb ourne, ED. and Murphy, JS, 1960, J. Exp. Med. 111: 387; Kilbourne, E.D., 1969, WHO Bull. 41: 643; Baez, M. et al., 1980, J. Infectious Dis. 141: 3 62; Robertson, JS. Et al., 1992, Biological 20: 213). This approach of reconstructing the A / PR / 8/34 strain can result in some influenza virus strains with improved growth characteristics, but many of the resulting reconstructed strains have limited growth in embryonated eggs. Show the possibility. Therefore, the current method for increasing the replication titer of newly found virus strains is not optimal, and an improved method for increasing virus yield in a preferred host for vaccine production is the antiviral vaccine It will be of great benefit to the development and manufacturing process. 3. Summary of the Invention INDUSTRIAL APPLICABILITY The present invention can be consistently and reliably propagated to a high titer in a host including but not limited to embryonated egg- and embryo-derived tissue culture cells, and can be used for production of an antiviral vaccine. Regarding influenza virus strains. The invention further relates to methods of making such high yielding viruses. More specifically, the high-yielding influenza virus of the present invention contains the portion of the virus that is virulent in the Mx host (ie, the host containing the Mx allele). The present invention is based, in part, on the surprising discovery that Mx mouse resistant influenza virus strains grow to higher titers in embryonated chicken eggs than specifically egg-matched virus strains. The methods provided for the production of high-yield virus strains include, firstly, reconstitution elicited by crossing a low-titre virus strain with a virus resistant to (ie virulent in) the Mx host. Including stock creation. Second, there is provided a method of making a high-yield virus strain, which constructs a high-yielding virus exhibiting a chimeric virus surface molecule such as a chimeric HA and / or NA surface molecule. The chimeric surface molecule comprises the cytoplasmic or cytoplasmic and transmembrane portion of the HA or NA molecule of the virus strain in Mx host, and at least the extracellular antigen of the HA or NA molecule from a newly isolated low titer virus strain. Contains parts. These methods can be used to generate high yielding influenza A, B or C virus strains. 4. Brief description of the drawings Figure 1. Diagram showing reconstitution of high-yield Mx-resistant parental lines (not marked with parallel lines) and recently isolated low-yield virus isolates (marked with parallel lines). The resulting reassorted virus contains HA and NA surface proteins from the low yielding parent, as well as the remaining viral components from the high yielding parent. Figure 2. FIG. 3 shows the interaction between surface proteins (HA and NA) of influenza virus and core structure. Figure 3. FIG. 6 shows the construction of high-yielding virus showing chimeric HA surface proteins. The two viruses shown have the extracellular domain or the extracellular and transmembrane domains (marked with parallel lines) from low yield virus strains. On the other hand, the remaining components of the virus are derived from the high yield strain produced in the Mx host strain. 5. Detailed Description of the Invention The present invention is based, in part, on the surprising discovery that virus strains produced in Mx mice grow to higher titers in embryonated chicken eggs than virus strains specifically adapted to eggs. Lindenmann (Lindenmann, J. et al., 1963, J. Immunol. 90: 942) found that the mouse A2G strain is resistant to influenza A and B viruses. This resistance was later found to be associated with the presence of the predominant Mx allele in several mouse strains, including the A2G strain. Only a few influenza viruses have been reported to be virulent in this mouse strain (Haller, O., 1981, "Current Topics in Microbiology and Immunology" 92, Henle, W., et al., Springer-. Verlag: Berlin, pp. 25-51). As shown in the example described in Section 6, influenza virus toxic in Mx-containing A2G mice behaves surprisingly as a high-yielding virus when grown in embryonated egg host. Therefore, as discussed further below, instead of the A / PR / 8/34 influenza virus strains currently used to generate potentially high yields of reassortant strains, viruses virulent in Mx hosts were used. Is used as the parent strain of the high-yield reconstructed virus strain of the present invention. In addition, as detailed below, portions of viral surface proteins (such as HA and / or NA proteins) derived from viral strains produced in Mx hosts are used as part of the chimeric high-yield viral strains of the invention. It The high yield reconstructs and methods of making chimeric virus strains of the invention are also described below. 5.1 High-yielding virus The high-yielding influenza virus of the present invention can grow to a high titer in a host. Hosts include, but are not limited to, embryonated eggs and the like, preferably embryonic tissue culture cells such as embryonated chicken eggs and chick embryos. Moreover, the high-yielding viruses of the present invention can grow to high titers in numerous other cells, including but not limited to mammalian and avian cell lines. At least a portion of each high-yielding influenza virus is derived from a component of the viral strain produced in the host that contains and expresses the predominant Mx allele. Such Mx host is preferably Mx mouse or Mx mouse cell line. Further, the Mx host can include, but is not limited to, a mammal, such as a ferret, that contains and expresses an Mx allele, any mammalian cell line, any avian, or avian cell line. . The Mx allele in these appropriate hosts may be endogenous to the host or may be introduced into the host using standard recombinant DNA techniques well known to those of skill in the art. High-yielding influenza viruses of the invention can include, but are not limited to, reassorted viruses and viruses displaying chimeric virus surface proteins (preferably chimeric HA and / or NA proteins). The high-yielding reconstituted influenza viruses of the present invention grow to high titers in both Mx hosts and embryonated egg and embryo-derived tissue culture cell hosts. The high-yielding reassortant virus must display on its surface viral surface proteins from the low titer parent strain, preferably chimeric HA and / or NA proteins. Because these proteins are major targets in the host immune response after infection. Therefore, the reassortant virus must contain at least genes encoding one or more viral surface proteins (such as HA and / or NA surface proteins) from the low titer parent strain. Furthermore, the high-yielding reconstructed virus of the present invention contains and expresses at least a part of the genome of the strain derived from the Mx host, which enables the strain to grow to a high titer in the Mx host. Such portions of the viral genome may include, but are not limited to: That is, all or any of the genes encoding the components of the RNA-specific RNA polymerase complex (PB1, PB2 and PA), the gene encoding the nucleocapsid-forming nuclear protein (NP), the matrix protein (M1, A gene encoding M2) and / or a gene encoding a nonstructural protein (NS1, NS2). The portion of the Mx host-derived parental strain genome contained within the high-yielding reassortant virus of the present invention is more than elicited by the low titer virus parent or the virus currently used to make the high-yield reassortant strain. Can also cause a low host interferon response, but this is not required. High-yielding viruses of the invention can include viruses that display chimeric virus surface proteins such as chimeric HA and / or NA surface proteins. Since the core structure of influenza virus is believed to interact with the cytoplasmic domains of surface proteins (such as HA and NA surface proteins), chimeric virus surface proteins containing cytoplasmic domains from high yield Mx resistant strains are unaltered viral surface proteins. May provide more preferred protein-protein interactions with the core structure from the Mx resistant virus strain than those that would provide. As a result, it offers a higher proliferative potential (see the diagram in FIG. 2 illustrating this concept). At least the extracellular domain of the chimeric virus surface protein must be derived from the extracellular portion of the low titer strain viral surface protein. The cytoplasmic or cytoplasmic and transmembrane regions of the chimeric virus surface protein must be derived from the viral surface proteins of the viral strain produced in the Mx host. Therefore, the portion of the viral genome that encodes the extracellular domain of the chimeric virus surface protein must be derived from the genome of a low titer virus strain, and also the cytoplasmic domain or cytoplasmic and transmembrane domains of the chimeric virus surface protein. The portion of the viral genome that encodes must be derived from the genome of the viral strain produced in the Mx host. In one embodiment, the high-yielding influenza virus of the present invention is a virus that is derived almost exclusively from a viral strain originally produced in the Mx mouse strain, the viral surface proteins (HA and / or HA) from a low titer viral strain. Or NA etc.). The portion of the low-titer strain-derived domain of the chimeric virus surface protein of the chimeric virus of the present invention can be modified so that the chimeric virus strain can grow to a higher titer in a host such as hatched chicken eggs. . The portion of the low titer strain-derived domain that can be modified includes, but is not limited to, the receptor binding site of the extracellular domain and / or the domain that affects the cleavability of surface proteins. Modifications can include, but are not limited to, amino acid insertions, deletions, or substitutions. In all cases, the modification does not substantially change the antigenicity of the virus strain of the invention, which is important for the protective host immune response. Amino acid substitutions may be conservative or non-conservative in nature. Conservative amino acid substitutions consist of replacing one or more amino acids in the low titer strain-derived domain of a viral surface protein with an amino acid having similar charge, size, and / or hydrophobic properties. For example, substitution of glutamic acid (E) with aspartic acid (D) and the like. Non-conservative substitutions consist of replacing one or more amino acids of the low titer strain-derived domain of a viral surface protein with an amino acid having a different charge, size, and / or hydrophobic character. For example, substitution of glutamic acid (E) with valine (V) and the like. Amino acid insertions can consist of a single amino acid residue or a stretch of residues that is 2 to 15 amino acids in length. One or more insertions can be incorporated into viral surface proteins from low titer strains, as long as the insertions do not substantially alter the antigenicity of the viral strain important for the protective immune response. Deletions of portions of viral surface proteins from low titer strains are also within the scope of the invention. Such a deletion consists of removing one or more amino acids from the protein sequence, unless the resulting deleted protein exhibits virtually altered antigenicity that does not elicit a protective immune response from the host. Deletions can include one contiguous portion of the peptide sequence, or one or more distinct portions. The high-yielding influenza virus of the present invention can be used as an antiviral vaccine. Such vaccines can be used in human as well as non-human hosts including, but not limited to, equine, porcine, avian hosts. Vaccination is carried out according to techniques well known to those skilled in the art. (For such techniques, see Palese, P. et al., 1992, US Pat. No. 5,166,057, which is incorporated herein by reference in its entirety). 5.2 Creation of high-yielding virus The method for producing the high-yielding virus of the present invention will be described below. Included are high-yielding reconstructed viruses and methods for making high-yielding viruses that display chimeric virus surface proteins. 5.2.1 Construction of high-yielding reconstructed virus To generate a high-yielding reassortant virus strain, a low titer parental influenza strain is crossed with the parental virus strain produced in the Mx host by standard mixed infection. Mixed infections are performed by co-infecting Mx resistant virus with various dilutions of low titer virus strains. Regarding influenza A, the high titer virus strain is PR8 strain (Haller, O., 1981, "Current Topics in Microbiology and Immunology", Henle, W. et al., Ed., Springer-Verlag: New York, pp. 25-. 51), but is not limited thereto. Other high yielding influenza A, B or C parental strains can be created by passing through the Mx host and selecting for strains that exhibit virulence and high growth characteristics in the Mx host. Mx hosts can include, but are not limited to: That is, the Mx mouse cell line such as A2G cell line and the Mx mouse line such as A2G mouse line (Lindenmann, J. et al., 1963, J. Immunol. 90: 942-951). Further, the Mx host can include, but is not limited to, any mammal (eg, a ferret), mammalian cell line, any avian, or avian cell line that contains and expresses the Mx allele. The Mx alleles in these appropriate hosts may be endogenous to the host or may be introduced into the host using standard recombinant DNA techniques well known to those of skill in the art. Animal or cell monolayers can be co-infected using standard techniques well known to those of skill in the art. In the case of whole animal infection, the host can be co-infected, for example by the intranasal or aerosol routes. The progeny reassortant virus resulting from the mixed infection can be used to pass through Mx hosts or to infect hosts such as cells from embryonated chicken eggs or embryos. Next, the growth characteristics of the reconstructed virus strains are evaluated, and strains showing high titer growth ability can function as the reconstructed virus of the present invention. Due to the fact that viral surface proteins are major targets of the host humoral response after infection, the high-yielding reassortant virus strains of the invention have at least one viral surface derived from a low-titer parental viral strain on their viral surface. The protein (such as HA or NA) has to be indicated (see figure 1 showing one embodiment of the reconstructed virus strain of the invention). It can be confirmed whether the virus surface protein of the reconstructed strain is derived from the low titer parent strain. The method assays the presence of viral surface proteins from low titer parent strains, for example, using antibodies raised against low titer viral surface proteins that do not cross-react with high yield parental viral surface proteins. It depends. Monoclonal or polyclonal antibody preparations can be used for these methods. Alternatively, the absence of HA or NA from the high yield parent strain can be assayed. The method is by contacting the reconstituted virus or viral protein with an antibody directed against the viral surface protein from the high titer parent strain that does not cross-react with the viral surface protein of the low yield parent strain. Further, viral proteins derived from low titer parent strains using a technique based on standard reverse transcriptase (RT): polymerase chain reaction (PCR; Mullis, KB, 1987, experimental embodiment described in US Pat. No. 4,683,202). The presence of the gene coding for can be assayed. In this case, a primer pair that specifically hybridizes to the nucleotide sequence present in the virus surface protein gene from the low titer parent strain, but does not hybridize to the sequence present in the virus surface protein gene from the high yield parent virus strain. Can be used. Therefore, PCR amplification using standard techniques (Innis, MA et al., 1990, PCR Protocols, Academic Press, NY) is a reconstructed strain containing the viral surface protein gene from the low titer parent strain as the starting material for the assay. Only when the RNA from which it is used produces an amplified fragment. Alternatively, a primer pair that specifically hybridizes to a nucleotide sequence present in a virus surface protein gene from a high-yield strain, but does not hybridize to the virus surface protein gene of a low-titer parent virus strain, The absence of the gene encoding the viral surface protein from Escherichia coli can also be assayed. Amplification does not occur if RNA from a reconstructed strain that does not contain the HA or NA gene from the high yield parent strain was used as the starting material for the assay. When creating a reassortant strain, instead of relying on the reassortment of completely random RNA gene segments, one can make a selection for the presence of viral surface proteins from the high yield parent strain. For example, the progeny virus obtained after the first mixed infection can be pretreated with antisera raised against the high yield parent strain. Such treatment neutralizes the effect of viruses displaying surface proteins from the high-yielding parent strain, inhibiting their ability to infect host cells. A virus sample treated with antiserum, which contains a population of infectious virus enriched for viruses displaying viral surface proteins from the low yield parent strain, is then used to infect an appropriate host. be able to. This method is a particularly useful technique in that it can easily neutralize the effect of antibodies to surface proteins when used to construct the virus of the present invention. The resulting reshaped virus strain can then be used to infect a host, such as cells from embryonated chicken eggs or embryos. At this point, the growth characteristics of the reconstructed strain in the host can be evaluated. Such growth properties can be assessed, for example, using methods well known to those of skill in the art, by measuring the number of PFU per volume of viral sample, or by assaying the hemagglutinin titer of the viral sample. 5.2.2 Construction of high-yielding chimeric reconstructed virus The generation of the inventive chimeric high-yielding virus described in clause 5.1 is described here. The steps involved in the production of such a chimeric virus are as follows: first, the construction of an RNA segment containing a gene encoding a chimeric virus surface protein; and second, the ribonucleoprotein (RNP) transfer for the construction of a high-yield strain. Operation. To construct a segment containing the chimeric virus gene, viral surface proteins of interest (such as HA and / or NA proteins) from both the high-yield strain originally selected in the Mx host and from the low-titer parent strain were selected. The coding RNA segment must be obtained. The method of obtaining these RNA segments uses standard techniques well known to those skilled in the art. See, for example, Palese and Schu lman, 1976, J. et al. Virol. 17: 876-884. The RNA segment encoding these genes is then isolated using techniques well known to those of skill in the art (Sambrooks et al., 1989, Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Springs Harbor Press, NY). Reverse transcribe to cDNA. Once the genes are in DNA form, standard recombinant DNA techniques can be used to isolate and combine the appropriate portions of each gene. Using HA virus surface proteins as an example, regions of the HA gene encoding the extracellular domain of HA proteins of low yield strains can be isolated using, for example, convenient restriction enzyme sites. This restriction enzyme site may be endogenous to the sequence, or by recombinant techniques (see, eg, Kunkel, 1985, Proc. Natl. Acad. Sci. USA. 82: 488) or other enzymatic or chemical means. It was created in the array. The portions of the HA gene that encode the cytoplasmic and transmembrane domains of the HA protein can then be isolated in the same manner. The portion of the cDNA encoding the extracellular domain of the low yield strain and the cDNA encoding the cytoplasmic and transmembrane domains of the high yield strain can then be ligated using standard enzymatic means. Alternatively, once the nucleotide sequences of the low yield strain and high yield strain HA genes are known, the chimeric viral genes of the invention can be synthesized using chemical means well known to those skilled in the art. In addition to the appropriate amino acid coding regions, the chimeric gene construct allows for efficient transcription of the viral polymerase binding site / promoter, the RNA polymerase binding site, and the chimeric RNA segment, suitable 3'and 5 known to those of skill in the art. 'Must include sequences such as regulatory sequences. The cDNA chimera construct can then be introduced into a system that transcribes cDNA into RNA of negative polarity. Such systems are well known to those of skill in the art and include, but are not limited to, T3, T7 and SP6 in vitro transcription systems and the like. The RNA chimera construct can be used directly. RNA with a negative polarity that encodes the chimeric virus surface NA protein can then be combined with the viral RNA polymerase complex. The complex may be isolated or made using recombinant techniques well known to those skilled in the art to form infectious RNP (ribonucleoprotein). RNP consists of viral RNA-specific RNA polymerase protein (P protein) and nucleoprotein (NP). The RNP can then be used with the "parental" virus to co-infect host cells. Suitable host cells may include, but are not limited to, Mx mouse cells such as the mouse A 2G cell line, or host cells derived from embryonated chicken eggs or embryos. In this case, the "parent" refers to the high-yield virus strain originally produced in the Mx strain. The techniques used to make infectious RNPs containing genes encoding chimeric proteins can be found in Palese et al. (Palease P. et al., 1992, US Pat. No. 5,166,057). After co-infection, a chimeric virus surface protein was demonstrated using essentially the same technique described in Section 5.2.1 above, and the remaining components were derived from the original high-yield Mx-resistant "parental" strain. The progeny virus that does this can be selected and characterized. 6. Example: High-yielding reconstituted influenza virus Production and identification This example describes a reconstituted influenza virus emerging in Mx mouse hosts that grows to high titers in hatched hen egg hosts. 6.1 Materials and methods Virus: The high-yielding Mx-resistant parent virus used was the PR8 virus strain ("Current Topics in Microbiology and Immunology", edited by Herle, W. et al., Springer-Verlag, New York, pp.25-51, 1981. Haller, O.). Recent influenza A virus isolates were used as low titer parental virus strains. Host system: A2G Mx mouse system (Lindenmann, J. et al., 1963, J. Immunol. 90: 942-951) and 11-day-old embryonated chicken eggs were used as host system. Reconstitution and identification procedure: Standard mixed infection method and hemagglutinin titer assay were utilized (Palese and Schulman, 1976, J. Virol. 17: 876-884). 6.2 result Experiments were performed to emerge high yielding reconstituted influenza A virus strains that can be used to produce large quantities of virus suitable for vaccine production. Therefore, the recently isolated influenza A strain, which only grows to a low titer in the embryonated egg host, and the high titer growth in Mx mice, were used as parent virus strains for the production of the reconstructed virus strain. The indicated influenza A virus strain PR8 was used. Low and high titer parental virus strains were co-infected into embryonated chicken eggs by the standard mixed infection method. The progeny reassortant virus produced by co-infection was then subcultured in embryonated egg host cells and the reassortant virus was isolated using a screen with antibodies to the HA and NA surface proteins of the PR8 parental strain. The results demonstrating that the reshaped virus produced in this experiment grows to high titers in embryonated egg host are shown in Table 1 below. The growth characteristics of virus strains were assayed using standard hemagglutinin titration methods. Thus, the recently isolated low-yielding influenza A virus was successfully used with the high-yielding PR8 virus, surprisingly resulting in a reconstructed strain that behaves as a high-yielding virus strain in the embryonated egg host. It was Obviously, many modifications and variations of the invention shown herein can be made without departing from its spirit and scope. The particular embodiments described above are presented by way of example only, and the present invention is limited only by the scope of the appended claims.

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Claims (1)

[Claims] 1. Increased titer in Mx host, hatched host or embryo-derived tissue culture cell host Influenza virus strains that can be propagated. 2. Influenza virus is influenza A or C virus strain, reconstructed The influenza virus according to claim 1, which is a virus strain or a chimeric virus strain. Ruth stock. 3. Virus strain derived from low titer parent virus strain and high titer Mx resistant virus strain The reconstructed or chimeric virus strain according to claim 2, which is 4. High titer of the Mx resistant parental virus strain is the Mx resistant parental virus strain in the Mx host. It is possible to grow to high titer in the Enzyme complex, nucleoprotein gene, matrix protein gene or nonstructural The reconstructed or chimeric virus strain according to claim 3, which is a protein gene. 5. A portion of the reconstructed virus strain derived from the low titer parental virus strain Surface protein, hemagglutinin protein or neuraminidase The reconstructed or chimeric virus strain according to claim 3, which is a protein. 6. The part of the reconstructed virus strain derived from the high titer Mx resistant parental virus strain Lower than host interferon response elicited by low titer parental virus strains 4. The reconstructed or chimeric mouse according to claim 3, which elicits a host interferon response. Illus strain. 7. Part of the reconstructed virus strain derived from the parental virus strain of low titer is the host immunity The reconstruction or key according to claim 3, which elicits a response. Melavirus strain. 8. Receptor binding domain of chimeric influenza virus surface protein or The chimeric infusate of claim 2, wherein the cleavability domain is modified. Ruenza virus strain. 9. Chimeric influenza virus surface protein insertion and deletion of amino acid residues Or a chimeric influenza virus according to claim 8, which is modified by substitution. stock. Ten. Reconstruction capable of growing to high titers in Mx host and in domesticated egg host A method of producing an influenza virus strain, the method comprising:     (A) Low titer parental influenza virus strain and high titer Mx resistance in a suitable host Co-infection with a sex influenza virus strain I'll make a strain, and     (B) Reconstructed influenza virus showing high titer growth ability in hatched chicken egg host Select stocks,   A method comprising: 11. Propagation to high titers in Mx hosts and in embryo-derived tissue culture cell hosts A method of producing a reconstituted influenza virus strain that comprises:     (A) Low titer parental influenza virus strain and high titer Mx resistance in a suitable host Co-infection with a sex influenza virus strain I'll make a strain, and     (B) Reconstructed influenza showing high titer growth ability in embryo-derived tissue culture cell host N. virus strain,   A method comprising: 12. Propagation to high titers in Mx hosts and in modified chicken egg hosts A method for producing a chimeric influenza virus strain capable of     (A) A suitable host cell is used as the chimeric influenza virus surface protein of claim 5. Ribonucleoprotein containing an RNA segment containing a gene encoding a protein To allow the gene to be expressed in a suitable host, And     (B) Chimeric influenza virus showing high titer growth ability in embryonated egg host Select stocks,   A method comprising: 13. Propagation to high titers in Mx hosts and in embryo-derived tissue culture cell hosts A method for producing a chimeric influenza virus strain capable of     (A) A suitable host cell is used as the chimeric influenza virus surface protein of claim 5. Ribonucleoprotein containing an RNA segment containing a gene encoding a protein To allow the gene to be expressed in a suitable host, And     (B) A chimeric influenza showing high titer growth ability in an embryo-derived tissue culture cell host N. virus strain,   A method comprising: 14. Anti-fluen containing the influenza virus of claim 1, 2 or 3. The virus vaccine.