CA2164946A1

CA2164946A1 - High yielding influenza viruses

Info

Publication number: CA2164946A1
Application number: CA002164946A
Authority: CA
Inventors: Peter Palese
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-06-11
Filing date: 1994-06-10
Publication date: 1994-12-22
Also published as: JPH08511424A; WO1994029439A1; EP0711340A4; EP0711340A1; AU7104194A

Abstract

The present invention relates to influenza virus strains which may reliably be propagated to high titer in hosts such as embryonated eggs. High yield strains, including reassortment influenza viruses and influenza viruses exhibiting chimeric viral surface molecules, such as chimeric HA and/or NA surface molecules, arc described. Further, methods for the production of such high yield influenza A, B, and/or C virus strains are described. These high yield viruses may be used in the production of anti-viral vaccines and the manufacturing of chimeric influenza viruses.

Description

W094/29439 ~ PCT~S94/06S41 .

HIG~ YIELDING INFLUENZA VIRU8E8 1. Introduction The present invention relates to influenza virus strains which may reliably be propagated to high titer in hosts such as embryonated eggs. High yield strains, including reassortment influenza viruses and influenza viruses exhibiting chimeric viral surface molecules, such as chimeric HA and/or NA surface molecules, are described. Further, methods for the production of such high yield influenza A, B, and/or C
virus strains are described. These high yield viruses may be used in the production of anti-viral vaccines and the manufacture of chimeric influenza viruses.

2. Background Of The Invention Anti-viral vaccine production presents many challenges. For example, an ever-changing group of influenza virus subtypes, such as, say, influenza A(H3N2) and A(HlN1), and influenza B, circulate within the general populati~n. Further, due to antigenic drift, a large amount of variation quickly develops within each influenza A subtype and within influenza B. Thus, it is neceCcAry to continually isolate influenza viruses and Acc~F5 their antigenic characteristics. Annually, then, such information is used in determining the composition of the influenza vaccine for the following reason (Robertson, J.S., et al. 1988, in Influenza Vaccines, Development and Perspectives, Giornale di Igiene e Medicina Preventiva 29:4 58).
Strains of influenza virus, newly isolated from patients, however, do not usually grow to high titers.
This is especially true when the viruses are propagated in chick embryonated eggs, the preferred W094/29~9 PCT~S94/06541 hosts for the production of large quantities of virus for use as killed virus vaccines.
In order to improve the growth characteristics of newly isolated influenza virus strains, attempts are often made to reassort the new strain with a high growth laboratory strain. For example, new influenza A virus strains are crossed with the high-yield influenza A/PR/8/34 virus strain to produce potentially high yield reassortment strains. Those reassortment viruses with high growth characteristics usually derive six genomic RNA segments from the high-yield parental virus strain and the hemagglutinin (HA) and neuraminidase (NA) segments from the newly ,5 isolated parental strain (Kilbourne, E.D. and Murphy, J.S., 1960, J. Exp. Med. 111:387; Kilbourne, E.D., 1969, WH0 Bull. 41:643; Baez, M. et al., 1980, J.
Infectious Dis. 141:362; Robertson, J.S. et al., 1992, Biological 20:213). Although this A/PR/8/34 strain reassortment approach may give some influenza virus strains with improved growth characteristics, many of the resulting reassortment strains will exhibit limited growth potential in embryonated eggs.
Current methods, therefore, for increasing the replication titer of newly discovered viral strains are not optimal, and improved methods for increasing viral yields in the preferred hosts, for vaccine production would be highly beneficial for the process of anti-viral vaccine development and manufacture.

3. SummarY Of The Invention The present invention relates to influenza strains which may consistently and reliably be propagated to high titer in hosts which include, but are not limited to embryonated eggs and embryo-derived tissue culture cells, and may be used in the W094/29~9 ~ 6 ~ 9 ~ ~ PCT~Sg~/06541 production of anti-viral vaccines. The invention further relates to methods for the production of such high yield viruses. More specifically, the high-yield influenza viruses of the invention contain portions of viruses which are virulent in Mx hosts, (i.e., hosts cont~; ni ng the Mx allele). The invention is based in part, on the surprising finding that Mx mouse resistant influenza virus strains grow to higher titers in embryonated eggs than do specifically egg-adap~ed viral strains.
The methods presented for the production of high yield viral strains include, first, the production of reassortment strains developed by crossing a low titer virus strain with a virus resistant to (i.e., virulent in) an Mx host. Second, methods for the production of high yield virus strains are presented wherein high yield viruses are constructed which exhibit chimeric viral surface molecules, such as chimeric HA and/or NA
surface molecules. The chimeric surface molecules contain the cytoplasmic or cytoplasmic and transmembrane portions of the HA or NA molecules of a viral strain in an Mx host, and at least the extracellular antigenic portion of the HA or NA
molecule from the newly isolated low titer virus strain. These methods may be utilized for the production of high yield influenza A, B, or C virus strains.

4. Brief Description Of The Fi~ures FIG. 1. Diagram depicting reassortment between a high yield Mx resistant parental strain (non-hatched) and a recently isolated, low yield virus isolate (hatched). The resulting reassortment virus shown contains HA and NA surface proteins derived from the W094/29~9 PCT~S94/06541 2~94~ ~

low yield parent and the rest of the viral components derived from the high yield viral parent.

FIG. 2. Diagram depicting the interaction of viral surface proteins (HA and NA) with the core structure of influenza viruses.

FIG. 3. Diagram depicting the construction of a high yield virus exhibiting a chimeric HA surface protein. The two viruses shown contain extracellular or extracellular and transmembrane domains derived from a low yield viral strain (hatched) while the remaining components of the virus are derived from a high yield strain developed in a Mx host strain.

5. Detailed Description Of The Invention The present invention is based, in part, on the surprising discovery that viral stains developed in Mx mice grow to higher titers in embryonated eggs than do specifically egg-adapted viral strains. Lindenmann (Lindenmann, J. et al., 1963, J. Immunol. 90:942) discovered that the A2G strain of mice is resistant to influenza A and B viruses. The resistance was later found to be associated with the presence of the dominant Mx allele present in several mouse lines, including the A2G strain. Only a few influenza viruses have been reported to be virulent in this strain of mice (Haller, O., 1981, in "Current Topics in Microbiology and Immunology 92, Henle, W. et al., Springer-Verlag: Berlin, pp. 25-51). As shown in the Working Example presented below in Section 6, influenza strains which are virulent in Mx-containing A2G mice surprisingly behave as high yield viruses when propagated in embryonated egg hosts.

W094/29~9 ~ PCT~S94/06541 Therefore, as discussed further below, instead of the A/PR/8/34 influenza strain currently used for generating potentially high yield reassortment strains, viruses which are virulent in Mx hosts are utilized as parent strains for the high yield reassortment virus strains of the invention. Further, as described in detail below, portions of the viral surface proteins, such as HA and/or NA proteins, derived from viral strains developed in Mx hosts are utilized as part of the chimeric high yield virus strains of the invention. Also described below are methods for the production of the high yield reassortment and chimeric virus strains of the invention.

5.l Hiqh Yield Viruses The high yield influenza viruses of the invention are capable of growing to high titer in hosts which include, but are not limited to such hosts as embryonated eggs, preferably embryonated hens eggs, and tissue culture cells derived from embryos such as chick embryos. In addition, the high yield viruses of the invention may be capable of growing to high titer in a large number of other cells, including, but not limited to mammalian and avian cell lines. At least a portion of each high yield influenza virus is derived from components of viral strains which have been developed in hosts which contain and express the dominant Mx allele. Such Mx hosts are preferably Mx mice or Mx mouse cell lines. Additionally, Mx hosts may include, but are not limited to any mammalian animal, ferret for example, any mammalian cell line, any avian animal, or avian cell line, which contains 35 and expresses the Mx allele. The Mx allele in these appropriate hosts may be endogenous to the host or W094/29~9 PCT~S94/06541 ~6~g may, alternatively, be introduced into the host using st~nA~rd recombinant DNA techni ques well known to those of ordinary skill in the art. The high yield influenza viruses of the invention may include, but are not limited to, reassortment viruses and viruses exhibiting chimeric viral surface proteins, preferably chimeric HA and/or NA proteins.
The high yield reassortment influenza viruses of the invention grow to high titer in both Mx hosts and in embryonated egg and embryo-derived tissue culture cell hosts. The high yield reassortment viruses should exhibit on their surfaces viral surface proteins, preferably HA and/or NA proteins, derived from the low titer parent because these proteins are major targets of the host immune response after infection. The reassortment viruses should contain, therefore, at least the gene(s) derived from the low titer viral parent encoding one or more viral surface proteins, such as the HA and/or NA surface proteins.
Further, the high yield reassortment viruses of the invention include and express at least those portions of the genome of the Mx host-derived strain which allow the strain to grow to high titer in the Mx host.
Such portions of the viral genome may include, but are not limited to, all or any of the genes encoding the components of the RNA-directed-RNA polymerase complex (PB1, PB2, and PA), the gene encoding the nucleoprotein (NP), which forms the nucleocapsid, the genes encoding the matrix proteins (M1, M2), and/or the genes encoding the nonstructural proteins (NS1, NS2). The portion of the Mx host-derived parental strain's genome contained within the high yield reassortment virus of the invention may, but is not required to, induce a lower host interferon response than the host interferon response elicited by the low W094/29~9 2 1 ~ ~ 9 ~ 6 PCT~S94/06541 titer viral parent, or viruses currently used to prepare high yield reassortment.
The high yield viruses of the invention may include viruses which exhibit chimeric viral surface proteins, such as chimeric HA and/or NA surface proteins. Because the core structures of influenza viruses are thought to interact with the cytoplasmic domains of the surface proteins, such as the HA and NA
surface proteins, chimeric viral surface proteins cont~;n;ng cytoplasmic domains derived from a high yield Mx resistant strain may provide more favorable protein-protein interactions with a core structure derived from a Mx resistant virus strain than unaltered viral surface proteins would, thus providing a higher growth potential (See FIG. 2 for a diagram depicting this concept). At least the extracellular portions of the chimeric viral surface proteins should be derived from the extracellular portions of the viral surface proteins of the low titer strain. The cytoplasmic or the cytoplasmic and transmembrane regions of the chimeric viral surface proteins should be derived from the viral surface proteins of a viral strain which was developed in the Mx host.
Accordingly, the portions of the viral genome encoding the extracellular domain(s) of the chimeric viral surface proteins should be derived from the genome of the low titer viral strain, and the portions of the viral genome enco~;~g cytoplasmic or cytoplasmic and transmembrane domains of the chimeric viral surface proteins should be derived from the genome of a viral strain which was developed in a Mx host. In one embodiment, the high yield influenza viruses of the invention may consist of a virus derived almost exclusively from a viral strain which was originally developed in a Mx mouse strain, and contains only the W094/29~9 PCT~S94/06541 extracellular domain of a viral surface protein, such as HA and/or NA, derived from a low titer viral strain.
Portions of the low titer strain-derived domains of the chimeric viral surface proteins of the chimeric viruses of the invention may be modified such that the chimeric virus strain is capable of growing to a higher titer in hosts such as embryonated eggs. The ~O portions of the low titer strain-derived domains which may be modified include, but are not limited to the extracellular domain's receptor-binding sites and/or those domains which affect the cleavability of the surface proteins. Modifications may include, but are not limited to amino acid insertions, deletions, or substitutions. In all cases, the modifications do not effectively change the antigenicity of the virus strains of the invention which is important for a protective host immune response.
Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the low titer strain-derived domains of the viral surface proteins with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids of the low titer strain-derived domains of the viral surface proteins with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.
Amino acid insertions may consist of single amino acid residues or stretches of residues ranging from 2 to 15 amino acids in length. One or more insertions W094/29~9 ~ PCT~S94/06541 = . ~ ......... .... .

may be introduced into the low titer strain-derived viral surface proteins so long as the insertions do not effectively change the antigenicity of the virus strain which is important for a protective immune response.
Deletions of portions of the low titer strain-derived viral surface proteins, are also within the scope of the invention. Such deletions consist of the removal of one or more amino acids from the protein-sequence, so long as the resulting deletion protein does not exhibit an effectively changed antigenicity which does not elicit a protective immune response from the host. Deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences.
The high yield influenza viruses of the invention may be used as anti-viral vaccines. Such vaccines may be u ed in human as well as non-human hosts, including, but not limited to equine, porcine, and avian hosts. Vaccine administration is performed according to te~hn;ques well known to those of ordinary skill in the art. tSee Palese, P. et al., 1992, U.S. Patent No. 5,166,057, which is incorporated herein by reference in its entirety, for such t~c~n;ques.) 5.2 Production Of Hiqh Yield Virus Presented below are methods for the production of the high yield viruses of the invention. Included are methods for the development and production of high yield reassortment viruses and high yield viruses which exhibit chimeric viral surface proteins.

W094l29~9 PCT~S94/06541 5.2.1 Production Of Hiqh Yield Reassortment Viruses In order to produce a high yield reassortment virus strain, a low titer parent influenza strain is crossed, in a standard mixed infection, with a parent virus strain developed in a Mx host. Mixed infections are performed by coinfecting Mx resistant virus with various dilutions of a low titer virus strain. For influenza A, high titer virus strains may include, but are not limited to the PR8 strain (Haller, O., 1981, in "Current Topics in Microbiology and Immunology", Henle, W. et al., eds., Springer-Verlag: New York, pp.
25-51). Other high yield influenza A, B, or C
parental strains may be developed by passage though Mx hosts and by selecting those strains which exhibit virulence and high growth characteristics in Mx hosts.
Mx hosts may include, but are not limited to Mx mouse cell lines such as the A2G cell line, and Mx mouse strains, such as the A2G mouse strain (Lindenmann, J.
et al., 1963, J. Immunol. 90:942-951). Additionally, Mx hosts may include, but are not limited to any mammalian animal, ferret, for example, mammalian cell line, any avian animal, or avian cell line which contains and expresses the Mx allele. The Mx allele in these appropriate hosts may be endogenous to the host or may, alternatively, be introduced in the host using stAn~Ard recombinant DNA techn; ques well known to those of ordinary skill in the art. Animals or monolayers of cells may be coinfected using standard t~r.hn; ques well known to those in the art. In the case of whole animal infections, hosts may, for example, be coinfected by intranasal or aerosol routes.
Progeny reassortment viruses resulting from mixed infections may be passaged through Mx hosts or may be used to infect hosts such as embryonated egg hosts or W094/29439 ~ ~3~ 4 PCT~S94/06541 .

embryo-derived cell hosts. The growth characteristics of the reassortment virus strains are then evaluated and those exhibiting high titer growth capabilities may act as reassortment viruses of the invention.
Due to the fact that the viral surface proteins are major targets of the host humoral response after infection, the high yield reassortment virus strains of the invention must exhibit on their viral surfaces at least one viral surface protein, such as HA or NA, which has been derived from the low titer parental viral strain (See FIG. 1 for a diagram depicting one embodiment of the reassortment virus strains of the invention). One may determine whether viral surface proteins of the reassortment strains are derived from the low titer parental strain by, for example assaying for their presence using antibodies raised against low titer viral surface proteins which do not cross react with the viral surface proteins of the high yield parental strain. Monoclonal or polyclonal antibody preparations may be used for these procedures.
Alternatively, one may assay for the absence of HA or NA proteins which have been derived from the high yield parental strains by contacting the reassortment virus or viral proteins with antibodies raised against the high titer parental strain-derived viral surface proteins which do not cross react with the viral surface proteins of the low yield parental strain.
In addition, one may assay for the presence of the genes enco~ing low titer-derived viral proteins by using a standard reverse transcriptase (RT):polymerase chain reaction (PCR; the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No.
4,683,202) based t~chn;que. In this instance, pairs of primers may be used which specifically hybridize to nucleotide sequences present in the low titer-derived W094/2g439 PCT~S94/06541 viral surface protein genes but do not hybridize to sequences present in the high yield parental virus strain-derived viral surface protein genes. PCR
amplification using st~n~Ard te~-hniques (Innis, M.A.
et al., eds., l99O, PCR Protocols, Academic Press, NY), therefore, will only yield an amplified fragment when RNA from a reassortment strain con~;n;ng low titer-derived viral surface protein genes is used as the starting material in the assay. Alternatively, one may assay for the absence of genes encoding viral surface proteins which have been derived from the high yield parental strains by utilizing primer pairs which specifically hybridize to nucleotide sequences present in the high yield parental strain-derived viral surface protein genes but do not hybridize to the viral surface protein genes of the low titer strain.
No amplification will occur when RNA from those reassortment strains which do not contain high yield parental strain derived HA or NA genes is used as the starting material for the assay.
When developing reassortment strains, one may select against the presence of high yield parental strain derived viral surface proteins, instead of relying on completely random reassortment of RNA gene segments. For example, progeny viruses obtained after an initial mixed infection may be pretreated with an antiserum raised against the viral surface proteins of the high yield parental strain. Such treatment will neutralize those viruses exhibiting the high yield parental strain derived surface proteins, inhibiting their ability to infect host cells. The antiserum-treated virus sample, which now contains a population of infectious viruses which are enriched for viruses that exhibit low yield parental strain-derived viral surface proteins, may then be used to infect W094/29~9 2 ~ PCT~S94/06S41 .

appropriate host cells. This is an especially useful techn i que when utilized in the construction of the viruses of the invention, in that neutralizing antibodies against surface proteins may readily be made.
The resulting reassortment virus strain may then be used to infect hosts such as embryonated egg or embryo-derived cell hosts, at which time growth characteristics of the reassortment strain in the host may be evaluated. Such growth characteristics may be evaluated by determining, for example, the number of PFUs per volume of viral sample or by assaying the hemagglutinin titer of the viral sample, using t~chn; ques well known to those of ordinary skill in the art.

5.2.2 Production of High Yield Chimeric Reassortment Viruses The production of the chimeric high yield viruses of the invention, described above in Section 5.1 is described here. The steps involved in the production of such chimeric viruses consists of, first, the construction of an RNA segment containing a gene ~ncoA;ng a chimeric viral surface protein, and, second, ribonucleoprotein (RNP) transfection to construct the high yield strain.
In order to construct the segment containing the chimeric viral gene, the RNA segments encoding the viral surface protein(s) of interest (such as the HA
and/or NA proteins) from both a high yield strain which was originally selected in an Mx host, and from a low yield strain must be obtained. Procedures for obtaining these RNA segments utilize standard 35 t~chn; ques well known to those of ordinary skill in the art. See, for example, Palese and Schulman, 1976, W094/29439 2 1 ~ ~9 ~ ~ PCT~S94/06541 J. Virol. 17:876-884, which is incorporated herein by reference in its entirety.
The RNA segment encoding these genes is then reverse-transcribed into cDNA, using ~echniques well known to those of skill in the art (Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Vols.
1-3, Cold Spring Harbor Press, NY). once the genes are present in a DNA form, the appropriate portions of each gene may be isolated and combined using stAn~rd recombinant DNA techniques. Using the HA viral surface protein gene as an example, the region of the HA gene which encodes the extracellular domain of the HA protein of a low yield strain, may be isolated by, for example, the use of a convenient restriction enzyme site endogenous to the sequence or engineered into the sequence by recombinant means (see, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA
82:488), or by other enzymatic or chemical means.
Next, the region of the HA gene encoding the cytoplasmic and transmembrane domains of the HA
protein may be isolated using the same types of procedures. The region of cDNA encoding the extracellular domain of the low yield strain and the cDNA encoding the cytoplasmic and transmembrane domains of the high yield strain may then be joined, using st~n~rd enzymatic means. Alternatively, once the nucleotide sequence of the HA gene of the low and high yield strains is known, the chimeric viral genes of the invention may be synthesized using chemical means well known to those of skill in the art.
In addition to the appropriate amino acid coding regions, the chimeric gene constructs should include those sequences, such as viral polymerase binding site/promoter, RNA polymerase-binding sites, and the appropriate 3' and 5' regulatory sequences well known W094/29~9 ~ PCT~S94/06541 to those of skill in the art which will allow efficient transcription of the chimeric RNA segments.
cDNA chimeric constructs may then be introduced into a system whereby the cDNA may be transcribed into RNA of a negative polarity. Such systems are well known to those of skill in the art and include, but are not limited to the T3, T7, and SP6 in vitro transcription systems and the like. RNA chimeric constructs may be utilized directly.
The negative polarity RNA encoding the chimeric viral surface NA proteins may then be combined with viral RNA polymerase complex, which may be isolated or may be produced using recombinant techniques, both of which are well known to those of skill in the art, to form infectious RNP (ribonucleoprotein) complex. RNPs comprise viral RNA-directed RNA polymerase proteins (the P proteins) and nucleoprotein (NP). The RNPs may then be used together with a "parent" virus to coinfect host cells. Appropriate host cells may include, but are not limited to Mx mouse cells, such as those of the mouse A2G cell line, or, alternatively, embryonated egg or embryo-derived host cells. The "parent" in this instance, is a high yield virus strain originally developed in a Mx strain. The t~chniques utilized for the production of infectious RNPs containing genes encoding chimeric proteins may be found in Palese et al. (Palese, P. et al., 1992, U.S. Patent No. 5,166,057). After coinfection, techniques essentially identical to those described above in Section 5.2.1 may be utilized to select for and characterize those progeny viruses which exhibit chimeric viral surface proteins and whose remaining components are derived from the original high yield MX
35 resistant "parental" strain.

W094/29~9 PCT~S94/06541

6. Example: Production and Characterization Of A High Yield Reassortment Influenza Virus In this example, a reassortment influenza virus developed in a Mx mouse host is described which grows to high titer in embryonated egg hosts.

6.1 Materials And Methods Viruses: The high yield Mx resistant parent virus used was the PR8 virus strain (Haller, O., 1981, in "Current Topics in Microbiology and Immunology;
Herle, W. et al., eds., Springer-Verlag: New York, pp.
25-51). A recent influenza A virus isolate was used as the low titer parent virus strain.
Host Strains: The A2G Mx mouse strain (Lindenmann, J. et al., 1963, J. Immunol. 90:942-951) and eleven day old embryonated hen's eggs were used as host strains.
Reassortment and characterization Procedures:
St~nAArd mixed infection procedures and hemagglutinin titer assays were utilized (Palese and Schulman, 1976, J. Virol. 17:876-884).

6.2 ~esults Bxperiments were performed to develop a high-yielding reassortment influenza A virus strain which may be utilized for the production of large amounts of virus suitable for vaccine production. To that end, a recently isolated influenza A strain which grows to only low titer in embryonated egg hosts and the influenza A virus strain, PR8, a virus strain which exhibits high titer growth in Mx mice, were used as the parent virus strains for the production of a reassortment virus strain. The low and high titer parent virus strains were coinfected into embryonated W094/29439 2 ~ PCT~S94/06S41 .

eggs in a standard mixed infection procedure. Progeny reassortment viruses produced from the coinfection were subsequently repA-ecAged in embryonated egg host cells, and reassortment viruses were isolated using an - antibody screen directed against the HA and NA surface proteins of the PR8 parent strain.
Presented in Table I, below, are results which demonstrate that the reassortment virus produced in this study grows to high titer in embryonated egg hosts. The growth characteristics of the virus strains were assayed using stAn~Ard hemagglutinin titer measurements. Thus, the recently isolated low yield influenza A virus was successfully used together with a high yield PR8 virus to produce a reassortment strain which, surprisingly, acts as a high yield virus strain in an embryonated egg host.

INFEUENZA A VIRU8 8TRAIN HE~ VTINATION (HA) TITER
Mx resistant strain 16,584 Recent isolate 128 Reassortment strain contA; n; ng HA 2,048 - 4,096 and NA of recent isolate and remaining genes derived from Mx resistant strain It is apparent that many modifications and variations of this invention as set forth here may be made without departing from the spirit and scope thereof. The specific embodiments described hereinabove are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A chimeric influenza virus strain exhibiting a recombinant influenza surface protein comprising an extracellular domain derived from an influenza virus strain which grows to low titer in an embryonated egg host or an embryo-derived tissue culture cell host, and a cytoplasmic domain derived from a high titer Mx resistant influenza virus strain so that the chimeric influenza virus strain grows to a higher titer in the embryonated egg host or the embryo-derived tissue culture cell host than the titer to which the low titer influenza virus strain grows in the embryonated egg host or the embryo-derived tissue culture cell host.

2. The chimeric influenza virus strain of Claim 1 wherein the recombinant influenza surface protein further comprises a transmembrane domain derived from the low titer influenza virus strain.

3. The chimeric influenza virus strain of Claim 1 wherein the recombinant influenza surface protein further comprises a transmembrane domain derived from the high titer Mx resistant influenza virus strain.

egg.

The chimeric influenza virus strain of Claim 1 wherein the embryo-derived tissue culture cell host is a chick embryo-derived tissue culture cell host.

6. The chimeric influenza virus strain of Claim 1 wherein the recombinant influenza surface protein is a recombinant hemagglutinin surface protein or a recombinant neuraminidase surface protein.

7. The chimeric influenza virus strain of Claim 1 wherein a cleavability domain of the recombinant influenza viral surface protein is modified by an amino acid residue insertion, deletion, or substitution.

8. The chimeric influenza virus strain of Claim 1 wherein a receptor-binding domain of the recombinant influenza viral surface protein is modified by an amino acid residue insertion, deletion, or substitution.

9. A method for the production of the chimeric influenza virus strain of Claim 1 comprising:

(a) constructing an RNA segment containing a gene encoding a recombinant influenza virus surface protein comprising an extracellular domain derived from an influenza virus strain which grows to low titer in an embryonated egg host or an embryo-derived tissue culture cell host, and a cytoplasmic domain derived from an Mx resistant influenza virus strain, so that the gene is capable of being expressed in a host cell;
(b) constructing a ribonucleoprotein containing the RNA
segment;
(c) transfecting a host cell with the ribonucleoprotein of step (b); and (d) selecting the chimeric influenza virus strain of Claim 1.

10. The method of Claim 9 wherein the recombinant influenza virus surface protein is a recombinant hemagglutinin surface protein or a recombinant neuraminidase surface protein.

11. The method of Claim 9 wherein the host cell is an Mx mouse cell, an embryonated egg cell, or an embryo-derived tissue culture cell.

12. The method of Claim 11 wherein the embryonated egg cell is an embryonated hen's egg cell.

13. The method of Claim 11 wherein the embryo-derived tissue culture cell is a chick embryo-derived tissue culture cell.

14. An anti-influenza virus vaccine comprising the influenza virus strain of Claim 1 and a pharmaceutically acceptable carrier.