Gross-Ref erersce to Related Applications

This application claims the benefit of the filing date of U.S. application No 62/892,241, filed on August 27, 2019, the disclosure of which is incorporated by reference herein.

Statement of Government Funding

This invention was made with government support under HHSN272201400008C awarded by the National Institutes of Health. The government has certain rights in the invention.

Background

Influenza is a major respiratory disease in some mammals including horses and is responsible for substantial morbidity and economic losses each year. In addition, influenza virus infections can cause severe systemic disease in some avian species, leading to death. The segmented nature of the influenza virus genome allows for reassortment of segments during virus replication in ce!is infected with two or more influenza viruses. The reassortment of segments, combined with genetic mutation and drift, can give rise to a myriad of divergent strains of influenza virus over time. The new strains exhibit antigenic variation in their hemagglutinin (HA) and/or neuraminidase (NA) proteins, and In particular the gene coding for the HA protein has a high rate of variability. The predominant current practice for the prevention of flu is vaccination. Most commonly, inactivated virus vaccines are used. As the influenza HA protein is the major target antigen for the protective immune responses of a host to the virus and is highly variable, the Isolation of influenza virus and the identification and characterization of the HA antigen in viruses associated with recent outbreaks is important for vaccine production. Based on prevalence and prediction, a vaccine is designed to stimulate a protective immune response against the predominant and expected influenza virus strains.

There are four general types of influenza viruses, Type A, Type B, Type G, and Type D, which are defined by the absence of serological cross reactivity between their interna! proteins. Influenza Type A viruses are further classified into subtypes based on antigenic and genetic differences of their glycoproteins, the HA and NA proteins. All the known HA and NA subtypes (H1 to H18 and N1 to N11 ) have been isolated from aquatic birds, which are thought to act as a natural reservoir for influenza.

Most influenza vaccines are produced in embryonated chicken eggs. However, the WHO- recommended Influenza vaccine strains often do not replicate efficiently in embryonated chicken eggs, requiring serial passages in eggs in order to ai!ow for adaptation of the virus. During adaptation and amplification in eggs, the hemagglutinin (HA) protein of influenza viruses often acquires egg-adapting mutations. These egg-adapting mutations in HA often alter the antigenicity of the viruses, resulting in vaccine viruses that are no longer optimally matched to the circulating virus strains.

Summary

As described herein, an influenza virus was passaged 7 times in eggs (in triplicate) to study the mutations that occurred in the 6 non-immunogenic viral segments during adaptation. Surprisingly, the virus acquired no HA mutations and instead had mutations in the NA, PB2, NP, and Ml proteins. The NA mutations were identical in a!i three experiments, and they included a deletion and 4 amino acid mutations. The NA mutations were tested alone and it was found that they, e.g., alone or in various combinations, were responsible for the effect, which permitted efficient growth in eggs without HA mutations.

The present disclosure thus relates to influenza mutations that prevent the acquisition of antigenicity-compromising mutations in the hemagglutinin (HA) segment of influenza virus during growth in eggs. The mutations in the neuraminidase (NA) protein of human influenza viruses were found to ‘stabilize’ the HA during egg-passages, e.g., in the presence of the mutations in NA, the HA protein did not acquire egg-adapting mutations. Those NA mutations may also increase the vaccine virus yield.

The disclosure provides isolated recombinant, e.g., reassortant, influenza viruses with selected amino acid residues or deletions at specified positions in NA

In one embodiment, the NA is selected to not encode a threonine at residue 32. in one embodiment, the NA is selected to not encode an aspartic acid (D) at position 147. In one embodiment, the NA is selected to not encode an asparagine (N) at residue 329. In one embodiment, the NA is selected to not encode a threonine (T) at residue 148 or residue 329 In one embodiment, the NA is selected to not encode a lysine (K) at residue 148 or residue 344. In one embodiment, the NA is selected to not encode a glycine (G) at residue 346. In one embodiment, the NA is selected to not encode a histidine (H) at residue 347. In one embodiment, the NA is selected to not encode an arginine (R) or an asparagine at residue 347. In one embodiment, the NA is selected to not encode a threonine at residue 369 In one embodiment, the NA is selected to not encode a NA having a threonine or lysine at position 148. In one embodiment, the NA is selected to not encode a NA having an aspartic acid at position 151. in one embodiment, the NA Is selected to not encode a NA having an asparagine at position 245. In one embodiment, the NA is selected to not encode a NA having a glycine at position 346. In one embodiment, the NA is selected to have a deletion of one or more of residues 46 to 50. The numbering for NA is based on N2. In one embodiment, the disclosure provides an isolated recombinant reassortant influenza virus having six “internal” viral segments from a vaccine influenza virus, e.g., PR8UW, a NA viral segment with one or more of the specified residues at particular positions or a deletion of specified residues, or any combination thereof, and a HA viral segment, e.g., any of H1-H18, e.g., from a circulating Influenza virus. Also provided are compositions comprising the recombinant influenza virus, pharmaceutical compositions such as vaccines.

Thus, for vaccine viruses that are to be grown or passaged in cells, e.g., in eggs, replacement of the residue at position 32, 147, 329, 347, or a deletion of one or more of residues 46 to 50, or any combination thereof, in NA, e.g., by mutation, or selection of a NA viral segment for a NA to not encode a threonine at residue 32, to not encode an aspartic acid at position 147, to not encode an asparagine at residue 329, to not encode a histidine at residue 347, to not encode a threonine at residue 369, or to have a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering is based on N2, may result In stabilization of HA and/or higher viral titers. In one embodiment, for vaccine viruses that are to be grown or passaged in ceiis, e.g., in eggs, replacement of the residue at position 148, 151 , 245, 346, or any combination thereof, in NA, e.g., by mutation, or selection of a NA viral segment for a NA to not encode a threonine or lysine at residue 148, to not encode an aspartic acid at position 151, to not encode an asparagine at residue 245, to not encode a glycine at residue 346, or any combination thereof, wherein the numbering is based on N2, may result in stabilization of HA and/or higher viral titers. In one embodiment, the disclosure provides an isolated recombinant Influenza virus comprising PA, PB1 , PB2, NP, NS, M, and HA viral segments and a NA viral segment that encodes an NA selected to not encode a threonine at residue 32, to not encode an aspartic acid at position 147, to not encode an asparagine at residue 329, to not encode a histidine at residue 347, to not encode a threonine at residue 369, or to have a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication In avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine at residue 32, does not have a deletion of residues 46 or 50, encodes an aspartic acid at position 147, encodes an asparagine at residue 329, encodes a histidine at residue 347, or any combination thereof. In one embodiment, the disclosure provides an isolated recombinant influenza virus comprising PA, PB1 , PB2, NP, NS, M, and HA viral segments and a NA viral segment that encodes an NA selected to not encode a threonine or lysine at residue 148, to not encode an aspartic acid at position 151 , to not encode an asparagine at residue 245, to not encode a glycine at residue 346, to not encode a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine, or lysine at residue 148, encodes an aspartic acid at position 151 , encodes an asparagine at residue 245, encodes a giycine at residue 346, any combination thereof. In one embodiment, the isolated recombinant influenza virus is a reassortant. In one embodiment, the NA viral segment encodes a NA that has at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to any one of SEQ ID Nos. 1-3, 30-38, 48-50, or 54. In one embodiment, the NA viral segment encodes a NA that has less than 100% amino acid sequence identity to SEQ ID NQ:2 or SEQ ID NO:3. In one embodiment, the NA viral segment encodes a N2, N3, N7, or N9 and the positions in N3, N7, or N9 with the specified residue(s) correspond to the specified positions in N2. In one embodiment, the NA viral segment encodes a N1, N4, N5, N6, N8, N10 or N11 and the positions in N1 , N4, N5, N6, N8, N10 or N11 with the specified residue(s) correspond to the specified positions in N2. In one embodiment, the residue at position 32 is A, I, G, or L In one embodiment, the deletion is a deletion of residues 46 to 50. In one embodiment, the residue at position 147 is N or Q. In one embodiment, the residue at position 148 is I or K. In one embodiment, the residue at position 151 is E, Q, H or K. In one embodiment, the residue at position 245 is S, T, A, I, G, or L. In one embodiment, the residue at position 329 is S, V, I, L, A, G, D or E. in one embodiment, the residue at position 344 is E, Q, N, H or D. in one embodiment, the residue at position 346 is V, I, A, S, T, L, or L. In one embodiment, the residue at position 347 is G, Q, N, S, T, Y, C or W. In one embodiment, the residue at posifion 369 is K, H, R, E, P, or D. In one embodiment, the HA is H1 , H3, H7, or H9. In one embodiment, the virus is an influenza A virus. In one embodiment, the PA,

PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or encode a polypeptide having at least 80%, 85%, 90%, 95%, or 99 amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39-44. In one embodiment, the PB2 has I,

A, L, or G at residue 147. in one embodiment, the virus is an influenza B virus. In one embodiment, the selected NA viral segment does not have an aspartic acid at position 147, does not have an asparagine at residue 329, and does not have an arginine or a histidine at residue 347. In one embodiment, the selected NA viral segment does not a threonine or lysine at position 148, does not have an aspartic acid at position 151 , and does not have an asparagine at position 245. In one embodiment, the selected NA viral segment has at least two of: N or Q at position 147, D or E at residue 329, or Q or G at residue 347. in one embodiment, the selected NA viral segment has at least two of: I, L, G or A at position 148, E or Q at position 151 , or S, I, T, V or G at position 245. In one embodiment, the selected NA viral segment has at least two of: I or L at position 148, E or Q at position 151 , or S, I, T, V or G at position 245. In one embodiment, the selected NA viral segment has N or Q at position 147, S, D or E at residue 329, and Q or G at residue 347. in one embodiment, the selected NA viral segment has N or Q at position 147, S, D or E at residue 329, and V, S, I or L at residue 346. in one embodiment, the residue at position 369 is K, H, R, E, P, or D. in one embodiment, the selected NA viral segment has I, L, G or A at position 148, E or Q at position 151 , S, I, T, V or G at position 245 and K, H, R, E, P, or D at position 369. In one embodiment, the selected NA viral segment has I or L at position 148, E or G at position 151, S, I, T, V or G at position 245 and K, H, R, E, P, or D at position 369. In one embodiment, the residue at position 369 is K, H, R, E, or D.

Further provided is an isolated recombinant nucleic acid, e.g., a vector such as a viral vector, comprising a nucleic acid sequence that encodes an influenza virus NA selected to not encode a threonine at residue 32, to have a deletion of one or more of residues 46-50, to not encode an aspartic acid at position 147, to not encode an asparagine at residue 329, or to not encode a histidine at residue 347, or any combination thereof, wherein the numbering is based on N2. In one embodiment, the isolated recombinant nucleic acid does not encode a threonine or lysine at residue 148, to not encode an aspartic acid at position 151, to not encode an asparagine at residue 245, to not encode a glycine at residue 346, to not encode a threonine at residue 369, or any combination thereof. In one embodiment, the NA has at least 95% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In one embodiment, the NA has less than 100% amino acid sequence identity to SEQ ID NQ:2 or SEQ ID NO:3. In one embodiment, the NA is a N2, N3, N7, or N9. In one embodiment, the NA is a N1 ,

N4, N5, N6, N8, N10 or N11. In one embodiment, the residue at position 32 is A, I, G, or L. In one embodiment, the deletion is a deletion of residues 46 to 50. In one embodiment, the residue at position 147 is N or Q. In one embodiment, the residue at position 329 is D or E. In one embodiment, the residue at position 347 is G, N, S, T, Y, C or W. In one embodiment, the residue at position 148 is I, L, G or A. In one embodiment, the residue at position 148 Is I or L. In one embodiment, the residue at position 151 is E, N or Q. In one embodiment, the residue at position 245 is S, T, I, L, A, N, or V. In one embodiment, the residue at position 369 is K, H, R, E, P, or D.

Also provided is a method to prepare influenza virus. The method includes contacting a cell with: a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operab!y linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA producfion comprising a promoter operabiy linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operabiy linked to an Influenza virus NS DNA linked to a transcription termination sequence, wherein the FBI, PB2, PA, NP, MS, and M DNAs in the vectors for vRNA production are from one or more influenza vaccine virus isolates, wherein the NA DMA in the vector for vRNA production encodes an NA selected to not encode a threonine at residue 32, to not encode an aspartic acid at position 147, to not encode an asparagine at residue 329, to not encode a histidine at residue 347, to not encode a threonine or lysine at residue 148, to not encode an aspartic acid at position 151 , to not encode an asparagine at residue 245, to not encode a glycine at residue 346, to not encode a threonine at residue 369, or to have a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering for NA residues is that for N2; and a vector for mRNA production comprising a promoter operab!y linked to a DNA segment encoding influenza virus PA, a vector for RNA production comprising a promoter operably linked to a DMA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DMA segment encoding Influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DMA segment encoding Influenza virus NP, and optionally comprising one or more of: a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding Influenza virus M1 , a vector for RNA production comprising a promoter operably linked to a DMA segment encoding influenza virus M2, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS1, or a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus. In one embodiment, the NA has at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to, for example, SEQ ID NO:1 SEQ ID NG:2, SEQ ID NO:3, SEQ ID NO:48 or SEQ ID NO:49. In one embodiment, the NA has at least 80%, 85%, 90%, 95%, or 99% amino acid sequence identity to, for example, SEQ ID Nos. 51-59 or 69-70. In one embodiment, the NA has less than 100% amino acid sequence Identity to SEQ ID NG:2 or SEQ ID NO:3. In one embodiment, the NA Is N2, N3,

N7, or N9. In one embodiment, the NA is N1, N4, N5, N6, N8, N10 or N11. In one embodiment, the residue at position 32 is A, I, G, or L In one embodiment, the deletion is a deletion of residues 46 to 50. In one embodiment, the residue at position 147 Is N or Q. In one embodiment, the residue at position 329 is

S, D or E. In one embodiment, the residue at position 347 is Q, N, S, T, Y, G or W. In one embodiment, the residue at position 148 is I, L, G or A. In one embodiment, the residue at position 148 is I or L In one embodiment, the residue at position 151 is E, N or Q. In one embodiment, the residue at position 245 is S,

T, I, L, A, N, or V. In one embodiment, the residue at position 329 is S, I, L, A, N, or V. In one embodiment, the residue at position 344 is E, Q, N, H or D. in one embodiment, the residue at position 346 is V, S, T, I, L, A, or V. In one embodiment, the residue at position 347 is G, S, T, L L, A, or V. in one embodiment, the residue at position 369 is K, H, R, E, P, or D. in one embodiment, the HA is H1, H3, H5, H7, or H9. in one embodiment, the virus is an influenza A virus. In one embodiment, PA, PB1 , PB2, NP, M, and NS viral segments have af least 85%, 85%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% , 85%, 90%, 95%, or 99% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44. in one embodiment, PB2 has I, A, L, or G at residue 147.

In one embodiment, HA is H2, H4, H5, H6, H8, or any of H10-H18. in one embodiment, the virus is an influenza B virus. Further provided is a method of immunizing an avian or a mammal with a composition having an effective amount of the virus described herein. In one embodiment, the composition comprises at least one other different influenza virus in one embodiment, the mammal is a human. In one embodiment, the composition is administered intranasally or via injection.

Thus, the invention provides a method to select for influenza viruses with enhanced replication in cell culture, e.g., enhanced replication in embryonated eggs. The method includes providing cells suitable for influenza vaccine production; serially culturing one or more influenza virus isolates in eggs; and isolating serially cultured virus with enhanced growth relative to the one or more isolates prior to serial culture. Also provided is a method to identify a NA that stabilizes HA and/or that confers altered growth of a recombinant influenza virus, e.g., in eggs. The method includes introducing one or more substitutions or deletions as described herein into a NA viral segment to yield a mutant NA viral segment; and optionally identifying whether the mutant NA viral segment, when present in a replication competent recombinant influenza virus, results in enhanced replication of the recombinant influenza virus in eggs and optionally inhibits HA mutations, relative fo a corresponding replication competent Influenza virus without the one or more substitutions and/or deletions in NA.

In one embodiment, the disclosure provides isolated influenza type A virus with a characteristic residue(s) and/or deletion, or a combination thereof, in NA described herein. In one embodiment, the isolated influenza type A virus with a characteristic residue(s) and/or deletion, or a combination thereof, has an NA amino acid sequence with at least 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to a polypeptide encoded by one of SEQ ID NOs:1, 2, 3, or 30-38. In one embodiment, the isolated influenza type A virus of the invention with a characteristic residue(s) and/or deletion, or a combination thereof, has an HA from any one of subtypes 1-18 of HA. in one embodiment the characteristic residue is a conservative substitution, e.g., relative to SEQ ID NQ:2 or SEQ ID NQ:3. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, ieucine, and iso!eucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chain is cysteine and methionine.

In one embodiment, conservative amino acid substitution groups are: threonine-vaiine-ieucine-iso!eucine- a!anine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine- glutamine.

In one embodiment, a mutation Is introduced Into a NA viral segment of an influenza virus isolate, e.g., via recombinant DNA techniques including site-specific mutagenesis, or replacing a portion of the NA coding sequence with a portion that includes the characteristic residue(s) or deletion. In one embodiment, a NA viral segment with a characteristic residue and/or deletion described herein is combined with a HA segment, and internal viral segments of an influenza vaccine virus.

The disclosure provides a plurality of Influenza virus vectors of the invention, e.g., those useful fo prepare reassortant viruses Including 6:1 :1 reassortants, 6:2 reassortants and 7:1 reassortants. A 6:1 :1 reassortant is an influenza virus with 6 interna! viral segments from a vaccine virus, a HA viral segment that is from a different (second) viral isolate than the vaccine virus, and a NA viral segment with a characteristic residue(s) and/or deletion, or a combination thereof, as described herein, which is from a different viral source than the HA segment and the vaccine virus; a 6:2 reassortant is an influenza virus with 6 internal viral segments from a vaccine virus, and a NA viral segment having a characteristic residue(s) and/or deletion, or a combination thereof, which segment is from the same source as the HA segment, and a HA viral segment from a different viral isolate than the vaccine virus; and a 7:1 reassortant, in one embodiment, is an influenza virus with 6 internal viral segments and a HA segment from a vaccine virus, and a NA segment that is modified to include the characteristic residue(s) and/or deletion, or a combination thereof, which NA segment is from a different viral source than the vaccine virus.

In one embodiment of the invention, the plurality includes vectors for vRNA production selected from a vector comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a operably linked to an influenza virus NS DNA linked to a transcription termination sequence. In one embodiment, the DNAs for vRNA production of PB1 , PB2, PA, NP, M, and NS, have sequences from an influenza virus that replicates to high fiters in cultured mammalian cells such as MDCK cells, Vero cells or PER.G6® cells or embryonated eggs, and/or from a vaccine virus, e.g., one that does not cause significant disease In humans. The DNA for vRNA production of NA may be from any NA, e.g., any of N1-N11 , and the DNA for vRNA production of HA may be from any HA, e.g., H1-H18. In one embodiment, the DNAs for vRNA production may be for an influenza B or G virus. For example, the DNAs for vRNA production include influenza B virus PA, PB1, PB2, NP, NS, and M or influenza B virus PA, PB1, PB2, NP, NS, M, and NA, wherein the vRNA for NA has a NA with a characteristic residue and/or deletion as described herein. The DNAs for vRNA production of NA and HA may be from different strains or isolates (6:1 :1 reassortants) or from the same strain or isolate (6:2 reassortants), or the NA or HA may be from the same strain or isolate as that for the interna! genes (7:1 reassortant). The plurality also includes vectors for mRNA production selected from a vector encoding influenza virus PA, a vector encoding Influenza virus PB1 , a vector encoding Influenza virus PB2, and a vector encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M, e.g., M1 and M2, HA or NA. The vectors encoding viral proteins may further include a transcription termination sequence.

Viruses that may provide the interna! genes for reassortants within the scope of the invention include viruses that have high fiters, e.g., titers of at least about 10 ⁵ PFU/mL, e.g., at least 10 ⁶ PFU/mL, 1Q ⁷ PFU/mL or 10 ⁸ PFU/mL; high titers in embryonated eggs, e.g., titers of at least about 1Q ⁷ EIDso/mL, e.g., at least 10 ³ EIDso/mL, 1Q ⁹ EIDso/mL or 10 ¹⁰ EIDso/mL; high titers in MDCK cells, e.g., titers of at least about 10 ⁷ PFU/mL, e.g., at least 10 ^s PFU/mL, or high titers in two of more of those host ce!!s.

Other reassortants with Interna! genes from other PR8 isolates or vaccine viruses may be employed in recombinant reassortant viruses. In one embodiment, the DNAs for the interna! genes for PB1, PB2, PA, NP, M, and MS encode proteins with substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NGs:24-29 or 39 to 44. As used herein, "substantially the same activity” includes an activity that is about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or more, or detectable protein level that is about 80%, 90% or more, the activity or protein level, respectively, of the corresponding full-length polypeptide in one embodiment, the nucleic acid a sequence encoding a polypeptide which is substantially the same as, e.g., having at least 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to, a polypeptide encoded by one of SEQ ID NQs:24-29 or 39 to 44. In one embodiment, the isolated and/or purified nucleic acid molecule comprises a nucleotide sequence which is substantially the same as, e.g., having at least 50%, e.g., 80%, 70%, 80% or 90%, including any integer between 50 and 100, or more contiguous nucleic acid sequence identity to one of SEQ ID NQs:24-29 and, in one embodiment, also encodes a polypeptide having at least 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to a polypeptide encoded by one of SEQ ID NOs:24-29 or 39 to 44. In one embodiment, the influenza virus polypeptide has one or more, for instance, 2, 5, 10, 15, 20 or more, conservative amino acids substitutions, e.g., conservative substitutions of up to 10% or 20% of the residues, relative to a polypeptide encoded by one of SEQ ID IMQs:24-29 or 39 to 44. Conservative amino acid substitutions refer fo the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and Isoleucine; a group of amino acids having aiiphatic-hydroxyi side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chain is cysteine and methionine in one embodiment, conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; !ysine-arginlne; alanine-valine; glutamic-aspartic; and asparagine-glutamine. In one embodiment, the influenza virus polypeptide has one or more, for instance, 2, 3 or 4, nonconservative amino acid substitutions, relative to a polypeptide encoded by one of SEQ ID NOs:24-29.

In one embodiment, the nucleic acid a sequence encoding a NA polypeptide which is substantially the same as, e.g., having at least 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to, one of SEQ ID Nos. 1-3 or 48-49, or a polypeptide encoded by one of SEQ ID NQs:51-59, or one of Accession Nos. ACP41107.1 (N1) (SEQ ID NO:36) AIK26357.1 (N7) (SEQ ID NO:37) ,ALH21372.1 (N9) (SEQ ID NO:45), or BAK86313.1 (N2) (SEQ ID NO:50), the sequences of which are incorporated by reference herein. In one embodiment, the isolated and/or purified nucieic acid molecule encodes a polypeptide having at least 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to an one of SEQ ID NQs:1 , 3, 30-35, or 48-49, one of Accession Nos. ACP41107.1 (N1) AIK26357.1 (N7), ALH21372.1 (N9), or BAK86313.1 (N2), or to a NA encoded by one of SEQ ID Nos. 51 -59, the sequences of which are incorporated by reference herein. In one embodiment, the influenza virus polypeptide has one or more, for instance, 2, 5, 10, 15, 20 or more, conservative amino acids substitutions, e.g., conservative substitutions of up to 10% or 20% of the residues, relative to SEQ ID NOs:1, 3, 30-35, 48-49, or one of Accession Nos. AGP41107.1 (N1) AIK26357.1 (N7), ALH21372.1 (N9), or BAK86313.1 (N2), or a NA encoded by one of SEQ ID Nos. 51-59, the sequences of which are incorporated by reference herein. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is giycine, aianine, valine, leucine, and isoieucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of a ino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chain is cysteine and methionine. In one embodiment, conservative amino acid substitution groups are: valine-leucine- isoieucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine-glutamine. In one embodiment, the influenza virus polypeptide has one or more, for instance, 2, 3 or 4, nonconservative amino acid substitutions, relative to a polypeptide having one of SEQ ID NOs:1 , 3, 30-35, 48-49, or one of Accession Nos. ACP41107.1 (N1) AIK26357.1 (N7),ALH21372.1 (N9), or BAK86313.1 (N2), or a NA encoded by one of SEQ ID Nos. 51-59, the sequences of which are incorporated by reference herein.

The invention thus includes the use of isolated and purified vectors or plasmids, which express or encode influenza virus proteins, or express or encode influenza vRNA, both native and recombinant vRNA. The vectors comprise influenza cDNA, e.g., influenza A (e.g., any influenza A gene including any of the 18 HA or 11 NA subtypes), B or C DNA {see Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2013), which is specifically Incorporated by reference herein). Any suitable promoter or transcription termination sequence may be employed to express a protein or peptide, e.g., a viral protein or peptide, a protein or peptide of a nonviral pathogen, or a therapeutic protein or peptide.

A composition or plurality of vectors of the invention may also comprise a heterologous gene or open reading frame of interest, e.g., a foreign gene encoding an immunogenic peptide or protein useful as a vaccine or in gene replacement, for instance may encode an epitope useful in a cancer therapy or vaccine, or a peptide or polypeptide useful In gene therapy. When preparing virus, the vector or plasmid comprising the gene or cDNA of interest may substitute for a vector or plasmid for an influenza viral gene or may be in addition to vectors or plasmids for all influenza viral genes. Thus, another embodiment of the invention comprises a composition or plurality of vectors as described above in which one of the vectors is replaced with, or further comprises, 5' influenza virus sequences optionally including 5' influenza virus coding sequences or a portion thereof, linked to a desired nucleic acid sequence, e.g., a desired cDNA, linked to 3' influenza virus sequences optionally including 3' influenza virus coding sequences or a portion thereof. In one embodiment, the desired nucleic acid sequence such as a cDNA is in an antisense (antigenomic) orientation. The introduction of such a vector in conjunction with the other vectors described above to a host ce!! permissive for influenza virus replication results in recombinant virus comprising vRNA corresponding to the heterologous sequences of the vector.

The promoter in a vector for vRNA production may be a RNA polymerase I promoter, a RNA polymerase li promoter, a RNA polymerase II! promoter, a T7 promoter, or a T3 promoter, and optionally the vector comprises a transcription termination sequence such as a RNA polymerase I transcription termination sequence, a RNA polymerase P transcription termination sequence, a RNA polymerase IN transcription termination sequence, or a ribozyme. Ribozymes within the scope of the invention include, but are not limited to, tetrahymena ribozymes, RNase P, hammerhead ribozymes, hairpin ribozymes, hepatitis ribozyme, as well as synthetic ribozymes. In one embodiment, the RNA polymerase i promoter is a human RNA polymerase I promoter. The promoter or transcription termination sequence in a vRNA or virus protein expression vector may be the same or different relative to the promoter or any other vector in one embodiment, the vector or piasmid which expresses influenza vRNA comprises a promoter suitable for expression in at least one particular host cell, e.g., avian or mammalian host cells such as canine, feline, equine, bovine, ovine, or primate cells including human cells, or for expression in more than one host.

In one embodiment, at least one vector for vRNA comprises a RNA polymerase P promoter linked to a ribozyme sequence linked to viral coding sequences linked to another ribozyme sequences, optionally linked to a R A polymerase II transcription termination sequence. In one embodiment, at least 2, e.g., 3, 4, 5, 6, 7 or 8, vectors for vRNA production comprise a RNA polymerase II promoter, a first ribozyme sequence, which Is 5' to a sequence corresponding to viral sequences including viral coding sequences, which is 5' to a second ribozyme sequence, which is 5' to a transcription termination sequence. Each RNA polymerase li promoter in each vRNA vector may be the same or different as the RNA polymerase li promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be the same or different as the ribozyme sequences in any other vRNA vector. In one embodiment, the ribozyme sequences In a single vector are not the same.

In one embodiment, at least one vector comprises sequences corresponding to those encoding FBI, PB2, PA, NP, M, or NS, or a portion thereof, having substantially the same activity as a corresponding polypeptide encoded by one of SEQ ID NOs:24-29 or 39 to 44, e.g., a sequence encoding a polypeptide with at least 80%, e.g., 85%, 90%, 92%, 95%, 98%, 99% or 100%, including any integer between 80 and 100, amino acid identity to a polypeptide encoded by one of SEQ ID NGs:24-29 Optionally, two vectors may be employed in place of the vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, e.g., a vector comprising a promoter operably linked to an Influenza virus M1 cDNA linked to a transcription termination sequence and a vector comprising a promoter operably linked to an influenza virus M2 cDNA linked to a transcription termination sequence.

A plurality of the vectors of the invention may be physically linked or each vector may be present on an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. In one embodiment, each vRNA production vector is on a separate plasmid. In one embodiment, each mRNA production vector is on a separate piasmid.

The invention also provides a method to prepare Influenza virus. The method comprises contacting a ceil with a plurality of the vectors of the invention, e.g., sequentially or simultaneously, in an amount effective to yield infectious influenza virus. The invention also includes isolating virus from a cell contacted with the plurality of vectors. Thus, the invention further provides isolated virus, as well as a host cell contacted with the plurality of vectors or virus of the invention in another embodiment, the invention includes contacting the cell with one or more vectors, either vRNA or protein production vectors, prior to other vectors, either vRNA or protein production vectors. In one embodiment, the promoter for vRNA vectors employed in the method is a RNA polymerase I promoter, a RNA polymerase II promoter, a RNA polymerase III promoter, a 13 promoter or a T7 promoter. In one embodiment, the RNA polymerase 1 promoter is a human RNA polymerase l promoter in one embodiment, each vRNA vector employed in the method is on a separate piasmid. In one embodiment, the vRNA vectors employed in the method are on one piasmid or on two or three different plasmids. In one embodiment, each mRNA vector employed in She method is on a separate plasmid. In one embodiment, the mRNA vectors for PA, FBI, PB2 and NP employed In the method are on one plasmid or on two or three different plasmids.

The methods of producing virus described herein, which do not require helper virus infection, are useful in viral mutagenesis studies, and in the production of vaccines (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot and mouth disease) and gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamyiase deficiency and central nervous system tumors). Thus, a virus for use in medical therapy (e.g., for a vaccine or gene therapy) is provided.

The invention also provides isolated viral polypeptides, and methods of preparing and using recombinant virus of the invention. The methods include administering to a host organism, e.g., a mamma!, an effective amount of the influenza virus of the invention, e.g., an inactivated virus preparation, optionally in combination with an adjuvant and/or a carrier, e.g., in an amount effective to prevent or ameliorate infection of an animal such as a mamma! by that virus or an antigenica!!y closely related virus in one embodiment, the virus is administered intramuscularly while in another embodiment, the virus is administered intranasaiiy. in some dosing protocols, all doses may be administered intramuscularly or intranasaiiy, while in others a combination of intramuscular and intranasal administration is employed.

The vaccine may further contain other isolates of influenza virus including recombinant influenza virus, other pathogen(s), additional biological agents or microbial components, e.g., to form a multivalent vaccine. In one embodiment, intranasai vaccination, for instance containing with inactivated influenza virus, and a mucosal adjuvant may induce virus-specific IgA and neutralizing antibody in the nasopharynx as we!i as serum IgG.

The influenza virus of the invention may employed with other anti-vira!s, e.g., amantadine, rimantadine, and/or neuraminidase inhibitors, e.g., may be administered separately in conjunction with those anti-vira!s, for instance, administered before, during and/or after.

Thus, the modified neuraminidase comprises at least one, or at least two, or at least three modifications, wherein the modification comprise one or more amino acids within positions 29-35, one or more amino acids within positions 44-52, one or more amino acids within positions 144-154, one or more amino acid positions within 240-250, one or more amino acids within positions 326-333, one or more amino acid positions within 344-350, one or more amino acid positions within 365-375, or combinations thereof, wherein the numbering is that for N2. in one embodiment, the NA comprises a deletion of at least one proi!ne, asparagine, giutarnine, valine, or a combination of a proi!ne, one or more asparagine(s), a glutamine, and a valine within positions 44-52; a substitution (replacement) of a threonine within positions 29-35; a substitution (replacement) of an threonine or an aspartic acid within positions 145-155; a substitution (replacement) of an asparagine within positions 240 to 250 or 326-333; a substitution (replacement) of a histidine within positions 345-350; or a combination thereof.

Brief Description of Figures

Figure 1. Nucleotide sequences for the virai segments of A ^/ Yokohama/2017/2003 (SEQ ID Nos. 4-11 ), and amino add sequence of the NA of A/Yokohama/2017/2003 (SEQ ID NO:3).

Figure 2. Amino acid sequence for the NA of A/Saitama/103/2014 (SEQ ID NO:2)

Figure 3. Nudeotide sequence of NA virai segment (SEQ ID NO: 12) and amino acid sequences for NA of mutant of A/Yokohama/2017/2003 (SEQ ID NO:1), and nudeotide sequence of other viral segments of the mutant (SEQ ID Nos.12-21) Figure 4 Graph showing titers in eggs of various reassortants with the PB2, M, NA and NP segments of mutant and wiid-type A/Yokohama/2017/2003. Virus inoculation: 2 x 10 ³ pfu/egg into allantoic fluid, 72 h incubation at 37°C.

Figure 5. Locations of the NA mutations on the 3D structure of N2 NA.

Figure 6 Graph showing titers in eggs for recombinant viruses with specific mutations found in the mutant of A/Yokohama/2017/2003 (“Y2017-M3L4”). Virus inoculation: 2 x 1Q ³ pfu/egg into allantoic fluid, 72 h incubation at 37°C.

Figure 7. Graph of virus titer in eggs for reassortants with two different backbones (PA, PB1, PB2, NP, NS and M) and two different HA and NA combinations (e.g , PB2-I504V, PB1- M40L/G180W, PA-R401K, NP-I116L, NS1-A30P/R118K; and NA of Y2017-M3L4 contains mutations: NA- T32A, D147N, N329D, H347Q and deletion of 46-5Qaa). Virus inoculation: 2 x 10 ³ pfu/egg into allantoic fluid, 72 h incubation at 37°C.

Figure 8. Amino acid sequence comparison of Yokohama ^/ 2017/2003 NA wiid-type (SEQ ID NO:3) and Y2017-M3L4 (SEQ ID NO:1).

Figure 9. Exemplary NA sequences for N3, N4, N6, N7, N8, and N9 (SEQ ID Nos. 30-35).

Figure 10. Exemplary sequences for the internal viral segments for a master vaccine strain (SEQ ID Nos. 39-44).

Figure 11. Exemplary NA sequences (SEQ ID Nos. 51-54).

Figure 12. Titers in eggs for various NA mutants.

Figure 13. Titers of HK4801 HA, Y2017-M3L4NA and HY-PR8 (PB2 C4U, I504V; PB1 C4U, M40L/G180W; PA C4U, R401 K; NP i 116L; NSA30P/R118K) and analyses for HA mutations in infected eggs over time.

Figure 14 shows data for viruses passaged in eggs that had certain NA mutants but did not resuit in substitutions In HA.

Figure 15 is a schematic of the positions of certain NA residues.

Figure 16 is a schematic of the positions of certain NA residues.

Figure 17 shows virus titers for egg passaged isolates (HK4801 NA (T148K, D151 E, H347G, and T369K)) conferred efficient replication in the allantoic cavity to viruses possessing either HK4801 HA or SingaporeOQ19 HA (HY-PR8 backbone).

Figure 18 shows egg titers for different combinations of selected residues at positions 153, 329, 347, and 369 in NA.

Figure 19 summarizes virus titers (HK4801 HA, Y2017-M3L4NA and HY-PR8 (PB2 C4U, I504V; PB1 C4U , M40L/G180W; PA C4U, R401 K; NP I116L; NS A30P/R118K) and HA status over time.

Figure 20 summarizes virus titers and HA status for viruses with different NAs.

Figure 21 provides inoculation and harvested virus titers In allantoic passages (HA- K189E/N158K A212T mutant virus).

Figure 22 shows detection of HA status after multiple passages.

Figure 23 shows egg titers for viruses with different NAs.

Figure 24 is an enlarged view of the NA activity center. Most egg-adapted mutations are located in/around the NA active site.

Figure 25. Locations of amino acid substitutions in the neuraminidase proteins of egg-adapted influenza A/Hong Kong/4801/2014(H3N2) and A/Alaska/232/2015(H3N2) (SEQ ID Nos. 51-52). Figure 26. Introduction of NA mutations (see Figure 25) into the NA of H3N2 viruses from fhe 2017/18 season (SEQ ID NQ:53) enhanced HY-PR8 backbone virus growth without HA mutations.

Figure 27. Mutations observed in NA mutant viruses (HY-PR8 backbone) in Figure 26 during egg passages.

Figure 28. Introduction of NA mutations into the NA of H3N2 viruses from the 2017/18 season enhanced HY-PR8 backbone virus growth without HA mutations.

Figure 29. Mutations observed in NA mutant viruses (HY-PR8 backbone) in Figure 28 during egg passages.

Figure 30. The HY-PR8 backbone virus possessing A/Yokohama/48/2018HA and A/Yokohama/48/2018NA (T148K, D151 E, N245S, H347G, and T369K) acquired the same NA-K148I mutation, and no HA mutations were detected (SEQ ID NQ:54).

Figure 31. A HY-PR8 backbone virus possessing A/Yokohama/48/2018HA and A/Yokohama/48/2018NA(T148I, D151 E, N245S, H347G, and T369K) only acquired the HA-435L mutation in the stem region.

Figure 32. HA-H435L locates to the stem region of the HA trimer. previous study reported that HA-H435L did not affect antigenicity (Kuwahara et a!., Jpn. J. Infect. Pis.. 2018).

Figure 33. Effect of introducing NA-T148I, D 151 E, N245S, H347G, and T369K into the NA of H3N2 viruses from the 2017/18 season.

Figure 34. Effect of introducing NA-T148I, D151 E, N245S, H347G, and T369K into the NA of H3N2 viruses from the 2018/19 season (SEQ ID Nos. 55-56).

Figure 35. Yokohama48NA (T148I, D151 E, N245S, H347G, and T369K) enhanced the growth of viruses possessing the HA of H3N2 viruses of the 2017-18 and 2018-19 seasons (SEQ ID Nos. 57-58).

Figure 36. Yokohama48NA (TΊ48I, D151 E, N245S, H347G, and Ϊ369K) has reduced s!alidase activity.

Figure 37 The growth of Kansas/14/2017 (SEQ ID NO:59) was enhanced by introducing the NA mutations T148I, D151 E, N245S, H347G, and T369K or by possessing Yokohama48NA (T148I, D151 E, N245S, H347G, and T369K).

Figure 38. Neutralization by human monoclonal IgG clone F045-092 against viruses possessing Aichi/2/68HA and wild-type or mutant NA from 2017-18 season H3N2 viruses.

Figure 39A. Position of sialic acid relative to residues in NA.

Figure 39B. Enlarged view of Figure 38A.

Figure 40. Exemplary NA sequences {SEQ ID Nos. 51-59) for modification and modified NA sequences (SEQ ID Nos 69-70). Figure 41. exemplary HA sequences (SEQ ID Nos. 60-68) from strains that were stabilized.

Figure 42. Exemplary NA residues in 6M virus which were found in egg-grown A/Hong Kong/4801 /2014 and A/Alaska/232/2015.

Figure 43. Viruses in which 6M residues were introduced into the NA of A/Delaware/33/2018 and A/Saint-Petersburg/RII-324S/2019 and viruses possessing Yokohama/147/2017NA(6M) NA enhanced the virus growth of HY-PR8-backbone virus possessing wild type HA of A/Delaware/33/2018 or A/Saint- Petersburg/RI!-324S/2Q19. Harvested viruses possessing each strain's NA(6M) or Yokohama/147/2017NA(6M) were sequenced however none had additional mutations in HA and NA.

Figure 44. Viruses in which 6M was introduced Into the NA of A/Tokyo/UT-GR85/2019 and A/Kanagawa/IC1820/2019 did not enhance HY-PR8~backbone virus growth. However, viruses possessing Yokohama/147/2017NA(6M) showed enhanced the virus growth of HY-PR8-backbone virus possessing wild type HA of A/Tokyo/UT-GR85/2019 or A/Kanagawa/IG1820/2019 without HA mutations. Harvested viruses possessing Yokohama/147/2017NA(6M) were sequenced however none had additional mutations in HA and NA.

Figure 45. Mutations observed in the HA and NA proteins of HY-PR8 backbone viruses possessing Yokohama! 47NA(6M) during 10 passages in eggs·

Figure 46. Location of HA mutations occurred during egg passages (shown in Figure 43) on the 3D structure of HA protein.

Figure 47. Location of NA mutations occurred during egg passages (shown in Figure 43) on the 3D structure of NA protein.

Figure 48. Reduced molecular weight of Yokohama147NA(6M). VP4G-induced VLPs bearing FLAG-iagged Yokohama147NA or Yokohama147NA(6M) were analyzed by immunoblotting with anti- FLAG and anti-VP40 antibodies.

Figure 49. Loss of glycosylation sites of NA protein due fo mutations.

Figure 50. Receptor-binding specificities of Yokohama147HA, Yokohama147NA, and Yokohamal 47NA(6M).

Figure 51. Introduction of 6M into Yokohamal 47NA decreased s!alidase activity.

Figure 52. HY-PR8 backbone virus possessing wild type HA and mutant NA(T148I, D151 E, N245S, T329S, K344E, H347G and T369K) (=6M+T148I+T329S+K344E) from A/Kansas/14/2017NA acquired none of HA and NA mutations during egg passages.

Figure 53. Location of NA mutations; T148I, D151 E, N245S, T329S, K344E, G346V, H347G and T369K on the 3D structure o! NA protein. 6M shown in purple and T148I, T329S, K344E shown in green.

Detailed Description

Definitions As used herein, She term “isolated” refers to in vitro preparation and/or isolation of a nucieic acid molecule, e.g , vector or plasmid, peptide or polypeptide (protein), or virus of the invention, so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus preparation Is generally obtained by in vitro culture and propagation, and/or via passage in eggs, and is substantially free from other infectious agents.

As used herein, “substantiaiiy purified” means the object species is the predominant species, e.g., on a molar basis it is more abundant than any other individual species in a composition, and preferably is at least about 80% of the species present, and optionally 90% or greater, e.g., 95%, 98%, 99% or more, of the species present in the composition.

As used herein, “substantially free” means below the level of detection for a particular infectious agent using standard detection methods for that agent.

A “recombinant” virus is one which has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes to the viral genome. Reassortant viruses can be prepared by recombinant or nonrecombinant techniques.

As used herein, the term "recombinant nucleic acid” or "recombinant DNA sequence or segment" refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in the native genome. An example of DNA "derived" from a source, would be a DNA sequence that is identified as a useful fragment, and which Is then chemically synthesized In essentially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the disclosure, by the methodology of genetic engineering.

As used herein, a "heterologous" influenza virus gene or viral segment is from an influenza virus source that is different than a majority of the other influenza viral genes or viral segments in a recombinant, e.g., reassortant, influenza virus.

The terms "isolated polypeptide", "isolated peptide" or "isolated protein" include a polypeptide, peptide or protein encoded by cDNA or recombinant RNA including one of synthetic origin, or some combination thereof.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule expressed from a recombinant DNA molecule in contrast, the term "native protein" is used herein to indicate a protein isolated from a naturally occurring (i.e., a nonreeombinani) source. Molecular biological techniques may be used to produce a recombinant form of a protein with identical properties as compared to the native form of the protein.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Alignments using these programs can be performed using the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnoiogy information (htp://www.ncbi.nim.nih.gov/). The algorithm may involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query

13 sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M {reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm may also perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm may be the smallest sum probability (P(M)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preierab!y less than about 0.01, and most preferably less than about 0.001.

The BLASTN program (tor nucleotide sequences) may use as defaults a wordiength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program may use as defaults a wordiength (W) of 3, an expecfation (E) of 10, and the BLOSUM62 scoring matrix. See htp://www.ncbi.n1 m.nih.gov. Alignment may also be performed manually by inspection.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Influenza Virus Structure and Propagation

Influenza A viruses possess a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode at least ten proteins. The influenza virus life cycle begins with binding of the hemagglutinin (HA) to sialic acid-containing receptors on the surface of the host cell, followed by receptor- mediated endocytosis. The low pH in late endosomes triggers a conformational shift in the HA, thereby exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates fhe fusion of the viral and endosomai membrane, and the matrix protein (M1) and RNP complexes are released Into the cytoplasm. RNPs consist of the nudeoprotein (NP), which encapsidates vRNA, and the viral polymerase complex, which is formed by the PA, PB1, and PB2 proteins. RNPs are transported into the nucleus, where transcription and replication take place. The RNA polymerase complex catalyzes three different reactions: synthesis of an mRNA with a 5' cap and 3' po!yA structure, of a full-length complementary RNA (cRNA), and of genomic vRNA using the cRNA as a template. Newly synthesized vRNAs, NP, and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane, where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a crucial roie late in infection by removing siaiic acid from siaiy!oiigosaccharides, thus releasing newly assembled virions from the ceil surface and preventing the self aggregation of virus particles. Although virus assembly involves protein-protein and protein-vRNA interactions, the nature of these interactions is largely unknown.

Although influenza B and C viruses are structurally and functionally similar to Influenza A virus, there are some differences. For example, influenza B virus does not have a M2 protein with ion channel activity but has BM2 and has a viral segment with both NA and NB sequences influenza C virus has only seven viral segments.

Cells That Can Be Used to Produce Virus

Any ceil, e.g., any avian or mammalian cell, such as avian eggs, a human, e.g., 293T or PER.C6© ceils, or canine, bovine, equine, feline, swine, ovine, rodent, for instance mink, e.g., MvLul cells, or hamster, e.g., CHO ceils, or non-human primate, e.g., Vero ceils, including mutant cells, which supports efficient replication of Influenza virus can be employed to isolate and/or propagate influenza viruses. Isolated viruses can be used to prepare a reassortant virus. In one embodiment, host ceils for vaccine production are continuous mammalian or avian cell lines or cel! strains. A complete characterization of the cells to be used, may be conducted so that appropriate tests for purity of the final product can be included. Data that can be used for the characterization of a cell includes (a) information on its origin, derivation, and passage history; (b) information on its growth and morphological characteristics; (c) results of tests of adventitious agents; (d) distinguishing features, such as biochemical, immunological, and cytogenetic patterns which allow the cells to be dearly recognized among other cell lines; and (e) results of tests for tumorigenicity. In one embodiment, the passage level, or population doubling, of the host ceil used is as low as possible.

In one embodiment, the cells are WHO certified, or certifiable, continuous cel! lines. The requirements for certifying such ce!! lines include characterization with respect to at least one of genealogy, growth characteristics, immunological markers, virus susceptibility tumorigenicity and storage conditions, as well as by testing in animals, eggs, and cell culture. Such characterization is used to confirm that the cells are free from detectable adventitious agents in some countries, karyoiogy may also be required. In addition, tumorigenicity may be tested in cells that are at the same passage level as those used for vaccine production. The virus may be purified by a process that has been shown to give consistent results, before vaccine production (see, e.g., World Health Organization, 1982).

Virus produced by the host cell may be highly purified prior to vaccine or gene therapy formulation. Generally, the purification procedures result in extensive removal of cellular DNA and other cellular components, and adventitious agents. Procedures that extensively degrade or denature DNA may also be used.

Influenza Vaccines

A vaccine includes an isolated recombinant influenza virus of the invention, and optionally one or more other isolated viruses including other isolated Influenza viruses, one or more Immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens, e.g., an immunogenic protein from one or more bacteria, non-influenza viruses, yeasf or fungi, or isolated nucleic acid encoding one or more viral proteins (e.g., DNA vaccines) Including one or more immunogenic proteins of the isolated influenza virus of the invention in one embodiment, the influenza viruses of the invention may be vaccine vectors for Influenza virus or other pathogens.

A complete virion vaccine may be concentrated by ultrafiltration and then purified by zonal centrifugation or by chromatography. Viruses other than the virus of the invention, such as those included in a multivalent vaccine, may be inactivated before or after purification using formalin or beta- propio!actone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine may be prepared as follows: using viral suspensions fragmented by treatment with detergent, the surface antigens are purified, by ultracentrifugation for example. The subunit vaccines thus contain mainly HA protein, and also NA. The detergent used may be cationic detergent for example, such as hexadecyi trimethyl ammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxychoiate (Laver & Webster, 1976); or a nonionic detergent such as that commercialized under the name TRITON X100. The hemagglutinin may also be Isolated after treatment of the virions with a protease such as brome!in, and then purified. The subunit vaccine may be combined with an attenuated virus of the invention in a multivalent vaccine.

A split vaccine comprises virions which have been subjected to treatment with agents that dissolve lipids. A split vaccine can be prepared as follows: an aqueous suspension of the purified virus obtained as above, inactivated or not, is treated, under stirring, by lipid solvents such as ethyl ether or chloroform, associated with detergents. The dissolution of the viral envelope lipids results in fragmentation of the viral particles. The aqueous phase is recuperated containing the split vaccine, constituted mainly of hemagglutinin and neuraminidase with their original lipid environment removed, and the core or its degradation products. Then the residual infectious particles are Inactivated if this has not already been done. The split vaccine may be combined with an attenuated virus of the Invention In a multivalent vaccine.

Inactivated Vaccines. Inactivated influenza virus vaccines are provided by inactivating rep!icated virus using known methods, such as, but noi limited to, formalin or b-propioiactone treatment. Inactivated vaccine types that can be used in the invention can include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. The WV vaccine contains intact, inactivated virus, while the SV vaccine contains purified virus disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

In addition, vaccines that can be used include those containing the isolated HA and NA surface proteins, which are referred to as surface antigen or subunit vaccines.

Live Attenuated Virus Vaccines. Live, attenuated influenza virus vaccines, such as those including a recombinant virus of the invention can be used for preventing or treating Influenza virus infection. Attenuation may be achieved in a single step by transfer of attenuated genes from an attenuated donor virus to a replicated isolate or reasserted virus according to known methods. Since resistance to influenza A virus is mediated primarily by the development of an immune response to the HA and/or NA glycoproteins, the genes coding for these surface antigens come from the reassorted viruses or clinical Isolates. The attenuated genes are derived from an attenuated parent in this approach, genes that confer attenuation generally do not code for the HA and NA glycoproteins.

Viruses (donor influenza viruses) are available that are capable of reproducibly attenuating influenza viruses, e.g., a cold adapted (ca) donor virus can be used for attenuated vaccine production. Live, attenuated reassortant virus vaccines can be generated by mating the ca donor virus with a virulent replicated virus. Reassortant progeny are then selected at 25°C (restrictive for replication of virulent virus), in the presence of an appropriate antiserum, which inhibits replication of the viruses bearing the surface antigens of the attenuated ca donor virus. Useful reassortants are: (a) infectious, (b) attenuated for seronegative non-adult mammals and immuno!ogicaily primed adult mammals, (c) immunogenic and (d) genetically stable. The immunogenicity of the ca reassortants parallels their level of replication. Thus, the acquisition of the six transferable genes of the ca donor virus by new wild-type viruses has reproducibly attenuated these viruses for use In vaccinating susceptible mammals both adults and non adult.

Other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as we!i as into coding regions. Such attenuating mutations can also be introduced into genes other than the HA or NA, e.g., the PB2 polymerase gene. Thus, new donor viruses can also be generated bearing attenuating mutations introduced by site-directed mutagenesis, and such new donor viruses can be used in the production of live attenuated reassortants vaccine candidates in a manner analogous to that described above for the ca donor virus. Similarly, other known and suitable attenuated donor strains can be reassorted with influenza virus to obtain attenuated vaccines suitable for use in the vaccination of mammals.

In one embodiment, such attenuated viruses maintain the genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolates. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking pathogenicity to the degree that the vaccine causes minima! chance of inducing a serious disease condition in the vaccinated mammal.

The viruses in a multivalent vaccine can thus be attenuated or inactivated, formulated and administered, according to known methods, as a vaccine to induce an immune response in an animal, e.g., a mammal. Methods are well-known in the art for determining whether such attenuated or inactivated vaccines have maintained similar antigenicity to that of the clinical isolate or high growth strain derived therefrom. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and nucleic acid screening (such as probe hybridization or PGR) to confirm that donor genes encoding the antigenic determinants (e.g., HA or NA genes) are not present in the attenuated viruses.

Pharmaceutical Compositions

Pharmaceutical compositions of the present Invention, suitable for inoculation, e.g., nasal, parenteral or ora! administration, comprise one or more influenza virus isolates, e.g., one or more attenuated or inactivated influenza viruses, a subunit thereof, isolated protein(s) thereof, and/or isolated nucleic acid encoding one or more proteins thereof, optionally further comprising sterile aqueous or non- aqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. The composition of the invention is generally presented in the form of individual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 pg, e.g., 30 to 100 pg, 0.1 to 2 pg, 0.5 to 5 pg, 1 to 10 pg, 10 pg to 20 pg , 15 pg to 30 pg, or 10 to 30 pg, of HA from each of the strains entering into their composition. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a single influenza virus, or a combination of influenza viruses, for example, at least two or three influenza viruses, including one or more reassortant(s).

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and Injectable organic esters such as ethyl oieate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

When a composition of the present invention is used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized.

Heterogeneity in a vaccine may be provided by mixing replicated influenza viruses for at least two influenza virus strains, such as 2-20 strains or any range or value therein. Vaccines can be provided for variations in a single strain of an influenza virus, using techniques known in the art.

A pharmaceutical composition according fo the present invention may further or additionally comprise at least one chemotherapeutic compound, for example, for gene therapy, immunosuppressants, anti-inflammatory agents or immune enhancers, and for vaccines, chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-a, interferon-b, interferon-y, tumor necrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor, or ganciclovir.

The composition can also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.

Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits) may be for either a “prophylactic” or “therapeutic” purpose. When provided prophy!acticai!y, the compositions of the invention which are vaccines are provided before any symptom or clinical sign of a pathogen infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent Infection. When provided prophylactically, the gene therapy compositions of the invention, are provided before any symptom or clinical sign of a disease becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with the disease.

When provided therapeutically, a viral vaccine is provided upon the detection of a symptom or clinical sign of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition Is provided upon the detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound(s) serves to attenuate a symptom or clinical sign of that disease.

Thus, a vaccine composition of the present invention may be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. Similarly, for gene therapy, the composition may be provided before any symptom or clinical sign of a disorder or disease is manifested or after one or more symptoms are detected.

A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered In a “therapeutically effective amount” if the amount administered is physiologically significant. A composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular Immune response against at least one strain of an infectious influenza virus.

The “protection” provided need not be absolute, i.e., the influenza infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of mammals. Protection may be limited to mitigating the severity or rapidity of onset of symptoms or clinical signs of the influenza virus Infection.

Pharmaceutical Administration

A composition of the present invention may confer resistance to one or more pathogens, e.g., one or more influenza virus strains, by either passive immunization or active immunization. In active immunization, an attenuated live vaccine composition is administered prophylactica!!y to a host (e.g., a mamma!), and the host's immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. A gene therapy composition of the present invention may yield prophylactic or therapeutic levels of the desired gone product by active immunization.

In one embodiment, the vaccine is provided to a mammalian female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother’s milk).

The present invention thus includes methods for preventing or attenuating a disorder or disease, e.g., an infection by at least one strain of pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a clinical sign or condition of the disease, or In the total or partial immunity of the Individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a clinical sign or condition of the disease, or in the total or partial immunity of the individual to the disease.

A composition having at least one influenza virus of the present invention, including one which is attenuated and one or more other isolated viruses, one or more Isolated viral proteins thereof, one or more isolated nucleic acid molecules encoding one or more viral proteins thereof, or a combination thereof, may be administered by any means that achieve the intended purposes.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperltonea!, intranasa!, ora! or transderrna! routes. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time

A typical regimen for preventing, suppressing, or treating an influenza virus related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

According to the present invention, an “effective amount” of a composition is one that is sufficient to achieve a desired effect. It is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not intended to limit the invention and represent dose ranges.

The dosage of a live, attenuated or killed virus vaccine for an animal such as a mammalian adult organism may be from about 1 G ² -10 ² °, e.g., 10 ³ -1 Q ¹² , 10 ² -1 Q ¹ °, 10 ⁵ -1 Q ¹¹ , 1 G ⁶ -1 Q ¹⁵ , 1 Q ² -10 ¹⁰ , or 10 ¹⁵ -10 ^so plaque forming units (PFU)/kg, or any range or value therein. The dose of one viral Isolate vaccine, e.g., in an inactivated vaccine, may range from about 0.1 to 1 G0G, e.g., 0.1 to 10 pg, 1 to 20 pg, 30 to 100 pg, 10 to 50 pg, 50 to 200 pg, or 150 to 300 pg, o! HA protein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccine may be standardized to contain a suitable amount, e.g., 0.1 pg to 1 pg, 0.5 pg to 5 pg, 1 pg to 10 pg, 10 pg to 20 pg, 15 pg to 30 pg, or 30 pg to 100 pg or any range or value therein, or the amount recommended by government agencies or recognized professional organizations. The quantity of NA can also be standardized, however, this glycoprotein may be labile during purification and storage.

The dosage of immunoreactive HA in each dose of replicated virus vaccine can be standardized to contain a suitable amount, e.g., 1-50 pg or any range or value therein, or the amount recommended by the U.S. Public Health Service (PHS), which is usually 15 pg, per component for older children >3 years of age, and 7.5 pg per component for children <3 years of age. The quantity of NA can also be standardized, however, this glycoprotein can be labile during the processor purification and storage (Kendal et al., 1980; Kerr et a!., 1975) Each 0.5-ml dose of vaccine may contain approximately 0.1 to 0.5 billion viral particles, 0.5 to 2 billion viral particles, 1 to 50 billion virus particles, 1 to 10 billion viral particles, 20 to 40 billion viral particles, 1 to 5 billion viral particles, or 40 to 80 billion viral particles. Exemplary Viruses

Useful modifications of influenza neuraminidase (NA) proteins are described herein that stabilize hemagglutinin (HA) protein during egg-passages of Influenza viruses that express those modified neuraminidase proteins. Modified nucleic acids are also described that encode such modified neuraminidase proteins. The modifications can include deletions, substitutions and combinations thereof within the neuraminidase protein and nucleic acid sequences. Viruses that express such modified neuraminidase proteins exhibit significantly reduced acquisition of antigenicity-compromising mutations in hemagglutinin (HA) during growth of influenza in eggs.

For example, in some cases the modified neuraminidase can have at least one, or at least two, or at least three modifications. Amino acid positions within Influenza neuraminidase proteins that can be modified include, for example, one or more amino acids within positions 29-35, one or more amino acids within positions 44-52, one or more amino acids within positions 144-154, one or more amino acid positions within 240-250, one or more amino acids within positions 328-333, one or more amino acid positions within 344-350, one or more amino acid positions within 365-375, and combinations thereof, based on N2 numbering. For example, the amino acid(s) can be any amino acid within these positions such as any of the amino acids listed in the table below.

Original Exemplary Alternative Residue Substitutions Substitutions Ala (A) val; !eu; ile Va! Arg (R) lys; gin; asn Lys Asn (N) gin; his; lys; arg Gin Asp ( Glu, Asn Glu, Asn Gys (C) Ser Ser Gin (Q) Asn Asn G!u (El Asp Asp G!y (G) Pro Pro His (H) asn; gin; lys; arg; gin; Arg; Gin lie (I) leu; val; met; ala; phe Leu nor!eucine

Leu (L) norieuclne; i!e; val; met; ala; lie phe

Lys (K) arg; gin; asn Arg Met (M) leu; phe; i!e Leu Phe (F) !eu; val; ile; ala Leu Pro (P) G!v G!y Ser (Si Thr Thr Thr (T) Ser, Ala Ser. Ais Trp (W) Tyr Tyr Tyr (Y) trp; phe; thr; ser Phe Va! fV) ile; leu; met; phe; a!a; Leu norieuclne

In some cases, a selected amino acid within positions 29-35, positions 44-52, positions 144-154, positions 326-333, positions within 344-350, positions within 365-375, can have a conservative substitution. However, In other cases, the selected amino acid within positions 29-35, positions 44-52, positions 144- ISO, positions 326-333, positions within 344-350, positions within 365-375, can have a non-conservative substitution.

For example, a modified neuraminidase can have a deletion of at least one proi!ne, asparagine, glutamine, valine, or a combination of a proline, one or more asparagine(s), a glutamine, and a valine within positions 44-52 of the modified neuraminidase. A modified neuraminidase can have a substitution (replacement) of a threonine within positions 29-35, where the replacement is any amino acid. A modified neuraminidase can have a substitution (replacement) of a threonine or an aspartic acid within positions 145-154 or 365 to 375, where the replacement is any amino acid. A modified neuraminidase can have a substitution (replacement) of an asparagine within positions 326-333, where the replacement is any amino acid. A modified neuraminidase can have a substitution (replacement) of a histidine within positions 345- 350, where the replacement is any amino acid. Exemplary substitutions (replacements) for various types of amino acids are provided in the table above.

One example of an influenza A virus (A/Yokohama/2013/2003(H3isi2)) neuraminidase protein sequence Is provided below

1 MNPNQKHTI GSVSLTISTI CFFMQ!AIL! TTVTLHFKQY 41 EFNSPPNNGV MLCEPTI!ER NITEIVYLTN TT!EKEICPK 81 LAEYRNWSKP QCNITGFAPF SKDNSIRLSA GGD!WVTREP 121 YVSCDPDKCY QFALGQGTTL NNVHSNDIVH DRTPYRTLLM 161 NELGVPFHLG TKQVCIAWSS SSCHDGKAWL HVGVTGDDEN 201 ATASFIYNGR LADSIVSWSK KILRTQESEC VGINGTCTVV 241 MTDGSASGKA DTKILFIEEG KIVHTSTLSG SAQHVEECSC 281 YPRYPGVRGV CRDNWKGSNR PIVDINIKDY SiVSSYVCSG 321 LVGDTPRK D SSSSSHGLDP NMEEGGHGVK GWAFDDGNDV 361 WMGRTISEKL RSGYETFKVI EGWSNPNSKL QINRQVIVDR 401 GNRSGYSGIF SVEGKSCINR GFYVELIRGR KQETEVLWTS 441 NSIVVFGGTS GTYGTGSWPD GAD!NLMP! (SEQ ID NQ:55)

Amino acids that can be modified to improve the stability of co-expressed HA are highlighted in boid and with underlining within the sequence shown above. A nucleic acid that encodes such an influenza A virus (A/Yokohama/2013/2003(H3N2)) neuraminidase protein sequence is shown below

1 AGCAAAAGCA GGAGTAAAGA TGAATCGAAA TCAAAAGATA 41 ATAAGGATTG GCTCTGTTTC CGTCACGATT TGCACAATAT 81 GGTTCTTGAT GGAAATTGGC ATCCTGATAA CTACTGTAAC 121 ATTGC ATTIC AAGCAATATG AATTCAACTC CCCCCCAAAC 161 AACCAAGTGA TGCTGTGTGA ACGAACAATA ATAGAAAGAA 201 ACATAAGAGA GATAGTGTAT CTGACGAAGA GCAGCATAGA 241 GAAGGAAATA TGCCCCAAAC TAG GAG A AT A CAGAAATTGG 281 TCAAAGCGGC AATGTAAGAT TACAGGATTT GCAGGTTTTT 321 GT AAGGAGAA TTCGATTGGG CTTTGCGGTG GTGGGGAGAT 361 CTGGGTGACA AGAGAACCTT ATGTGTCATG CGATCCTGAC 401 AAGTGTTATC AATTTGCCCT TGGACAGGGA ACAAC ACT AA 441 AGAAGGTGCA TTCAAATGAC ATAGTACATG ATAGGAGCGC 481 TTATGGGACG GTATTGATGA ATGAGTTGGG TGTTGCATTT 521 GATGTGGGGA CGAAGGAAGT GTGCATAGCA TGGTGCAGCT 561 CAAGTTGTCA CGATGGAAAA GCATGGCTGC ATGTTTGTGT 601 AACGGGGGAT GATGAAAATG GAAGTGCTAG CTTGAT T TAG 641 AATGGGAGGG TTGCAGATAG TATTGTTTGA TGGTGCAAAA 681 AAATCCTCAG GACCCAGGAG TCAGAATGCG TTTGTATCAA 721 TGGAACTTGT ACAGTAGTAA TGAGTGATGG GAGTGCTTCA 761 GGAAAAGCTG ATACTAAAAT ACTATTCATT GAGGAGGGGA 801 AAATTGTTCA TACT AG GAGA TTATCAGGAA GTGCTGAGCA 841 TGTCGAGGAG TGCTCCTGTT ATGGTCGATA TCCTGGTGTC 881 AGATGTGTGT GCAGAGACAA CTGGAAAGGC TCGAATAGGG 921 CGATGGTAGA TATAAAGAT A AAGGATTATA GGATTGTTTG 961 GAGTTATGTG TGGTCAGGAG TTGTTGGAGA GAGACGCAGA 1001 AAAAACGACA GCTCCAGGAG TAGCCATTGG TTGGATCCAA 1041 AGAATGAGGA AGGTGGTCAT GGAGTGAAAG GCTGGGCCTT 1081 TGATGATGGA AATGAGGTGT GGATGGGAAG AAGGATGAGC 1121 GAGAAGTTAC GGTGAGGATA TGAAACGTTC AAAGTCATTG 1161 AAGGCTGGTC CAACCCTAAC TCCAAATTGC AGATAAATAG 1201 GGAAGTCATA GTTGACAGAG GTAACAGGTG GGGTTATTCT 1241 GGTATTTTGT GTGTTGAAGG CAAAAGGTGG ATCAATCGGT 1281 GCTTTTATGT GGAGTTGATA AGGGGAAGAA AACAGGAAAC 1321 TGAAGTCTTG TGGAGCTCAA ACAGTATTGT TGTGTTTTGT 1361 GGCAGGTGAG GTACATATGG AAGAGGGTGA TGGCGTGATG 1401 GGGCGGAGAT GAATGTGAT G GCTATATAAG GTTTCGCAAT 1441 TTT AGAAAAA AACTGCTTGT TTGTAGT (SEQ ID NO:56)

Modifications at the specified positions in neuraminidase can confer enhanced growth of the virus.

Another example of an infiuenza A virus (A/Yokohama/47/2002(H1 N2))) neuraminidase sequence is shown below, with positions of modifications highlighted in bold and with underlining.

10 20 30 40 50

MNPNQKim GSVSLTIATI GFLMQIAILV TTVTLHFKQY EGNSPPNNQV

60 70 80 90 100

MLCEPT!IER NITEiVYLTN TTIEKEIGPK LAEYRNWSKP QGNITGFAPF 110 120 130 140 150

SKDNSIRLSA GGD!WVTREP YVSCDPDKGY QFALGQGTTL NNGHSNDTVH 160 170 180 190 200

DRTPYRTLLM NELGVPFHLG TKQVCIAWSS SSCHDGKAWL HVCVTGDDGN 21 Q 220 230 240 250

ATASF!YNGR LVDSIGSWSK KILP.TQESEG VCINGTCTVV MTDGSASGKA 260 270 280 290 300

DTKILFIEEG KiVHTSLLSG SAQHVEECSC YPRYPGVRCV CRDNWKGSNR 310 320 330 340 350

PiVD!NVKDY S!VSSYVCSG LVGDTPRK D SSSSSHGLDP NNEEGGHGVK 360 370 380 390 400

GWAFDDGNDV WMGRTISEKL RSGYETFKVI EGWSKPNSKL QINRQVIVDR 410 420 430 440 450

GNRSGYSGIF SVEGKSCiNR CFYVELIRGR NQETEVLWTS NSIWFGGTS 460

GTYGTGSWPD GAD!NLMP!

(SEQ ID NO: 57)

Amino acids that can be modified to improve the stability of co-expressed HA are highlighted in bold and with underlining within the sequence shown above.

In some cases, in one or more modifications can also be introduced into HA, PA, PB1, PB2, NP, M1 , M2, NS2, PB1-F2, PA-X, and/or NS1 proteins (and nucleic acids encoding such proteins).

Enhanced growth of the virus when passaged through embryonated chicken eggs or cultured cells is observed when the modified NA proteins are expressed and such expression can result in significantly higher viral liters. Thus, the invention provides a method for making influenza viruses with enhanced replication in ce!! culture or embryonated eggs. The method includes providing ceils suitable for Influenza vaccine production; modifying nucleic acids encoding the neuraminidase; and isolating virus strains with enhanced growth relative to the one or more unmodified viral isolates. In some cases, a method for making influenza viruses with enhanced replication in cel! culture can involve, serially culturing one or more influenza virus isolates in embryonated chicken eggs; and isolating serially cultured virus with enhanced growth relative to the one or more isolates prior to serial culture. In some cases, the viruses can be grown or passaged within ceils in culture, e.g., MDCK or Vero cells.

The modified neuraminidases can be expressed in a variety of influenza strains. For example, A''Puerio Rico/8/34 (H1N1), “PR8,” virus often serves as the genetic backbone for generation of inactivated influenza vaccines. Some vaccine strains based on PR8 backbone can replicate to relatively iow titers in eggs and cell culture, resulting in delayed vaccine production and vaccine shortage. However, expression of the modified neuraminidases described herein can improve replication of the PR8 (and other) influenza strains.

In one embodiment of the invention, vectors for vRNA production can include a vector comprising a promoter operab!y linked to a modified NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a operably linked to an influenza virus NS DNA linked to a transcription termination sequence in one embodiment, the DMAs for vRNA production of PB1 , PB2, PA, NP, M, and NS, have sequences from an influenza virus that replicates to high titers in cultured mammalian cells such as MDGK cells, Vero ceils or PER.C6® cells or embryonated eggs, and/or from a vaccine virus, e.g., one that does not cause significant disease in humans. The DMA for vRNA production of NA may be from any NA, e.g., any of N1 -N11 , and the DNA for vRNA production of HA may be from any HA, e.g., H1-H18. In one embodiment, the DMAs for vRNA production may be for an influenza B or C virus. The DNAs for vRNA production of NA and HA may be from different strains or isolates (6:1 :1 reassortants) or from the same strain or isolate (6:2 reassortants), or the NA may be from the same strain or isolate as that for the internal genes (7:1 reassortant). Vectors for mRNA production can include a vector encoding a modified NA, a vector encoding influenza virus PA, a vector encoding influenza virus PB1, a vector encoding influenza virus PB2, and a vector encoding influenza virus NP, and optionally one or more vectors encoding NP, NS, M, e.g., M1 and M2, HA or NA. The vectors encoding viral proteins may further include a transcription termination sequence.

Other reassortants with Internal genes from other PP.8 isolates or vaccine viruses may be employed in recombinant reassortant viruses of the invention. In particular, 5:1 :2 reassortants having UW-PR8 PB1 , PB2, PA, NP, and M (“5”) and PR8(Cam) NS (Ί”); 6:1 :1 reassortants having UW-PRS (modified) NA, PB1 , PB2, PA, NP, and M (“6”) and PR8(Cam) NS (“1”); and 7:1 reassortants having UW- PP.8 PB1 , PB2, PA, NP, M, (modified) NA, and NS (“7”) may be employed.

The neuraminidases that can be modified can have sequences that vary from those described herein. However, in some cases, the modified neuraminidases can have substantially the same activity as a corresponding polypeptide described by sequence herein. As used herein, "substantially the same activity" includes an activity that is about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or more activity, or a detectable protein level that is about 80%, 90% or more protein level, of the corresponding protein described herein. In one embodiment, the nucleic acid encodes a polypeptide which is substantially the same as, e.g., having at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to a polypeptide encoded by one of sequences described herein in one embodiment, the Isolated and/or purified nucleic acid molecule comprises a nucleotide sequence which is substantially the same as, e.g., having at least 50%, e.g., 60%, 70%, 80% or 90%, including any integer between 50 and 100, or more contiguous nucleic acid sequence identity to one of the nucleic acid sequences described herein. In one embodiment, a nucleic acid also encodes a polypeptide having at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99%, including any integer between 80 and 99, contiguous amino acid sequence identity to a polypeptide described herein.

In one embodiment, a modified influenza virus neuraminidase polypeptide has one or more, for instance, 2, 5, 10, 15, 20 or more, conservative amino acids substitutions, e.g., conservative substitutions of up to 10% or 20% of 2, 5, 10, 15, 20 or more, of a combination of conservative and non-conservative amino acids substitutions, e.g., conservative substitutions of up to 10% or 20% of the residues, or relative to a polypeptide with one of the sequences disclosed herein.

The invention thus includes the use of isoiafed and purified vectors or piasmids, which express or encode influenza virus proteins, or express or encode influenza vRNA, both native and recombinant vRNA. The vectors comprise influenza cDNA, e.g., influenza A (e.g., any influenza A gene including any of the 18 HA or 11 NA subtypes), B or G DNA (see Fields Virology (Fields et ai. (eds.), Lippincott, Williams and Wickens (2006), which is specifically Incorporated by reference herein). Any suitable promoter or transcription termination sequence may be employed to express a protein or peptide, e.g., a viral protein or peptide, a protein or peptide of a nonvira! pathogen, or a therapeutic protein or peptide.

A composition or plurality of vectors of the invention may also comprise a heterologous gene or open reading frame of interest, e.g., a foreign gene encoding an Immunogenic peptide or protein useful as a vaccine or in gene replacement, for instance, may encode an epitope useful in a cancer therapy or vaccine, or a peptide or polypeptide useful In gene therapy. When preparing virus, the vector or plasmid comprising the gene or cDNA of interest may substitute for a vector or plasmid for an influenza viral gene or may be in addition to vectors or plasmids for all Influenza viral genes. Thus, another embodiment of the invention comprises a composition or plurality of vectors as described above in which one of the vectors is replaced with, or further comprises, 5' influenza virus sequences optionally Including 5' influenza virus coding sequences or a portion thereof, linked to a desired nucleic acid sequence, e.g., a desired cDNA, linked to 3’ influenza virus sequences optionally Including 3' influenza virus coding sequences or a portion thereof in one embodiment, fhe desired nucleic acid sequence such as a cDNA is in an antisense (antigenomic) orientation. The introduction of such a vector in conjunction with the other vectors described above to a host ceil permissive for influenza virus replication results in recombinant virus comprising vRNA corresponding to the heterologous sequences of the vector.

The promoter in a vector for vRNA production may be a R.NA polymerase I promoter, a RNA polymerase II promoter, a RNA polymerase III promoter, a T7 promoter, or a T3 promoter, and optionally the vector comprises a transcription termination sequence such as a RNA polymerase I transcription termination sequence, a RNA polymerase II transcription termination sequence, a RNA polymerase III transcription termination sequence, or a rlbozyme. Ribozymes within the scope of the invention include, but are nof limited to, tetrahymena ribozymes, RNase P, hammerhead ribozymes, hairpin ribozymes, hepatitis ribozyme, as well as synthetic ribozymes. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter.

The promoter or transcription termination sequence in a vRNA or virus protein expression vector may be the same or different relative to the promoter or any other vector. In one embodiment, the vector or plasmid which expresses influenza vRNA comprises a promoter suitable for expression in at least one particular host cell, e.g., avian or mammalian host cells such as canine, feline, equine, bovine, ovine, or primate cells including human cells, or for expression in more than one host.

In one embodiment, at least one vector for vRNA comprises a RNA polymerase II promoter linked to a ribozyme sequence linked to viral coding sequences linked to another ribozyme sequences, optionally linked to a RNA polymerase H transcription termination sequence. In one embodiment, at least 2, e.g., 3, 4, 5, 6, 7 or 8, vectors for vRNA production comprise a RNA polymerase H promoter, a first ribozyme sequence, which Is 5' to a sequence corresponding to viral sequences including viral coding sequences, which is 5' to a second ribozyme sequence, which is 5' to a transcription termination sequence. Each RNA polymerase II promoter in each vRNA vector may be the same or different as the RNA polymerase II promoter in any other vRNA vector. Similarly, each ribozyme sequence in each vRNA vector may be the same or different as the ribozyme sequences in any other vRNA vector. In one embodiment, the ribozyme sequences in a single vector are not the same.

In one embodiment, the invention provides a plurality of influenza virus vectors for a reassortant, comprising a vector for vRNA production comprising a promoter operab!y linked to a modified influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the DNAs for the modified NA, PB1, PB2, PA, NP, NS, and M are from one or more Influenza vaccine seed viruses and contain two or more of the characteristic residues at the specified position(s); and a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1 , a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding Influenza virus HA, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1 , a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS1 , or a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, at least one vector comprises sequences corresponding to those encoding PB1, PB2, PA, NP, M, or NS, or a portion thereof, having substantially the same activity as a corresponding polypeptide described herein or encoded by a nucleic acid described herein. Optionally, two vectors may be employed in place of the vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, e.g., a vector comprising a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence and a vector comprising a promoter operably linked to an influenza virus M2 cDNA linked to a transcription termination sequence.

A plurality of the vectors of the invention may be physically linked or each vector may be present on an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. In one embodiment, each vRNA production vector is on a separate plasmid. In one embodiment, each rnRNA production vector Is on a separate plasmid.

The invention also provides a method to prepare influenza virus. The method comprises contacting a ceil with a plurality of the vectors of the invention, e.g., sequentially or simultaneously, in an amount effective to yield infectious influenza virus. The invention also includes isolating virus from a ceil contacted with the plurality of vectors. Thus, the invention further provides isolated virus, as well as a host cell contacted with the plurality of vectors or virus of the invention. In another embodiment, the invention includes contacting the cell with one or more vectors, either vRNA or protein production vectors, prior to other vectors, either vRNA or protein production vectors. In one embodiment, the promoter for vRNA vectors employed in the method is a RNA polymerase I promoter, a RNA polymerase II promoter, a RNA polymerase IN promoter, a T3 promoter or a T7 promoter. In one embodiment, the RNA polymerase I promoter is a human RNA polymerase I promoter. In one embodiment, each vRNA vector employed in the method is on a separate plasmid. In one embodiment, the vRNA vectors employed in the method are on one plasmid or on two or three different plasmids. In one embodiment, each mRNA vector employed in the method is on a separate plasmid. In one embodiment, the mRNA vectors for PA, PB1, PB2 and NP employed In the method are on one plasmid or on two or three different plasmids.

Exemplary viral sequences for a master vaccine strain (PR8UW)

HA

AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAGGCAAACCTACTGGTCCTGT TATGTGCACT

TGCAGCTGCAGATGCAGACACAATATGTATAGGCTACCATGCGAACAATTCAACCGA CACTGTTGAC

ACAGTACTGGAGAAGAATGTGAGAGTGACACACTGTGTTAACGTGCTGGAAGACAGC GAGAACGGAA AACT AT GT AG ATT AAAAGG AAT AGCGCC ACT ACAATT GGGG AAAT GT AAC AT GGGCGG AT GGCT CTT GGGAAACCCAGAATGCGAGCGACTGCTTGCAGTGAGATCATGGTCCTACATTGTAGAAAC ACCAAAG T CTG AGAAT GGAAT AT GTT AT CCAGG AG ATTT CAT CG ACT AT G AGGAGCTG AGGG AGCAATT GAGCT CAGTGT CAT CATT CG AAAG ATT CG AAAT ATTT CCCAAAG AAAGCT C ATGGCCCAACCACAAC AGAAAC GGAGTAAGGGCAGGATGCTGGGATGAGGGGAAAAGGAGTTTTTAGAGAAATTTGGTATGG GTGAGGG AGAAGGAGGGCTCATACCCAAAGGTGAAAAATTCTTATGTGAACAAAAAAGGGAAAGAAG TCCTTGT ACT GTGGGGT ATT CAT C ACGCGGCT AACAGTAAGG AACAACAGAAT GT CT AT GAG AAT G AAAATGCTT AT GT GT CT GT AGTG ACTT CAAATT AT AACAGGAG ATTT ACCCCGG AAAT AGCAG AAAG ACCCAAAGT A AGAGAT CAAGCT GGGAGGAT GAACT ATT ACTGGACCTTGCT AAAACCCGGAGACACAATAAT ATTT G AGGGAAATGGAAATCTAATAGGACGAATGTATGGTT CGGACTGAGTAGAGGGTTTGGGTGCGGCAT CATCAGCTGAAACGGATCAATGCATGAGTGTAACAGGAAGTGTGAAAGAGCGCTGGGAGC TATAAAG AGG AGT CT GCCTT AGCAG AAT AT ACAGCGAGTGACAAT AGG AG AGT GCGCAAAAT ACGT CAGG AGT G GCAAATTGAGGATGGTTACAGGACTAAGGAAGATTCCGTCCATTGAATCCAGAGGTGTAT TTGGAGG CATTGCCGGTTTTATTGAAGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCA TGATCAG AATGAAGAGGGATCAGGCTATGCAGGGGATCAAAAAAGCACAGAAAATGCGATTAACGGG ATTACAA ACAAGGT GAACACTGTT AT CGAGAAAATGAACATTCAATT GACAGCT GTGGGT AAAGAATT CAACAAA TTAGAAAAAAGGATGGAAAATTTAAATAAAAAAGTTGATGATGGATTTCTGGACATTTGG ACATATAAT GCAGAATTGTTAGTTCTACTGGAAAATGAAAGGACTCTGGATTTCCATGAGTCAAATGTG AAGAATCT GTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATCGGAAATGGATGTTT TGAGTTCT ACCACAAGT GT GAC AAT G AAT GCAT GG AAAGT GT AAG AAATGGG ACTT AT GATT AT CCCAAAT ATT CA GAAGAGTCAAAGTTGAACAGGGAAAAGGTAGATGGAGTGAAATTGGAATCAATGGGGATC TATCAGA TTCTGGCGATCTACTCAACTGTGGCCAGTTCACTGGTGCTTTTGGTCTCCCTGGGGGCAA TCAGTTT CT GG AT GT GTT CT AAT GG AT CTTTGCAGTGCAGAAT ATGCAT CTG AGATT AG AATTT CAGAG AT AT GA GGAAAAACACCCTT GTTT CT ACT (SEQ ID NO:22)

NA

AGCAAAAGGAGGGGTTTAAAAT GAAT CGAAAT CAGAAAATAAT AAGCATTGGAT GAAT GT GT CT GGT A GT CGGACT AATTAGCCT AAT ATTGCAAAT AGGG AAT AT AAT CT CAAT ATGG ATT AGCCATT CAATT CAA ACT GG AAGT CAAAACCAT ACTGG AAT ATGCAACC AAAACAT CATT ACGT AT AAAAAT AGCACCT GGGT AAAGG ACACAACTT GAGT GAT ATTAACGGGGAATTGAT GT CTTT GT GCGAT GCGTGGGT GGGCT AT AT ACAGCAAAGAGAATAGGATAAGAATTGGTTGCAAAGGAGAGGTTTTTGTGATAAGAGAGC GCTTTATT T G ATGTT CT GAGTT GG AATGCAGG AGCTTTTTT GT G ACGCAAGGTGGCTT ACT GAAT G ACA AGCATT G AAGT GGGACTGTT AAGGACAGAAGCGCTT AT AGGGCCTT AATGAGCTGCGCT GT GGGT GAAGCT CG GTCCCCGTACAATTCAAGATTTGAATCGGTTGCTTGGTCAGCAAGTGCATGTCATGATGG CATGGGC TGGCTAACAATCGGAATTTCAGGTCCAGATAATGGAGCAGTGGCTGTATTAAAATACAAC GGCATAAT AACTGAAACCATAAAAAGTTGGAGGAAGAAAATATTGAGGACACAAGAGTCTGAATGTGC CTGTGTAA AT GGTT CAT GTTTT ACT AT AAT G ACT GAT GGCCCG AGTG AT GGGCT GGCCT CGT ACAAAATTTT CAAG AT CGAAAAGGGG AAGGTT ACT AAAT CAAT AGAGTT GAATGCACCT AATT CT CAGT AT G AGG AATGTT C CT GTT ACCCT GAT ACCGGCAAAGT GAT GT GT GT GTGCAGAGACAATT GGCAT GGTT CGAACCGGCCA

GCGGCGTCCGGAAGATGGAAGAGGCAGGTGTGGTCCAGTGTATGTTGATGGAGCAAA CGGAGTAAA GGGATTTTGATATAGGTATGGTAATGGTGTTTGGATAGGAAGGAGCAAAAGTGACAGTTC GAGACAT GGGTTTGAGATGATTTGGGATCCTAATGGATGGACAGAGACTGATAGTAAGTTCTGTGTG AGGGAAG AT GTT GT GGGAAT GAGT GATT GGT CAGGGT AT AGCGGAAGTTT GGTT CAACAT CGT G AGCT GAGAGG GGT AG ACT GT ATGAGGGCGTGCTT GT GGGTT GAATT AAT GAGGGGACG AGCTAAAG AAAAAAGAAT G TGGAGTAGTGCGAGCAGCATTTCTTTTTGTGGGGTGAATAGTGATAGTGTAGATTGGTCT TGGCGAGA CGGTGCTGAGTTGCCATTGAGCATTGACAAGTAGTCTGTTCAAAAAACTCCTTGTTTCTA GT (SEQ ID NO:23)

AGCGAAAGCA GGTACTGATG CAAAATGGAA GATTTTGTGC GACAATGCTT GAATGCGATG ATTGTGGAGG TTGGGGAAAA AACAAT GAAA GAGTATGGGG AGGAGCTGAA AATGGAAACA AACAAATTTG CAGGAATATG CAGT CACTI G GAAGTATGCT TCATGTATTC AGATTTTCAG TTCATCAATG AGCAAGGCGA GTCAATAATG GTAGAACTTG GTGATCGAAA TGCACTTTTG AAGCACAGAT TTGAAATAAT GGAGGGAAGA GATGGCACAA TGGCGTGGAC AGTAGTAAAC AGTATTTGGA ACACTACAGG GGCTGAGAAA GCAAAGTTT C TACCAGATTT GTATGATTAC AAGGAGAATA GATT CAT CG A AATTGGAGTA ACAAGGAGAG AAGTTCAGAT ATACTATCTG GAAAAGGCCA AT AAAATT AA ATCTGAGAAA ACACACATCC ACATTTT CT C GTTCACTGGG GAAGAAATGG CCACAAAGGC AGACTACACT CTCGATGAAG AAAGCAGGGC TAGGATCAAA ACCAGAGTAT TCAGCATAAG ACAAGAAATG GGCAGGAGAG GGCTGTGGGA TTCGTTTGGT GAGTCGGAGA GAGGAGAAGA GAGAATTGAA GAAAGGTTTG AAATGAGAGG AAGAATGGGG AAGGTTGGCG ACGAAAGT CT CGCGCGGAAG TTGTCGAGCG TTGAAAATTT TAGAGCGTAT GTGGATGGAT TGGAAGCGAA CGGGTACATT GAGGGGAAGG TGTCTGAAAT GT GCAAAGAA

GGGCGTCGCT GTTCTGAGGG GTGCAAATTG CTGGTGATGG ATGGCTTAAA ATTAAGCATT GAGGACGCAA GTGATGAAGG AGAGGGAATA GCGCTATATG ATGGAATGAA ATGCATGAGA AGATTGTTTG GATGGAAGGA AGGGAATGTT GTTAAACCAG ACGAAAAGGG AATAAATCCA AATT ATCTTC TGTCATGGAA GGAAGTACTG GCAGAACTGC AGGACATTGA GAATGAGGAG AAAATTGGAA AG ACT AAAAA TATGAAGAAA AGAAGTGAGC TAAAGTGGGC ACTTGGT GAG AACATGGCAC CAGAAAAGGT AGACTTTGAC GACTGTAAAG ATGTAGGTGA TTTGAAGGAA TATGATAGTG ATGAACCAGA ATTGAGGTCG CTTGCAAGTT GG ATT CAG AA TGAGTTTAAC AAGGCATGCG AACT GACAG A TTCAAGCTGG ATAGAGCTCG ATGAGATTGG AGAAGATGTG GCTGCAATTG AACAGATTGC AAGCATGAGA AGGAATTATT TGACATCAGA GGTGTCTCAC TGCAGAGCCA CAGAATACAT AATGAAGGGA GTGTACATCA ATACTGCGTT GCTTAATGCA TCTTGTGCAG CAATGGATGA TTTGCAATTA ATTCGAATGA TAAGCAAGTG TAGAACTAAG GAGGGAAGGC GAAAGACCAA CTTGTATGGT TTCATCATAA AAGGAAGATG GCACTT AAGG AATGACACCG ACGTGGTAAA CTTTGTGAGC ATGGAGTTTT CTCTCACTGA CCCAAGACTT G AAGG AC AT A AATGGGAGAA GTACTGTGTT CTTGAGATAG GAGATATGCT T AT AAGAAGT GCCATAGGCC AGGTTTCAAG GCCCATGTTC TTGTATGTGA GAAGAAATGG AACCTCAAAA ATTAAAATGA AATGGGGAAT GGAGATGAGG CGTTGCGTCG TGCAGTGAGT TC A AG A AATT GAGAGTATGA TTGAAGCTGA GTGCTGTGTC AAAGAGAAAG AGATGAGCAA AGAGTT GTTT GAGAACAAAT GAGAAACAT G GCGCATTGGA GAGTGCGCGA AAGGAGTGGA GGAAAGTTGC ATTGGGAAGG TCTGCAGGAC TTTATTAGCA AAGTCGGTAT TCAACAGCTT GTATGGATCT GCACAACTAG AAGG ATTTT G AGGTGAATCA AGAAAAGTGC TTGTTATGGT TCAGGGTCTT AGGGACAACG TGGAAGCTGG GAGCTTTGAT CTTGGGGGGG TATATGAAGC AATTGAGGAG TGCGTGATTA ATGAT CGCTG GGTTTTGGTT AATGCTTCTT GGTTCAACTC CTTCCTTAGA GATGCATTGA GTTAGTTGT G GCAGTGGTAC TATTTGCTAT CCATACTGTC C AAAAAAGT A CGTTGTTTCT ACT (SEQ ID NO:24) AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACCTTACTTTTCTTAAAAGTG CCAGCACA

AAATGCT AT AAGCACAACTTT GCGTT AT ACT GG AG ACCCT GCTT ACAGGCAT GGG AGAGGAACAGGA T ACACGAT GGATACTGT CAACAGGAGACAT CAGTACTCAGAAAAGGGAAGAT GGACAACAAACACCG AAACTGGAGCACGGGAACT GAAGCGG ATTG AT GGGGCACTGCGAGAAGACAATG AAGCAAGT GGTT A TGGCGAAAGAGATTGTGTATTGGAGGCGATGGCTTTGCTTGAGGAATCGCATCGTGGTAT TTTTGAAA ACTCGTGTATTGAAACGATGGAGGTTGTTGAGGAAAGAGGAGTAGACAAGCTGAGAGAAG GCGGAGA G AGCT AT G ACTGG ACT CT AAAI AG AAACGAACGT GGTGCAACAGGATTGGCGAAG AGAAT AG AAGT G TTCAGATCAAATGGCGTCAGGGCGAATGAGTGTGGAAGGGTCATAGACTTGGTTAAGGAT GTAATGG AGTGAATGAACAAAGAAGAAATGGGGATGAGAAGTCATTTTCAGAGAAAGAGAGGGGTGA GAGAGAA TATGACTAAGAAAATGATAACAGAGAGAAGAATGGGTAAAAAGAAGCAGAGATTGAAGAA AAGGAGTT ATCTAATTAGAGCATTGAGCGTGAAGAGAATGAGCAAAGATGCTGAGAGAGGGAAGCTAA AAGGGAG AGCAATTGGAAGGCGAGGGATGGAAATAAGGGGGTTTGTATAGTTTGTTGAGACACTGGG AAGGAGT ATATGTGAGAAACTTGAACAATCAGGGTTGCCAGTTGGAGGCAATGAGAAGAAAGCAAAG TTGGCAA AT GIT GT AAGG AAG AT GAT G ACCAATT CT GAGG ACACCG AACTTT CTTT C AGCAT GACT GG AG AT AAG AGCAAATGGAACGAAAATCAGAATCGTCGGATGTTTTTGGCGATGATCACATATATGACC AGAAATGA GCCCG AATGGTT CAG AAAI GTT CT AAGT ATTGCT CCAAT AAT GTT CT CAAAGAAAATGGGG AGACTGG G AAAAGGGT AT AT GTTT G AG AGCAAG AGT AT GAAACTT AG AACT CAAAT ACGTGGAG AAATGCT AGCA AGCAT GG ATTT GAAAT ATTT CAAT GATT CAACAAGAAAG AAG ATTG AAAAAAT GCG ACGGGT GTT AAT A G AGGGG ACT GCAT CATTG AGGCGT GGAAT GAT G ATGGGCAT GTT CAAT AT GTT AAGCAGT GTATT AG GGGTCTGCATGGTGAATCTTGGAGAAAAGAGATAGAGGAAGACTAGTTACTGGTGGGATG GTCTTGA AT CCT CT GAGG ATTTTGCT CT GATT GTG AAT GCAGCGAAT CAT GAAGGGATT CAAGCCGGAGT CG AC

AG GT AC ATTT G A ATT C AC AAGTTTTTT CT ATGGTT ATG GGTTTGTTG CG AATTT CAG CAT G G AG CTT CC CAGTTTTGGGGT GTCTGGG AT C AAGG AGT CAGCGG ACAT G AGTATT GG AGTT ACT GT CAT CAA AAAC AAT AT GAT AAAGAAT GAT CTTGGT CCAGCAACAGCT CAAATGGCCCTT CAGTTGTT CAT CAAAG ATT A CAGGT ACACGT AGCGATGGCAT AT AGGT G ACACAGAAAT ACAAAGCGG AAGAT GATTT GAAAT AAAGA AAGTGTGGGAGCAAACCCGTTCGAAAGCTGGACTGCTGGTCTCCGACGGAGGCCCAAATT TA ACAA GATT AG AAAT CT GCACATT CGT G AAGT CTGGCT AAAAT GGG AATT G ATGG ATG AGG ATT AGCAGGGG CGTTT ATGGAAGCGAGT G AAGCGATTTGT CAGGCAT AAAGAAATT G AAT GAAT GAACAAT GGAGT GAT GATGCCAGGACATGGTCCAGCCAAAAACATGGAGTATGATGCTGTTGCAACAACACACTC CTGGATC CCCAAAAGAAATCGATCCATCTTGAATACAAGTCAAAGAGGAGTAGTTGAGGATGAACAA ATGTAGCA AAGGTGCTGCAATTTATTTGAAAAATTCTTCCCCAGCAGTTGATAGAGAAGACCAGTCGG GATATCCA GTATGGTGGAGGCTATGGTTTCCAGAGCCCGAATTGATGCACGGATTGATTTCGAATGTG GAAGGAT AAAGAAAGAAGAGTT CACT G AGAT GAT GAAGAT CT GTT CCACCATT GAAG AGCT CAG ACGGCAAAAA T AGTG AATTT AGCTTGT GCTT CAT G AAAAAATGCCTT GTTT CT ACT {SEQ ID NO:25)

PB2

AGCGAAAGCA G GTG A ATT AT ATTG A AT ATG GAAAGAATAA AAGAACTACG AAATCTAATG TCGCAGTCTC GCACCCGCGA GAT ACT CACA AAAAGCACCG TGGACGATAT GGGCATAATG AAG AAGT ACA CATCAGGAAG ACAGGAGAAG AACCCAGCAC TTAGGATGAA AT GG AT GAT G GCAATGAAAT ATGCAATTAC AGCAGACAAG AGGATAACGG AAAT GATT CC TG AG AG AAAT GAGCAAGGAC AAACTTT AT G GAGTAAAATG AATGATGCCG GAT CAGACGG AGTGATGGTA TCACCTCTGG CTGTGACATG GTGGAATAGG AATGGACCAA TAACAAATAC AGTTCATTAT CCAAAAA CT ACAAAAGTTA TTTTG AAAGA GTGGAAAGGC TAAAGCATGG AACGTTTGGC CCTGTCCATT TTAGAAACCA AGT CAAAAT A CGTCGGAGAG TTG AGAT AAA TCCTGG CAT GCAGATCTGA GTGGCAAGGA GGCACAGGAT GTAATCATGG AAGTTGTTTT CCCTAACGAA GTGGGAGCCA GGATACTAAC ATGGGAATCG CAAGTAACGA TAACC AAAGA GAAGAAAGAA GAACTCCAGG ATTG CAAAAT TTGTCCTTTG ATGGTTGCAT ACATGTTGGA GAGAGAACTG GTCCGCAAAA CG AG ATT CCT CCCAGTGGCT GGTGGAACAA GCAGTGTGTA CATTG A AGTG TTGCATTTGA GTCAAGGAAC ATGCTGGGAA CAGATGTATA GTCGAGGAGG GGAAGTGAGG AATGATGATG TTGATGAAAG CTTGATTATT GCTGGTAGGA AC AT AGTG AG AAGAGCTGCA GTATGAGCAG ATGGAGTAGC AT GTTT ATTG GAGATGTGCC ACAGGACACA GATTGGTGGA ATT AGG AT GG TAGAGATGCT T AGG CAG A AC CCAACAGAAG AGCA AGGCGT GGATATATGC AAGGGTGCAA TGGGACTGAG AATTAGCTCA TCCTTCAGTT TTGGTGGATT CAGATTTAAG AGAAGAAGCG GATCATCAGT CAAGAGAGAG GAAGAGGTGC TTACGGGCAA T CTT CAAAGA TTGAAGATAA GAGTGCATGA GGGATATGAA GAGTTGACAA TGGTTGGGAG AAGAGCAACA GCCATACTCA GAAAAGCAAC CAGGAGATTG ATTCAGCTGA TAGTGAGTGG GAGAGACGAA CAGTGGATTG CCGAAGGAAT AATTGTGGCC ATGGTATTTT CACAAGAGGA TTGTATGATA AAAGGAGTCA GAGGTGATCT GAATTTGGTG AATAGGGCGA ATCAACGATT GAATGCTATG GATGAAGTTT TAAGACATTT TCAGAAGGAT GCGAAAGTGC TTTTTCAAAA TTGGGGAGTT GAAGCTATGG AGAATGTGAT GGGAATGATT GGGATATTGC CGGACATGAC TCGAAGCATC GAGATGTGAA TGAGAGGAGT GAGAATGAGC AAAATGGGTG TAGATGAGTA GTCGAGCACG

GTAGTGTGTC CGGAGGAGGT CAGTGAAAGA GAGGGAACAG AG AAACT GAG AATAAGTTAG TCATCGTCAA TGATGTGGGA GATTAATGGT CGTGAATCAG TGTTGGTGAA TACGT ATGAA T GGATCATCA GAAAGTGGGA AAGTGTTAAA ATTCAGTGGT CCCAGAACGG TACAATGGTA TACAATAAAA TGGAATTTGA AGCATTTGAG TCTTTAGTAG GTAAGGCCAT TAGAGGGCAA TACAGTGGGT TTGTAAGAAC TCTGTTGGAA GAAATGAGGG ATGTGGTTGG GAGATTTGAT ACCGCAGAGA TAATAAAAGT TGTTCGCTTC GCAGCCGGTC CACCAAAGGA AAGTAGAATG CAGTTCTGCT CATTTACTGT GAATGTGAGG GGATCAGGAA TGAGAATAGT TGTAAGGGGC AATTCTCCTG TATTCAACTA TAACAAGGGC AGGAAGAGAG TCACAGTTGT CGGAAAGGAT GCTGGCACTT TAAGTGAAGA GCGAG AT GAA GGCACAGGTG GAGTGGAGTG CGCTGTTCTG AGGGGATTCC TCATTGTGGG CAAAGAAGAC AAGAGATATG GGGCAGCAGT AAGGATGAAT GAAGTGAGCA AGCTTGCGAA AGGAGAGAAG GCTAATGTGG TAATTGGGCA AGGAGACGTG GTGTTGGTAA TGAAAGGGAA ACGGGACTCT AGGATAGTTA CTGACAGCGA GACAGCGACG AAAAGAATTC GGATGGCCAT CAATTAGTGT CGAATAGTTT AAAAAGGACC TTGTTTCTAC T (SEQ

AGCAAAAGGA GGGTAGATAA TGACTGAGTG AGTGACATCA AAATCATGGG GTCTGAAGGG AGGAAAGGAT GTTAGGAACA GATGGAGAGT GATGGAGAAG GGCAGAATGC GAGTGAAATG AGAGCATCGG TCGGAAAAAT GATTGGTGGA ATTGGACGAT TCTACATGCA AATGTGGACG GAACTGAAAC TGAGTGATTA TGAGGGAGGG TTGATCGAAA AGAGGTTAAG AATAGAGAGA ATGGTGGTCT CTGGTTTTGA GGAAAGGAGA AATAAATAGC TTGAAGAAGA TGCGAGTGCG GGGAAAGATC CTAAGAAAAC TGGAGGACCT ATATACAGGA GAGTAAAGGG AAAGTGGATG AGAGAACTCA TCGTTTATGA CAAAGAAGAA ATAAGGCGAA TCTGGCGCCA AGGTAAT AAΪ GGTGACGATG CAAGGGCTGG TCTGACTCAG ATGATGATCT GGCATTCCAA TTTGAAT GAT GCAAGTTATG AGAGGACAAG AGCTGTTGTT CGCACCGGAA TGGATGCGAG GATGTGGTCT CTGATGCAAG GTTCAACTCT CGCTAGGAGG TCTGGAGCGG GAGGTGCTGC AGTCAAAGGA GTTGGAACAA TGGTGATGGA ATTGGTCAGA ATGATCAAAC GTGGGATCAA TGATCGGAAC TTCTGGAGGG GTGAGAATGG ACGAAAAACA AGAATTGCTT ATGAAAGAAT GTGCAACATT GT CAAAGGGA AATTT CAAAC TGCTGCACAA AAAGCAAT GA TGGATGAAGT GAGAGAGAGC CGGAACGCAG GGAATGCTGA GTTGGAAGAT CTCACTTTTC TAGCACGGTG TGGACTGATA TTGAGAGGGT CGGTTGGTCA CAAGTCGTGC CTGCGTGCGT GTGTGTATGG AGCTGCCGTA GCCAGTGGGT ACGACTTTGA AAGGGAGGGA TACTCTGTAG TCGGAATAGA CCCTTTCAGA CTGCTTCAAA ACAGCCAAGT GTACAGCCTA ATCAGAGCAA ATGAGAATCC AGGACACAAG AGTGAAGTGG TGTGGATGGC ATGCCATTCT GCCGCATTTG AAGATCTAAG AGTATTAAGG TTGATGAAAG GGAGGAAGGT GGTCGCAAGA GGGAAGCTTT CGAGT AGAGG AGTTGAAATT GCTTGCAATG AAAAT AT GG A GACTATGGAA TCAAGTAGAC TTGAACTGAG AAGGAGGTAC TGGGCGATAA GGACGAGAAG TGGAGGAAAC AGCAATCAAC AGAGG GCATG TGCGGGCGAA ATCAGCATAG AACCTAGGTT GTCAGT ACAG AGAAATGTCG GTTTTGACAG AAGAACCATT ATGGGAGCAT TCAATGGGAA TACAGAGGGG AGAACATGTG AGATGAGGAG GGAAATGATA AGGATGATGG AAAGTGGAAG ACGAGAAGAT GTGTGTTTGC AGGGGCGGGG AGTGTTCGAG CTGTGGGAGG AAAAGGCAGC GAGGCCGATC GTGCCTTCGT TTGACATGAG T AATGAAGG A TCTTATTTCT TGGGAGACAA TG GAG AG GAG TAGGACAATT AAAGAAAAAT AGGCTTGTTT CTAGT (SEQ ID NQ:27)

M

AGCAAAAGCA GGTAGATATT GAAAGATGAG TCTTGTAACG GAGGTCGAAA CGTACGTAGT GTCTATCATC CCGTGAGGCC CCCTCAAAGC CGAGATCGCA CAGAGACTTG AAGATGTCTT TGCAGGGAAG AACACGGATC TTGAGGTTCT GATGGAATGG CTAAAGACAA GACCAATCGT GTCACGTCTG ACTAAGGGGA TTTTAGGATT TGTGTTCACG CTCACCGTGG CCAGTGAGCG AGGACTGCAG CGTAGACGCT TTGTCGAAAA TGCCCTTAAT GGGAACGGGG ATCCAAATAA CATGGAGAAA GCAGTTAAAG TGTATAGGAA GCTGAAGAGG GAGATAAGAT TGCATGGGGC GAAAGAAATG T GACT GAG IT ATTCTGGTGG TGGACTTGGC AGTTGTATGG GCGTCATATA

CAACAGGATG GGGGCTGTGA CGACTGAAGT GGCATTTGGG CTGGTATGTG GAAGCTGTGA AGAGATTGGT GAGTCGCAGG ATCGGTCTGA TAGGGAAATG GTGACAACAA GCAATCGAGT AATGAGAGAT GAGAACAGAA TGGTTTTAGC CAGGAGTACA GCTAAGGCTA TGGAGGAAAT GGCTGGATGG AGTGAGGAAG GAGCAGAGGG GATGGAGGTT GCTAGTGAGG CTAGACAAAT GGTGGAAGCG ATGAGAAGCA TTGGGAGTGA TGCTAGCTCC AGTGGTGGTC TGAAAAATGA TCTTGTTGAA AATTTGGAGG CCTATGAGAA ACGAATGGGG GTGGAGATGC AACGGTTCAA GTGATCCTCT CACTATTGCC GCAAATATCA TTGGGATGTT GCACTTGACA TTGTGGATTC TTGATCGTCT TTTTTTCAAA TGCATTTACC GT GGCTTTAA ATACGGACTG AAAGGAGGGG GTTCTACGGA AGGAGTGCCA AAGTGTATGA GGGAAGAATA TCGAAAGGAA CAGCAGAGTG CTGTGGATGC TGACGATGGT CATTTTGTCA GCATAGAGCT GGAGTAAAAA AGTACCTTGT TTCTACT (SEQ ID NO:28)

MS

AGCAAAAGCA GGGTGACAAA AACATAATGG ATGCAAACAC TGTGTCAAGG TTTCAGGTAG ATTGCTTTCT TTGGCATGTC CGCAAAGGAG TTGCAGACGA AGAACTAGGC GATGGCGCAT TCCTTGATCG GGTTCGCCGA GATCAGAAAT CCCTAAGAGG AAGGGGGAGT ACTCTCGGTC TGGACATCAA GAGAGCCAGA CGTGCTGGAA AGCAGATAGT GGAGCGGATT GTGAAAGAAG AATGGGATGA GGGACTTAAA ATGAGCATGG CCTCTGTACC TGCGTCGCGT TACCTAACTG ACATGACTGT TGAGGAAATG TGAAGGGACT GGTGCATGGT GATAGCGAAG CAGAAAGTGG GAGGCGCTGT TTGTATGAGA ATGGACGAGG CGATGATGGA TAAGAAGATG ATACTGAAAG CGAACTTGAG TGTGATTTTT GAGCGGGTGG AGACTGTAAT ATTGCTAAGG GCTTTGAGCG AAGAGGGAGG AATTGTTGGG G AAATTT GAG GATTGCGTTC TCTTGCAGGA CATAGTGGTG AGGATGTCAA AAATGCAGTT GGAGTGCTGA TCGGAGGACT TGAATGGAAT GATAAGAGAG TTGGAGTCTG TGAAACTGTA GAGAGATTGG GTTGGAGAAG CAGTAATGAG AATGGGAGAG CTGCACTGAG T CGAAAACAG AAACGAGAAA TGGCGGGAAG AATTAGGTCA GAAGTTT GAA GAAATAAGAT GGTTGATTGA AGAAGTGAGA GAGAAACTGA AGATAAGAGA GAATAGTTTT

GAGGAAATAA CATTTATGGA AGCGTTAGAT GTATT GGTTG AAGTGGAGGA AGAGATAAGA AGTTTCTGGT TTCAGGTTAT TTAGTACTAA AAAAGACCCT TGTTT CTACT {SEQ ID NO:29)

Exemplary Neuraminidase Modifications

Materials Viruses: Y2017: A/Yokohama/2017/2003 (H3N2)

HK4801 : A/Hong Kong/4801 /2014(H3N2)

Y2Q17-M3L.4: Y2017 passaged 7 times in eggs HY-PR8: high yield PR8 (H1 N1)

Results Y2017 virus was passaged 7 times in eggs (3 times in the amniotic cavity, fo!lowed by 4 times in the alianioic cavity). A progeny virus, Y2017-M3L4, grew efficiently in the allantoic cavity (10 ⁷ to about 10 ^s PFU/mL), whereas the original Y2017 virus did not grow at aii (<10 PFU/mL).

Mutations observed In Y2017-M3L4 virus were as follows: Table 1

A comparison of the growth ability of mutant Y2017 viruses, generated by reverse genetics, in allantoic fluid revealed that NA mutations were responsible for the high growth of Y2Q17- M3L4 virus (Figure 4) A plasmid with PB2-T 147I was used for virus generation (PB2-T 147I, V344L and PB2-T147I, V344L, E358K were not analyzed). Mutations were not observed in the HAgene of the virus possessing a mutated NA segment and its other genes from wild-type Y2017 after replication in a!!antoic fluid {Figure 4).

Figure 5 shows the location of the NA mutations in Y2017-M3L4 in a 3D mode!.

Comparison of the growth ability of Y2017 viruses with NA mutations revealed that NA-D147N, N329D, and H347Q generally contributed to the increased growth ability in allantoic fluid (Figure 6).

The NA of Y2017-M3L4 allowed virus possessing HK4801 HA to replicate efficiently in the allantoic cavity and the HY-PR8 backbone further enhanced the growth of this virus (Figure 7).

In summary, described herein are influenza virus mutations that inhibit (e.g., prevent) the acquisition of antigenicity-compromising mutations In the hemagglutinin (HA) protein of influenza during growth In eggs and/or allow for enhanced replication. In one embodiment, the mutations are within the neuraminidase (NA) viral segment of human influenza viruses, and the mutant NA proteins stabilize the HA protein during egg-passages. Thus, in the presence of the mutant NA proteins, the HA protein does not acquire egg-adapting mutations. In some cases, the respective mutations in NA can also increase the yield of vaccine viruses.

Analysis of the growth capability of NA mutant viruses revealed that NA-D147N, N329D, and H347Q contribute to the increased growth capability of the viruses in allantoic fluid (Figure 12). HA mutations were not observed in the virus possessing HK4801HA, Y2017-M3L4NA, and the HY-PR8 backbone (Figure 13) after 3 passages in the allantoic cavity.

By passaging an HY-PR8 backbone virus possessing HK4801 NA (T148K and the saturated mutations N329X and H347X) and HK4801 HA in eggs, a virus possessing HK48Q1NA (T148K, D151 E, H347G, and T369K) emerged that replicated efficiently in the allantoic cavity (Figure 14;

4M=T 148K, D151 E, H347G, and T369K) HA mutations were not observed during passages in eggs (1 x in the amniot!c cavity then 5x in the allantoic cavity).

HK48G1 NA (T148K, D151 E, H347G, and T369K) conferred efficient replication in the aiiantoic cavity to HY-PR8 backbone viruses possessing either HK4801 HA or SingaporeOOI 9HA. Virus inoculation: 2 x 1 Q ³ pfu/egg into aiiantoic fluid, 72 h incubation at 37°C (Figure 16).

The HA coding nucleic acid sequence and NA coding nucleic acid and amino acid sequences for SingaporeOOI 9 are as follows:

A''S/ngrapofe/INFiNH-16-Q019/2016(H3N2) HA atgaagactatcattgctttgagctacattctatgtctggttttcgctcaaaaaattcct ggaaatgacaatagcacggcaacgctgtgccttgggcacca tgcagtaccaaacggaacgatagtaaaaacaatcacaaataaccgaattgaagttactaa tgctactgagttggttcagaattcctcaataggtgaa atatgcgacaatcctcatcagatccttgatgaagagaactgcacactaatagatactcta ttgggagaccctcaatgtgatggctttcaaaataaaaa atgggacctttttgttgaacgaagcaaagcctacagcaactgttacccttatgatgtgcc ggattatgcctcccttaggtcactagttgcctcatccggca cactggagtttaaaaatgaaagcttcaattggactggagtcactcaaaacggaacaagtt ctgcttgcataaggggatctagtagtagtttctttagtag attaaattggttgacccacttaaactacacatatccagcattgaacgtgactatgccaaa caaggaacaatttgacaaattgtacatttggggggttca ccacccgggtacggacaaggaccaaatcttcctgtatgctcaatcatcaggaagaatcac agtatctaccaaaagaagccaacaagctgtaatcc caaatatcggatciagacccagaafaagggatatccctagcagaataagcatciattgga caaiagtaaaaccgggagacatacfttigataacag cacagggaatcfaatigctccfaggggitacticaaaatacgaagigggaaaagctcaat aatgagatcagatgcacccattggcaaaigcaagict gaatgcatcactccaaatggaagcattcccaaigacaaaccaitccaaaatgtaaacagg atcacatacggggcctgtcccagatatgtiaagcaf agcacictgaaattggcaacaggaatgcgaaatgfaccagagaaacaaactagaggcaia ittggcgcaafagcgggtttcatagaaaaiggttg ggagggaatggtggatggttggtacggtttcaggcatcaaaattctgagggaagaggaca agcagcagatctcaaaagcactcaagcagcaatc gatcaaatcaatgggaagctgaataggttgatcggaaaaaccaacgagaaattccatcag attgaaaaagaattctcagaagtagaaggaagag ttcaagaccttgagaaatatgttgaggacactaaaatagatctctggtcatacaacgcgg agcttcttgttgccctggagaaccaacatacaattgatct aactgacicagaaatgaacaaacigitigaaaaaacaaagaagcaacigagggaaaaigc tgaggatatgggaaaiggitgtitcaaaaiaiacc acaaatgtgacaatgcctgcatagaatcaataagaaatgaaacttatgaccacaatgtgt acagggatgaagcattgaacaaccggttccagatca agggagitgagctgaagtcaggatacaaagatiggatcctaiggatttcctiigccaiai catgiittitgcitigtgttgciitgtiggggitcaicatgigggc cigccaaaagggcaacaitagatgcaacaittgcaittga (SEQ ID NO:46)

A/S/ngapore/INFINH-16-0019/2016(H3N2) NA atgaatccaaatcaaaagataataacgattggctctgttctctcaccatttccacaatat gcttcttcatgcaaattgccatcctgataactactgtaacat tgcattcaagcaatatgaatcaactcccccccaaacaaccaagtgatgctgtgigaacca acaataatagaaagaaacataacagagatagigt atttgaccaacaccaccatagagaaggaaatatgccccaaaccagcagaatacagaaatt ggtcaaaaccgcaatgtggcatacaggatitgca ccttctctaaggacaattcgattaggctttccgctggtggggacatctgggtgacaagag aaccttatgtgtcatgcgatcctgacaagtgttatcaatt† gcccttggacagggaacaacactaaacaacgtgcattcaaataacacagtacgtgatagg accccttatcggactctattgatgaatgagtgggtgt tcctttccatctgaggaccaagcaagtgtgcatagcatggtccagctcaaattgtcacga tagaaaagcatggctgcatatttgtataacgggagatg ataaaaatgcaactactagcttcatttacaatgggaggcttatagatagtgttgtttcat agtccaaagatattctcaggacccaggagtcagaatgcgtt tgtatcaatggaacttgtacagtagtaatgactgatggaaatgctacaggaaaagctgat actaaaatactattcattgaggaggggaaaatcgttcat actagcaaattgtcaggaagtgctcagcatgtcgaagagtgctcttgctatcctcgatat cctggtgtcagatgtgtctgcagagacaactggaaagg atccaaccggcccatcgtagatataaacataaaggatcataacattgtttccaattatgt gtgttcaggacttgttgaagacacacccagaaaaaacg acagctccagcagtagccattgtttgaatcctaacaatgaagaaggtggtcatggagtga aaggctgggcctttgatgatggaaatgacgtgtggat ggggagaacaatcaacgagacgtcacgcttagggtatgaaaccttcaaagtcgttgaagg ctggtccaaccctaagtccaaattgcagataaata ggcaagtcatagttgacagaggtgataggtccggttattctggtattttctctgttgaag gcaaaagctgcatcaatcggtgcttttatgtggagttgattag gggaagaaaagaggaaactgaagtciigiggaccicaaacagtatgitgtgtitigtggc acctcaggtacataiggaacaggcicatggcctgaig gggcggaccicaatcicatgcaiaiaiaa (SEQ ID NO:47) which encodes

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K I L F S E E G K I V H T S K t SG S A <3 H V E S € S G ¥ P R Y P Q V R CV € R D N W 0 SN R P i V D I N S K □ H S ί V 8 S Y V OS 8 L V 3 OT P R K N D S S S S3 H CL R P N N E E 38 H 8 ¥ K 8 AF GNOVWMGRTi £TSrfl<3YETFKVV£GWSNP 8KlOINR VIVDR aDReOYSGIrSVEGKeCiNRCrYVELmGRKEETEVlWTSNSIYVFCGTS G T Y G T G S W POGAOLNLMHI (SEQ ID NO:48),

MA mutations T153N, M329T, and T369K aiiowed A/Saitama/102/2014 (H3N2) to replicate efficiently in the allantoic cavity (Kuwahara et ai., 2018) Therefore, the effect of introducing NA-T153N, N329T (or D), T369K, and H347Q into HK48Q1NA (T148K) was examined. Figure 18 reports on virus titers for different combinations of NA residues identified in screenings. Figures 19 and 20 report on virus titers for viruses with different combinations of selected NA residues.

Alaska/232/2015_HY-PR8 (H3N2) WT/mutant virus were passaged in eggs and HA and NA segments sequenced. Alaska WT (a more recent H3N2 virus where WT has 245N, prior to 2015 H3N2 WT viruses had 245S), HA-R142S, -K189E viruses did not get mutations in HA, even after 3 amniofic and 10 allantoic passages. HA-K189E/N158KA212T mutant did not get mutations in HA, but had some mutations in NA which exhibited improved growth in eggs since r6 (Figure 21). The difference of NA mutations between p4 (normal growth) (NA-N245S mutation, virus grows more than 1000 fold better than with NA-245N) and p6 (better growth) was G346V (Figure 22). Therefore, G348V may also contribute to adaptation to eggs. The NA for A/Alaska/232/2015 has the following sequence: mnpnqkiiti gsvsitisti cffmqiaiii iivt!hfkqy efnsppnnqv m!eepiiier niieivyltn ttiekeicpk paeyrnwskp qcgitgfapf skdnslr!sa ggdiwvirep yvscdpdkcy qfalgqgtil nnvhsnntvr drtpyrtllm ne!gvpfh!g tkqvciawss sschdgkaw! hvcitgddkn atasfiyngr !vdswswsk di!rtqesee vcingtctw mtdgrsatgka dtkilfieeg kivhtsklsg saqhveecsc yprypgvrcv crdnwkgsnr pivdinikdh sivssyvcsg ivgdtprknd ssssshclnp nneegghgvk gwafddgndv wmgrtinets r!gyeifkvv egwsnpkskl qinrqvivdr gdrsgysgif svegkscinr cfyve!irgr keetevlwts nsiwfegts gtygtgswpd gad!nlmhi (SEQ ID NO:49)

IMA pHH21 plasmids were constructed: Alaska NA-T148K/D151 E/N245S (found in E4); Alaska NA-G346V; and Alaska NA-T148K/D151 E/N245S/G346V (found in E6) Mutant NAs were combined with WT Alaska HA or HY-PR8 backbone. Eggs were inoculated with the same dosage of WT/mutant Alaska viruses and harvested viruses titrated (Figure 23). NA-T148K/D151 E/N245S/G346V mutant virus grew to a higher titer than WT virus but the single mutation G346V did not increase virus growth compared to WT. These results suggested that a combination of G346V and one (or two to three) other mutations, e.g., 3 mutations such as T14SK, D151 E and N245S, may be important for virus Alaska virus to grow efficiently in eggs. Harvested virus samples with high titer (>5 Log 10 PFU/mL) were sequenced however none had additional mutations in HA and NA.

The invention will be described by the following non-limiting examples.

Example S

As shown in Figures 25-28, certain substitutions in N2 stabilized HA (e.g., did not allow for substitutions in HA) for up to about 8 passages in eggs in various H3N2 isolates from different influenza seasons. However, HA substitutions were found in some but not all isolates passaged in eggs for 10 passages (Figure 29). Unexpectedly another change in NA (1481) was correlated with stabilizing HA even after more than 8 passages. The presence of that additional change in some cases resulted In a HA change but that change, in the stem region of HA (Figures 32), is unlikely to alter antigenicity.

1481 or 148K was introduced into the NA along with other substitutions that were identified (Figures 33-35). The addition of 1481 did not substantially alter virus flier in eggs while 148K In some cases impacted titer. Interestingly, viruses having a NA with 1481 in combination with other changes had reduced sia!idase activity (Figure 36). For example, viruses having a NA with 1481, 151 E, 245S, 347G, and 369K and 1481, 150S, 151 E, 245S, 347G and 369K, as wei! as viruses having a NA with 148K, 151 E, 245S, 347G and 369K, had reduced sialidase activity.

Example ll

Mutations in the influenza surface glycoprotein neuraminidase (NA) confer efficient replication to recent human H3N2 viruses in eggs without the acquisition of mutations at the antigenic sites of the other surface glycoprotein, (hemagglutinin) HA. With NA mutations, the mutant NAs recognize sialic acid linked to galactose via alpha 2-3 linkages (Siaa2-3 Gal) prevalent on epithelial cells In the chorioallantoic membrane in chicken eggs. The NA mutations allow the viruses to attach and enter cells even under conditions where the interaction between HA and its receptor is inhibited (Figure 38), suggesting that the mutant NA serves as a receptor-binding protein in place of HA.

By possessing an HA protein with disrupted or no receptor-binding activity, the disclosed mutant NA may confer to influenza viruses such as H3N2 viruses efficient growth in embryonated chicken eggs without the acquisition of any egg-adaptive HA mutations at antigenic sites. Because HA does not encounter selective pressure to recognize the Siao2-3 Gai receptor in the presence of the mutant NA, the amino acid residues around the HA receptor-binding pocket remain unchanged during passages in embryonated chicken eggs.

To this end, HA proteins are constructed that lacked or possessed limited receptor- binding activity but retained their antigenicity by Introducing three mutations, e.g., Y98F, VV153A, and H183F, at sites located Inside the receptor-binding pocket that would not affect the antigenicity of the HA (Figures 39A-B).

Example ill

Exemplary NA residues were found in egg-grown A' ^' Hong Kong/4801 /2014 and A/Alaska/232/2015 (“6M”). introducing 6M mutations into the NA of A/Yokohama/48/2018 and A/Yokohama/147/2017 enhanced HY-PR8-backbone virus growth. Therefore, the effect of introducing 6M mutations into other strains and the etfect of possessing Yokohama147NA(6M) on the growth of viruses possessing HA from other strains was examined (Figures 43-44) Harvested viruses possessing each strain's NA(6M) or Yokohama/147/2017NA(6M) were sequenced. None had additionai mutations in HA and NA were observed.

Viruses possessing A/Yokohama/147/2017 NA(6M) acquired HA-D225G and K27E after passage 10. HA-K27Eis located in the stem region of HA protein, suggesting that K27E was unlikely to alter HA antigenicity. HA-D225G is located near receptor binding site. However, the reactivity of an H3N2 virus possessing HA-D225N with ferret antiserum differed from that of the wild-type virus by only two-fold, suggesting that HA-D225G alone was unlikely to alter HA antigenicity substantially (Chambers et a!., Cell Rep. 2015).

It was analyzed whether the viruses possessing Yokohama147NA(6M) and HA from other strains can also replicate without acquiring HA mutations at major antigenic sites during egg passages. During egg passages, many of the tested viruses acquired HA-D225G but none of them acquired HA mutations at major antigenic sites. G479E, K453N, E484G (located in the stem region) and R545K (located in the cytoplasmic tail) were unlikely to alter HA antigenicity (Figure 45).

The mechanism of how the NA(6M) mutant viruses can replicate efficiently in eggs was investigated. VP40-induced VLPs bearing FLAG-tagged Yokohama147NA or Yokohama147NA(6M) were prepared. Immunoblotting analysis with anti-FLAG and anti-VP40 antibodies showed reduced molecular weight of Yokohama147NA(6M) protein compared to that of wiid-type Yokohama147NA protein (Figure 48). Figure 49 shows another western blotting analysis suggesting the loss of g!ycosy!ation site of mutant NA protein due to the introduction of 6M mutations.

Next the receptor-binding specificities of Yokohama147HA, Yokohama147NA, and Yokohama147NA(6M) were analyzed using in a glycan microarray containing a library of a2-3 and a2-6 sialosides, including N-!inked glycans representative of fhose found on chorioallantoic membranes of eggs. The analysis showed Yokohama147NA(6M) bound to a2-3 sialosides found on chorioallantoic membranes of eggs (Figure 50). It was determined whether 6M mutations alter the NA sialidase activity (Figure 51). Ebola VP40- based VLPs bearing Yokohama147NA or NA(6'n') were serially diluted, incubated with the siaiidase substrate 4-MUNANA, and the released 4-MU was quantified to assess sialidase activity. The analysis revealed that introduction of 6M into Yokohama! 47NA decreased its sialidase activity. To identify further NA mutations that can allow viruses replicate efficiently in eggs without depending on HA receptor binding activity, HY-PR8 backbone viruses were generated that possess HA(de! RBS) and A/Kansas/14/2017NA(6M) and then were passaged in eggs. During the passages, a mutant NA was obtained (T148I, D151 E, N245S, T329S, K344E, G346V, H347G and T369K) (=6M+T148I+T329S+K344E). HY-PR8 backbone viruses possessing wild type HA and NA(6M+T148I+T329S+K344E) from A/Kansas/14/2017 were prepared and then analyzed to determine if the virus acquired the HA mutations during passages in eggs. The virus possessing NA(6M+T148I+T329S+K344E) did not acquire any of HA and A mutations during 10 egg passages (Figure 52).

Examo!e IV

In one embodiment, an isolated recombinant influenza virus is provided comprising a selected NA viral segment encoding a plurality of selected residues or a deletion of residues in NA, wherein the selected NA viral segment does not encode a NA having a threonine (T) or lysine (K) at residue 148, and does not encode a threonine at residue 32, an aspartic acid (D) at position 151, an asparagine (N) at position 245, an asparagine at residue 329, a glycine (G) at position 346, a histidine at residue 347, or includes a NA having a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering Is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148 and has one or more of a threonine at residue 32, does not have a deletion of residues 46 to 50, an aspartic acid at position 147, an aspartic acid at residue 151 , an asparagine at residue 245, an asparagine at residue 329, a glycine at residue 346, a histidine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode a NA having a threonine at residue 148, and does not encode a NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding Influenza virus that has a NA that encodes a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 389, or any combination thereof in one embodiment, the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, and does not encode a NA having an aspartic acid at position 151 , an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering Is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, an aspartic acid at residue 151 , an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment encodes a NA having an isoieucine (I), leucine (L), glycine or alanine (A) at residue 148. In one embodiment, the isolated recombinant influenza virus is a reassortant. In one embodiment, the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In one embodiment, the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEG ID NQ:2. In one embodiment, the NA viral segment encodes a N2, N3, N7, or N9. In one embodiment, the NA viral segment encodes a N1 , N4, N5, N6, N8, N10 or N11. In one embodiment, the residue at position 32 is A, I, G, or L wherein the deletion is a deletion of residues 46 to 50, wherein the residue at position 147 is N or glutamine (Q), wherein the residue at position 329 is D or glutamic acid E, or wherein the residue at position 346 is serine (S), T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L. In one embodiment, the residues at position 346 is V, S, I or L. In one embodiment, the residue at position 148 is I. in one embodiment, the residue at position 151 is E, N or Q. In one embodiment, the residue at position 245 Is S, T, I, L, A, N, W, Y, P, V, or G. In one embodiment, the residue at position 347 is G, Q, S, T, Y, C or W. In one embodiment, the residue at position 369 is K, H, R, E, P, or D. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 347 is G, Q, S, T, Y, C or W, or any combination thereof. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 347 is G or Q, or any combination thereof. In one embodiment, the residue at position 148 is K, R or H, the residue at position 151 Is E, N or Q, the residue at position 245 Is S, T, I, L, A, W, Y, P, V, or G, or any combination thereof in one embodiment, the residue at position 148 is K, R or H, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, or V, and/or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, or any combination thereof. In one embodiment, the selected NA viral segment does not encode a NA having a threonine at position 148, does not encode a NA having an aspartic acid at position 151 , does not encode a NA having an asparagine at position 245, does not encode a NA having a histidine, arginine or an asparagine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode a NA having an aspartic acid at position 147, does not encode a NA having an asparagine at residue 329, does not encode a NA having a histidine, arginine or asparagine at residue 347, or any combination thereof. In one embodiment, the selected NA viral segment does not encode a NA having a threonine at position 148, does not encode a NA having an aspartic acid at position 151, does not encode a NA having an asparagine at position 245, does not encode a NA having a glycine at position 346, or any combination thereof. In one embodiment, the HA is H1, H3, H5, H7, or H9. In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1 , PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44. In one embodiment, the PB2 has I, A, L, or G at residue 147.

Also provided, in one embodiment, is an isolated recombinant nucleic acid comprising a nucleic acid sequence for an influenza virus NA viral segment that encodes a NA having a plurality of selected residues or a deletion of residues, wherein the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, and does not encode a threonine at residue 32, an aspartic acid at position 151 , an asparagine at position 245, an asparagine at residue 329, a glycine at position 346, a histidine at residue 347, or include a NA having a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering is based on N2. In one embodiment, the selected NA viral segment does not encode a NA having a threonine at residue 148, and does not encode a NA having an aspartic acid at position 151 , an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, and does not encode a NA having an aspartic acid at position 151, an asparagine at position 245, a histidine at residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication In avian eggs or has a reduction In HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, an aspartic acid at residue 151 , an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA viral segment encodes a NA having an isoleucine (I), leucine (L), glycine (G) or alanine (A) at residue 148. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NG:1, SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the NA is a N2, N3, N7, or N9. In one embodiment, the NA is a N1 , N4, N5, N6, N8, N10 or N11. In one embodiment, the HA is H1 , H2, H3, H5, H7, or H9. In one embodiment, the residue at position 32 is A, I, G, or L, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A,W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A,W, Y, P, V, or G, the residue at position 329 is S, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 Is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NG:49, or at least 90% amino acid sequence identity to a NA encoded by one of SEQ ID Nos. 51-59 or 69-70.

In one embodiment, a method to prepare influenza virus is provide comprising: contacting a cell with: a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNA production are from one or more influenza vaccine virus isolates, wherein the NA DMA in the vector for vRNA production encodes a MA having a plurality of selected residues or a deletion of residues, wherein the selected NA viral segment does not encode one or more of: a threonine or lysine at residue 148, a threonine at residue 32, an aspartic acid at position 151 , an asparagine at position 245, an asparagine or threonine at residue 329, a lysine at residue 344 a glycine at position 346, a histidine at residue 347, or Include a NA having a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering is based on M2; and a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1 , a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for rnRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1 , a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus. In one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NQ:3, SEQ ID NQ:48 or SEQ ID NO:49. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, the HA is HI, H3, H7, or H9. In one embodiment, the HA is H2, H4, H5, H6, H8, or any of H10-H18. In one embodiment, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 347 is G, Q, N, S, T, Y, G or W, or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V. In one embodiment, the residue at position 151 is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A,W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, D, E, or R, or any combination thereof. In one embodiment, the PA, PB1 , PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44.

Further provided is a method of immunizing an avian or a mammal, comprising: administering to the avian or the mammal a composition having an effective amount of the virus described herein. In one embodiment, the composition comprises at least one other different influenza virus. In one embodiment, the amma! is a human. In one embodiment, the composition is administered infranasaiiy or via injection.

Viruses described herein may be passaged in eggs or other cells.

Exemplary backbone viral segments include but are not limited to: PB2, M202L, F323L; PB1, Q247H; PA, K142N; NP, R74K; M,V97A, Y1G0H; and NS, K55E, or PB2, 1504V; FBI, M4QL/G180W: PA, R401 K; NP, 11161. and NS1, A30P/R118K.

Example V

In one embodiment, a method to decrease influenza HA binding to cells is provided that includes altering one or more residues in the HA binding pocket of HA that binds to sialic acid on allantoic membranes. In one embodiment, nucleic acid encoding the HA is altered in one embodiment, the HA is H1 , H3, H7, or H9. In one embodiment, the HA is H2, H4, H5, H6, H8, or any of H10-H18. In one embodiment, the residue at position 98, 153 or 183 of HA is altered based on the numbering of H3 HA. in one embodiment, the residue at position 98 is not Y. In one embodiment, the residue at position 153 is not W. In one embodiment, the residue at position 183 is not H. In one embodiment, the residue at position 98 is F, G, I, V, T, H, W, or L. In one embodiment, the residue at position 153 is A, G, I, V, T, or L in one embodiment, the residue at position 183 is F, A, G, I, L, V, Y, W, P, or T.

In one embodiment, a method to prepare an influenza virus that binds to ceils via Influenza neuraminidase is provided that includes providing a vector comprising a recombinant nucleic acid molecule comprising sequences for an influenza virus HA segment from a first influenza virus isolate, which segment encodes an HA with an amino acid other than tyrosine at position 98 in HA1 , other than tryptophan at position 153 in HA1 , other than histidine at position 183 in HA1, or any combination thereof, wherein the numbering for HA1 residues is that for H3; modifying the HA segment to encode F, G, I, V, T, H, W, or L at position 98, encode A, G, I, V, T, or L at position 153, encode F, A, G, I, L V, Y, W, P, or T at position 183, or any combination thereof, thereby yielding a modified HA segment; and contacting a ce!! with a vector comprising promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or ifs complement operably linked to an influenza virus PB1 segment DMA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PB2 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to the modified HA segment linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NP segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields fu!! length, genomic influenza virus RNA or its complement operably linked to an influenza virus NA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus M segment DNA linked to a transcription termination sequence, and a vector comprising a promoter that yields full length, genomic Influenza virus RNA or its complement operably linked to an influenza virus NS segment DNA linked to a transcription termination sequence; and a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding Influenza virus PA, a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus PB1 , a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus M1 , a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus M2, or a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus NS1 or a vector comprising a promoter that yields rnRNA operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus that binds to cells via the NA.

Example VI

In one embodiment, an isolated recombinant influenza virus comprising a selected NA viral segment encoding a plurality of selected residues, a HA viral segment, and one or more of a PB1 viral segment, a PB2 viral segment, a PA viral segment, a NP viral segment, a M viral segment and a NS viral segment. In one embodiment, the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid (D) at position 151 , does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and/or does not encode a threonine at position 369, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication In avian eggs, has reduced sia!idase activity, has increased binding to certain sialic acid residues and/or has a reduction in HA mutations when grown In avian eggs relative to a corresponding Influenza virus that has a NA that encodes a threonine or lysine at residue 148, encodes an aspartic acid at residue 151, encodes an asparagine at residue 245, encodes a threonine at residue 329, encodes a lysine at residue 344, encodes a glycine at residue 346, encodes a histidine at residue 347, or encodes a threonine at position 369, or any combination thereof. In one embodiment, the NA segment of the recombinant virus has at position 329 a serine (S), valine (V), alanine (A), G, cysteine (C), methionine (M), isoleucine (!) or leucine (L) or at position 346 a V, S, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L. in one embodiment, the NA segment of the recombinant virus has at position 148 an I. in one embodiment, the NA segment of the recombinant virus has at position 151 an E, N or Q. in one embodiment, the NA segment of the recombinant virus has at position 245 a S, T, i,

L, A, W, Y, P, V, or G. in one embodiment, the NA segment of the recombinant virus has at position 329 a S, I, L, A, W, Y, P, V, or G. In one embodiment, the NA segment of the recombinant virus has at position 344 an E, H, D, N or Q. in one embodiment, the NA segment of the recombinant virus has at position 346 a V, S, T, I, L, A, W, Y, or P. in one embodiment, the NA segment of the recombinant virus has at position 347 a G, Q, S, T, Y, G or W. In one embodiment, the NA segment of the recombinant virus has at position 369 a K, H, R, E, P, or D. In one embodiment, the recombinant virus is a reassortant. In one embodiment, the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NQ:48, or SEQ ID NQ:49, or has at least 90% amino acid sequence identity to a NA encoded by any one of SEQ ID Nos. 51-59. In one embodiment, the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEQ ID NQ:2. In one embodiment, the NA viral segment encodes a N2, N3, N7, or N9. In one embodiment, the NA viral segment encodes a N1 , N4, N5, N6, N8, N10 or N11. In one embodiment, the HA is H2 or H3 In one embodiment, the virus is an influenza A virus. In one embodiment, the PA, PB1 , PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44. In one embodiment, PB2 has I, A, L, or G at residue 147.

Further provided is an isolated recombinant nucleic acid comprising a nucleic acid sequence for an influenza virus NA viral segment that encodes a NA having a plurality of selected residues, wherein the selected NA viral segment, does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151 , does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and/or does not encode a threonine at position 369, wherein the numbering is based on N2. in one embodiment, the NA has at least 90% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NQ:2, SEQ ID NO:3, SEQ ID NQ:48, or SEG ID NO:49, or at least 90% amino acid sequence identity to a NA encoded by one of SEG ID Nos. 51-59. in one embodiment, the NA is a N2, N3, N7, or N9. in one embodiment, the NA is a N1 , N4, N5, N6, N8, N10 or N11. In one embodiment, the residue at position 148 is I, the residue at position 329 is S, the residue at position 151 is E, N or G, the residue at position 245 is S, T, I, L, A, V, or G, the residue at position 347 is

G, G, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, i, L, or V, the residue at position 369 is K,

H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 151 is E, N or G, the residue at position 148 is I or K, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof.

In one embodiment, a method to prepare influenza virus is provided. The method includes contacting a ceil with a vector for vRNA production comprising a promoter operabiy linked to an ini!uenza virus PA DMA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an Influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector tor vRNA production comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1 , PB2, PA, NP, NS, and M DNAs in the vectors for vRNA production are from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes a NA having a plurality of selected residues, wherein the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151 , does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and/or does not encode a threonine at position 369, wherein the numbering is based on N2; and a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding Influenza virus PB2, and a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding Influenza virus NP, and optionally a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operahiy linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus. In one embodiment, the NA has at least 90% amino acid sequence Identity to SEQ ID NO:1, SEQ ID NO:2, SEG ID NO:3, SEG ID NG:48 or SEQ ID NO:49 or at least 90% amino acid sequence identity to a NA encoded by one of SEG D Nos. 51-59. In one embodiment, the NA is N2, N3, N7, or N9. In one embodiment, the HA is H1, H2, H3, H7, or H9. In one embodiment, HA is H2, H4, H5, H6, H8, or any of H10-H18. In one embodiment, the residue at position 329 is S, A, I , L or G, the residue at position 347 is G, Q, N, S, T, Y,

C or W, or the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V In one embodiment, the residue at position 151 is E, N or G, the residue at position 148 is K, H, D or E, the residue at position 245 Is S, T, I,

L, A,W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is V, S, T, P, Y, W, A, N, I, or L, the residue at position 369 is K, H, D, E, or R, or any combination thereof. In one embodiment, PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence Identity to a polypeptide encoded by SEG ID Nos. 24 to 29 or 39 to 44. Also provided Is Isolated virus prepared by the method.

The recombinant virus may be employed in a method of immunizing an avian or a mammal, which includes administering to the avian or the mamma! a composition having an effective amount of the virus. In one embodiment, the composition comprises at least one other different influenza virus in one embodiment, the mammal is a human in one embodiment, the composition is administered intranasa!!y or via Injection.

Example Vli

In one embodiment, an isolated recombinant influenza virus comprising a selected NA viral segment encoding a plurality of selected residues or a deletion of residues in NA is provided. The virus includes the selected NA vlra! segment encoding the plurality of selected residues, a HA viral segment, and one or more of a PB1 virai segment, a PB2 viral segment, a PA viral segment, a NP viral segment a M viral segment and a NS viral segment. In one embodiment, the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151 , does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs, has reduced siaiidase activity, enhanced binding to a2-3 siaiosides, or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, encodes an aspartic acid at residue 151 , encodes an asparagine at residue 245, encodes a threonine at residue 329, encodes a lysine at residue 344, encodes a glycine at residue 346, encodes a histidine at residue 347, and encodes a threonine at position 369. In one embodiment, .the selected NA viral segment does nof encode a NA having a threonine or lysine at residue 148, does not encode a threonine at residue 32, does not encode an aspartic acid at position 151, does not encode an asparagine at position 245, does not encode an asparagine or a threonine at residue 329, does not encode a iysine at position 344, does not encode a giycine at position 346, does not encode a histidine at residue 347, and/or does not encode a threonine at residue 369, or indudes a NA having a deletion of one or more of residues 46 to 50, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs, has reduced siaiidase activity, enhanced binding to a2-3 sialosides, or has a reduction In HA mutations when grown in avian eggs relative to a corresponding Influenza virus that has a NA that encodes a threonine or iysine at residue 148 and a threonine at residue 32, does not have a deletion of residues 46 to 50, has an aspartic acid at position 147, has an aspartic acid at residue 151, has an asparagine at residue 245, has an asparagine or threonine at residue 329, has a glycine at residue 346, has a histidine at residue 347, has a threonine at residue 369, or any combination thereof. In one embodiment, the selected NA segment encodes two or more of positions 148, 151 , 245, 329, 344, 347, or 369 having lysine or isoieucine at residue 148, glutamic acid at residue 151, serine, threonine, giycine, alanine, leucine or Isoieucine at residue 245 or serine, giycine, alanine, leucine or isoieucine residue 329, glutamic acid, aspartic acid, glutamine, asparagine or histidine at residue 344, valine, leucine, Isoieucine, threonine or serine at reside 346, giycine, alanine, valine, leucine, isoieucine or threonine at residue 347, or Iysine, histidine, aspartic acid or glutamic acid at residue 369. In one embodiment, wherein the selected NA segment encodes two or more of positions 148, 151 , 245, 329, 344, 347, or 369 having isoieucine (I) at residue 148, glutamic acid at residue 151 , serine, threonine, leucine or isoieucine at residue 245 or serine, leucine or isoieucine at residue 329, glutamic acid, aspartic acid or histidine at residue 344, valine, leucine, or isoieucine at reside 346, glycine, a!anine, valine, ieucine, or isoieucine at residue 347, or iysine, aspartic acid or g!utamic acid at residue 369. In one embodiment, the selected NA segment does not encode threonine at residue 148, does not encode asparagine at residue 245, does not encode threonine at residue 369, does not encode aspartic acid at residue 151, does not encode a Iysine at residue 344, does not encode giycine at reside 346, does not encode histidine at residue 347, and does not encode threonine at residue 369. in one embodiment, he selected NA segment encodes lysine or isoieucine (I) at residue 148, encodes glutamic acid (E) at residue 151 , encodes serine (S), threonine, glycine, alanine (A), ieucine (L) or isoieucine at residue 245, encodes serine, giycine, alanine, ieucine or isoieucine at residue 329, encodes glutamic acid, arginine (R), aspartic acid (D) or histidine at residue 344, encodes valine, Ieucine, isoieucine, threonine or serine at reside 346, encodes giycine, alanine, valine, ieucine, isoieucine or threonine af residue 347, or encodes iysine, histidine, aspartic acid or glutamic acid at residue 369. in one embodiment, Jhe selected NA viral segment does not encode a NA having a threonine at residue 148, and does not encode a NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, Isoieucine or ieucine at residue 346, a histidine af residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding Influenza virus that has a NA that encodes a threonine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a histidine at residue 347, and a threonine at residue 369, or any combination thereof; or wherein the selected NA vira! segment does not encode a NA having a threonine or iysine at residue 14-8, and does not encode a NA having an aspartic acid at position 151, an asparagine at position 245, a valine, serine, isoieucine or ieucine at residue 346, a histidine ai residue 347, or a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, an aspartic acid at residue 151, an asparagine at residue 245, a glycine at residue 346, a histidine at residue 347, and a threonine at residue 369, or any combination thereof. In one embodiment Jhe selected NA viral segment encodes a NA having an isoieuclne, leucine, glycine or alanine at residue 148. In one embodiment, the residue at position 32 is A, I, G, or L, the deletion is a deletion of residues 46 to 50, wherein the residue at position 147 is N or glutamine (Q), wherein the residue at position 329 is D or glutamic acid, or wherein the residue at position 346 is serine, T, pro!ine (P), tyrosine (Y), tryptophan (W), A, N, I, or L. In one embodiment, Jhe residue at position 148 is I, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, N, W, Y, P, V, or G, the residue at position 347 is G, Q, S, T, Y, G or W, the residue at position 369 Is K, H,

R, E, P, or D, or any combination thereof. In one embodiment, he residue at position 329 is serine, valine, alanine, G, cysteine (C), methionine (M), isoleucine or leucine or wherein the residue at position 346 is V,

S, T, proline (P), tyrosine (Y), tryptophan (W), A, N, I, or L. In one embodiment, he residue at position 148 is I, the residue at position 151 is E, N or Q, the residue at position 245 Is S, T, I, L, A, W, Y, P, V, or G, the residue at position 329 is S, I, L, A, W, Y, P, V, or G, the residue at position 344 is E, H, D, N or Q, the residue at position 346 is V, S, T, I, L, A, W, Y, or P, the residue at position 347 is G, G, S, T, Y, C or W, or the residue at position 369 is K, H, R, E, P, or D. In one embodiment, Jhe isolated recombinant influenza virus Is a reassortant. In one embodiment, he NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEG ID NO:1 ,SEQ ID NQ:2, SEQ ID NO:3, SEG ID NO:48, or SEQ ID NO:49, or has at least 90% amino acid sequence identity to a NA encoded by any one of SEQ ID Nos. 51-59. In one embodiment, he NA viral segment encodes a N2, N3, N7, or N9 NA. In one embodiment, the NA viral segment encodes a N1 , N4, N5, N6, N8, N10 or N11 NA. In one embodiment, the recombinant virus has a H1 , H2, H3, H5, H7, or H9 HA. In one embodiment, the isolated recombinant influenza virus is an influenza A virus. In one embodiment, the PA, PB1 , PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEG ID Nos. 24 to 29 or 39 to 44. In one embodiment, the PB2 has I, A, S.., or G. in one embodiment, the virus has one or more of PB2-I504V, PB1-M4QL/G180W, PA-R401 K, NP-I116L, or NS1-A30P/R118K. in one embodiment, the virus has PB2-I504V, PB1-M40L/G180W, PA-R401 K, NP-I116L, and NS1-A30P/R118K.

In one embodiment, an isolated recombinant nucleic acid is provided comprising a nucleic acid sequence for an influenza virus NA viral segment that encodes a NA having a plurality of selected residues or a deletion of residues, wherein the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, and does not encode a threonine at residue 32, an aspartic acid at position 151 , an asparagine at position 245, an asparagine or threonine at residue 329, a glycine at position 346, a histidine at residue 347, or include a NA having a deletion of one or more of residues 46 to 50, or any combination thereof, or wherein the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151 , does not encode an asparagine at position 245, does not encode a threonine at position 329, does not encode a lysine at position 344, does not encode a giycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at position 369, wherein the numbering is based on N2. In one embodiment, the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode a NA having an aspartic acid at position 151, does not encode a NA having an asparagine at position 245, does not encode a NA having a glycine at residue 346, does not encode a NA having a histidine at residue 347, or does not encode a NA having a threonine at residue 369, or any combination thereof. In one embodiment, wherein the residue at position 151 is E, N or Q. In one embodiment, the residue at position 148 is I, L, V, A, or G. in one embodiment, the residue at position 245 is S, T, i, L, A, W, Y, P, V, or G or at position 329 is S, i, L, A, W, Y, P, V, or G. in one embodiment, the residue at position 347 is G, G, S, or T. in one embodiment, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V. In one embodiment, the residue at position 369 is K, H, R, E, P, or D. In one embodiment, the residue at position 32 is A, i, G, or L, the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 151 is E, N or G, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, G, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the residue at position 151 Is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 347 is G, Q, S, or T, the residue at position 346 is S, T, P, Y, W, A, N, I, L, or V, the residue at position 369 is K, H, R, E, P, or D, or any combination thereof. In one embodiment, the NA has at least 90% amino acid sequence identity to SEG ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NQ:48, or SEQ ID NG:49, or at least 90% amino acid sequence identity to a NA encoded by one of SEQ ID Nos. 51-59. In one embodiment, the NA is a N2, N3, N7, or N9. In one embodiment, the NA Is a N1 , N4, N5, N6, N8, N10 or N 11.

In one embodiment, a method to prepare influenza virus is provided. The method Includes contacting a cel! with a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1 , PB2, PA, NP, NS, and M DNAs in the vectors for vRNA production are from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes a NA having a plurality of selected residues or a deletion of residues, wherein the selected NA viral segment does not encode a NA having a threonine or lysine at residue 148, does not encode an aspartic acid at position 151 , does not encode an asparagine at position 245, does not encode an asparagine or threonine at residue 329, does not encode a lysine at position 344, does not encode a glycine at position 346, does not encode a histidine at residue 347, and does not encode a threonine at residue 369, or any combination thereof, wherein the numbering is based on N2; and a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding Influenza virus PA, a vector for RNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segmeni encoding influenza virus PB2, and a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operabiy linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding Influenza virus NA, a vector for RNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1 , a vector for mRNA production comprising a promoter operably linked to a DNA segmeni encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus. In one embodiment, the NA has at least 90% amino acid sequence Identity to SEQ ID NQ:1 , SEQ ID NO:2, SEQ ID NQ:3, SEQ ID NO:48 or SEQ ID NO:49 or at least 90% amino acid sequence identity to a NA encoded by one of SEQ D Nos. 51-59. in one embodiment, the NA Is N2, N3, N7, or N9. In one embodiment, the HA is H2 or H3. In one embodiment, .the residue at position 329 is S, the residue at position 347 is G, and the residue at position 346 Is V. In one embodiment, the residue at position 151 Is E, N or Q, the residue at position 148 is I, L, V, A, or G, the residue at position 245 is S, T, I, L, A, V or G, the residue at position 344 is E, D, N, H or Q, the residue at position 347 Is G, L, I, V, A, S, or T, the residue at position 346 is V, S, T, A, N, I, L, or V, the residue at position 369 is K, H, D, E, or R, or any combination thereof. In one embodiment, the PA, PB1, PB2, NP, M, and NS viral segments have at least 85% nucleic acid sequence Identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44. Also provided is isolated virus prepared by the method.

In one embodiment, a method of immunizing an avian or a mammal is provided, comprising: administering to the avian or the mammal a composition having an effective amount of the virus. In one embodiment, the composition comprises at least one other different influenza virus. In one embodiment, the mamma! is a human. In one embodiment, the composition is administered intranasal!y or via injection.

Further provided is a method comprising passaging the virus in eggs.

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AN publications, patents and patent applications are Incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it wiii be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.