High titer recombinant influenza viruses with enhanced replication in vero cells

An isolated recombinant reassortant influenza virus having enhanced replication in Vero cells, prepared by:

• providing a vector comprising sequences for a HA gene segment that does not encode an aspartic acid or glutamic:
• acid at position 117 in HA2, wherein the numbering for HA2 residues is that for H1 HA2;
• altering the residue at position 117 in HA2 in the HA to aspartic acid or glutamic acid;
• contacting cells with one or more vectors for expression of vRNAs for PA, PB1, PB2, NP, NS, M, HA and NA gene segments, wherein the vector for expression of HA vRNA comprises the sequences for a HA gene segment with the altered residue;
• and
• isolating from the cells recombinant reassortant influenza virus having enhanced replication in Vero cells.

A method to prepare an influenza virus with enhanced replication in Vero cells, comprising:

• providing a vector comprising a recombinant nucleic acid molecule comprising sequences for an influenza virus HA gene segment from a first influenza virus isolate, which segment encodes an HA with an alanine, asparagine, arginine or lysine at position 117 in HA2, wherein the numbering for HA2 residues is that for H1 HA2;
• modifying the HA gene segment to encode an aspartic acid or glutamic acid at position 117in HA2, thereby yielding a modified HA segment;
• and
• contacting a cell 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 its complement operably linked to an influenza virus PB1 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 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 full 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 mRNA operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M2, or a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS1 or a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS2;
• in an amount effective to yield infectious influenza virus.