Helper-free Rescue of Recombinant Negative Strand RNA Virus Systems

A method for producing a population of infectious influenza virus particles for preparation of a vaccine, comprising:

• growing a selected reassortant influenza virus to produce a population of recombinant infectious virus particles and recovering said population of infectious influenza virus particles,
• wherein said reassortant influenza virus was produced in cultured cells in the absence of a helper virus by expression vectors that direct the expression of genomic vRNA or antigenomic vRNA (cRNA) segments derived from at least two different strains of influenza virus to produce a virus particle having a selected genome and that is a reassortant of said at least two different strains
• wherein substantially all of the infectious influenza virus particles of the resulting population contain said selected genome.

A method for producing an influenza virus comprising:

• (a) producing in a cell all of the genomic RNA of an influenza virus by introducing into the cell DNA encoding at least one of said genomic RNA or cRNA corresponding to said genomic RNA of the virus, and
• (b) producing in the cell RNA dependent polymerase and nucleoprotein and assembling in the cell the influenza virus, said cell being free of helper virus.

A method for producing a population of infectious influenza virus particles for preparation of a vaccine, comprising:

• growing a selected reassortant influenza virus to produce a population of recombinant infectious virus particles comprising the HA-encoding and NA-encoding vRNA segments of a first influenza strain and the non-HA and non-NA encoding vRNA segments of at least one influenza strain different from said first influenza strain and recovering said population of infectious influenza virus particles,
• wherein the infectious influenza virus particles of the resulting population contain substantially the same genome genome.