Influenza virus reassortment

A method of preparing an influenza virus comprising the steps of

• a) preparing one or more expression construct(s) which comprise(s) coding sequences for expressing at least one segment of an influenza virus genome;
• b) introducing into a cell one or more expression construct(s) which encode(s) the viral segments of an influenza virus, wherein at least one expression construct is the expression construct prepared in step (a);
• and
• c) culturing the cell in order to produce a reassortant influenza virus from the expression construct(s) introduced in step (b);
• wherein steps (a) to (c) are performed in a time period of 100 hours or less.

A method of preparing a reassortant influenza virus, comprising:

• a) providing a synthetic expression construct which comprises coding sequences for expressing at least one segment of an influenza virus genome by (i) synthesising a plurality of overlapping fragments of the synthetic expression construct, wherein the overlapping fragments span the complete synthetic expression construct, and (ii) joining the fragments to provide the synthetic expression construct;
• b) introducing into a cell which is not 293T one or more expression construct(s) which encode(s) the viral segments required to produce an influenza virus, wherein at least one expression construct is the synthetic expression construct prepared in step (a);
• and
• c) culturing the cell in order to produce a reassortant influenza virus from the viral segments introduced in step (b);
• wherein steps (a) to (c) are performed in a time period of 124 hours or less.

A method of preparing an influenza virus, comprising:

• a) providing a synthetic expression construct which comprises coding sequences for expressing at least one segment of an influenza virus genome by (i) synthesising a plurality of overlapping fragments of the synthetic expression construct, wherein the overlapping fragments span the complete synthetic expression construct, and (ii) joining the fragments to provide the synthetic expression construct;
• b) introducing into a cell one or more expression construct(s) which encode(s) the viral segments of an influenza virus, wherein at least one expression construct is the synthetic expression construct prepared in step (a);
• c) culturing the cell in order to produce a reassortant influenza virus from the viral segments introduced in step (b);
• and
• d) contacting a cell which is of the same cell type as the cell used in step (c) with the virus produced in step (c) to produce further reassortant influenza virus;
• wherein steps (a) to (c) are performed in a time period of 124 hours or less.

A method of preparing an influenza vaccine, comprising:

• a) contacting a cell with a reassortant influenza virus prepared by the method of any preceding claim;
• b) culturing the cell in order to produce an influenza virus;
• and
• c) preparing a vaccine from the influenza virus produced in step (b).

A method of preparing a synthetic expression construct which encodes a viral segment from an influenza virus, comprising:

• a) providing the sequence of at least part of the coding region of the HA or NA segment from an influenza virus;
• b) identifying the HA and/or NA subtype of the influenza virus from which the coding region is derived;
• c) providing a UTR sequence from an influenza virus with the same HA or NA subtype as the subtype identified in step (b);
• and
• d) preparing a synthetic expression construct which encodes a viral segment comprising the coding sequence and the UTR.