Methods to produce bunyavirus replicon particles

A method for generating a recombinant, non-spreading bunyavirus replicon particle, the method comprising:

• A) culturing a eukaryotic cell with growth medium;
• B1) expressing T7 polymerase in the eukaryotic cell;
• B2) expressing bunyavirus Gn protein and bunyavirus Gc proteins, and optionally bunyavirus NSm protein, in the eukaryotic cell, wherein said Gn and Gc proteins may form a heterodimeric polyprotein (GnGc);
• and wherein upon the optional expression of NSm protein, a heterotrimeric polyprotein (NSmGnGc) may form;
• B3) transforming or transducing the eukaryotic cell with a vector comprising cDNA that encodes a bunyavirus L genome segment, wherein said L genome segment cDNA is flanked by a T7 promoter and cDNA of a ribozyme sequence;
• B4) transforming or transducing the eukaryotic cell with a vector comprising cDNA that encodes at least part of a bunyavirus S genome segment, wherein said S genome segment cDNA comprises at least the N-gene and the 3′ and 5′ UTRs, wherein said S genome segment cDNA is flanked by a T7 promoter and cDNA of a ribozyme sequence;
• and, optionally,
• B5) transforming or transducing the eukaryotic cell with a vector comprising cDNA that encodes a bunyavirus M genome segment from which the GnGc coding region has been functionally inactivated, wherein said M genome segment cDNA is flanked by a T7 promoter and cDNA of a ribozyme sequence;
• C) following steps B1-B5, the eukaryotic cell is maintained in the growth medium and recombinant, non-spreading bunyavirus replicon particles are generated and isolated from the growth medium;
• wherein the sequence of steps B1, B2, B3, B4, B5 is random and all or part of these steps may be performed simultaneously.