FIG 1.

Schematic detailing the assembly of the cDNA copy of the M41-CK genome. The construction of the cDNA encoding the full-length copy of the M41-CK genome was a multi-step process which started with a recombinant vaccinia virus encoding the cDNA BeauR-Rep-M41-Struct (43). Using this hybrid IBV cDNA construct, the Beau-R sequence (blue) encoding 5′UTR nsp 2 and 3 was deleted and sequentially replaced, using two RT-PCR-generated and one chemically synthesized cDNA fragment, with the corresponding M41-CK sequence (red), generating an intermediate hybrid IBV cDNA denoted M41-5′UTR-nsp3-Beau-nsp4-16-M41-S-poly(A). At the immediate 5′ end of the IBV cDNA, this intermediate hybrid contained a T7 RNA promoter sequence for generating infectious RNA. The remaining Beau-R replicase sequence was deleted from the intermediate IBV cDNA construct and sequentially replaced, using four chemically synthesized fragments, with the corresponding M41-CK polymerase sequence, leading to the generation of a full-length cDNA copy of the M41-CK genome under the control of a T7 RNA promoter with a hepatitis delta ribozyme-T7 terminator sequence immediately adjacent to a 25-nucleotide poly(A) tail within the vaccinia virus genome.