Using DNA Shuffling to Create Novel Infectious Bronchitis Virus S1 Genes: Implications for S1 Gene Recombination

We employed the staggered extension process (StEP) to shuffle the S1 genes from four infectious bronchitis virus (IBV) strains representing four unique serotypes. Upon creating a shuffled S1 gene library, we randomly selected 25 clones and analyzed them by DNA sequencing. In total, eleven clones contained novel S1 gene recombinants. Based on sequence data, each recombinant was unique and contained a full-length open reading frame. The average number of crossovers per recombinant was 5 and the average number of point mutations was 1.3, leading mostly to non-synonymous amino acid changes. No recombinant contained sequences from all four parental genes and no recombinant contained any sequence from the distantly related Delaware 072 strain. Our data suggests that recombination between distantly related IBV strains within the S1 gene probably does not readily occur. This finding is extremely important in light of the common industry vaccination practice of mixing different live-attenuated IBV strains.