Molecular tools for the study, diagnosis, and control of infectious bronchitis virus
Callison, Scott Andrew
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We developed molecular tools for the study, diagnosis, and control of infectious bronchitis virus. A hammerhead ribozyme targeted to the IBV nucleocapsid mRNA was designed, synthesized, and in vitro analyzed. At a concentration of 0.5 or 10 µM, the ribozyme, designated IBVNRIBOZYME, effectively cleaved target RNAs in trans. Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and real-time RT-PCR. The amount of target RNA continually declined over a five-hour period. We employed the staggered extension process (StEP) to shuffle the S1 genes from four infectious bronchitis virus (IBV) strains representing four unique serotypes. We produced 11 recombinat S1 genes. Each recombinant was unique and contained a fulllength open reading frame. The average number of crossovers per recombinant was 5 and the average number of point mutations was 1.3. No recombinants contained sequences from all four parental genes, but several contained sequences from three of the parental S1 genes.