Experimental confirmation of recombination upstream of the S1 hypervariable region of infectious bronchitis virus

Chimeric infectious bronchitis virus (IBV) genomes with cross-over sites in the S1 gene were generated by co-infection with two distinct IBV strains. Recombinant viruses were collected from chicken embryos, embryonic cultured cells and chickens co-infected with Ark99 and Mass41 strains and purified by differential centrifugation. The recombinant S1 genes were identified by reverse transcription polymerase chain reaction (RTPCR) using heterologous primers and confirmed by nucleotide sequencing. The recombinants with Ark99 5′ and Mass41 3′ sequences were identified following the in vitro, in ovo and in vivo co-infections. Mixed RNA extracted from Ark99 and Mass41 did not produce RTPCR products with these primers at the PCR conditions used. Cross-over sites within the amplified 580 (Mass41) or 604 (Ark99) bases of the 5′ S1 gene could only be detected between nucleotides 50 and 155. While this region, lying upstream of the S1 hypervariable region, corresponded with sites commonly identified in naturally occurring isolates, recombination sites identified in these studies could not be detected within the HVR of S1 of the genomes of chimeric viruses.