EXPERIMENTAL CONFIRMATION OF RECOMBINATION UPSTREAM OF THE S1 HYPERVARIABLE REGION OF INFECTIOUS-BRONCHITIS VIRUS

Chimeric infectious bronchitis virus (IBV) genomes with cross-over sit es in the S1 gene were generated by co-infection with two distinct IBV strains. Recombinant viruses were collected from chicken embryos, emb ryonic cultured cells and chickens co-infected with Ark99 and Mass41 s trains and purified by differential centrifugation. The recombinant SI genes were identified by reverse transcription polymerase chain react ion (RTPCR) using heterologous primers and confirmed by nucleotide seq uencing. The recombinants with Ark99 5' and Mass41 3' sequences were i dentified following the in vitro, in ovo and in vivo co-infections. Mi xed RNA extracted from Ark99 and Mass41 did not produce RTPCR products with these primers at the PCR conditions used. Cross-over sites withi n the amplified 580 (Mass41) or 604 (Ark99) bases of the 5' S1 gene co uld only be detected between nucleotides 50 and 155. While this region , lying upstream of the S1 hypervariable region, corresponded with sit es 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. (C) 1997 Elsevier Science B .V.