Detection of infectious bronchitis virus by multiplex polymerase chain reaction and sequence analysis.

A multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to amplify the S1 and S2 genes of vaccine and recent Taiwanese isolates of infectious bronchitis virus (IBV). DNA fragments of 228 and 400 base pairs in length were amplified among IBV isolates in multiplex PCR, suggesting that there were no apparent deletions or insertions in these regions. No PCR products were amplified from unrelated avian viruses and negative controls. The results suggested that multiplex PCR provided a specific and sensitive approach for identification of IBV isolates. Sequence analysis of the hypervariable region (HVR) of S1 gene exhibited high variations among Taiwanese IBV isolates. The TWI and TWII groups were about 84-98 and 94-99% identity within the groups. American strains were most divergent sharing only 60% homology with TWI and TWII Taiwanese strains. The Mass group varied 0-10% among each other and had over 70% homology with TWI and TWII Taiwanese strains. A phylogenetic tree based on the nucleotide sequences of the HVR of S1 gene revealed that Taiwanese IBV isolates had evolved into three groups (TWI, TWII, and Mass). This suggested that there were multiple groups of viruses cocirculating in Taiwan.