Molecular typing of N gene and 3′ untranslated region of IBV field isolates and vaccine strains using RT-PCR and RFLP

Whole nucleocapsid (N) gene and 3′ untranslated region (UTR) of infectious bronchitis virus (IBV) vaccines and Iranian field isolates were amplified using reverse transcription and polymerase chain reaction (RT-PCR). The amplified fragments were subjected to digestion using two restriction endonuclease enzymes, Alu I and Mnl I. Five different restriction fragment length polymorphism (RFLP) patterns were generated using both enzymes, classifying IBV strains into five similar groups. Based on RT-PCR and RFLP analysis of the N gene and 3′ UTR, IBV vaccine strains H52, H120 and MA5 all from the same serotype generated identical patterns using both enzymes. Vaccine strain and field isolate of 4/91 also had the same pattern distinct from other IBVs. The IB88 vaccine strain and two other field isolates MNS-7862-1 and Ur1/09 had three different RFLP patterns distinguishable from each other and the other IBVs. In conclusion, RT-PCR and RFLP analysis of the N gene and 3′ UTR could be employed as a useful method for differentiating IBV strains especially in cases where S1 gene amplification is not successful because of its highly variable nature among different IBVs.