Identification of infectious bursal disease virus quasispecies in commercial vaccines and field isolates of this double-stranded RNA virus.

Quasispecies of infectious bursal disease virus (IBDV) vaccine and wild-type strains were identified using real-time RT-PCR at a region of the viral genome known for sequence variability. The LightCycler (Idaho Technology, Inc.) and hybridization probe system (Roche, Molecular Biochemicals) were used. An anchor probe labeled with LightCycler Red 640 and mutation probe labeled with fluorescein were designed using the Del-E IBDV sequence. The sequence of the mutation probe included nucleotides in the hydrophilic B region of VP2 that are important to a viral neutralizing epitope. This Del-E mutation probe was allowed to hybridize to the RT-PCR products following amplification and its temperature of dissociation (T(m)) from each viral template was determined using the LightCycler melting peak analysis. The observed T(m) for the Del-E mutation probe with its homologous virus, Del-E, was usually 65.5 degrees C but ranged from 65 to 66.4 degrees C. Peak melting temperatures for the test viruses were inversely proportional to the number of mutations observed between the Del-E mutation probe and target virus sequence. All the IBDV vaccine strains tested and all but two of the wild-type strains exhibited more than one melting peak, indicating that genetic subpopulations or quasispecies of the viruses were present in the samples. Since the mutation probe was located at a site which encodes a neutralizing epitope of the virus, it is possible that the genetic differences observed are translated into antigenic changes in this VP2 epitope and contribute to antigenic diversity in the quasispecies cloud.