Recent trends in the molecular diagnosis of infectious bursal disease viruses.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. Two serotypes of this double-stranded RNA virus exist but only serotype 1 viruses cause disease in chickens. Detection and strain identification of IBDV is important because antigenic subtypes found within serotype 1 make it necessary to tailor vaccination programs to the antigenic type found in the bird's environment. Because conventional virus isolation and characterization are not practical for routine diagnosis of IBDV, antigen-capture enzyme-linked immunosorbent assay (ELISA) and molecular assays based on reverse transcription-polymerase chain reaction (RT-PCR) technology were developed. Compared with antigen-capture ELISA, RT-PCR assays have greater versatility and are more sensitive and specific. Strain identification has been accomplished using a variety of post-RT-PCR assays, including restriction enzyme digestion of the RT-PCR products. The resulting restriction fragment length polymorphisms (RFLP) are used to differentiate viruses into molecular groups that correlate with antigenic and pathogenic types. Recently, two types of real-time RT-PCR have been used to identify and differentiate strains of IBDV. Both methods use distance-dependent interaction between two dye molecules, known as fluorescence resonance energy transfer (FRET). The dye molecules are attached to one or more nucleotide probes that detect specific nucleotide sequences of the virus. Our laboratory has used a two-probe assay to identify single-nucleotide mutations among IBDV strains. A mutation probe is used in this assay to detect substitution mutations in a region of the viral genome that encodes a neutralizing epitope of the virus. These assays are accurate, reliable and inexpensive compared with conventional RT-PCR because they do not require RFLP or other labor-intensive post-RT-PCR assays to distinguish viral strains.