Molecular detection and differentiation of infectious bursal disease virus.

Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription-polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.