Development of Differential RT-PCR Assays and Molecular Characterization of the Complete VP1 Gene of Five Strains of Very Virulent Infectious Bursal Disease Virus

Several antigenic and pathogenic subtypes of infectious bursal disease virus (IBDV) have been identified worldwide. Simple and quick differential diagnostic assays are vital for implementing control and prevention strategies for infectious bursal disease (IBD). Currently reverse transcriptase-polymerase chain reaction/restriction fragment length polymorphism analysis (RT-PCR/RFLP) and real-time RT-PCR detection have been used. However, the RT-PCR/RFLP analysis is time consuming and not feasible for assaying large numbers of samples, and the real-time PCR is expensive. The reliable indicator for very virulent (vv) IBDV (vvIBDV) remains in vivo pathogenicity testing because of the lack of a virulence marker. In this study simple RT-PCR assays were developed for differentiating various types of IBDV. Two sets of primers specific for serotype 2 and vvIBDV were designed based on the sequences of segment A and B of IBDVs. Initially a total of 25 previously characterized virus strains including 11 serotype 1 classics, 4 serotype 1 variants, and 5 serotype 1 vv and 5 serotype 2 strains were used to validate the differential RT-PCR assays. The results indicated that primer set 1 specifically amplified a 415 bp RT-PCR product for the serotype 2 viruses, and primer set 2 specifically amplified a 715 RT-PCR product for vvIBDV except for two vv Taiwan strains. To further confirm the specificity of primer set 2 for the vvIBDVs, 20 field samples suspected to be vvIBDVs from different geographic locations around the world were tested. All but one suspected vv Korean strain (91108) tested positive by this primer set. To understand the molecular basis for the failure to detect these viruses with primer set 2, a complete VP1 gene of two vv Taiwan (PT and IL) strains along with two vv Turkey (OA and OE) and one vv Holland (Hol) strain was sequenced and phylogenetically analyzed with 11 other strains retrieved from GenBank. The results showed that PT and IL were closely related to the classic strain IM with nucleotide (nt) similarities of 98.6% and 97.7%, respectively. The two Taiwan strains clustered together with the classic and variant IBDVs in the unrooted neighbor-joining phylogenetic tree, indicating independent evolution of these strains from the rest of the vv isolates. The RT-PCR assays developed in this study could differentiate vvIBDVs from classic strains and serotype 1 from serotype 2 IBDVs with a high degree of sensitivity and specificity and are fast, simple, and inexpensive.