Characterization of field strains of infectious bursal disease virus (IBDV) using molecular techniques
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This study was aimed to apply different molecular techniques in the genotyping of field strains of infectious bursal disease virus (IBDV) currently present in the United States and in some other countries. The different techniques included the reverse transcription-polymerase chain reaction / restriction fragment length polymorphism (RTPCR/ RFLP), heteroduplex mobility assay (HMA), nucleotide and amino acid sequence analysis, and riboprobe in situ hybridization (ISH). From 150 samples analyzed from the United States, 80% exhibited RFLP identical to the variant Delaware E strain, other strains detected included Sal-1, D-78, Lukert, PBG-98, Delaware A, GLS IBDV standard challenge strain -like (STC-like). The analysis of the deduced amino acid sequence of the VP2 hypervariable region from six strains classified as Delaware variant E, revealed some amino acid substitutions that make them somewhat different from the original variant E strain isolated in the mid 1980s. The isolate 9109 was classified as a standard strain, but, it exhibited a unique RFLP pattern characterized by the presence of the Ssp I restriction site characteristic of the very virulent IBDV (vvIBDV) strains. The pathogenic properties of this isolate were compared to those of isolate 9865 (variant strain) and the Edgar strain. All three strains induced subclinical disease, however by in situ hybridization some differences in the tissue tropism were observed. The viral replication of the variant isolate 9865 was more restricted to the bursa of Fabricius. Isolate 9109 and the Edgar strain were also observed in thymus, cecal tonsils, spleen, kidney and proventriculus. A variety of inactivated IBDV strains received from Latin America were detected by RT-PCR/RFLP. The more interesting findings include the presence in Mexico and Venezuela of IBDV strains with unknown RFLP patterns, and the observation of RFLP patterns indicative of vvIBDV strains in Brazil and Dominican Republic. Finally, the HMA was evaluated as a method for genotyping IBDV. The HMA was able to differentiate between standard, antigenic variants and very virulent strains. Minor differences between antigenic variants were also detected. The results obtained by HMA were similar to that obtained with RFLP and phylogenetic analysis.