| Four field isolates (70, 586, 1174, SEA-5) of infectious bursal disease virus (IBDV) with distinct reverse transcription polymerase chain reaction restriction fragment length polymorphism. (RT/PCR-RFLP) patterns were serially passed 24 or 25 times in 10-day-old specific pathogen free (SPF) chick embryos by chorioallantoic membrane (CAM) inoculation. The nucleotide base sequence of a 697-bp fragment of genome segment-A containing the partial coding sequence of VP2, and the entire hyper-variable (H-V) region was determined for each of the field and embryo passed viruses. Following virus passage six nucleotide residues, specifying five amino acid changes, occurred between the original field viruses and their chick-embryo (CE) origin ancestors. All of the nucleotide base changes were isolated to the H-V region. In three of the four CE passed viruses amino acid residue 249 was shifted from lysine to asparagine. Additional individual residue changes occurred at amino acids 222 (serine → leucine) and 281 (glutamine → valine). It is likely that the amino acid residue changes, particularly residue 249, are adaptive for virus growth in the chick embryo. CE-pass-1 or CE-Pass-24/25 virus challenge of three-week-old SPF chicks demonstrated no difference in pathogenicity between these virus pairs as determined by clinical symptoms, bursa; bodyweight indexes, and histopathologic lesion scoring. Attempts to adapt the original field and various CE-passed virus isolates for growth in baby grivet monkey kidney cells (BGM-70), chick embryo fibroblast (CEF), and CEF cell cultures prepared from virus infected embryos were not successful.; A ssRNA internal control test reagent for assessing the performance of individual IBDV RT/PCR-RFLP diagnostic test reactions was also prepared. The internal control was differentiable by size, co-amplifiable with genomic IBDV RNA, and free of the restriction enzyme sites used for RFLP analysis of IBDV RT/PCR product. |
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