Infectious bursal disease live vaccines: Changes in the virus population during serial passage in chickens and chicken embryo fibroblast cells

Two attenuated infectious bursal disease virus strains used as commercial l ive vaccine were passaged five successive times in specific-pathogen-free c hickens and chicken embryo fibroblast (CEF) cells. Both attenuated strains increased in virulence during the passage in susceptible chickens as eviden ced by the decrease in bursa/body weight ratios. A direct nucleotide sequen ce analysis of the VP2 hypervariable domain amplified by the reverse transc ription-polymerase chain reaction revealed that the nucleotide at position 890 (T) in both strains was A after the passage in chicken. In addition, th e nucleotide at position 830 (A) was T or C after the subsequent passage in CEF cells. Because of the nucleotide differences, the amino acid residue a t position 253 (His) in both vaccines was Gin after the passage in chickens , and the amino acid residue Gin was changed back to His during the subsequ ent passage in CEF cells. The digestion of the amplified fragment with rest riction endonucleases Stul and Ncol, which recognize the sequence differenc e at position 890, showed that the population of the virus that had amino a cid Gin at position 253 was gradually increased during the passage in chick ens. Conversely, the population of the virus that had amino acid His at pos ition 253 was gradually increased during the subsequent passage in CEF cell s.