Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain

A recombinant virus, CVI/rpp38, was developed from the Marek's disease viru s (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by tra nsfection of CV1988/Rispens-infected chich embryo fibroblasts (CEFs) with p lasmid pHA25 DNA containing pp38 gene from GA strain of MDX Monoclonal anti body (MAb)H19 which reacts with pp38 from CA but not with that fi om CVI988 was used to screen for recombinant viruses in transfected cell culture pla tes by immunofluorescent assay (IFA). A positive plaque was isolated, propa gated and purified from cell-free virus particles after sonication of infec ted CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in 1FA bu t also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequenc e of the mutant virus showed a substitution of G at position 320 by A resul ting in a change of an amino acid residue from arginine to glutamine. Compa rison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 a nd CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 ep itope in pp38. This mutant is potentially useful to further explore the bio logical function of pp38 and its H19 epitope.