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 virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chich embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H 19 which reacts with pp38 from GA but not with that from CV1998 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CV1988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by A resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CV1988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified HI 9 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.