title: Molecular analysis of pseudorabies viral vaccines and their rapid differentiation from wild-type isolates using DNA-amplified glycoprotein I and thymidine kinase gene segment polymorphisms.
abstract: Several conventional and genetically recombinant modified-live viral (MLV) vaccines are used to control pseudorabies virus infections (Aujeszky's disease, PRV) in swine. Differentiating vaccinal PRV (V-PRV) from wild PRV (WT-PRV) is important for herd health, regulatory and forensic purposes, and for studies of PRV latency and epidemiology. All PRV vaccines used currently contain glycoprotein I (gI) and/or thymidine kinase (TK) gene deletions, whereas WT-PRV typically contain intact gI and TK genes. Utilizing these differences we developed an effective but simple differential polymerase chain reaction (PCR) approach based upon the amplification of gI and TK gene polymorphisms. The primary immunoreactive epitope-encoding region of the gI gene and nearly the entire TK gene were amplified and analyzed using nested PCR procedures. TK and gI PCR products were cleaved with Sal I and Sac I, and Nco I restriction enzymes respectively. PCR product and restriction fragment length polymorphisms enabled most V-PRV to be clearly distinguished from each other, and all of them, as a group, clearly differentiated from typical WT-PRV. Mixtures of V-PRV and WT-PRV could be identified as such. The uncommon but occasional occurrence of atypical WT-PRV containing altered gI and/or TK genes indicates the need for interpretive caution, particularly if aberrant gene segment polymorphisms are observed. This rapid and precise molecular approach will facilitate regulatory monitoring, epidemiological investigations, diagnostic differentiation, purity testing and latency/recrudescence studies with the class of biologicals and offers a model for similar analyses of other MLV biologicals as well.
authors.affiliation: Diagnostic Virology Laboratory, U.S. Department of Agriculture, Ames, IA 50010.
authors.forename: J B
authors.initials: JB
authors.lastname: Katz
authors.forename: J C
authors.initials: JC
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D006021:Glycophorins / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011557:Pseudorabies / Q000517:prevention & control; D013552:Swine; D013553:Swine Diseases / Q000517:prevention & control; D013937:Thymidine Kinase / Q000235:genetics; D014614:Vaccines, Synthetic / Q000302:isolation & purification; D014765:Viral Vaccines / Q000302:isolation & purification*
chemical_list: D004279:DNA, Viral; D006021:Glycophorins; D014614:Vaccines, Synthetic; D014765:Viral Vaccines; D013937:Thymidine Kinase