A strategy for precision of genotyping of Epstein-Barr virus by polymerase chain reaction: application for studying Hodgkin's lymphoma.
Previous studies on the genotyping of Epstein-Barr virus (EBV) have been based on the analysis of a single gene locus. The assignment of genotype of an isolate could easily be over-looked with this assay. Our strategy for precision of EBV genotyping has exploited the existence of two families of EBV strains (type A and B) that can be distinguished at three divergent gene loci (EBNA-2, EBNA-3C, and EBER). To precisely determine the genotype of EBV in Hodgkin's disease (HD), we designed primers and simultaneously analysed these three gene loci that distinguish type A and B viruses by the polymerase chain reaction (PCR) technique. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of PCR-amplified products or the mobility shifts in single-strand conformation polymorphism (SSCP) analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. Fifteen EBV-infected cell lines were analysed and a good correlation between EBNA-2 and EBNA-3C typing results was found. In contrast, approximately 33% of the cell lines analysed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
J C
JC
Tumor Virology Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia 30333.
Lin
S C
SC
Mar
E C
EC
D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens; D005814:Genes, Viral; D005838:Genotype; D004854:Herpesvirus 4, Human / Q000145:classification*; D006689:Hodgkin Disease / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens