Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease.
To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. 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 the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed 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. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.
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Molecular Biology Section, Centers for Disease Control, Atlanta, GA 30333.
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D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D019309:Epstein-Barr Virus Nuclear Antigens; D005814:Genes, Viral; D005838:Genotype; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006689:Hodgkin Disease / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000235:genetics*; D012269:Ribosomal Proteins
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C067169:RPL22 protein, human