Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions

We describe a case of acute, disseminated Epstein-Barr virus (EBV) infection which was analyzed for the cellular distribution of viral replication by automated, colorimetric in situ DNA hybridization using a single, synthetic, terminally biotin-labeled oligonucleotide probe composed of 23 consecutive nucleotides selected from the EBV Not I region. The GC-rich, Not I region is a 125-base pair sequence that is repeated in tandem an average of 12.6 times in the EBV genome. The synthetic sequence had 91% base homology with another EBV genomic tandem repeat, the 102-base pair Pst I region, which is also GC-rich, has an overall 70% homology with the Not I region, and is reiterated about 25 times in the viral DNA. Disseminated EBV infection was detected in nuclei of atypical lymphocytes in several organs, including lung, bronchus, trachea, spleen, liver, and stomach, with the probes. In addition, the synthetic oligomer compared favorably with a significantly more expensive, nick translated, biotinylated probe cloned from the BAM HI-V (W), large internal repeat region. This 3.0-kilobase pair (kbp) sequence is repeated an average of 11 times in EBV. Although both probes identified regions repeated multiple times in the virus, and each confirmed an identical tissue distribution for the infection, the signal obtained with the Not I/Pst I probe was more intense and confined to the nuclei of fewer lymphocytes than the general, more weakly distributed signal obtained with the probe from the large internal repeat region. Consistent positive cellular staining was obtained with the Not I/Pst I probe in both EBV-infected control tissue culture cells and in formalin-fixed, paraffin-embedded tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)