Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction.
We designed a highly sensitive and specific polymerase chain reaction assay for the detection of Epstein-Barr virus (EBV)-related sequences in B-cell DNA of EBV-seropositive healthy individuals. By using this assay, we were able to amplify at least 10 copies of a plasmid containing the BamHI-W region, which is repeated up to 11 times within the EBV genome, in the presence of 1 microgram of EBV-negative DNA, indicating that one virus genome was detectable from 150,000 cells. In 15 of 16 tested individuals, EBV-related sequences were found frequently when the DNA from 10(6) B lymphocytes was examined and 1 microgram of DNA was used in each polymerase chain reaction. When the results of amplifying the diluted plasmid were used as a semiquantitative standard, the number of EBV genomes detected could be estimated to range between 50 and less than 1 from 10(6) B lymphocytes. The results of our study will provide the basis for further investigations of the characteristics of the latent carrier state in healthy EBV-seropositive individuals, such as the determination of the number of virus copies per cell and expression of antigens.
Wagner
H J
HJ
Institute of Immunology and Transfusion Medicine, University of Lübeck Medical School, Germany.
Bein
G
G
Bitsch
A
A
Kirchner
H
H
D000914:Antibodies, Viral / Q000097:blood; D001402:B-Lymphocytes / Q000382:microbiology*; D001483:Base Sequence; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000276:immunology / Q000382:microbiology*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D015342:DNA Probes; D004279:DNA, Viral