Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes

Abstract

The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 x 10(6) copies of the adenovirus genome/10(7) cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form.

FIG. 1.

Real-time PCR detection of human species C adenovirus DNA. (A) Nucleotide sequences of the primers and TaqMan probe are to the hexon region of Ad5. (B) The species C viruses as well as representatives from each of the other human adenovirus species were amplified by real-time PCR using the hexon primers, and the products were run on an ethidium bromide-stained 1.8% agarose gel. Numbers indicate the adenovirus serotype tested. M, marker; −, negative water control; +, Ad2 positive control DNA. (C) Purified Ad2 DNA was serially 10-fold diluted, and duplicates of each dilution (from 5 × 10⁷ to 5 copies) were tested. The fluorescence intensity (RFU = relative fluorescent units) collected in real time for each sample was plotted against the number of PCR cycles. The horizontal line indicates the fluorescence threshold setting, which is set at 10 standard deviations above the baseline emission.

FIG. 2.

Distribution and quantitation of adenovirus DNA per 10⁷ lymphocytes in 42 tonsil and adenoid samples from 35 donors. Real-time PCR was performed on DNA purified from Ficoll-purified lymphocytes from tonsils (T) and adenoids (A). Cellular input DNA amounts were normalized to quantities of GAPDH DNA between samples being compared. Individual donors were tested two to five times, and the results are presented as mean and standard error of the mean for all experiments. (Inset) Number of Ad genomes/10⁷ cells as a function of donor age. Each point represents a single sample. Horizontal lines are the average number of genomes for each age group in which more than two samples were tested.

FIG. 3.

Detection of proximal (E1A) and distal (Fiber) regions of the adenovirus genome in tonsil and adenoid lymphocytes. (A) Relative location of E1A, hexon, and fiber genes within the adenovirus genome. (B) Nested PCR for adenovirus E1A DNA (172-bp product). (C) Nested PCR for adenovirus fiber DNA (175-bp product). Samples from all patients shown were positive for hexon by real-time PCR, except for the sample from patient 31, which was negative. Nested PCR was performed on DNA purified from Ficoll-purified lymphocytes. +, positive control using either 50 or 5 copies of Ad2 DNA as template; −, negative water control.

FIG. 4.

Analysis of adenovirus DNA in separated lymphocyte subpopulations. (A) Ficoll-purified lymphocyte populations were purified from adenoid lymphocytes with directly conjugated Dynal magnetic beads to CD4 and CD8 together or CD19 as detailed in Materials and Methods. The resulting cells were then stained with fluorescent antibodies and analyzed on the flow cytometer for purity. CD2 was used as a pan-T-cell marker. CD20 was used as a pan-B-cell marker. (B) Real-time PCR for hexon was performed on 5 μl of DNA prepared from the purified CD4⁺ CD8⁺, CD19⁺, and unseparated (All) cells. Adenovirus DNA values in samples were normalized by referring the viral copy number to the actual amount of input cellular DNA as estimated by quantification of the GAPDH gene in each sorted population.

FIG. 5.

Analysis of Ad DNA in separated lymphocyte subpopulations. (A) Lymphocyte populations were purified from adenoid lymphocytes with Dynal magnetic beads to CD4 and CD8 together or CD19. The beads were then removed from the CD4⁺ CD8⁺-selected cells as detailed in Materials and Methods, and the cells were further purified with MACS magnetic beads to CD3. The purified cell populations were stained with fluorescent antibodies and analyzed on the flow cytometer for purity. (B) Real-time PCR for hexon was performed on DNA prepared from the purified CD3⁺ CD4⁺ CD8⁺, CD19⁺, and unseparated (All) cells. Cells that were CD4⁺ CD8⁺ but CD3⁻ were also tested. Adenovirus DNA levels in samples were normalized by referring the viral copy number to the actual amount of input cellular DNA estimated by quantification of the GAPDH gene in each sorted population. (Inset) Nested PCR for adenovirus fiber DNA was performed on the unseparated (All), CD4⁺ CD8⁺ CD3⁺, and CD19⁺ samples, and the PCR products were run on a 1.8% agarose gel stained with ethidium bromide. M, marker. (C) Lymphocyte populations were purified from adenoid lymphocytes with Dynal magnetic beads to CD19. T cells were directly obtained with MACS magnetic beads to CD3. The resulting cells were then analyzed for purity by flow cytometry. (D) Real-time PCR for hexon was performed on DNA prepared from the purified CD3⁺, CD19⁺, and unseparated cells (All). (Inset) Nested PCR for adenovirus fiber DNA was performed on the same samples, and the PCR products were run on a 1.8% agarose gel stained with ethidium bromide. M, marker.