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. 2011;6(9):e24859.
doi: 10.1371/journal.pone.0024859. Epub 2011 Sep 30.

Adenoviruses in lymphocytes of the human gastro-intestinal tract

Soumitra Roy  1 , Roberto Calcedo, Angelica Medina-Jaszek, Martin Keough, Hui Peng, James M Wilson
Affiliations

Adenoviruses in lymphocytes of the human gastro-intestinal tract

Soumitra Roy et al. PLoS One. 2011.

Abstract

Objective: Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects.

Methods: The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA.

Results: Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested.

Conclusion: Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.

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Conflict of interest statement

Competing Interests: SR is an inventor on patents licensed to various biopharmaceutical companies. JMW is a consultant to ReGenX Holdings, and is a founder of, holds equity in, and receives a grant from affiliates of ReGenX Holdings; in addition, he is an inventor on patents licensed to various biopharmaceutical companies, including affiliates of ReGenX Holdings. This work was also partially funded by a commercial source (GlaxoSmithKline). This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Detection of adenoviral DNA by nested PCR.
Top – Diagram showing the region of the adenoviral DNA polymerase gene that was amplified. Primers designed to hybridize with sequences conserved across species A–F were used to amplify a 1.5 kb region. The product was used as template for a 2nd round of PCR using similarly conserved primers located internal to the first primer set that amplified a 258 bp product. Bottom – Agarose gel showing products of the 1st round PCR (upper gel panel) showing positivity (1.5 kb product) in some samples (arrow). The products of the first round were used for a 2nd nested PCR (lower gel panel).
Figure 2
Figure 2. Alignments of the amplified 199 bp sequences contained internal to the primers of the 258 nested PCR products.
The donor from whom each sequence was identified (as described in Tables 1 and 2) is indicated and corresponds to donor numbers in Tables 1 and 2. (The letter following the donor number identifies the source of the DNA used for analysis: T- intestinal tissue, L- LPLs, L*- IELs. All sequences were assigned to one of four adenoviral species, (HAdV-B, HAdV-C, HAdV-E, or HAdV-F) using the neighbor-joining algorithm of Saitou and Nei and the resulting alignments with one of the reference HAdV species (red font) is shown. Regions of identity across all sequences are shaded yellow; regions that are common to the majority of sequences are shaded blue. Sequences that were obtained more than once were assigned to one of 11 “sequence types”. The donor sample that yielded each sequence type is shown in the table.
Figure 3
Figure 3. Two instances of multiple adenoviral DNA sequences amplified from a single donor.
Sequences amplified from donors 5 and 13 and the source of the DNA from which sequences were amplified is shown in the tables. The phylogenetic relationships between these sequences as computed using the nearest neighbor algorithm of Saitou and Nei is also shown. The calculated distance value for each sequence is shown in parentheses (Vector NTI, AlignX module).
Figure 4
Figure 4. Copy numbers of adenovirus DNA (per million diploid genomes) detected by quantitative PCR in lamina propria lymphocyte (LPLs).
Data for unfractionated (total LPLs) as well as CD4+ and CD8+ fractionated lymphocytes are shown. Each data point represents a single sample. The mean and standard deviation for each data set are indicated.
Figure 5
Figure 5. Detection of the adenoviral transcription.
RNA was isolated from IELs and LPLs from 11 subjects (table, upper left) and subjected to reverse transcription followed by PCR. The adenoviral L3b transcript (shown in green in the diagram, upper right; hexon orf shown in orange) harbors a tri-partite leader comprised of three widely separated leader exons followed by the last exon encoding the hexon protein. The PCR primers (as shown by arrowheads) were designed to hybridize to conserved sequences in the first leader and to the last exon. The approximately 300 base-pair product from reactions that were positive were cloned and sequenced. The sequences obtained from the different samples were assigned to adenoviral species B, C, or E (table) and aligned to a reference adenovirus sequence (HAdV-7, HAdV-2, and HAdV-4, respectively) for each species. Regions of identity across all sequences are shaded yellow; regions that are common to the majority of sequences are shaded blue. The exon boundaries are indicated by vertical lines. The putative hexon start codon is indicated by an arrow below the alignments. One of the species B sequences (sequence 48-2) contains a second leader that represents an alternate splicing pattern from that seen with the other sequences.
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