Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

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

Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors

Redox active tyrosines in photosystem II: role in proton coupled electron transfer reactions

Proton coupled electron transfer reactions often involve tyrosine residues, because when oxidized, the phenolic side chain deprotonates. Tyrosine Z (YZ) is responsible for extracting electrons in a stepwise fashion from the oxygen evolving-complex in order to build enough potential to oxidize water. This process requires that each step YZ must deprotonate and reprotonate in order to maintain the high midpoint potential that is necessary to oxidize the oxygen-evolving complex, which makes YZ highly involved in proton coupled electron transfer reactions. In this thesis YZ has been studied within oxygen-evolving photosystem II utilizing electron paramagnetic resonance spectroscopy to monitor the tyrosyl radical that is formed upon light excitation. Kinetic analysis of YZ has shed light on the factors that are important for PSII to carry out water oxidation at the oxygen-evolving complex. Most notably the strong hydrogen-bonding network and the midpoint potential of YZ have been shown to be integral aspects of the water splitting reactions of PSII. By studying YZ within oxygen-evolving PSII, conclusions are readily applied to the native system.PhDCommittee Chair: Bridgette A. Barry; Committee Member: Adegboyega K. Oyelere; Committee Member: Facundo Fernandez; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Mostafa El-Saye

Redox Control and Hydrogen Bonding Networks: Proton-Coupled Electron Transfer Reactions and Tyrosine Z in the Photosynthetic Oxygen-Evolving Complex

In photosynthetic oxygen evolution, redox active tyrosine Z (YZ) plays an essential role in proton-coupled electron transfer (PCET) reactions. Four sequential photooxidation reactions are necessary to produce oxygen at a Mn₄CaO₅ cluster. The sequentially oxidized states of this oxygen-evolving cluster (OEC) are called the S_<i>n</i> states, where <i>n</i> refers to the number of oxidizing equivalents stored. The neutral radical, YZ•, is generated and then acts as an electron transfer intermediate during each S state transition. In the X-ray structure, YZ, Tyr161 of the D1 subunit, is involved in an extensive hydrogen bonding network, which includes calcium-bound water. In electron paramagnetic resonance experiments, we measured the YZ• recombination rate, in the presence of an intact Mn₄CaO₅ cluster. We compared the S₀ and S₂ states, which differ in Mn oxidation state, and found a significant difference in the YZ• decay rate (<i>t</i>_1/2 = 3.3 ± 0.3 s in S₀; <i>t</i>_1/2 = 2.1 ± 0.3 s in S₂) and in the solvent isotope effect (SIE) on the reaction (1.3 ± 0.3 in S₀; 2.1 ± 0.3 in S₂). Although the YZ site is known to be solvent accessible, the recombination rate and SIE were pH independent in both S states. To define the origin of these effects, we measured the YZ• recombination rate in the presence of ammonia, which inhibits oxygen evolution and disrupts the hydrogen bond network. We report that ammonia dramatically slowed the YZ• recombination rate in the S₂ state but had a smaller effect in the S₀ state. In contrast, ammonia had no significant effect on YD•, the stable tyrosyl radical. Therefore, the alterations in YZ• decay, observed with S state advancement, are attributed to alterations in OEC hydrogen bonding and consequent differences in the YZ midpoint potential/p<i>K</i>_a. These changes may be caused by activation of metal-bound water molecules, which hydrogen bond to YZ. These observations document the importance of redox control in proton-coupled electron transfer reactions

Copy numbers of adenovirus DNA (per million diploid genomes) detected by quantitative PCR in lamina propria lymphocyte (LPLs).

<p>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.</p

Detection of adenoviral DNA in intestinal tissue fragments.

<p>DNAs extracted from intestinal tissue from 58 donors were tested for the presence of adenoviral DNA by nested PCR. The sex (M/F) and age of the donor at the time of biopsy or autopsy is shown. When positive, the 199 bp sequence of the adenoviral DNA polymerase gene that was amplified was used to assign the species shown. The “sequence type” is shown in parenthesis after the assigned species in case of sequences isolated more than once and corresponds to the “sequence types” shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024859#pone-0024859-g002" target="_blank">Figure 2</a>.</p

Detection of adenoviral DNA by nested PCR.

<p>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).</p

Detection of adenoviral DNA in intestinal lymphocytes.

<p>DNAs from intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from 24 donors were tested for the presence of adenoviral DNA by nested PCR. The sex (M/F) and age of the donor at the time of biopsy or autopsy is shown. When positive, the sequence of the adenoviral DNA polymerase gene that was amplified was used to assign the species shown (the result of the analysis of whole tissue DNA is also shown). All the identified sequences from any particular individual sample are indicated. The “sequence type” is shown in parentheses after the assigned species in case of sequences isolated more than once and corresponds to the “sequence types” shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024859#pone-0024859-g002" target="_blank">Figure 2</a>.</p