Epidemiology of Human Adenoviruses: A 20-Year Retrospective Observational Study in Hospitalized Patients in Bern, Switzerland

Akello, Joyce Odeke; Kamgang, Richard; Barbani, Maria Teresa; Suter-Riniker, Franziska; Leib, Stephen L; Ramette, Alban

Viral DNAwas extracted from 200 μL of either original sample or cell culture supernatant of HAdV positive samples with the Boom extraction method 20 or the NUCLISENS easyMAG (bioMérieux, Geneva, Switzerland) extractor, as per manufactures instructions, and finally eluted in 50 μL. A PCR procedure that targeted the hypervariable regions 1-7 of the hexon gene was performed, according to Sarantis and colleagues. 21 M13 universal priming tails (forward, 5′-TGTAAAACGAC GGCCAGT-3′; and reverse, 5′-CAGGAAACAGCTATGA CC-3′) were added to the Sarantis and colleagues primers (forward, 5′-CTGATGTACTACAACAGCACTGGCAACA TGGG-3′; and reverse 5′-GCGTTGCGGTGGTGGTTAA ATGGGTTTACGTTGTCCAT-3′). A standard protocol of the ZymoTaq Hotstart DNA PCR was applied, consisting of 2× Zymo reaction buffer, 0.2 mM dNTP mix, 0.5 µM of each primer, 5 U/µL ZymoTaq DNA Polymerase, 10 µL of DNA template and water up to 50 µL. Thermal cycling conditions were 95°C for 10 mins, 37 cycles at 95°C for 30 secs, 51°C for 30 secs, and 72°C for 1 min, with a final extension at 72°C for 7 mins. PCR products were resolved and visualized by electrophoresis on 1% agarose gel stained with RedSafe DNA Gel Stain (Invitrogen, Switzerland). Expected amplicon sizes ranged from 602 to 630 bp. Samples with PCR product band of the expected size were purified using 1.8× Agencourt Ampure XP beads (Beckman Coulter, Nyon, Switzerland), quantified by Qubit 3.0 fluorometer (ThermoFisher Scientific, Reinach, Switzerland), diluted to concentration of 180 ng in total reaction volume of 12 µL and then submitted to Microsynth AG (Balgach, Switzerland) for Sanger sequencing. All sequence data were deposited to the European Nucleotide Archive, under project reference PRJEB36200.