Cultivation for 21 Days Should Be Considered to Isolate Respiratory Adenoviruses from Samples Containing Small Numbers of Adenoviral Genomes

Adenovirus types 1, 2, and 3 can usually be isolated in only a short time, although occasionally it may take longer. This phenomenon has been explained empirically as being due to the viral load in the sample, although to date there has been no experimental confirmation of this. In this study we therefore tried to establish a correlation between the quantity of respiratory adenovirus genome in the clinical sample and the time required for its isolation. The correct choice of sensitive cell line is important for this purpose, thus we compared the sensitivity of three different cell lines (HeLa, A549, and RD), and found A549 to be the most sensitive to adenoviruses 1–3. Stored clinical samples (n=21) containing adenoviruses 1–3 were diluted to make solutions containing between 10 and 108 copies/µL of adenovirus genome (n=242). These diluted clinical samples were then inoculated into A549 cells, which were cultivated for 21 days and the results compared to the number of viral genomes in each cultivated sample. Adenoviruses could be isolated from all samples (41/41) containing ≥106 copies/µL within 6 days, whereas samples containing 10 and 102 copies/µL required cultivation for 12.6±3.8 and 11.2±3.8 days (mean±S.D.), respectively, before adenoviruses could be isolated. A cultivation time of 21 days should therefore be considered for the isolation of respiratory adenoviruses from samples containing