PMC:3537549 / 19143-22461 JSONTXT

{"project":"2_test","denotations":[{"id":"22906110-16054243-143530977","span":{"begin":2912,"end":2914},"obj":"16054243"}],"text":"Viruses and viral inhibition assays\nVSV Indiana strain (Institute of Virology, Vetsuisse Faculty, University of Zurich, Switzerland), FCoV Wellcome strain (a generous gift from Prof. A. Kipar, University of Liverpool, Great Britain), FPV (kindly provided by Prof. U. Truyen, University of Leipzig, Germany), FHV ZH5-04 strain and FCV F9 strain (kindly provided by Veterinaria AG, Zurich, Switzerland) were titrated on both CrFK and fcwf-4 cells. Viral stock dilutions inducing 95% cytopathic effect (CPE) after 24 h (72 h for FCoV and FPV) were selected for inhibition experiments in order to ensure proper measurement of inhibitory effects. Monolayers of CrFK and fcwf-4 cells in 96-well plates were incubated for 24 h with 100 μL of the supernatants produced with PBMCs from cats of groups 1, 2 and 3. With the exception of assays carried out with FPV, viral inhibition experiments were conducted simultaneously and with supernatants thawed an equal number of times. The treated cells were then inoculated with virus (VSV, FCV, FHV, FCoV) or trypsinized with 0.05% trypsin-EDTA (Gibco®, Invitrogen) and allowed to settle in viral suspension (FPV), and inhibition assays were carried out after 24 h (72 h for FCoV and FPV) according to the procedure described previously[62]. Briefly, supernatants were discarded and cell debris was removed from the wells by 3 cycles of washing with Hank’s balanced salt solution (HBSS) (Gibco, Invitrogen) and shaking on an orbital shaker for 15 s. Remaining cells were fixed with 5% formalin and stained with a crystal violet solution. For spectrophotometric measurements, 100% methanol was added to the dried out wells and absorbance was read at 595 nm on a SpectraMax Plus 384 microtiter plate reader (Molecular Devices, Bucher Biotec AG, Basel, Switzerland). Viral inhibition rates were calculated with the following formula:Mean optical densityODvalues of duplicate wells treated with SupernatantMean OD values of quadruplicate wells treated with medium alone\nFeLV-A Glasgow-1 strain (a generous gift from Prof. M. Hosie and O. Jarret, University of Glasgow, Great Britain) was titrated on FEA cells, and the lowest stock dilution leading to productive infection of the cells after 48 h was used for inhibition assays. Experiments were carried out in 96-well plates and cells were treated with 100 μL of supernatants or relevant controls immediately prior to inoculation. Every second day thereafter, 50 μL culture medium was replaced by the same volume of fresh supernatant. At appropriate time points, cells and supernatants were harvested and total nucleic acid was extracted from both the cells and supernatants using the MagNA Pure LC DNA Isolation Kit I and MagNA Pure LC Instrument (Roche Diagnostics). Viral replication in supernatants and proviral loads in cells were measured by real-time RT-PCR and real-time PCR respectively, with assays previously described[63]. The time course experiments were conducted with supernatants derived from PBMCs of three cats and the measurements on day 8 post inoculation were carried out with material derived from two additional cats. In order to facilitate interpretation of the figures illustrating measurements of viral RNA loads, 45 cycles-cycle threshold (Ct) values were calculated and means of duplicate wells are depicted."}