Virus contamination of each cell line was investigated by Real-Time PCR assay. HCMV (Human Cytomegalovirus), HIV (Human Immunodeficiency Virus), HSV-1 (Herpes Simplex Virus1), HSV-2 (Herpes Simplex Virus2), EBV (Epstein-Barr Virus), HBV (Hepatitis B Virus) and HCV (Hepatitis C Virus) were examined by artus® RG PCR Kit (Qiagen, Milan, Italy), according to the manufacturer’s instructions. HHV-6 (Human Herpesvirus 6), HHV-7 (Human Herpesvirus 7), HHV-8 (Human Herpesvirus 8) and HPV (Human Papilloma Virus) were evaluated by a Real Quality PCR kit (AB AnaliticaSrl, Padova, Italy), following the manufacturer’s instructions. Finally, Influenza virus type A was examined using the primers M-for (5’-AGATGAGTCTTCTAACCGAGGTCG-3’), M-rev (5’-TGCAAAAACATCTTCAAGTCTCTG-3’) proposed by van de Brand and colleagues , the probe INF-M (5’-TET- TCAGGCCCCCTCAAAGCCGA-BHQ1-3’, ) and the QuantiTect Virus kit (Qiagen), according to the manufacturer’s instructions. Positive and negative controls, provided by the manufacturer, have been included in each session.
In vitro testing for adventitious agents was performed in compliance with the European Pharmacopoeia recommendations (Cell substrates for the production of vaccines for human use) . Cell culture samples and cell cryolisates were investigated on MRC-5, RK13.6 and LCC-MK2 cells grown in 24-well plates for adventitious viruses with the ability to induce cytopathic effect (CPE) (co-culture and cryolisate method). Briefly, 0.1 ml of each sample was inoculated on cell monolayers and, following adsorption for 30 minutes, cells were fed with the specific growth medium containing 3% (v/v) of FBS and incubated at 37°C in 5% CO2. After 7 days of growth, medium was renewed, while monolayers were observed daily for CPE for 14 days. On day 14, treated LCC-MK2 cells were removed from the incubator and tested for haemadsorption of guinea pig and chicken erythrocytes. Briefly, cell monolayers were washed and duplicate wells were overlaid with 0.5 ml of 0.5% (v/v) of guinea pig and chicken erythrocytes and, after 30 minutes of incubation at room temperature, examined for adsorption. As positive control H/A/WSN/33 (VIR RE RSCIC 50) influenza virus was used.
Cell cultures were also examined for the presence of retroviruses, using the Reverse Transcriptase Assay, colorimetric kit (ROCHE, Basel, Switzerland) for the quantitative determination of the viral Reverse Transcriptase (RT) activity. According to manufacturer’s instructions, a calibration curve was prepared from HIV-1 RT included in the kit. For the lysis of the retroviruses, 40 μl of supernatant were mixed with 40 μl of Lysis Buffer. After 30 minutes of incubation at room temperature, 20 μl of the reaction mixture were added to each reaction and HIV-1 RT standard tube. Finally, samples were incubated at 37°C for 15 h. Samples and HIV-1 RT dilutions were transferred into the wells of the MP modules and incubated for 1 h at 37°C. The solution was completely removed and the strip was rinsed 5 times with 250 μl of Washing Buffer per well for 30 s. 200 μl of anti-DIG-POD working solution were added and incubated for 1 h at 37°C. The solution was completely removed and the washing steps were repeated. Finally, 200 μl of ABTS Substrate Solution were added and the plate was incubated at room temperature for 30 minutes. The absorbance was measured at 405 nm by using a Gen5 microplate reader (Biotek, Milan, Italy) and the effective RT activity was extrapolated from the standard curve. Full paper
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