Journal: Journal of Virology
Article Title: Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms
doi: 10.1128/JVI.00827-17
Figure Lengend Snippet: 25HC inhibits PRRSV penetration. (A) Inactivated assay. PRRSV and 25HC were incubated at 37°C for 3 h and PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h, and then the media were replaced by a mixture of 25HC (12.5 μM) or ethanol (EtOH) and PRRSV (2, 20, and 200 PFU/well). After incubation at 4°C for another 2 h, cells were washed with precooled PBS, covered with overlay medium (1.8% [wt/vol] Bacto agar mixed 1:1 with 2× DMEM containing 0.05 mg/ml neutral red), incubated at 37°C for a further 72 h, and examined by a plaque assay. N.S., not significant. (B) Adsorption assay. PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h, and then the media were replaced by a mixture of 25HC (12.5 μM) or ethanol (dilution) and PRRSV (3, 30, and 300 PFU/well). After incubation at 4°C for another 2 h, the cells were washed with precooled PBS, and then cells were treated as described for panel A and plaques were counted directly. (C) Penetration assay. PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h and then incubated for another 2 h at 4°C with PRRSV at different doses (1, 10, and 100 PFU/well). The virus-containing medium was replaced by fresh medium containing 25HC (12.5 μM) or ethanol, and the temperature was shifted to 37°C for 3 h. Cells then were treated as described for panel A and plaques were counted directly. (D) Replication assay. PK-15CD163 cells were incubated with PRRSV (MOI of 1.0) for 6 h, the cell-free virus particles were removed, and cells were cultured in fresh medium containing 25HC (12.5 μM). At 7, 8, 9, and 10 hpi, the infected cells were collected for qRT-PCR to detect the level of negative-sense PRRSV RNA. (E) Release assay. PK-15CD163 cells were infected with PRRSV (MOI of 1.0). At 18 hpi, the inocula were replaced by fresh medium containing 25HC (12.5 μM). Culture media were harvested at 15, 30, 45, and 60 min after medium switching and titrated by a plaque assay. All results are means ± standard deviations from three independent experiments performed in triplicate.
Article Snippet: After incubation at 4°C for another 2 h, cells were washed with precooled PBS, covered with overlay medium (1.8% [wt/vol] Bacto agar mixed 1:1 with 2× DMEM containing 0.05 mg/ml neutral red), incubated at 37°C for a further 72 h, and examined by a plaque assay.
Techniques: Incubation, Cell Culture, Plaque Assay, Adsorption, Virus, Infection, Quantitative RT-PCR, Release Assay
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![25HC inhibits PRRSV penetration. (A) Inactivated assay. PRRSV and 25HC were incubated at 37°C for 3 h and PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h, and then the media were replaced by a mixture of 25HC (12.5 μM) or ethanol (EtOH) and PRRSV (2, 20, and 200 PFU/well). After incubation at 4°C for another 2 h, cells were washed with precooled PBS, covered with overlay medium (1.8% <t>[wt/vol]</t> <t>Bacto</t> agar mixed 1:1 with 2× <t>DMEM</t> containing 0.05 mg/ml neutral red), incubated at 37°C for a further 72 h, and examined by a plaque assay. N.S., not significant. (B) Adsorption assay. PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h, and then the media were replaced by a mixture of 25HC (12.5 μM) or ethanol (dilution) and PRRSV (3, 30, and 300 PFU/well). After incubation at 4°C for another 2 h, the cells were washed with precooled PBS, and then cells were treated as described for panel A and plaques were counted directly. (C) Penetration assay. PK-15CD163 cells cultured in six-well plates were prechilled at 4°C for 1 h and then incubated for another 2 h at 4°C with PRRSV at different doses (1, 10, and 100 PFU/well). The virus-containing medium was replaced by fresh medium containing 25HC (12.5 μM) or ethanol, and the temperature was shifted to 37°C for 3 h. Cells then were treated as described for panel A and plaques were counted directly. (D) Replication assay. PK-15CD163 cells were incubated with PRRSV (MOI of 1.0) for 6 h, the cell-free virus particles were removed, and cells were cultured in fresh medium containing 25HC (12.5 μM). At 7, 8, 9, and 10 hpi, the infected cells were collected for qRT-PCR to detect the level of negative-sense PRRSV RNA. (E) Release assay. PK-15CD163 cells were infected with PRRSV (MOI of 1.0). At 18 hpi, the inocula were replaced by fresh medium containing 25HC (12.5 μM). Culture media were harvested at 15, 30, 45, and 60 min after medium switching and titrated by a plaque assay. All results are means ± standard deviations from three independent experiments performed in triplicate.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9739/pmc05599739/pmc05599739__zjv9991829210005.jpg)