Infinity3 microscope camera

The assays evaluate the antiviral activity of the compound when administered before, during or after HSV-2 infection. Cells were incubated with different concentrations of A-3302-B (from 100 μM to 0.4 μM) in a 24-well plate at 37 °C for 2 h before infection (pretreatment), for 2 h during infection (cotreatment) or 24 h after removal of virus inoculum (post-treatment). Cells were concurrently infected with HSV-2 at MOI of 0.001 PFU/cell and treated for plaque reduction assay. Plaque size was measured with an Axiovert 200 inverted microscope (Zeiss, Jena, Germany) equipped with Infinity3 microscope camera (Lumenera, Ottawa, ON, Canada) and Infinity Capture software (Lumenera), and was analyzed with Infinity Analyze software (Lumenera) and ImageJ software (Bethesda, MD, USA).

The anti-herpetic activity of the drug formulations was evaluated via a plaque reduction assay. Vero cells were seeded at a 1.0 × 10⁵/well density in 24-well plates. The following day, cells were infected with HSV-1, HSV-2 or HSV-2 ACV-r at a multiplicity of infection (MOI) of 0.001 PFU/cells. After a 2 h incubation at 37 °C, the viral inoculum was removed and the cells were treated with serial dilutions of the drug formulations (from 14.8 µM to 0.23 µM) in DMEM containing 1.2% methylcellulose (Sigma-Aldrich). VACV was tested in parallel as a reference. At 24 h post-infection (hpi) (for HSV-2 and HSV-2 ACV-r) or 48 hpi (for HSV-1), the cells were subjected to fixing and staining with 0.1% crystal violet in 20% ethanol, and the viral plaques were microscopically counted. The inhibition of infectivity by the antiviral treatment was calculated as percentage of viral plaques in the treated wells in comparison to the untreated controls. The size of HSV-2 plaques was determined in at least 10 microscopic fields for each compound concentration (VACV, SBEβCD-ND-VACV and SBEβCD-ND) using an Axiovert 200 inverted microscope (Zeiss, Jena, Germany), Infinity3 microscope camera (Lumenera, Ottawa, ON, Canada) and Infinity Capture software 6.3 (Lumenera). Data were analyzed with Infinity Analyze software 7.0 (Lumenera) and ImageJ software 1.5 (Bethesda, MD, USA).