Lsrii flow cytometry

The lentiviral production and infection procedures were carried out as reported previously with minor modifications.34 Briefly, cells were transfected with the following lentivirus plasmids: psPAX2 packaging plasmid, pMD2G envelope plasmid (Addgene), pWPI‐vector ctrl, or pWPI‐ARQs (‐ARQ13, ‐ARQ25 or ‐ARQ35). Lentiviral plasmids carrying the GFP gene were co‐transfected with psPAX2 and pMD2G into HEK293T cells at a ratio of 3:2:4 with lipofectamine 2000 (Invitrogen) per the manufacturer's instructions. After 6 hrs, the media was replaced with fresh DMEM/10% FBS, and the cells were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO₂ for 48 hrs. Media containing virus was collected by centrifugation and filtered through a 0.45‐μm filter. Media containing 0.8 mg/ml polybrene (Sigma‐Aldrich) was then added to culture dishes containing 10⁶ HEC‐1A cells. After 16‐hrs infection, the media containing the virus was replaced with fresh DMEM/10% FBS medium, and the cells were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO₂ for 48 hrs. Infected cells were then collected and analysed. The green fluorescence protein (GFP) + cells were measured with flow cytometry (BD, CA, USA, LSR II Flow Cytometry) to determine infection efficiencies. GFP+ cells with infection efficiencies greater than 85% were subjected to the following experiments.

Serum cytokine assays were performed as previously described (18 (link)). The levels of cytokines (baboon IL-6, TNF-α, IL-1β) in baboon serum were measured by cytometric bead array (CBA) with a human Inflammatory Cytokine Kit (No. 551811; BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. LSR II flow cytometry (BD Biosciences) was used to collect data, which were analyzed using CBA analysis software (BD Biosciences). Levels of pig IL-6 in baboon serum were measured by ELISA using a porcine IL-6 Quantikine ELISA Kit (No. P6000B; R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. Optical density was measured by using a Wallac Victor3 1420 Multilabel Counter (Perkin Elmer, Waltham, MA) at 450nm, with the correction wavelength set at 540nm or 570nm.

Confluent monolayers of Vero SUA cells (from Prof. Stacey Efstathiou, NIBSC, UK) were pre-treated with 100 μg/mL cycloheximide or 50 μM acyclovir, or left untreated, for one hour at 37°C with 5% CO₂. The cells were infected at an MOI of 5 either with or without drug as appropriate. After incubation for 1 hour at 37°C with 5% CO₂, the unabsorbed virus was removed and replaced with fresh media containing the appropriate drug. The cells were then incubated for six h at 37°C, 5% CO₂, after which time media containing cycloheximide was removed and replaced with 5 μg/mL actinomycin D. The cells were incubated for a further four hours at 37°C, 5% CO₂. The cells were then harvested, fixed with 1% paraformaldehyde, and the expression of eGFP was assessed using flow cytometry using a LSR-II flow cytometry (BD biosciences). Flow cytometry analysis was performed with the aid of FlowJo 8.7.1 software (Treestar).

ABCG2 efflux analysis was performed as described previously by Goodell et al. [28 (link)]. Briefly, 10⁶ cells were detached and washed, then incubated in DMEM containing 2% FBS and 5 µg/mL Hoechst33342 dye (Sigma-Aldrich) for 90 min at 37 °C, either alone or in the presence of 50 µM verapamil (Sigma-Aldrich). After certain incubation time periods, the cells were washed with ice-cold 1× PBS and then resuspended with 1× PBS supplemented with 2% FBS and 2 µg/mL propidium iodide (Sigma-Aldrich) at 4 °C for 5 min to exclude dead cells. The cells were analyzed using flow cytometry (BD, CA, USA, LSR II Flow Cytometry) with dual wavelength analysis (Hoechst-blue, 424–444 nm; Hoechst-red, 675 nm) after excitation with 350 nm UV light.