Foetal calf serum

Cell culture was performed using Vero cells as recommended by the OIE protocol [18 ]. Briefly, 102 positive capripox virus PCR samples were inoculated to a 25 cm³ cell culture flask (Corning, Mediatech Inc., Virginia, USA) of 80% confluent monolayer for penetration at 37 °C for 2 h, then washed thrice where maintenance medium was added with 2% foetal calf serum (Corning, Corning, Mediatech Inc., Virginia, USA) and 1% antibiotics (Gibco, USA). The cultures were incubated at 37 °C with 5%CO₂ with frequent changing of medium every 2 days. Cytopathic effect (CPE) was recorded daily under an inverted microscopic (Zeiss Axiovert 4 °C, Germany) for 14 days. Negative culture was considered when there was absence of CPE following two or more blind passages.

Dulbecco’s modified Eagle’s medium, foetal calf serum and penicillin/streptomycin were from Corning (Corning, NY, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sphingosine-1-phosphate (S1P, cod.62570) and methanandamide (N-(2-hydroxy-1R-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, cod.157182-49-5) were from Cayman Chemicals (Ann Arbor, MI, USA); N-arachidonoyl-ethanolamine (anandamide (AEA), cod. A0580) and 2-arachidonoil-glycerol (2-AG, cod. A8973) were from Sigma-Aldrich (St. Louis, MO, USA). AEA-d8 and 2-AG-d8 were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and were used as internal standards. The selective antagonists of CB₁ SR141716A (SR1, cod. SML0800), of CB₂ SR144528 (SR2, cod. SML1899) and of GPR55 ML193 trifluoroacetate (ML193, cod. SML1340) were from Sigma-Aldrich (St. Louis, MO, USA). The TRPV1 selective antagonist 5′-iodoresiniferatoxin (I-RTX, cod. 1362) was from TOCRIS (Bristol, UK). For molecular biology studies RevertAid H Minus First Strand cDNA Synthesis Kit, from Thermo Scientific (Waltham, MA, USA), and SensiFASTTM SYBR Lo-ROX kit, from Bioline (London, UK) were used.