For the third experiment (experiment 3) carried out at Anses Ploufragan, France, Large White pigs were obtained from a local high health status farm and the average weight at the

first immunisation was 11 kg. All pigs were maintained at high security facilities throughout the experiment. The first experiment at Pirbright was performed under Home Office licence PPL 70-6369. Experiments at Ploufragan were performed according to the animal welfare experimentation agreement given by the Direction des Services Vétérinaires des Côtes d’Armor (AFSSA registration number B-22-745-1), under the responsibility of Marie-Frédérique buy Wortmannin Le Potier (agreement number 22-17). Briefly, pigs were intramuscularly inoculated

with 104 TCID50 of non-virulent ASFV isolate OURT88/3 and boosted intramuscularly 3 weeks later with 104 HAD50 of virulent ASFV Everolimus supplier isolate of OURT88/1. Pigs were then challenged 3 weeks later with 104 HAD50 of either Benin 97/1 or virulent Uganda 1965 intramuscularly. ASFV-inoculated pigs were monitored for body temperature and other clinical symptoms and these were recorded and scored according to the clinical scoring system shown in Supplementary Table 1. Weight gain was also recorded in the experiments carried out at Ploufragan. All pigs were examined post-mortem either when the pigs died or at the termination of the experiments. Tissues were collected for further analysis. Peripheral blood was analysed at different days post-immunisation for the presence of ASFV by quantitative PCR (qPCR) as described previously [22]. Samples which tested positive by qPCR were further analysed by cytopathic and/or haemadsoption assay (HAD) using standard pig bone marrow cells in 96 well plate [23] and [24]. Spleen, tonsil, retropharyngeal and ileocaesal Resminostat lymph nodes from post-mortem tissues were also analysed for the presence of ASFV by qPCR and HAD. Virus detected from tissue samples

by qPCR was expressed as copy number per mg tissue and by HAD as HAD50. Development of T cell immune responses to ASFV after immunisation was analysed by IFN-γ ELISPOT and proliferation assays as described previously [25]. All ASFV isolates used as antigens for T cell assays were prepared by culture in porcine bone marrow cells, and ASFV titres were determined by qPCR [22] and adjusted to give the equivalent of 105 HAD50/ml. Uninfected porcine bone marrow culture supernatants were used as negative control antigen. The development of ASFV specific antibodies was analysed using a competition ASF ELISA kit (INGENASA PPA3 COMPPAC), and the antibody titre was expressed as log 2 dilution of end point which gives 50% competition.