Because of the loss of pigs after the OURT88/1 boost, only four pigs were subsequently challenged with virulent Uganda

1965. Two of these developed transient pyrexia and low viraemia. Pig 1834 had a temperature at day 6 of 40.3 °C, and the virus genome was detected at 227 copies/ml and virus at 1.75 HAD50/ml; pig 1845 had a temperature at day 7 of 40.6 °C and the virus genome was detected at 633 copies/ml; and virus at 2 HAD50/ml. The other two pigs challenged with virulent Uganda 1965 isolate showed no clinical signs and no virus was detected in blood by qPCR or HAD assay. Five pigs were challenged with Benin 97/1, two pigs (1811, 1844) developed typical ASF (Fig. 3C and D) and were terminated at days 6 and 7 respectively before developing severe disease. The remaining pigs (1809, 1829, 1837) did not develop pyrexia or other ASF clinical signs but occasionally virus genome was detected Fulvestrant by qPCR at concentrations up to 323 copies/ml but virus was not detected by HAD assay. The two groups of naïve pigs challenged with either virulent Uganda 1965 or Benin 97/1 all developed severe clinical signs of ASF with VE-821 cell line high viraemia (up to 5.37 × 107 genome copies/ml; virus up to 7.25 HAD50), and either died or were terminated within 8 days of challenge (Fig. 3). Post-mortem

examination confirmed severe ASF in these control pigs (see summary in Supplementary Table 2). In the third experiment, 7 immune pigs were

generated and 6 of these were challenged with Benin 97/1. One pig (474) showed pyrexia from 2 weeks after the first immunisation (Supplementary Fig. 1C). This pig was euthanised before the OURT88/1 boost. Post-mortem examination of this pig revealed a dark enlarged spleen characteristic of ASFV infection and Montelukast Sodium virus DNA was detected from the spleen and retropharyngeal lymph node (RLN) by qPCR (8790 and 41000 virus genome copies/mg tissue respectively) and by cytopathic effect in cultures of porcine macrophages. HAD was not observed in these cultures, indicating that the replicating virus was non-HAD, as expected for the OURT88/3 isolate. Six pigs each of the immune and non-immune groups were challenged with Benin 97/1. All of the immunised pigs were protected from challenge without showing any clinical signs or development of viraemia (Fig. 2C and D). Low copy numbers of the virus genome were detected by qPCR, but not HAD, in spleen and RLN of pig 55 at the termination of the experiment but not in any other lymphoid tissues and blood in this pig, or in any tissues from the other immunised and challenged pigs. In contrast, high copy numbers of virus genome and of virus were detected in blood (up to 5.62 × 108 virus genome copies/ml; virus up to 8.3 HAD50/ml) and tissues (virus ∼7 HAD50/mg of tissue) were detected from all lymphoid tissues in all of the non-immune pigs challenged (see summary in Supplementary Table 2).