N(pro) of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein N(pro) of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE(-), N(pro) acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE(-) vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE(-) vaccine virus in pigs, the IRF3-degrading function of N(pro) was not recovered. Therefore, we examined whether restoring the ability of N(pro) to block IFN-α/β induction of both the avirulent and moderately virulent GPE(-)-derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in N(pro) regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional N(pro) significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional N(pro) influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.