Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus

•Mutations in attenuated chimeric virus mediating virulence are identified.•Substitutions T745I and M979K are responsible for the acquisition of pathogenicity.•Substitutions T745I and M979K enhance the replication of chimeric virus vSM/CE2.

Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2 containing the E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain Shimen (vSM) was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To investigate the molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant (vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of vSM/CE2-p11 were observed. Based on reverse genetic manipulation of the chimeric cDNA clone pSM/CE2, the mutated viruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were rescued. The data from infection of pigs demonstrated that the M979K amino acid substitution was responsible for pathogenicity. Studies in vitro indicated that T745I and M979K increased infectious virus production and replication. Our results indicated that two residues located at sites 745 and 979 within E2 play a key role in determining the replication in vitro and pathogenicity in vivo of chimeric CSFV vSM/CE2.