Antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on E2 glycoprotein of classical swine fever virus

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, ⁷⁵³RYLASLHKKALPTSV⁷⁶⁷, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues ⁷⁵³RYLASLHKKALPTSV⁷⁶⁷ on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues ⁷⁶¹K, ⁷⁶³L and ⁷⁶⁴P were critical for the reactivity with mAbs. In addition, the up- and downstream residues ⁷⁵³R, ⁷⁵⁴Y, ⁷⁵⁵L and ⁷⁶⁵T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif ⁷⁵³RYLASLHKKALPT⁷⁶⁵, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.     

Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: essential features for infectious particle formation

The nonstructural protein NS2-3 of pestiviruses undergoes tightly regulated processing. For bovine viral diarrhea virus it was shown that uncleaved NS2-3 is required for infectious particle formation while cleaved NS3 is essential for genome replication. To further investigate the functions of NS2-3 and NS4A in the pestivirus life cycle, we established T7 RNA polymerase-dependent trans-complementation for p7-NS2-3-4A of classical swine fever virus (CSFV). Expression of NS2-3 and NS4A in trans restored the production of infectious particles from genomes lacking NS2-3 expression. Co-expression of cleaved NS4A was essential. None of the enzymatic activities harbored by NS2-3 were required for infectious particle formation. Importantly, expression of uncleavable NS2-3 together with NS4A rescued infectious particles from a genome lacking NS2, demonstrating that cleaved NS2 per se has no additional essential function. These data indicate that NS2-3 and NS3, each in association with NS4A, have independent functions in the CSFV life cycle.