Classical Swine Fever Virus (CSFV) E2 glycoprotein is a major inducer of neutralizing antibodies and protective immunity in swine. E2 mediates virus adsorption to the target cell, and harbors genetic determinants associated with virus virulence. CSFV E2 also contains between residues 829 and 837 a discrete epitope (TAVSPTTLR) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related Pestiviruses Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV). In this report, a CSFV infectious clone of the virulent Brescia isolate (BICv) was used to progressively mutate the mAb WH303 epitope of CSFV E2 to the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). While the resulting virus mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) demonstrated in vitro growth characteristics similar to those of parental BICv, mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and a significant decrease in plaque size relative to parental BICv. Immunohistochemical reactivity with WH303 was lost only in T3v, T4v and T5v. Interestingly, progressive mutation of the WH303 epitope had an additive effect on attenuation for the virus in swine, with mutants T1v, T2v or T3v inducing progressively milder but invariably lethal CSF, T4v inducing only mild and transient clinical disease, and T5v inducing no disease. Swine infected with either T4v or T5v showed decreased virus replication in tonsils, draining lymph node, spleen and kidney and a significant reduction in virus shedding. Finally, T5v-infected animals were protected from clinical disease when challenged with virulent Brescia virus at 3 or 21 days post T5v inoculation. These results indicate that amino acid residues 830 to 834 of E2 are critical for virulence of CSFV in swine and that engineering at this locus may provide basis for a rationally designed live attenuated CSF vaccine.
