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Title: IDENTIFICATION OF A NOVEL VIRULENCE DETERMINAT WITHIN THE E2 STRUCTURAL GLYCOPROTEIN OF CLASSICAL SWINE FEVER

Author
item RISATTI, GUILLERMO - UNIV OF CONNECTICUT
item Holinka-Patterson, Lauren
item CARRILLO, CONSUELO - USDA APHIS, PIADC
item KUTISH, GERALD - UNIV OF CONNECTICUT
item LU, ZHIQIANG - DHS, PIADC
item FERNANDEZ-SAINZ, IGNACIO - ORISE, PIADC
item Borca, Manuel

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2006
Publication Date: 11/10/2006
Citation: Risatti, G., Holinka, L.G., Carrillo, C., Kutish, G.F., Lu, Z., Fernandez-Sainz, I., Borca, M.V. 2006. Identification of a novel virulence determinat within the e2 structural glycoprotein of classical swine fever. Virology, 11/10/2006, Vol 355, Issue i, pages 94-101.

Interpretive Summary: Classical Swine Fever Virus (CSFV) E2 glycoprotein is one of the structural proteins of the virus. E2 is a major inducer of neutralizing antibodies and protective immunity in swine. Also, E2 mediates virus adsorption to the target cell, and harbors genetic determinants associated with virus virulence. CSFV E2 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, we present a series of mutants where the WH303 epitope is progressively mutated. Mutant viruses showed, along with the lost of reactivity against mAB WH303, progressive attenuation. These findings constitute the first mapping of a discrete genetic determinant associated with virulence into E2 protein of any of the pestiviruses. Interestingly, animals infected with some of the mutated viruses were protected from clinical disease when challenged with virulent Classical Swine Fever virus at 3 or 21 days post infection indicating that mutated viruses may provide basis for a rationally designed live attenuated CSF vaccine.

Technical Abstract: 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.