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AssigneeThe United States of America as represented by the Secretary of Agriculture
InventorsManuel V. Borca, Guillermo R. Risatti, Daniel L. Rock
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Transposon linker insertion mutagenesis of a full-length infectious clone of the highly pathogenic classical swine fever virus (CSFV) isolate Brescia (pBIC) was used to identify genetic determinants of CSFV virulence and host range. A virus mutant, RB-C22 (RB-C22v), possessing a 19-residue tag insertion at the carboxyl end of E1 was constructed. RB-C22v and the parental virus pBIC (pBICv) exhibited similar growth characteristics on primary porcine macrophage cell cultures although RB-C22v produced significantly smaller plaques on SK6 cell cultures. In vivo, RB-C22v was markedly attenuated in swine. In contrast with pBIC infection, where mortality was 100%, all RB-C22v-infected pigs survived infection remaining clinically normal. Additionally, chimeras of the Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Chimeras 138.8v and 337.14v, chimeras containing the E2 glycoprotein of CS and chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, were attenuated in swine. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. The combined results indicate a significant role for E1 glycoprotein and E2 glycoprotein in swine virulence.
A recombinant classical swine fever virus comprising DNA which has been modified to encode a CSFV E1 glycoprotein comprising a 19 mer peptide insertion.
A recombinant classical swine fever virus comprising DNA encoding CSFV E2 glycoprotein which has been modified by replacing the E2 gene of the highly pathogenic strain Brescia which the E2 gene from vaccine strain CS, a modification resulting in attenuation of CSFV.
An isolated nucleic acid encoding an antigenic CSFV E1 glycoprotein wherein said glycoprotein comprises a 19 mer peptide insertion.
An isolated polypeptide having the sequence set forth in SEQ ID NO:28.
An isolated antigenic CSFV E1 glycoprotein having the sequence set forth in SEQ ID NO:30.
An immunogenic composition comprising a viable recombinant classical swine fever virus comprising DNA encoding CSFV E2 glycoprotein which has been modified by replacing the E2 gene of the highly pathogenic strain Brescia with the E2 gene from vaccine strain CS, a modification resulting in attenuation of CSFV.
An immunogenic composition comprising a viable recombinant classical swine fever virus comprising DNA modified to encode a CSFV E1 glycoprotein comprising a 19 mer peptide insertion.
A method of producing a recombinant classical swine fever virus comprising DNA modified to encode a CSFV E1 glycoprotein comprising a 19 mer peptide insertion.
A method of producing a recombinant classical swine fever virus comprising DNA encoding CSFV E2 glycoprotein which has modified by replacing the E2 gene of the highly pathogenic strain Brescia with the E2 gene from vaccine strain CS, a modification resulting in attenuation of CSFV.
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