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AssigneeThe United States of America as represented by the Secretary of Agriculture
InventorsManuel V. Borca, Guillermo R. Risatti
Estimating the term…
E1, along with Erns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Our previous studies indicated that glycosylation status of either E2 or Erns strongly influence viral virulence in swine. Here, we have investigated the role of E1 glycosylation of highly virulent CSFV strain Brescia during infection in the natural host. The three putative glycosylation sites in E1 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E1 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all three putative glycosylation sites in E1. Single mutations of each of the E1 glycosylation sites showed that CSFV amino acid N594 (E1.N3 virus), as well the combined mutation of N500 and N513 (E1.N1N2 virus) resulted in BICv attenuation. Infection of either E1.N1N2 or E1.N3 viruses were able to efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. These results, along with those demonstrating the role of glycosylation of Erns and E2, suggest that manipulation of the pattern of glycosylation could be a useful tool for development of CSF live-attenuated vaccines.
An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule that encodes a genetically modified classical swine fever virus (CSFV) mutant of the highly pathogenic native Brescia strain, wherein said CSFV mutant encodes a genetically modified E1 glycoprotein set forth in SEQ ID NO:3, wherein mutated non-glycosylated amino acid alanine in the sequence AVTS (SEQ ID NO:9) has replaced glycosylated asparagine in the sequence NVTS (SEQ ID NO:8) at position 500 and mutated non-glycosylated amino acid alanine in the sequence ACTP (SEQ ID NO:11) has replaced glycosylated asparagine in the sequence NCTP (SEQ ID NO:10) at position 513, said mutated amino acids having a glycosylation pattern different from the amino acids present in positions 500 and 513 of said highly pathogenic native Brescia strain, wherein said genetically modified CSFV mutant is attenuated and unable to produce a pathogenic CSFV infection in a porcine animal.
A recombinant classical swine fever virus comprising DNA encoding a mutated CSFV E1 glycoprotein having the sequence identified by SEQ ID NO:
A method of producing an attenuated recombinant classical swine fever virus comprising DNA encoding a modified CSFV E1 glycoprotein, comprising:
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