Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2006
Publication Date: 1/1/2007
Citation: Rissatti, G., Holinka, L.G., Fernandez-Sainz, I., Carrillo, C., Lu, Z., Borca, M.V. 2007. N-linked glycosylation status of classical swine fever virus strain brecia e2 glycoprotein influences virulence in swine. Journal of Virology. Vol. 81, No.2, P. 924-933. Interpretive Summary: E2 is the most important of the four structural proteins of Classical Swine Fever Virus (CSFV). E2 has been involved in diverse viral functions as the attachment to the target cell during the infection as well as the induction of a protective immune response during vaccination. Actually, E2 is a heavily glycosidated protein, although the role of this glycosilation in the biology of the virus is completely unknown. In this report we analyzed the function of the glycosilation of E2 in the capacity of the virus to produced disease in swine. We showed that a mutated virus, containing partially elimination of the glycosilation in E2, is completely attenuated in swine. This mutated virus, although attenuated, is able to induce a strong immune response in the infected animals which resulted protected against the challenge as early as 3 days after being infected with the virus mutant. Thus, this newly developed virus could potentially be the basis for the production of a live attenuated vaccine strain of CSFV.
Technical Abstract: E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Although E2 have been involved in virus attachment to target cells, the induction of a protective immune response as well in the process of viral pathogenesis, the role of glycosylation in the functionality of the protein has never been addressed. CSFV E2 has seven putative glycosylation sites, all of them highly conserved among CSFV isolates. In this study, we used oligonucleotide site directed mutagenesis of a highly virulent CSFV strain Brescia E2 infectious clone to construct a panel of virus mutants to investigate whether the removal of each of the putative glycosylation sites in E2 glycoprotein could affect viral virulence/pathogenesis in swine. We found that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when A185 in the mutant virus was substituted back to wild type N, producing a viable virus ('O1N1-N6/N3v) that was completely attenuated in swine. Fine mapping of the glycosylation site responsible for the 'O1N1-N6/N3v attenuation demonstrated that single substitution of E2 amino acid residue N116 to A (E2N1 virus) renders an attenuated virus with a decreased virus replication and virus shedding. Additionally, E2N1 virus protected swine from challenge with virulent Brescia virus at 3 and 28 days post-infection.