RESUMEN
E(rns) glycoprotein, along with E(1) and E(2), is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E(rns) is a heavily glycosylated protein involved in several functions, including virus attachment and entry to target cells, production of neutralizing antibodies, and virulence. The role of added glycans to CSFV strain Brescia E(rns) on virus virulence was assessed in swine. A panel of virus mutants was constructed and used to investigate whether the removal of each of seven putative glycosylation sites in the E(rns) glycoprotein would affect viral virulence in swine. Only N269A/Q substitution rendered attenuated viruses (N1v/N1Qv) that, unlike BICv and other mutants, produced a transient infection in swine characterized by mild symptoms and decreased virus shedding. Notably, N1v efficiently protected swine from challenge with virulent BICv at 3 and 21 days post-infection suggesting that glycosylation of E(rns) could be modified for development of CSF live-attenuated vaccines.
Asunto(s)
Virus de la Fiebre Porcina Clásica/patogenicidad , Mutación , Polisacáridos/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Células Cultivadas , Peste Porcina Clásica/fisiopatología , Peste Porcina Clásica/prevención & control , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Glicosilación , Macrófagos/virología , Mutagénesis Sitio-Dirigida , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , VirulenciaRESUMEN
Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). In vitro growth of mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) was similar to parental BICv, while mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence.