Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine.

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.

Cerebral and renal phaeohyphomycosis in a dog infected with Bipolaris species.

Mycotic meningoencephalitis in dogs may manifest as a primary disease of the central nervous system or as a part of disseminated infection. Fungi belonging to the genus Bipolaris are saprophytic plant pathogens and can cause disease in humans. The authors report a case of Bipolaris infection in a dog with granulomatous meningoencephalitis, nephritis, and vasculitis. The clinical and histological features resembled those of the more common aspergillosis, thus warranting confirmation by molecular methods. Polymerase chain reaction and sequence analysis identified Bipolaris from the brain lesion, indicating its involvement in the disease. To the authors' knowledge, this is the first reported case of meningoencephalitis caused by this fungus in a domestic animal.

Alteration of the N-linked glycosylation condition in E1 glycoprotein of Classical Swine Fever Virus strain Brescia alters virulence in swine.

E1, along with E(rns) and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or E(rns) affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by means of site directed mutagenesis of a CSFV Brescia infectious clone (BICv) and their effect on virulence assessed in swine. Removal of all three putative glycosylation sites in E1, at CSFV positions N500, N513 and N594, yielded nonviable progeny, while single or dual site mutants excluding N594 were viable. Individual N594A (E1.N3 virus) or combined N500A/N513A (E1.N1N2 virus) substitutions resulted in BICv attenuation. Furthermore infection with E1.N3 or E1.N1N2 viruses efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. As previously observed with E(rns) and E2 and here with E1 data suggest that modification of glycosylation patterns could be used for developing CSFV live-attenuated vaccines.