N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

Abstract

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 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 E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…This is reflected in numerous reports in which mutations of different functional domains in the structural proteins C, E rns , E1, and E2 and in the nonstructural proteins p7 and NS4B were shown to reduce the pathogenicity in pigs, indicating that these genes may be involved in determining CSFV virulence (7,8,9,10,11,12,13,32,36,37,38,39,40,46,54).…”

“…Comparison of the genomes of the GPE Ϫ vaccine virus and the parent ALD virus revealed 225 nucleotide substitutions and 46 amino acid differences (22), which did not provide any conclusive information on the molecular basis of the attenuation. Numerous reports suggest that the viral proteins N pro , C, E rns , E1, E2, p7, and NS4B may be involved in determining the virulence of CSFV in pigs (7,8,9,10,11,12,13,32,36,37,38,39,40,43,46,54). So far, however, all conclusions on CSFV virulence determinants rely on mutations that result in downregulation of pathogenicity.…”

Genetic clustering of recent classical swine fever virus isolates from Karnataka, India revealed the emergence of subtype 2.2 replacing subtype 1.1

“…Among the seven glycosylation sites identified [21], the five sites at aa 805-807, 810-812,874-876, 918-920 and 949-951 were highly conserved in all 11 isolates along with reference isolates. The remaining two potential glycosylation sites at aa 777-779 and 986-988 were conserved in present isolates but varied among the reference isolates.…”

Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine

“…E2 has been implicated, along with E rns (8) and E1 (9), in viral adsorption to host cells (10,11). Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence (12)(13)(14)(15)(16).…”