The E2 Glycoprotein of Classical Swine Fever Virus Is a Virulence Determinant in Swine

Abstract

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.Classical swine fever (CSF) is a highly contagious and often fatal hemorrhagic disease of swine and is caused by Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae (5). Intermittent CSF outbreaks in Europe and other parts of the world result in significant economic losses. While infection with highly virulent CSFV strains can cause acute CSF characterized by high morbidity and mortality, isolates of moderate to low virulence induce a prolonged, chronic disease (35).CSFV is a small enveloped virus with a single-stranded, 12.5-kb RNA genome of positive polarity. The CSFV genome contains a single open reading frame encoding an approximately 4,000-amino-acid polyprotein which through cellular and viral protease-mediated co-and posttranslational processing gives rise to the following 11 to 12 final cleavage products: NH 2 -Npro-C-E rns -E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (23). Protein C and the glycoproteins E rns , E1, and E2 represent structural components of the virion (28). E1 and E2 are anchored to the envelope by their carboxy termini, with E rns loosely associated with the envelope. E rns and E2 are present as homodimers linked by disulfide bridges on the surfaces of CSFV virions, whereas E2 is also found dimerized with E1 (28, 37, 38). The genetic basis of CSFV virulence and host range remains poorly understood (35).Development of infectious CSFV cDNA clones has enabled genetic approaches for defining mechanisms of viral replication and pathogenesis. Infectious clones (IC) of the attenuated CSFV C-strain and the pathogenic Alfort/Tübingen and Eystrup strains have been constructed, enabling identification of E rns and N pro as virulence factors in swine and of the role of different E rns mutations in virus attenuation (17, 19). Additionally, attempts to mutate and in...

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

“…Swine kidney cells (SK6) (17), free of BVDV, were cultured in Dulbecco's minimal essential medium (DMEM) (Gibco, Grand Island, NY) with 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO). CSFV strain Brescia was propagated in SK6 cells and used for the construction of an infectious cDNA clone (IC) (9). Growth kinetics was assessed on primary swine macrophage cell cultures prepared as described by Zsak et al (24).…”

“…E1 has been implicated (21), along with E rns and E2 (4), in viral adsorption to host cells. Importantly, different modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (3,6,9,10,11,12,15,20).In lysates of CSFV-infected cells, E2 glycoprotein forms homodimers and heterodimers with E1 glycoprotein (14,22) via disulfide bridges between cysteine residues. Formation of these disulfide-linked heterodimers also occurs in CSFV virions (19), suggesting that the interaction between envelope proteins plays an important role in virus assembly, virion maturity, and consequently in efficiency of viral infection.…”

“…E1 has been implicated (21), along with E rns and E2 (4), in viral adsorption to host cells. Importantly, different modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (3,6,9,10,11,12,15,20).…”

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

“…E2 has been implicated, along with E rns (14) and E1 (43), in viral adsorption to host cells; indeed, chimeric pestiviruses exhibit infectivity and cell tropism phenotypes consistent with those of the E2 gene donor (18,40). Modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (21,29,31,41).…”

“…(37), free of BVDV, were cultured in Dulbecco's minimal essential medium (Gibco, Grand Island, NY) with 10% fetal calf serum (Atlas Biologicals, Fort Collins, CO). CSFV strain Brescia was propagated in SK6 cells and used for the construction of an infectious cDNA clone (29). Growth kinetics was assessed on primary swine macrophage cultures prepared as described by Zsak et al (46).…”