Chimeric classical swine fever viruses containing envelope protein ERNS or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response

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.

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confidence: 99%

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confidence: 99%

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

Risatti

Holinka

Sainz

et al. 2007

“…Additionally, attempts to mutate and inactivate E rns in the attenuated C-strain resulted in a virus atypically cytopathic for cell cultures (10, 39). Thus, CSFV IC hold great promise for identifying viral proteins or protein domains functioning in viral virulence and host range and for rationally engineering marker live attenuated CSF vaccines (17,19,21,30,31).Here we have used chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS to identify genetic determinants of CSFV virulence and host range. Results demonstrate that the CSFV E2 glycoprotein is a major determinant of virulence in swine.…”

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confidence: 99%

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

Risatti

Borca

Kutish

et al. 2005

133

108

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...

“…E2 is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable (7). 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).…”

Section: Importancementioning

confidence: 99%

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

Holinka

Largo

Gladue

et al. 2016

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a WimleyWhite interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond ( 869 CKWGGNWTCV 878 , named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872 GG 873 dipeptide, as well as two aromatic residues ( 871 W and 875 W) accessible to solvent. Reverse genetics utilizing a fulllength infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. IMPORTANCEThis report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs. C lassical swine fever (CSF) is a highly contagious disease of swine caused by CSF virus (CSFV), a small enveloped virus with a positive-sense, single-strand RNA genome (1). The approximately 12.5-kb CSFV genome contains a single open reading frame that encodes a polyprotein composed of 3,898 amino acids that ultimately yields up to 12 final cleavage pr...