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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.

SUBMITTER: Risatti GR

PROVIDER: S-EPMC1075681 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

Risatti G R GR Borca M V MV Kutish G F GF Lu Z Z Holinka L G LG French R A RA Tulman E R ER Rock D L DL

Journal of virology 20050301 6

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 chi ...[more]

PMID: 15731272

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Project description: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 Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, as well as two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length 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. IMPORTANCE:This 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.

Project description:The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197-206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

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The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

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The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

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