Mutations in Classical Swine Fever Virus NS4B Affect Virulence in Swine

Abstract

NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.

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.…”

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence

“…However, NS4B is a poorly characterized protein and its roles in CSFV replication and pathogenesis are not well understood. A putative Toll/interleukin-1 receptor-like domain was identified in the C-terminal region of NS4B (Fernandez-Sainz et al, 2010). Mutations in this domain of NS4B in the highly virulent Brescia strain resulted in an attenuated phenotype along with enhanced activation of TLR-7-induced genes.…”

An Overview of the Immune Evasion Strategies Adopted by Different Viruses with Special Reference to Classical Swine Fever Virus

“…Several studies on different pestiviruses have revealed that NS4B is an endoplasmic reticulum (ER)-associated integral membrane protein that contains four putative transmembrane domains flanked by cytoplasmic N-and C-terminal regions [119,120,121,122]. Interaction of CSFV NS4B with molecular components of the immune system has also been reported [123].…”

“…The synthesis of NS4B protein during viral replication in the tonsil down regulates the expression of IL-6 and this is especially true with CSFV strain Brescia [123]. Swine Leukocyte Antigen I (SLA I) molecules present the endogenous peptides to activate the CD8 + T cells that control viral replication within cells.…”