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

Abstract

c Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE ؊ was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE ؊ vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE ؊ /P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.

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

“…8). This suggests that the virulence of CSFV is more likely a multi-genic trait determined by a complex interplay of several viral proteins or genes acting in concert as already postulated in previous studies (Leifer et al, 2013;Tamura et al, 2012). To the best of our knowledge, this is the first study to show that the N-terminal domain of CSFV NS4B can confer enhanced pathogenicity to a low virulent virus, which was related to an enhanced viral replication.…”

“…The CSFV genome and the viral proteins N pro , E2 and NS4B of the GPE 2 vaccine strain and of the highly virulent Eystrup strain are depicted schematically. The amino acid differences related to virulence reported previously (Tamura et al, 2012(Tamura et al, , 2014 and the five additional residues in the N-terminal half of NS4B (2377, 2391, 2398, 2399 and 2414) evaluated in the present study (#) are shown. Numbering starts with the methionine encoded by the AUG start codon.…”

“…The N-terminal domain of NS4B of the highly virulent CSFV strain Eystrup confers enhanced pathogenicity to a low virulent GPE 2 -derived virus Our previous studies demonstrated the combination of four amino acid residues in N pro (D 136 ), E2 (A 830 ) and NS4B (A 2475 /V 2563 ) can confer pathogenicity to the CSFV GPE 2 vaccine strain in pigs (Tamura et al, 2012(Tamura et al, , 2014 (Fig. 1).…”

“…Our previous study demonstrated that the two amino acid residues that differ between the vaccine strain GPE 2 and its parental strain ALD in the predicated trans-membrane domains of NS4B can modulate viral genome replication and influence the pathogenicity of the virus in pigs (Tamura et al, 2012). Here, we generated GPE 2 -derived virus carrying the complete NS4B of the Eystrup strain, in order to determine whether the additional five amino acid differences in the N-terminal domain of NS4B may contribute to the different pathogenicity in pigs.…”

“…In BVDV, NS4B can act as a modulator of virus cytopathogenicity (Qu et al, 2001). Our previous study demonstrated that two amino acid residues within the predicted trans-membrane domains of NS4B are involved in viral genome replication and that they contribute to the pathogenicity of CSFV in pigs (Tamura et al, 2012). These two residues are a part of the seven amino acids that differ in NS4B between the highly virulent Eystrup strain and the GPE 2 vaccine strain.…”