N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

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.     

Determinants of virulence of classical swine fever virus strain Brescia

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB (476)R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB (476)R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB (476)S variant into a His residue did not affect virulence. However, the (710)His in the C1.1.1/CoBrB virus, together with adaptive mutations (276)R, (476)R, and (477)I in E(rns), resulted in reduced virulence in pigs. These results indicated that mutations in E(rns) and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E(rns) may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the (710)L-->H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.     

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

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.     

The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man₈GlcNAc₂ isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.     

Varicella-zoster virus glycoprotein E is a critical determinant of virulence in the SCID mouse-human model of neuropathogenesis

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.     

Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B

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.     

Novel swine virulence determinant in the left variable region of the African swine fever virus genome

Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.     

Classical swine fever virus p7 protein is a viroporin involved in virulence in swine

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.     

Classical swine fever virus glycoprotein E rns is an endoribonuclease with an unusual base specificity

The glycoprotein E(rns) of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for E(rns). Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5' of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that E(rns) is a highly active endoribonuclease (k(cat)/K(m) = 2 x 10(6) to 10 x 10(6) M(-1) s(-1)) with a strong affinity to NpU containing single-stranded RNA substrates (K(m) = 85 to 260 nM).     

Mutations abrogating the RNase activity in glycoprotein E(rns) of the pestivirus classical swine fever virus lead to virus attenuation

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV). Amino acid exchanges or deletions introduced by site-directed mutagenesis into the putative active site of the RNase residing in the glycoprotein E(rns) of CSFV abolished the enzymatic activity of this protein, as demonstrated with an RNase test suitable for detection of the enzymatic activity in crude cell extracts. Incorporation of the altered sequences into an infectious CSFV clone resulted in recovery of viable viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. These RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable for at least seven passages. In contrast to animals inoculated with an RNase-positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cells, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchanges of both codons 297 and 346 showed that all these RNase-negative mutants were attenuated. All viruses with mutations affecting codon 346 were completely apathogenic, whereas those containing only changes of codon 297 consistently induced clinical symptoms for several days, followed by sudden recovery. Analyses of reisolated viruses gave no indication for the presence of revertants in the infected animals.     

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus

Applied Microbiology and Biotechnology - Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs....     

Biosynthesis of classical swine fever virus nonstructural proteins

Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced low-level concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.     

Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E(rns) (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553-9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser(476) and an HS-dependent recombinant virus (S-RT virus) with Arg(476) were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E(rns), E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg(476) was mutated back to Ser(476). Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E(rns) S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.     

The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors

It has been reported that genes encoding antigens of bacterial and viral pathogens can be expressed in plants and are shown to induce protection antibodies. The structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes, has been expressed in different systems. Here, we report the expression of CFSV E2 gene in tobacco chloroplasts. Mice immunized with leaf extracts elicited specific antibodies. This indicated that the expressed E2 proteins had a certain degree of immunogenicity. To our knowledge, this is the first report showing induction of protective antibody in response to classical swine fever virus (CSFV) by immunization with antigen protein E2 expressed in tobacco chloroplasts, which will open a new way to protection from CSFV by plant chloroplasts as bioreactors.     

Expression and functional characterization of classical swine fever virus E(rns) protein

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with unusual RNase activity. Recently, E(rns) was found to have a new function of counteracting the beta-interferon (IFN-beta) induction pathway. In this study, wildtype ErnsSM and two mutated E(rns) proteins ErnsH297k and ErnsH346k were expressed in insect cells and purified for RNase activity and function analysis. RNase activity assay in vitro demonstrated that only wildtype E(rns) protein had RNase activity. However, both wildtype ErnsSM and the two mutated E(rns)ErnsH297k and ErnsH346k as exogenous proteins had a block effect on Newcastle disease virus (NDV)-mediated IFN-beta promoter induction.