Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection.
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The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E(rns) and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E(rns)E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E(rns) fragment (E(rns)(aa 109-160)) or an E2 fragment (E2(aa 1-221)) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E(rns)E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi. (+info)
N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction.
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Classical swine fever virus (CSFV) protects cells from double-stranded (ds) RNA-mediated apoptosis and IFN-alpha/beta induction. This phenotype is lost when CSFV lacks N(pro) (DeltaN(pro) CSFV). In the present study, we demonstrate that N(pro) counteracts dsRNA-mediated apoptosis and IFN-alpha/beta induction independently of other CSFV elements. For this purpose, we generated porcine SK-6 and PK-15 cell lines constitutively expressing N(pro) fused to the enhanced green fluorescent protein (EGFP). The survival of the SK6-EGFP-N(pro) cell line after polyinosinic polycytidylic acid [poly(IC)] treatment was comparable to that of CSFV-infected SK-6 cells and was significantly higher than the survival of the parent cell line. In PK-15 cells, the presence of EGFP-N(pro) prevented the DeltaN(pro) CSFV- and poly(IC)-mediated IFN-alpha/beta production. Importantly, N(pro) also inhibited IFN-alpha and IFN-beta promoter-driven luciferase expression in human cells and blocked IFN-alpha/beta induction mediated by Newcastle disease virus. This establishes a novel function for N(pro) in counteraction of the IFN-alpha/beta induction pathway. (+info)
Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.
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Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations. (+info)
Nictitating membrane as a potentially useful postmortem diagnostic specimen for classical swine fever.
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The gold standard for diagnosis of classical swine fever (CSF) is cell culture virus isolation combined with reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent antibody test (FAT) in cryosections of tonsils, spleen, various lymph nodes, ileum, and kidney. Autolytic and heterolytic samples render correct FAT evaluation difficult and can even yield false-negative or ambiguously positive results. To extend the spectrum of CSF diagnostic specimens, the authors tested whether the nictitating membrane (NM) might be a useful adjunct diagnostic specimen in wild boars and domestic pigs. To accomplish this, results of virus isolation, FAT, and RT-PCR were compared on NM samples and lymphoid tissues, which are the routine specimens of choice for CSF diagnosis. Wild boars (n = 30) and domestic pigs (n = 8) were experimentally challenged with various CSF virus (CSFV) strains or isolates of different virulence. The FAT revealed CSFV antigen in surface and tubular adenoid epithelium as well as in lymphatic follicles of the NM. In wild boars and domestic pigs with CSF, a strong agreement was found between results of FAT, virus isolation, and RT-PCR on NM and lymphoid tissues. These results suggest that NM is a useful additional specimen that can provide valuable data for postmortem diagnosis of CSF. The NM is relatively easy to sample at necropsy, and postmortem autolysis and heterolysis of this tissue is minimal compared with internal organs. (+info)
An African swine fever virus gene with homology to DNA ligases.
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Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase. (+info)
Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells.
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Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-alpha/beta), which in macrophages has been related to the presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus-host cell interaction has been analyzed. Higher levels of detectable dsRNA were produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and cp CSFV induced IFN-alpha/beta in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and no IFN-alpha/beta was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro deletion mutants induced an increase in DC maturation and IFN-alpha/beta production-for both ncp and cp viruses-despite reduced replication efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-alpha/beta induction and DC maturation, requires both regulated dsRNA levels and the presence of viral Npro. (+info)
Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine.
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Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine. (+info)
Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo.
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Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-gamma ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from naive animals. In addition to the IFN-gamma responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible--activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL. (+info)