Antibody responses of pigs to defined Erns fragments after infection with classical swine fever virus

Antibody responses of pigs to defined Erns fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA). Selection of various E(rns) fragments was based on an immunodominant Erns region encompassing three overlapping antigenic regions, amino acids 65 to 145 (Erns(aa)65-145) (AR1), 84 to 160 (Erns(aa)84-160) (AR2), and 109 to 220 (Erns(aa)109-220) (AR3), identified earlier by our group (M. Lin, E. Trottier, J. Pasick, and M. Sabara, J. Biochem., in press). Defined Erns fragments, including AR1, AR2, AR3, Erns(aa)65-160 (AR12), Erns(aa)84-220 (AR23), Erns(aa)65-220 (AR123), Erns(aa)109-145 (the consensus region defined by the three overlapping regions), and Erns(aa)109-160 (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV. Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the Erns fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100%. Detection of antibody responses to refolded Erns(aa)109-145 and Erns(aa)109-160 by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational. When cutoff values were raised to give a specificity of 100%, four Erns fragments (AR2, AR23, Erns(aa)109-145, and Erns(aa)109-160) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%). Erns(aa)109-145 and Erns(aa)109-160 were capable of detecting antibody responses in infected pigs as early as 7 days postinfection. Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection. An advantage of such a test would be its utilization for serological survey in a classical swine fever-free country (e.g., Canada) in biocontainment level 2 laboratories.     

Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene

Summary.  A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge. Accepted May 29, 2001 Received April 11, 2001     

An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7_E2alf. After transfection of in vitro-transcribed CP7_E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7_E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7_E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and "marker vaccine" properties of the generated chimeric CP7_E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 10(7) TCID(50), CP7_E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-E(RNS)-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7_E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7_E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV "marker vaccine".     

Laboratory infection with Zika virus after vaccination against yellow fever

Summary One of the authors contracted a Zika virus infection during laboratory work. Subsequent studies revealed an immunological response of the anamnestic type due to preceding yellow fever vaccinations which rendered difficult etiological diagnosis had it not been possible to isolate the virus from the serum sample collected during the acute phase of the disease.The authors comment on the possible importance of similar cases encountered in tropical countries when dignosis is based merely on the serological conversion from the acute phase to the convalescent phase of the disease.     

Immunity against NS3 protein of classical swine fever virus does not protect against lethal challenge infection

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. In the European Union the virus has been eradicated from the domestic pig population and prophylactic immunization has been banned. Nevertheless, intervention immunizations using marker vaccines are one possibility to deal with reintroduced CSFV. At present, baculovirus-expressed E2 protein is used as such a marker vaccine. However, this vaccine cannot fully protect against viral spread; hence the use of another subunit, or of a combination of two or more subunits, might be beneficial. Therefore the immunological effects of nonstructural protein 3 (NS3) on the humoral as well as the cellular arms of the immune system were investigated. Although effectors of both sides of the immune system were stimulated by application of recombinant NS3 protein, no protection against lethal CSFV challenge could be achieved.     

Use of recombinant protein E2 of the classical swine fever virus for immunization of animals

Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.     

Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses.     

Phylogenetic comparison of classical swine fever virus in China.

An N-terminal fragment of the E2 gene of classical swine fever (CSF) virus encoding major immunogenic sites was amplified by RT-PCR directly from 110 clinical specimens representing 109 epizootic sites during the last decade in China. Phylogenetic relationships between these viruses as well as 20 reference strains were determined by comparison of their nucleotide sequences. A phylogenetic tree showed that 103 of the 110 field viruses (93.6%) were clustered within group 2 and subdivided into three subgroups, while the remaining seven viruses (6.4%), along with two Chinese reference strains, Shimen and HCLV (attenuated vaccine strain), were clustered into subgroup 1.1 within group 1. However, none of the Chinese CSF viruses were members of subgroup 1.2 (represented by reference strain Brescia). This is the first report on the distribution of CSF virus genotypes in China. Results indicated that CSF viruses predominating in recent epizootics within China are genetically divergent from the reference strain Shimen and the vaccine strain HCLV.     

Phylogenetic analysis of classical swine fever virus in Taiwan

Summary. Two envelope glycoprotein (E^rns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the E^rns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (E^rns–E2) displayed better resolution than that based on the complete open reading frame.     

Evidence of natural recombination in classical swine fever virus

Classical swine fever (CSF) virus, one member of the family Flaviviridae is the pathogen of CSF, an economically important and highly contagious disease of pigs. Although homologous recombination has been demonstrated in many other members of the family, it is unknown whether there is recombination in natural populations of CSFV. To detect possible recombination events, we performed a phylogenetic analysis of 25 full-length CSFV strains isolated all over the world. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning. A mosaic virus, CSFV 39 (AF407339) isolated in China was found. And its two putative parental-like strains CSFV Shimen (AF333000) and GXWZ02 (AY367767) were identified. Our work revealed that homologous recombination occurred in natural CSFV populations, generating genetic diversity. This would provide some insights for the role homologous recombinant plays in CSFV evolution.     

Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus

The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with anti-CSFV antibodies. Based on the CnC2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with CSFV vaccine rapidly. The chimeric protein used as a detector was labeled with colloidal gold. Staphylococcal protein A (SPA) and anti-CnC2 monoclonal antibodies (mAbs) were blotted onto the nitrocellulose membrane as the test and control lines, respectively. The strip assay could be performed within 5min, which did not require any special equipment or skills. Through testing sera against various strains of CSFV, the sensitivity of the strip was determined to be 97.0% (65/67) and the specificity was 100% (98/98). The strip results were consistent with those of the existing commercial ELISA kit, and their correlation coefficient was 0.935. In conclusion, the immunochromatographic strip was an acceptable method for surveying CSFV-antibody titers in pigs.     

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses

An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.     

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.     

Evolution of T lymphocytes and cytokine expression in classical swine fever (CSF) virus infection

This study characterized the cell-mediated immune response in pigs inoculated with the Alfort 187 isolate of classical swine fever (CSF) virus. Quantitative changes in the T-lymphocyte population (CD3(+), CD4(+) and CD8(+)) and qualitative changes in cytokine expression (IL-2, IL-4 and IFNgamma) by these cells in serum, thymus and spleen were demonstrated. These changes coincided spatially and temporally with previously described quantitative and qualitative changes in monocyte-macrophage populations, thus demonstrating the contribution of the two cell populations to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1 cell-mediated immune response in the early and middle stages of the experiment, giving way to a type 2 immune response towards the end of the experiment; these findings, which accorded with the serological results and lymphopenia, may influence the delayed humoral response characteristic of CSF.     

Analysis of synonymous codon usage in classical swine fever virus

Using the complete genome sequences of 35 classical swine fever viruses (CSFV) representing all three genotypes and all three kinds of virulence, we analyzed synonymous codon usage and the relative dinucleotide abundance in CSFV. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in CSFV. Furthermore, we observed that the relative abundance of dinucleotides in CSFV is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as the subgenotypes and aromaticity, also influence the codon usage variation among the genomes of CSFV. This study represents the most comprehensive analysis to date of CSFV codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.     

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.     

Characterization of antibody responses against a neutralizing epitope on the glycoprotein E2 of classical swine fever virus

The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies.