A self-assembling nanoparticle: Implications for the development of thermostable vaccine candidates.

Effective controls on viral infections rely on the continuous development in vaccine technology. Nanoparticle (NP) antigens are highly immunogenic based on their unique physicochemical properties, making them molecular scaffolds to present soluble vaccine antigens. Here, viral targets (113-354 aas) were genetically fused to N terminal of mi3, a protein that self-assembles into nanoparticles composed of 60 subunits. With transmission electron microscopy, it was confirmed that target-mi3 fusion proteins which have insertions of up to 354 aas in N terminal form intact NPs. Moreover, viral targets are surface-displayed on NPs as indicated in dynamic light scattering. NPs exhibit perfect stability after long-term storage at room temperature. Moreover, SP-E2-mi3 NPs enhance antigen uptake and maturation in dendritic cells (DCs) via up-regulating marker molecules and immunostimulatory cytokines. Importantly, in a mouse model, SP-E2-mi3 nanovaccines against Classical swine fever virus (CSFV) remarkably improved CSFV-specific neutralizing antibodies (NAbs) and cellular immunity related cytokines (IFN-γ and IL-4) as compared to monomeric E2. Specially, improved NAb response with more than tenfold increase in NAb titer against both CSFV Shimen and HZ-08 strains indicated better cross-protection against different genotypes. Collectively, this structure-based, self-assembling NP provides an attractive platform to improve the potency of subunit vaccine for emerging pathogens.

Efficacy of Oral Vaccine against Classical Swine Fever in Wild Boar and Estimation of the Disease Dynamics in the Quantitative Approach.

Classical swine fever virus (CSFV) in the wild boar population has been spreading in Japan, alongside outbreaks on pigs, since classical swine fever (CSF) reemerged in September 2018. The vaccination using oral bait vaccine was initially implemented in Gifu prefecture in March 2019. In the present study, antibodies against CSFV in wild boar were assessed in 1443 captured and dead wild boars in Gifu prefecture. After the implementation of oral vaccination, the increase of the proportion of seropositive animals and their titer in wild boars were confirmed. Quantitative analysis of antigen and antibodies against CSFV in wild boar implies potential disease diversity in the wild boar population. Animals with status in high virus replication (Ct < 30) and non- or low-immune response were confirmed and were sustained at a certain level after initial oral vaccination. Through continuous vaccination periods, the increase of seroprevalence among wild boar and the decrease of CSFV-positive animals were observed. The epidemiological analysis based on the quantitative virological outcomes could provide more information on the efficacy of oral vaccination and dynamics of CSF in the wild boar population, which will help to improve the implementation of control measures for CSF in countries such as Japan and neighboring countries.

Signature of genome wide gene expression in classical swine fever virus infected macrophages and PBMCs of indigenous vis-a-vis crossbred pigs.

The genetic basis of differential host immune response vis-à-vis transcriptome profile was explored in PBMCs of indigenous (Ghurrah) and crossbred pigs after classical swine fever vaccination and in monocyte derived macrophages (MDMs) challenged with virulent classical swine fever (CSF) virus. The humoral immune response (E2 antibody) was higher (74.87%) in crossbred than indigenous pigs (58.20%) at 21st days post vaccination (21dpv). The rate of reduction of ratio of CD4+/CD8+ was higher in crossbred pigs than indigenous pigs at 7th days post vaccination (7dpv). The immune genes IFIT1, IFIT5, RELA, NFKB2, TNF and LAT2 were up regulated at 7dpv in RNA seq data set and was in concordance during qRT-PCR validation. The Laminin Subunit Beta 1 (LAMB1) was significantly (p ≤ 0.05) down-regulated in MDMs of indigenous pigs and consequently a significantly (p ≤ 0.01) higher copy number of virulent CSF virus was evidenced in macrophages of crossbred pigs than indigenous pigs. Activation of LXR:RXR pathway at 60 h post infection (60hpi) in MDMs of indigenous versus crossbred pigs inhibited nuclear translocation of NF-κB, resulted into transrepression of proinflammatory genes. But it helped in maintenance of HDL level by lowering down cholesterol/LDL level in MDMs of indigenous pigs. The key immune genes (TLR2, TLR4, IL10, IL8, CD86, CD54, CASP1) of TREM1 signaling pathway were upregulated at 7dpv in PBMCs but those genes were downregulated at 60hpi in MDMs indigenous pigs. Using qRT-PCR, the validation of differentially expressed, immunologically important genes (LAMB1, OAS1, TLR 4, TLR8 and CD86) in MDMs revealed that expression of these genes were in concordance with RNA-seq data.

Serum Lipidomics Analysis of Classical Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro.

Lipids metabolism plays a significant role in cellular responses to virus pathogens. However, the impact of lipids metabolism in CSFV infection is not yet confirmed. In the present study, for the fist time, we performed serum lipidomics analysis of piglets infected with CSFV based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and identified 167 differentially expressed lipid metabolites. Interestingly, free fatty acids (FFAs) accumulated significantly in these metabolites, accompanied by an increase in sphingolipids and a decrease in glycerolipids and glycerophospholipids, suggesting that CSFV infection markedly changed the serum lipid metabolism of piglets. FFAs are the principal constituents of many complex lipids and are essential substrates for energy metabolism. Based on this, we focused on whether FFAs play a prominent role in CSFV infection. We found that CSFV infection induced FFAs accumulation in vivo and in vitro, which is due to increased fatty acid biosynthesis. Meanwhile, we discovered that alteration of cellular FFAs accumulation by a mixture of FFAs or inhibitors of fatty acid biosynthesis affects progeny virus production in vitro. Furthermore, in the absence of glucose or glutamine, CSFV still has replication capacity, which is significantly reduced with the addition of fatty acid beta oxidation inhibitors, suggesting that the process of FFAs enter the mitochondria for beta oxidation to produce ATP is necessary for virus replication. Finally, we demonstrated CSFV induced FFAs accumulation results in impaired type I IFN signaling-mediated antiviral responses by down-regulating RIG-I-like receptors (RLRs) signaling molecules, which may represent a mechanism of CSFV replication. Taken together, these findings provide the first data on lipid metabolites during CSFV infection and reveal a new view that CSFV infection requires FFAs to enhance viral replication.

Evaluation of surface glycoproteins of classical swine fever virus as immunogens and reagents for serological diagnosis of infections in pigs: a recombinant Newcastle disease virus approach.

Classical swine fever (CSF) is an important viral disease of domestic pigs and wild boar. The structural proteins E2 and Erns of classical swine fever virus (CSFV), which participate in the attachment of the virion to the host cell surface and its subsequent entry, are immunogenic. The E2 and Erns proteins are used for diagnosis and the development of vaccines against CSFV infection in swine. Newcastle disease virus (NDV) has been successfully used as a viral vector to express heterologous proteins. In the present study, the E2 and Erns proteins of CSFV were expressed in cell culture as well as embryonated chicken eggs, using recombinant NDV (rNDV). Rescued rNDV expressing the E2 and Erns proteins induced the production of CSFV-neutralizing antibodies upon intranasal vaccination of pigs. Serum samples from vaccinated animals were found to neutralize both homologous and heterologous CSFV strains. Furthermore, rNDV expressing the E2 and Erns proteins of CSFV was used to develop an indirect ELISA, which was used to measure the the antibody titers of randomly collected serum samples. The results suggested that the ELISA based on rNDV-expressed E2 and Erns proteins could be used to screen for CSFV infections. This study shows that rNDV-based expression of CSFV antigens is potentially applicable for development of vaccines and diagnostic tests for CSFV infection. This approach could be an economically favorable alternative to the existing vaccine and diagnostics for CSFV in pigs.

Classical swine fever in Victorian domestic pigs: evidence of disease freedom.

OBJECTIVE: Australia is currently regarded as free of classical swine fever (CSF), a highly contagious disease of pigs caused by a pestivirus. This study aimed to provide additional evidence that the Victorian domestic pig population is free of CSF. DESIGN: A structured representative sero-prevalence survey of Victorian domestic pigs at slaughter. METHOD: Three-hundred and ninety-one pigs from 23 holdings were sampled at the time of slaughter between March 2016 and October 2017. RESULTS: All samples were negative for CSF virus Ab on ELISA. Because of uncertainty in the sensitivity of the CSF Ab ELISA, estimates of the true prevalence of CSF were calculated using Bayesian methods. The median and upper bound of the 95% credible intervals for the true prevalence of CSF was zero when the diagnostic sensitivity of the CSF Ab ELISA was assumed to range from 0.75 to 0.95. CONCLUSION: These results provide evidence that the population of domestic pigs in Victoria in 2016-2017 was free of CSF.

E2 and Erns isotype-specific antibody responses in serum and oral fluid after infection with classical swine fever virus (CSFV).

Oral fluid sampling for the detection of classical swine fever virus infection provides a relatively inexpensive method for conducting active CSF surveillance. The purpose of this study was to detect CSFV nucleic acid and antibody in serum and oral fluid samples in a group of 10 pigs infected with the moderate CSFV strain, Paderborn. Based on clinical signs, outcome, and other results, pigs were placed into one of three disease outcome groups; Acute, Chronic and Recovered. Oral fluid and serum samples were analyzed for the presence of CSFV nucleic acid along with E2 and Erns surface protein-specific IgM, IgG and IgA responses. The results were summarized into a timeline of detection events beginning with the appearance of E2-IgM in serum (3 DPI) followed by CSFV nucleic acid in serum (6 DPI), CSFV nucleic acid in oral fluid (8 DPI), E2-IgG in serum (20 DPI), and E2-IgG in oral fluid (24 DPI). The results show that a combination of molecular and serological analyses of oral fluid can be incorporated into CSF surveillance.

Virulence evaluation of classical swine fever virus subgenotype 2.1 and 2.2 isolates circulating in China.

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and Erns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while Erns antibody was detectable in 4-56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay.

The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses.

Detection of Classical swine fever virus infection by individual oral fluid of pigs following experimental inoculation.

We evaluated the use of oral fluid as an alternative to serum samples for Classical swine fever virus (CSFV) detection. Individual oral fluid and serum samples were collected at different times post-infection from pigs that were experimentally inoculated with CSFV Alfort 187 strain. We found no evidence of CSFV neutralizing antibodies in swine oral fluid samples under our experimental conditions. In contrast, real-time reverse transcription-polymerase chain reaction could detect CSFV nucleic acid from the oral fluid as early as 8 d postinfection, which also coincided with the time of initial detection in blood samples. The probability of CSFV detection in oral fluid was identical or even higher than in the corresponding blood sample. Our results support the feasibility of using this sampling method for CSFV genome detection, which may represent an additional cost-effective tool for CSF control.

African and classical swine fever situation in Ivory-Coast and neighboring countries, 2008-2013.

This study was conducted from 2008 to 2013 to determine the animal health status of Ivory Coast and neighboring countries (Burkina Faso, Ghana, Togo and Benin) for African swine fever (ASF) and classical swine fever (CSF), and to assess the risk factors for ASF introduction in Ivory Coast. Ivory Coast had probably been free from ASF from 1998 to 2014 when it was re-introduced in this country. However, the ASF virus was found in all neighboring countries. In contrast, no evidence of CSF infection was found so far in Ivory Coast and neighboring countries. To assess the risk of ASF reintroduction in Ivory Coast, we surveyed 59 modern pig farms, and 169 pig owners in 19 villages and in two towns. For the village livestock, the major risk factor was the high frequency of pig exchanges with Burkinabe villages. In the commercial sector, many inadequate management practices were observed with respect to ASF. Their identification should enable farmers and other stakeholders to implement a training and prevention program to reduce the introduction risk of ASF in their farms.

A Promising Trigene Recombinant Human Adenovirus Vaccine Against Classical Swine Fever Virus.

Classical swine fever (CSF) vaccine based on HAdV-5 had achieved an efficient protection in swine. Both classical swine fever virus (CSFV) E0 glycoprotein and E2 glycoprotein were the targets for neutralizing antibodies and related to immune protection against CSF. Interleukin-2 (IL2), as an adjuvant, also had been used in CSF vaccine research. In this study, coexpression of the CSFV E0, E2, and IL2 genes by HAdV-5 (rAdV-E0-E2-IL2) was constructed and immunized to evaluate its efficacy. Three expressed genes had been sequentially connected with foot-and-mouth disease virus 2A (FMDV 2A). The vaccine was administered by intramuscular inoculation to CSFV-free pigs (10(8) TCID50) twice at triweekly intervals. No adverse clinical signs were observed in any of the pigs after vaccination. The vaccine induced strong humoral and cellular responses that led to complete protection against clinical signs of lethal CSFV infection, viremia, and shedding of challenge virus. The rAdV-E0-E2-IL2 is a promising, efficient, and safe marker vaccine candidate against CSFV.

Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals.

Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

BACKGROUND: Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. OBJECTIVE: The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. ANIMALS AND METHODS: In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. RESULTS: Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. CONCLUSIONS: This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.

Classical swine fever virus infection modulates serum levels of INF-α, IL-8 and TNF-α in 6-month-old pigs.

Several studies have highlighted the important role of cytokines in disease development of classical swine fever virus (CSFV) infection. In the present study, we examined the kinetics of 7 porcine cytokines in serum from pigs infected with 3 different CSFV strains. Based on the clinical picture in 6-month-old Danish pigs, the strains used for inoculation were classified as being of low (Bergen), low to moderate (Eystrup) and moderate to high (Lithuania) virulence. The cytokines interferon-alpha (INF-α), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) showed increased levels after CSFV infection with more or less comparable course in the 3 groups. However, the cytokine level peaked with a 2-3 days delay in pigs infected with the low virulent strain compared to those infected with a moderately or highly virulent strain. These findings may indicate that INF-α, IL-8 and TNF-α are involved in the immune response during CSFV infection with strains of different virulence.

Real-time SPR characterization of the interactions between multi-epitope proteins and antibodies against classical swine fever virus.

The envelope glycoprotein E2 is the major immunodominant protein of the classical swine fever virus and can induce neutralizing antibodies and protective host-immune responses in infected swine. We designed, expressed, and purified multi-epitope protein (GST-BT22) that contains a tandem repeat of the E2 antigenic-determinant residues 693-704, 770-780, and 826-843, each of which is separated by a GGSSGG sequence. In the same manner, we also designed, expressed, and purified a second protein (GST-BT23) that contains a C-terminal sequence consisting of residues 1446-1460 from the classical swine fever virus nonstructural protein NS2-3 separated from the GST-BT22 sequence by a GGSSGG sequence. Western blotting of GST-BT22 and GST-BT23 with serum from a swine that had been experimentally infected with the virus showed that the proteins reacted with anti-serum, whereas GST did not. Surface plasmon resonance was used to quantify the affinities of GST-BT22 and GST-BT23 for serum antibodies (K(a) = 4.31 × 10(8) and 5.01 × 10(8), respectively). GST, used as a control, was reacted an order of magnitude less strongly than did GST-BT22 and GST-BT23. Surface plasmon resonance, therefore, appears to be a sensitive and precise method for epitope evaluation and can be used to characterize the immunogenicity of a recombinant protein.

Disseminated intravascular coagulation does not play a major role in the pathogenesis of classical swine fever.

Classical swine fever (CSF) is a multi-systemic disease that can be accompanied by severe haemorrhagic lesions. The underlying pathogenetic mechanisms are still far from being understood, though disseminated intravascular coagulation (DIC) was discussed as a major factor. In the presented study, the direct thrombin inhibitor hirudin was used in an attempt to elucidate the role of the coagulation system in the pathogenesis of CSF-induced haemorrhagic lesions. Two groups of piglets (n=5) were infected with highly virulent CSF virus (CSFV) strain CSF0634. One group underwent daily treatment with hirudin, the other served as untreated challenge infection control. Assessment of clinical signs using a clinical score system, coagulation tests, and blood counts were performed daily. Both groups developed acute-lethal CSF with haemorrhagic lesions. Although changes in the coagulation system were seen in the late stages of CSFV infection, our results strongly suggest that DIC does not present the crucial event in the pathogenesis of haemorrhagic lesions.