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An experimental infection with a classical swine fever virus in weaner pigs. II. The use of serological data to estimate the day of virus introduction in natural outbreaks.

In a companion paper a logistic regression model of seroprevalence over time was developed on the basis of data obtained during an experimental infection of weaner pigs with classical swine fever (CSF) virus. The model was applied to seroprevalence data from three outbreaks of the 1993-1994 epizootic to test whether the model could predict correctly the day of virus introduction into the herd. It was concluded that the logistic regression model has potential as a tool to estimate in retrospect the day CSF virus was introduced into a pig herd, which in turn may assist in identification of risk factors implicated in the further spread of the disease.

Animals↗

Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard.

Knowledge of the sensitivity of diagnostic tests for infectious diseases under field conditions can be used to design a surveillance program that increases the effectiveness of the control policy. In this study, the sensitivity of tests for the detection of classical swine fever (CSF) virus (CSFV) under field conditions was estimated without knowledge of the true disease status of the animals tested. During the CSF epidemic of 1997-1998 in The Netherlands, tonsil samples from pigs of CSF suspect farms were collected for laboratory diagnosis of CSE These specimens were tested in a fluorescence antibody test (FAT1) for the presence of CSFV antigen. When at least 1 specimen in a particular sample series from a farm was positive, this farm was declared CSFV infected. Specimens of that series, either FAT1 negative (98) or FAT1 positive (127), were subsequently tested again (FAT2). After that, a suspension was made of the remaining tissue, and this suspension was evaluated with a virus isolation test. In total, 225 tonsil specimens were examined. A statistical model was formulated, and the sensitivity of the 3 tests and the prevalence of positive specimens in the sample were estimated by the method of maximum likelihood. The sensitivity of the FAT1, the test that was used for confirmation of CSFV infection in a pig herd, was approximately 78% (95% confidence interval [CI] = 62-92%). The effectiveness of the selection process of animals on the farm by the veterinarian was estimated to be 77% (64-87%). The sensitivity of the combination of FAT1 and FAT2 (60%) indicates that at least 5 animals should be selected on a CSF-suspect farm to gain a detection probability for CSFV of 99%.

Animal Husbandry↗

Airborne transmission of classical swine fever virus under experimental conditions.

Sixty-one pigs were housed in an isolation unit with three compartments and five pens. Each compartment had its own ventilation system resulting in air currents flowing from compartment A (pens 1 to 3) towards compartment B (pen 4), but not towards compartment C (pen 5). Classical swine fever virus was introduced by the experimental inoculation of one pig in the middle pen (pen 2) of compartment A. The virus infected the pigs in pen 4, following the prevalent air currents, and the compartmentalisation had only a retarding effect on the transmission of the virus. The absence of infection in the pigs in pen 5, which was not different from pen 4 except for the ventilation system, indicates that the spread of virus was affected by the air currents.

Air Microbiology↗

Development of new concepts in vaccines and their effects on the control of infectious diseases (classical swine fever in particular).

Molecular biology and technological advances in DNA recombination have ushered in a new era in vaccinology. In particular, "deleted" vaccines have emerged over the past 10 years. The first such vaccines were used to protect pigs against Aujeszky's disease. The same principles were subsequently applied to the development of vaccines against classical swine fever (CSF), relaunching the debate on whether to use sanitary or medical measures. The decision about the measures to be applied depends on several criteria and factors such as the capabilities of the vaccines to be used in an emergency situation and the local epidemiological situation. Under the light of the last CSF serious epizootic which appeared in Europe in 1997, the effects of the different types of measures are discussed.

Animals↗

Does porcine reproductive and respiratory syndrome virus potentiate classical swine fever virus infection in weaner pigs?

Fifteen 6-week-old crossbred weaners weighing about 12 kg each were randomly divided into three groups of five animals each. One group of pigs was inoculated first with porcine reproductive and respiratory syndrome (PRRS) virus and then 3 days later with CSF virus. The second group received classical swine fever (CSF) virus, while the third group was inoculated with PRRS virus only. The aim of the experiment was to determine whether a primary PRRS virus infection influences the clinical outcome of experimentally induced CSF in young pigs. The PRRS virus infected weaners developed mild respiratory symptoms and recovered completely. All five weaners which were inoculated with CSF virus only showed severe clinical signs typical of the acute form of CSF. One pig had to be killed 15 days post-inoculation (p.i.); the remaining four died between the 18th and 22nd day p.i. The clinical course of the animals inoculated with both viruses was slightly different from that of the pigs that received only CSF virus. Four out of five pigs from the PRRS/CSF group became febrile and viraemic earlier than the animals which received CSF virus only. These pigs had to be killed 15-17 days post CSF virus inoculation. One animal in this group survived the acute phase of CSF and recovered completely. It was concluded that the observed divergences of the clinical courses would not have been noticed under field conditions. Therefore these findings cast doubt on the relevance of PRRS virus infection potentiating significantly the clinical outcome of CSF in young pigs.

Animals↗

Prevalence of antibodies to classical swine fever, Aujeszky's disease, porcine reproductive and respiratory syndrome, and bovine viral diarrhoea viruses in wild boars in Croatia.

During the hunting season in February 1999, a total of 44 blood samples were collected from wild boars shot in the area of Moslavacka gora. These blood samples were examined by enzyme immunoassay for the presence of antibodies to classical swine fever (CSFV), Aujeszky's disease (ADV), bovine viral diarrhoea (BVDV), and porcine reproductive and respiratory syndrome (PRRSV) viruses. Out of 44 serum samples examined, 17 (38.63%) were positive for CSFV, 24 (54.54%) were positive for ADV and two (4.54%) were positive for BVDV. All sera were negative for PRRSV. The results, recorded for the first time in Croatia, supported the hypothesis that wild boar act as a potential reservoir of CSFV, ADV and BVDV, and thus have a role in the epidemiology of these diseases.

Animals↗

Classical swine fever virus interferes with cellular antiviral defense: evidence for a novel function of N(pro).

Classical swine fever virus (CSFV) replicates efficiently in cell lines and monocytic cells, including macrophages (MPhi), without causing a cytopathic effect or inducing interferon (IFN) secretion. In the present study, the capacity of CSFV to interfere with cellular antiviral activity was investigated. When the porcine kidney cell line SK-6 was infected with CSFV, there was a 100-fold increased capacity to resist to apoptosis induced by polyinosinic-polycytidylic acid [poly(IC)], a synthetic double-stranded RNA. In MPhi, the virus infection inhibited poly(IC)-induced alpha/beta IFN (type I IFN) synthesis. This interference with cellular antiviral defense correlated with the presence of the viral N(pro) gene. Mutants lacking the N(pro) gene (DeltaN(pro) CSFV) did not protect SK-6 cells from poly(IC)-induced apoptosis, despite growth properties and protein expression levels similar to those of the wild-type virus. Furthermore, DeltaN(pro) CSFV did not prevent poly(IC)-induced type I IFN production in MPhi but rather induced type I IFN in the absence of poly(IC) in both MPhi and the porcine kidney cell line PK-15, but not in SK-6 cells. With MPhi and PK-15, an impaired replication of the DeltaN(pro) CSFV compared with wild-type virus was noted. In addition, DeltaN(pro) CSFV, but not wild-type CSFV, could interfere with vesicular stomatitis virus replication in PK-15 cells. Taken together, these results provide evidence for a novel function associated with CSFV N(pro) with respect to the inhibition of the cellular innate immune system.

Animals↗

Disease prevention and preparedness: the Food and Agriculture Organization Emergency Prevention System.

In 1994, the Food and Agriculture Organization undertook to revitalise its activities in the control of transboundary animal disease by establishing a new special programme known as the Emergency Prevention System (EMPRES) against transboundary animal and plant pests and diseases. The emphasis of the EMPRES livestock component is placed on pre-empting outbreaks and losses experienced by agriculture through the enhancement of local capacity to detect and react rapidly to plague events. EMPRES concentrates on the co-ordination of the Global Rinderpest Eradication Programme--a time-bound eradication programme--whilst addressing the progressive control of the most serious epidemic diseases within a broad framework of emergency preparedness. Programme activities are discussed in relation to early warning, early reaction, facilitating research and co-ordination. In addition to rinderpest, particular attention has been paid to contagious bovine pleuropneumonia, a re-emerging disease in Africa targeted for strategic attention, and foot and mouth disease, for which co-ordinated regional control in Latin America and South-East Asia has been initiated. Tactical responses to other disease emergencies such as African swine fever, classical swine fever (hog cholera), Rift Valley fever, peste des petits ruminants and lumpy skin disease are described.

Animal Diseases↗

[The eradication of African swine fever in Brazil, 1978-1984].

The African swine fever episode in Brazil was due to trade and tourism between Spain, Portugal and Brazil, at a time when outbreaks were on the rise in Europe. The eradication of the disease, the slaughter of pigs, the elimination of the carcasses and the isolation of affected farms were given wide media coverage, and had a major socio-economic impact. It was forbidden to raise pigs in garbage dumps or to give them feed considered hazardous. Analyses performed in Brazil as well as national and international investigations by researchers from reference laboratories concluded that the disease had spread from Rio de Janeiro to other states, as is stated in official reports. Following emergency measures, a control programme was implemented, leading to enhanced quality in the pig farming sector. The authors describe epidemiological surveillance of African swine fever, classical swine fever and related diseases, biosafety in swine farming, and the emergency action plan comprising animal health training for veterinarians and social workers. The results of the eradication programme were excellent, despite the controversy over compulsory sacrifice in a country with serious social problems. In 2004, Brazil was the fourth largest pork producer and exporter, with an output of 2.679 million tons and exports of 508,000 tons to international markets with very high standards.

African Swine Fever↗

Porcine interleukin-3 enhances DNA vaccination against classical swine fever.

DNA vectors can be used to deliver vaccine antigens that stimulate effective protective immunity in mice, but in larger, outbred animal species, the protective efficacy is lower or large doses of DNA are required. These data demonstrate that porcine interleukin-3 (IL-3) when delivered to pigs by DNA vector or in low doses as recombinant protein, can enhance antibody responses to classical swine fever virus antigen expressed from co-delivered DNA, and improve the protective efficacy of the DNA vaccine. The effect was further enhanced when IL-3 was expressed as a fusion protein with the potyvirus coat protein. The adjuvant effect of IL-3 was compared to that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 was shown to be at least as efficacious as GM-CSF. The response to IL-3 is novel and suggests, that at least in pigs, IL-3 could be used as an adjuvant for DNA vaccines.

Adjuvants, Immunologic↗

Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus.

Our previous study proved that the N-terminal (aa693-711) of glycoprotein E2 contained sequential neutralizing epitopes. In this study, four candidate epitope-vaccines (EVs) were separately prepared and evaluated. Among them, epitope-vaccine EV-BC1a (BC1a: aa693-699) induced high level of epitope-specific neutralizing antibodies and exhibited similar protective capability with that induced by Chinese vaccine strain (C-strain). These results confirmed CKEDYRY (aa693-699) as a principal sequential neutralizing epitope on E2 N-terminal. Moreover, these findings also indicate that epitope-vaccine is a potent candidate strategy for marker vaccine against classical swine fever virus (CSFV).

Amino Acid Sequence↗

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines.

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Animals↗

[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.

Animals↗

The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator.

The cDNA of the nucleocapsid (core) protein of classical swine fever virus (CSFV) was generated by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a eukaryotic expression vector. The effect of the recombinant core protein on the transcriptional regulation of cellular as well as viral promoters was studied. Using transient transfection assay, our results demonstrated that the core protein can activate the promoter of human heat shock protein 70 gene, and suppressed the SV40 early promoter. These findings indicate that the core protein appears to function not only as a viral structural protein but also as a regulator of gene expression. The implications of core proteins on the viral maturation are discussed.

Animals↗

[Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product].

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P. pastoris culture contained protein E2. The results of the study on the immunological activity indicated that the protein E2 expressed in P. pastoris can elicit animal bodies to produce antibodies against protein E2.

Animals↗

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library.

Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and Erns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289-293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of Erns (aa 117-119) was identified for two independent Erns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.

Amino Acid Motifs↗

RNase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies.

The structural glycoprotein E0 of classical swine fever virus (CSFV) possesses an intrinsic RNase activity. Here we present the first comprehensive biochemical characterization of E0, using a recombinant glycoprotein expressed in insect cells. We were able to show that the presence of neither carbohydrate moieties nor disulfide bonds is a prerequisite for RNase activity. In addition, virus-neutralizing and nonneutralizing anti-E0 monoclonal antibodies were tested for their ability to influence RNase activity. In these experiments, the antibodies which effectively blocked the infection of STE cells also exerted a high degree of E0 RNase inhibition. This correlation suggests that the RNase activity of CSFV E0 plays a role in the viral life cycle.

Animals↗