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The thesis describes epidemiological research carried out to improve the control of classical swine fever virus (CSFV) epidemics. First, control measures and strategies are investigated in order to enable quick and adequate action upon CSFV detection. CSFV transmission experiments are described, as well as a mathematical model of CSFV transmission between herds, both used to assess the effectiveness of the E2 subunit marker vaccines with respect to CSFV transmission. Quantification of the effectiveness was done with the 'basic reproduction ratio' Ri (Rh), defined as the number of animals (herds) that is infected by one typical infectious animal (herd) in a completely susceptible population. The transmission experiments pointed out that Ri decreases to below the threshold value 1 as of three weeks after vaccination until at least six months after vaccination. Already one week after vaccination of a pig herd, virus entry will lead to only minor outbreaks. It appeared that the presence of maternal antibodies at the time of vaccination may reduce the antibody levels at later age, but these lower levels still keep Ri below 1. Because vertical transmission is not completely blocked by the vaccine, persistently infected piglets might be born on vaccinated herds. These herds cannot be clinically detected and might thus be a risk for further virus spread. Detection will be possible if some animals, e.g. the breeding sows, on those herds remain unvaccinated. Whether that would lead to an effective control strategy was tested in a mathematical model of CSFV transmission. The model was based on the Dutch CSFV epidemic of 1997/1998, with a moderately virulent virus strain in a pig dense area with relatively many multiplier herds. The model results point out that an effective control strategy (Rh < 1) requires a complete prohibition of transport of unvaccinated animals. Moreover, in addition to the control measures that are obliged by EU legislation (like the tracing of infectious contacts and hygiene measures), the virus transmission between herds should be halved, e.g. by vaccinating 50% of all pig herds. If all animals but the breeding sows would be vaccinated, the demands for a successful control strategy are well met. In addition to the investigation of control measures and strategies, a tool was tested, which had been developed to analyse the effectiveness of the control strategy in an ongoing epidemic. The tool was designed to use data of an ongoing epidemic for estimation of Rh and the number of infected, yet undetected herds. The only data needed for the calculations are the numbers of detected herds in each week of the epidemic. Unfortunately, the only result the model could generate was whether Rh was smaller or larger than 1. Further mathematical research is needed to understand the shortcomings. Until then, some extra data might be used, e.g. an estimate of the number of infected herds at the day of the first detection, or an estimate of the average time between infection of a herd and detection.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-gamma-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-gamma producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence. ; This research was supported by grant AGL2012-38343 from Spanish government. Author who received the funding: Llilianne Ganges (LG). S. Muñoz-González received predoctoral fellowship FI-DGR 2014 from AGAUR, Generalitat de Catalunya.

Classical swine fever (CSF) is one of the most important viral diseases of domestic pigs (Sus scrofa domesticus) and wild boar (Sus scrofa). For at least 4 decades, several European Union member states were confronted with outbreaks among wild boar and, as it had been shown that infected wild boar populations can be a major cause of primary outbreaks in domestic pigs, strict control measures for both species were implemented. To guarantee early detection and to demonstrate freedom from disease, intensive surveillance is carried out based on a hunting bag sample. In this context, virologic investigations play a major role in the early detection of new introductions and in regions immunized with a conventional vaccine. The required financial resources and personnel for reliable testing are often large, and sufficient sample sizes to detect low virus prevalences are difficult to obtain. We conducted a simulation to model the possible impact of changes in sample size and sampling intervals on the probability of CSF virus detection based on a study area of 65 German hunting grounds. A 5-yr period with 4,652 virologic investigations was considered. Results suggest that low prevalences could not be detected with a justifiable effort. The simulation of increased sample sizes per sampling interval showed only a slightly better performance but would be unrealistic in practice, especially outside the main hunting season. Further studies on other approaches such as targeted or risk-based sampling for virus detection in connection with (marker) antibody surveillance are needed.

Classical swine fever (CSF) is among the most important diseases of domestic pigs and causes great socio-economic losses. Therefore, control of CSF is given high priority within the European Union, including financial support of concerted control actions in candidate and in potential candidate countries. Unfortunately, from some of these countries information on the CSF situation and related data is very limited. This study was undertaken to gather all available information on the domestic pig population and husbandry, and of the CSF situation in domestic pigs and wild boar in South-Eastern European countries that have recently joined or are applying to join the European Union. A characteristic feature of pig production in Eastern Europe is that most of them are in backyard holdings. Although mandatory vaccination is carried out in most of these countries, sporadic CSF outbreaks still occur. Little is still known about the CSF situation in wild boar. In addition, molecular epidemiology of 97 CSF virus isolates available from these countries, from outbreaks that occurred between 1994 and 2007, was performed. Most of the isolates were from Romania and Bulgaria. Genetic typing showed that almost all isolates (with exception of Croatian and of the Macedonian isolates) belonged to genotype 2.3. On the basis of these sequences, and additional sequences from outbreaks in Eastern and Western European countries taken from the database held at the European Union Reference Laboratory (EURL), two clusters could be distinguished within subtype 2.3. They were tentatively named 2.3.1 and 2.3.2. (C) 2010 Elsevier B.V. All rights reserved

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a notifiable disease of economic importance that causes severe leukopenia, fever, and hemorrhagic disease in domesticated pigs and wild boar across the globe. CSFV has been shown to antagonize the induction of type I interferon (IFN), partly through a function of its N-terminal protease (N(pro)), which binds interferon regulatory factor 3 (IRF3) and targets it for proteasomal degradation. Additionally, N(pro) has been shown to antagonize apoptosis triggered by the double-stranded RNA (dsRNA) homolog poly(I·C); however, the exact mechanism by which this is achieved has not been fully elucidated. In this study, we confirm the ability of N(pro) to inhibit dsRNA-mediated apoptosis and show that N(pro) is also able to antagonize Sendai virus (SeV)-mediated apoptosis in PK-15 cells. Gene-edited PK-15 cell lines were used to show the dsRNA-sensing pathogen recognition receptors (PRRs) TLR3 and RIG-I specifically respond to poly(I·C) and SeV, respectively, subsequently triggering apoptosis through pathways that converge on IRF3 and culminate in the cleavage of caspase-3. Importantly, this IRF3-mediated apoptosis was found to be dependent on transcription-independent functions of IRF3 and also on Bax, a proapoptotic Bcl-2 family protein, through a direct interaction between the two proteins. Deletion of IRF3, stable expression of N(pro), and infection with wild-type CSFV were found to antagonize the mitochondrial localization of Bax, a key hallmark of the intrinsic, mitochondrial pathway of apoptosis. Together, these findings show that N(pro)'s putative interaction with IRF3 is involved not only in its antagonism of type I IFN, but also dsRNA-mediated mitochondrial apoptosis. IMPORTANCE Responsible for severe hemorrhagic disease in domestic pigs and wild boar, classical swine fever is recognized by the World Organisation for Animal Health (OIE) and European Union as a notifiable disease of economic importance. Persistent infection, ...

Classical Swine Fever (CSF) virus has been confirmed for the first time in pig in Bali. The object of thisstudy was suspected CSF cases diagnosed at the diagnostic laboratory assistantship of the Faculty ofVeterinary Medicine, Udayana University, in 2007-2008. Total number of cases was 12. Case recordsincluded the signalment of case (breed, age, body weight, and the origin of respective case), clinical signs,post-mortem lesions, and histological pictures. CSF virus was confirmed using the standardized reversetranscriptase-polymerase chain reaction (RT-PCR) for CSF from European Union. One RT-PCR productwas sequenced. CSF virus was confirmed in seven out of 12 cases (58%). The cDNA sequence wasconfirmed to be specific of CSF E2 protein coding region with 98% homology to one isolate from China thatwas available in GeneBank. Further works are recommended to elucidate the sensitivity of RT-PCR, toclarify some differential diagnose, and to find out the genetic variation of CSF virus in Bali.Key words: classical swine fever virus, Bali, RT-PCR

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 Erns-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 Erns 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 Erns 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.

The introduction of classical swine fever virus (CSFV) into a country free of disease without vaccination may have huge consequences in terms of both disease spread and economic losses. More quantitative insight into the main factors determining the probability of CSFV introduction (PCSFV) is needed to optimally use resources for the prevention of CSFV introduction. For this purpose a spreadsheet model was constructed that calculates the annual PCSFV into member states of the European Union (EU). The scenario pathway approach was used as most probabilities in the model are very small. Probability distributions were used to take into account inherent variability of input parameters. The model contained pathways of CSFV introduction including the import of pigs and pork products, returning livestock trucks, and contacts with wild boar. All EU member states were included as possible sources of CSFV. Default results for the Netherlands showed a mean overall annual PCSFV of approximately 0.06, indicating that the Netherlands can expect CSFV introduction on average once every 18 years from the pathways and countries included in the model. Almost 65% of this probability could be attributed to the pathway of returning livestock trucks. The most likely sources of CSFV introduction were Germany, Belgium, and the United Kingdom. Although the calculated probabilities were rather low when compared with expert estimates and recent history, the most likely causes of CSFV introduction indicated by the model were considered to be realistic. It was therefore concluded that the model is a useful tool to structure and analyze information for decision making concerning the prevention of CSFV introduction.

Classical swine fever (CSF) is a highly contagious viral disease of domestic swine and wild boars. The aim of the study was to examine samples of lymphoid tissues and brain for the presence of Classical Swine Fever Virus (CSFV) antigen in piglets that originate from vaccinated and unvaccinated sows, in order to compare the intensity of pathological lesions in conditions of different immunological status. A total of 20 crossbreed piglets of both sexes, aged 45 days were divided in three groups (G1, G2 and G3). Piglets of G1 originated from sows vaccinated with a live vaccine containing attenuated C strain CSFV. Piglets of G2 originated from unvaccinated sows. Two piglets of G3 originated from vaccinated and two piglets from unvaccinated sows and they served as the control group. All the piglets from G1 and G2 were intramuscularly inoculated with 2×105 TCDI/50 of CSFV, Baker strain and they died by the 22nd day post inoculation. Immunohistochemistry was applied for immunolabeling E2 (gp55) glycoprotein of CSFV in the tonsils, mandibular lymph nodes, ileocecal valve and brain of all tested piglets. Differences were observed in the severity of lesions in the lymphoid tissue and the brain between G1 and G2 piglets. The present level of colostral antibodies was not able to protect piglets in G1 from the fatal outcome of the disease. Among others, this finding may also have an impact on vaccination policy in the future. Having in mind that vaccination of all domestic pigs with attenuated C-strain vaccine is still a mandatory control measure for CSF in Serbia, soon a non-vaccination policy should be harmonized with EU directives.

Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression. ; This research was supported by grants AGL2013–48998 and AGL2015–66907 from the Spanish government. S.M and A.C had

Three dendrimeric peptides were synthesized in order to evaluate their immunogenicity and their potential protection against classical swine fever virus (CSFV) in domestic pigs. Construct 1, an optimized version of a previously used dendrimer, had four copies of a B-cell epitope derived from CSFV E2 glycoprotein connected to an also CSFV-derived T-cell epitope through maleimide instead of thioether linkages. Construct 2 was similarly built but included only two copies of the B-cell epitope, and in also bivalent construct 3 the CSFV T-cell epitope was replaced by a previously described one from the 3A protein of foot-and-mouth disease virus (FMDV). Animals were inoculated twice with a 21-day interval and challenged 15days after the second immunization. Clinical signs were recorded daily and ELISA tests were performed to detect antibodies against specific peptide and E2. The neutralising antibody response was assessed 13days after challenge. Despite the change to maleimide connectivity, only partial protection against CSFV was again observed. The best clinical protection was observed in group 3. Animals inoculated with constructs 2 and 3 showed higher anti-peptide humoral response, suggesting that two copies of the B-cell epitope are sufficient or even better than four copies for swine immune recognition. In addition, for construct 3 higher neutralizing antibody titres against CSFV were detected. Our results support the immunogenicity of the CSFV B-cell epitope and the cooperative role of the FMDV 3A T-cell epitope in inducing a neutralising response against CSFV in domestic pigs. This is also the first time that the FMDV T-cell epitope shows effectivity in improving swine immune response against a different virus. Our findings highlight the relevance of dendrimeric peptides as a powerful tool for epitope characterization and antiviral strategies development. ; The research in CReSA was supported by grant AGL2015-66907 from the Spanish government. J.A. B. had a pre-doctoral fellowship FPI-MINECO 2016 from Spanish government. S. M. had a pre-doctoral fellowship FI-DGR 2014 from AGAUR, Generalitat de Catalunya. Work at CBMSO was supported by grants AGL2014-52395-C2-01 (MINECO, Spain) and S2013/ABI-2906-PLATESA (Comunidad Autónoma de Madrid). Work at UPF was funded by AGL2014-52395-C2-02 (MINECO, Spain)

Three dendrimeric peptides were synthesized in order to evaluate their immunogenicity and their potential protection against classical swine fever virus (CSFV) in domestic pigs. Construct 1, an optimized version of a previously used dendrimer, had four copies of a B-cell epitope derived from CSFV E2 glycoprotein connected to an also CSFV-derived T-cell epitope through maleimide instead of thioether linkages. Construct 2 was similarly built but included only two copies of the B-cell epitope, and in also bivalent construct 3 the CSFV T-cell epitope was replaced by a previously described one from the 3A protein of foot-and-mouth disease virus (FMDV). Animals were inoculated twice with a 21-day interval and challenged 15days after the second immunization. Clinical signs were recorded daily and ELISA tests were performed to detect antibodies against specific peptide and E2. The neutralising antibody response was assessed 13days after challenge. Despite the change to maleimide connectivity, only partial protection against CSFV was again observed. The best clinical protection was observed in group 3. Animals inoculated with constructs 2 and 3 showed higher anti-peptide humoral response, suggesting that two copies of the B-cell epitope are sufficient or even better than four copies for swine immune recognition. In addition, for construct 3 higher neutralizing antibody titres against CSFV were detected. Our results support the immunogenicity of the CSFV B-cell epitope and the cooperative role of the FMDV 3A T-cell epitope in inducing a neutralising response against CSFV in domestic pigs. This is also the first time that the FMDV T-cell epitope shows effectivity in improving swine immune response against a different virus. Our findings highlight the relevance of dendrimeric peptides as a powerful tool for epitope characterization and antiviral strategies development. ; The research in CReSA was supported by grant AGL2015-66907 from the Spanish government. J.A. B. had a pre-doctoralfellowship FPI-MINECO 2016 from Spanish government. S. M.had a pre-doctoral fellowship FI-DGR 2014 from AGAUR, Gen-eralitat de Catalunya. Work at CBMSO was supported by grantsAGL2014-52395-C2-01 (MINECO, Spain) and S2013/ABI-2906-PLATESA (Comunidad Autónoma de Madrid). Work at UPF wasfunded by AGL2014-52395-C2-02 (MINECO, Spain).

Classical swine fever (CSF) is one of the most important diseases of pigs. Although prophylactic vaccination is banned within the European Union, emergency vaccination, allowing differentiation of vaccinated from infected animals, is an option for disease control. Up to now, these strategies are based on antibody detection. In this context, conventional modified live vaccines are not suitable. A promising perspective could be genetic differentiation of vaccinated from infected animals where field virus strains are differentiated from vaccine viruses by sequence differences. This concept could also be used with marker vaccines. To this end, a set of real-time reverse transcription-polymerase chain reaction (RT-PCR) assays was developed and validated. Specific primers and probes were designed for detection of the C-strain "Riems" vaccine virus or the chimeric marker vaccine candidate CP7_E2alf. A heterologous internal positive control was also included. The assays were then multiplexed to detect simultaneously either CSF field virus, C-strain "Riems", and the internal control or CSF field virus, CP7_E2alf, and the internal control. To validate both systems, samples from vaccination/challenge trials were tested. Only samples from vaccinated animals were found to be positive, while all samples from wild type virus-infected animals and a broad test panel of different pestiviruses were negative. Field application of the "C-strain Riems" specific assay was proven with wild boar samples from surveillance programs in Germany and France. In conclusion, ready-to-use RT-PCR sets are presented as reliable tools for genetic differentiation of vaccinated from infected animals for CSFV eradication strategies. (c) 2009 Elsevier B.V. All rights reserved

Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application. (C) 2009 Elsevier Ltd. All rights reserved