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Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.

Infection of pigs with the African swine fever virus (ASFV) leads to a devastating hemorrhagic disease with a high mortality of up to 100%. In this study, a CD2v gene deletion was introduced to a genotype IX virus from East Africa, ASFV-Kenya-IX-1033 (ASFV-Kenya-IX-1033-∆CD2v), to investigate whether this deletion led to reduced virulence in domestic pigs and to see if inoculation with this LA-ASFV could induce protective immunity against parental virus challenge. All pigs inoculated with ASFV-Kenya-IX-1033-ΔCD2v survived inoculation but presented with fever, reduced appetite and lethargy. ASFV genomic copies were detected in only one animal at one time point. Seven out of eight animals survived subsequent challenge with the pathogenic parental strain (87.5%) but had mild to moderate clinical symptoms and had a gross pathology compatible with chronic ASFV infection. All mock-immunised animals developed acute ASF upon challenge with ASFV-Kenya-IX-1033 and were euthanised upon meeting the humane endpoint criteria. ASFV genome copy numbers after challenge were similar in the two groups. ASFV-Kenya-IX-1033-∆CD2v is therefore a useful tool to investigate the development of immunity to ASFV genotype IX, but safety concerns preclude its use as a candidate vaccine without further attenuation.

African swine fever (ASF) is among the most devastating viral diseases of pigs and wild boar worldwide. In recent years, the disease has spread alarmingly. Despite intensive research activities, a commercialized vaccine is still not available, and efficacious live attenuated vaccine candidates raise safety concerns. From a safety perspective, inactivated preparations would be most favourable. However, both historical and more recent trials with chemical inactivation did not show an appreciable protective effect. Under the assumption that the integrity of viral particles could enhance presentation of antigens, we used gamma irradiation for inactivation. To this means, gamma irradiated ASFV "Estonia 2014" was adjuvanted with either Polygen™ or Montanide™ ISA 201 VG, respectively. Subsequently, five weaner pigs per preparation were immunized twice with a three-week interval. Six weeks after the first immunization, all animals were challenged with the highly virulent ASFV strain "Armenia 2008". Although ASFV p72-specific IgG antibodies were detectable in all vaccinated animals prior challenge, no protection could be observed. All animals developed an acute lethal course of ASF and had to be euthanized at a moderate humane endpoint within six days. Indeed, the vaccinated pigs showed even higher clinical scores and a higher inner body temperature than the control group. However, significantly lower viral loads were detectable in spleen and liver of immunized animals at the time point of euthanasia. This phenomenon suggests an immune mediated disease enhancement that needs further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pikalo, Porfiri, Akimkin, Roszyk, Pannhorst, Kangethe, Wijewardana, Sehl-Ewert, Beer, Cattoli and Blome.)

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade. The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera "CP7_E2alf"), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain "Roesrath" and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and E rns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid. Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. However, until recently, the available live vaccines did not allow a serological marker concept that is essentially important to circumvent long-term trade restrictions. In 2014, a new live attenuated marker vaccine, Suvaxyn ® CSF Marker (Zoetis), was licensed by the European Medicines Agency. This vaccine is based on pestivirus chimera "CP7_E2alf" that carries the main immunogen of CSF virus "Alfort/187", glycoprotein E2, in a bovine viral diarrhea virus type 1 backbone ("CP7"). This review summarizes the available data on design, safety, efficacy, marker diagnostics, and its possible integration into control strategies.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. These vaccines have usually outstanding efficacy and safety but lack differentiability of infected from vaccinated animals (DIVA or marker strategy). In contrast, the first generation of E2 subunit marker vaccines shows constraints in efficacy, application, and production. To overcome these limitations, new generations of marker vaccines are developed. A wide range of approaches have been tried including recombinant vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. At present, especially two new constructs have been intensively tested, the adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine candidate "rAdV-SFV-E2" and the pestivirus chimera "CP7_E2alf". The later was recently licensed by the European Medicines Agency. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point shows still weaknesses and it is important to embed vaccination in a well-established vaccination strategy and a suitable diagnostic workflow. In summary, conventional vaccines are a standard in terms of efficacy. However, only vaccines with DIVA will allow improved eradication strategies e.g. also under emergency vaccination conditions in free regions. To answer this demand, new generations of marker vaccines have been developed and add now to the tool box of CSF control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) remains one of the most important viral diseases that impact on sustainable pig production world wide. To control the disease in either endemic situations or in case of large, high-impact contingencies, safe and highly efficacious live attenuated vaccines exist since decades. However, until recently, the available live vaccines did not allow a serological marker concept that would be important to circumvent long-term trade restrictions. Recently, a new live attenuated marker vaccine, Suvaxyn ® CSF Marker (Zoetis), was licensed by the European Medicines Agency (EMA). To supplement the data that are necessary for the design of appropriate vaccination strategies, a trial was carried out with single "emergency-type" vaccination of two pregnant sows. Focus was laid on the kinetics of maternally derived antibodies (MDA) in the screening assays of their offspring that would be used in case of a CSF outbreaks, i.e. CSFV E2 and E rns antibody ELISA. Neutralization peroxidase linked antibody assays were carried out to allow a rough estimate of protection. Upon vaccination with Suvaxyn ® CSF Marker 21days before farrowing, MDAs were measurable in all piglets born to the vaccinated sows. The E2 ELISA reactivities showed an almost linear decrease over 10 weeks after which all piglets were tested negative in the ELISA again. No problems were observed in DIVA assays (E rns antibodies) when heat-inactivated sera were used. The protective effect of MDA needs further investigations as the titers were found to be lower than reported for C-strain vaccines., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recently, CP7_E2alf (SuvaxynCSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.

Over the last decade, pestivirus chimaera CP7_E2alf has proven to be a most promising marker vaccine candidate against classical swine fever (CSF). To provide further background data for the risk assessment towards licensing and release, especially on presence of the vaccine chimaera in faeces, urine, and organs of the male reproductive tract, supplementary studies were carried out under controlled laboratory conditions. In detail, the shedding and dissemination pattern of Suvaxyn(®) CSF Marker ("CP7_E2alf") was assessed in 12 adult boars after single intramuscular vaccination with a tenfold vaccine dose. Four and seven days post vaccination, six animals were subjected to necropsy and triplicate samples were obtained from reproductive and lymphatic organs as well as urine, faeces, blood, and several additional organs and matrices. The sampling days were chosen based on pre-existing data that indicated the highest probability of virus detection. Upon vaccination, neither local nor systemic adverse effects were observed in the experimental animals. It was confirmed that primary replication is restricted to the lymphatic tissues and especially the tonsil. While viral genome was detectable in several samples from lymphatic tissues at four and seven days post vaccination, infectious virus was only demonstrated at four days post vaccination in one tonsil sample and one parotid lymphnode. Sporadic detection at a very low level occurred in some replicates of liver, lung, bone marrow, and salivary gland samples. In contrast, viral genome was not detected in any sample from reproductive organs and accessory sex glands, in faeces, urine, or bile. The presented data on the dissemination of the vaccine virus CP7_E2alf in adult boars are supplementing existing safety and efficacy studies and indicate that the use of the vaccine is also safe in reproductive boars., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E(rns), and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E(rns) antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E(rns) ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E(rns) antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

African swine fever (ASF) is among the most devastating viral diseases of pigs. In recent years, the disease has spread alarmingly. Despite intensive research activities, promising vaccine candidates are still lacking. For this reason, a study was undertaken to re-assess inactivated ASFV preparations with state-of-the-art adjuvants. Inactivated preparations of ASF virus (ASFV) "Armenia08" were adjuvanted with either Polygen™ or Emulsigen(®)-D, respectively, and used to immunize six weaner pigs two times with a three-week interval. Six weeks after the first immunization, animals were challenged with the homologues highly virulent ASFV. Although ASFV-specific antibodies were detectable in all but one vaccinated animal prior to challenge, no protective effect of immunization was observed. All animals developed acute-lethal ASF and had to be euthanized within eleven days post challenge. A slightly accelerated clinical course in vaccinees could even indicate an antibody dependent enhancement, which could also influence efficacy of other vaccine approaches., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called 'faunistic-hunting farms' in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3-35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future., (Published by Elsevier Ltd.)

Classical swine fever (CSF) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig industry. In most countries with industrialized pig production, prophylactic vaccination against CSF is banned, and all efforts are directed towards eradication of the disease, e.g. by culling of infected herds and animal movement restrictions. Nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks. For this application, potent vaccines are needed that allow differentiation of infected from vaccinated animals. Among the promising candidates for next generation marker vaccines is the chimeric pestivirus CP7_E2alf. Efficacy studies are usually carried out using highly virulent CSFV strains of genotype 1 that do not mirror the current field situation where strains of genotype 2 predominate. To prove that CP7_E2alf also protects against these strains, efficacy was assessed after single oral vaccination of wild boar and single intramuscular vaccination of domestic pigs using challenge models with recent CSFV strains and the highly virulent strain "Koslov" (genotype 1.1). It could be demonstrated that CP7_E2alf pilot vaccine batches for intramuscular and oral use were able to protect pigs from challenge infection with a highly virulent CSFV. Moreover, solid protection was also achieved in case of challenge infection with recent field strains of genotypes 2.1 and 2.3. Thus, broad applicability under field conditions can be assumed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) in wild boar repeatedly appeared in different federal states of the Federal Republic of Germany since 1995, from which it has been successfully eradicated sometimes fast, sometimes in a more time taking way using oral immunization as a main element of control. Since 2005 the cases focused solely on North Rhine-Westphalia and Rhineland-Palatinate. In the present study, therefore, the situation of CSF in wild boar has been closely investigated concerning the period 2005 to 2012 in these two regions. It is noteworthy that in this period two different variants of the virus subtype 2.3 occurred in two regionally defined areas of the "Eifel" and "Westerwald" as well as in the "Pfalz". The two Federal States have undertaken extensive oral vaccination campaigns and surveillance activities, which enabled an assessment of the existing virus prevalence and serological prevalence in the different regions. After an initial high serological prevalence, caused probably by interaction of infection and vaccination, the serological levels stabilized seasonally adjusted in a range from 50 to 60% in almost all areas. The vaccination campaigns have been maintained by both Federal States over a period of at least 2.5 years after virus has been detected for the last time. In consequence Germany as a whole has been recognized for the first time to be officially free from CSF in wild boar. By genotyping of virus isolates it has been demonstrated that the virus changed over time and played a role in the outbreak area "Westerwald".

The use of vaccines is still one of the most effective tools to control infectious diseases. Up to now, conventional vaccines are employed in the majority of cases. Drawbacks of these established vaccines include the lack of differentiability of infected from vaccinated animals (DIVA or marker strategy), limitations in the efficacy spectrum, and constraints and restrictions in production. For this reason, new vaccines, which do not show these disadvantages, are under development, especially for notifiable diseases such as classical swine fever (CSF). In principle, the following modern vaccine types can be differentiated: recombinant attenuated vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/ RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point often shows weaknesses. Alternative vaccine candidates are so far only prototypes and licensing is only a medium term possibility. Moreover, most of these vaccines are genetically engineered and can be problematic in terms of licensing and the public's acceptance. In conclusion, conventional vaccines still present the standard, especially in terms of efficacy. Yet, only vaccines with DIVA properties are feasible for the control of CSF. Thus, development and assessment of alternative vaccines is of paramount importance. The present overview summarizes concepts and vaccine types using the example of classical swine fever. It also recapitulates their advantages and disadvantages as well as their limitations.

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7 weeks and 50% at the age of 10 weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8 weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4 weeks before farrowing with one dose of C-strain vaccine., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Due to the tremendous socio-economic impact of classical swine fever (CSF) outbreaks, emergency vaccination scenarios are continuously under discussion. Unfortunately, all currently available vaccines show restrictions either in terms of marker capacities or immunogenicity. Recent research efforts were therefore directed at the design of new modified live marker vaccines. Among the most promising candidates the chimeric pestiviruses "CP7_E2alf" and "flc11" were identified. Within an international research project, these candidates were comparatively tested in challenge experiments after a single oral vaccination. Challenge infection was carried out with highly virulent CSF virus strain "Koslov", 14 or 21 days post vaccination (dpv), respectively. Safety, efficacy, and marker potential were addressed. All assessments were done in comparison with the conventional "gold standard" C-strain "Riems" vaccine. In addition to the challenge trials, multiple vaccinations with both candidates were performed to further assess their marker vaccine potential. All vaccines were safe and yielded full protection upon challenge 21 days post vaccination. Neither serological nor virological investigations showed major differences among the three vaccines. Whereas CP7_E2alf also provided clinical protection upon challenge at 14 days post vaccination, only 50% of animals vaccinated with flc11, and 83% vaccinated with C-strain "Riems" survived challenge at this time point. No marked differences were seen in protected animals. Despite the fact that all multiple-vaccinated animals stayed sero-negative in the accompanying marker test, the discriminatory assay remains a weak point due to delayed or inexistent detection of some of the vaccinated and subsequently infected animals. Nevertheless, the potential as live marker vaccines could be confirmed for both vaccine candidates. Future efforts will therefore be directed at the licensing of "Cp7_E2alf" as the first live marker vaccine for CSF., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf was tested in a "duration of immunity" trial according to the World Organisation for Animal Heath (OIE) guidelines. To this means, 15 weaner pigs were either orally or intramuscularly vaccinated with a single dose of CP7_E2alf vaccine produced under Good Laboratory Practice (GLP) conditions. Ten additional pigs were included as controls. Six months later, all animals were oronasally challenged with highly virulent CSF virus (CSFV) strain "Koslov". Upon vaccination, all but one orally and all intramuscularly vaccinated pigs developed rising and later on stable CSFV glycoprotein E2-specific antibodies. In contrast, no CSFV E(rns)-specific "marker" antibodies were detectable prior to challenge infection. None of the co-housed control animals seroconverted. Upon challenge infection, all seropositive animals were protected from lethal challenge, whereas all control animals and the non-responder developed severe signs of CSF. One control animal recovered, the others had to be euthanised due to animal welfare reasons between days 4 and 7 post challenge infection. All protected animals showed quickly rising neutralizing antibodies reaching high titres by the end of the trial. At the end of the trial, the marker ELISA was positive for most challenged animals that survived the CSFV infection (27 out of 30). Using reverse transcription polymerase chain reaction, low level genome detection was seen in all vaccinated animals between days 4 and 10 post challenge infection, but no virus could be isolated from any samples of these animals. The OIE guidelines require seroconversion in at least 8 out of 10 vaccinated animals. This requirement was fulfilled. Moreover, only control animals should die. With this requirement, only the intramuscular vaccination fully complied as one orally vaccinated pig did not respond. Concluding, CP7_E2alf induced stable antibodies that led to protection from lethal challenge with highly virulent CSFV strain "Koslov" six months after vaccination, with the exception of one non-responder after oral vaccination., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chimeric pestivirus CP7_E2alf is a promising live marker vaccine candidate against classical swine fever. Prior to a possible application in the field, several safety aspects have to be addressed. Due to the fact that CP7_E2alf is based on a bovine viral diarrhea virus backbone, its behavior in ruminants is of particular interest. In the framework of this study, its innocuousness in non-target species was addressed by inoculation of calves, young goats, lambs, and rabbits. To this means, high titres of CP7_E2alf were applied orally to three animals of each species. Additional animals were left as unvaccinated contact controls. During the study, all animals remained clinically healthy, and neither fever nor leukopenia were observed. Virus could not be isolated from purified white blood cells or from nasal or faecal excretions. Moreover, none of the animals (inoculated or contact control) seroconverted. In the target species, innocuousness, shedding and transmission of vaccine virus was addressed in different animal trials that were carried out primarily for the purpose of efficacy, potency or duration of immunity studies. In all experiments, CP7_E2alf proved to be completely safe for the vaccinees and unvaccinated contact controls. Furthermore, no shedding or transmission was detected in any of the experiments. Even after parental vaccination, vaccine virus genome was barely detectable in blood or organ samples of vaccinated animals. Thus, CP7_E2alf can be regarded as completely safe for both target and non-target species., (Copyright © 2011 Elsevier Ltd. 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.

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.

Abstract: Classical swine fever (CSF) is one of the most important diseases of pigs. Vaccination in the European Union is limited to emergency situations. Currently, vaccination for the purpose of disease control is carried out in wild boar populations. Wild boar are in most cases vaccinated using an oral bait vaccine based on the live modified vaccine virus C-strain “Riems”. A real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for differentiation of C-strain “Riems” vaccine virus from CSF virus (CSFV) field isolates was published previously. In this real-time RT-PCR system differentiation is based on two nucleotide difference one at the 3′ end of each of the primer-binding sites in the ERNS encoding genome region. During extensive diagnostic use of this protocol in an outbreak of CSF in wild boar in Germany, some C-strain positive field samples were found to give negative results in the C-strain “Riems” specific real-time RT-PCR, but positive results in a pan-CSFV real-time RT-PCR system. Moreover, sequencing of C-strain “Riems” vaccine batches for intramuscular use revealed differences in the ERNS encoding region. This led to the assumption that mutations in the corresponding primer-binding site of the C-strain specific system had appeared in the field, and possibly also during manufacturing of different vaccine batches. To test this hypothesis and restore sensitivity, a new primer set for detection of the possible C-strain virus quasi species was designed and tested. [Copyright &y& Elsevier]