[Show abstract][Hide abstract] ABSTRACT: Bacterial and viral infections are still a major threat of human health. The increasing resistance of bacterial isolates to common antibiotics and the lack of new compounds reaching the clinic leading to serious problems in health care. The variability of different virus families with individual entry pathways and replication strategies make the development of suitable therapeutics with cross-species activity complicated. Furthermore, the infections often cause each other, so that an initial virus infection is frequently accompanied by a bacterial ‘superinfection’ with severe consequences.
We developed a new class of compounds based on polypeptides, which exhibit broad spectrum antiviral activity with simultaneous inhibition of important bacterial pathogenicity factors such as lipopolysaccharide (LPS, endotoxin) and lipoproteins. Here, we summarise recent results and discuss them in the context of the progress made in the field of polypeptides as novel anti-infective therapeutic agents.
Frontiers in Clinical Drug Research: Anti-Infectives, Edited by Atta-ur-Rahman, 01/2014: chapter Anti-Infective Polypeptides for Combating Bacterial and Viral Infections: pages 3-31; Bentham Science Publshers., ISBN: 978-1-60805-855-6
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The E(rns) glycoprotein of classical swine fever virus (CSFV) has been studied in detail concerning biochemical and functional properties, whereas less is known about its antigenic structure. In order to define epitopes recognized by CSFV-specific antibodies, the binding sites of seven E(rns)-specific monoclonal antibodies were investigated. Mapping experiments using chimeric E(rns) proteins, site-directed mutagenesis and an overlapping peptide library identified one antigenic region located between amino acids (aa) 55 to 110 on the E(rns) protein of CSFV Alfort/187. The domain comprises three linear motifs ⁎(64)TNYTCCKLQ(72), (73)RHEWNKHGW(81), and (88)DPWIQLMNR(96), respectively, and two aa at position 102 and 107 that are crucial for the interaction with antibodies. Additionally, the presentation of the epitope in a correct conformation is mandatory for an efficient antibody binding. These findings allow a better understanding of the organization and the structure of the E(rns) and provide valuable information with regard to the development of E(rns)-based diagnostic tests.
[Show abstract][Hide abstract] ABSTRACT: Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447(Δc)), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447(Δc). Upon infection of the natural host, Vp447(Δc) was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.
Journal of virological methods 08/2011; 175(2):170-4. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Due to its strong impact on economics and trading the Foot-and-Mouth-Disease (FMD) is one of the most important animal diseases within animal husbandry. Because no recent specific field observation for FMD exists in Germany, the risk assessment needs validated epidemiological models to prepare decision tools for FMD-outbreak management. The aim of this investigation was therefore to prepare a risk assessment for different transmission pathways to use for FMD-models in future. To prepare a FMD-transmission model the risk was assessed within a highly animal densed region in Germany by means of an expert survey. For each transmission pathway an assessment was given in the categories low, medium, high and severe. Some pathways were rated homogenously between the experts, but some were rated heterogeneously. Therefore areas were identified with common rating as well as areas, where further investigations to specify FMD-models are necessary.
Berliner und Münchener tierärztliche Wochenschrift 03/2010; 123(3-4):89-95. · 0.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The inter-laboratory comparison tests for classical swine fever (CSF) laboratory diagnosis organised by the European Community Reference Laboratory for CSF are regularly performed within European Union Member States. The objective of this study was to evaluate the results of the inter-laboratory comparison tests carried out over the last decade, from 1998 until 2007, by using a statistical approach. A set of five or six lyophilised sera was sent to participants. These included sera containing CSF antibodies, sera containing antibodies against ruminant pestiviruses, sera containing CSF virus and negative sera. This study focused on the results of the diagnostic reference methods for CSF: the neutralisation test for the detection of CSF antibodies (including its interpretation) and virus isolation for the detection of CSF virus. For the detection of CSF antibodies, results were closest to what was expected by the Community Reference Laboratory when only neutralisation tests were performed. The percentage of correct results decreased as soon as the results of CSF antibody enzyme-linked immunosorbent assay were included or when sera with antibodies to ruminant pestiviruses were added to the panel. The results for the detection of CSF antibodies are still valid today, as no additional method has been introduced recently. Regarding CSF virus detection, CSF virus isolation is well established but on the way to being superseded as the reference test by reverse-transcription polymerase chain reaction.
Revue scientifique et technique (International Office of Epizootics) 12/2009; 28(3):1091-101. · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical Swine Fever (CSF) has caused several outbreaks in EU Member States with grave economic consequences. Several times the diagnosis of CSF was made too late partially due to non-specific clinical signs which did not raise suspicion for CSF. Virulence of CSF virus isolates (CSFV) still remains a subject of discussion and speculation as sufficient knowledge is still not available. Six uncharacterised CSFV isolates from 1996 to 2007 were assessed in animal experiments for their clinical virulence in order to broaden the knowledge about circulating CSFV and thereby assist disease eradication. A clinical (CS) and pathological score was applied and further extended by additional parameters to a modified CS (mCS) including case fatality, antibody production and leukocyte count. The unknown CSFV isolates could be classified as moderately or highly virulent. The inclusion of additional parameters, especially case fatality, into the mCS gave a more reliable classification of virulence, proving that there are clinical signs and laboratory parameters of blood which can be recognised. Therefore a subclinical course of infection is unlikely, especially in weaner pigs.
[Show abstract][Hide abstract] ABSTRACT: For the important livestock pathogens classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), cytopathogenic (cp) and non-cp viruses are distinguished according to the induction of apoptosis in infected tissue culture cells. However, it is currently unknown whether cp CSFV differs from non-cp CSFV with regard to virulence in the acutely infected host. In this study, we generated helper virus-independent CSFV Alfort-Jiv, which encompasses sequences encoding domain Jiv-90 of cellular J-domain protein interacting with viral protein (Jiv). Expanding the knowledge of BVDV, our results suggest that Jiv acts as a regulating cofactor for the nonstructural (NS) protein NS2 autoprotease of CSFV and initiates NS2-3 cleavage in trans. For Alfort-Jiv, the resulting expression of large amounts of NS3 correlated with increased viral RNA synthesis and viral cytopathogenicity. Moreover, both cp Alfort-Jiv and the parental non-cp CSFV strain Alfort-p447 efficiently replicate in cell culture. Animal experiments demonstrated that in contrast to parental non-cp Alfort-p447, infection with cp Alfort-Jiv did not cause disease in pigs but induced high levels of neutralizing antibodies, thus elucidating that cp CSFV is highly attenuated in its natural host. In contrast to virulent Alfort-p447, the attenuated CSFV strain Alfort-Jiv induces the expression of cellular Mx protein in porcine PK-15 cells. Accordingly, the remarkable difference between cp and non-cp CSFV with regard to the ability to cause classical swine fever in pigs correlates with different effects of cp and non-cp CSFV on cellular antiviral defense mechanisms.
Journal of Virology 10/2008; 82(19):9717-29. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study the effect of control measures implemented during the classical swine fever (CSF) epidemic in wild boar in the Eifel region of the German federal state of Rhineland-Palatinate from 1999 to 2005 was assessed. During the first 3 years after official confirmation of virus detection these measures comprised intensive hunting, especially of young animals and hygiene measures. Subsequently oral immunisation (o.i.) using a modified live virus vaccine was introduced as an additional control tool. All shot wild boar from the restricted area were tested virologically and serologically for CSF. The laboratory results from over 110,000 animals accompanied by information about age, gender and geographical origin of the animals were collected in a relational database. In total about 82% of all virologically positive wild boars were piglets, thus confirming the importance of this age group in the perpetuation of the epidemic. An analysis of the hunting bag showed that piglets were underrepresented compared to older animals throughout the eradication programme. This finding indicated that hunters did not comply with the control strategy of intense targeting of young animals. Before as well as after the implementation of o.i. a significantly higher virological prevalence and a significantly lower serological prevalence were observed in piglets compared to yearlings and adults. Shortly after the beginning of the vaccination campaign in February 2002 CSFV prevalence decreased significantly whereas the serological prevalence increased markedly in all age classes. In order to test the influence of age and vaccination on the serological prevalence a logistic regression model was used. Our results strongly suggest that under the field conditions in the Eifel region vaccination against CSFV had a crucial influence on the increase of seroprevalence rate and the elimination of CSFV. The last virus-positive pig was found 13 months after start of o.i.
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a highly contagious disease causing major losses in pig populations almost worldwide. The disease occurs in many regions of Asia, Central and South America and parts of Europe and Africa. Some countries have eradicated the disease (Australia, USA, Canada, within the EU), yet it keeps recurring sporadically (South Africa, Germany, Netherlands, England). The causative virus is a member of the genus Pestivirus, family Flaviviridae. The first diagnosis of CSF is based on the recognition of clinical signs by the veterinarian in the field and by post mortem findings. Many signs are not exclusively associated with CSF and they may vary with the strain of virus, age and health status of the pigs. Since clinical signs may be confused with other pig diseases, laboratory diagnosis of CSF is indispensable. Both the Office International des Epizooties (OIE) and the European Union, have approved diagnostic manuals establishing sampling methods and diagnostic procedures for the confirmation of the disease. In this review, experiences with current tests will be analyzed and complemented with new developments, with emphasis on the polymerase chain reaction after reverse transcription of the RNA genome (RT-PCR).
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is a highly contagious viral disease of pigs. According to the OIE classification of diseases it is classified as a notifiable (previously List A) disease, thus having the potential for causing severe socio-economic problems and affecting severely the international trade of pigs and pig products. Effective control measures are compulsory, and to expose weaknesses a reliable tracing of the spread of the virus is necessary. Genetic typing has proved to be the method of choice. However, genotyping involves the use of multiple software applications, which is laborious and complex. The implementation of a sequence database, which is accessible by the World Wide Web with the option to type automatically new CSF virus isolates once the sequence is available is described. The sequence to be typed is tested for correct orientation and, if necessary, adjusted to the right length. The alignment and the neighbor-joining phylogenetic analysis with a standard set of sequences can then be calculated. The results are displayed as a graph. As an example, the determination is shown of the genetic subgroup of the isolate obtained from the outbreaks registered in Russia, in 2005. After registration (Irene.firstname.lastname@example.org) the database including the module for genotyping are accessible under http://viro08.tiho-hannover.de/eg/eurl_virus_db.htm.
Journal of Virological Methods 04/2007; 140(1-2):95-9. · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study analysed the transport behaviour of the glycoprotein E2 of Bovine viral diarrhea virus (BVDV) expressed from recombinant vesicular stomatitis virus (rVSV). E2 protein was found to be retained at an intracellular compartment. A chimeric protein containing the membrane anchor and cytoplasmic tail of the VSV G protein, E2-G(MT), was transported to the cell surface. Only the latter protein was incorporated into rVSV particles in significant amounts. A soluble form of E2 lacking the membrane anchor, E2(MTdel), appeared to be affected in conformational stability. In contrast to both membrane-anchored forms of E2, expression of the soluble form was detectable only by immunofluorescence microscopy but not by Western blotting. These results are in agreement with reports of intracellular retention of the E2 protein due to a retention signal in the membrane anchor. However, in another analysis of E2 expressed from rVSV, E2 protein was reported to be transported to the cell surface and incorporated into VSV particles [Grigera, P. R., Marzocca, M. P., Capozzo, A. V. E., Buonocore, L., Donis, R. O. & Rose, J. K. (2000). Virus Res 69, 3-15]. Reasons for these contradictory results are discussed.
Journal of General Virology 02/2007; 88(Pt 1):157-65. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper summarises the views of a European group of scientists involved in the control of bovine viral diarrhoea virus (BVDV), as part of a European Union Thematic Network. The group concludes that the technical tools and the knowledge needed to eradicate BVDV are at hand, as proven by successful national control schemes in several European countries. A generic model for BVDV control is presented, which includes biosecurity, elimination of persistently infected animals and surveillance as central elements. These elements are termed 'systematic', in contrast to control efforts without clear goals and surveillance to evaluate progress. The network concludes that a systematic approach is needed to reach a sustainable reduction in the incidence and prevalence of BVDV in Europe. The role of vaccines in systematic control programmes is considered as an additional biosecurity measure, the effect of which should be evaluated against cost, safety and efficacy. It is also concluded that active participation by farmers' organisations is a strong facilitator in the process that leads up to the initiation of control, and that public funding to support the initiation of organised BVD control programmes can be justified on the basis of expected wider societal benefits, such as animal welfare and reduction in the use of antibiotics. If applied successfully, the focus on biosecurity in systematic BVD control programmes would also reduce the risk of the introduction and spread of other epizootic and zoonotic agents, thereby improving both cattle health and welfare in general, as well as increasing the competitiveness of the cattle industry.
Revue scientifique et technique (International Office of Epizootics) 01/2007; 25(3):961-79. · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rapid and accurate diagnosis is of the utmost importance in the control of epizootic diseases such as classical swine fever (CSF), and efficacious vaccination can be used as a supporting tool. While most of the recently developed CSF vaccines and diagnostic kits are mostly validated according to World Organisation for Animal Health (OIE) standards, not all of the well-established traditional vaccines and diagnostic tests were subject to these validation procedures and requirements. In this report, data were compiled on performance and validation of CSF diagnostic tests and vaccines. In addition, current strategies for differentiating infected from vaccinated animals are reviewed, as is information on the control of CSF in wildlife. Evaluation data on diagnostic tests were kindly provided by National Reference Laboratories for CSF in various European countries.
Revue scientifique et technique (International Office of Epizootics) 01/2007; 25(3):1025-38. · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several European countries have initiated national and regional control-and-eradication campaigns for bovine viral diarrhea virus (BVDV). Most of these campaigns do not involve the use of vaccines; in Germany, vaccination is used only in states in which it is considered necessary because of high BVDV prevalence. In European countries without organized BVDV control programs, vaccination is commonly used to control BVDV. Diagnostic test strategies are fundamental to all control-and-eradication campaigns; therefore, the purpose of this review is to describe how the available diagnostic tests are combined into test strategies in the various phases of control-and-eradication campaigns in Europe. Laboratory techniques are available for BVDV diagnosis at the individual animal level and at the herd level. These are strategically used to achieve 3 main objectives: 1) initial tests to classify herd status, 2) follow-up tests to identify individual BVDV-infected animals in infected herds, and 3) continued monitoring to confirm BVDV-free status. For each objective or phase, the validity of the diagnostic tests depends on the mode of BVDV introduction and duration of infection in test-positive herds, and on how long noninfected herds have been clear of BVDV. Therefore, the various herd-level diagnostic tools--such as antibody detection in bulk milk or in blood samples from young stock animals, or BVDV detection in bulk milk--need to be combined appropriately to obtain effective strategies at low cost. If the individual diagnostic tests are used with due consideration of the objectives of a specific phase of a BVDV control program, they are effective tools for controlling and eradicating BVDV in regions not using vaccination and where vaccination is a part of the control or eradication program.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 10/2006; 18(5):427-36. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to characterize the effect of an experimental infection with the classical swine fever (CSF) virus on libido and ejaculate parameters of adult boars. Four boars 10 month old were infected with a CSF field isolate (Visbek/Han95). Semen was collected every second day after infection and daily during the pyrexic phase. The only clinical signs in the boars were an increase in body temperature, but never above 39.9 degrees C and a temporally reduction of food intake. The libido was always good, so semen collection was performed in three boars without difficulty and the semen quality was always in the range of the minimum requirements for sperm that is used for artificial insemination. Although one boar had a good libido only a sperm free ejaculate could be collected on one day. The results show that a CSF virus infection of adult boars hardly causes any clinical symptoms and that sperm can be obtained despite fever. Insemination boars may thus be of special epidemiological relevance for the dissemination of the CSF virus.
DTW. Deutsche tierärztliche Wochenschrift 08/2006; 113(7):251-5. · 0.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bovine viral diarrhoea (BVD) control/eradication programmes based on the test and removal of persistently infected cattle without use of vaccination were first introduced by the Scandinavian countries in the early 1990s. Within the last 10 years the programmes have proven to be very successful and have served as a blueprint for several other European regions. However, in areas with high cattle densities, intense animal trade and high BVD prevalence this control approach is risky, because there is a high probability that herds, which have been cleared of persistently infected (PI) animals and have become partly or fully susceptible to reintroduction of the virus, will come in contact with a BVD virus (BVDV) infected animal. A combination of the test and removal strategy with subsequent systematic vaccination of cattle could overcome this problem. The goals of vaccination in such a programme is protection against reintroduction of BVDV into herds free from PI cattle and foetal protection of pregnant animals accidentally exposed to the virus. Two-step vaccination is based on the use of inactivated BVDV-1 vaccine for priming followed by a live attenuated vaccine booster 4 weeks later. The immune response elicited by such a vaccination scheme has proven to be long lasting and foetal infection after challenge with BVDV-1 and BVDV-2 was prevented in pregnant animals 5 months after vaccination. These findings suggest that the implementation of a two-step vaccination in the initial phase of control programmes in addition to test and removal of PI animals in areas with high cattle densities and endemic BVD is practical and efficacious.