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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.
Veterinary Microbiology 10/2012; · 3.33 Impact Factor
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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.
Virology 08/2012; 433(1):45-54. · 3.35 Impact Factor
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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.
PLoS Pathogens 03/2012; 8(3):e1002598. · 9.13 Impact Factor
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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
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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.
Veterinary Microbiology 12/2010; 146(3-4):276-84. · 3.33 Impact Factor
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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. · 5.40 Impact Factor
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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.
Veterinary Microbiology 05/2008; 132(1-2):29-38. · 3.33 Impact Factor
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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).
Vaccine 08/2007; 25(30):5524-30. · 3.77 Impact Factor
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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.greiser-wilke@tiho-hannover.de) 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. · 2.01 Impact Factor
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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. · 3.36 Impact Factor
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ABSTRACT: For several decades after the first description of bovine viral diarrhea and its causative agent (BVDV) the economic impact of the infection was underestimated and in addition there were no suitable diagnostics and procedures for a systematic control at hand. Today, there are several estimates on the real economic impact of the infection and during the last 15 years the serological and virological laboratory diagnosis of BVDV infections has improved. Also, successful procedures aimed at eradicating BVDV infections by using a strict test and removal policy for animals persistently infected (PI) with BVDV accompanied by movement restrictions for infected herds have been implemented in the Scandinavian countries. The success of these efforts has encouraged other European countries to follow the same procedures. However, the Scandinavian control strategy might-for a number of reasons-not be acceptable for all European countries. In such cases, the test and removal strategy, with its fundamental elements of biosecurity, removal of PI animals and monitoring of herd status, in combination with systematic vaccination, might be an acceptable compromise. The impact of the BVDV-free status of regions and nations on international trade is not yet clear. In any case, biosecurity measures will be of utmost importance for individual control programs as well as multiple control programs to co-exist in Europe.
Animal Health Research Reviews 07/2005; 6(1):63-74.
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ABSTRACT: The most widely used vaccines for the control of classical swine fever (CSF) in countries where it is endemic are live attenuated virus strains, which are highly efficacious, inducing virtually complete protection against challenge with pathogenic virus. In the European Union (EU), the combination of prophylactic mass vaccination and culling of infected pigs in endemic regions has made it possible to almost eradicate the disease. However, it is not possible to discriminate between infected and vaccinated animals, thus hampering disease control measures that rely on serology. Therefore, vaccination was banned at the end of 1990 before the internal common market was established in the EU. Vaccination is allowed only in severe emergencies. In addition, there are strict restrictions on the international trade in pig products from countries using vaccination. To circumvent these problems, marker vaccines which allow differentiation of infected from vaccinated animals (DIVA) have been developed. There are several approaches, ranging from protective peptides, single expressed proteins, naked DNA and chimeric viruses. To date, two subunit vaccines based on the E2 glycoprotein are commercially available and have been tested extensively for their efficacy. The accompanying discriminatory tests are based on an ELISA detecting another viral glycoprotein, the E(rns). The subunit vaccines were found to be less efficacious than live attenuated vaccines. In addition, the currently available discriminatory tests do not provide high enough specificity and sensitivity. Although there is an urgent need for more advanced marker vaccines and better discriminatory tests, the development of new DIVA vaccines against CSF is hampered by the small market potential for these products.
Animal Health Research Reviews 01/2005; 5(2):223-6.
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ABSTRACT: The intracellular transport of the surface glycoprotein E2 of bovine viral diarrhoea virus was analysed by expressing the cloned gene in the absence of other viral proteins. Immunofluorescence analysis and surface biotinylation indicated that E2 is located in an early compartment of the secretory pathway and not transported to the cell surface. In agreement with this result, E2 was found to contain only high-mannose oligosaccharide side-chains but no N-glycans of the complex type. To define the intracellular localization signal of the E2 protein, chimeric proteins were generated. E2 chimeras containing the MT (membrane anchor plus carboxy-terminal domain) of the G protein of vesicular stomatitis virus (VSV) or of the F protein of bovine respiratory syncytial virus (BRSV) were transported to the cell surface. On the other hand, VSV G protein containing the MT domain of E2 was detected only in the ER, indicating that this domain contains an ER localization signal. A chimeric E2 protein, in which not the membrane anchor but only the carboxy-terminal end was replaced by the corresponding domain of the BRSV F protein, was also localized in the ER. Therefore, it was concluded that the membrane anchor contains the ER localization signal of E2. Interestingly, the ER export signal within the VSV G protein cytoplasmic tail was found to overrule the ER localization signal in the E2 protein membrane anchor.
Journal of General Virology 06/2004; 85(Pt 5):1101-11. · 3.36 Impact Factor
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ABSTRACT: Various monoclonal antibodies (MAbs) that recognize cell surface proteins on bovine cells were previously shown to efficiently block infection with bovine viral diarrhea virus (BVDV) (C. Schelp, I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess, Arch. Virol. 140:1997-2009, 1995). With one of these MAbs, a 50- to 58-kDa protein was purified from calf thymus by immunoaffinity chromatography. Microchemical analysis of two internal peptides revealed significant sequence homology to porcine and human CD46. The cDNA of bovine CD46 (CD46(bov)) was cloned and further characterized. Heterologously expressed CD46(bov) was detected by the MAb used for purification. A putative function of CD46(bov) as a BVDV receptor was studied with respect to virus binding and susceptibility of nonpermissive cells. While the expression of CD46(bov) correlated well with the binding of [(3)H]uridine-labeled BVDV, the susceptibility of cells nonpermissive for BVDV was not observed. However, the expression of CD46(bov) resulted in a significant increase in the susceptibility of porcine cells to BVDV. These results provide strong evidence that CD46(bov) serves as a cellular receptor for BVDV.
Journal of Virology 03/2004; 78(4):1792-9. · 5.40 Impact Factor
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ABSTRACT: The economic impact of BVDV infections has led a number of countries in Europe to start eradication or control programmes. While in both cases the primary step is identification and elimination of persistently infected (PI) animals, the strategy applied thereafter is dependent on the density and seroprevalence of the regional cattle population. One of the first countries to design and implement an eradication programme was Sweden in 1993, a country with a relatively low cattle density and no vaccination. For screening, an indirect antibody ELISA for serum, milk and bulk milk samples is being used. The basics of the Swedish model are no vaccination, voluntary participation, and financing of the entire scheme by the subscribing farmers. BVDV-free herds are certified and permanently checked. While in 1993 only about 35% of the herds were seronegative, about 87% were BVDV-free in 2001. The aim of control programmes in high density areas with high seroprevalence is to minimize economic losses by reducing the incidence of PI animals and thereby virus circulation (German model). Participation is voluntary, and parts of the costs are carried by the public animal insurance (Tierseuchenkasse). Screening is performed using an antigen capture ELISA with blood or serum. In Lower Saxony, for example, a herd is declared BVDV unsuspicious if all animals up to 36 months are BVDV antigen negative and the female offspring older than six months is vaccinated twice (an inactivated vaccine is used for basic immunization, and an attenuated live virus vaccine for boosting).
Biologicals 07/2003; 31(2):113-8. · 1.70 Impact Factor
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ABSTRACT: Located between the open reading frames encoding the matrix (M) and the fusion (F) protein the morbillivirus genome contains an unusually large non-coding intercistronic region (M-F UTR) of up to 5.6% of the full length genome. Any function(s) of this region have largely remained obscure. Here, we analyze the M-F UTR and the proximal coding part of the downstream F gene of several recent canine distemper morbillivirus (CDV) wild-type (wt) isolates and vaccine strains. While the F gene coding part appeared to be highly conserved (about 93% homology), a considerable degree of strain-specific variation of up to 21.4% was evident when comparing the M-F UTR. Phylogenetic analysis revealed a co-circulation of several contemporary CDV genotypes within a close geographic range (central Europe). A remarkably distinct CDV wt lineage, so far detected only in mustelids, is displayed. A rather non-scattered pattern of mutations within the M-F UTR suggested superimposition of RNA sequence and/or secondary structure constraints. Extensive folding in the long (460 nt) and moderately GC-rich 5-UTR of the F mRNA was evident, particularly around the putative F protein translation initiation codon (AUG461 of the Onderstepoort vaccine strain). The region immediately preceding the putative F initiation site also harbored the only mutation unique to both vaccine strains within the F-5UTR (position 455: Awt vs. Cvac). The putative F protein start codon, AUG461, was found to be mutated to AUA or GUA in all wt isolates analyzed and in another vaccine strain (Rockborn). Possible consequences for F protein translation initiation in wt CDV are discussed.
Virus Genes 10/1998; 17(3):259-270. · 1.85 Impact Factor
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ABSTRACT: Infections with the bovine virus diarrhoea (BVD) virus are endemic with high seroprevalence in many countries of the European Union (EU). The significant economic damage caused by BVD infections has led to a paradigm shift with respect to a possible control. In some EU Member States control programmes have been initiated mostly on a voluntary basis and some compulsory. The most important element of all control efforts is the identification and removal of persistently infected (PI) animals. The subsequent steps depend on the respective seroprevalence and cattle density. Sweden was one of the first countries to introduce a national control program (1993), that is now being used as standard procedure in other countries. The starting position for the program was comparatively favorable since the country's cattle density is low and vaccination was not allowed. BVD infected herds were screened using a bulk milk ELISA and subsequently the PI animals in positive herds were identified and removed. The goal of the control program is the cattle population's certified freedom of BVD. The Scandinavian model is not applicable for most regions of Germany, since BVD virus prevalence and cattle density are unfavorably high. Here the primary goal is to minimize the economic losses caused by BVD and to lower the infective pressure. Therefore a Federal guideline was issued and some Federal States have provided additional regulations for compensation of PI animals and additional costs, respectively. Primary goal of the guideline is the eradication of PI animals and the systematic vaccination of all female offspring in order to avoid further economic damage and the emergence of new PI animals in case of re-infection of the herd. Goal of this strategy is the BVD unsuspicious herd with a high immune status.
Berliner und Münchener tierärztliche Wochenschrift 116(5-6):222-6. · 0.82 Impact Factor
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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 123(3-4):89-95. · 0.82 Impact Factor
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ABSTRACT: In spite of differences in etiology, viral haemorrhagic diseases share similarities in their pathogenesis. Characteristic for these diseases are thrombocytopenia, petechia and increased vascular leakage. Most lesions can be attributed to cytokine-mediated interactions triggered by infected and activated monocytes and macrophages, rather than by virus-induced direct cell damage. Causative agents of viral hemorrhagic diseases are enveloped RNA viruses. In most cases, they are transmitted to humans from their animal hosts by rodents or arthropod vectors (Arboviruses). Due to the clinical picture, the acute lethal form of classical swine fever (CSF) is also considered as a viral haemorrhagic disease. CSF is caused by an RNA virus in the family Flaviviridae, and members of the Suidae family are the only ones clinically affected. It is a highly contagious, therefore notifiable disease. In contrast to other viral hamorrhagic diseases, it is mainly transmitted oro-nasally by contact with infected pigs, or by contaminated items (semen, swill feed, clothing). The present survey summarizes analogies between classical representatives of viral haemorrhagic fevers, and recapitulates current knowledge concerning the pathogenesis of classical swine fever.
Berliner und Münchener tierärztliche Wochenschrift 124(1-2):36-47. · 0.82 Impact Factor
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