New insights into the antigenic structure of the glycoprotein E(rns) of classical swine fever virus by epitope mapping.

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.

Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control.

The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics.

[Pathogenesis of classical swine fever--similarities to viral haemorrhagic fevers: a review]. / Pathogenese der Klassischen Schweinepest--Parallelen zu viralen hämorrhagischen Fiebern: eine Ubersicht.

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.

Classical swine fever virus in South-Eastern Europe--retrospective analysis of the disease situation and molecular epidemiology.

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.

Genetic diversity of Porcine reproductive and respiratory syndrome virus (PRRSV) in selected herds in a pig-dense region of North-Western Germany.

The Porcine reproductive and respiratory syndrome virus (PRRSV) can persist for several weeks in infected pigs, and readily spreads from viraemic pigs to other pigs as well as to other herds. In regions with dense pig populations such as the North-Western part of Germany the virus is endemic. The aim of this study was to analyse the genetic diversity of PRRSV found in 18 herds in this region, which had pigs with respiratory or reproductive failures, and where PRRSV had been demonstrated by RT-PCR. Phylogenetic trees were calculated using the complete nucleotide sequences of the ORF5. Of the 65 samples sequenced, five PRRSV from four herds were of the North American (NA; Type 2) genotype, and 60 of the European (EU; Type 1) genotype. To ascertain if the field PRRSV varied with time and to monitor the health condition of the herds they were revisited 2 years later. Although only two herds still reported clinical signs, PRRSV was found by RT-PCR in 10 of the 18 herds. Phylogenetic analysis showed that of the 23 PRRSV sequenced, 15 were of the European (EU) genotype. The EU genotype isolates from both samplings could be assigned to one of 12 clusters. There was no indication for the existence of herd specific clusters. ORF5 sequence identities between PRRSV from one herd in one cluster were either 100%, or had single base exchanges. These data indicate that the mutation rates for the European field isolates are similar to that found for the NA genotype vaccine strain used in Germany.

Frequency of PRRS live vaccine virus (European and North American genotype) in vaccinated and non-vaccinated pigs submitted for respiratory tract diagnostics in North-Western Germany.

The frequency of PRRSV corresponding to live vaccines and wild-type was determined in 902 pigs from North-Western Germany submitted for post-mortem examination. Overall, 18.5% of the samples were positive for the EU wild-type virus. EU genotype vaccine virus was detected in 1.3% and the NA genotype vaccine virus in 8.9% of all samples. The detection of the EU vaccine was significantly higher in pigs vaccinated with the corresponding vaccine (OR=9.4). Pigs vaccinated with NA genotype had significantly higher detection chances for the corresponding vaccine virus when compared to non-vaccinated animals (OR=3.34) animals, however, NA vaccine was also frequently detected in non-vaccinated pigs. Concluding, the dynamics of NA genotype vaccine and EU wild-type virus corresponds with studies on PRRSV spread in endemically infected herds. The potential of spontaneous spread of the NA genotype vaccine should be considered in the planning of eradication programs.

Formation of bovine viral diarrhea virus E1-E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains.

The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins Erns, E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not Erns were infectious, indicating that Erns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1-E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1-E2 heterodimers are essential for BVDV entry.

First isolation of EBLV-2 in Germany.

In Europe, rabies in bats is caused by European Bat Lyssavirus (EBLV) type 1 (EBLV-1) or type 2 (EBLV-2) which form two distinct genotypes (gt 5 and 6) within the genus Lyssavirus of the family of Rhadoviridae. Spill-over infections of EBLV in humans have caused fatal rabies encephalitis and highlighted the relevance of this wildlife disease for public health. The vast majority of the 831 European bat rabies cases reported between 1977 and 2006 were identified as EBLV-1. Only few virus isolates originating from Switzerland, The Netherlands and the United Kingdom were characterized as EBLV-2. Here we report the first EBLV-2 case detected in Germany in a Daubenton's bat (Myotis daubentonii) in August 2007. The bat showed clinical signs of disorders of the central nervous system and subsequently tested positive for rabies. The virus was isolated and characterized as EBLV-2 based on its antigen pattern and by nucleotide sequencing. Phylogenetic analysis indicated an association to EBLV-2 isolates from Switzerland which correlates with the origin of the bat close to the Swiss border.

Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae).

Salivary proteins of hematophagous Culicoides spp. are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted Culicoides saliva. In the current study, a collection method for midge saliva was developed. Over a 3-d period, 3- to 5-d-old male and female Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were repeatedly placed onto the collection system and allowed to deposit saliva into a filter. Salivary products were eluted from the filters and evaluated by gel electrophoresis and mass spectrometry as well as by intradermal testing and determination of clotting time. Gel electrophoresis revealed approximately 55 protein spots displaying relative molecular masses from 5 to 67 kDa and isoelectric points ranging from 4.5 to 9.8. The majority of molecular species analyzed by mass spectrometry showed high convergence with salivary proteins recently obtained from a cDNA library of Culicoides sonorensis Wirth & Jones, including proteins involved in sugarmeal digestion, defense, and coagulation inhibition as well as members of the D7 family and unclassified salivary proteins. In addition, the proteome analysis revealed a number of peptides that were related to proteins from insect species other than Culicoides. Intradermal injection of the saliva in human skin produced edema, vasodilatation, and pruritus. The anticoagulant activity of the saliva was demonstrated by significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.

Diagnostic methods for detection of Classical swine fever virus--status quo and new developments.

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).

Molecular characterization of the env gene from Brazilian field isolates of Bovine leukemia virus.

Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions. In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found in the aminoacid sequences of the gp51 protein were localized in the G and H epitopes. Using the SIFT software, it was predicted that they should not alter the protein functions. Phylogenetic analyses showed that partial or complete env gene sequences grouped in three or four phylogenetic clusters, respectively. The sequences from the Brazilian isolates had similar mutation rates as compared to samples from other countries, and belonged to at least two phylogenetic clusters.

A sequence database allowing automated genotyping of Classical swine fever virus isolates.

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.

A one-step multiplex real-time RT-PCR for detection and typing of bovine viral diarrhea viruses.

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5'-untranslated region of the viral genome. The real-time assay was able to detect 10-100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group.

In the last decade, several studies were performed to characterise bovine viral diarrhoea virus (BVDV) isolates and define genetic groups by genotyping. Much data is now available from GenBank, predominantly sequences from the 5' untranslated region (5'-UTR). In order to find out whether genetic grouping of isolates from different countries could be harmonised, 22 new isolates from five countries were analysed in combination with published sequences. Eighteen of these isolates were typed as BVDV genotype 1 (BVDV-1), and one isolate from Argentina and three isolates from Brazil were typed as BVDV-2. BVDV-1 isolates were clustered into five previously defined genetic groups: BVDV-1a, b, d, e and f. Two isolates from Finland and one from Egypt formed a group which was tentatively labelled as BVDV-1j, since statistical support was low. By using a fragment of the Npro gene for typing, we found that these isolates fall into the same group as a deer strain, and are statistically significant. Some Swiss BVDV strains taken from GenBank were found in a new genetic group which was designated as BVDV-1k. The BVDV-2 isolates included in this study seemed to fall into two genetic groups.

Phocine distemper in German seals, 2002.

Approximately 21,700 seals died during a morbillivirus epidemic in northwestern Europe in 2002. Phocine distemper virus 1 was isolated from seals in German waters. The sequence of the P gene showed 97% identity with the Dutch virus isolated in 1988. There was 100% identity with the Dutch isolate from 2002 and a single nucleotide mismatch with the Danish isolate.

Phylogenetic analysis of the E2 gene of classical swine fever viruses from Lao PDR.

The E2 genes of 21 classical swine fever viruses (CSFV) were genetically characterized and compared with reference CSF viruses. The viruses originated from CSF outbreaks that occurred in the Lao People's Democratic Republic (Lao PDR) during 1997 though to 1999. All viruses characterized belonged to genogroup 2 and were members of subgroups 2.1 and 2.2. Results demonstrated a geographic delineation between subgroups 2.1 that was only found in the North-Central region, and subgroup 2.2 that was mostly found in the South-Central regions of Lao PDR. Although it was not possible to determine the origin of these viruses, it is probable that they may have been introduced to Lao PDR following cross-border trade. Alternatively, they have evolved independently of other viruses in the region.

The surface glycoprotein E2 of bovine viral diarrhoea virus contains an intracellular localization signal.

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.

Vaccination against classical swine fever virus: limitations and new strategies.

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.

Characterization of the horse (Equus caballus) IGHA gene.

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse ( Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5' end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.

[Perspectives on BVD eradication in Germany]. / Perspektiven der BVD-Bekämpfung in Deutschland.

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.