The European Classical Swine Fever Virus Database: Blueprint for a Pathogen-Specific Sequence Database with Integrated Sequence Analysis Tools.

Molecular epidemiology has become an indispensable tool in the diagnosis of diseases and in tracing the infection routes of pathogens. Due to advances in conventional sequencing and the development of high throughput technologies, the field of sequence determination is in the process of being revolutionized. Platforms for sharing sequence information and providing standardized tools for phylogenetic analyses are becoming increasingly important. The database (DB) of the European Union (EU) and World Organisation for Animal Health (OIE) Reference Laboratory for classical swine fever offers one of the world's largest semi-public virus-specific sequence collections combined with a module for phylogenetic analysis. The classical swine fever (CSF) DB (CSF-DB) became a valuable tool for supporting diagnosis and epidemiological investigations of this highly contagious disease in pigs with high socio-economic impacts worldwide. The DB has been re-designed and now allows for the storage and analysis of traditionally used, well established genomic regions and of larger genomic regions including complete viral genomes. We present an application example for the analysis of highly similar viral sequences obtained in an endemic disease situation and introduce the new geographic "CSF Maps" tool. The concept of this standardized and easy-to-use DB with an integrated genetic typing module is suited to serve as a blueprint for similar platforms for other human or animal viruses.

Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study.

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 "Margarita" and the recent field isolate CSF1058 "Pinar del Rio". Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

Close relationship of ruminant pestiviruses and classical Swine Fever virus.

To determine why serum from small ruminants infected with ruminant pestiviruses reacted positively to classical swine fever virus (CSFV)-specific diagnostic tests, we analyzed 2 pestiviruses from Turkey. They differed genetically and antigenically from known Pestivirus species and were closely related to CSFV. Cross-reactions would interfere with classical swine fever diagnosis in pigs.

Efficacy of marker vaccine candidate CP7_E2alf against challenge with classical swine fever virus isolates of different genotypes.

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.

Morphogenesis of pestiviruses: new insights from ultrastructural studies of strain Giraffe-1.

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.

First molecular identification and characterization of classical swine fever virus isolates from Nepal.

Classical swine fever (CSF) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. Here, for the first time, molecular identification and characterization of CSFV isolates from two recent outbreaks in Nepal are presented. Analysis of full-length E2-encoding sequences revealed that these isolates belonged to CSFV subgenotype 2.2 and had highest genetic similarity to isolates from India. Hence, for CSFV, Nepal and India should be regarded as one epidemiological unit. Both Nepalese isolates exhibited significant sequence differences, excluding a direct epidemiological connection and suggesting that CSFV is endemic in that country.

Classical swine fever virus isolates from Cuba form a new subgenotype 1.4.

Identification and classification of classical swine fever virus (CSFV) on the basis of nucleotide sequencing and phylogenetic analysis have become an important tool to study the epidemiology and to control CSF disease. According to phylogenetic analyses of short sequences from the 5'nontranslated region (150 nt) and the E2 (190 nt), most CSFV isolates from South and Central America have been assorted to the subgenotypes 1.1 and 1.3, while CSFV isolates from Cuba have been allocated to subgenotype 1.2. Here we demonstrate that determination and comparison of full-length E2 sequences as well as of the sequences encoding for N(pro), C, E(rns), E1 and E2 (3361 nt) do not support segregation of Cuban CSFV isolates to subgenotype 1.2. In fact, our analysis revealed that the Cuban isolates are more divergent from other so far known CSFV subgenotype 1 isolates and form a novel separate subgenotype that is proposed to be designated subgenotype 1.4.

Complete genome sequence of a novel pestivirus from sheep.

We report here the complete genome sequence of pestivirus strain Aydin/04-TR, which is the prototype of a group of similar viruses currently present in sheep and goats in Turkey. Sequence data from this virus showed that it clusters separately from the established and previously proposed tentative pestivirus species.

Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences.

Molecular epidemiology has proven to be an essential tool in the control of classical swine fever (CSF) and its use has significantly increased during the past two decades. Phylogenetic analysis is a prerequisite for virus tracing and thus allows implementing more effective control measures. So far, fragments of the 5´NTR (150 nucleotides, nt) and the E2 gene (190 nt) have frequently been used for phylogenetic analyses. The short sequence lengths represent a limiting factor for differentiation of closely related isolates and also for confidence levels of proposed CSFV groups and subgroups. In this study, we used a set of 33 CSFV isolates in order to determine the nucleotide sequences of a 3508-3510 nt region within the 5´ terminal third of the viral genome. Including 22 additional sequences from GenBank database different regions of the genome, comprising the formerly used short 5´NTR and E2 fragments as well as the genomic regions encoding the individual viral proteins Npro, C, Erns, E1, and E2, were compared with respect to variability and suitability for phylogenetic analysis. Full-length E2 encoding sequences (1119 nt) proved to be most suitable for reliable and statistically significant phylogeny and analyses revealed results as good as obtained with the much longer entire 5´NTR-E2 sequences. This strategy is therefore recommended by the EU and OIE Reference Laboratory for CSF as it provides a solid and improved basis for CSFV molecular epidemiology. Finally, the power of this method is illustrated by the phylogenetic analysis of closely related CSFV isolates from a recent outbreak in Lithuania.