Immanuel Leifer

Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany.

Classical swine fever (CSF) has caused significant economic losses in industrialized pig production, and is still present in some European countries. Recent CSF outbreaks in Europe were mainly associated with strains of genogroup 2 (subgroup 2.3). Although there are extensive datasets regarding 2.3 strains, there is very little information available on longer fragments or whole classical swine fever virus (CSFV) genomes. Furthermore, there are no detailed analyses of the molecular epidemiology of CSFV wild boar isolates available. Nevertheless, complete genome sequences are supportive in phylogenetic analyses, especially in affected wild boar populations. Here, German CSFV strains of subgroup 2.3 were fully sequenced using two different approaches: (i) a universal panel of CSFV primers that were developed to amplify the complete genome in overlapping fragments for chain-terminator sequencing; and (ii) generation of a single full-length amplicon of the CSFV genome obtained by long-range RT-PCR for deep sequencing with next-generation sequencing technology. In total, five different strains of CSFV subgroup 2.3 were completely sequenced using these newly developed protocols. The approach was used to study virus spread and evolutionary history in German wild boar. For the first time, the results of our study clearly argue for the possibility of a long-term persistence of genotype 2.3 CSFV strains in affected regions at an almost undetectable level, even after long-term oral vaccination campaigns with intensive monitoring. Hence, regional persistence in wild boar populations has to be taken into account as an important factor in the continual outbreaks in affected areas.

Generation of recombinant pestiviruses using a full-genome amplification strategy

Complete genome amplification of viral RNA provides a new tool for the generation of modified viruses. We have recently reported a full-genome amplification strategy for recovery of pestiviruses (Rasmussen et al., 2008). A full-length cDNA amplicon corresponding to the Border disease virus-Gifhorn genome was generated by long RT-PCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived.

Modified live marker vaccine candidate CP7_E2alf provides early onset of protection against lethal challenge infection with classical swine fever virus after both intramuscular and oral immunization.

Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application.

Approaches to define the viral genetic basis of classical swine fever virus virulence

Classical swine fever (CSF), a highly contagious disease of pigs caused by the classical swine fever virus (CSFV), can lead to important economic losses in the pig industry. Numerous CSFV isolates with various degrees of virulence have been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe peracute hemorrhagic fever with nearly 100% mortality. Knowledge of the molecular determinants of CSFV virulence is an important issue for effective disease control and development of safe and effective marker vaccines. In this review, the latest studies in the field of CSFV virulence are discussed. The topic of virulence is addressed from different angles; nonconventional approaches like codon pair usage and quasispecies are considered. Future research approaches in the field of CSFV virulence are proposed.

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.

Clustering of classical swine fever virus isolates by codon pair bias

BackgroundThe genetic code consists of non-random usage of synonymous codons for the same amino acids, termed codon bias or codon usage. Codon juxtaposition is also non-random, referred to as codon context bias or codon pair bias. The codon and codon pair bias vary among different organisms, as well as with viruses. Reasons for these differences are not completely understood. For classical swine fever virus (CSFV), it was suggested that the synonymous codon usage does not significantly influence virulence, but the relationship between variations in codon pair usage and CSFV virulence is unknown. Virulence can be related to the fitness of a virus: Differences in codon pair usage influence genome translation efficiency, which may in turn relate to the fitness of a virus. Accordingly, the potential of the codon pair bias for clustering CSFV isolates into classes of different virulence was investigated.ResultsThe complete genomic sequences encoding the viral polyprotein of 52 different CSFV isolates were analyzed. This included 49 sequences from the GenBank database (NCBI) and three newly sequenced genomes. The codon usage did not differ among isolates of different virulence or genotype. In contrast, a clustering of isolates based on their codon pair bias was observed, clearly discriminating highly virulent isolates and vaccine strains on one side from moderately virulent strains on the other side. However, phylogenetic trees based on the codon pair bias and on the primary nucleotide sequence resulted in a very similar genotype distribution.ConclusionClustering of CSFV genomes based on their codon pair bias correlate with the genotype rather than with the virulence of the isolates.

Characterization of C-strain “Riems” TAV-epitope escape variants obtained through selective antibody pressure in cell culture

Classical swine fever virus (CSFV) C-strain “Riems” escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.