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Abstract 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.

Abstract Background The 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. Results The 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. Conclusion Clustering of CSFV genomes based on their codon pair bias correlate with the genotype rather than with the virulence of the isolates.

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.