Classical Swine Fever Virus Genome Research Articles

Organization and diversity of the 3'-noncoding region of classical swine fever virus genome.

Specific PCR primers were selected to amplify a 359 bp DNA fragment flanking the 3'-part of the polymerase gene and the 3'-noncoding (3'-NC) region of the genome of classical swine fever virus (CSFV). In RT-PCR the selected fragment was amplified from the genomes of 27 viral strains collected from Europe, America and Asia over a period of a half century as well as from three vaccine strains of CSFV. Eight PCR products were sequenced using an automatic sequencing device. Nucleotide sequence analysis was performed by computer programs DNASTAR and PHYLIP. The comparative studies revealed that the 3'-NC region contains a variable region of nucleotides which is located immediately after the stop codon TGA or TAA. Furthermore, a strongly conserved constant region was identified near to the extreme 3'-terminus. Two imperfect repeated sequences were found both in the variable and in the constant regions. In addition, the variable region was characterized by the occurrence of a 50 bp long poly AT track. Phylogenetic analysis with different mathematical approaches (MegAlign, Neighbor-Joining method, Maximum Likelihood, Parsimony) revealed that the studied CSFV strains were clustered into two main phylogenetic groups. Group I was comprised of the reference strain Brescia, together with old American and European field isolates and a Brazilian vaccine strain. Group II included the reference strain Alfort (Tübingen) and recent European field strains. The Congenital Tremor strain formed a distinct lineage which, although being highly divergent, was more closely related to group I than to group II. In conclusion, the present phylogenetic grouping yielded very similar results as previous studies base on comparison of the E2 (gp55) region. The agreement of the phylogenetic analysis in the two distinct regions confirms the reliability of the genetic grouping of CSFV strains into two main genogroups.

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter. Cleavage at an SrfI restriction site introduced at the exact 3' end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription. This RNA proved to be infectious after transfection into porcine kidney cells. Infectivity was not increased after capping of the synthetic RNA. Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody. RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells. A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells. An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP. The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis.