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.