In vitro adaptation and genome analysis of a sub-subgenotype 2.1c isolate of classical swine fever virus.

Classical swine fever (CSF) still causes substantial economic losses in the pig industry in China. This study reports the isolation and characterization of a field CSF virus named GD53/2011 from pig kidney tissue collected during a CSF outbreak in Guangdong province, China. Phylogenetic analysis based on the full-length E2 gene sequence revealed that this isolate belongs to CSFV sub-subgenotype 2.1c. To further understand the replication characteristics, GD53/2011 was subsequently adapted in PK-15 cells, and its full-length genome was sequenced. After adaptation in PK-15 cells, the titer of GD53/2011 was significantly increased from 10(3.39) TCID50/ml at passage 6 (F6) to 10(8.50) TCID50/ml at passage 46 (F46) with the peak titer obtained at 48 h post-inoculation. Sequence comparison revealed that the E(rns) gene at passages 6, 15, and 25 of GD53/2011 was identical to that in the original tissue, but one amino acid substitution (S476R) was detected at passages 35 and 46. Furthermore, E2 gene sequences at passages 6, 15, 25, 35, and 46 was found identical to that in the original tissue, indicating that the E2 gene was stable during CSF virus adaptation in PK-15 cells. Full-length protein sequence comparison of GD53/2011 with other 2.1 sub-subgenotype isolates showed that Core and NS5A, rather than E2, are more genetically variable. Taken together, a field CSFV strain GD53/2011 was isolated, fully sequenced, and adapted to high growth titer in PK-15 cells, which might be suitable for future studies on CSFV infection, replication, and vaccine development.