A recombinant classical swine fever virus stably expresses a marker gene

Abstract

The gene coding for bacterial chloramphenicol acetyltransferase (CAT) was inserted in frame into the viral Npro gene of the full-length cDNA clone pA187-1 of the classical swine fever virus (CSFV) strain Alfort/187. RNA transcribed in vitro from the resulting plasmid was transfected into SK-6 porcine kidney cells. Infectious progeny virus vA187-CAT recovered from transfected cells had growth characteristics indistinguishable from those of parental virus vA187-1. In cells infected with vA187-CAT the predicted fusion protein, CAT-Npro, was detected, and it retained the enzymatic activities of both CAT and Npro. The CAT gene remained stably inserted in the viral genome after 10 virus passages. Thus, marker virus vA187-CAT represents a useful tool for quantitative analysis of viral replication and gene expression.

FIG. 1

Schematic representation of the genome of vA187-CAT. The authentic genes of the CSFV genome are shown as open boxes, and the CAT gene is shown as a shaded box. The lower part of the figure shows the insertion site and flanking sequences of the CAT gene in detail. The nucleotide numbers correspond to the numbering of the CSFV Alfort/187 genome and the CAT gene sequence.

FIG. 2

Comparative growth kinetics of vA187-1 and vA187-CAT. A total of 2 × 10⁶ SK-6 cells were infected at the indicated MOIs. (A) Virus titers in the cleared supernatant after freezing and thawing of infected cells. The values indicated at time zero represent the titers of the respective inoculates. (B) Expression of CAT and E2 proteins as measured by CAT and E2 ELISAs. The cells were lysed in 1 ml of hypotonic buffer and tested in duplicate. The y axis is logarithmic for better comparison with the corresponding virus titers shown in panel A. The detection limit of the CAT ELISA (0.1 ng/ml) is shown as a dashed line.

FIG. 3

Virus titers (A) and CAT (B) and E2 (C) expression during passage of vA187-CAT. For each passage of vA187-CAT (virus passage) and of cells persistently infected with vA187-CAT (cell passage), the cell culture supernatant was titrated and the hypotonic cell lysate was analyzed by CAT and E2 ELISAs. The y axis is logarithmic in panels B and C for better comparison with the corresponding virus titers shown in panel A.

FIG. 4

RT-PCR of viral RNA after 10 passages of vA187-CAT. Viral RNA was extracted from cell culture supernatants and RT was carried out with primer HR3. (A) PCR with primers CAT-SmaL and CAT-SmaR resulting in a CAT gene-specific product of 672 bp; (B) PCR with primers Pest 1 and N^pro-R1 flanking the site of insertion of the marker gene. The lengths of the specific products are 1,231 and 568 bp for vA187-CAT and vA187-1, respectively. Lanes 1, vA187-CAT, P2 stock virus; lanes 2, vA187-CAT, virus passage 10; lanes 3, vA187-CAT, persistently infected cells, passage 10; lanes 4, vA187-1; lanes 5, mock-infected SK-6 cells; lanes 6, pA187-CAT DNA; lanes 7, pA187-1 DNA.

FIG. 5

Western blot analysis of proteins extracted from SK-6 cells infected with vA187-CAT. Specific proteins in hypotonic cell lysates were detected with either anti-CAT antibodies (A) or anti-N^pro rabbit serum (B). Molecular weight markers (in thousands) are shown on the left. Lanes 1, CAT protein; lanes 2 to 6, lysates of SK-6 cells infected with vA187-1 (lanes 2), vA187-CAT, P2 stock virus (lanes 3), vA187-CAT, virus passage 10 (lanes 4), vA187-CAT, persistently infected cells, passage 10 (lanes 5), and lysate of mock-infected SK-6 cells (lanes 6). Arrows indicate the CAT-N^pro fusion (f), CAT (c), and N^pro (n).