A cell line resistant to avian leukosis virus subgroup B infection

Abstract

The expression of env proteins that bind to viral cell receptors on avian leukosis virus (ALV)-susceptible cells can block ALV infection. In this study, we constructed a cell line (DF-1/B) by expressing the ALV-B env protein in DF-1 cells. PCR, immune fluorescence assay, Western blot, and immune electron microscopy results showed that the env gene can be stably expressed in DF-1cells and the env protein could be detected on the DF-1 cell membrane. An antiviral experiment concluded that the DF-1/B cell line could be resistant to 1 × 104 TCID50 ALV-B virus infection, but had no inhibitory effect on other subgroup ALV. This means that the DF-1/B cell line is specifically resistant to ALV-B and can be used as a tool for ALV-B diagnosis.

Keywords: env protein; ALV-B; cell line.

Figure 1.

Zeocin selection of cell lines. (A) After zeocin selection, the transfected DF-1 cells formed a single cell colony within 10 to 15 D. The single cell colony appeared to increase in size over the following 6 to 10 D, and the differences in the size of the cell clones are shown on days 16 (B) and 20 (C). (D) The cells had nearly grown to a monolayer by approximately 21 D in culture. Magnification is ×400 for (A) and 200 × for (B–D).

Figure 2.

(A) PCR amplification of the env gene from DF-1/B cells. (M) DNA marker 3; (DF-1/B): Genomic DNA extracted from DF-1/B cells; (DF-1): Genomic DNA extracted from DF-1 cells. (B) Verification of the stability of the ALV-B env gene in DF-1/B cells during passages. (M) DNA marker3; Lanes 1–8: ALV-B env gene cell-culture passage levels 15, 25, 30, 35, 40, 45, 50, 60, respectively.

Figure 3.

(A) PCR amplification of the ALV-B env gene in DF-1/B cells. (M) DNA marker3; (1): RNA extracted from DF-1/B cells; (2): Genomic DNA extracted from DF-1/B cells; (3) RNA extracted from DF-1 cells. (B) Levels of ALV-B env gene transcription in DF-1/B cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of 2 independent experiments, both performed in triplicate.

Figure 4.

Detection of ALV-B env protein in DF-1/B cells by IFA. (A) No green fluorescence was observed in DF-1 cells, which served as negative controls. (B) Green fluorescence was observed in DF-1/B cells. Magnification is × 100 for (A) and (B).

Figure 5.

Western blot analysis of env protein expression in DF-1/B cells. (DF-1/B): DF-1/B cell lysate; (DF-1): DF-1 cell lysate was used as a negative control. ALV-B env protein in the cell lysates were detected with monoclonal antibodies for ALV-B at a dilution of 1:200. β-actin cell lysates was also detected using actin antibodies for control for equal loading. A IRDye 800-conjugated anti-mouse IgG (1:10,000; Rockland Immunochemicals, Limerick, PA) diluted in PBS was used as the secondary antibody.

Figure 6.

The result of antiviral experiment. Five different virus titers from 10¹ TCID₅₀ to 10⁵ TCID₅₀ per 0.1 ml of ALV-B were inoculated per well in 24-well cell culture plates containing 1 mL 1.7 × 10⁵cells/well DF-1/B cells in triplicate. (A) ALV-B (CD08) replication was inhibited in DF-1/B, but not DF-1 cells. (B) Replication of ALV-J (CHN06) was not inhibited in either DF-1/B or DF-1 cells. (C) Infection by clinically isolated unidentified subgroups ALV, one of them was blocked in DF-1/B, but not in DF-1cells. In A–C, viral p27 protein levels determined by ELISA were reported. Black lines mean S/P = 0.2.

Figure 7.

Electron microscopy image of DF-1/B cells that were pre-incubated with TBS (pH7.4) and subsequently incubated with 10 nm colloidal gold-labeled goat anti-mouse IgG. These samples served as negative controls. (A) TEM × 25,000, (B) TEM × 100,000. (C) Electron microscopy image of DF-1/B cells that were pre-incubated with goat anti-mouse EGFP antibodies and monoclonal antibodies for ALV-B individually and subsequently incubated with 10 nm colloidal gold-labeled goat anti-mouse IgG. TEM × 250,000. The arrows point to gold particles.