A chicken liver cell line efficiently supports the replication of ALV-J possibly through its high level viral receptor and efficient protein expression system

Abstract

In this study, we identified a chicken liver cell line (LMH) which could strongly support the replication of ALV-J (Subgroup J of avian leukosis virus) with high viral titer. Notably, ALV-J was efficiently detected by ELISA in LMH cells 1 day before DF1 cells. In comparison with DF1 cells, LMH cells not only expressed higher levels of ALV-J receptor chNHE-1, but also possessed a more efficient protein expression system for foreign genes. Thus, LMH cells could be a novel tool to shorten the ALV-J eradication approach and accelerate studies on the pathogenesis and oncogenesis of ALV-J.

Figure 1

The viral replication kinetics of ALV-J in DF1 and LMH cells. A Viral growth curve of ALV-J. DF1 and LMH cells were infected with ALV-J GY03 at MOI 0.001 respectively, and the supernatant of the infected cells were collected at the indicated time points and titrated by TCID₅₀; B comparison of viral rescuing efficiency in DF1 and LMH cells. DF1 and LMH cells were transfected with 4 μg of ALV-J infectious clone respectively and the supernatant of the transfected cells were collected at the indicated time points and titrated by TCID₅₀.

Figure 2

Comparison of the ALV-J detection with ELISA between DF1 and LMH cells. DF1 and LMH cells were infected with ALV-J GY03 and GY07 at MOI 0.001 (A, C) or MOI 0.0001 (B, D) respectively, and the supernatant of the infected cells were collected at the indicated time points and the p27 antigen in the supernatant was detected by ELISA.

Figure 3

Comparison of clinical sample detection with ELISA between DF1 and LMH cells. DF1 and LMH cells were inoculated with homogenates of chicken liver and spleen samples, respectively, and the supernatant of the infected cells were collected at 6 dpi (A), 7 dpi (B) or 8 dpi (C). The p27 antigen in the supernatant was detected by ELISA.

Figure 4

Expression level of ALV-J receptor chNHE-1 and protein expression level for foreign genes in DF1 and LMH cells. A Transcription level of chNHE-1 in DF1 and LMH cells were detected with real-time PCR. B Western blot analysis for the expression of chNHE-1 in the DF1 and LMH cells. Lane 1, DF1 cells; lane 2, LMH cells. The gray ratio values of chNHE-1 to GAPDH are shown. C–E Western blot analysis for DF1 cells and LMH cells transfected with 1 μg pc-ALV-p27, pc-ALV-J-env and pc-eGFP respectively, at 12 h and 24 h. Lane 1 and 2, DF1 and LMH cells transfected with the plasmids indicated for 12 h, respectively; Lane 3 and 4, DF1 and LMH cells transfected with the indicated plasmids for 24 h, respectively.