Detection and analysis of bovine foamy virus infection by an indicator cell line

Abstract

Aim: To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. Methods: BFV infection was induced by the co‐culture method or the transient transfection of the infectious BFV plasmid [pCMV (cytomegalovirus) ‐ BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein‐positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. Results: The titer of BFV in fetal bovine lung cells was 4‐5‐folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co‐culture method, and in the meantime was significantly higher than that produced by the infectious clone pCMV‐BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. Conclusions: BICL can be used for the titration of the BFV viral infection in non‐cytopathic condition. In addition, we provide important evidence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

“…To sensitively and conveniently monitor BFV infectivity, a new bovine cell-based reporter cell line was constructed. Similar to the BHK-21-based BICL BFV titration cells [ 37 ], bovine MDBK reporter cells were constructed. Infection of these cells with BFV is associated with BFV Tas/Bel1 transactivator expression, triggering both viral and Gfp expression, allowing easy and sensitive detection of BFV infection.…”

“…The baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection. The BFV indicator cell line (BICL) and MDBK indicator cell line (MICL) for BFV were derived from BHK and MDBK cells, respectively, and contained a green fluorescent protein (Gfp) expression cassette under the control of the BFV long-terminal repeat (LTR) promoter [ 37 ]. Both were cultured in DMEM supplemented with 10% FHS, 1% penicillin-streptomycin and 200 µg/mL G418 and used for titration of wild-type (wt) BFV and selected variants.…”

The Influence of Envelope C-Terminus Amino Acid Composition on the Ratio of Cell-Free to Cell-Cell Transmission for Bovine Foamy Virus

“…BHK-21, Cf2Th, HEK293T, BFVL (BHK21-derived indicator cells containing a luciferase gene under the control of the BFV LTR) [30], and BICL (BHK21-derived indicator cells containing an enhanced green fluorescent protein under the control of the BFV LTR) cells [31,32] were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher, Waltham, MA, USA) containing 10% fetal bovine serum (GE Healthcare, Cincinnati, OH, USA) and 1% penicillin-streptomycin (Thermo Fisher, Waltham, MA, USA) at 37 °C in a 5% CO 2 atmosphere. BFV3026 was stored in our lab and cultured with Cf2Th and BICL cells.…”