Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvβ6 Cell Line

The LFBK-αvβ6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvβ6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvβ6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 μM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvβ6 cultures (renamed WRL-LFBK-αvβ6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvβ6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.


INTRODUCTION
LFBK-α V β 6 cells are a porcine kidney cell line that was transduced to express the bovine α V β 6 integrin receptor in order to provide a highly sensitive, continuous cell line for the propagation of foot-and-mouth disease virus (FMDV) [(1); correction (2)]. LaRocco et al. (1) demonstrated that over-expression of α V β 6 integrin, a known cellular receptor for FMDV, enhanced susceptibility to a range of FMDVs relative to other continuous cell lines (i.e., BHK, IB-RS-2, MVPK, LFBK, and LK), whilst maintaining sensitivity to vesicular viruses that are clinically indistinguishable from FMD. Subsequently, Fukai et al. (3) demonstrated that LFBK-α V β 6 cells have similar susceptibility to non-epithelium FMDV clinical samples as ZZ-R 127 cells.
We previously validated the use of LFBK-α V β 6 cells for diagnostic purposes within the World Reference Laboratory for FMD (WRLFMD) (4). Our results demonstrated that LFBK-α V β 6 cells had similar analytical sensitivity to FMDV epithelium suspensions as primary bovine thyroid cells (BTY), which had previously been identified as the most sensitive cell system for FMDV isolation (5). Additionally, the LFBK-α V β 6 cells had enhanced susceptibility to porcine-adapted FMDVs compared to IB-RS-2 cells (4). Unfortunately, the LFBK-α V β 6 cell line is persistently infected with a non-cytopathic bovine diarrhea virus (BVDV; family Flaviviridae, genus Pestivirus) (Rodriguez, personal communication), which complicates the use of these cells due to concerns about crosscontamination of other cell lines and downstream applications, including vaccine production and preparation of challenge viruses for in vivo studies.
The presence of a non-cytopathic BVDV in cells is not a novel occurrence. Multiple studies have documented noncytopathic BVDV strains present in fetal bovine serum (6)(7)(8)(9)(10), which likely lead to the subsequent contamination of numerous cell lines, including many non-bovine cultures (11)(12)(13). Given the negative impact of BVDV in the cattle industry and as a laboratory contaminant, numerous studies have evaluated chemical compounds on their ability to inactivate or inhibit BVDV [reviewed in (14)]. One aromatic cationic compound, DB772, has been shown to prevent and eliminate non-cytopathic BVDV from persistently infected fetal fibroblast cells without causing cytotoxicity (15,16), as well as having antiviral properties in vivo (17).
The aim of this study was to eradicate BVDV from LFBKα V β 6 cells using DB772, following similar procedures to those described in Givens et al. (16). Unclarified cell suspensions collected during and after DB772 treatment were tested for the presence of BVDV genome using a pestivirus real-time RT-PCR assay (rRT-PCR). Cell line sensitivity after treatment with DB772 was assessed by performing comparative titrations with a range of vesicular viruses alongside the original LFBK-α v β 6 cell line.

Cells and Treatment With DB772
The LFBK-α v β 6 cell line [(1), correction (2)], supplied by the Plum Island Animal Disease Center (New York, USA), was maintained as previously described (4). Briefly, LFBK-α v β 6 cells (passage 19) were grown in a 175 cm 2 cell filter-cap tissue culture flask (Cellstar, Greiner Bio-One) at 37 • C in the presence of 5% CO 2 until the monolayer reached 90-100% confluency. The cell monolayer was washed with 15 mL sterile phosphate buffer saline (PBS; Severn Biotech), followed by 15 mL 0.25% trypsin-EDTA (Gibco). After removing the trypsin, the flask was incubated at 37 • C until the cells dissociated from the flask surface. The cells were then resuspended in 25 mL Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% bovine serum (BS; certified BVDV negative, Gibco) (referred to DMEM + BS). Two 50 mL Falcon tubes each received 1 mL of the cell suspension and were centrifuged at 340 g for 5 min at 4 • C. The pelleted cells were resuspended in 5 mL of DMEM + BS (untreated control) or 4 µM DB772 DMEM + BS, transferred to 25 cm 2 filter-cap tissue culture flasks (TPP Techno Plastic Products) and incubated at 37 • C in the presence of 5% CO 2 . After 24 h, the media was removed from the flasks and replaced with the corresponding 5 mL DMEM + BS with or without DB772. The cells were incubated at 37 • C in the presence of 5% CO 2 until the monolayers reached 90-100% confluency.
Subsequent passages were performed following the procedure above, including using 1 mL of cell suspensions (split ratio of 1:4), but with the other volumes adjusted for the use of 25 cm 2 flasks (i.e., 3 mL sterile PBS, 3 mL 0.25% trypsin-EDTA, resuspension of cells in 4 mL DMEM + BS, and 5 mL DMEM + BS with or without DB772). At each passage, 125 µL of the cell suspension was added to 325 µL of MagMAX lysis buffer (ratio 5:13; Applied Biosystems, Thermo Fisher Scientific) and stored at −80 • C until testing. Treatment with DB772 continued for 9 passages (passages 20-28), after which the cells were maintained in DMEM + BS, identical to the untreated cells. To differentiate the two cultures, the LFBK-α v β 6 cells treated with 4 µM DB772 were renamed WRL-LFBK-α v β 6 .

BVDV Detection
RNA was purified from unclarified cell suspensions using the MagMAX-96 viral RNA isolation kit (Applied Biosystems, Thermo Fisher Scientific) on a Kingfisher Flex extraction robot (Thermo Fisher Scientific) using an automated protocol. All extractions were performed in triplicate, including a BVDV genome positive and a negative control per extraction plate. The positive control was prepared from LFBK-α v β 6 cells and generated a value of ∼23 C T , and the negative control was a negative pig epithelium suspension (i.e., control regularly used with routine vesicular disease diagnostic testing and which contains porcine genomic material).
Purified RNA was tested by rRT-PCR using the EXPRESS One-Step Superscript qRT-PCR kit (Invitrogen, Thermo Fisher Scientific). Each well of the rRT-PCR consisted of 5 µL of RNA template and 15 µL of master mix [10 µL of EXPRESS mix, 1 µL each of forward and reverse primer (20 µM), 0.5 µL of probe (15 µM), 0.5 µL ROX reference dye (diluted 1-in-10 with nuclease free water) and 2 µL Taq polymerase]. Primers and probes targeting the conserved 5 ′ non translated region of the pestivirus genome (19) were used, as recommended by the OIE Manual (20) for the detection of BVDV. The following cycling conditions were used on a 7,500 Fast Real-time PCR instrument using the fast setting (Applied Biosystems, Thermo Fisher Scientific): 50 • C for 15 min, 95 • C for 20 s, then 50 cycles of 95 • C for 3 s and 60 • C for 30 s. Samples with C T a value ≤40 were considered positive (20). using the Spearman-Karber method and expressed as Log 10 TCID 50 /mL. Three independent titrations were performed per virus and average viral titers were compared between cell lines using t-tests (GraphPad Prism 9.1.0).

DISCUSSION
In this study, the aromatic cationic compound DB772 was tested at 4 µM to eliminate the non-cytopathic BVDV infection in the LFBK-α v β 6 cell line. After three passages in the presence of DB772, BVDV was undetected and remained undetected throughout subsequent cell passages, even after treatment with DB772 was stopped (Figure 1). The analytical sensitivity of the WRL-LFBK-α v β 6 culture was comparable to the original BVDVinfected LFBK-α v β 6 cell line when titrations of FMDV, SVDV, VSV, VESV, and SVV were tested ( Table 1). These data support the use of WRL-LFBK-α v β 6 for FMD and vesicular disease diagnostics, with the findings in agreement to those previously reported in Gray et al. (4). It is possible that fewer than nine passages with DB772 could have been sufficient to eliminate BVDV from LFBKα v β 6 cells (i.e., stopping after three passages); however, rRT-PCR testing was not conducted immediately after each passage to confirm the levels of BVDV genome detection. Nonetheless, the presence of DB772 did not appear to have an immediate or long-term effect on the cells (i.e., no cytotoxicity and no difference in viral sensitivity). Cell growth was slower in the presence of DB772, but rebounded after subsequent passaging in DMEM + BS.
These results support those of Givens et al. (16), demonstrating that DB772 can be used to eradicate BVDV from persistently infected cells, and highlights the potential use of DB772 for other high value cell lines with non-cytopathic BVDV. Treatment with DB772 and the subsequent elimination of BVDV from LFBK-α v β 6 cells did not alter the ability of these cells to support the growth of FMDV and other vesicular viruses. The BVDV-free cell culture, WRL-LFBK-α v β 6 , can be handled using standard precautions associated with cell culture practices, now that the risk of cross-contaminating other cell lines and/or reagents with BVDV is removed. The elimination of BVDV from these cells is also an advantage for preparing challenge strains for in vivo studies and vaccine preparations.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
VM conceived the study. VM and AG developed the methodology. AG and EH conducted the experiments with supervision from BW and VM. AG analyzed these data. BW and VM wrote the manuscript. DK obtained funding. All authors read and approved the manuscript content.