This study investigated replacing primary kidney cells with cell lines for virus isolation assays for foot-and-mouth disease virus (FMDV). In test 1, titers in lamb kidney cells, pig kidney cells, and ZZ-R cells were almost equal, while lower titers were found in BHK-21 cells. In test 2, the highest titers were detected in LFBK cells. The study concluded that IBRS-2 and ZZ-R cells were as sensitive as primary cells and could replace them. LFBK cells were found to be more sensitive than primary cells but current LFBK cells have a pestivirus contamination limiting their use. LFBK cells without pestivirus would be the preferred

1. Comparison of use of primary cells and cell
lines for virus isolation assays for FMDV
P.L. Eblé, K. Weerdmeester, A. Dekker. Wageningen Bioveterinary Research, Houtribweg 39, 8221RA Lelystad, the Netherlands.
Background
For virus isolation (VI) of FMDV primary kidney cells (ovine (lamb,
LK-2) or porcine (PK-2)) are used at WBVR. Stocks of these cells
are prepared from kidneys obtained from 6-8 week old lambs or
piglets and stored in liquid nitrogen until use. We mainly perform
VI for research purposes (e.g. materials of animal experiments,
testing of disinfectants) and then the FMD strains that we use are
known beforehand. Further, we use VI to confirm positive RT-PCR
results of suspect field cases (‘unknown’ FMD strains).
Results
In test 1, titres in LK-2, PK-2 and ZZ-R cells are almost equal
whereas lower titres in BHK-21 cells were detected. There is
however variation between strains. In test 2, the highest titres
are detected on LFBK cells.
Objective
We investigated if replacement of the primary cells by cell lines
in our VI assay is feasible. This would reduce the number of
experimental animals, is less laborious and might be better
reproducible
References
• Brehm, K.E., Ferris, N.P., Lenk, M., Riebe, R., and Haas, B. (2009). Highly sensitive fetal goat
tongue cell line for detection and isolation of foot-and-mouth disease virus. Journal of Clinical
Microbiology 47(10), 3156-3160.
• LaRocco, M., Krug, P.W., Kramer, E., Ahmed, Z., Pacheco, J.M., Duque, H., et al. (2013). A
Continuous Bovine Kidney Cell Line Constitutively Expressing Bovine αVβ6 Integrin Has
Increased Susceptibility to Foot-and-Mouth Disease Virus. Journal of Clinical Microbiology
51(6), 1714-1720. doi: 10.1128/jcm.03370-12.
This study was funded by the Ministry of Agriculture, Nature and Food Quality, the Netherlands
project WOT-01-003-11
Wageningen Bioveterinary Research
P.O. Box 65, 8200 AB Lelystad, the Netherlands
Contact: Phaedra.Eble@wur.nl
T + 31 (0)320 238680, M +31 (0)6 53924754
Materials and Methods
Titrations of FMD viruses were performed in plaque count assays.
Ten-fold dilution series of the samples (200 µl, tested in
duplicate) were allowed to adsorb for 1h on monolayers of cells in
a six-well tissue culture plate and maintenance medium
containing 1% methylcellulose was added. After 2 days of
incubation at 370C in a humified atmosphere containing 5% CO2
the plates were washed in tap water with citric acid. Monolayers
were rinsed with tap water and stained with amido-black. Plaques
were counted macroscopically. Virus titres are expressed as log10
plaque forming units (PFU) per ml.
In test 1 we used cell adapted (VNT) viruses (to mimic ‘known’
viruses) and compared the sensitivity of the primary cells with
that of two cell lines (BHK-21, ZZ-R). In test 2 we used cattle
challenge viruses (to mimic ‘unknown’ viruses) and compared the
sensitivity of primary lamb kidney cells with that of cell lines
IBRS-2 and LFBKαVβ6 (LFBK).
Table 1: Results cell adapted viruses (log10 PFU/ml)
Figure 1: Preparation of primary cells and a plaque count assay
Conclusion / Discussion
IBRS-2 and ZZ-R cells are as sensitive as our primary cells and
could replace them. LFBK cells are more sensitive as compared to
our primary cells. Since the ZZ-R cells are difficult to handle (i.e.
to produce larger stocks), it would be difficult to replace our
primary cells with ZZ-R cells in case we need larger quantities.
LFBK cells would be the best option for replacement of the
primary cells. However, the current LFBK cells have a pestivirus
contamination, which limits their use. LFBK cells without
pestivirus contamination would be the preferred choice for
replacement of our primary cells.
Table 2: Results cattle challenge viruses (log10 PFU/ml)