Immortalization of primary lamb kidney cells towards a continuous cell line for propagation of foot and mouth disease virus

Abstract

Foot and mouth disease (FMD) is a highly contagious disease of great economic importance. The disease is caused by foot and mouth disease virus (FMDV). Diagnosis of FMD is done through tests such as virus isolation (VI), enzyme linked immunosorbent assay (ELISA), virus neutralisation tests (VNT) and polymerase chain reaction (PCR) platforms. Tests such as VI and VNT require the use of cells that are susceptible to infection with FMDV. Cells that can be used for FMDV culture include primary cells and cell lines developed for diagnostic applications. Cells such as primary lamb kidney cells have been shown to be very sensitive to infection with FMDV, but the cells are short lived, laborious to prepare, and give variable results due to the use of different batches. Notwithstanding, the sensitivity of primary lamb kidney cells to infection by FMDV can be taken advantage of and developed into a cell line. Primary lamb kidneys cells’ lifespan can be increased through exogenous introduction of telomerase reverse transcriptase (TERT). In this study, transduction was conducted on primary lamb kidney cells at 80% confluency using recombinant lentivirus to over express TERT. The lentivirus construct included puromycin antibiotic resistance, thus, the cells were subjected to antibiotic selection, and surviving cells were expanded further. The over expression of TERT was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using a comparative ΔΔct method. The differences in the cycle thresholds for detection of TERT between primary cells and transduced cells showed increased expression of TERT in transduced cells. This increased expression however waned with increased cell passages. Encouragingly, the transduced cells were able to maintain similar morphology to primary cells over successive passages. The growth rate results also showed that primary cells’ growth regressed with progressive passaging as that of transduced cells increased. Analysis of TCID50/mL results for SAT and O serotypes of FMDV demonstrated no significant differences between primary and transduced cells. Virus isolation of SAT and O serotypes of FMDV gave similar time intervals to reach 100% cytopathic effect. These results demonstrate promise that the transduced cells could be used as a possible replacement for primary lamb kidney cells. Future work however must determine the extent of passaging that can be undertaken on transduced cells before there are notable changes to properties of the transduced cells. The transduced cells would also need to be validated using a broader range of virus serotypes and strain.