Quantification of background expression of interferon beta in cell culture of Siberian bat (Myotis sibiricus)

Background. The study of the immune response of these mammals to viral infections is necessary to reveal the fundamental mechanisms of the circulation of zoonotic infections in nature. There is a hypothesis about the constantly “on” activity of the interferon pathway proteins, developed evolutionarily in bats to counteract viral infections. We assessed the expression of interferon beta as a marker of the innate immune system in kidney cells of the Siberian bat (Myotis sibiricus, Kastschenko, 1905) MdbK3-14.

The aim. Evaluation of the background level of interferon beta (IFN-β) gene expression in bat cells as a marker of the activity of the mammalian innate immune system.

Materials and methods. MdbK3-14 cells were grown in 24-well plates. Cell monolayers were detached with trypsin solution and total RNA was isolated. The concentration of mRNA of IFN-β gene transcripts and reference genes beta actin (ACTB) and succinate dehydrogenase subunit A (SDHA) was determined by one-step multiplex RT-qPCR and confirmed by RT-dPCR.

Results. Specific primers with a probe for detecting mRNA of the IFN-β gene in bat cells were designed. The detection of SDHA and IFN-β gene transcripts was stable both in RT-qPCR (CV = 0.5 % and CV = 0.2 %, respectively) and in RT-dPCR (CV = 0.8 % and CV = 1.4 %, respectively). In addition, stable detection of ACTB mRNA was achieved using RT-dPCR (CV = 0.8 %), but the average variability value for actin using RT-qPCR exceeded the permissible value (CV = 3.6 % with an acceptable CV ≤ 2 %). The results of quantitative determination in RT-qPCR and RT-dPCR correlated with each other. The expression levels of IFN-β in MdbK3-14 cells averaged 0.97 ± 0.15 relative units in RT-qPCR and 0.13 ± 0.05 relative units in RT-dPCR.

Conclusions. In the absence of immune stimulation, background expression of IFN-β occurs in the M. sibiricus kidney cell line.

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