Novel cell line derived from natural host Apodemus Peninsulae for in-vitro studies of tick-borne encephalitis virus

Abstract

Purpose: The purpose of the research was to develop a natural host-derived cell line for the study of the host specificity of tick-borne encephaitis virus and evalute the reproduction of virus in this cell line. Methods & Materials: The cell line was established from trypsinized homogenates of kidneys of female Apodemus peninsulae (Korean field mouse). Cell line was characterized by cariotyping and by nucleotide sequence analysis of the cytochrome B gene and the D-loop region of mitochondrial genome. Cells were infected with the tick-borne encephalitis virus (TBEV) isolate 92 M belonging to Siberian subtype. Tissue cultures were sampled at 0, 4, 8, 12, 16, 20 and 24 hours (hpi) as well as after 4 and 7 days post infection (dpi). TBEV reproduction dymamics was evaluated by plaque titration of supernatant medium from infected cells. Replication of intracellular RNA was measured by quantitative PCR. Production of TBEV antigen was evaluated using ELISA test. Porcine embryo kidney cell line SPEV was used in parallel as reference. All experiments were performed in triplicates. Results: The adhesive diploid cell line was obtained by serial passages of primary culture and designated as ApnK. Sequencing confirmed the species origin of the cells. This cell line was used for experiments at passage 30. The growth of TBEV infectivity started at 12 hpi and concentration of infectious virus gradually increased up to 4 dpi. Afterwards it stabilized with titres of 7 lg PFU/ml. The synthesis of intracellular genomic RNA was firstly confirmed at 8 hpi and the dynamics was similar to the infectivity growth reaching the plateau at 4 dpi with concentration of 8 lg copies/mkl. TBEV antigen was detected in cell supernatants at 20 hpi and the dynamics corresponded to those of infectivity growth and RNA replication. In all experiments the reproduction of TBEV in ApnK cells was slowly than in SPEV cells up to 4 dpi when they equilibrated. Conclusion: The stabile cell line of natural host of TBEV, A. peninsulae, was established. This cell line susceptible for TBEV infection, however the reproduction of the virus is restricted in comparison to convenient in vitro model, porcine embryo kidney cell line SPEV