Establishment of MDCK/FX Cell for Efficient Replication of Influenza Viruses

Background: Replication of influenza virus to high titer is a prerequisite for successful cell-based vaccine production. Entry of virus into the cell depends on the cleavage of the hemagglutinin precursor (HA0) protein mediated by trypsin. Objectives: The aim of the present study was to apply a technique to establish MDCK/FX manipulated cell, which may provide a new platform for developing influenza vaccine based on the cell culture approaches. Methods: Chicken embryo FX expressed into the pCDNA3.1 vector was transfected into the MDCK cell line. The longevity of the generated cell and the viable cell density were evaluated for 17 passages prior to virus inoculation. Then, the ability of MDCK/FX cell for efficient replication of H9N2 influenza virus was evaluated by viral titration and quantitative RT-PCR. Results: RT-PCR data revealed that FX was stably expressed in the cell after the subsequent passages without any change in the rate of culture’s confluency. Growth kinetic of H9N2 virus analysis demonstrated that MDCK/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in MDCK cells supplemented with TPCK-trypsin. Quantification of influenza infectious particles in the cell culture revealed the equivalents viral RNA copies and viral titers. Conclusions: The results indicated a potential application for the MDCK/FX in influenza virus replication procedure and related studies.