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Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines
1Department of Bacteriology, School of Medicine, Hiroshima University, Japan.
Abstract:
Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(-) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(-) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA0) to HA1 and HA2, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(-) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs.
Insights
MDCK(+) cells support influenza A and B virus replication without trypsin, unlike MDCK(-) cells. This difference is due to endogenous proteases activating hemagglutinin, aiding influenza virus isolation and drug screening.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Influenza virus replication requires hemagglutinin (HA) cleavage by proteases.
- Trypsin is commonly used to activate HA for influenza virus propagation in cell culture.
- Endogenous proteases in cell lines can potentially support influenza virus replication.
Purpose of the Study:
- To investigate the role of endogenous proteases in influenza A and B virus replication in two MDCK cell lines.
- To compare the trypsin-dependency of influenza virus replication in MDCK(-) and MDCK(+) cells.
- To characterize the properties of the endogenous protease(s) involved.
Main Methods:
- Multi-cycle replication and plaque formation assays of influenza A and B viruses.
- Assessment of hemagglutinin (HA) cleavage in MDCK(-) and MDCK(+) cells.
- Determination of trypsin-dependency for viral replication.
Main Results:
- MDCK(+) cells supported multi-cycle replication and plaque formation of influenza A and B viruses without exogenous trypsin.
- MDCK(-) cells required trypsin for influenza A virus replication but not for influenza B virus.
- Influenza virus replication correlated with HA0 cleavage to HA1 and HA2, indicating endogenous protease activity.
- The protease in MDCK(+) cells activates HA for both influenza A and B, while MDCK(-) cells' protease activates only influenza B HA.
- The MDCK(+) cell protease is suggested to be a cell surface-associated serine protease.
Conclusions:
- MDCK(+) cells possess endogenous proteases capable of activating HA for both influenza A and B viruses, enabling trypsin-independent replication.
- MDCK(-) cells have endogenous proteases that activate HA only for influenza B viruses, requiring trypsin for influenza A.
- The MDCK(+) cell line is a valuable tool for isolating influenza viruses from clinical samples and for screening anti-influenza drugs targeting protease activity.