Related Experiment Video
Updated: May 7, 2026
Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells
Published on: November 22, 2017
Heterogeneity of the MDCK cell line and its applicability for influenza virus research
Vladimir Y Lugovtsev1, Darya Melnyk, Jerry P Weir
1Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.
Insights
MDCK cell clones vary in their ability to support influenza virus replication. Type-1 cells efficiently replicate influenza A and B viruses, while Type-2 cells require trypsin for influenza A virus replication due to hemagglutinin activation differences.
Area of Science:
- Virology
- Cell Biology
Background:
- MDCK cells are widely used for influenza virus research.
- Heterogeneity within MDCK cell populations can impact experimental outcomes.
Purpose of the Study:
- To characterize single-cell clones derived from MDCK cells.
- To evaluate their suitability for influenza virus research, focusing on replication efficiency.
Main Methods:
- Isolation and characterization of MDCK single-cell clones.
- Morphological analysis using light microscopy.
- Glycan profiling using peanut agglutinin (PNA).
- Influenza virus infection assays and multicycle replication studies.
- Western blot analysis for hemagglutinin cleavage.
Main Results:
- Three distinct MDCK cell morphotypes (Type-1 and Type-2) were identified, differing in PNA-binding glycans.
- Type-1 clones supported multicycle replication of influenza A and B viruses without exogenous trypsin.
- Type-2 clones required trypsin for influenza A virus replication due to impaired hemagglutinin cleavage.
- Influenza B virus replication was less dependent on trypsin in Type-2 cells, though plaque size varied.
- All clones supported influenza virus infection and possessed both α2-3 and α2-6 sialylated glycans.
Conclusions:
- Original MDCK cell populations comprise diverse cell types with differential capacities for influenza virus replication.
- MDCK cell clone selection is critical for influenza A and B virus research, particularly concerning hemagglutinin activation.
- Understanding MDCK cell heterogeneity is essential for reproducible influenza virus studies.
Abstract:
Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA). All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1) and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2-3 and α2-6). The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed). HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to support replication of influenza A and B viruses.
