Figure 1.
Microphotographs of the parental MDCK and cloned cell lines.
A.Original MDCK (ATCC-CCL-34, NBL-2; +7 passages). B. Type-1 cells (clone 4F2; +5 passages after cloning): gentle polymorphic cells with a visible nucleus and very fine almost indistinguishable intercellular space, cells often develop large cytoplasmic area. C. Type-2 cells (clone 3D7; +4 passages after cloning): polygonal cells with almost invisible nuclei; the intercellular space is well developed, and contributes to a specific a mosaic-like appearance of the monolayer. D. Formation of the numerous liquid-filled structures, “domes”, visible as rounded refracting blisters on the monolayer of the Type-3 cells (clone 3E11; +11 passages after cloning).
Figure 2.
Evaluation of the level of the surface expression of the PNA-specific carbohydrates.
The level of cell surface expression of PNA-specific glycans was evaluated by flow cytometry using FITC-labeled PNA. Each panel shows intensity of fluorescence (X axis) in relation to the normalized cell counts (Y axis) of cells treated by the fluorescent probe (green peak), in comparison with an unstained control cells (gray peak). Gates indicate the percentage of “All-Positive” cells, and “High-Positive” cell. The mean fluorescence is shown in the upper right corner of each panel and summarized in the table included in the right corner of the figure. Data from one representative experiment.
Figure 3.
Detection of α2–3 or α2–6 sialylated glycans on the cell surface.
Detection of the of influenza virus receptors on cell surface, the carbohydrates terminally sialylated via α2–3 or α2–6 glycosidic linkage, was performed by flow cytometry. Sialic acid moieties with α2–3 glycosidic linkage were detected by biotinylated lectin MAA from Maackia Amurensis (MAL-II) in combination with Streptavidin conjugated with R-Phycoerythrin (PE); glycans with α2–6 linkage were detected by FITC-labeled SNA (Sambucus Nigra (Elderberry) Bark Lectin). Sample preparation and procedure are described in Material and Methods. Quadrant Q1: MAA(α2–3)-positive cells; Quadrant Q2: MAA(α2–3)+SNA(α2–6)-positive (double-positive) cells; Quadrant Q3: SNA(α2–6)-positive cells; Quadrant Q4: unstained cells [control]. Data from one representative experiment.
Figure 4.
Infectivity titers of influenza virus strains in MDCK clones.
Infectivity titers (TCID50, log10/0.1 ml) of reference viruses were determined using different cell clones and parental MDCK. Each panel represents infectivity titers for the indicated virus strain. Blue bars represent Type-1 cells; red bars – Type-2 cells; green bars – Type-3 cells. Values represent Mean ± SE (data from two independent experiments). Asterisk (*) indicates a significant difference (P<0.05) in comparison with the values in the parental MDCK cells (black bars).
Figure 5.
Plaque size of virus variants with α2–3 or α2–6 binding preferences in MDCK clones.
Variants of influenza B virus strain B/Victoria/504/2000 with binding preferences for sialylated glycans with either α2–3 (orange bars) or α2–6 (green bars) glycosidic linkage were used as probes to investigate the cell clones for their support of replication of viruses with different receptor-binding properties. Bars represent the diameter (mm) of virus plaques in the corresponding cell clones 96 hours post infection, at 33°C. Values represent the Mean ± SE from two independent experiments. Asterisk (*) indicates a significant difference (P<0.01) in comparison with the corresponding values in the parental MDCK cells.
Figure 6.
Plaque size of influenza A viruses in MDCK clones with and without trypsin.
Panel A (upper panel): A/Brisbane/59/2007 IVR-148 (H1N1 seasonal). Panel B (middle panel): A/Uruguay/716/2007 X-175C (H3N2 seasonal). Panel C (lower panel): rg-A/Vietnam/1203/2004 (H5N1, low-pathogenic). Bars represent the diameter (mm) of virus plaques in parental MDCK and the corresponding cell clones 96 hours post infection, at 33°C. Blue bars represent plaque size without trypsin; pink bars represent plaque size in the presence of the trypsin in the overlaying agar media. Values represent the Mean ± SE from two independent experiments. Asterisk (*) indicates a significant difference (P<0.01) in comparison with the corresponding values in the parental MDCK cells.
Table 1.
Phenotype of MDCK clones and their characterization as substrates for influenza A and B viruses.
Figure 7.
Growth kinetics of influenza viruses in MDCK clones (without exogenous trypsin).
Growth kinetics of influenza A and B viruses in MDCK clones were determined by infectivity titration (TCID50, log10/0.1 ml) of the cell culture media samples collected at different time-points post infection. Culture of the corresponding MDCK clones (confluent cell monolayer) were infected at MOI 0.000001, and incubated at 33°C until complete destruction of the monolayer due to CPE. Panel A (upper panel): A/Brisbane/59/2007 IVR-148 (H1N1, seasonal). Panel B (lower panel): B/Hubei-Wujiagang/158/2009.
Figure 8.
The HA1/HA2 cleavage profile of the HA of A/Brisbane/59/2007 IVR-148 (H1N1, seasonal) and B/Hubei-Wujiagang/158/2009 grown in the corresponding cell clones (without addition of trypsin to the culture media) was evaluated by SDS-PAGE (reducing conditions) followed by Western blot with rabbit polyclonal antibodies specific to HA1 or HA2 portions of the viral hemagglutinin. Panel A: Western blot of H1N1 with anti-HA1 antibodies. Panel B: Western blot of H1N1 with anti-HA2 antibodies. Panel C: Western blot of B/Hubei with anti-HA2 antibodies. The protein bands corresponding to the non-cleaved HA0 migrate at the level of 80 kDa; bands representing HA1 and HA2 molecules migrate at the level of ∼58 kDa and 28–30 kDa, respectively.