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(concentration, μg/mL)Fluorescence intensity of (×103)a Estimated number of Fluo residues per one virion (×103)b Initial labeling solution Labeling solution after 1-2 h incubation with the virus Labeled virus after purification fluo-Ad-DOPE (25) 3.5 ± 1.0 8.0 ± 1.0 3.0 ± 0.1 2.8 ± 0.1 fluo-Ad-DOPE (50) 6.0 ± 1.0 12.0 ± 1.0 5.0 ± 0.1 6.5 ± 0.1 fluo-Ad-DOPE (100) 8.5 ± 1.0 18.0 ± 1.0 7.0 ± 0.1 12.0 ± 0.1 FITC (100) 1500.0 ± 1.0 1500.0 ± 1.0 15.0 ± 0.1 1.0 ± 0.1 a: The value was normalized for equal concentration of virus particles; b: Since the amount of protein in the sample was known, the mass of a single virion was taken as 1×10-12 mg (Ruigrok R, et al., 1984). Table 1. Fluorescent labelling efficiency of A/FPV/Rostock/34 (H7N1) influenza virus
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Influenza virus Label (concentration, μg/mL) % of NA activity compared to the respective unlabeled virusa A/Puerto Rico/8/34 (H1N1) fluo-Ad-DOPE (25) 31.3 ± 4.1* fluo-Ad-DOPE (50) 18.4 ± 3.6* fluo-Ad-DOPE (100) 23.1 ± 3.8* biot-CMG2 -DOPE (25) 41.2 ± 5.9* biot-CMG2 -DOPE (50) 53.1 ± 7.3* biot-CMG2 -DOPE (100) 48.8 ± 4.0* FITC (100) 53.1 ± 4.0* A/duck/France/46/82 (H1N1) fluo-Ad-DOPE (50) 96.4 ± 4.4 FITC (100) 72.3 ± 5.1* A/mallard/Pennsylvania/10218/84 (H5N2) fluo-Ad-DOPE (50) 97.5 ± 3.8 FITC (100) 95.2 ± 4.8 A/FPV/Rostock/34 (H7N1) fluo-Ad-DOPE (25) 92.1 ± 6.1 fluo-Ad-DOPE (50) 95.4 ± 5.0 fluo-Ad-DOPE (100) 98.3 ± 4.5 FITC (100) 43.5 ± 3.7* A/California/07/09 (H1N1) biot-CMG2 -DOPE (50) 92.7 ± 9.9 A/Perth/16/09 (H3N2) biot-CMG2 -DOPE (50) 96.3 ± 5.6 B/Brisbane/60/08 biot-CMG2 -DOPE (50) 94.1 ± 6.4 a Unlabeled viruses were incubated with PBS instead of fluo-Ad-DOPE, biot-CMG2-DOPE, or FITC at the same labeling conditions that were used for labeling with the respective dyes, and their NA activities were taken as 100%.
* P < 0.05 compared to the respective unlabeled virus.Table 2. NA activity of influenza viruses after labeling with fluo-Ad-DOPE, biot-CMG2-DOPE, or FITC
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Viruses Label
(concentration, μg/mL)Reciprocal HA titer Virus yield Plaque sizec log10 TCID50/mla log10 PFU/mLb A/California/07/09 fluo-Ad-DOPE (50) 640 7.9 ± 0.1 6.7 ± 0.2 0.5 ± 0.1 (H1N1) biot-CMG2 -DOPE (50) 320 7.7 ± 0.1 6.7 ± 0.4 0.6 ± 0.2 – 640 8.1 ± 0.3 6.6 ± 0.4 0.5 ± 0.2 A/Puerto Rico/8/34 fluo-Ad-DOPE (50) 1024 8.4 ± 0.1 8.8 ± 0.4 0.8 ± 0.1 (H1N1) biot-CMG2 -DOPE (50) 1024 8.1 ± 0.3 8.5 ± 0.3 0.9 ± 0.1 – 1024 8.0 ± 0.5 8.7 ± 0.4 0.8 ± 0.2 A/Perth/16/09 fluo-Ad-DOPE (50) 160 8.2 ± 0.2 7.8 ± 0.6 2.5 ± 0.4 (H3N2) biot-CMG2 -DOPE (50) 160 8.5 ± 0.1 8.1 ± 0.9 2.4 ± 0.5 – 160 8.3 ± 0.1 7.9 ± 0.5 2.8 ± 0.5 B/Brisbane/60/08 fluo-Ad-DOPE (50) 320 8.0 ± 0.4 6.8 ± 0.3 0.1 ± 0.1 biot-CMG2 -DOPE (50) 640 8.1 ± 0.2 6.5 ± 0.2 0.2 ± 0.1 – 640 7.8 ± 0.5 6.7 ± 0.2 0.2 ± 0.1 a: Values are the log10 TCID50/ml ± SD from three independent determinations. TCID50 values were determined in MDCK cells with 10-fold serial diluted viruses, incubated for 72h at 37℃ (or at 33℃ for influenza B viruses), and calculated by the Reed-Muench method (Reed LJ, et al., 1938); b: Values are the log10 PFU/mL ± SD from three independent determinations. PFU was determined in MDCK cells by plaque assay after 3days of incubation at 37℃ with 10-fold serial diluted viruses (or at 33℃ for influenza B viruses); c: Values are mean plaque diameter (mm) ± SD as measured by using the Finescale comparator. Table 3. Growth characteristics of human influenza viruses labeled with fluo-Ad-DOPE or biot-CMG2-DOPE in vitro
Figure 6 个
Table 3 个