Figure 6个  Table 3
    • Label
      (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

    • 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

    • 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