DOI: 10.1128/JCM.41.2.742-750.2003
Article Figures & Data
Figures
- FIG. 1.
Detection of NI-resistant influenza viruses in a mixture of wild-type and resistant viruses by the CL assay and FA-2. Wild-type and mutant viruses were mixed in the following ratios based on enzyme activity: 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100. The results from one representative assay are displayed for each pair of viruses: R292K (A/H3N2), H274Y (A/H1N1), E119V (A/H3N2), R152K (B), and wild type (wt).
Tables
- TABLE 1.
Summary of three influenza virus NI assaysa
Assay format and condition Characteristic of: CL assay FA-1 FA-2 Virus titration Yes Yes No Volume of virus and buffer (μl) 50 20 50 Substrate 5 μl of NA-STAR (100 μM) 30 μl of MUNANA (100 μM) NA Titration conditions 15 min, 37°C, shaking 1 h, 37°C NA Inhibition assay Signal/noise ratio 40:1 2:1 NA Volume of virus (μl) 40 10 50 μl (1/10 dilution) Volume of inhibitor (μl) 10 10 50 Inhibitor concn (nM) 0.028-550 0.0038-1,000 0.01-10,000 Inhibitor preincubation 30 min, RT 30 min, 37°C 45 min, RT Substrate 5 μl of NA-STAR (100 μM) 30 μl of MUNANA (100 μM) 50 μl of MUNANA (200 μM) Incubation 15 min, 37°C shaking 1 h, 37°C 2 h, 37°C Stop solution 55 μl of Sapphire II enhancer 150 μl of 0.14 M NaOH in 83% ethanol 100 μl of 0.14 M NaoH in 83% ethanol Substrate half-life 5 min Hours Hours Assay duration 1 h 2 h 30 min 2 h 45 min Instrumentation NORTHSTAR Luminometer FluoroCount fluorometer FluoroCount fluorometer ↵ a All assays were done with 96-well microtiter plates. RT, room temperature; NA, not applicable.
- TABLE 2.
Zanamivir IC50 values for each clinical isolate in three NI assays
Influenza virus type Isolate IC50 (nM) in the following assay: CL assay (n = 12) FA-1 (n = 12) FA-2 (n = 6) Mean (SD) CV Rangea (fold change) Mean (SD) CV Rangea (fold change) Mean (SD) CV Rangea (fold change) B 97012351 1.00 (0.14) 13.90 0.76-1.26 (0.7) 6.77 (10.14) 149.89 0.51-26.40 (50.8) 4.66 (3.14) 67.37 0.69-7.67 (10.1) 97012942 1.07 (0.17) 15.88 0.81-1.35 (0.7) 5.28 (8.84) 167.42 0.65-27.70 (41.6) 5.19 (3.65) 70.26 0.53-8.22 (14.5) 97013118 1.40 (0.37) 26.58 1.04-2.51 (1.4) 4.49 (8.34) 185.89 0.76-29.80 (38.2) 5.35 (3.70) 69.23 0.70-9.21 (12.1) 97013406 1.03 (0.19) 18.48 0.73-1.29 (0.8) 5.44 (8.40) 154.55 0.81-23.70 (28.3) 2.89 (1.75) 60.55 0.39-5.25 (12.5) 98010075 1.11 (0.13) 12.11 0.91-1.34 (0.5) 7.72 (10.54) 136.45 0.56-30.00 (52.6) 4.83 (3.22) 66.73 0.67-7.59 (10.3) A/H1N1 98010152 0.89 (1.36) 151.87 0.27-4.94 (17.3) 1.86 (0.86) 45.98 0.76-3.20 (3.2) 1.21 (0.63) 51.67 0.21-1.92 (8.1) 98013212 0.27 (0.04) 15.24 0.22-0.34 (0.5) 1.32 (0.74) 56.01 0.39-2.49 (5.4) 0.50 (0.27) 53.46 0.16-0.74 (3.6) 98014203 0.26 (0.03) 12.40 0.19-0.30 (0.6) 1.91 (1.19) 62.32 0.48-3.84 (7.0) 1.40 (1.11) 79.26 0.19-2.86 (14.1) 98026506 0.26 (0.07) 25.40 0.17-0.41 (1.6) 2.53 (1.64) 64.94 0.60-5.74 (8.6) 0.89 (0.48) 54.45 0.13-1.35 (9.4) 98027803 0.45 (0.68) 150.39 0.22-2.61 (10.9) 5.15 (5.82) 112.98 0.42-18.40 (42.8) 0.81 (0.53) 65.52 0.11-1.32 (11.0) A/H3N2 97011746 1.63 (0.68) 41.41 0.22-2.24 (9.0) 3.96 (4.77) 120.47 1.21-14.40 (10.9) 2.96 (1.88) 63.44 0.50-4.29 (7.4) 97011803 1.70 (0.72) 42.40 0.26-2.36 (8.1) 2.88 (1.72) 59.67 1.03-6.56 (5.4) 2.55 (1.67) 65.65 0.37-4.03 (9.9) 97012203 1.82 (0.43) 23.53 1.21-2.90 (1.4) 5.26 (6.47) 123.08 1.06-18.80 (16.7) 2.93 (2.06) 70.19 0.35-5.41 (60.2) 98010134 1.63 (0.27) 16.64 1.29-2.07 (0.6) 6.08 (7.75) 127.54 0.85-21.40 (24.2) 1.86 (1.23) 66.22 0.32-3.15 (8.8) 98026522 2.07 (0.92) 44.25 1.54-4.94 (2.2) 13.11 (14.02) 106.98 1.77-43.80 (23.7) 6.22 (4.40) 70.69 0.89-11.00 (11.4) ↵ a Ranges are given as minimum to maximum.
- TABLE 3.
Oseltamivir carboxylate (GS4071) IC50 values for each clinical isolate in three NI assays
Influenza virus type Isolate IC50 (nM) in the following assay: CL assay (n = 12) FA-1 (n = 12) FA-2 (n = 6) Mean (SD) CV Rangea (fold change) Mean (SD) CV Rangea (fold change) Mean (SD) CV Rangea (fold change) B 97012351 3.99 (0.85) 21.34 2.89-5.80 (1.0) 13.29 (4.52) 33.99 7.85-26.00 (2.4) 67.88 (22.74) 33.49 43.60-107.00 (1.5) 97012942 3.63 (0.73) 20.07 2.86-5.57 (0.9) 13.05 (1.85) 14.20 9.81-15.70 (0.6) 65.75 (19.01) 28.92 44.40-96.80 (1.2) 97013118 4.81 (1.75) 36.45 2.99-9.35 (2.1) 12.81 (3.34) 26.11 7.24-19.70 (1.7) 71.05 (23.02) 32.40 45.90-114.00 (1.5) 97013406 5.32 (2.46) 46.26 0.54-8.75 (15.2) 23.23 (9.74) 41.92 0.37-36.20 (96.8) 88.87 (12.48) 14.04 68.10-104.00 (0.5) 98010075 3.58 (0.59) 16.49 2.55-4.53 (0.8) 13.55 (4.20) 30.98 8.49-22.40 (1.6) 64.32 (19.98) 31.07 41.00-94.90 (1.3) A/H1N1 98010152 0.48 (0.17) 35.06 0.36-0.98 (1.7) 2.16 (1.33) 61.76 0.66-4.26 (5.4) 3.56 (1.06) 29.75 2.04-4.71 (1.3) 98013212 0.47 (0.13) 28.46 0.37-0.82 (1.2) 2.82 (4.14) 146.85 0.45-14.60 (31.4) 3.42 (0.81) 23.74 2.19-4.17 (0.9) 98014203 0.47 (0.09) 19.35 0.36-0.64 (0.8) 2.19 (1.02) 46.67 1.11-4.41 (3.0) 6.78 (5.79) 85.47 1.96-16.90 (7.6) 98026506 0.43 (0.10) 22.81 0.29-0.65 (1.2) 4.68 (6.77) 144.60 0.93-24.50 (25.3) 3.70 (1.44) 38.80 1.38-5.27 (2.8) 98027803 0.40 (0.29) 73.91 0.01-1.05 (104) 5.31 (6.65) 125.21 1.73-25.30 (13.6) 2.79 (1.43) 51.24 0.81-5.21 (5.4) A/H3N2 97011746 0.54 (0.16) 29.37 0.32-0.90 (1.8) 0.84 (0.54) 63.88 0.33-1.90 (4.8) 1.77 (0.30) 17.02 1.41-2.13 (0.5) 97011803 0.52 (0.14) 26.64 0.41-0.94 (1.3) 0.78 (0.30) 38.29 0.37-1.19 (2.2) 1.60 (0.25) 15.90 1.25-1.85 (0.5) 97012203 0.37 (0.07) 18.21 0.25-0.53 (1.1) 0.92 (1.04) 112.21 0.20-3.13 (14.7) 0.96 (0.13) 13.89 0.75-1.11 (0.5) 98010134 0.41 (0.03) 7.55 0.37-0.48 (0.3) 0.44 (0.18) 40.86 0.07-0.72 (9.3) 1.05 (0.20) 19.02 0.77-1.25 (0.6) 98026522 0.30 (0.10) 32.96 0.22-0.59 (1.7) 0.23 (0.24) 103.75 0.07-0.94 (12.4) 0.75 (0.16) 21.81 0.59-0.99 (0.7) ↵ a Ranges are given as minimum to maximum.
- TABLE 4.
Inhibitory concentrations of zanamivir and oseltamivir for parental and mutant NAs of influenza viruses recovered in clinical trials
Isolate NA IC50 (nM) in the following assay with the indicated druga: CL assay FA-1 FA-2 Zanamivir Oseltamivir Zanamivir Oseltamivir Zanamivir Oseltamivir Mean SD Fold change Mean SD Fold change Mean SD Fold change Mean SD Fold change Mean SD Fold change Mean SD Fold change B/Memphis/20/96 152R 3.5 0.8 6.7 0.3 3.2 0.2 13.3 2.1 5.8 0.7 92.2 4.4 152K 33.7 5.8 9.6 509.2 154.0 76 473.1 163.9 147.8 412.7 71.0 31 255.5 42.7 44.1 1,154 290 12.5 A/Texas/36/91 (H1N1) 274H 0.5 0.05 0.4 0.04 0.5 0.1 0.8 0.04 0.8 0.0 1.5 0.1 274Y 0.7 0.05 1.4 253.9 20.2 634.8 0.8 0.2 1.6 282.8 12.3 353.5 1.0 0.1 1.3 680.0 61.5 453.3 A/Sydney/5/97 (H3N2) 292R 1.8 0.1 0.4 0.04 1.2 0.1 0.4 0.01 3.5 0.2 0.9 0.1 292K 6.7 0.4 3.7 3,877 517 9,693 29.4 2.8 24.5 4,576 1,633 11,440 47.0 4.3 13.4 7,271 1,195 8,079 A/Wuhan/359/95- like (H3N2) 119E 0.7 0.05 0.3 0.01 1.6 0.5 1 0.7 6.1 1.7 0.5 0.1 119V 1.3 0.1 1.9 15.6 2.8 52 1.4 0.2 0.9 106.4 11.1 106.4 6.1 0.4 1 167.7 30.0 335.4 ↵ a IC50 values are the mean of six independent assays. Fold change is the fold change in IC50 values for the mutant virus compared to the wild-type virus.
