*
Fig. 4.1 Alignment of amino acids at envelope protein of DV2 PL046 strain and DV3 H87 strain.
The residues lined the β-OG binding pocket were marked with rectangles: residues 48-52, 126-128, and 268-279.
E
E E E
E E
E E
E E
E
E E
E
E E
E
E
A
Tetracycline mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 106.44 7.76 106.44±7.76
50 μM 121.55 4.96 121.55±4.96
100 μM 119.45 7.61 119.45±7.61
300 μM 85.55 18.19 85.55±18.19
500 μM 40.52 22.41 40.52±22.41
700 μM 23.48 20.79 23.48±20.79
1000 μM 12.86 13.02 12.86±13.02
B
Fig. 4.2 Effect of tetracycline on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of tetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM tetracycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve. The IC50
value of tetracycline is 457.89 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
106.44 121.55 119.45
85.55
40.52
23.47
12.86 0
50 100 150
10 50 100 300 500 700 1000
PFU (%)
Drug concentration (μM)
IC50=457.89 μM
A
B
Fig. 4.3 Effect of tetracycline on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of tetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM tetracycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve. The IC50
value of tetracycline is 333.85 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
95.61 100.46
100.46
54.79
26.49 27.99
11.33 0
50 100 150
0 100 200 300 400 500 600 700 800 900 1000 1100
PFU (%)
Drug concentration (μM)
Tetracycline mean sd mean±sd 0 μM 100 0.0 100±0.0
10 μM 95.61 9.66 95.61±9.66
50 μM 100.46 7.54 100.46±7.54
100 μM 100.46 7.83 100.46±7.83
300 μM 54.79 15.26 54.79±15.26
500 μM 26.49 12.86 26.49±12.86
700 μM 27.989 10.96 27.99±10.96
1000 μM 11.33 12.72 11.33±12.72
IC50=333.85 μM
A
Doxycycline mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 126.82 15.13 126.82±15.13
50 μM 45.20 6.92 45.20±6.92
100 μM 14.82 6.92 14.83±6.92
200 μM 5.90 3.00 5.90±3.00
300 μM 1.69 1.35 1.69±1.35
500 μM 0.57 0.54 0.57±0.54
700 μM 0 0 0
B
Fig. 4.4 Effect of doxycycline on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of doxycycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM doxycycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve. The IC50
value of tetracycline is 47.64 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
126.82
45.20 14.83
5.90 1.69
0 0
50 100 150
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=47.64 μM
A
Doxycycline mean sd mean±sd
0 uM 100 0.0 100±0.0
10 uM 116.25 5.57 116.25±5.57 50 uM 156.28 30.98 156.28±30.98 100 uM 84.48 15.5 84.48±15.5 200 uM 48.94 27.16 48.94±27.16 300 uM 25.48 8.21 25.48±8.21 500 uM 16.40 0.79 16.40±0.79 700 uM 6.58 1.14 6.58±1.14 B
Fig. 4.5 Effect of doxycycline on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of doxycycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM doxycycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve. The IC50
value of tetracycline is 197.02 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
116.25 156.28
84.47
48.94 25.48
16.40
6.58 0
50 100 150 200
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=197.02 μM
A
Chlortetracycline mean sd mean±sd
0 μM 100 0.0 100±00
10 μM 101.13 2.65 101.13±2.65
50 μM 48.92 9.98 48.91±9.98
100 μM 27.81 4.08 27.81±4.08
200 μM 13.34 3.62 13.34±3.62
300 μM 6.94 2.59 6.94±2.59
500 μM 6.39 3.21 6.39±3.21
700 μM 2.16 3.12 2.16±3.12
B
Fig. 4.6 Effect of chlortetracycline on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of chlortetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM chlortetracycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve.
The IC50 value of chlortetracycline is 49.17 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration.
The numbers by the curve indicate the percentage of PFU of individual concentrations.
101.13
48.91 27.81
13.34
6.94 6.39 2.16
0 50 100 150
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=49.17 μM
A
Chlortetracycline mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 98.67 10.58 98.67±10.58
50 μM 60.11 11.85 60.11±11.85
100 μM 59.52 8.29 59.52±8.29
200 μM 34.60 13.98 34.60±13.98
300 μM 17.81 9.75 17.81±9.75
500 μM 12.04 9.25 12.04±9.25
700 μM 3.95 2.47 3.95±2.47
B
Fig. 4.7 Effect of chlortetracycline on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of chlortetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM chlortetracycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve.
The IC50 value of chlortetracycline is 138.20 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration.
The numbers by the curve indicate the percentage of PFU of individual concentrations.
98.67 60.11
59.52
34.60
17.81
12.04 0 3.95
50 100 150
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=138.20 μM
A
Rolitetracycline mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 128.38 19.01 128.38±19.01
50 μM 171.93 30.86 171.93±30.86
100 μM 160.02 35.83 160.02±35.83
200 μM 54.41 15.6 54.41±15.6
300 μM 26.14 13.38 26.14±13.38
500 μM 5.26 2.95 5.26±2.95
700 μM 0.92 1.58 0.92±1.58
B
Fig. 4.8 Effect of rolitetracycline on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of rolitetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM rolitetracycline as 100%, the relative percentage of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve.
The IC50 value of tetracycline is 215.60 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
128.38 171.93
160.02
54.41 26.14
5.26 0.92
0 50 100 150 200 250
0 100 200 300 400 500 600 700 800 900 1000
PFU (%)
Drug concentration (μM)
IC50=215.60 μM
A
Rolitetracycline mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 100.99 18.50 100.99±18.50
50 μM 119.13 12.03 119.13±12.03
100 μM 110.74 6.84 110.74±6.84
300 μM 68.95 17.21 68.95±17.20
500 μM 40.28 23.56 40.28±23.56
700 μM 13.34 10.34 13.34±10.34
B
Fig. 4.9 Effect of rolitetracycline on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of rolitetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM rolitetracycline as 100%, the relative percentage of the PFUs were calculated. The result is average of 2 experiments. (B) The response curve. The IC50 value of rolitetracycline is 432.19 μM. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations.
100.99 119.13
110.74
68.95
40.28
13.34 0
50 100 150
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=432.19 μM
A
Kanamycin mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 103.19 17.59 103.19±17.59
50 μM 100.06 5.81 100.06±5.81
100 μM 107.75 5.24 107.75±5.24
300 μM 111.28 13.51 111.28±13.51
500 μM 104.34 6.13 104.34±6.13
700 μM 122.1 24.72 122.10±24.72
B
Fig. 4.10 Effect of kanamycin on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of kanamycin at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM kanamycin as 100%, the relative percentage of the PFUs were calculated. The result is the average of 2 experiments. (B) The response curve. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
103.195
100.06
107.75 111.28 104.34 122.10
0 50 100 150 200
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50= NA
A
Kanamycin mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 88.05 20.07 88.05±20.07
50 μM 85.84 3.01 85.84±3.01
100 μM 90.48 4.73 90.48±4.73
200 μM 83.70 1.60 83.70±1.60
300 μM 91.75 7.70 91.75±7.70
500 μM 87.50 5.36 87.50±5.36
700 μM 86.06 0.94 86.06±0.94
B
Fig. 4.11 Effect of kanamycin on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of kanamycin at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM kanamycin as 100%, the relative percentage of the PFUs were calculated. The result is the average of 2 experiments. (B) The response curve. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
88.05
85.84 90.48
83.70
91.75 87.50
86.06
0 50 100 150
0 100 200 300 400 500 600 700 800
PFU (%)
Drug concentration (μM)
IC50=NA
A
9-amino-1,2,3,4- tetrahydroacridine hydrochloride hydrate
mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 99.05 1.35 99.05±1.35
50 μM 93.72 4.23 93.72±4.23
100 μM 69.23 17.7 69.23±17.7
200 μM 68.59 3.74 68.59±3.74
300 μM 53.68 4.24 53.68±4.24
B
Fig. 4.12 Effect of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate as 100%, the relative percentage of the PFUs were calculated. The result is the average of 2 experiments. (B) The response curve.
The y axis shows the percentage of the amount of plaque formation compared with control.
The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
99.05 93.72
69.23 68.59
53.68
0 50 100 150
0 50 100 150 200 250 300 350
PFU (%)
Drug concentration (μM)
IC50=NA
A
9-amino-1,2,3,4- tetrahydroacridine hydrochloride hydrate
mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 88.81 3.22 88.81±3.22
50 μM 86.60 2.43 86.60±2.43
100 μM 74.41 2.73 74.41±2.73
200 μM 74.09 13.78 74.09±13.78
300 μM 68.33 23.19 68.33±23.19
500 μM 63.22 8.83 63.22±8.83
B
Fig. 4.13 Effect of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate as 100%, the relative percentage of the PFUs were calculated. The result is the average of 2 experiments. (B) The response curve.
The y axis shows the percentage of the amount of plaque formation compared with control.
The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
88.81 86.60
74.41 74.09
68.33
63.22
0 50 100
0 100 200 300 400 500 600
PFU (%)
Drug concentration (μM)
IC50= NA
A
Berberine mean sd mean±sd
0 μM 100 0.0 100±0.0
10 μM 105.26 8.62 105.26±8.62
50 μM 132.49 6.94 132.49±6.94
100 μM 153.91 12.78 153.91±12.78
200 μM 125.94 17.38 125.94±17.38
300 μM 70.13 3.85 70.13±3.85
B
Fig. 4.14 Effect of berberine on DV2 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of berberine at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM berberine as 100%, the relative percentage of the PFUs were calculated.
The result is the average of 2 experiments. (B) The response curve. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
105.26
132.49 153.91
125.94
70.13
0 50 100 150 200
0 50 100 150 200 250 300 350
PFU (%)
Drug concentration (μM)
IC50= NA
A
Berberine mean sd mean±sd 0 μM
10 μM 94.38 14.04 94.38±14.04
50 μM 113.37 4.25 113.37±4.25
100 μM 95.68 12.21 95.68±12.21
B
Fig. 4.15 Effect of berberine on DV3 plaque formation using BHK-21 mammalian cells.
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of berberine at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates added 0 μM berberine as 100%, the relative percentage of the PFUs were calculated.
The result is the average of 2 experiments. (B) The response curve. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
94.38
113.37
95.68
0 50 100 150
0 20 40 60 80 100 120
PFU (%)
Drug concentration (μM)
IC50= NA
Fig. 4.16 The cell morphology of BHK-21 when adding the different concentrations of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate with no virus.
Control: no 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate.
10 μM
50 μM 100 μM
200 μM
500 μM
300 μM
700 μM
Control 0 μM
Fig. 4.17 The cell morphology of BHK-21 when adding the different concentrations of berberine.
Control: no berberine.
0 μM
Control
700 μM 300 μM
500 μM 200 μM
100 μM 50 μM
10 μM
(A)
(B)
Fig. 4.18 The percentage of cell numbers of BHK-21 when adding the different concentrations of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate and berberine.
To count the cell numbers on each plate in the absence or presence of serial dilutions of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (A) / berberine (B) cultured for 7 days. Using the cell numbers from the culture plates without 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate / berberine as 100%, the relative percentage of the cell numbers was calculated. The y axis shows the percentage of the amount of cell numbers compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of cell numbers of individual concentrations.
111.31
Appendix 1
Map of pcDNA3
Neo: Neomycin, resistance gene; Amplicillin: Amplicillin resistance gene; PCMV: Human cytomegalovirus (CMV) immediate early promoter; RBS: ribosome binding site; T7 promoter:
the T7 transcription promoter.
pcDNA3
5446 bp
Ampicillin
T7 promoter RBS
Neomycin
P CM V
Appendix 2
Maps of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis
(From 徐婕琳 and 楊馥嘉, Yang laboratory) Neo: Neomycin, resistance gene; Amplicillin: Amplicillin resistance gene; PCMV: Human cytomegalovirus (CMV) immediate early promoter; RBS: ribosome binding site; T7 promoter:
the T7 transcription promoter; NS2A: nonstructural protein 2A; NS2B: nonstructural protein 2B; NS4A: nonstructural protein 4A; NS4B: nonstructural protein 4B. HA3His6: the HAHis tag at the C-terminus.
pNS2A-HAHis
Appendix 3
Effect of tetracycline on DV2 plaque formation using BHK-21 mammalian cells
(A)
Tetracycline mean sd mean±sd
10 μM 110.18 9.07 110.18±9.07
100 μM 101.19 5.32 101.19±5.32 1000 μM 104.05 4.19 104.05±4.19
(B)
(A) To count the PFUs on each plate in the absence or presence of serial dilutions of tetracycline at a fixed number plaque formation units (PFUs). Using the number of PFUs from the culture plates without tetracycline as 100%, the relative percentages of the PFUs were calculated. The result is the average of 3 experiments. (B) The response curve. The y axis shows the percentage of the amount of plaque formation compared with control. The x axis denotes the drug concentration. The numbers by the curve indicate the percentage of PFU of individual concentrations. NA: not applicable.
(From 杜育穎，Yang laboratory)
110.18
101.19 104.05
0 50 100 150
0 200 400 600 800 1000 1200
PFU%
Drug concentration (μM)
IC50= NA