Antiviral protocols for dengue

1. 1
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2. DENGUE VIRUS
DENGUE VIRUS
CULTURING
High
throughput
Screening
assay
Plaque
reduction
Assay
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3. DENGUE
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 A mosquito borne virus
 Belongs to family Flaviviridae
 Carriers are
 Aedes aegypti
 Aedes albopictus
 Endemic in more than hundred countries
 Southeast Asia
 Southern and Central America
 Carribean and south pacific regions
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4. DENGUE
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 2.5 billions people are living in areas at risk for

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dengue
50-100 millions infections/annum
500,000 hospitalizations/annum
24,000 deaths/annum
No effective antiviral therapy or vaccines are
available for it
Dengue infection ranges from dengue fever to
dengue shock syndrome
The therapy “if” exists is purely SUPPORTIVE
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5. DENGUE
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 Four related but distinct serotypes exist for dengue and
transmit through mosquito bite
 Its enveloped single stranded RNA virus
 Its genome encodes a single polyprotein that is cleaved


Cotranslationally
Post translationally
By host
 Virus encoded protease
 3 structural, 7 non structural proteins

 But NS3 protease
and NS5 RNA polymerase
are best targets for antiviral drug therapy
development

As experienced for HCV and HIV
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6. Dengue virus culturing
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 Requirements:
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Vero cells (African green monkey kidney cells)
BHK-21 cells (baby hamster kidney cells)
DENV-2 (serotype 2) NGC (New Guinea C) strain
Eagle’s minimum essential medium (MEM) without L glutamine.
MEM without L -glutamine and sodium
bicarbonate
Heat-inactivated fetal calf serum (FCS).
200 mM L -glutamine.
Gentamicin solution (50 mg/mL).
100 mM sodium pyruvate
MEM nonessential amino acids
0.05 % trypsin–EDTA.
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7. Cells, virus and reagents
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 Vero cell growth medium (MEM/10 % FCS):

Eagle’s MEM supplemented with 10 % FCS, 2 mM
L-glutamine, and 0.02 mg/mL gentamicin.
 Vero cell maintenance medium (MEM/2 % FCS):

Same as the growth medium but supplemented with 2 %
FCS.
 BHK-21 cell growth medium:
 Eagle’s MEM supplemented with 10 % FCS, 2 mM L glutamine , 1× MEM nonessential amino acids, 1× sodium
pyruvate, and 0.02 mg/mL gentamicin.
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8. Cells, virus and reagents
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 BHK-21 cell maintenance medium
 supplemented with 2 % FCS.
 BHK-21 cell maintenance medium
 MEM without L -glutamine and sodium
bicarbonate
 Dulbecco’s phosphate-buffered saline (D-PBS), (no
calcium chloride, no magnesium chloride)
 10 % formalin in D-PBS.
 1 % crystal violet solution (Sigma-Aldrich).
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9. Cells, virus and reagents
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 1.5 % methylcellulose in water:

Weigh 1.5 g of methylcellulose in an Erlenmeyer flask and add 100 mL
of deionized H2O into the flask. Place the cap loosely on the bottle.
 Put a stirring bar into the flask and place on a magnetic stirrer. Heat the
flask under stirring until boiling.Then allow the flask to cool to
room temperature. Continue stirring the flask overnight.
 Trypan blue stain
 ATPLite™ 1-step kit
 Reference compounds:
(a) Interferon α-2a (Roferon A, Roche).
(b) 6-Azauridine (Sigma-Aldrich).
(c) 2′-C-Methylcytidine (Sigma-Aldrich).
(d) Ribavirin (Sigma-Aldrich).
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10. Consumables
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1. 6-well, flat-bottom plates:

Corning ® Costar ® cell culture plates.
2. Nunc ® TripleFlasks (F8542, Sigma-Aldrich) .
3. Greiner Lumitrac 384-well plate (VWR).
4. White-view 384-well plates

(white wall and transparent bottom)
5. Absorbent pad.
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11. Equipments
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1.
2.
3.
4.
5.
6.
7.
Centrifuge.
Magnetic stirrer.
Multidrop combi liquid dispenser (Thermo
Scientifi c).
ViewLux™ (a luminescent reader)
Cell counter (Coulter Electronics LTD) (optional).
Microplate shaker.
Light-box.
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12. 12
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13. Viral culturing
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 Procedure:
 1. Seed Vero cells in T175 flasks using the Vero cell growth
medium


and incubate at 37 °C in a humidified 5 % CO 2 incubator overnight .
The cell monolayer should be sub-confluent (~90 %) on the following
day.
 2. On the following day:
 prepare a viral dilution at a multiplicity of infection (MOI) of
0.1 TCID 50 /cell in MEM/2 % FCS.

Prepare 15 mL of virus dilution for each flask to be infected.
 3. Remove the old culture medium from flasks and wash the
sub-confluent cells once with D-PBS.
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14. Viral culturing
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 4.
Add 15 mL of the viral dilution to each flask.
 5. Swirl the flask gently to spread the viral inoculum evenly to the
whole-cell monolayer.
 6. Incubate the cells at 37 °C, 5 % CO 2 , for 2 h with occasional
swirling.
 7. Discard the viral inoculum.
 8.
Rinse the infected cells once with D-PBS.
 9. Add 18 mL of fresh MEM/2 % FCS to each flask

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incubate the infected cells at 37 °C, 5 % CO 2 ,
wait for 5–6 days until the viral cytopathic effect (CPE) reaches ~90–100
%.
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15. Viral culturing
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 10. Collect the supernatant into sterile 50 mL Falcon tubes
and centrifuge at 3,700 × g and 4 °C for 10 min to remove the
cell debris.
 11. After centrifugation;

pool the supernatants from all falcon tubes into a sterile bottle or a
fresh T175 flask.
 12. Add heat-inactivated 20%FCS to the harvested
supernatant
 Aliquot 1 mL to each cryovial and store in a −80 °C freezer.
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16. Plaque reducing assay
 1. Prepare a BHK-21 cell
suspension at a density of
1.0 × 10 5 cells/mL in BHK21 cell growth medium.
 2. Seed 6-well plates with
3mL of the BHK-21 cell
suspension per well


Final value 3 × 10 5 cells/well.
Prepare duplicate wells for
each test compound
concentration.
 3. Incubate the plate at 37 °C,
5 % CO 2 , overnight.
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 4. On the following day, check
Baby hamster BHK-21 cells with a
the
microscope.

The cell monolayer should be
sub-confluent (~90 %).
 5. Thaw a DENV stock quickly
at 37 °C in a water-bath with
shaking.

Make an appropriate dilution
of the virus (100 PFU /mL) in
the BHK-21 cell maintenance
medium for plates.
 6. Remove the old culture
medium by inverting the plate
over absorbent pad.
 7. Rinse the cells with D-PBS
once.

17. Plaque reducing assay
 8. Add 1 mL of diluted
virus into each well (100
PFU per well).
 9. Incubate the plates at
37 °C, 5 % CO 2 , for 1.5–2
h.

Swirl the plates
periodically to spread the
virus evenly over the cell
monolayer.
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 10. During the incubation
period;

perform 5 or 10 fold serial dilutions
of test compounds in BHK21 cell
growth media
 Prepare methylcellulose
overlay :
 Test wells:
 1.5% Methyl cellulose in water
+ test compund + MEM Cell
maintenance media 2% FCS
 Control wells:
 1.5% MC in water+ MEM cell
maintenance media 2% FCS
 Prepare enough volumes for the
duplicate wells.

18. Plaque reducing assay
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Remove the viral inoculums from
the plates by inverting the plates over
an absorbent pad.
11.
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17.
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18.
12. Add 3 mL/well of the MC overlay
containing various test compounds
to the corresponding wells of 6-well
plates.
13. Add 3 mL of MC overlay containing
no compound to the control wells.
14. Incubate
the plates at 37 °C, 5 %
CO 2 , for 4 days.
Stain by adding 2–3 drops of 1.0
% crystal violet to each well.
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Remove excess of crystal violet by
rinsing the plates in a gentle tap water
flow.
 20. Air-dry the plates.
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21. Place
the plates upside down on
a light-box(plaque counter)
15. Remove the methylcellulose overlay
from plates by inverting the plate over an
absorbent pad.
16.
Add 2 mL of 10 % formalin to
each well to fix the cells

Incubate at room temperature for 10
min.
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Leave at room temperature for 5–10 min.
19.
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Discard the formalin
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Count the plaques in each well using
a colony counter, if more wells used
then average it
22. Enter the data
in a standard
statistical program, such as
GraphPad Prism

Calculate, EC 50 of test compounds

19. High-throughput antiviral assay for screening inhibitors of DENV
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 1. Seed enough Vero cells based on
the number of test plates to be
tested.
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The cells should be confluent at the
day of antiviral experiment
. We typically culture the Vero cells
in triple flasks.

three parallel growth surfaces with
a total culture area of 500 cm 2 .
 2. Discard the old cell culture
medium from a triple flask and rinse
three cell monolayers with 60 mL DPBS.

3. Add 15 mL of 0.05 % trypsin–
EDTA to a triple flask and spread
the solution over all three surfaces.
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 4. Pour off excess of trypsin and
incubate the flask at 37 °C for 2–3
min.
 5. Dislodge cells by tapping flask with
the palm of hand.
 6. Add 30 mL of Vero cell
maintenance medium (MEM/2 %
FCS) to the flask.

Rock the flask to dislodge the cells
 7. Collect the cell suspension in a
sterile Falcon tube.

20. High-throughput antiviral assay for screening DENV inhibitors
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.
 8 Count the cells using either a cell counter or a trypan blue stain.
 9. For Activity plate:
 Prepare 5.8 mL of cell suspension, 15microlitre for each well

density of 1 × 10 5 cells/ mL for each 384-well (the optimal number of cells in
each well is 1,500
 10. For Toxicity plate:Prepare 11.6 mL of cell suspension, 30 μL for each well
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at a density of 5 × 10 4 cells/mL for each 384-well toxicity plate
. Thaw a DENV-2 stock quickly in a 37 °C water-bath with shaking.
 Make an appropriate dilution of the virus in MEM/2 % FCS to result in an
MOI of 0.1 TCID 50 /cell.
 Prepare 5.3 mL viral dilution for each 384-well activity plate (15 μL/well).
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21. High-throughput antiviral assay for screening DENV
inhibitors
 12. In a biosafety cabinet, use
a multidrop combi liquid
dispenser for dispensing
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 Toxicity test plates:

 ACTIVITY TEST PLATES:
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(a) Dispense 15 μL of MEM/2 %
FCS into the wells of columns 23
and 24 (cell controls).
(b) Dispense 15 μL of cell
suspension into all wells of the
test plate.
(c) Dispense 15 μL of the virus
dilution into all wells of columns
1–22.
(d) Dispense test compound/s
15 μL into all wells of colunms 118
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
(a) Dispense 30 μL of
MEM/2 % FCS into the wells
of columns 23 and 24
(medium controls).
(b) Dispense 30 μL of cell
suspension into all wells of
columns 1–22.

22. High-throughput antiviral assay for screening inhibitors of DENV
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 13. Incubate the plates at 37 °C, 5 % CO 2 , for 5–6 days.
 14. After incubation, check the plates with reference
compounds under an inverted microscope.
 The CPE in the virus control wells should reach ~100 %.
 The ATP levels in compound-treated and untreated cells are
measured by a luminescent assay using an ATPLite™ 1-Step
kit.
 We use ViewLux™ to measure the luminescence.
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23. High-throughput antiviral assay for screening inhibitors of DENV
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
The following procedures are recommended by the manufacturer:
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(a) Warm up all reagents to room temperature before use.
(b) Reconstitute the lyophilized substrate solution by adding
the appropriate volume of buffer to the substrate bottle.
Mix the contents by inversion and leave the solution to stand
for 5 min
(c) Add 40 μL of the reconstituted reagent to each well of
activity and toxicity plates using a multidrop combi liquid
dispenser
(d) Shake the plates for 2 min at 700 rpm using an orbital
microplate shaker.
(e) Program Viewlux to take a 0.1-s integrated reading of each
plate.
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24. High-throughput antiviral assay for screening
DENV inhibitors
 15. Export the test results to
an Excel sheet.
 Calculate the percentage of
inhibition based on the
reduction of the luminescent
signals in the compoundtreated wells relative to the
cell controls.
 If duplicate or quadruplicate
wells are used for each drug
concentration, average the
luminescent readings in those
wells.
 Calculate the EC 50 and CC
50 of test compounds using
computer software, such as
GraphPad Prism.
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