cell based antiviral assays for screening and profiling of anti dengue agents

Shahan Ullah Mphil Pharmacology

1

Shahan UllahMPhil Pharmacology

[email protected]

Cell-based Antiviral assays for screening and profiling

inhibitors against dengue virus

Institute of Basic Medical SciencesKhyber Medical University Peshawar

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Shahan Ullah Mphil Pharmacology 2

DENGUE VIRUS CULTURING

Plaque reduction

Assay

High throughput Screening

assay

DENGUE VIRUS


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DENGUE

A mosquito borne virusBelongs to family FlaviviridaeCarriers 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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DENGUE

2.5 billions people are living in areas at risk for dengue

50-100 millions infections/annum500,000 hospitalizations/annum24,000 deaths/annumNo effective antiviral therapy or vaccines are

available for itDengue infection ranges from dengue fever to

dengue shock syndromeThe therapy “if” exists is purely SUPPORTIVE


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DENGUE

Four related but distinct serotypes exist for dengue and transmit through mosquito bite

Its enveloped single stranded RNA virusIts 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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Dengue virus culturing

Requirements: 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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Cells, virus and reagents

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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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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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 kitReference 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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Consumables

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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Equipments

1. Centrifuge.2. Magnetic stirrer.3. Multidrop combi liquid dispenser (Thermo

Scientifi c).4. ViewLux™ (a luminescent reader)5. Cell counter (Coulter Electronics LTD)

(optional).6. Microplate shaker.7. Light-box.


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Viral culturing

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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Viral culturing

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 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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Viral culturing

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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Plaque reducing assay

1. Prepare a BHK-21 cell suspension at a density of 1.0 × 10 5 cells/mL in BHK-21 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.

4. On the following day, check the BHK-21 cells with a 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.


Baby hamster


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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.

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.

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11. Remove the viral inoculums from the plates by inverting the plates over an absorbent pad.

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.

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.

17. Discard the formalin

18. Stain by adding 2–3 drops of 1.0 % crystal violet to each well. Leave at room temperature for 5–10 min.

19. Remove excess of crystal violet by rinsing the plates in a gentle tap water flow.

20. Air-dry the plates. 21. Place the plates upside

down on a light-box(plaque counter) 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



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High-throughput antiviral assay for screening inhibitors of DENV

1. Seed enough Vero cells based on the number of test plates to be tested. 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 D-PBS.

3. Add 15 mL of 0.05 % trypsin–EDTA to a triple flask and spread the solution over all three surfaces.

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.

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High-throughput antiviral assay for screening DENV inhibitors

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 at a density of 5 × 10 4 cells/mL for each 384-well toxicity plate

11. 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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12. In a biosafety cabinet, use a multidrop combi liquid dispenser for dispensing

ACTIVITY TEST PLATES: (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 1-18

Toxicity test plates: (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.


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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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The following procedures are recommended by the manufacturer: (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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15. Export the test results to an Excel sheet.

Calculate the percentage of inhibition based on the reduction of the luminescent signals in the compound-treated 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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cell based antiviral assays for screening and profiling of anti dengue agents

Education

infected cells

cell debris

seed vero cells

vero cell growthmedium

virus dilution

erlenmeyer flask

viral culturing

vero cell growth medium