Micro- -plaque assay of influenza virus plaque assay of influenza - - PowerPoint PPT Presentation

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Micro- -plaque assay of influenza virus plaque assay of influenza - - PowerPoint PPT Presentation

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VIRGIL Antiviral Course VIRGIL Antiviral Course 3- -6 October 2006 6 October 2006 3 Micro- -plaque assay of influenza virus plaque assay of influenza virus Micro sensitivity to neuraminidase inhibitors sensitivity to neuraminidase

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SLIDE 1

Mikhail Matrosovich and Tatyana Matrosovich Mikhail Matrosovich and Tatyana Matrosovich

VIRGIL Antiviral Course VIRGIL Antiviral Course 3 3-

  • 6 October 2006

6 October 2006

Micro Micro-

  • plaque assay of influenza virus

plaque assay of influenza virus sensitivity to neuraminidase inhibitors sensitivity to neuraminidase inhibitors

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SLIDE 2

Influenza virus receptors: sialic acids Influenza virus receptors: sialic acids

HA binds to sialic acid (Sia) HA binds to sialic acid (Sia)

1

Neu5Ac

M.Matrosovich M.Matrosovich

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SLIDE 3

Functions of influenza virus neuraminidase (NA) Functions of influenza virus neuraminidase (NA)

Early in infection: Early in infection: Late in infection: Late in infection:

Removes Removes receptors from receptors from virus progeny virus progeny and cell and cell surface surface Promotes virus Promotes virus release and release and spread spread Destroys Destroys mucin mucin inhibitors inhibitors and decoy and decoy receptors receptors Promotes Promotes virus entry virus entry into cell into cell

Neu5Ac

M.Matrosovich M.Matrosovich

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SLIDE 4

Inhibition of NA impaires virus release and spread Inhibition of NA impaires virus release and spread

Normal virus release (top) Normal virus release (top) and release in the presence of and release in the presence of NA inhibitor (bottom) NA inhibitor (bottom) From Gubareva et al., 2000 From Gubareva et al., 2000

M.Matrosovich M.Matrosovich

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SLIDE 5

Plaque reduction assay Plaque reduction assay

NA inhibitor decreases size of plaques produced NA inhibitor decreases size of plaques produced by influenza virus by influenza virus From Bantia et al., 1998 From Bantia et al., 1998

M.Matrosovich M.Matrosovich

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SLIDE 6

Pitfalls of plaque assays Pitfalls of plaque assays

  • 1. Assays in
  • 1. Assays in MDCK cells

MDCK cells do not correlate with virus do not correlate with virus sensitivity to NA inhibitors in vivo sensitivity to NA inhibitors in vivo

  • 2. Plaque assays under agar overlays are cumbersome
  • 2. Plaque assays under agar overlays are cumbersome

and cannot be performed in 96 and cannot be performed in 96-

  • well plates

well plates

New assay solves these problems New assay solves these problems

M.Matrosovich M.Matrosovich

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SLIDE 7
  • I. Viral sensitivity to NAI in MDCK cells do not
  • I. Viral sensitivity to NAI in MDCK cells do not

correlate with sensitivity in vivo correlate with sensitivity in vivo

Viruses with drug Viruses with drug-

  • sensitive NA can be

sensitive NA can be resistant resistant Viruses with drug Viruses with drug-

  • resistant

resistant mutations in HA and NA can mutations in HA and NA can display sensitivity display sensitivity

Laboratory cells do not mimic receptors in human airway epitheli Laboratory cells do not mimic receptors in human airway epithelium um

M.Matrosovich M.Matrosovich

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SLIDE 8

How to model influenza virus receptors of human How to model influenza virus receptors of human airway tissues in a laboratory cell line? airway tissues in a laboratory cell line?

Cell line with high Cell line with high concentration of 6 concentration of 6-

  • linked

linked sialic acids is required sialic acids is required

Laboratory cells

M.Matrosovich M.Matrosovich

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SLIDE 9

Preparation of a cell line for resistance assay: Preparation of a cell line for resistance assay:

  • verexpression of SIAT1 in MDCK cells
  • verexpression of SIAT1 in MDCK cells

SIAT1 SIAT1 (beta (beta-

  • galactoside a2,6

galactoside a2,6-

  • sialyltransferase) generates 6

sialyltransferase) generates 6-

  • linked sialic acid receptors recognized by human influenza virus

linked sialic acid receptors recognized by human influenza viruses es

M.Matrosovich M.Matrosovich

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SLIDE 10

Influenza viruses are more sensitive to Influenza viruses are more sensitive to NA NA inhibitor in MDCK inhibitor in MDCK-

  • SIAT1 cells than in MDCK cells:

SIAT1 cells than in MDCK cells:

A/Sydney/5/97 (H3N2) A/Sydney/5/97 (H3N2)

M.Matrosovich M.Matrosovich

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SLIDE 11

Viral plaque assays Viral plaque assays

General cellular stain Immuno General cellular stain Immuno-

  • staining

staining detects destroyed cells detects destroyed cells detects infected cells detects infected cells

Under liquid medium, Under liquid medium, the plaques are not the plaques are not localised and cannot localised and cannot be counted be counted

M.Matrosovich M.Matrosovich

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SLIDE 12

Known overlays Known overlays

  • Gels (agar,

Gels (agar, agarose agarose) )

Time and labor consuming; heated agar can damage Time and labor consuming; heated agar can damage cells; cannot be used in 96 cells; cannot be used in 96-

  • well plates

well plates

  • “

“Semi Semi-

  • liquid

liquid” ” overlays (solutions of

  • verlays (solutions of

methylcellulose, methylcellulose, tragacanth tragacanth gum, etc) gum, etc)

High High viscousity viscousity --

  • -> particularly difficult to handle

> particularly difficult to handle in in microplate microplate format format

M.Matrosovich M.Matrosovich

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SLIDE 13

Our approach: Our approach: Thixotropic Thixotropic gels gels

The viscosity decreases as shear rate increases The viscosity decreases as shear rate increases (Examples: yogurt, ketchup) (Examples: yogurt, ketchup)

M.Matrosovich M.Matrosovich

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SLIDE 14

Avicel AvicelTM

TM (FMC

(FMC BioPolymer BioPolymer) )

  • Microcrystalline water insoluble cellulose

Microcrystalline water insoluble cellulose

  • Particles (~0,2 uM)

Particles (~0,2 uM) form a network of weak form a network of weak hydrogen bonds that hydrogen bonds that account for thixotropic account for thixotropic properties of Avicel properties of Avicel dispersions dispersions

  • Low viscosity

Low viscosity (~ 100 (~ 100-

  • 200 mPa.s at 1,5%

200 mPa.s at 1,5% Compare to 3000 mPa.s for 1,5% solution of Compare to 3000 mPa.s for 1,5% solution of methylcellulose) methylcellulose)

M.Matrosovich M.Matrosovich

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SLIDE 15

Avicel AvicelTM

TM (FMC

(FMC BioPolymer BioPolymer) )

Standardised commercial product: Standardised commercial product: Widely used as vehicle for the preparation of Widely used as vehicle for the preparation of pharmaceutical suspensions and emulsions pharmaceutical suspensions and emulsions

M.Matrosovich M.Matrosovich

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SLIDE 16

Plaque assays under Avicel Plaque assays under Avicel vs vs agar agar

influenza virus A/Memphis/14/96 (H1N1) influenza virus A/Memphis/14/96 (H1N1)

  • Plaques are bigger; size can be controlled

Plaques are bigger; size can be controlled

  • As low as 0.3% ( ! ) of Avicel is still sufficient to localize

As low as 0.3% ( ! ) of Avicel is still sufficient to localize plaques plaques

  • More plaques under Avicel than under agar

More plaques under Avicel than under agar

M.Matrosovich M.Matrosovich

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SLIDE 17

Avicel vs. methylcellulose, MDCK Avicel vs. methylcellulose, MDCK-

  • SIAT1 cells

SIAT1 cells

M.Matrosovich M.Matrosovich

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SLIDE 18

Plaque formation by different human and avian viruses Plaque formation by different human and avian viruses

M.Matrosovich M.Matrosovich

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SLIDE 19

Assay variants in 96 Assay variants in 96-

  • well plate

well plate

Viral inoculum Viral inoculum was removed was removed before adding before adding Avicel overlay Avicel overlay

  • Avicel overlay

Avicel overlay was added w/o was added w/o removing removing inoculum inoculum

No need to remove viral inoculum: easier to No need to remove viral inoculum: easier to perform, lower chances of cross perform, lower chances of cross-

  • contamination

contamination

M.Matrosovich M.Matrosovich

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SLIDE 20

Detecting drug Detecting drug-

  • resistant viruses

resistant viruses

MDCK MDCK-

  • SIAT1, 96

SIAT1, 96-

  • well format

well format

The viruses were kindly provided by The viruses were kindly provided by Robert Webster Robert Webster

M.Matrosovich M.Matrosovich

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SLIDE 21

Step 1. Step 1. Seed MDCK Seed MDCK-

  • SIAT1 cells in 96

SIAT1 cells in 96-

  • well plate

well plate

M.Matrosovich M.Matrosovich

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SLIDE 22

Step 2. Step 2. Wash the cells 3 Wash the cells 3-

  • 4 times with serum

4 times with serum-

  • free medium

free medium

M.Matrosovich M.Matrosovich

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SLIDE 23

Step 3. Step 3. Add 10 Add 10-

  • fold serial dilutions of the drug, 50 ul/well

fold serial dilutions of the drug, 50 ul/well

Drug concentration, Drug concentration, uM uM

H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12

0.004 0.04 0.4 4 40 400

M.Matrosovich M.Matrosovich

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SLIDE 24

Step 4. Step 4. Add 3 Add 3-

  • fold serial dilutions of the virus, 50 ul/well

fold serial dilutions of the virus, 50 ul/well

Drug concentration, Drug concentration, uM uM

H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12

. 4 . 4 . 4 4 4 4

Dilution 1 Dilution 1 Dilution 3 Dilution 3 Dilution 9 Dilution 9 Dilution 27 Dilution 27

M.Matrosovich M.Matrosovich

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SLIDE 25

Step 5. Step 5. Mix, incubate 1 Mix, incubate 1-

  • 2 h for initiation of infection

2 h for initiation of infection

Drug Drug

H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12

. 4 . 4 . 4 4 4 4

VIRUS VIRUS Dilution 1 Dilution 1 Dilution 3 Dilution 3 Dilution 9 Dilution 9 Dilution 27 Dilution 27

M.Matrosovich M.Matrosovich

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SLIDE 26

Step 6. Step 6. Add Avicel overlay medium, 100 ul/well Add Avicel overlay medium, 100 ul/well

Drug Drug

H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12

. 4 . 4 . 4 4 4 4

VIRUS VIRUS Dilution 1 Dilution 1 Dilution 3 Dilution 3 Dilution 9 Dilution 9 Dilution 27 Dilution 27

M.Matrosovich M.Matrosovich

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SLIDE 27

Step 7. Step 7. Incubate for 20 Incubate for 20-

  • 48 h to allow formation of plaques

48 h to allow formation of plaques

Drug Drug

H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12

. 4 . 4 . 4 4 4 4

VIRUS VIRUS Dilution 1 Dilution 1 Dilution 3 Dilution 3 Dilution 9 Dilution 9 Dilution 27 Dilution 27

M.Matrosovich M.Matrosovich

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SLIDE 28

Step 8. Step 8. Fix and immunostain to visualise plaques: Fix and immunostain to visualise plaques:

  • remove overlay medium, incubate with 4% paraformaldehyde,

remove overlay medium, incubate with 4% paraformaldehyde, 30 min at 4 oC 30 min at 4 oC

  • permeabilize the cells with 0.5% Triton

permeabilize the cells with 0.5% Triton-

  • X

X-

  • 100, 10 min

100, 10 min

  • incubate with primary antibodies (anti

incubate with primary antibodies (anti-

  • NP

NP-

  • A or

A or -

  • NP

NP-

  • B), 1 h

B), 1 h

  • incubate with HRP

incubate with HRP-

  • labeled secondary antibodies, 1 h

labeled secondary antibodies, 1 h

  • incubate with precipitate

incubate with precipitate-

  • forming peroxidase substrate,

forming peroxidase substrate, 30 min 30 min

  • let dry and analyse

let dry and analyse

M.Matrosovich M.Matrosovich

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SLIDE 29

VIRUS VIRUS Dilution 1 Dilution 1 Dilution 3 Dilution 3 Dilution 9 Dilution 9 Dilution 27 Dilution 27

Drug concentration, Drug concentration, uM uM

M.Matrosovich M.Matrosovich

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SLIDE 30

Post Post-

  • treatment virus is NAI

treatment virus is NAI-

  • resistent

resistent

M.Matrosovich M.Matrosovich

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SLIDE 31