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 inhibitors Mikhail Matrosovich and Tatyana Matrosovich Mikhail Matrosovich and Tatyana Matrosovich
Influenza virus receptors: sialic acids Influenza virus receptors: sialic acids 1 Neu5Ac HA binds to sialic acid (Sia) HA binds to sialic acid (Sia) M.Matrosovich M.Matrosovich
Functions of influenza virus neuraminidase (NA) Functions of influenza virus neuraminidase (NA) Early in infection: Late in infection: Late in infection: Early in infection: Neu5Ac Destroys Destroys Removes Removes mucin receptors from mucin receptors from inhibitors virus progeny inhibitors virus progeny and decoy and cell and decoy and cell receptors surface receptors surface Promotes Promotes virus Promotes Promotes virus virus entry virus entry release and release and into cell into cell spread spread M.Matrosovich M.Matrosovich
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
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
Pitfalls of plaque assays Pitfalls of plaque assays 1. Assays in MDCK cells MDCK cells do not correlate with virus do not correlate with virus 1. Assays in 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- -well plates well plates and cannot be performed in 96 New assay solves these problems New assay solves these problems M.Matrosovich M.Matrosovich
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- -resistant resistant Viruses with drug Viruses with drug sensitive NA can be mutations in HA and NA can sensitive NA can be mutations in HA and NA can resistant resistant display sensitivity display sensitivity Laboratory cells do not mimic receptors in human airway epithelium um Laboratory cells do not mimic receptors in human airway epitheli M.Matrosovich M.Matrosovich
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 Laboratory cells concentration of 6- concentration of 6 -linked linked sialic acids is required sialic acids is required M.Matrosovich M.Matrosovich
Preparation of a cell line for resistance assay: Preparation of a cell line for resistance assay: overexpression of SIAT1 in MDCK cells overexpression of SIAT1 in MDCK cells SIAT1 (beta (beta- -galactoside a2,6 galactoside a2,6- -sialyltransferase) generates 6 sialyltransferase) generates 6- - SIAT1 linked sialic acid receptors recognized by human influenza viruses es linked sialic acid receptors recognized by human influenza virus M.Matrosovich M.Matrosovich
Influenza viruses are more sensitive to NA NA Influenza viruses are more sensitive to inhibitor in MDCK- -SIAT1 cells than in MDCK cells: SIAT1 cells than in MDCK cells: inhibitor in MDCK A/Sydney/5/97 (H3N2) A/Sydney/5/97 (H3N2) M.Matrosovich M.Matrosovich
Viral plaque assays Viral plaque assays General cellular stain Immuno- -staining staining General cellular stain Immuno 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
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- -well plates well plates cells; cannot be used in 96 - “ “Semi Semi- -liquid liquid” ” overlays (solutions of overlays (solutions of - methylcellulose, tragacanth tragacanth gum, etc) gum, etc) methylcellulose, High viscousity viscousity -- --> particularly difficult to handle > particularly difficult to handle High in microplate microplate format format in M.Matrosovich M.Matrosovich
Our approach: Thixotropic Thixotropic gels gels Our approach: The viscosity decreases as shear rate increases The viscosity decreases as shear rate increases (Examples: yogurt, ketchup) (Examples: yogurt, ketchup) M.Matrosovich M.Matrosovich
TM (FMC Avicel TM (FMC BioPolymer BioPolymer) ) Avicel - 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
TM (FMC Avicel TM (FMC BioPolymer BioPolymer) ) Avicel 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
Plaque assays under Avicel vs vs agar agar Plaque assays under Avicel 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
Avicel vs. methylcellulose, MDCK- -SIAT1 cells SIAT1 cells Avicel vs. methylcellulose, MDCK M.Matrosovich M.Matrosovich
Plaque formation by different human and avian viruses Plaque formation by different human and avian viruses M.Matrosovich M.Matrosovich
Assay variants in 96- -well plate well plate Assay variants in 96 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- -contamination contamination perform, lower chances of cross M.Matrosovich M.Matrosovich
Detecting drug- -resistant viruses resistant viruses Detecting drug MDCK- -SIAT1, 96 SIAT1, 96- -well format well format MDCK The viruses were kindly provided by The viruses were kindly provided by Robert Webster Robert Webster M.Matrosovich M.Matrosovich
Step 1. Step 1. Seed MDCK- -SIAT1 cells in 96 SIAT1 cells in 96- -well plate well plate Seed MDCK M.Matrosovich M.Matrosovich
Step 2. Step 2. Wash the cells 3- -4 times with serum 4 times with serum- -free medium free medium Wash the cells 3 M.Matrosovich M.Matrosovich
Step 3. Step 3. Add 10- -fold serial dilutions of the drug, 50 ul/well fold serial dilutions of the drug, 50 ul/well Add 10 H G F E D C B A 1 2 3 4 5 6 7 8 9 10 11 12 0.004 0.04 400 0.4 40 0 0 4 Drug concentration, uM uM Drug concentration, M.Matrosovich M.Matrosovich
Step 4. Step 4. Add 3- -fold serial dilutions of the virus, 50 ul/well fold serial dilutions of the virus, 50 ul/well Add 3 H G F E D C B A 1 Dilution 1 Dilution 1 2 3 4 Dilution 3 Dilution 3 5 6 7 Dilution 9 Dilution 9 8 9 10 Dilution 27 Dilution 27 11 12 4 0 4 0 0 0 4 0 0 . . . 0 0 0 0 0 4 4 4 Drug concentration, uM uM Drug concentration, M.Matrosovich M.Matrosovich
Step 5. Step 5. Mix, incubate 1- -2 h for initiation of infection 2 h for initiation of infection Mix, incubate 1 H G F E D C B A VIRUS VIRUS 1 2 Dilution 1 Dilution 1 3 4 5 Dilution 3 Dilution 3 6 7 8 Dilution 9 Dilution 9 9 10 11 Dilution 27 Dilution 27 12 4 0 4 0 0 0 4 0 0 . . . 0 0 0 0 0 4 4 4 Drug Drug M.Matrosovich M.Matrosovich
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