Plaque inhibition assay

Extracts that exhibited significant inhibitory activity, defined as >50% inhibition of CPE at 100 (xg/mL, were advanced into secondary screening, which includes confirmation of activity observed during the primary screen using an expanded range of concentrations (dose response), plaque inhibition assay, and a one-step growth inhibition and testing of additional influenza viruses. As shown in Fig. 1.2a, the quantitative dose response assay was used to assess the potency of the most two... 

Sensitivity of passaged virus to drug is then determined in a plaque inhibition assay, in a microtiter CPE inhibition assay in liquid culture, or in a yield reduction assay. 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

A virus in both sialidase inhibition and plaque-reduction assays. Alkyl ethers up to twelve carbon atoms in length exhibited similar inhibitory activity to Zanamivir against influenza A virus sialidase, however, showed a pronounced improvement in plaque-reduction assay compared to the parent triol 1. Alkylation of the C-7 hydroxyl with two-carbon substituents bearing terminal hydroxyl, amino, azido, and acetamido groups yielded inhibitors 61-64 and did not significantly affect the binding and had similar potency to that of ethyl or propyl ethers 65 or 66 (Fig. 9). 

The NA inhibitory activities of the carbocyclic analogues were evaluated in two assay systems (O Table 2). The intrinsic activity of each compound was assessed by measuring the inhibition of enzymatic activity, and compounds that exhibited potent NA inhibitory activity were further evaluated in cell culture by a plaque reduction assay using an influenza A (HlNl) sfrain. 

Test virus for the resistant phenotype in an enzyme inhibition assay, or in a plaque assay, either when the virus appears more sensitive or after 10 passages. 

M. oleifera extracts inhibits plaque formation of anti-herpes simplex vims type 1 (HSV-1) more than 50% at 100 ag/ml in a plaque reduction assay (55). M. oleifera extracts are also effective against thymidine kinase-deficient HSV-1 and phosphonoacetate-resistant HSV-1 vims strains. The extract ofM. oleifera at a dose of 750 mg/kg body weight per day significantly delays the development of skin lesions, prolongs the mean survival times and reduces the mortality of HSV-1 infected mice. Compared to the synthetic compound acyclovir, M. oleifera extracts delay the development of skin lesions and has mean survival times as acyclovir. A polysaccharide from hot aqueous extract of mature pods (fruits) of M oleifera with a structural repeating unit [->4)-a-D-GlCp(l->] has immunoenhancing properties (76). 

Plaque inhibition or reduction assays Only for viruses which form plaques in suitable cell systems. Titration of a limited viruses number or residual viruses infectivity after extracellular action of the tested compound. The tested compound must be in a non-toxic dose or cytotoxicity should be eliminated by dilution or filtration before the titration. 

Plaque assay When a virus particle initiates an infection upon a layer or lawn of host cells which is growing spread out on a flat surface, a zone of lysis or growth inhibition may occur which results in a clearing of the cpll growth. This clearing is called a plaque, and it is assumed that each plaque has originated from one virus particle. 

Inhibiting the production of the Aft peptides represents the most direct approach to curtailing their potential to accumulate as amyloid plaques, by inhibiting either the ft-sec-retase at step 1 or the y-secretase at step 2. Because these are enzymatic reactions with measurable products, a biochemical assay using a purified enzyme preparation can be integrated into an HTS platform, facilitating the rapid evaluation of large numbers of compounds for inhibition of the enzymatic activity. 

Zanamivir (2) is a potent competitive inhibitor of viral neuraminidase glycoprotein, which is essential in the infective cycle of both influenza A and B viruses. It inhibits a wide range of influenza A and B types in vitro as well as in vivo. The concentrations of inhibiting in vitro plaque formation of influenza A and B virus by 50% in Madin-Darby canine kidney (MDCK) cells were 0.004-0.014 p.mol/L in laboratory-passaged strains, and 0.002-16 p.mol/L in assays of clinical isolates. Due to its low bioavailability, it is delivered by inhalation via the Diskhaler , 10 mg twice daily, or intranasally 2-4 times daily for 5 days. After an intravenous dose of 1 -16 mg, the median elimination half-life was ti/2 = 7 h, the volume of distribution at steady state was Vdss = 16 L, and 90% of the dose was excreted unchanged in the urine. After intranasal and inhaled (dry powder) administration, maximum serum concentrations occurred within 2h and the terminal phase half-lives were 3.4 and 2.9 h, respectively. The bioavailabilities were 10 and 25%, respectively, and 20% after inhalation of zanamivir (2) by nebulizer. 

The assay for interferon involves incubating cells overnight with increasing dilutions of interferon and then challenging the cells with, say, vesicular stomatitis virus (VSV) at 20 p.f.u. per cell. Twenty hours later the culture fluids are harvested and assayed for VSV using a plaque assay ( 14.3.1) on mouse cells. The greatest dilution of interferon which inhibits virus yield by 3.2 fold (0.5 log10) contains 1 unit of interferon (Baron, 1969). 

The cell-wall sulphated PS of the red microalga Porphyridium spp. also appears to be a good candidate for the development of an anti-HSV drug [74,75]. Treatment of cells with 1 pg/ml PS resulted in 50% inhibition of HSV infection as measured by the plaque assay. Inhibition of the production of new viral particles was also shown when pre-infected cell cultures were treated with the PS. It seems therefore that the PS is able to inhibit viral infection by preventing adsorption of the virus into the host cells and/or by inhibiting the production of new viral particles inside the host cells. The cell-wall sulphated PS of this red microalga Porphyridium spp. also had impressive antiviral activity against VZV [74]. 

The cytotoxicity of distamycin derivatives was estimated on the basis of the morphological modifications induced in HeLa cell cultures, after incubation for 40 h in Hanks saline solution + 0.5 % lactalbumin hydrolysate + 5 % calf serum (HLS). Assay on vaccinia virus Cultures of HeLa cells (grown in HLS medium) or mouseembryo cells (grown in HLS medium plus 0.1 % yeastolate) infected with vaccinia virus (Strain WR/ATCC) were used. Preliminary assays were made according to Herrmann et al.° Subsequent studies were carried out by assessing the inhibition of plaque formation (ECP) as well as the inhibition of infectious virus production in test tube cultures treated with the compounds for 40 h after the absorption of the virus. 

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