Plaque-reduction-assay

Fig. 1.2 a Microtiter plate illustrating dose response activity of an extract of the red alga Gigartina skottsbergii (A4) as weU as unrelated and ineffective extract in well F2. b Plaque reduction assay depicting a marked reduction of virus replication in cells treated with extract A4 (50 p-g/mL) compared with No Drug or the unrelated ineffective extract F2. The effect of A4 is similar to that of the control drug ribavirin... 

Pyrazolo[3,4-, pyrimidines 427 and 428 were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds provide remarkable evidence that they are very specific for human enteroviruses, in particular coxsackie viruses <2004BML2519>. 

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... 

Fig. 9. Zanamivir derivatives modified through alkylation of the C-7 hydroxyl group. Values in parentheses reflect IC50 values against influenza A virus in a plaque-reduction assay, and are relative to a reference value of 1.0 for Zanamivir (1).

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. 

Fig. 1. Plaque reduction assay of varicella-zoster virus. (A) Varicella-zoster virus plaques in Mewo cell sheet. The upper row has no drug present. Note toxicity at 100 xM for all compounds tested. (B) Efficacy of compounds A, B, and C. All have IC50 values of <1 pM.

Determination of the Drug-Susceptibility Profile of the Drug-Resistant Strains by Cytopathic Effect (CPE) Reduction Assay or Plaque Reduction Assay... 

The CPE or plaque reduction assays can be performed faster, have less variability than the DNA hybridization assay, do not need special materials and equipment, and avoid the need to handle radioisotopes and nuclear waste. The DNA hybridization technique may be quite useful for confirmatory purposes but not for large-scale evaluation of clinically or in vitro isolated drug-resistant strains. The CPE reduction assay and the plaque reduction assay are the most commonly used tests to evaluate the drug-susceptibility profile of drug-resistant strains, for HSV, VZV, or CMV. 

One of the first standardized phenotypic tests was the HeLa CD4+ plaque reduction assay (4). Although this assay is easy to perform, inexpensive and has a high reproducibility, it is rather labor-intensive and is only suitable for syncytium-inducing (SI) strains. Most patient isolates contain nonsyncytium-inducing (NSI) strains or a mixture of SI and NSI strains. The necessary in vitro cultivation of patient isolates to obtain a high-titer standardized Sl-inocu-lum selects for a subpopulation of SI variants. 

In most labs that perform sensitivity tests on clinical HIV isolates, the HeLa CD4+ plaque reduction assay has been replaced by the more generally useful peripheral blood mononuclear cell (PBMC)-based culture system. More than 80% of clinical isolates can be grown in PBMCs, but selection of variant strains cannot be excluded. Japour et al. (5) developed a widely used standardized protocol for drug susceptibility testing in PBMCs. This protocol can be considered the present standard and is called ACTG-DoD protocol (AIDS Clinical Trials Group, Department of Defense). It is described in Subheading 2. 

Larder, B. A., Chesebro, B., and Richman, D. D. (1990) Susceptibilities of zidovudine-susceptible and -resistant human immunodeficiency virus isolates to antiviral agents determined by using a quantitative plaque reduction assay. Antimicrob. Agents Chemother. 34,436 141. 

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). 

Jordan and Seet studied the antiviral effects of Amphotericin B Methyl Ester (AME), an antimicrobial agent, on several vimses including VACV in a plaque reduction assay [64], The concentration of AME resulting in a 50% inactivation of the plaque forming units, after a 60 min exposure at 35°C, was reported to be 5.0 pg/mL. The authors suggested lipid components in the host cell membrane which incorporate into the viral envelope may serve as a susceptible site for AME. 

Antiviral effects of Amphotericin B Methyl Ester by a plaque reduction assay [64]). 

Toxicity of glutaraldehyde and oxidized spermine using a plaque reduction assay [4]. 

Table I. Comparative Anti-HSV Activity of FMAU and FEAU in Plaque Reduction Assays in Vero Cells...

Using a plaque reduction assay, the ability of each compound to prevent viral growth is summarized in Table 8.2. 

Table 8.4 Plaque-reduction assay results for the platinum-tilorone polymers...

De Logu et al. (2000) investigated the inactivation of HSV-1 and HSV-2 and the prevention of cell-to-cell virus spread by the EO of the Asteraceae Santolina insularis. The plaque-reduction assay showed an IC50 values of 0.88 p.g/mL for HSV-1 and 0.7 xg/mL for HSV-2, respectively, whereas another test on Vero cells showed a cytotoxic concentration (CC50) of 112 p.g/mL, which leads to a CC50/IC50 ratio of 127 for HSV-1 and 160 for HSV-2. These findings indicate that the antiviral effect of the EO was caused by direct virucidal effects. There was no antiviral activity detected in a postattachment assay. Dne to attachment assays it was shown that virus adsorption was not reduced. Additionally, the reduction of plaqne formation assay indicated that the EO reduced cell-to-cell transmission of both HSV-1 and HSV-2. 

Primo et al. (2001) examined in vitro the antiviral activity of the EO from the Lamiaceae Minthostachys verticillata (Griseb.) Epling against HSV-1 and PrV using the viral plaque-reduction assay. The EO influences HSV-1 and PrV multiplication, an activity which is attributed to the main constituents of the EO, namely menthone (39.5%) and especially pulegone (44.6%). The therapeutic index values attained 10.0 and 9.5 for HSV-1 and PrV, respectively. 

A lyophylisate of the expressed sap of . purpurea in cultures of mice L-cells (clone 929) at a concentration of 10 ig/ml to 100 Llg/ml exhibited no direct antiviral activity against encephalomyocarditis virus (EMC virus) or vesicular-stomatitis-virus (VSV). An antiviral effect was only observed when the sap was added together with DEAR dextran. DEAE dextran itself showed no effect. The authors concluded an interferon-like activity [145]. By the colorimetric assay according to Finter and by the Plaque-Reduction-Assay it could be shown, that mice L-929 cells or HeLa cells became resistant for 24 h against influenza-, herpes- and vesicular-stomatitis-virus by 50-80%, when the cells were pretreated 4-6 h with 20 ig/ml of an . purpurea expressed sap preparation. Together with hyaluronidase, no effect was observed. The active component could not be inactivated by heat (60-80 C) [146]. 

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