Phenotypic assays use a variety of end-point measurements to determine whether a virus is inhibited by an antiviral drug or demonstrates drug resistance. Some of these end-point measurements include a reduction in the number of plaques, inhibition of viral DNA synthesis, a reduction in the yield of a viral structural protein, or a reduction of the enzymatic activity of a functional protein. As mentioned, an advantage of phenotypic assays is that they are much better for assessing the combined effect of multiple-resistance mutations on drug susceptibility. This is useful for assaying viruses such as hepatitis B virus (HBV), human immunodeficiency virus type 1 (HIV-1), and human cytomegalovirus (HCMV), which acquire resistance mutations in multiple genes. The disadvantages of phenotypic assays are that they are labor intensive, expensive, and require weeks to perform.

Plaque Reduction Assay

The plaque reduction assay (PRA) is the standard method of antiviral susceptibility testing to which new methods are compared. CLSI has developed standardized PRA protocols for antiviral susceptibility testing of HSV. This test is based on the principle of inhibition of viral plaque formation in the presence of an antiviral agent. The concentration of antiviral drug that inhibits plaque formation by 50% is the IC50; that is, the 50% inhibitory concentration and 50% effective concentration.

DU Assay

The dye uptake (DU) assay has been used for years in the susceptibility testing of HSV. When the virus is in the presence of an antiviral drug, only cells that are alive and viable take up a vital dye called neutral red. Following infection with HSV, the relative amount of dye bound to viable cells compared with that bound to uninfected cells determines the extent of the viral lytic activity. The drug concentration that inhibits viral lytic activity by 50% is the IC50.

DNA Hybridization

The DNA hybridization assay measures the effect of antiviral reagents on the synthesis of viral DNA. The assay semiquantitatively measures how much viral DNA is produced in the presence of antiviral agents compared with how much is produced in the absence of antiviral agents; the IC50 is calculated from these comparisons. These assays have been successfully used for susceptibility testing of HSV, varicella zoster virus (VZV), and HCMV.

Enzyme Immunoassay

The enzyme immunoassay (EIA) uses spectrophotometry analysis to quantitatively measure the amount of viral activity. The concentration of antiviral agent that reduces the amount of absorbance by 50% compared with the absorbance values of a viral control is the IC50. This method of susceptibility testing has been used for influenza A, HSV, and VZV.

Flow Cytometry

 Flow cytometry readily distinguishes drug-resistant iso lates from drug-susceptible isolates; in addition, it is time saving, and it has been used to detect viral susceptibility in HCMV infections and in treatment with the drug ganciclovir. It uses a fluorochrome-labeled monoclonal anti body to an HCMV early antigen. Flow cytometry is used to quantitate the number of virus-infected cells; the instrument quantitates the number of antigen-positive cells in the absence and the presence of antiviral com pounds. The advantages of this assay are that it can be automated; it is time-saving; and it is easier to use and less objective than other phenotypic susceptibility assays.

Neuraminidase Inhibition Assay

The neuraminidase inhibition assay is used to detect neuraminidase (NA) inhibition resistance when the drugs oseltamivir and zanamivir are used to treat influenza A and influenza B infections. Oseltamivir and zanamivir act by inhibiting the influenza viral protein neuraminidase. Resistance to these drugs is measured by incubating cultured influenza isolates containing neuraminidase with varying concentrations of the drugs. A fluorogenic substrate is then added, allowing the fluorescence to be quantitated by a fluorimeter. The IC50 is calculated by comparing the activity of viral NA to a control reaction that does not use any neuraminidase inhibitors.

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مواضيع ذات صلة

Methods of Antiviral Susceptibility Testing: Influenza

Methods of Antiviral Susceptibility Testing: Genotypic Susceptibility Assays

Pathogenesis and Pathology of Rabies Virus

Properties of the Rabies Virus

Coronavirus Infections in Humans

Properties of Coronaviruses

Congenital Rubella Syndrome

Postnatal Rubella

Hendra Virus and Nipah Virus Infections

Measles (Rubeola) Virus Infections

Rhabdoviruses

Retroviruses