Antiviral Resistance Testing

Abstract

The number of antiviral drugs approved for clinical use has dramatically increased in the past 25 years. However, the use of the drugs may result in the development of resistance mutations in viral genes targeted for therapy. As a result, antiviral resistance testing is commonplace in many molecular diagnostic laboratories and has become a useful tool for the clinical management of viral infections that can be treated with antivirals. Antiviral resistance testing involves identification of the mutations using sequencing methods (genotyping) and determination of their effect on viral drug susceptibility using in‐vitro cell‐based assays (phenotyping). For viruses with well characterised resistance mutations, such as human immunodeficiency virus, genotypic methods that are fast and easier to perform are sufficient. However, phenotypic methods or a combination of both methods provide a comprehensive strategy for other viruses.

Key Concepts:

  • The majority of antiviral drugs target virus‐specific proteins important for viral replication.

  • Antiviral resistance develops via the emergence of mutations in viral genes targeted for therapy.

  • There are two main methods for the measurement of antiviral resistance namely, genotypic antiviral resistance testing and phenotypic drug susceptibility testing.

  • Genotypic antiviral resistance testing identifies mutations associated with drug resistance using sequencing techniques.

  • Genotypic interpretation systems (GISs) are rule‐based computational tools that rely on a database of sequence, clinical and laboratory information to translate the mutational pattern in amplified sequence into drug susceptibility levels using arbitrary scores.

  • Phenotypic drug susceptibility testing is a direct measure of the ability of a virus isolate to replicate in the presence of a specific drug using an in‐vitro cell culture system.

  • The clinical significance and utility of detecting minority drug resistance mutations present at less than 20% of the population has yet to be fully determined.

Keywords: antiviral resistance; antiviral drug; genotypic assay; phenotypic assay; HIV; HBV; HCV; influenza; herpesviruses

Figure 1.

Schematic of the two methods used for antiviral resistance testing of virus isolated from a patient undergoing antiviral therapy. Genotypic antiviral resistance testing (GART) involves the identification of drug resistance mutations (red circle) using sequencing methods. Phenotypic drug susceptibility testing directly measures the susceptibility of a virus isolate to a specific drug using an in‐vitro cell culture system. It determines the concentration of drug required to inhibit the replication of the virus isolated from the patient by 50% (IC50) compared with that of reference wild‐type virus. The dose–response graph shows a shift in the curve of patient‐derived resistant virus (red) compared with wild‐type virus (black) representing an approximately 20‐fold decrease in drug susceptibility (IC50 value) of patient virus.

Figure 2.

Genotypic interpretation report. An example of a genotypic interpretation report from the Stanford HIV drug resistance database showing a report on an HIVRT sequence containing an NRTI (M184V) and NNRTI (K103N) mutation. The report has different sections including: ‘summary data’ showing the segment of sequence being analysed and subtyped; ‘drug resistance interpretation’ showing the mutations identified and the susceptibility of the sequence to different drugs; and ‘mutation scoring’ showing the susceptibility score of a mutation to individual drugs, from 0 to 70 (the higher the score, the higher the level of resistance each mutation is known to cause as determined from clinical and laboratory data). A negative score indicates that the mutation causes hypersusceptibility.

Reproduced with permission from http://hivdb.stanford.edu/. © Stanford HIV drug resistance database.
Figure 3.

An overview of virus drug susceptibility assays. (a) Single round replication assay used to analyse HIV drug susceptibility. Segments of virus genome, for example, protease and RT, are amplified from patient‐derived virus and cloned into recombinant test vectors that have an inactivated envelope gene and express a reporter gene (e.g., luciferase, shown in yellow). The test vector together with a vector expressing virus envelope glycoprotein (red) is cotransfected into cells to produce pseudotyped recombinant virus particles capable of a single round of replication. The pseudoviruses are then used to infect target cells in the presence of different concentrations of drug and infectivity is analysed by detecting luciferase light emission using a luminometer. The results are then analysed using dose–response inhibition curves to determine the drug concentration required to inhibit viral replication by 50% (IC50). (b) Plaque reduction and enzyme inhibition assays used to analyse herpesviruses and influenza virus drug susceptibility. Viruses are isolated using cell culture techniques from a sample collected from an infected patient. For herpesviruses, the cultured virus is titrated and used to infect target cells in the presence of different concentrations of drug and viral infectivity is assessed by plaque reduction assay. For influenza virus, the cultured virus is titrated and incubated with different concentrations of drug and enzymatic activity assessed by adding a fluorescent or chemiluminescent substrate, which is detected using a fluorometer or luminometer, respectively. The results obtained from the assays are then analysed using dose–response inhibition curves to determine the drug concentration required to inhibit viral replication by 50% (IC50).

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Mbisa, Jean Lutamyo(Sep 2013) Antiviral Resistance Testing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024795]