Chapter 8: Antiviral Agents and Resistance

Viruses are composed of either DNA or RNA, a protein coat (capsid), and, in many, a lipid or lipoprotein envelope. The nucleic acid codes for enzymes involved in replication and for several structural proteins. Viruses use molecules (eg, amino acids, purines, pyrimidines) supplied by the cell and cellular structures (eg, ribosomes) for synthetic functions. Thus, one of the challenges in the development of antiviral agents is identification of the steps in viral replication that are unique to the virus and not used by the normal cell. Among the unique viral events are attachment, penetration, uncoating, RNA-directed DNA synthesis (reverse transcription) or RNA-directed RNA synthesis (RNA viruses), and assembly and release of the intact virion. Each of these steps may have complex elements with the potential for inhibition. For example, assembly of some virus particles requires a unique viral enzyme, protease, and this has led to the development of protease inhibitors. A general scheme for the points of action of antiviral agents is shown in Figure 8–1.

In some cases, antiviral agents do not selectively inhibit a unique replicative event but inhibit DNA polymerase. Inhibitors of this enzyme take advantage of the fact that the virus is synthesizing nucleic acids more rapidly than the cell; therefore, there is relatively greater inhibition of viral than cellular DNA.

In many acute viral infections, especially respiratory ones, the bulk of viral replication has already occurred when symptoms are beginning to appear. Initiating antiviral therapy at this stage is unlikely to make a major impact on the illness. For these viruses, immuno- or chemoprophylaxis, rather than therapy, is a more logical approach. However, other viral infections are characterized by ongoing viral replication and do benefit from viral inhibition, such as human immunodeficiency virus (HIV) infection and chronic hepatitis B and C.

The principal antiviral agents in current use are discussed according to their modes of action. Their features are summarized in Table 8–1.

Inhibitors of Attachment

Attachment to a cell receptor is a virus-specific event. Antibodies can bind to the extracellular virus and prevent this attachment. However, although therapy with antibody is useful in prophylaxis, it has been minimally effective in treatment.

Inhibitors of Cell Penetration and Uncoating

Amantadine and rimantadine are symmetric amines, which are thought to inhibit viral uncoating as their primary antiviral effect.

Rimantadine differs from amantadine by the substitution of a methyl group for a hydrogen ion. They are extremely selective, with activity against only influenza A, where they act as inhibitors of the viral M2 protein. They have been used either as prophylaxis or for therapy. Unfortunately, since 2001, the rates of resistance to amantadine/rimantadine have increased so sharply (up to 100% for some strains) that they are no longer routinely recommended.

Pharmacology and Toxicity

Both amantadine and rimantadine are available only as oral preparations. The pharmacokinetics of the two agents is quite different. Amantadine is excreted by the kidney without being metabolized, and its dose must be decreased in patients with impaired renal function. In contrast, rimantadine is metabolized by the liver, then excreted in the kidney, and dosage adjustment for renal failure is not necessary. The major toxicity is in the CNS—seizures, somnolence, etc.

Neuraminidase Inhibitors

Oseltamivir and zanamivir are antiviral agents that inhibit the neuraminidase of influenza A and B viruses. The neuraminidase cleaves terminal sialic acid from glycoconjugates and plays a role in the release of virus from infected cells. Zanamivir is given by inhalation using a specially designed device. Oseltamivir phosphate is the oral prodrug of oseltamivir, a drug comparable to zanamivir in antineuraminidase activity.

Treatment with either oseltamivir or zanamivir reduces influenza symptoms, shortens the course of illness by 0.5 to 1.5 days, and reduces the rate of complications.

Inhibitors of Nucleic Acid Synthesis

At present, most antiviral agents are nucleoside analogs that are active against virus-specific nucleic acid polymerases or reverse transcriptases and have much less activity against analogous host enzymes. Some of these agents serve as nucleic acid chain terminators after incorporation into nucleic acids.

Idoxuridine and Trifluorothymidine

Idoxuridine (5-iodo-2′-deoxyuridine, IUdR) is a halogenated pyrimidine that blocks nucleic acid synthesis by being incorporated into DNA in place of thymidine and producing a nonfunctional molecule. It is phosphorylated by cellular thymidine kinase to the active compound, which inhibits both viral and cellular DNA polymerase. The resulting host toxicity precludes systemic administration in humans. Idoxuridine can be used topically as effective treatment of herpetic infection of the cornea (keratitis). Trifluorothymidine, a related pyrimidine analog, is effective in treating herpetic corneal infections, including those that fail to respond to IUdR. Trifluorothymidine has largely replaced IUdR.

Acyclovir

This antiviral agent differs from the nucleoside guanosine by having an acyclic (hydroxyethoxymethyl) side chain. The key to its benefit is that it must be phosphorylated by viral thymidine kinase to be active. Therefore, the compound is essentially nontoxic because it is not phosphorylated or activated in uninfected host cells. Viral thymidine kinase catalyzes the phosphorylation of acyclovir to a monophosphate. From this point, host cell enzymes complete the progression to the diphosphate and, finally, the triphosphate. Acyclovir triphosphate inhibits viral replication by competing with guanosine triphosphate and inhibiting the function of the virally encoded DNA polymerase. The selectivity and minimal toxicity of acyclovir is aided by its 100-fold or greater affinity for viral DNA polymerase than for cellular DNA polymerase. A second mechanism of viral inhibition results from incorporation of acyclovir triphosphate into the growing viral DNA chain. This causes termination of chain growth because there is no 3′-hydroxy group on the acyclovir molecule to provide attachment sites for additional nucleotides.

Activity of acyclovir against herpesviruses directly correlates with the capacity of the virus to induce a thymidine kinase. Susceptible strains of herpes simplex virus types 1 and 2 (HSV-1 and -2) are the most active thymidine kinase inducers and are the most readily inhibited by acyclovir. Cytomegalovirus (CMV) induces little or no thymidine kinase and is not inhibited. Varicella-zoster and Epstein-Barr viruses are between these two extremes in terms of both thymidine kinase induction and acyclovir susceptibility.

Resistant strains of HSV have been recovered from immunocompromised patients, including patients with acquired immunodeficiency syndrome (AIDS); in most instances, resistance results from mutations in the viral thymidine kinase gene, rendering it inactive in phosphorylation. Resistance may also result from mutations in the viral DNA polymerase. Remarkably, resistant virus has rarely been recovered from immunocompetent patients, even after years of drug exposure and frequent usage.

Pharmacology and Toxicity

Acyclovir is available in three forms: topical, oral, and parenteral. Topical acyclovir is rarely used. The oral form has low bioavailability (~10%), but achieves concentrations in blood that inhibit HSV and, to a lesser extent, varicella-zoster virus (VZV). Intravenous acyclovir is used for serious HSV infection (eg, congenital, encephalitis) as well as for VZV infection in immunocompromised patients. Because acyclovir is excreted by the kidney, the dosage must be reduced in patients with renal failure. Central nervous system toxicity and renal toxicity have been reported in patients treated with prolonged high intravenous doses. Despite its mechanism of action acyclovir is remarkably free of bone marrow toxicity, even in patients with hematopoietic disorders—a feature attributable to the absence of its phosphorylation (ie, activation) in uninfected host cells.

Treatment and Prophylaxis

Acyclovir is effective in the treatment of primary HSV mucocutaneous infections or for severe recurrences in immunocompromised patients. The agent is useful in neonatal herpes and encephalitis, infection in immunocompromised patients and for varicella in older children or adults. Acyclovir is beneficial against herpes zoster in elderly patients or any patient with eye involvement. In patients with frequent severe genital herpes, the oral form is effective in preventing recurrences. Because it does not eliminate the virus from the host, it must be taken daily to be effective. Acyclovir is minimally effective in the treatment of recurrent genital or labial herpes in otherwise healthy individuals.

Valacyclovir, Famciclovir, and Penciclovir

Valacyclovir is a prodrug of acyclovir that is, better absorbed and, therefore, can be used in lower and less frequent dosage (bioavailability ~60%). When absorbed, it becomes acyclovir. It is currently approved for use in HSV and VZV infections. Dosage adjustment is necessary in patients with impaired renal function.

Famciclovir is similar to acyclovir in its structure and requirement for phosphorylation, but differs slightly in its mode of action. After absorption, the agent is converted to penciclovir, the active moiety, which inhibits viral DNA polymerase. However, it does not irreversibly terminate DNA replication. Famciclovir is currently approved for the treatment of HSV and VZV infections. Penciclovir is approved for topical treatment of recurrent herpes labialis.

Ganciclovir

Ganciclovir (DHPG), a nucleoside analog of guanosine, differs from acyclovir by a single carboxyl side chain. This structural change confers approximately 50 times more activity against CMV than acyclovir. Acyclovir has low activity against CMV because it is not well phosphorylated in CMV-infected cells due to the absence of the gene for thymidine kinase in CMV. However, ganciclovir is active against CMV because another viral-encoded phosphorylating enzyme (UL97) is present in CMV-infected cells that is, capable of phosphorylating ganciclovir and converting it to the monophosphate. Then, cellular enzymes convert it to the active compound, ganciclovir triphosphate, which inhibits the viral DNA polymerase (UL 54). Since ganciclovir can be phosphorylated in normal, uninfected, host cells, toxicity, especially neutropenia, frequently limits therapy. Discontinuation of therapy is necessary in patients whose neutrophils do not increase during dosage reduction or in response to cytokines. Thrombocytopenia (platelet count less than 20 000/mm³) occurs in approximately 15% of patients. Ganciclovir is also active against herpes simplex and VZ viruses but is not the drug of choice for these viruses due to toxicity.

Oral ganciclovir is available, but is inferior to the intravenous form. Oral valganciclovir, a prodrug of ganciclovir, has improved bioavailability and is equivalent to the intravenous form.

Clinical Use

Administration of ganciclovir or valganciclovir is indicated for the prevention or treatment of active CMV infection in immunocompromised patients, but other herpesviruses (particularly HSV-1, HSV-2, and VZV) are also susceptible. Because patients with AIDS with severe CMV infection frequently have concurrent illnesses caused by other herpesviruses, treatment with ganciclovir may benefit associated HSV and VZV infections.

Resistance

After several months of continuous ganciclovir therapy for treatment of CMV, between 5% and 10% of patients with AIDS excrete resistant strains of CMV. In almost all isolates, a mutation is found in the phosphorylating gene (UL97), and in a lesser number a mutation may also be found in the viral DNA polymerase (UL 54). Most of these strains remain sensitive to foscarnet, which may be used as an alternate therapy. If only a UL97 mutation is present, the strains remain susceptible to the nucleotide analog cidofovir (see later in chapter); however, if the CMV strain has a ganciclovir-induced mutation in DNA polymerase (UL 54), the virus is cross-resistant to cidofovir. Ganciclovir resistance has been noted in transplant recipients, in patients with lung or liver transplants, and those requiring prolonged prophylaxis or treatment.

Nucleotide Analogs: Cidofovir

The first example of the nucleotide analogs is cidofovir. This compound has a phosphonate group attached to the molecule and appears to the cell as a nucleoside monophosphate, in effect, a nucleotide. Cellular enzymes then add two phosphate groups to generate the active compound. In this form, the drug inhibits both viral and cellular nucleic acid polymerases, but selectivity is provided by its higher affinity for the viral enzyme.

Nucleotide analogs do not require phosphorylation, or activation, by a viral-encoded enzyme and remain active against viruses that are resistant due to mutations in codons for these enzymes, for example, a UL97 mutant CMV. Resistance to cidofovir can, of course, develop with mutations in the viral DNA polymerase, UL54. An additional feature of cidofovir is a very prolonged half-life as a result of slow clearance by the kidneys.

Cidofovir is approved for intravenous therapy of CMV retinitis, and maintenance treatment may be given as infrequently as every 2 weeks. In addition, it is occasionally used to treat severe, disseminated adenovirus and BK virus infections although its efficacy for these is unproven. Nephrotoxicity is a serious complication of cidofovir treatment, and patients must be monitored carefully for evidence of renal impairment.

Foscarnet

Foscarnet, also known as phosphonoformate, is a pyrophosphate analog that inhibits viral DNA polymerase by blocking the pyrophosphate-binding site of the viral DNA polymerase and preventing cleavage of pyrophosphate from deoxyadenosine triphosphate. This action is relatively selective; CMV DNA polymerase is inhibited at concentrations less than 1% of that required to inhibit cellular DNA polymerase. Unlike such nucleosides as acyclovir and ganciclovir, foscarnet does not require phosphorylation to be an active inhibitor of viral DNA polymerases. This biochemical fact becomes especially important with regard to viral resistance, because the principal mode of viral resistance to nucleoside analogs is a mutation that eliminates phosphorylation of the drug in virus-infected cells. Thus, foscarnet can usually be used to treat patients with ganciclovir-resistant CMV and acyclovir-resistant HSV. Excretion is entirely renal without a hepatic component, and dosage must be decreased in patients with impaired renal function. Multiple metabolic abnormalities occur as evidence of toxicity.

Interferons

Interferons are host cell-encoded proteins synthesized in response to double-stranded RNA (dsRNA) that circulate to protect uninfected cells by inhibiting viral protein synthesis. Ironically, interferons harvested in tissue culture were the first antiviral agents, but their clinical activity was disappointing. Recombinant DNA techniques now allow relatively inexpensive large-scale production of interferons by bacteria and yeasts.

Interferon α is beneficial in the treatment of chronic active hepatitis B and C infection, although its efficacy is often transient. Combinations of interferon-α with lamivudine, famciclovir, and certain nucleotides have been evaluated for treatment of hepatitis B but are being supplanted by newer drugs. Topical or intralesional interferon application is beneficial in the treatment of human papilloma virus infections. Parenteral use can cause symptomatic systemic toxicity (eg, fever, malaise), partly because of its effect on host cell protein synthesis.

Ribavirin

Ribavirin is another analog of the nucleoside guanosine. Unlike acyclovir, which replaces the ribose moiety with a hydroxymethyl acyclic side chain, ribavirin differs from guanosine in that the base ring is incomplete and open. Similar to other nucleoside analogs, ribavirin must be phosphorylated to mono-, di-, and triphosphate forms, but cellular enzymes can carry out each of these, thus, heightening the risk of toxicity. Ribavirin is active against a broad range of viruses in vitro, but its in vivo activity is limited. The mechanism of the antiviral effect of ribavirin is not as clear as that of acyclovir. It is an inhibitor of RNA polymerase, and it also inhibits inosine monophosphate dehydrogenase—an enzyme important in the synthetic pathway of guanosine. Yet another mode of action is by decreasing synthesis of the mRNA 5′ cap because of interference with both guanylation and methylation of the nucleic acid base.

Aerosol administration enables ribavirin to reach concentrations in respiratory secretions up to 10 times greater than necessary to inhibit respiratory syncytial virus (RSV) replication and substantially higher than those achieved with oral administration. Problems encountered with aerosolized ribavirin include precipitation of the agent in tubing used for administration and exposure of healthcare personnel. Thus, its use for RSV infection is not generally recommended.

Oral and intravenous forms have been used for patients with Lassa fever and infections with other arenaviruses, although studies have been limited. In a recent trial of hantavirus treatment, ribavirin was ineffective. A reversible anemia has been associated with oral administration of ribavirin and, in preclinical studies, it was teratogenic, mutagenic, and gonadotoxic.

There are now more than 30 antivirals for HIV in six different drug classes. Antiviral treatment is now recommended for everyone infected by HIV. This recommendation is a departure from the past when it was felt that treatment could wait while patients showed clinical or laboratory abnormalities attributed to HIV. For example, it was once felt that treatment could be withheld until the CD4 lymphocyte count was less than 200/mL. Treatment is beneficial at all stages of infection including primary, early infection, that is, within the first six months. In general, treatment consists of at least two different drug classes. These regimens are referred to as HAART (highly active antiretroviral treatment).

1. Fusion Inhibitors. Enfuvirtide is a synthetic peptide (36 amino acids) which inhibits the fusion of HIV-1 with CD4 cells. The latter is a complex process, including viral attachment and coreceptor binding and is necessary for subsequent viral entry into a cell. As with other HIV antagonists, it should only be used in combination with other classes of HIV inhibitors. There is no oral form, and it is usually reserved for patients failing other therapies.

2. Entry Inhibitors. CCR5 is a molecule very similar to CD4 that acts as a viral receptor. Maraviroc blocks the predominant route of viral entry by interfering with the attachment of HIV gp 120 with the CCR5 receptor on the CD4 lymphocyte surface. Maraviroc is an oral drug which, like all anti-HIV agents, should not be used alone. Resistance may develop by the virus adapting to another receptor, CXCR4.

3. Nucleoside Reverse Transcriptase Inhibitors (NRTIs). Zidovudine (AZT), a nucleoside analog of thymidine, inhibits the reverse transcriptase of HIV by terminating the developing DNA chain. As with other nucleosides, AZT must be phosphorylated; host cell enzymes carry out the process. The basis for the relatively selective therapeutic effect of AZT is that HIV reverse transcriptase is more than 100 times more sensitive to AZT than is host cell DNA polymerase. Nonetheless, toxicity frequently occurs.

   AZT was the first useful treatment for HIV infection, but now is recommended for use only in combination with other inhibitors of HIV replication.Toxicity includes malaise, nausea, and bone marrow toxicity. All hematopoietic components may be depressed, but they usually reverse with discontinuation of the drug or dose reduction. Resistance is associated with one or more mutations in the HIV reverse transcriptase gene.

   A series of oral compounds similar to AZT have been developed and are used in combination with other HIV antivirals. Although they have similar mechanisms of action, their side effects may differ. These compounds include didanosine (ddI, dideoxyinosine) which has serious adverse effects of treatment including peripheral neuropathy and pancreatitis; both conditions are dose-related.

   Stavudine (D4T) is another nucleoside analog that inhibits HIV replication by terminating the growth of the chain of viral nucleic acid. D4T is well absorbed and has a high bioavailability. Adverse effects include headache, nausea and vomiting, asthenia, confusion, and elevated serum transaminase and creatinine kinase. A painful sensory peripheral neuropathy that appears to be dose-related may occur. D4T should be used only in combination with other anti-HIV agents.

   Lamivudine (3TC), another oral nucleoside reverse transcriptase inhibitor, is a comparatively safe and usually well-tolerated agent. It is used in combination with AZT or other nucleoside analogs.

   Abacavir, tenofovir, and emtricitabine are newer oral nucleoside reverse transcriptase inhibitors which, like those discussed earlier, should only be used in combination with other classes of HIV antivirals. They appear to be less toxic than older NRTIs especially for “mitochondrial toxicity” manifest as myopathy, neuropathy, hepatic failure, and lactic acidosis.

4. Nonnucleoside Reverse. Transcriptase Inhibitors Certain oral compounds that are not nucleoside analogs also inhibit HIV reverse transcriptase by binding to it and preventing conversion of HIV RNA, not HIV DNA. Several compounds, such as nevirapine, efavirenz, etravirine, and rilpivirine have been evaluated alone or in combination with other nucleosides. They are collectively referred to as nonnucleoside reverse transcriptase inhibitors (NNRTIs). These compounds are very active against HIV-1, do not require cellular enzymes to be phosphorylated, and bind to, essentially, the same site on reverse transcriptase. Cross-resistance does not occur between nucleoside RT inhibitors and NNRTIs, but does occur between one NNRTI and another. Unfortunately, drug resistance readily emerges with even a single passage of virus in the presence of drug in vitro and in vivo. Thus, NNRTIs should be used only in combination regimens with other drugs active against HIV.

5. Protease Inhibitors. Additional oral agents that inhibit HIV are the protease inhibitors. These agents block the action of the viral-encoded enzyme protease, which cleaves polyproteins to produce viral proteins. Inhibition of this enzyme leads to blockage of viral assembly and release. The protease inhibitors are potent suppressors of HIV replication in vitro and in vivo, particularly when combined with other antiretroviral agents. These drugs do not require intracellular phosphorylation for activation.

   In late 1995, saquinavir was the first protease inhibitor to receive approval. Ritonavir, indinavir, nelfinavir, darunavir, fosamprenavir, and tipranavir are other potent protease inhibitors that have since been released. These drugs may cause hepatotoxicity as all agents inhibit P450, resulting in important drug interactions. They also appear to cause lipodystrophy. Because drug resistance to all protease inhibitors develops, these agents should not be used alone without other anti-HIV drugs. Lopinavir is a protease inhibitor which is marketed in combination with ritonavir. Atazanavir, another protease inhibitor is usually prescribed with ritonavir to increase serum concentration of atazanavir. Ritonavir, itself, is a “booster,” increasing the effectiveness of other protease inhibitors (PIs).

6. Integrase Inhibitors. HIV integrase aids the insertion of viral DNA into host cell DNA. This occurs after the viral reverse transcriptase (RNA/DNA-dependent DNA polymerase) produces double-stranded viral DNA. This step is key to the cell becoming a permanent carrier. Three integrase inhibitors, raltegravir, elvitegravir, and dolutegravir are approved for use in the United States. They are oral and are used in combination with other classes of antiretrovirals.

Acute hepatitis is not usually treated since most infections will resolve on their own. Treatment is reserved for patients with chronic active hepatitis B especially those with cirrhosis, liver inflammation, and high viral loads. The mainstays of treatment are: (1) interferons (interferon-α and Peg-IFN) or (2) inhibitors of hepatitis B DNA replication.

Interferon

In general, interferon slows the replication of the virus and/or enhances immune responses. Pegylated interferon can be given parenterally weekly, compared with thrice weekly for interferon-α and is the interferon of choice. Both products have a high incidence of side effects, with an “influenza-like” syndrome being very common. A 48-week course of pegylated interferon-based treatment is successful, that is, viral DNA and HBsAg clearance in only 3% to 7% of patients and even then is not a cure due to intrahepatic viral persistence.

Nucleoside/Nucleotide (NUC) Analog Inhibitors

Lamivudine was the first inhibitor of hepatitis B DNA polymerase (reverse transcriptase [RT]) to be employed clinically. It has been followed—and supplanted—with similar molecules that are less prone to resistance development. Of these, entecavir and tenofovir have become the preferred agents for monotherapy due to their potency and very low rates of resistance development. The other polymerase inhibitors should not be used as monotherapy because of the ease with which resistance may develop. Unfortunately, NUC-based therapy only clears virus and HBsAg in 1% of patients.

As of 2016 there are at least 11 different FDA-approved antivirals for the treatment of hepatitis C. These include interferon and ribavirin as well as direct acting antivirals (DAA) which include protease polymerase and the NS5A phosphoprotein antivirals. These are used in various combinations, but usually with at least two different drugs to enhance efficacy and/or reduce the development of resistance. Most of the studied combinations no longer require the use of ribavirin, thereby avoiding its associated anemia. The use of interferon is disappearing, a welcome development to eliminate unpleasant side effects and parenteral treatment. Recommended treatments vary with the genotype of the patient’s virus. Genotype 1A, the cause of approximately 70% of cases in the United States is the most difficult to eradicate. The presence of cirrhosis also determines which combination regimen is recommended as does the subtype of genotype 1 (A vs B) and resistance due to prior treatment. Cures, defined as undetectable viral RNA for at least 12 weeks following the completion of treatment, may occur in over 90% of patients. This is referred to as a sustained viral response (SVR) and is 97% to 100% predictive of a cure.

Viral genomes and their replication, as well as the mechanisms of action of the available antiviral agents, have been intensively studied. Accordingly, an understanding of resistance to antiviral drugs has evolved; investigation of resistance mechanisms has shed light on the function of specific viral genes and the central role of gene mutations. For example, it has become clear that a common mechanism of resistance to nucleosides (eg, acyclovir and ganciclovir) by herpesviruses consists of mutations in the viral-induced enzyme responsible for phosphorylating the nucleoside. For HSV, this is thymidine kinase; for CMV, this gene is designated UL97.

The likelihood of resistant mutants results from at least four factors:

1. Rate of viral replication. Herpesviruses, especially CMV and VZV, do not replicate as rapidly as HIV and hepatitis B and C viruses. Higher rates of replication are associated with higher rates of spontaneous mutations.

2. Selective pressure of the drug. The greater the drug exposure, the more rapid the emergence of resistant mutants up to a point. With still greater drug exposure, viral replication and resistant mutants decrease until viral replication ceases.

3. Rate of viral mutations. In addition to viral replication, the rate of mutations differs among different viruses. In general, single-stranded RNA viruses (eg, HIV and influenza) have more rapid rates of mutation than double-stranded DNA viruses (eg, HSV).

4. Rates of mutation in differing viral genes. For example, within the herpesviruses, the genes for phosphorylating nucleosides (eg, UL97) are more susceptible to mutation than the viral DNA polymerase.

Resistance to antiviral agents may be detected in several ways:

• Phenotypic. This is the traditional method of growing virus in tissue culture in medium containing increasing concentrations of an antiviral agent. The concentration of the agent that reduces viral replication by 50% is the end point, and is referred to as the inhibitory concentration (IC₅₀). The IC₅₀ of resistant virus is higher than that of susceptible virus. The degree of viral replication is obtained by counting viral plaques (ie, equivalent to viral “colonies”) or by measuring viral antigen or nucleic acid concentration. Unfortunately, phenotypic assays are very time-consuming, requiring days to weeks for completion. IC₅₀ values increase as the percentage of the viral population with the mutation increases.

• Genotypic. When the exact mutation or deletion responsible for antiviral resistance is known, it is possible to sequence the viral gene or detect it with restriction enzymepatterns. These tests are rapid but require knowledge of the expected mutation, and they do not provide quantitation of the percentage of the viral population harboring the mutation. If only 1% to 5% of the population has the mutation, this result may not be detected.

• Viral quantitation in response to treatment. Various methods of quantitating virus (eg, culture, polymerase chain reaction, antigen assay) provide a means of assessing the decline of viral titer in response to treatment with an antiviral agent. These assays are rapid, and do not require knowledge of the expected mutation. If no decline occurs despite adequate dosage and compliance, viral resistance may be responsible. Likewise, if viral titer initially decreases but subsequently recurs and/or increases, then resistance may have developed.