RNA viruses use error-prone RdRp (mutation rate ~10–4 to 10–6 per nucleotide per cycle). In HIV: ~10 billion virions/day → every possible single-point mutation generated many times daily in one patient. Resistant variants pre-exist before therapy as minority variants. Drug eliminates wild-type virus → resistant minority expands to dominance. This is why combination therapy is required: simultaneous resistance to 2–3 mechanistically diverse agents requires multiple independent mutations — occurring at rates many orders of magnitude lower than a single mutation.

Low fitness cost: resistant strain replicates as well as wild-type → spreads without drug pressure. Influenza S31N (adamantane resistance) swept from 2% to 96% global prevalence in one season through natural transmission. High fitness cost: resistant variant replicates poorly → reverts when drug removed; rarely transmitted. DTG/BIC INSTI resistance mutations: high fitness cost → near-zero transmitted resistance. Fitness cost determines whether resistance is a patient-level problem or a public health crisis.

Sequences viral gene regions → identifies mutations at resistance-associated codons. Standard Sanger sequencing: detects mutations in >20% of viral population. Next-generation sequencing (NGS): detects minority variants at 1–5% frequency. Results interpreted against curated databases (Stanford HIVdb, Rega, ANRS). Perform while on failing regimen — wild-type rapidly outcompetes resistant variants after treatment stops; resistance mutations may become undetectable within weeks of drug discontinuation.

Directly measures IC50 of resistant virus vs. wild-type in cell culture. Result: fold-change in susceptibility. Most informative when novel mutation combinations make genotypic interpretation uncertain. Limitations: expensive, slow (2–3 weeks), requires viral culture. Virtual phenotype: computational prediction of phenotypic result from genotypic data using large paired databases — faster and cheaper than true phenotype.

TK mutation (most common): acyclovir, valacyclovir, penciclovir all lose activity (all require TK). Foscarnet and cidofovir retain activity (do not need TK). DNA polymerase mutation: may reduce acyclovir AND foscarnet susceptibility (both act on polymerase). Cidofovir may retain activity (different binding site). Clinical rule: acyclovir-resistant HSV → switch to foscarnet. If foscarnet also fails → cidofovir.

NS5A RASs (positions 28, 30, 31, 93): clinically relevant for 1st-gen agents (ledipasvir, elbasvir). Velpatasvir and pibrentasvir retain activity against most NS5A RASs → baseline testing not routine for SOF/VEL or GLE/PIB. NS5B sofosbuvir: S282T resistance — severe fitness cost, never documented clinically despite cell culture evidence. Sofosbuvir is the pharmacological anchor precisely because of this absolute resistance barrier.