Consecutive A (H3N2) influenza virus isolates from 2 influenza virus-infected immunodeficient patients treated with amantadine were examined using a novel polymerase chain reaction (PCR)-restriction analysis for resistance to this antiviral compound. The data indicate that immunodeficient patients may shed resistant viruses for prolonged periods and with different drug resistance mutations present at different times. This PCR-restriction technique allows rapid detection of amantadine- or rimantadine-resistant strains.

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We provide evidence that the quasispecies nature (extreme genetic heterogeneity) of foot-and-mouth disease virus is relevant to the virus evading an immune response.A monoclonal antibody neutralizing the viral infectivity ( Foot-and-mouth disease virus (FMDV) is a picornavirus that causes the economically most important viral disease of cattle and other cloven-hooved animals (1, 2). As for most other RNA genomes, FMDV populations are genetically heterogeneous (3-5) and show the potential for very rapid evolution (6, 7). A direct consequence of the genetic variability of FMDV is its remarkable antigenic diversity (1, 2). Attempts to quantify such diversity and classify FMDV isolates have included the early serological subtyping (8) and the more recent analyses of reactivity with monoclonal antibodies (mAbs) (9)(10)(11). Knowledge of the types and frequency of occurrence of amino acid substitutions that lead to variations in viral epitopes is relevant to the design of new synthetic vaccines, whose efficacy may be hampered by the rapid antigenic variation of RNA viruses in nature (7,(10)(11)(12) In recent years, we have emphasized that FMDV shows a quasispecies structure (3-5), probably shared by most other RNA genetic elements (6, 7, 12, 24). This concept, which originated in theoretical work by Eigen and his colleagues (25)(26)(27), was first shown experimentally to adequately describe populations of phage QP3 (28) and later other RNA genomes as well (reviewed in refs. 6, 7, 12, and 24). According to the quasispecies structure, each FMDV genome population includes one or several "master" sequence(s)-which may nevertheless represent a small proportion of molecules (4, 24, 28)-and a "mutant spectrum" consisting of a distribution of single and multiple residue mutants (4,12,24,27,28). The proportion of each mutant depends on the frequency with which it arises by mutation and on its competitive growth with all other variants (present and arising) in the replicating population (26-28). Mutant viruses with chemically conservative amino acid substitutions, prone to behave in a quasineutral fashion, are more likely to be represented in a viral quasispecies than variants with mutations that adversely affect fitness even if viable, as documented with phage QB (28). In the present study we show that chemically conservative amino acid substitutions fixed at the main antigenic determinant of FMDV C during one epizootic-and that are also found frequently among other FMDV isolatesgreatly affected the interaction with one neutralizing mAb (n-mAb), SD6. This antibody recognizes an epitope surrounding a conserved amino acid sequence of VP1, proposed to be a receptor binding site (17, 18, 44). The effect of these naturally occurring amino acid changes has been mimicked with substituted synthetic peptides, which bind n-mAb SD6 to the same extent as complete variant viruses, thus providing an in vitro assay for epitopic variation of FMDV. MATERIALS AND METHODSViruses. The origin of the FMDV field isolates (C-S isolates) has b...