  The sequence diversity of viral populations within individual hosts is the
starting material for selection and subsequent evolution of RNA viruses such as
foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS)
performed on a Genome Analyzer platform (Illumina), this study compared the
viral populations within two bovine epithelial samples (foot lesions) from a
single animal with the Inoculum used to initiate experimental infection.
Genomic sequences were determined in duplicate sequencing runs, and the
consensus sequence determined by NGS, for the Inoculum, was identical to that
previously determined using the Sanger method. However, NGS reveals the fine
polymorphic sub-structure of the viral population, from nucleotide variants
present at just below 50% frequency to those present at fractions of 1%. Some
of the higher frequency polymorphisms identified encoded changes within codons
associated with heparan sulphate binding and were present in both feet lesions
revealing intermediate stages in the evolution of a tissue-culture adapted
virus replicating within a mammalian host. We identified 2,622, 1,434 and 1,703
polymorphisms in the Inoculum, and in the two foot lesions respectively: most
of the substitutions occurred only in a small fraction of the population and
represent the progeny from recent cellular replication prior to onset of any
selective pressures. We estimated an upper limit for the genome-wide mutation
rate of the virus within a cell to be 7.8 x 10-4 per nt. The greater depth of
detection, achieved by NGS, demonstrates that this method is a powerful and
valuable tool for the dissection of FMDV populations within-hosts.
