| Abstract: | Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.The viral population within each human immunodeficiency virus (HIV)-infected individual is highly diverse and constantly evolving (2, 3). However, our understanding of the viral population is based largely on the consensus sequence of the dominant circulating virus because the full diversity of the viral population is extremely difficult to characterize. One recent study showed that despite viral fitness recovery in vitro, recovery was not correlated with changes observed in the consensus sequence of HIV. Instead, increased fitness correlated with general viral heterogeneity (5). This finding suggests that by limiting our studies to consensus sequences, we are missing many aspects of viral evolution that influence fitness, drug resistance, and immune evasion, among other characteristics. Studies that examined minor viral variants have provided new insights into HIV transmission and pathogenesis, with direct implications for HIV treatment (7, 13). Unfortunately, traditional techniques to identify rare variants, such as molecular cloning, single-genome amplification, or quantitative real-time (qRT)-PCR, are either labor intensive or restricted to the detection of single variants, limiting their widespread use (8, 11, 12, 14).New second-generation technologies have radically altered DNA sequencing. Recent work by our group and others has employed pyrosequencing for targeted ultradeep sequencing of short regions of the viral genome, including CD8+ T-lymphocyte epitopes and regions of known drug resistance mutations, demonstrating a practical method to identify extremely low-frequency viral variants (4, 15). While sequencing short regions is appropriate in certain circumstances, the region of interest must be identified in advance, and the effect of mutations in that region on the remaining genome is ignored. Studying the heterogeneity of HIV across the entire genome may provide insights into interactions between minor variants, improve our understanding of HIV evolution, and ultimately provide insights into disease pathogenesis.In this study, we combined pyrosequencing with a transposon-based fragmentation method to allow powerful ultradeep sequencing of the full-length HIV and simian immunodeficiency virus (SIV) genomes, demonstrating a new and highly practical approach to study the complexity of the viral population within a host and identify minor variants on a genome-wide scale. While this study applied pyrosequencing to immunodeficiency viruses, this approach could be applied to any viral pathogen. |
