Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region

Prevalence of WHO Transmitted Drug Resistance Mutations by Deep Sequencing in Antiretroviral-Naïve Subjects in Hunan Province, China

There are few data on the prevalence of WHO transmitted drug resistance mutations (TDRs) that could affect treatment responses to first line antiretroviral therapy (ART) in Hunan Province, China.Determine the prevalence of WHO NRTI/NNRTI/PI TDRs in ART-naïve subjects in Hunan Province by deep sequencing.ART-naïve subjects diagnosed in Hunan between 2010-2011 were evaluated by deep sequencing for low-frequency HIV variants possessing WHO TDRs to 1% levels. Mutations were scored using the HIVdb.stanford.edu algorithm to infer drug susceptibility.Deep sequencing was performed on samples from 90 ART-naïve subjects; 83.3% were AE subtype. All subjects had advanced disease (average CD4 count 134 cells/mm3). Overall 25.6%(23/90) of subjects had HIV with major WHO NRTI/NNRTI TDRs by deep sequencing at a variant frequency level ≥ 1%; 16.7%(15/90) had NRTI TDR and 12.2%(11/90) had a major NNRTI TDR. The majority of NRTI/NNRTI mutations were identified at variant levels 1%.ART-naïve subjects from Hunan Province China infected predominantly with subtype AE frequently possessed HIV variants with WHO NRTI/NNRTI TDRs by deep sequencing that would affect the first line ART used in the region. Specific mutations conferring nevirapine high-level resistance were identified in 7.8% of subjects. The majority of TDRs detected were at variant levels <5% likely due to subjects having advanced chronic disease at the time of testing. PI TDRs were identified frequently, but were found in isolation and at low variant frequency. As PI/r use is infrequent in Hunan, the existence of PI mutations likely represent AE subtype natural polymorphism at low variant level frequency