We are using the single-genome sequencing (SGS) technology we developed previously to analyze and understand the accumulation of genetic variation in gag/pol and env. We have made significant advances in additional assay development and have extended studies to a number of different patient groups, including chronically infected patients, both naive and on therapy, as well as in primary and early human immunodeficiency virus (HIV) infection (in collaboration with J. Margolick, E. Daar, and S. Kottilil), and in long-term nonprogressors (in collaboration with Mens). As a result, we are obtaining a more comprehensive picture of HIV genetic variation in vivo in the presence or absence of drug resistance. We have expanded analytic approaches to HIV population genetics using SGS and we have developed new technologies. The SGS approach, as developed in the DRP, is rapidly becoming the standard approach to investigate HIV populations, with a number of groups and large networks employing the technique, notably the Center for HIV/AIDS Vaccine Immunology (CHAVI). We continue to investigate the utility of the approach, and expand applications. We have collaborated with M. Jordan to compare HIV population structure as determined by SGS or standard cloning methodologies. The results demonstrate concordance between methods but also identify certain discrepancies requiring additional study. We have also collaborated with W.-S. Hu in an in-depth investigation of intersubtype recombination, demonstrating adaptive effects at distant sites. As resistance to integrase inhibitors increases, and NIH clinics are enrolling more such patients, we are preparing to extend SGS to study the integrase sequence as well. The DRP is also developing new technologies to investigate HIV-1 genetic variation. With the Translational Research Unit we have investigated the use of investigating massively parallel pyrosequencing techniques (to study HIV population genetics. Although such ultra-deep technology has been used to study HIV-1, the utility of the approach remains uncertain, because it is not clear whether the approach can accommodate a highly genetically diverse virus population and yield accurate phylogenetic data. The DRP has an extensive database of single-genome sequences from a large cohort of well-characterized patients. These single-genome sequences will provide the gold standard to compare results of pyrosequencing and determine the utility of massively parallel sequencing in genetic analysis of HIV-1 populations. We have also developed useful quality control procedures. In initial studies, we identified improvements that are essential to prevent assay-induced recombination; these optimization procedures enable pyrosequencing to be used reliably to investigate recombination and epistasis in genetically diverse populations. We are also investigating the use additional next generation sequencing approaches, including Illumina technology to obtain fine structure analysis of HIV populations in vivo. In efforts to expand our capability to perform next generation sequencing, we have obtained additional IATAP funds to acquire digital droplet PCR instrument, which will expand our capability for quantification of viral nucleic acids, We are collaborating with the Translational Research Unit to adapt dd PCR technology to sequence HIV amplified in the ddPCR process. This advance will significantly expand our single genome sequencing capability. Understanding of the expansion of genetic diversity following infection from a genetically limited to a highly diverse population has useful implications for applicability in understanding the HIV epidemic. Based on our understanding of genetic variation in acute and chronically infected individuals, we developed a new bioinformatics algorithm to discriminate between recently and chronically infected individuals based exclusively on population-based commercial genotyping data. Development of this algorithm has yielded the invention report EIR #238-2009. Field testing is currently in development, and we anticipate that this technique will be of broad epidemiologic utility in investigating incidence rates of HIV-1 infection. The development of these techniques has led to new insights in HIV population dynamics in understanding the effects of antiretroviral therapy, the nature of replication in natural suppression of HIV, and population dynamics of non-subtype B HIV populations. The nature of HIV-1 populations in patients undergoing antiretroviral therapy remains uncertain, and we are conducting an extensive genetic analysis of HIV-1 before and after initiation of antiretroviral therapy (completed Protocol 97-I-0082, new Protocol 08-I-0221). These results will yield new information regarding the nature and timing of genetic bottlenecks occurring during antiretroviral therapy. Analysis of HIV-1 sequences at relatively low viremia has been limited by technical issues in amplifying the relatively few HIV-1 sequences present in plasma during therapy. We have successfully adapted the SGS procedure to obtain acceptable numbers of sequences from patients suppressed on antiretroviral therapy. In collaboration with M. Polis and D. Persaud (NIH Bench to Bedside Award, 2006), we are analyzing genetic variation in patients enrolled in Protocol 97-I-0082 (now 08-I-0221; F. Maldarelli, PI) who have been suppressed on antiretroviral therapy for prolonged (greater than 8 y) periods. Initial analyses demonstrate that HIV does not undergo a genetic bottleneck upon initiation of antiretroviral therapy; despite 100-10,000 fold decline in levels of peripheral viremia, no significant decreases in genetic diversity were detected in the first 1-2 y of therapy. These data indicate common source of virus infecting short lived cells (responsible for greater than 90-99% of virus produced prior to therapy) and longer lived cells (responsible for virus produced 1-2 years after therapy is initiated). After prolonged therapy, emergence of predominant clones (as previously noted by Bailey et al.) was detected in the majority (7/8) patients. These data suggest that the non-clonal populations slowly decayed over time or that the clonal population increased by cellular expansion. We are also applying population genetics approaches to quantify the emergence of drug resistance mutations in rebound viremia in patients undergoing antiretroviral therapy. We are specifically investigating the relative roles of mutation and selection in development of resistance to AZT and NNRTI, as well as quantifying the role of APOBEC mediated mutations in the emergence of the M184I mutation conferring resistance to 3TC, FTC, and ddI. We are collaborating with A. Pau and H.C. Lane on a new study of HIV drug resistance in individuals undergoing antiretroviral therapy. We will be using the laboratory techniques and analytic approaches we have developed within the DRP to investigate mechanisms of resistance in clinical samples. We are collaborating with M. Kearney and J. Mellors to conduct an indepth analysis of HIV population genetics using sequencing data they generated from the ACTG 5142. Finding from this analysis will provide new information regarding position specific genetiv variation in HIV RT. [Corresponds to Project 3 in the October 2011 site visit report of the Clinical Retrovirology Section, HIV Drug Resistance Program]