Data release: targeted systematic literature search for tick and tick-borne pathogen distributions in six countries in sub-Saharan Africa from 1901 to 2020.

BACKGROUND: Surveillance data documenting tick and tick-borne disease (TBD) prevalence is needed to develop risk assessments and implement control strategies. Despite extensive research in Africa, there is no standardized, comprehensive review. METHODS: Here we tackle this knowledge gap, by producing a comprehensive review of research articles on ticks and TBD between 1901 and 2020 in Chad, Djibouti, Ethiopia, Kenya, Tanzania, and Uganda. Over 8356 English language articles were recovered. Our search strategy included 19 related MeSH terms. Articles were reviewed, and 331 met inclusion criteria. Articles containing mappable data were compiled into a standardized data schema, georeferenced, and uploaded to VectorMap. RESULTS: Tick and pathogen matrixes were created, providing information on vector distributions and tick-pathogen associations within the six selected African countries. CONCLUSIONS: These results provide a digital, mappable database of current and historical tick and TBD distributions across six countries in Africa, which can inform specific risk modeling, determine surveillance gaps, and guide future surveillance priorities.

HIV Expression in Infected T Cell Clones.

The principal barrier to an HIV-1 cure is the persistence of infected cells harboring replication-competent proviruses despite antiretroviral therapy (ART). HIV-1 transcriptional suppression, referred to as viral latency, is foremost among persistence determinants, as it allows infected cells to evade the cytopathic effects of virion production and killing by cytotoxic T lymphocytes (CTL) and other immune factors. HIV-1 persistence is also governed by cellular proliferation, an innate and essential capacity of CD4+ T cells that both sustains cell populations over time and enables a robust directed response to immunological threats. However, when HIV-1 infects CD4+ T cells, this capacity for proliferation can enable surreptitious HIV-1 propagation without the deleterious effects of viral gene expression in latently infected cells. Over time on ART, the HIV-1 reservoir is shaped by both persistence determinants, with selective forces most often favoring clonally expanded infected cell populations harboring transcriptionally quiescent proviruses. Moreover, if HIV latency is incomplete or sporadically reversed in clonal infected cell populations that are replenished faster than they are depleted, such populations could both persist indefinitely and contribute to low-level persistent viremia during ART and viremic rebound if treatment is withdrawn. In this review, select genetic, epigenetic, cellular, and immunological determinants of viral transcriptional suppression and clonal expansion of HIV-1 reservoir T cells, interdependencies among these determinants, and implications for HIV-1 persistence will be presented and discussed.

The largest HIV-1-infected T cell clones in children on long-term combination antiretroviral therapy contain solo LTRs.

Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked.

Application of ultrasensitive digital ELISA for p24 enables improved evaluation of HIV-1 reservoir diversity and growth kinetics in viral outgrowth assays.

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

Current HIV/SIV Reservoir Assays for Preclinical and Clinical Applications: Recommendations from the Experts 2022 NIAID Workshop Summary.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Second, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.

HIV-1 usurps transcription start site heterogeneity of host RNA polymerase II to maximize replication fitness.

HIV-1 relies on host RNA polymeraseII (Pol II) to transcribe its genome and uses multiple transcription start sites (TSS), including three consecutive guanosines located near the U3-R junction, to generate transcripts containing three, two, and one guanosine at the 5' end, referred to as 3G, 2G, and 1G RNA, respectively. The 1G RNA is preferentially selected for packaging, indicating that these 99.9% identical RNAs exhibit functional differences and highlighting the importance of TSS selection. Here, we demonstrate that TSS selection is regulated by sequences between the CATA/TATA box and the beginning of R. Furthermore, we have generated two HIV-1 mutants with distinct 2-nucleotide modifications that predominantly express 3G RNA or 1G RNA. Both mutants can generate infectious viruses and undergo multiple rounds of replication in T cells. However, both mutants exhibit replication defects compared to the wild-type virus. The 3G-RNA-expressing mutant displays an RNA genome-packaging defect and delayed replication kinetics, whereas the 1G-RNA-expressing mutant exhibits reduced Gag expression and a replication fitness defect. Additionally, reversion of the latter mutant is frequently observed, consistent with sequence correction by plus-strand DNA transfer during reverse transcription. These findings demonstrate that HIV-1 maximizes its replication fitness by usurping the TSS heterogeneity of host RNA Pol II to generate unspliced RNAs with different specialized roles in viral replication. The three consecutive guanosines at the junction of U3 and R may also maintain HIV-1 genome integrity during reverse transcription. These studies reveal the intricate regulation of HIV-1 RNA and complex replication strategy.

Deep Sequencing Analysis of Individual HIV-1 Proviruses Reveals Frequent Asymmetric Long Terminal Repeats.

Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.

HIV drug resistance in various body compartments.

PURPOSE OF REVIEW: HIV drug resistance testing using blood plasma or dried blood spots forms part of international guidelines. However, as the clinical utility of assessing drug resistance in other body compartments is less well established, we review this for blood cells and samples from other body compartments. RECENT EVIDENCE: Although clinical benefit is not clear, drug resistance testing in blood cells is often performed when patients with suppressed plasma viral loads require a treatment substitution. In patients with HIV neurocognitive disease, cerebral spinal fluid (CSF) drug resistance is rarely discordant with plasma but has nevertheless been used to guide antiretroviral drug substitutions. Cases with HIV drug resistance in genital fluids have been documented but this does not appear to indicate transmission risk when blood plasma viral loads are suppressed. SUMMARY: Drug-resistant variants, which may be selected in tissues under conditions of variable adherence and drug penetration, appear to disseminate quickly, and become detectable in blood. This may explain why drug resistance discordance between plasma and these compartments is rarely found. Partial compartmentalization of HIV populations is well established for the CSF and the genital tract but other than blood plasma, evidence is lacking to support drug resistance testing in body compartments.

Plasma SARS-CoV-2 RNA Levels as a Biomarker of Lower Respiratory Tract SARS-CoV-2 Infection in Critically Ill Patients With COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n = 33, r = 0.83, P < 10-9) and then declined in parallel in available serial samples except in nonsurvivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early after ICU admission.

A Pre-Vaccination Baseline of SARS-CoV-2 Genetic Surveillance and Diversity in the United States.

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in simultaneously collected longitudinal samples from mechanically-ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (r=0.83, p<10 -8 ) and then declined in parallel except in non-survivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early in severe disease.

Therapeutic implications of ongoing alveolar viral replication in COVID-19.

In patients with moderate-to-severe COVID-19 pneumonia, an aberrant post-viral alveolitis with excessive inflammatory responses and immunothrombosis underpins use of immunomodulatory therapy (eg, corticosteroids and interleukin-6 receptor antagonism). By contrast, immunosuppression in individuals with mild COVID-19 who do not require oxygen therapy or in those with critical disease undergoing prolonged ventilation is of no proven benefit. Furthermore, a window of opportunity is thought to exist for timely immunosuppression in patients with moderate-to-severe COVID-19 pneumonia shortly after clinical presentation. In this Viewpoint, we explore the shortcomings of a universal immunosuppression approach in patients with moderate-to-severe COVID-19 due to disease heterogeneity related to ongoing SARS-CoV-2 replication, which can manifest as RNAaemia in some patients treated with immunotherapy. By contrast, immunomodulatory therapy has overall benefits in patients with rapid SARS-CoV-2 clearance, via blunting of multifaceted, excessive innate immune responses in the lungs, potentially uncontrolled T-cell responses, possible autoimmune responses, and immunothrombosis. We highlight this therapeutic dichotomy to better understand the immunopathology of moderate-to-severe COVID-19, particularly the role of RNAaemia, and to refine therapy choices.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir.

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

HIV-1 Persistence in Children during Suppressive ART.

There is a growing number of perinatally HIV-1-infected children worldwide who must maintain life-long ART. In early life, HIV-1 infection is established in an immunologically inexperienced environment in which maternal ART and immune dynamics during pregnancy play a role in reservoir establishment. Children that initiated early antiretroviral therapy (ART) and maintained long-term suppression of viremia have smaller and less diverse HIV reservoirs than adults, although their proviral landscape during ART is reported to be similar to that of adults. The ability of these early infected cells to persist long-term through clonal expansion poses a major barrier to finding a cure. Furthermore, the effects of life-long HIV persistence and ART are yet to be understood, but growing evidence suggests that these individuals are at an increased risk for developing non-AIDS-related comorbidities, which underscores the need for an HIV cure.

Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

HIVIntact: a python-based tool for HIV-1 genome intactness inference.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

2020 SARS-CoV-2 diversification in the United States: Establishing a pre-vaccination baseline.

In 2020, SARS-CoV-2 spread across the United States (U.S.) in three phases distinguished by peaks in the numbers of infections and shifting geographical distribution. We investigated the viral genetic diversity in each phase using sequences publicly available prior to December 15 th , 2020, when vaccination was initiated in the U.S. In Phase 1 (winter/spring), sequences were already dominated by the D614G Spike mutation and by Phase 3 (fall), genetic diversity of the viral population had tripled and at least 54 new amino acid changes had emerged at frequencies above 5%, several of which were within known antibody epitopes. These findings highlight the need to track the evolution of SARS-CoV-2 variants in the U.S. to ensure continued efficacy of vaccines and antiviral treatments. ONE SENTENCE SUMMARY: SARS-CoV-2 genetic diversity in the U.S. increased 3-fold in 2020 and 54 emergent nonsynonymous mutations were detected.

CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5' LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5' LTR promoter.