Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons.

Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical (n = 9) or nearly identical (n = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2-13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.

A bioinformatic analysis of T-cell epitope diversity in SARS-CoV-2 variants: association with COVID-19 clinical severity in the United States population.

Long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the identification of T-cell epitopes affecting host immunogenicity. In this computational study, we explored the CD8+ epitope diversity estimated in 27 of the most common HLA-A and HLA-B alleles, representing most of the United States population. Analysis of 16 SARS-CoV-2 variants [B.1, Alpha (B.1.1.7), five Delta (AY.100, AY.25, AY.3, AY.3.1, AY.44), and nine Omicron (BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ.1, BQ.1.1, XBB.1, XBB.1.5)] in analyzed MHC class I alleles revealed that SARS-CoV-2 CD8+ epitope conservation was estimated at 87.6%-96.5% in spike (S), 92.5%-99.6% in membrane (M), and 94.6%-99% in nucleocapsid (N). As the virus mutated, an increasing proportion of S epitopes experienced reduced predicted binding affinity: 70% of Omicron BQ.1-XBB.1.5 S epitopes experienced decreased predicted binding, as compared with ~3% and ~15% in the earlier strains Delta AY.100-AY.44 and Omicron BA.1-BA.5, respectively. Additionally, we identified several novel candidate HLA alleles that may be more susceptible to severe disease, notably HLA-A*32:01, HLA-A*26:01, and HLA-B*53:01, and relatively protected from disease, such as HLA-A*31:01, HLA-B*40:01, HLA-B*44:03, and HLA-B*57:01. Our findings support the hypothesis that viral genetic variation affecting CD8 T-cell epitope immunogenicity contributes to determining the clinical severity of acute COVID-19. Achieving long-term COVID-19 immunity will require an understanding of the relationship between T cells, SARS-CoV-2 variants, and host MHC class I genetics. This project is one of the first to explore the SARS-CoV-2 CD8+ epitope diversity that putatively impacts much of the United States population.

Temporary increase in circulating replication-competent latent HIV-infected resting CD4+ T cells after switch to an integrase inhibitor based antiretroviral regimen.

BACKGROUND: The principal barrier to an HIV cure is the presence of the latent viral reservoir (LVR), which has been understudied in African populations. From 2018 to 2019, Uganda instituted a nationwide rollout of ART consisting of Dolutegravir (DTG) with two NRTI, which replaced the previous regimen of one NNRTI and the same two NRTI. METHODS: Changes in the inducible replication-competent LVR (RC-LVR) of ART-suppressed Ugandans with HIV (n = 88) from 2015 to 2020 were examined using the quantitative viral outgrowth assay. Outgrowth viruses were examined for viral evolution. Changes in the RC-LVR were analyzed using three versions of a Bayesian model that estimated the decay rate over time as a single, linear rate (model A), or allowing for a change at time of DTG initiation (model B&C). FINDINGS: Model A estimated the slope of RC-LVR change as a non-significant positive increase, which was due to a temporary spike in the RC-LVR that occurred 0-12 months post-DTG initiation (p < 0.005). This was confirmed with models B and C; for instance, model B estimated a significant decay pre-DTG initiation with a half-life of 6.9 years, and an ∼1.7-fold increase in the size of the RC-LVR post-DTG initiation. There was no evidence of viral failure or consistent evolution in the cohort. INTERPRETATION: These data suggest that the change from NNRTI- to DTG-based ART is associated with a significant temporary increase in the circulating RC-LVR. FUNDING: Supported by the NIH (grant 1-UM1AI164565); Gilead HIV Cure Grants Program (90072171); Canadian Institutes of Health Research (PJT-155990); and Ontario Genomics-Canadian Statistical Sciences Institute.

Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates in southeast Louisiana reveals persistent genetic clusters spanning multiple locations.

BACKGROUND: We investigated 51 g-negative carbapenem-resistant Enterobacterales (CRE) isolates collected from 22 patients over a five-year period from six health care institutions in the Ochsner Health network in southeast Louisiana. METHODS: Short genomic reads were generated using Illumina sequencing and assembled for each isolate. Isolates were classified as Enterobacter spp. (n = 20), Klebsiella spp. (n = 30), and Escherichia coli (n = 1) and grouped into 19 different multi-locus sequence types (MLST). Species and patient-specific core genomes were constructed representing ∼50% of the chromosomal genome. RESULTS: We identified two sets of patients with genetically related infections; in both cases, the related isolates were collected > 6 months apart, and in one case, the isolates were collected in different locations. On the other hand, we identified four sets of patients with isolates of the same species collected within 21 days from the same location; however, none had genetically related infections. Genes associated with resistance to carbapenem drugs (blaKPC and/or blaCTX-M-15) were found in 76% of the isolates. We found three blaKPC variants (blaKPC-2, blaKPC-3, and blaKPC-4) associated with four different Enterobacter MLST variants, and two blaKPC variants (blaKPC-2, blaKPC-3) associated with seven different Klebsiella MLST variants. CONCLUSIONS: Molecular surveillance is increasingly becoming a powerful tool to understand bacterial spread in both community and clinical settings. This study provides evidence that genetically related infections in clinical settings do not necessarily reflect temporal associations, and vice versa. Our results also highlight the regional genomic and resistance diversity within related bacterial lineages.

HIV-1 subtypes maintain distinctive physicochemical signatures in Nef domains associated with immunoregulation.

BACKGROUND: HIV subtype is associated with varied rates of disease progression. The HIV accessory protein, Nef, continues to be present during antiretroviral therapy (ART) where it has numerous immunoregulatory effects. In this study, we analyzed Nef sequences from HIV subtypes A1, B, C, and D using a machine learning approach that integrates functional amino acid information to identify if unique physicochemical features are associated with Nef functional/structural domains in a subtype-specific manner. METHODS: 2253 sequences representing subtypes A1, B, C, and D were aligned and domains with known functional properties were scored based on amino acid physicochemical properties. Following feature generation, we used statistical pruning and evolved neural networks (ENNs) to determine if we could successfully classify subtypes. Next, we used ENNs to identify the top five key Nef physicochemical features applied to specific immunoregulatory domains that differentiated subtypes. A signature pattern analysis was performed to the assess amino acid diversity in sub-domains that differentiated each subtype. RESULTS: In validation studies, ENNs successfully differentiated each subtype at A1 (87.2%), subtype B (89.5%), subtype C (91.7%), and subtype D (85.1%). Our feature-based domain scoring, followed by t-tests, and a similar ENN identified subtype-specific domain-associated features. Subtype A1 was associated with alterations in Nef CD4 binding domain; subtype B was associated with alterations with the AP-2 Binding domain; subtype C was associated with alterations in a structural Alpha Helix domain; and, subtype D was associated with alterations in a Beta-Sheet domain. CONCLUSIONS: Recent studies have focused on HIV Nef as a driver of immunoregulatory disease in those HIV infected and on ART. Nef acts through a complex mixture of interactions that are directly linked to the key features of the subtype-specific domains we identified with the ENN. The study supports the hypothesis that varied Nef subtypes contribute to subtype-specific disease progression.

Comparing antimicrobial resistant genes and phenotypes across multiple sequencing platforms and assays for Enterobacterales clinical isolates.

INTRODUCTION: Whole genome sequencing (WGS) of bacterial isolates can be used to identify antimicrobial resistance (AMR) genes. Previous studies have shown that genotype-based AMR has variable accuracy for predicting carbapenem resistance in carbapenem-resistant Enterobacterales (CRE); however, the majority of these studies used short-read platforms (e.g. Illumina) to generate sequence data. In this study, our objective was to determine whether Oxford Nanopore Technologies (ONT) long-read WGS would improve detection of carbapenem AMR genes with respect to short-read only WGS for nine clinical CRE samples. We measured the minimum inhibitory breakpoint (MIC) using two phenotype assays (MicroScan and ETEST) for six antibiotics, including two carbapenems (meropenem and ertapenem) and four non-carbapenems (gentamicin, ciprofloxacin, cefepime, and trimethoprim/sulfamethoxazole). We generated short-read data using the Illumina NextSeq and long-read data using the ONT MinION. Four assembly methods were compared: ONT-only assembly; ONT-only assembly plus short-read polish; ONT + short-read hybrid assembly plus short-read polish; short-read only assembly. RESULTS: Consistent with previous studies, our results suggest that the hybrid assembly produced the highest quality results as measured by gene completeness and contig circularization. However, ONT-only methods had minimal impact on the detection of AMR genes and plasmids compared to short-read methods, although, notably, differences in gene copy number differed between methods. All four assembly methods showed identical presence/absence of the blaKPC-2 carbapenemase gene for all samples. The two phenotype assays showed 100% concordant results for the non-carbapenems, but only 65% concordance for the two carbapenems. The presence/absence of AMR genes was 100% concordant with AMR phenotypes for all four non-carbapenem drugs, although only 22%-50% sensitivity for the carbapenems. CONCLUSIONS: Overall, these findings suggest that the lack of complete correspondence between CRE AMR genotype and phenotype for carbapenems, while concerning, is independent of sequencing platform/assembly method.

Temporary increase in circulating replication-competent latent HIV-infected resting CD4+ T cells after switch to an integrase inhibitor based antiretroviral regimen.

The principal barrier to an HIV cure is the presence of a latent viral reservoir (LVR) made up primarily of latently infected resting CD4+ (rCD4) T-cells. Studies in the United States have shown that the LVR decays slowly (half-life=3.8 years), but this rate in African populations has been understudied. This study examined longitudinal changes in the inducible replication competent LVR (RC-LVR) of ART-suppressed Ugandans living with HIV (n=88) from 2015-2020 using the quantitative viral outgrowth assay, which measures infectious units per million (IUPM) rCD4 T-cells. In addition, outgrowth viruses were examined with site-directed next-generation sequencing to assess for possible ongoing viral evolution. During the study period (2018-19), Uganda instituted a nationwide rollout of first-line ART consisting of Dolutegravir (DTG) with two NRTI, which replaced the previous regimen that consisted of one NNRTI and the same two NRTI. Changes in the RC-LVR were analyzed using two versions of a novel Bayesian model that estimated the decay rate over time on ART as a single, linear rate (model A) or allowing for an inflection at time of DTG initiation (model B). Model A estimated the population-level slope of RC-LVR change as a non-significant positive increase. This positive slope was due to a temporary increase in the RC-LVR that occurred 0-12 months post-DTG initiation (p<0.0001). This was confirmed with model B, which estimated a significant decay pre-DTG initiation with a half-life of 7.7 years, but a significant positive slope post-DTG initiation leading to a transient estimated doubling-time of 8.1 years. There was no evidence of viral failure in the cohort, or consistent evolution in the outgrowth sequences associated with DTG initiation. These data suggest that either the initiation of DTG, or cessation of NNRTI use, is associated with a significant temporary increase in the circulating RC-LVR.

The Persistence of HIV Diversity, Transcription, and Nef Protein in Kaposi's Sarcoma Tumors during Antiretroviral Therapy.

Epidemic Kaposi's sarcoma (KS), defined by co-infection with Human Herpes Virus 8 (HHV-8) and the Human Immunodeficiency Virus (HIV), is a major cause of mortality in sub-Saharan Africa. Antiretroviral therapy (ART) significantly reduces the risk of developing KS, and for those with KS, tumors frequently resolve with ART alone. However, for unknown reasons, a significant number of KS cases do not resolve and can progress to death. To explore how HIV responds to ART in the KS tumor microenvironment, we sequenced HIV env-nef found in DNA and RNA isolated from plasma, peripheral blood mononuclear cells, and tumor biopsies, before and after ART, in four Ugandan study participants who had unresponsive or progressive KS after 180-250 days of ART. We performed immunohistochemistry experiments to detect viral proteins in matched formalin-fixed tumor biopsies. Our sequencing results showed that HIV diversity and RNA expression in KS tumors are maintained after ART, despite undetectable plasma viral loads. The presence of spliced HIV transcripts in KS tumors after ART was consistent with a transcriptionally active viral reservoir. Immunohistochemistry staining found colocalization of HIV Nef protein and tissue-resident macrophages in the KS tumors. Overall, our results demonstrated that even after ART reduced plasma HIV viral load to undetectable levels and restored immune function, HIV in KS tumors continues to be transcriptionally and translationally active, which could influence tumor maintenance and progression.

Absence of antibody responses to SARS-CoV-2 N protein in COVID-19 vaccine breakthrough cases.

Understanding the risk factors for breakthrough coronavirus disease 2019 (COVID-19) (BC19) is critical to inform policy. Herein, we assessed Delta (Lineage B.1.617.2) variant-specific effectiveness of the BNT162b2 (Pfizer) vaccine and characterized Delta-driven BC19 cases (fully vaccinated individuals who get infected) with known-time-since-vaccination. In this longitudinal prospective study (January 21-October 30, 2021), 90 naïve and 15 convalescent individuals were enrolled at the initiation of vaccination. Samples from 27 unvaccinated individuals with previous laboratory-confirmed COVID-19 diagnosis were collected at a single time point. Longitudinal serology profile (antibodies against severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] S and N proteins) and live-virus-based neutralization capacities were assessed while controlling for age. Sex, age, history of reactions to the COVID-19 vaccine, and viral neutralization capacities were identified as significant risk factors for breakthrough COVID-19. At 8 months postvaccination, male sex, individuals ⩾65 years of age, and individuals who experienced noticeable side effects with the COVID-19 vaccine were at 5.47 (p-value = 0.0102), 4.33 (p-value = 0.0236), and 4.95 (p-value = 0.0159) fold greater risk of BC19 as compared to their peers, respectively. Importantly, every five-fold increase in viral neutralization capacities (by live-virus-based assays) was significantly associated with ~4-fold reduction in the risk occurrence of breakthrough COVID-19 (p-value = 0.045). Vaccine boosting remarkably increased these viral neutralization capacities by 16.22-fold (p- value = 0.0005), supporting the importance of the BNT162b2 booster in efforts to control the incursion of future variants into the population at large. Strikingly, BC19 cases exhibited a delayed/absent antibody response to the N protein, suggesting limited exposure to the virus. Since antibodies against N protein are widely used to evaluate the extent of virus spread in communities, our finding has important implications on the utility of existing serological diagnostic and surveillance for COVID-19.

Distinct HIV-1 Population Structure across Meningeal and Peripheral T Cells and Macrophage Lineage Cells.

HIV-1 sequence population structure among brain and nonbrain cellular compartments is incompletely understood. Here, we compared proviral pol and env high-quality consensus single-molecule real-time (SMRT) sequences derived from CD3+ T cells and CD14+ macrophage lineage cells from meningeal or peripheral (spleen, blood) tissues obtained at autopsy from two individuals with viral suppression on antiretroviral therapy (ART). Phylogenetic analyses showed strong evidence of population structure between CD3+ and CD14+ virus populations. Distinct env variable-region characteristics were also found between CD3+ and CD14+ viruses. Furthermore, shared macrophage-tropic amino acid residues (env) and drug resistance mutations (pol) between meningeal and peripheral virus populations were consistent with the meninges playing a role in viral gene flow across the blood-brain barrier. Overall, our results point toward potential functional differences among meningeal and peripheral CD3+ and CD14+ virus populations and a complex evolutionary history driven by distinct selection pressures and/or viral gene flow. IMPORTANCE Different cell types and/or tissues may serve as a reservoir for HIV-1 during ART-induced viral suppression. We compared proviral pol and env sequences from CD3+ T cells and CD14+ macrophage lineage cells from brain and nonbrain tissues from two virally suppressed individuals. We found strong evidence of viral population structure among cells/tissues, which may result from distinct selective pressures across cell types and anatomic sites.

SARS-CoV-2 genomic surveillance as an evidence-based infection control approach in an offshore petroleum employee population.

BACKGROUND: Industrial hygienists (IH) in the oil and gas business instituted an extraordinary number of safety protocols to limit spread of SARS-CoV-2 onto offshore platforms in the Gulf of Mexico. We used genomic surveillance to provide actionable information concerning the efficacy of their efforts. METHODS: Over 6 months, employees at a single company were serology and PCR tested during a 1-5 day predeployment quarantine and when postdeployment symptoms were reported. From each positive test (n = 49), SARS-CoV-2 genomes were sequenced. Phylogenetic analysis was used to investigate the epidemiology of transmissions. RESULTS: Genomic surveillance confirmed 2 viral strains were infecting 18 offshore workers. Genomic data combined with epidemiological data suggested that a change in quarantine protocols contributed to these outbreaks. A pre-deployment outbreak involved a WHO variant of interest (Theta) that had infected 4 international workers. Two additional predeployment clusters of infections were identified. CONCLUSIONS: Our findings support that IH quarantine/testing protocols limited viral transmissions, halted offshore outbreaks, and stopped the spread of a variant of interest. The study demonstrates how genomic data can be used to understand viral transmission dynamics in employee populations and evaluate safety protocols in the offshore oil and gas industry.

Outbreak of P.3 (Theta) SARS-CoV-2 emerging variant of concern among service workers in Louisiana.

During routine industrial quarantine/premobilization procedures, four individuals who recently traveled from the Philippines tested positive for SARS-CoV-2. Subsequent genomic analysis showed that all four were infected with a relatively rare Variant of Interest (P.3, Theta) derived from a single origin. This demonstrates the importance of on-going genomic surveillance of SARS-CoV-2.

Ultradeep HIV-1 Proviral Envelope Sequencing Reveals Complex Population Structure within and between Brain and Splenic Tissues.

Understanding tissue-based HIV-1 proviral population structure is important for improving treatment strategies for individuals with HIV-associated neurological disorders (HAND). Previous analyses have revealed HIV-1 envelope (env) population structure between brain and peripheral tissues as well as Env functional differences, especially in individuals with HAND. Furthermore, population structure has been detected among different anatomical locations in the brain itself, although such patterns are inconsistent across individuals and less strongly associated with the presence/absence of HAND. Here, we utilized the Pacific Biosciences single-molecule real-time (SMRT) high-throughput technology to generate thousands of sequences for each tissue, along with phylogenetic and distance-based analyses, to investigate env sequences from paired brain and spleen samples from eight individuals with/without HAND. To account for the high error rate associated with SMRT sequencing, we used a clustering approach to identify high-quality consensus sequences representative of ≥10 reads ("HQCS10"). In parallel, we characterized variable regions from nonclustered sequences to identify potential functional differences. We found evidence for significant population structure between brain and spleen tissues, as well as among brain tissues and within the same brain tissue, in individuals both with and without HAND. Variable region analysis showed differences in length and charge among brain and nonbrain tissues as well as within the brain, suggesting possible functional differences. Our results demonstrate the complexity of HIV-1 env structure/gene flow among tissues and support the concept that selective pressures in different tissue microenvironments drive viral evolution and adaptation. IMPORTANCE Understanding the evolution of HIV-1 in the brain compared to other tissues is important for improving treatment strategies for individuals with HIV-associated neurological disorders (HAND). We utilized high-throughput sequencing technology to generate thousands of full-length env sequences from paired brain and spleen samples from eight individuals with/without HAND. We found significant viral population structure for participants both with and without HAND, providing robust evidence for the brain as a compartmentalized tissue and potentially a viral reservoir. We also found striking genetic differences between virus populations, even from the same tissue, suggesting the potential for functional differences and the possibility for multiple evolutionary pathways that result in similar tropisms and/or other tissue-adapted characteristics. Our results demonstrate the complexity of viral population structure within the brain and suggest that analysis of peripheral blood samples alone may not be fully informative with respect to improving strategies to treat or eradicate HIV-1.

Emergence of an early SARS-CoV-2 epidemic in the United States.

The emergence of the COVID-19 epidemic in the United States (U.S.) went largely undetected due to inadequate testing. New Orleans experienced one of the earliest and fastest accelerating outbreaks, coinciding with Mardi Gras. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large-scale events accelerate transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana had limited diversity compared to other U.S. states and that one introduction of SARS-CoV-2 led to almost all of the early transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras, and the festival dramatically accelerated transmission. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate epidemics.

Emergence of an early SARS-CoV-2 epidemic in the United States.

The emergence of the early COVID-19 epidemic in the United States (U.S.) went largely undetected, due to a lack of adequate testing and mitigation efforts. The city of New Orleans, Louisiana experienced one of the earliest and fastest accelerating outbreaks, coinciding with the annual Mardi Gras festival, which went ahead without precautions. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large, crowded events may have accelerated early transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana initially had limited sequence diversity compared to other U.S. states, and that one successful introduction of SARS-CoV-2 led to almost all of the early SARS-CoV-2 transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras and that the festival dramatically accelerated transmission, eventually leading to secondary localized COVID-19 epidemics throughout the Southern U.S.. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate COVID-19 epidemics on a local and regional scale.

Molecular surveillance of methicillin-resistant Staphylococcus aureus genomes in hospital unexpectedly reveals discordance between temporal and genetic clustering.

BACKGROUND: The objective of this study was to identify sources and linkages among methicillin-resistant Staphylococcus aureus infections using whole-genome sequencing (WGS). METHODS: A total of 56 samples were obtained from all patients with a confirmed MRSA infection over 6 months at University of Florida-Health Jacksonville. Samples were cultured and sequenced; data was analyzed on an automated cloud-based platform. Genetic Clusters were defined as <40 single nucleotide polymorphisms. Temporal Clusters were defined as ≥5 MRSA cases over 3 days. RESULTS: We found 7 Genetic Clusters comprising 15 samples. Four Genetic Clusters contained patients with non-overlapping stays (3-10 weeks apart), 3 of which contained patients who shared the same Unit. We also found 5 Temporal Clusters comprising 23 samples, although none of the samples were genetically related. DISCUSSION: Results showed that temporal clustering may be a poor indicator of genetic linkage. Shared epidemiological characteristics between patients in Genetic Clusters may point toward previously unidentified hospital sources. Repeated observation of related strains is also consistent with ongoing MRSA transmission within the surrounding high-risk community. CONCLUSIONS: WGS is a valuable tool for hospital infection prevention and control.

Persistence of HIV transmission clusters among people who inject drugs.

OBJECTIVE: We investigated the duration of HIV transmission clusters. DESIGN: Fifty-four individuals newly infected at enrollment in the ALIVE cohort were included, all of whom had sequences at an intake visit (T1) and from a second (T2) and/or a third (T3) follow-up visit, median 2.9 and 5.4 years later, respectively. METHODS: Sequences were generated using the 454 DNA sequencing platform for portions of HIV pol and env (HXB2 positions 2717-3230; 7941-8264). Genetic distances were calculated using tn93 and sequences were clustered over a range of thresholds (1--5%) using HIV-TRACE. Analyses were performed separately for individuals with pol sequences for T1 + T2 (n = 40, 'Set 1') and T1 + T3 (n = 25; 'Set 2'), and env sequences for T1 + T2 (n = 47, 'Set 1'), and T1 + T3 (n = 30; 'Set 2'). RESULTS: For pol, with one exception, a single cluster contained more than 75% of samples at all thresholds, and cluster composition was at least 90% concordant between time points/thresholds. For env, two major clusters (A and B) were observed at T1 and T2/T3, although cluster composition concordance between time points/thresholds was low (<60%) at lower thresholds for both sets 1 and 2. In addition, several individuals were included in clusters at T2/T3, although not at T1. CONCLUSION: Caution should be used in applying a single threshold in population studies where seroconversion dates are unknown. However, the retention of some clusters even after 5 + years is evidence for the robustness of the clustering approach in general.

HIV-1 Subtype Distribution and Diversity Over 18 Years in Rakai, Uganda.

The Rakai Community Cohort Study in south central Uganda has surveyed people aged 15-49 since 1994. Antiretroviral therapy (ART) was introduced in 2004. HIV p24 and gp41 subtype distribution and viral diversity were studied from blood samples collected at three surveys in 1994-1995, 2002-2003, and 2008-2009, which were compared with a new survey round from 2011 to 2012. These included 1364 HIV+ individuals. For both p24 and gp41 domains, the genetic diversity within subtypes A and D was significantly increasing in the pre-ART era and decreased between the last two survey rounds in the ART era (p < .01). This study suggests that despite ongoing mixing of viral subtypes, an association with the introduction of ART to a reduction of intra-subtype viral genomic diversity may be occurring, which can be explored in ongoing studies.

Recombination Analysis of Near Full-Length HIV-1 Sequences and the Identification of a Potential New Circulating Recombinant Form from Rakai, Uganda.

The Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium has been vital in the generation and examination of near full-length HIV-1 sequences generated from Sub-Saharan Africa. In this study, we examined a subset (n = 275) of sequences from Rakai, Uganda, collected between August 2011 and January 2015. Sequences were initially screened with COMET for subtyping and then evaluated using bootscanning and phylogenetic inference. Among 275 sequences, 38.6% were subtype D, 19.3% were subtype A, 2.9% were subtype C, and 39.3% were recombinant. The recombinants were structurally diverse in the number of breakpoints observed, the location of recombinant segments, and represented subtypes, with AD recombinants accounting for the majority of all recombinants (29.8%). Within the AD subpopulation, we identified a potential new circulating recombinant form in five individuals where the polymerase gene was subtype D and most of env was subtype A (D-A junctures at HXB2 6760 and 8709). While the breakpoints were identical for the viruses from these individuals, the viral fragments did not cluster together. These results suggest selection for a viral strain where properties of the subtype A and subtype D portions of the virus confer a survival advantage. The continued study of recombinants will increase our breadth of knowledge for the genetic diversity and evolution of HIV-1, which can further contribute to our understanding toward a universal HIV-1 vaccine.

Longitudinal Antibody Responses in People Who Inject Drugs Infected With Similar Human Immunodeficiency Virus Strains.

BACKGROUND: Multiple factors influence the human immunodeficiency virus (HIV) antibody response produced during natural infection, leading to responses that can vary in specificity, strength, and breadth. METHODS: People who inject drugs identified as recently infected with HIV (n = 23) were analyzed for clustering of their viral sequences (genetic distance, <2%). Longitudinal antibody responses were identified for neutralizing antibody (Nab) potential, and differences in antibody subclass, specificity, and Fc receptor ligation using pseudovirus entry and multiplexed Fc array assays, respectively. Responses were analyzed for differences between subject groups, defined by similarity in the sequence of the infecting virus. RESULTS: Viral sequences from infected individuals were grouped into 3 distinct clusters with 7 unclustered individuals. Subjects in cluster 1 generally had lower antibody response magnitudes, except for antibodies targeting the V1/V2 region. Subjects in clusters 2 and 3 typically had higher antibody response magnitudes, with the Fv specificity of cluster 2 favoring gp140 recognition. NAb responses differed significantly between clusters for 3 of 18 pseudoviruses examined (P < .05), but there were no differences in overall NAb breadth (P = .62). DISCUSSION: These data demonstrate that individuals infected with similar viral strains can generate partially similar antibody responses, but these do not drastically differ from those in individuals infected with relatively unrelated strains.