Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic.

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Seafood Wholesale Market. Here, we analyze environmental qPCR and sequencing data collected in the Huanan market in early 2020. We demonstrate that market-linked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity is consistent with market emergence and find increased SARS-CoV-2 positivity near and within a wildlife stall. We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and raccoon dogs, previously identified as possible intermediate hosts. We also detect animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them with those from farms and other markets. This analysis provides the genetic basis for a shortlist of potential intermediate hosts of SARS-CoV-2 to prioritize for serological and viral sampling.

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

The origins of SARS-CoV-2: A critical review.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

The role of socio-economic disparities in the relative success and persistence of SARS-CoV-2 variants in New York City in early 2021.

Socio-economic disparities were associated with disproportionate viral incidence between neighborhoods of New York City (NYC) during the first wave of SARS-CoV-2. We investigated how these disparities affected the co-circulation of SARS-CoV-2 variants during the second wave in NYC. We tested for correlation between the prevalence, in late 2020/early 2021, of Alpha, Iota, Iota with E484K mutation (Iota-E484K), and B.1-like genomes and pre-existing immunity (seropositivity) in NYC neighborhoods. In the context of varying seroprevalence we described socio-economic profiles of neighborhoods and performed migration and lineage persistence analyses using a Bayesian phylogeographical framework. Seropositivity was greater in areas with high poverty and a larger proportion of Black and Hispanic or Latino residents. Seropositivity was positively correlated with the proportion of Iota-E484K and Iota genomes, and negatively correlated with the proportion of Alpha and B.1-like genomes. The proportion of persisting Alpha lineages declined over time in locations with high seroprevalence, whereas the proportion of persisting Iota-E484K lineages remained the same in high seroprevalence areas. During the second wave, the geographic variation of standing immunity, due to disproportionate disease burden during the first wave of SARS-CoV-2 in NYC, allowed for the immune evasive Iota-E484K variant, but not the more transmissible Alpha variant, to circulate in locations with high pre-existing immunity.

Random-Effects Substitution Models for Phylogenetics via Scalable Gradient Approximations.

Phylogenetic and discrete-trait evolutionary inference depend heavily on an appropriate characterization of the underlying character substitution process. In this paper, we present random-effects substitution models that extend common continuous-time Markov chain models into a richer class of processes capable of capturing a wider variety of substitution dynamics. As these random-effects substitution models often require many more parameters than their usual counterparts, inference can be both statistically and computationally challenging. Thus, we also propose an efficient approach to compute an approximation to the gradient of the data likelihood with respect to all unknown substitution model parameters. We demonstrate that this approximate gradient enables scaling of sampling-based inference, namely Bayesian inference via Hamiltonian Monte Carlo, under random-effects substitution models across large trees and state-spaces. Applied to a dataset of 583 SARS-CoV-2 sequences, an HKY model with random-effects shows strong signals of nonreversibility in the substitution process, and posterior predictive model checks clearly show that it is a more adequate model than a reversible model. When analyzing the pattern of phylogeographic spread of 1441 influenza A virus (H3N2) sequences between 14 regions, a random-effects phylogeographic substitution model infers that air travel volume adequately predicts almost all dispersal rates. A random-effects state-dependent substitution model reveals no evidence for an effect of arboreality on the swimming mode in the tree frog subfamily Hylinae. Simulations reveal that random-effects substitution models can accommodate both negligible and radical departures from the underlying base substitution model. We show that our gradient-based inference approach is over an order of magnitude more time efficient than conventional approaches.

Integrated Genomic and Social Network Analyses of SARS-CoV-2 Transmission in the Healthcare Setting.

BACKGROUND: Infection prevention (IP) measures are designed to mitigate the transmission of pathogens in healthcare. Using large-scale viral genomic and social network analyses, we determined if IP measures used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic were adequate in protecting healthcare workers (HCWs) and patients from acquiring SARS-CoV-2. METHODS: We performed retrospective cross-sectional analyses of viral genomics from all available SARS-CoV-2 viral samples collected at UC San Diego Health and social network analysis using the electronic medical record to derive temporospatial overlap of infections among related viromes and supplemented with contact tracing data. The outcome measure was any instance of healthcare transmission, defined as cases with closely related viral genomes and epidemiological connection within the healthcare setting during the infection window. Between November 2020 through January 2022, 12 933 viral genomes were obtained from 35 666 patients and HCWs. RESULTS: Among 5112 SARS-CoV-2 viral samples sequenced from the second and third waves of SARS-CoV-2 (pre-Omicron), 291 pairs were derived from persons with a plausible healthcare overlap. Of these, 34 pairs (12%) were phylogenetically linked: 19 attributable to household and 14 to healthcare transmission. During the Omicron wave, 2106 contact pairs among 7821 sequences resulted in 120 (6%) related pairs among 32 clusters, of which 10 were consistent with healthcare transmission. Transmission was more likely to occur in shared spaces in the older hospital compared with the newer hospital (2.54 vs 0.63 transmission events per 1000 admissions, P < .001). CONCLUSIONS: IP strategies were effective at identifying and preventing healthcare SARS-CoV-2 transmission.

Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Accuracy in Near-Perfect Virus Phylogenies.

Phylogenetic trees from real-world data often include short edges with very few substitutions per site, which can lead to partially resolved trees and poor accuracy. Theory indicates that the number of sites needed to accurately reconstruct a fully resolved tree grows at a rate proportional to the inverse square of the length of the shortest edge. However, when inferred trees are partially resolved due to short edges, "accuracy" should be defined as the rate of discovering false splits (clades on a rooted tree) relative to the actual number found. Thus, accuracy can be high even if short edges are common. Specifically, in a "near-perfect" parameter space in which trees are large, the tree length $\xi$ (the sum of all edge lengths) is small, and rate variation is minimal, the expected false positive rate is less than $\xi/3$; the exact value depends on tree shape and sequence length. This expected false positive rate is far below the false negative rate for small $\xi$ and often well below 5% even when some assumptions are relaxed. We show this result analytically for maximum parsimony and explore its extension to maximum likelihood using theory and simulations. For hypothesis testing, we show that measures of split "support" that rely on bootstrap resampling consistently imply weaker support than that implied by the false positive rates in near-perfect trees. The near-perfect parameter space closely fits several empirical studies of human virus diversification during outbreaks and epidemics, including Ebolavirus, Zika virus, and SARS-CoV-2, reflecting low substitution rates relative to high transmission/sampling rates in these viruses.[Ebolavirus; epidemic; HIV; homoplasy; mumps virus; perfect phylogeny; SARS-CoV-2; virus; West Nile virus; Yule-Harding model; Zika virus.].

Hepatitis C Virus in people with experience of injection drug use following their displacement to Southern Ukraine before 2020.

BACKGROUND: Due to practical challenges associated with genetic sequencing in low-resource environments, the burden of hepatitis C virus (HCV) in forcibly displaced people is understudied. We examined the use of field applicable HCV sequencing methods and phylogenetic analysis to determine HCV transmission dynamics in internally displaced people who inject drugs (IDPWID) in Ukraine. METHODS: In this cross-sectional study, we used modified respondent-driven sampling to recruit IDPWID who were displaced to Odesa, Ukraine, before 2020. We generated partial and near full length genome (NFLG) HCV sequences using Oxford Nanopore Technology (ONT) MinION in a simulated field environment. Maximum likelihood and Bayesian methods were used to establish phylodynamic relationships. RESULTS: Between June and September 2020, we collected epidemiological data and whole blood samples from 164 IDPWID (PNAS Nexus.2023;2(3):pgad008). Rapid testing (Wondfo® One Step HCV; Wondfo® One Step HIV1/2) identified an anti-HCV seroprevalence of 67.7%, and 31.1% of participants tested positive for both anti-HCV and HIV. We generated 57 partial or NFLG HCV sequences and identified eight transmission clusters, of which at least two originated within a year and a half post-displacement. CONCLUSIONS: Locally generated genomic data and phylogenetic analysis in rapidly changing low-resource environments, such as those faced by forcibly displaced people, can help inform effective public health strategies. For example, evidence of HCV transmission clusters originating soon after displacement highlights the importance of implementing urgent preventive interventions in ongoing situations of forced displacement.

Introduction and Establishment of SARS-CoV-2 Gamma Variant in New York City in Early 2021.

BACKGROUND: Monitoring the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is an important public health objective. We investigated how the Gamma variant was established in New York City (NYC) in early 2021 in the presence of travel restrictions that aimed to prevent viral spread from Brazil, the country where the variant was first identified. METHODS: We performed phylogeographic analysis on 15 967 Gamma sequences sampled between 10 March and 1 May 2021, to identify geographic sources of Gamma lineages introduced into NYC. We identified locally circulating Gamma transmission clusters and inferred the timing of their establishment in NYC. RESULTS: We identified 16 phylogenetically distinct Gamma clusters established in NYC (cluster sizes ranged 2-108 genomes); most of them were introduced from Florida and Illinois and only 1 directly from Brazil. By the time the first Gamma case was reported by genomic surveillance in NYC on 10 March, the majority (57%) of circulating Gamma lineages had already been established in the city for at least 2 weeks. CONCLUSIONS: Although travel from Brazil to the United States was restricted from May 2020 through the end of the study period, this restriction did not prevent Gamma from becoming established in NYC as most introductions occurred from domestic locations.

Discovery of Novel Herpes Simplexviruses in Wild Gorillas, Bonobos, and Chimpanzees Supports Zoonotic Origin of HSV-2.

Viruses closely related to human pathogens can reveal the origins of human infectious diseases. Human herpes simplexvirus type 1 (HSV-1) and type 2 (HSV-2) are hypothesized to have arisen via host-virus codivergence and cross-species transmission. We report the discovery of novel herpes simplexviruses during a large-scale screening of fecal samples from wild gorillas, bonobos, and chimpanzees. Phylogenetic analysis indicates that, contrary to expectation, simplexviruses from these African apes are all more closely related to HSV-2 than to HSV-1. Molecular clock-based hypothesis testing suggests the divergence between HSV-1 and the African great ape simplexviruses likely represents a codivergence event between humans and gorillas. The simplexviruses infecting African great apes subsequently experienced multiple cross-species transmission events over the past 3 My, the most recent of which occurred between humans and bonobos around 1 Ma. These findings revise our understanding of the origins of human herpes simplexviruses and suggest that HSV-2 is one of the earliest zoonotic pathogens.

Incorporating metadata in HIV transmission network reconstruction: A machine learning feasibility assessment.

HIV molecular epidemiology estimates the transmission patterns from clustering genetically similar viruses. The process involves connecting genetically similar genotyped viral sequences in the network implying epidemiological transmissions. This technique relies on genotype data which is collected only from HIV diagnosed and in-care populations and leaves many persons with HIV (PWH) who have no access to consistent care out of the tracking process. We use machine learning algorithms to learn the non-linear correlation patterns between patient metadata and transmissions between HIV-positive cases. This enables us to expand the transmission network reconstruction beyond the molecular network. We employed multiple commonly used supervised classification algorithms to analyze the San Diego Primary Infection Resource Consortium (PIRC) cohort dataset, consisting of genotypes and nearly 80 additional non-genetic features. First, we trained classification models to determine genetically unrelated individuals from related ones. Our results show that random forest and decision tree achieved over 80% in accuracy, precision, recall, and F1-score by only using a subset of meta-features including age, birth sex, sexual orientation, race, transmission category, estimated date of infection, and first viral load date besides genetic data. Additionally, both algorithms achieved approximately 80% sensitivity and specificity. The Area Under Curve (AUC) is reported 97% and 94% for random forest and decision tree classifiers respectively. Next, we extended the models to identify clusters of similar viral sequences. Support vector machine demonstrated one order of magnitude improvement in accuracy of assigning the sequences to the correct cluster compared to dummy uniform random classifier. These results confirm that metadata carries important information about the dynamics of HIV transmission as embedded in transmission clusters. Hence, novel computational approaches are needed to apply the non-trivial knowledge collected from inter-individual genetic information to metadata from PWH in order to expand the estimated transmissions. We note that feature extraction alone will not be effective in identifying patterns of transmission and will result in random clustering of the data, but its utilization in conjunction with genetic data and the right algorithm can contribute to the expansion of the reconstructed network beyond individuals with genetic data.

StrainHub: a phylogenetic tool to construct pathogen transmission networks.

SUMMARY: In exploring the epidemiology of infectious diseases, networks have been used to reconstruct contacts among individuals and/or populations. Summarizing networks using pathogen metadata (e.g. host species and place of isolation) and a phylogenetic tree is a nascent, alternative approach. In this paper, we introduce a tool for reconstructing transmission networks in arbitrary space from phylogenetic information and metadata. Our goals are to provide a means of deriving new insights and infection control strategies based on the dynamics of the pathogen lineages derived from networks and centrality metrics. We created a web-based application, called StrainHub, in which a user can input a phylogenetic tree based on genetic or other data along with characters derived from metadata using their preferred tree search method. StrainHub generates a transmission network based on character state changes in metadata, such as place or source of isolation, mapped on the phylogenetic tree. The user has the option to calculate centrality metrics on the nodes including betweenness, closeness, degree and a new metric, the source/hub ratio. The outputs include the network with values for metrics on its nodes and the tree with characters reconstructed. All of these results can be exported for further analysis. AVAILABILITY AND IMPLEMENTATION: strainhub.io and https://github.com/abschneider/StrainHub.

Addressing Ethical Challenges in US-Based HIV Phylogenetic Research.

In recent years, phylogenetic analysis of HIV sequence data has been used in research studies to investigate transmission patterns between individuals and groups, including analysis of data from HIV prevention clinical trials, in molecular epidemiology, and in public health surveillance programs. Phylogenetic analysis can provide valuable information to inform HIV prevention efforts, but it also has risks, including stigma and marginalization of groups, or potential identification of HIV transmission between individuals. In response to these concerns, an interdisciplinary working group was assembled to address ethical challenges in US-based HIV phylogenetic research. The working group developed recommendations regarding (1) study design; (2) data security, access, and sharing; (3) legal issues; (4) community engagement; and (5) communication and dissemination. The working group also identified areas for future research and scholarship to promote ethical conduct of HIV phylogenetic research.

The Relationship Between the Human Immunodeficiency Virus-1 Transmission Network and the HIV Care Continuum in Los Angeles County.

BACKGROUND: Public health action combating human immunodeficiency virus (HIV) includes facilitating navigation through the HIV continuum of care: timely diagnosis followed by linkage to care and initiation of antiretroviral therapy to suppress viral replication. Molecular epidemiology can identify rapidly growing HIV genetic transmission clusters. How progression through the care continuum relates to transmission clusters has not been previously characterized. METHODS: We performed a retrospective study on HIV surveillance data from 5226 adult cases in Los Angeles County diagnosed from 2010 through 2014. Genetic transmission clusters were constructed using HIV-TRACE. Cox proportional hazard models were used to estimate the impact of transmission cluster growth on the time intervals between care continuum events. Gamma frailty models incorporated the effect of heterogeneity associated with genetic transmission clusters. RESULTS: In contrast to our expectations, there were no differences in time to the care continuum events among individuals in clusters with different growth dynamics. However, upon achieving viral suppression, individuals in high growth clusters were slower to experience viral rebound (hazard ratio 0.83, P = .011) compared with individuals in low growth clusters. Heterogeneity associated with cluster membership in the timing to each event in the care continuum was highly significant (P < .001), with and without adjustment for transmission risk and demographics. CONCLUSIONS: Individuals within the same transmission cluster have more similar trajectories through the HIV care continuum than those across transmission clusters. These findings suggest molecular epidemiology can assist public health officials in identifying clusters of individuals who may benefit from assistance in navigating the HIV care continuum.

FAVITES: simultaneous simulation of transmission networks, phylogenetic trees and sequences.

MOTIVATION: The ability to simulate epidemics as a function of model parameters allows insights that are unobtainable from real datasets. Further, reconstructing transmission networks for fast-evolving viruses like Human Immunodeficiency Virus (HIV) may have the potential to greatly enhance epidemic intervention, but transmission network reconstruction methods have been inadequately studied, largely because it is difficult to obtain 'truth' sets on which to test them and properly measure their performance. RESULTS: We introduce FrAmework for VIral Transmission and Evolution Simulation (FAVITES), a robust framework for simulating realistic datasets for epidemics that are caused by fast-evolving pathogens like HIV. FAVITES creates a generative model to produce contact networks, transmission networks, phylogenetic trees and sequence datasets, and to add error to the data. FAVITES is designed to be extensible by dividing the generative model into modules, each of which is expressed as a fixed API that can be implemented using various models. We use FAVITES to simulate HIV datasets and study the realism of the simulated datasets. We then use the simulated data to study the impact of the increased treatment efforts on epidemiological outcomes. We also study two transmission network reconstruction methods and their effectiveness in detecting fast-growing clusters. AVAILABILITY AND IMPLEMENTATION: FAVITES is available at https://github.com/niemasd/FAVITES, and a Docker image can be found on DockerHub (https://hub.docker.com/r/niemasd/favites). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

HIV-TRACE (TRAnsmission Cluster Engine): a Tool for Large Scale Molecular Epidemiology of HIV-1 and Other Rapidly Evolving Pathogens.

In modern applications of molecular epidemiology, genetic sequence data are routinely used to identify clusters of transmission in rapidly evolving pathogens, most notably HIV-1. Traditional 'shoe-leather' epidemiology infers transmission clusters by tracing chains of partners sharing epidemiological connections (e.g., sexual contact). Here, we present a computational tool for identifying a molecular transmission analog of such clusters: HIV-TRACE (TRAnsmission Cluster Engine). HIV-TRACE implements an approach inspired by traditional epidemiology, by identifying chains of partners whose viral genetic relatedness imply direct or indirect epidemiological connections. Molecular transmission clusters are constructed using codon-aware pairwise alignment to a reference sequence followed by pairwise genetic distance estimation among all sequences. This approach is computationally tractable and is capable of identifying HIV-1 transmission clusters in large surveillance databases comprising tens or hundreds of thousands of sequences in near real time, that is, on the order of minutes to hours. HIV-TRACE is available at www.hivtrace.org and from www.github.com/veg/hivtrace, along with the accompanying result visualization module from www.github.com/veg/hivtrace-viz. Importantly, the approach underlying HIV-TRACE is not limited to the study of HIV-1 and can be applied to study outbreaks and epidemics of other rapidly evolving pathogens.

Size, Composition, and Evolution of HIV DNA Populations during Early Antiretroviral Therapy and Intensification with Maraviroc.

Residual viremia is common during antiretroviral therapy (ART) and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial and monitored for a median of 107 weeks. Participants started ART with (n = 9) or without (n = 9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 env, gag, and pol (454 Roche) was performed on longitudinally collected plasma and peripheral blood mononuclear cell (PBMC) samples while on ART. Sequence data were analyzed for evidence of evolution by (i) molecular diversity analysis, (ii) nonparametric test for panmixia, and (iii) tip date randomization within a Bayesian framework. There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (-0.08 ± 0.01 versus -0.09 ± 0.01 log10 copies/week in MVC+ versus MVC- groups; P = 0.62). All participants had low-level residual viremia (median, 2.8 RNA copies/ml). Across participants, medians of 56 (interquartile range [IQR], 36 to 74), 29 (IQR, 25 to 35), and 40 (IQR, 31 to 54) haplotypes were generated for env, gag, and pol regions, respectively. There was no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed, and potential low-level viral replication should always be considered in cure strategies.IMPORTANCE Residual viremia is common among HIV-infected people on ART. It remains controversial if this viremia is a consequence of propagating infection. We hypothesized that molecular evolution would be detectable during viral propagation and that therapy intensified with the entry inhibitor maraviroc would demonstrate less evolution. We performed a randomized double-blinded treatment trial with 18 acutely infected men (standard ART versus standard ART plus maraviroc). From longitudinally collected blood plasma and cells, levels of HIV DNA and cell-free HIV RNA were quantified by droplet digital PCR, and HIV DNA (env, gag, and pol coding regions) was deep sequenced (454 Roche). Investigating people who started ART during the earliest stages of their HIV infection, when viral diversity is low, provides an opportunity to detect evidence of viral evolution. Despite using a battery of analytical techniques, no clear and consistent evidence of viral propagation for over 90 weeks of observation could be discerned.