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.

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.

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.

Characterization of Learning, Motivation, and Visual Perception in Five Transgenic Mouse Lines Expressing GCaMP in Distinct Cell Populations.

To study the mechanisms of perception and cognition, neural measurements must be made during behavior. A goal of the Allen Brain Observatory is to map the activity of distinct cortical cell classes underlying visual and behavioral processing. Here we describe standardized methodology for training head-fixed mice on a visual change detection task, and we use our paradigm to characterize learning and behavior of five GCaMP6-expressing transgenic lines. We used automated training procedures to facilitate comparisons across mice. Training times varied, but most transgenic mice learned the behavioral task. Motivation levels also varied across mice. To compare mice in similar motivational states we subdivided sessions into over-, under-, and optimally motivated periods. When motivated, the pattern of perceptual decisions were highly correlated across transgenic lines, although overall performance (d-prime) was lower in one line labeling somatostatin inhibitory cells. These results provide important context for using these mice to map neural activity underlying perception and behavior.

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.

A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes the cortical activity of nearly 60,000 neurons from six visual areas, four layers, and 12 transgenic mouse lines in a total of 243 adult mice, in response to a systematic set of visual stimuli. We classify neurons on the basis of joint reliabilities to multiple stimuli and validate this functional classification with models of visual responses. While most classes are characterized by responses to specific subsets of the stimuli, the largest class is not reliably responsive to any of the stimuli and becomes progressively larger in higher visual areas. These classes reveal a functional organization wherein putative dorsal areas show specialization for visual motion signals.

Mouse color and wavelength-specific luminance contrast sensitivity are non-uniform across visual space.

Mammalian visual behaviors, as well as responses in the neural systems underlying these behaviors, are driven by luminance and color contrast. With constantly improving tools for measuring activity in cell-type-specific populations in the mouse during visual behavior, it is important to define the extent of luminance and color information that is behaviorally accessible to the mouse. A non-uniform distribution of cone opsins in the mouse retina potentially complicates both luminance and color sensitivity; opposing gradients of short (UV-shifted) and middle (blue/green) cone opsins suggest that color discrimination and wavelength-specific luminance contrast sensitivity may differ with retinotopic location. Here we ask how well mice can discriminate color and wavelength-specific luminance changes across visuotopic space. We found that mice were able to discriminate color and were able to do so more broadly across visuotopic space than expected from the cone-opsin distribution. We also found wavelength-band-specific differences in luminance sensitivity.

Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines.

Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.