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.

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.

Clinical associations of patients with anti-3-hydroxy-3-methylglutaryl CoA reductase antibody-associated immune-mediated necrotising myopathy.

BACKGROUND: Anti-3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) antibodies are associated with a subtype of immune-mediated necrotising myopathy (IMNM). AIMS: To determine clinical associations of anti-HMGCR antibodies for anti-HMGCR-associated IMNM (HMGCR-IMNM) among a cohort of patients in Western Australia and to determine whether serial HMGCR antibody levels parallel disease activity. METHODS: Adult patients with positive anti-HMGCR antibodies detected by enzyme-linked immunosorbent assay between January 2015 and November 2019 were included. Symptoms, examination findings, imaging findings and blood test results were reviewed retrospectively using patient records and laboratory database results. RESULTS: Among 26 patients with positive anti-HMGCR antibodies, 23 were diagnosed with HMGCR-IMNM representing a positive predictive value (PPV) of 88%. Myopathy was frequently severe at diagnosis with limb weakness graded as Medical Research Council score 3 or below in 78% of patients, bulbar muscle weakness in 39% and an average creatine kinase (CK) at diagnosis of 7986 U/L. The majority (83%) required at least two therapies to maintain remission, 48% had at least one flare of disease and 57% did not achieve CK normalisation. Correlation between CK and anti-HMGCR antibody level at diagnosis was low (r = 0.04). Anti-HMGCR antibodies fell with treatment in 10 of 12 patients, but remained persistently positive in 83% of patients. CONCLUSIONS: The PPV of anti-HMGCR antibodies for HMGCR-IMNM in this Western Australian cohort is 88%. Patients typically present with proximal limb weakness, dysphagia and markedly elevated CK, and, despite multiagent immunosuppression, a significant number of patients have evidence of persistent biochemical myositis. Anti-HMGCR antibodies did not correlate with CK levels at diagnosis.

Editorial Commentary: Blood Flow Restriction Therapy Prior to Anterior Cruciate Ligament Reconstruction: Small Effects Require Accurate Measurements.

A recent research study showed that blood flow restriction (BFR) therapy was safe and well tolerated but failed to demonstrate efficacy as a modality that provides greater gains in quadriceps strength when added to a standard home program in patients awaiting anterior cruciate ligament (ACL) reconstruction. Despite employing a validated method of measurement, the results were highly variable, indicating the need for measurements with sufficient accuracy to detect the small, but potentially meaningful, gains in quadriceps strength that's been attributed to BFR. The results inform future investigations of BFR prior to ACL surgery by demonstrating the need for accurate methods of measurements when the anticipated effects are small.

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.

Self-Reported Prevalence, Magnitude, and Methods of Rapid Weight Loss in Male and Female Competitive Powerlifters.

ABSTRACT: Nolan, D, Lynch, AE, and Egan, B. Self-reported prevalence, magnitude, and methods of rapid weight loss in male and female competitive powerlifters. J Strength Cond Res 36(2): 405-410, 2022-Rapid weight loss (RWL) is common practice in weight category sports, but no empirical data exist documenting the weight-making practices of competitive strength athletes. This study investigated the self-reported prevalence, magnitude, and methods of RWL used by male and female powerlifters when preparing for competition. Competitive powerlifters (n = 321; M/F, 194/127) completed an anonymous online questionnaire previously validated for assessment of methods of RWL. Respondents were categorized by their federation's respective antidoping policy, weigh-in procedure, and degree of assistive equipment allowed, in addition to their use or not of RWL. Subgroup analyses were performed on the largest category of respondents (n = 200, M/F, 117/83; ≤2-hour weigh-in, drug-tested, "raw") based on sex, weight category, and competitive status. Prevalence of RWL was 85.8%, with an average RWL of 3.0 ± 1.9% body mass and an RWL score of 25.1 ± 7.4. Neither sex nor weight category influenced the RWL score, but in male athletes, a lower RWL score (22.7 ± 6.3) was reported in athletes in the lowest tertile of the Wilks score (p = 0.015). Frequencies of "always use" were reported as 54.0% for fluid restriction and 49.0% for water loading. Coaches (37.5%) and online resources (35.0%) were "very influential" on RWL practices in these athletes, while doctors (85.5%) and dieticians (63.0%) were reported to be "not influential." The prevalence of RWL is high in competitive powerlifting, and the methods used are akin to other weight category sports, but the reported RWL scores are lower than reported in combat sports with longer recovery periods after weigh-in.

Characteristics and Future Direction of Tibialis Posterior Tendinopathy Research: A Scoping Review.

Background and Objectives: Tibialis posterior tendon pathologies have been traditionally categorized into different stages of posterior tibial tendon dysfunction (PTTD), or adult acquired flatfoot deformity (AAFD), and more recently to progressive collapsing foot deformity (PCFD). The purpose of this scoping review is to synthesize and characterize literature on early stages of PTTD (previously known as Stage I and II), which we will describe as tibialis posterior tendinopathy (TPT). We aim to identify what is known about TPT, identify gaps in knowledge on the topics of TPT, and propose future research direction. Materials and Methods: We included 44 studies and categorized them into epidemiology, diagnosis, evaluation, biomechanics outcome measure, imaging, and nonsurgical treatment. Results: A majority of studies (86.4%, 38 of 44 studies) recruited patients with mean or median ages greater than 40. For studies that reported body mass index (BMI) of the patients, 81.5% had mean or median BMI meeting criteria for being overweight. All but two papers described study populations as predominantly or entirely female gender. Biomechanical studies characterized findings associated with TPT to include increased forefoot abduction and rearfoot eversion during gait cycle, weak hip and ankle performance, and poor balance. Research on non-surgical treatment focused on orthotics with evidence mostly limited to observational studies. The optimal exercise regimen for the management of TPT remains unclear due to the limited number of high-quality studies. Conclusions: More epidemiological studies from diverse patient populations are necessary to better understand prevalence, incidence, and risk factors for TPT. The lack of high-quality studies investigating nonsurgical treatment options is concerning because, regardless of coexisting foot deformity, the initial treatment for TPT is typically conservative. Additional studies comparing various exercise programs may help identify optimal exercise therapy, and investigation into further nonsurgical treatments is needed to optimize the management for TPT.

Delayed Expression of PD-1 and TIGIT on HIV-Specific CD8 T Cells in Untreated HLA-B*57:01 Individuals Followed from Early Infection.

While the relationship of protective human leukocyte antigen (HLA) class I alleles and HIV progression is well defined, the interaction of HLA-mediated protection and CD8 T-cell exhaustion is less well characterized. To gain insight into the influence of HLA-B*57:01 on the deterioration of CD8 T-cell responses during HIV infection in the absence of antiretroviral treatment, we compared HLA-B*57:01-restricted HIV-specific CD8 T-cell responses to responses restricted by other HLA class I alleles longitudinally after control of peak viremia. Detailed characterization of polyfunctionality, differentiation phenotypes, transcription factor, and inhibitory receptor expression revealed progression of CD8 T-cell exhaustion over the course of the infection in both patient groups. However, early effects on the phenotype of the total CD8 T-cell population were apparent only in HLA-B*57-negative patients. The HLA-B*57:01-restricted, HIV epitope-specific CD8 T-cell responses showed beneficial functional patterns and significantly lower frequencies of inhibitory receptor expression, i.e., PD-1 and coexpression of PD-1 and TIGIT, within the first year of infection. Coexpression of PD-1 and TIGIT was correlated with clinical markers of disease progression and declining percentages of the T-bethi Eomesdim CD8 T-cell population. In accordance with clinical and immunological deterioration in the HLA-B*57:01 group, the difference in PD-1 and TIGIT receptor expression did not persist to later stages of the disease.IMPORTANCE Given the synergistic nature of TIGIT and PD-1, the coexpression of those inhibitory receptors should be considered when evaluating T-cell pathogenesis, developing immunomodulatory therapies or vaccines for HIV, and when using immunotherapy or vaccination for other causes in HIV-infected patients. HIV-mediated T-cell exhaustion influences the patient´s disease progression, immune system and subsequently non-AIDS complications, and efficacy of vaccinations against other pathogens. Consequently, the possibilities of interfering with exhaustion are numerous. Expanding the use of immunomodulatory therapies to include HIV treatment depends on information about possible targets and their role in the deterioration of the immune system. Furthermore, the rise of immunotherapies against cancer and elevated cancer incidence in HIV-infected patients together increase the need for detailed knowledge of T-cell exhaustion and possible interactions. A broader approach to counteract immune exhaustion to alleviate complications and improve efficacy of other vaccines also promises to increase patients' health and quality of life.

Brain tissue transcriptomic analysis of SIV-infected macaques identifies several altered metabolic pathways linked to neuropathogenesis and poly (ADP-ribose) polymerases (PARPs) as potential therapeutic targets.

Despite improvements in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders (HAND) remain prevalent in subjects undergoing therapy. HAND significantly affects individuals' quality of life, as well as adherence to therapy, and, despite the increasing understanding of neuropathogenesis, no definitive diagnostic or prognostic marker has been identified. We investigated transcriptomic profiles in frontal cortex tissues of Simian immunodeficiency virus (SIV)-infected Rhesus macaques sacrificed at different stages of infection. Gene expression was compared among SIV-infected animals (n = 11), with or without CD8+ lymphocyte depletion, based on detectable (n = 6) or non-detectable (n = 5) presence of the virus in frontal cortex tissues. Significant enrichment in activation of monocyte and macrophage cellular pathways was found in animals with detectable brain infection, independently from CD8+ lymphocyte depletion. In addition, transcripts of four poly (ADP-ribose) polymerases (PARPs) were up-regulated in the frontal cortex, which was confirmed by real-time polymerase chain reaction. Our results shed light on involvement of PARPs in SIV infection of the brain and their role in SIV-associated neurodegenerative processes. Inhibition of PARPs may provide an effective novel therapeutic target for HIV-related neuropathology.

Integrating finite element modelling and 3D printing to engineer biomimetic polymeric scaffolds for tissue engineering.

The suitability of a scaffold for tissue engineering is determined by a number of interrelated factors. The biomaterial should be biocompatible and cell instructive, with a porosity and pore interconnectivity that facilitates cellular migration and the transport of nutrients and waste products into and out of the scaffolds. For the engineering of load bearing tissues, the scaffold may also be required to possess specific mechanical properties and/or ensure the transfer of mechanical stimuli to cells to direct their differentiation. Achieving these design goals is challenging, but could potentially be realised by integrating computational tools such as finite element (FE) modelling with three-dimensional (3D) printing techniques to assess how scaffold architecture and material properties influence the performance of the implant. In this study we first use Fused Deposition Modelling (FDM) to modulate the architecture of polycaprolactone (PCL) scaffolds, exploring the influence of varying fibre diameter, spacing and laydown pattern on the structural and mechanical properties of such scaffolds. We next demonstrate that a simple FE modelling strategy, which captures key aspects of the printed scaffold's actual geometry and material behaviour, can be used to accurately model the mechanical characteristics of such scaffolds. We then show the utility of this strategy by using FE modelling to help design 3D printed scaffolds with mechanical properties mimicking that of articular cartilage. In conclusion, this study demonstrates that a relatively simple FE modelling approach can be used to inform the design of 3D printed scaffolds to ensure their bulk mechanical properties mimic specific target tissues.

The Utility of Whole Body 18F-FDG PET-CT in Diagnosing Isolated Cardiac Sarcoidosis: The Western Australian Cardiac Sarcoid Study.

BACKGROUND: It is reported that up to 29-52% of patients with cardiac sarcoidosis (CS) may have isolated cardiac sarcoidosis (ICS). The wide variation in prevalence may be related to the diagnostic methods for assessing extracardiac involvement. Whole-body 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) imaging is a useful and increasingly used technique for screening for extracardiac involvement in cases of suspected ICS. This study aims to determine the rate of isolated cardiac involvement with clinically manifest CS using cardiac 18F-FDG PET-CT. METHODS: We performed a retrospective analysis of data in the West Australian Cardiac Sarcoid (WACaS) Database. After cardiologist review and workup, all cases of proven or probable CS, based on either current Heart Rhythm Society criteria for the diagnosis of CS or local expert consensus were included. Only patients who underwent whole body 18F-FDG PET-CT were included in the final analysis. RESULTS: Fifty-two (52) cases of CS were identified. Data on symptoms, imaging findings, treatment and outcomes were collected. Of the 42 patients who underwent diagnostic 18F-FDG PET-CT, 32 demonstrated changes consistent with CS. Of the 32, 69% were male, mean age 50 years at diagnosis. Only 3/32 (9.4%) patients had ICS. Pulmonary involvement occurred in 91% with varied involvement in other organs. The mean number of extracardiac sites at diagnosis was 2.2. CONCLUSIONS: This study demonstrates the utility of 18F-FDG PET-CT in diagnosing extracardiac organ involvement in cases of CS. With the use of this modality, ICS may be rarer than previously reported.

IgE blockade in autoimmunity: Omalizumab induced remission of bullous pemphigoid.

Bullous pemphigoid (BP) is a blistering dermopathy and a prototypic antibody-mediated autoimmune disease. Detection of IgG autoantibodies against hemidesmosomal proteins BP180 and/or BP230 are diagnostic and levels can correlate with disease activity. Therapies include corticosteroids and oral immunosuppressants, while intravenous immunoglobulin and rituximab are reserved for treatment resistant cases. Here we describe a patient with severe BP which was refractory to standard first line therapy, intravenous immunoglobulin and rituximab induced depletion of peripheral B cells. Use of the monoclonal anti-IgE antibody omalizumab resulted in rapid resolution of blistering despite ongoing high levels of anti-skin IgG antibodies. To our knowledge this is the first case of BP responsive to omalizumab after failure of rituximab to be reported. This case adds to emerging data on omalizumab as a novel BP treatment as well as providing new evidence of an independent role for autoreactive IgE-mediated inflammation in the formation of BP skin lesions.

Population pharmacokinetic-pharmacodynamic modelling of the relationship between testosterone and prostate specific antigen in patients with prostate cancer during treatment with leuprorelin.

AIMS: This investigation aimed to quantitatively characterize the relationship between the gonadotropin-releasing hormone agonist leuprorelin, testosterone (T) and prostate specific antigen (PSA) concentrations over time, to aid identification of a target T concentration that optimises the balance of the benefits of T suppression whilst reducing the risk of side effects related to futile over-suppression. METHODS: Data from a single dose study to investigate the effect of leuprorelin in a 6-month depot formulation on T and PSA in prostate cancer patients were analysed using a population pharmacokinetic-pharmacodynamic modelling approach. The developed model was qualified using external data from 3 studies, in which the effect of different formulations of leuprorelin on T and PSA was evaluated in prostate cancer patients. RESULTS: The effect of leuprorelin on the relationship between T and PSA was adequately characterized by the Romero model with minor modifications, combined with a turnover model to describe the delay in response between T and PSA. The data were significantly better described when assuming a minimum PSA level that is independent on the treatment-related reduction in T, as compared to a model with a proportional reduction in PSA and T. CONCLUSIONS: The model-based analysis suggests that on a population level, reducing T concentrations below 35 ng/dL does not result in a further decrease in PSA levels (>95% of the minimal PSA level is reached). More data are required to support this relationship in the lower T and PSA range.

Pre-exposure prophylaxis for HIV prevention during pregnancy and lactation: forget not the women and children.

Pregnancy is known to be a time of increased susceptibility to acquiring to human immunodeficiency virus (HIV) infection and this increased maternal risk places the unborn child at risk of vertical transmission. Pre-exposure prophylaxis (PrEP) involves the provision of antiretroviral therapy to an HIV-negative individual with ongoing risk of HIV exposure to limit the likelihood of HIV transmission. The inclusion of PrEP as part of a comprehensive strategy is recognised as an effective and safe means of reducing HIV infection in serodiscordant couples, thereby reducing the risk of vertical transmission of HIV. Current data suggest that PrEP is safe to continue during pregnancy and breastfeeding in HIV-negative women who remain vulnerable to acquiring HIV. The recent Pharmaceutical Benefits Scheme subsidisation of PrEP has reduced the financial and practical obstacles of PrEP provision, and a subsequent increase in patient awareness and acceptance of PrEP is expected. The framework for appropriately identifying and managing at-risk pregnant and lactating women requiring PrEP is poorly defined and warrants further clarification to better support clinicians and this patient group. This review discusses the current recommendations highlighting the gaps in the guidelines and makes some recommendations for future guideline development.

Evidence for Mother-to-Child Transmission of Zika Virus Through Breast Milk.

Zikavirus (ZIKV) is an emerging viral pathogen that continues to spread throughout different regions of the world. Herein we report a case that provides further evidence that ZIKV transmission can occur through breastfeeding by providing a detailed clinical, genomic, and virological case-based description.

Insights into the Impact of CD8+ Immune Modulation on Human Immunodeficiency Virus Evolutionary Dynamics in Distinct Anatomical Compartments by Using Simian Immunodeficiency Virus-Infected Macaque Models of AIDS Progression.

A thorough understanding of the role of human immunodeficiency virus (HIV) intrahost evolution in AIDS pathogenesis has been limited by the need for longitudinally sampled viral sequences from the vast target space within the host, which are often difficult to obtain from human subjects. CD8+ lymphocyte-depleted macaques infected with simian immunodeficiency virus (SIV) provide an increasingly utilized model of pathogenesis due to clinical manifestations similar to those for HIV-1 infection and AIDS progression, as well as a characteristic rapid disease onset. Comparison of this model with SIV-infected non-CD8+ lymphocyte-depleted macaques also provides a unique opportunity to investigate the role of CD8+ cells in viral evolution and population dynamics throughout the duration of infection. Using several different phylogenetic methods, we analyzed viral gp120 sequences obtained from extensive longitudinal sampling of multiple tissues and enriched leukocyte populations from SIVmac251-infected macaques with or without CD8+ lymphocyte depletion. SIV evolutionary and selection patterns in non-CD8+ lymphocyte-depleted animals were characterized by sequential population turnover and continual viral adaptation, a scenario readily comparable to intrahost evolutionary patterns during human HIV infection in the absence of antiretroviral therapy. Alternatively, animals that were depleted of CD8+ lymphocytes exhibited greater variation in population dynamics among tissues and cell populations over the course of infection. Our findings highlight the major role for CD8+ lymphocytes in prolonging disease progression through continual control of SIV subpopulations from various anatomical compartments and the potential for greater independent viral evolutionary behavior among these compartments in response to immune modulation.IMPORTANCE Although developments in combined antiretroviral therapy (cART) strategies have successfully prolonged the time to AIDS onset in HIV-1-infected individuals, a functional cure has yet to be found. Improvement of drug interventions for a virus that is able to infect a wide range of tissues and cell types requires a thorough understanding of viral adaptation and infection dynamics within this target milieu. Although it is difficult to accomplish in the human host, longitudinal sampling of multiple anatomical locations is readily accessible in the SIV-infected macaque models of neuro-AIDS. The significance of our research is in identifying the impact of immune modulation, through differing immune selective pressures, on viral evolutionary behavior in a multitude of anatomical compartments. The results provide evidence encouraging the development of a more sophisticated model that considers a network of individual viral subpopulations within the host, with differing infection and transmission dynamics, which is necessary for more effective treatment strategies.

Brain-specific HIV Nef identified in multiple patients with neurological disease.

HIV-1 Nef is a flexible, multifunctional protein with several cellular targets that is required for pathogenicity of the virus. This protein maintains a high degree of genetic variation among intra- and inter-host isolates. HIV Nef is relevant to HIV-associated neurological diseases (HAND) in patients treated with combined antiretroviral therapy because of the protein's role in promoting survival and migration of infected brain macrophages. In this study, we analyzed 2020 HIV Nef sequences derived from 22 different tissues and 31 subjects using a novel computational approach. This approach combines statistical regression and evolved neural networks (ENNs) to classify brain sequences based on the physical and chemical characteristics of functional Nef domains. Based on training, testing, and validation data, the method successfully classified brain Nef sequences at 84.5% and provided informative features for further examination. These included physicochemical features associated with the Src-homology-3 binding domain, the Nef loop (including the AP-2 Binding region), and a cytokine-binding domain. Non-brain sequences from patients with HIV-associated neurological disease were frequently classified as brain, suggesting that the approach could indicate neurological risk using blood-derived virus or for the development of biomarkers for use in assay systems aimed at drug efficacy studies for the treatment of HIV-associated neurological diseases.

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis.

UNLABELLED: The emergence of a distinct subpopulation of human or simian immunodeficiency virus (HIV/SIV) sequences within the brain (compartmentalization) during infection is hypothesized to be linked to AIDS-related central nervous system (CNS) neuropathology. However, the exact evolutionary mechanism responsible for HIV/SIV brain compartmentalization has not been thoroughly investigated. Using extensive viral sampling from several different peripheral tissues and cell types and from three distinct regions within the brain from two well-characterized rhesus macaque models of the neurological complications of HIV infection (neuroAIDS), we have been able to perform in-depth evolutionary analyses that have been unattainable in HIV-infected subjects. The results indicate that, despite multiple introductions of virus into the brain over the course of infection, brain sequence compartmentalization in macaques with SIV-associated CNS neuropathology likely results from late viral entry of virus that has acquired through evolution in the periphery sufficient adaptation for the distinct microenvironment of the CNS. IMPORTANCE: HIV-associated neurocognitive disorders remain prevalent among HIV type 1-infected individuals, whereas our understanding of the critical components of disease pathogenesis, such as virus evolution and adaptation, remains limited. Building upon earlier findings of specific viral subpopulations in the brain, we present novel yet fundamental results concerning the evolutionary patterns driving this phenomenon in two well-characterized animal models of neuroAIDS and provide insight into the timing of entry of virus into the brain and selective pressure associated with viral adaptation to this particular microenvironment. Such knowledge is invaluable for therapeutic strategies designed to slow or even prevent neurocognitive impairment associated with AIDS.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

UNLABELLED: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.