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.

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.

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.

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.

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

UNLABELLED: HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. IMPORTANCE: It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.

Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques.

UNLABELLED: While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (~92 days) before they were detected in gp120 (~182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains. IMPORTANCE: The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology.

Anti-α4 antibody treatment blocks virus traffic to the brain and gut early, and stabilizes CNS injury late in infection.

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28+, RNA+) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV - RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.

The meningeal lymphatic system: a route for HIV brain migration?

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.

Spatiotemporal dynamics of simian immunodeficiency virus brain infection in CD8+ lymphocyte-depleted rhesus macaques with neuroAIDS.

Despite the success of combined antiretroviral therapy in controlling viral replication in human immunodeficiency virus (HIV)-infected individuals, HIV-associated neurocognitive disorders, commonly referred to as neuroAIDS, remain a frequent and poorly understood complication. Infection of CD8(+) lymphocyte-depleted rhesus macaques with the SIVmac251 viral swarm is a well-established rapid disease model of neuroAIDS that has provided critical insight into HIV-1-associated neurocognitive disorder onset and progression. However, no studies so far have characterized in depth the relationship between intra-host viral evolution and pathogenesis in this model. Simian immunodeficiency virus (SIV) env gp120 sequences were obtained from six infected animals. Sequences were sampled longitudinally from several lymphoid and non-lymphoid tissues, including individual lobes within the brain at necropsy, for four macaques; two animals were sacrificed at 21 days post-infection (p.i.) to evaluate early viral seeding of the brain. Bayesian phylodynamic and phylogeographic analyses of the sequence data were used to ascertain viral population dynamics and gene flow between peripheral and brain tissues, respectively. A steady increase in viral effective population size, with a peak occurring at ~50-80 days p.i., was observed across all longitudinally monitored macaques. Phylogeographic analysis indicated continual viral seeding of the brain from several peripheral tissues throughout infection, with the last migration event before terminal illness occurring in all macaques from cells within the bone marrow. The results strongly supported the role of infected bone marrow cells in HIV/SIV neuropathogenesis. In addition, our work demonstrated the applicability of Bayesian phylogeography to intra-host studies in order to assess the interplay between viral evolution and pathogenesis.

Longitudinal analysis of intra-host simian immunodeficiency virus recombination in varied tissues of the rhesus macaque model for neuroAIDS.

Human immunodeficiency virus intra-host recombination has never been studied in vivo both during early infection and throughout disease progression. The CD8-depleted rhesus macaque model of neuroAIDS was used to investigate the impact of recombination from early infection up to the onset of neuropathology in animals inoculated with a simian immunodeficiency virus (SIV) swarm. Several lymphoid and non-lymphoid tissues were collected longitudinally at 21 days post-infection (p.i.), 61 days p.i. and necropsy (75-118 days p.i.) from four macaques that developed SIV-encephalitis or meningitis, as well as from two animals euthanized at 21 days p.i. The number of recombinant sequences and breakpoints in different tissues and over time from each primate were compared. Breakpoint locations were mapped onto predicted RNA and protein secondary structures. Recombinants were found at each time point and in each primate as early as 21 days p.i. No association was found between recombination rates and specific tissue of origin. Several identical breakpoints were identified in sequences derived from different tissues in the same primate and among different primates. Breakpoints predominantly mapped to unpaired nucleotides or pseudoknots in RNA secondary structures, and proximal to glycosylation sites and cysteine residues in protein sequences, suggesting selective advantage in the emergence of specific recombinant sequences. Results indicate that recombinant sequences can become fixed very early after infection with a heterogeneous viral swarm. Features of RNA and protein secondary structure appear to play a role in driving the production of recombinants and their selection in the rapid disease model of neuroAIDS.

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.

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.

Efficient transmission and persistence of low-frequency SIVmac251 variants in CD8-depleted rhesus macaques with different neuropathology.

Infection of CD8-depleted rhesus macaques with the genetically heterogeneous simian immunodeficiency virus (SIV)mac251 viral swarm provides a rapid-disease model for simian acquired immune deficiency syndrome and SIV-encephalitis (SIVE). The objective was to evaluate how the diversity of the swarm influences the initial seeding of the infection that may potentially affect disease progression. Plasma, lymphoid and non-lymphoid (brain and lung) tissues were collected from two infected macaques euthanized at 21 days post-infection (p.i.), as well as longitudinal specimens and post-mortem tissues from four macaques followed throughout the infection. About 1300 gp120 viral sequences were obtained from the infecting SIVmac251 swarm and the macaques longitudinal and post-mortem samples. Phylogenetic and amino acid signature pattern analyses were carried out to assess frequency, transmission dynamics and persistence of specific viral clusters. Although no significant reduction in viral heterogeneity was found early in infection (21 days p.i.), transmission and replication of SIV variants was not entirely random. In particular, two distinct motifs under-represented (<4 %) in the infecting swarm were found at high frequencies (up to 14 %) in all six macaques as early as 21 days p.i. Moreover, a macrophage tropic variant not detected in the viral swarm (<0.3 %) was present at high frequency (29-100 %) in sequences derived from the brain of two macaques with meningitis or severe SIVE. This study demonstrates the highly efficient transmission and persistence in vivo of multiple low frequency SIVmac251 founder variants, characterized by specific gp120 motifs that may be linked to pathogenesis in the rapid-disease model of neuroAIDS.