Zika virus diversity in mice is maintained during early vertical transmission from placenta to fetus, but reduced in fetal bodies and brains at late stages of infection.

Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, within-host diversity in ZIKV populations during vertical transmission has not been well studied. Here, we used the established anti-interferon treated Rag1-/- mouse model of ZIKV vertical transmission to compare genomic variation within ZIKV populations in matched placentas, fetal bodies, and fetal brains via RNASeq. At early stages of vertical transmission, the ZIKV populations in the matched placentas and fetal bodies were similar. Most ZIKV single nucleotide variants were present in both tissues, indicating little to no restriction in transmission of ZIKV variants from placenta to fetus. In contrast, at later stages of fetal infection there was a sharp reduction in ZIKV diversity in fetal bodies and fetal brains. All fetal brain ZIKV populations were comprised of one of two haplotypes, containing either a single variant or three variants together, as largely homogenous populations. In most cases, the dominant haplotype present in the fetal brain was also the dominant haplotype present in the matched fetal body. However, in two of ten fetal brains the dominant ZIKV haplotype was undetectable or present at low frequencies in the matched placenta and fetal body ZIKV populations, suggesting evidence of a strict selective bottleneck and possible selection for certain variants during neuroinvasion of ZIKV into fetal brains.

Identification of an autotransporter peptidase of Rickettsia rickettsii responsible for maturation of surface exposed autotransporters.

Members of the spotted fever group rickettsia express four large, surface-exposed autotransporters, at least one of which is a known virulence determinant. Autotransporter translocation to the bacterial outer surface, also known as type V secretion, involves formation of a ß-barrel autotransporter domain in the periplasm that inserts into the outer membrane to form a pore through which the N-terminal passenger domain is passed and exposed on the outer surface. Two major surface antigens of Rickettsia rickettsii, are known to be surface exposed and the passenger domain cleaved from the autotransporter domain. A highly passaged strain of R. rickettsii, Iowa, fails to cleave these autotransporters and is avirulent. We have identified a putative peptidase, truncated in the Iowa strain, that when reconstituted into Iowa restores appropriate processing of the autotransporters as well as restoring a modest degree of virulence.

RNA Editing as a General Trait of Ebolaviruses.

RNA editing has been discovered as an essential mechanism for the transcription of the glycoprotein (GP) gene of Ebola virus but not Marburg virus. We developed a rapid transcript quantification assay (RTQA) to analyze RNA transcripts generated through RNA editing and used immunoblotting with a pan-ebolavirus monoclonal antibody to confirm different GP gene-derived products. RTQA successfully quantified GP gene transcripts during infection with representative members of 5 ebolavirus species. Immunoblotting verified expression of the soluble GP and the transmembrane GP. Our results defined RNA editing as a general trait of ebolaviruses. The degree of editing, however, varies among ebolaviruses with Reston virus showing the lowest and Bundibugyo virus the highest degree of editing.

Shared transcriptional profiles of atypical B cells suggest common drivers of expansion and function in malaria, HIV, and autoimmunity.

Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD+IgMlo and IgD-IgG+ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

Genome-Scale Transcript Analyses of Human Neutrophils.

Transcriptome analyses of unicellular and multicellular organisms have changed fundamental understanding of biological and pathological processes across multiple scientific disciplines. Over the past 15 years, studies of polymorphonuclear leukocyte (PMN or neutrophil) gene expression on a global scale have provided new insight into the molecular processes that promote resolution of infections in humans. Herein we present methods to analyze gene expression in human neutrophils using Affymetrix oligonucleotide microarrays and next-generation sequencing. Notably, the procedures utilize commercially available reagents and materials and thus represent a standardized approach for evaluating PMN transcript levels.

Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach.

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10-300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion.

Mitochondrial Respiration Is Impaired during Late-Stage Hamster Prion Infection.

Mitochondria are crucial to proper neuronal function and overall brain health. Mitochondrial dysfunction within the brain has been observed in many neurodegenerative diseases, including prion disease. Several markers of decreased mitochondrial activity during prion infection have been reported, yet the bioenergetic respiratory status of mitochondria from prion-infected animals is unknown. Here we show that clinically ill transgenic mice overexpressing hamster prion protein (Tg7) infected with the hamster prion strain 263K suffer from a severe deficit in mitochondrial oxygen consumption in response to the respiratory complex II substrate succinate. Characterization of the mitochondrial proteome of purified brain mitochondria from infected and uninfected Tg7 mice showed significant differences in the relative abundance of key mitochondrial electron transport proteins in 263K-infected animals relative to that in controls. Our results suggest that at clinical stages of prion infection, dysregulation of respiratory chain proteins may lead to impairment of mitochondrial respiration in the brain.IMPORTANCE Mitochondrial dysfunction is present in most major neurodegenerative diseases, and some studies have suggested that mitochondrial processes may be altered during prion disease. Here we show that hamster prion-infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits. Tg7 mice infected with the 263K hamster prion strain have little or no signs of mitochondrial dysfunction at the disease midpoint but suffer from a severe deficit in mitochondrial respiration at the clinical phase of disease. A proteomic analysis of the isolated brain mitochondria from clinically affected animals showed that several proteins involved in electron transport, mitochondrial dynamics, and mitochondrial protein synthesis were dysregulated. These results suggest that mitochondrial dysfunction, possibly exacerbated by prion protein overexpression, occurs at late stages during 263K prion disease and that this dysfunction may be the result of dysregulation of mitochondrial proteins.

Simtuzumab treatment of advanced liver fibrosis in HIV and HCV-infected adults: results of a 6-month open-label safety trial.

BACKGROUND: Chronic liver injury can result in fibrosis that may progress over years to end-stage liver disease. The most effective anti-fibrotic therapy is treatment of the underlying disease, however when not possible, interventions to reverse or slow fibrosis progression are needed. AIM: The aim of this study was to study the safety and tolerability of simtuzumab, a monoclonal antibody directed against lysyl oxidase-like 2 (LOXL2) enzyme, in subjects with hepatitis C virus (HCV), human immunodeficiency virus (HIV), or HCV-HIV co-infection and advanced liver disease. METHODS: Eighteen subjects with advanced liver fibrosis received simtuzumab 700 mg intravenously every 2 weeks for 22 weeks. Transjugular liver biopsies were performed during screening and at the end of treatment to measure hepatic venous pressure gradient (HVPG) and to stage fibrosis. RESULTS: Treatment was well-tolerated with no discontinuations due to adverse events. No significant changes were seen in HVPG or liver biopsy fibrosis score after treatment. Exploratory transcriptional and protein profiling using paired pre- and post-treatment liver biopsy and serum samples suggested up-regulation of TGF-ß3 and IL-10 pathways with treatment. CONCLUSION: In this open-label, pilot clinical trial, simtuzumab treatment was well-tolerated in HCV- and HIV-infected subjects with advanced liver disease. Putative modulation of TGF-ß3 and IL-10 pathways during simtuzumab treatment merits investigation in future trials.

Identification of Glial Activation Markers by Comparison of Transcriptome Changes between Astrocytes and Microglia following Innate Immune Stimulation.

The activation of astrocytes and microglia is often associated with diseases of the central nervous system (CNS). Understanding how activation alters the transcriptome of these cells may offer valuable insight regarding how activation of these cells mediate neurological damage. Furthermore, identifying common and unique pathways of gene expression during activation may provide new insight into the distinct roles these cells have in the CNS during infection and inflammation. Since recent studies indicate that TLR7 recognizes not only viral RNA but also microRNAs that are released by damaged neurons and elevated during neurological diseases, we first examined the response of glial cells to TLR7 stimulation using microarray analysis. Microglia were found to generate a much stronger response to TLR7 activation than astrocytes, both in the number of genes induced as well as fold induction. Although the primary pathways induced by both cell types were directly linked to immune responses, microglia also induced pathways associated with cellular proliferation, while astrocytes did not. Targeted analysis of a subset of the upregulated genes identified unique mRNA, including Ifi202b which was only upregulated by microglia and was found to be induced during both retroviral and bunyavirus infections in the CNS. In addition, other genes including Birc3 and Gpr84 as well as two expressed sequences AW112010 and BC023105 were found to be induced in both microglia and astrocytes and were upregulated in the CNS following virus infection. Thus, expression of these genes may a useful measurement of glial activation during insult or injury to the CNS.

Effect of sofosbuvir and ribavirin treatment on peripheral and hepatic lipid metabolism in chronic hepatitis C virus, genotype 1-infected patients.

UNLABELLED: Hepatitis C virus (HCV) modulates intrahepatic cholesterol biosynthetic pathways to promote viral replication. Chronic HCV infection is associated with altered metabolism, including dyslipidemia and insulin resistance (IR), which contributes to disease progression and influences response to therapy. To further understand the impact of HCV infection on host metabolism, we examined changes in serum lipid profiles and intrahepatic expression of lipid-related genes during interferon (IFN)-free treatment of chronic HCV, genotype 1 infection with sofosbuvir and ribavirin (RBV), and explored associations with treatment outcome. Serum lipids (total cholesterol, low-density lipoprotein [LDL], high-density lipoprotein [HDL], and triglycerides [TGs]) and hemoglobin A1C (HbA1C) were measured during treatment, while gene expression of lipid-related genes was assessed using paired pre- and end-of-treatment (EOT) liver biopsies from 8 patients (n=7 sustained virologic response [SVR]; n=1 relapse) and unpaired EOT liver biopsies from 25 patients (n=17 SVR; n=8 relapse). Serum LDL concentration and particle size increased early in therapy, whereas TG concentration and very-low-density lipoprotein particle size decreased concomitantly, irrespective of treatment outcome. Whereas LDL increased in patients regardless of treatment outcome, average LDL concentration was lower at baseline and post-treatment in patients who relapsed. Analysis of paired liver biopsies revealed altered expression of genes associated with lipid transport, assembly, and signaling. In unpaired EOT liver biopsies, intrahepatic expression of fatty acid metabolism and lipid transport genes was lower in patients who experienced treatment relapse. CONCLUSION: Clearance of HCV using an IFN-free antiviral regimen results in rapid changes in peripheral and intrahepatic metabolic pathways, implicating a direct effect of HCV replication on lipid homeostasis.

Proteomics analysis of amyloid and nonamyloid prion disease phenotypes reveals both common and divergent mechanisms of neuropathogenesis.

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP(C)) into a pathological isoform termed PrP(Sc). In wild-type mice, PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP(Sc) typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP(Sc) accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP(C) anchoring to the plasma membrane correlated not only with the type of PrP(Sc) deposition but also with unique biochemical pathways associated with pathogenesis.

Endogenous intrahepatic IFNs and association with IFN-free HCV treatment outcome.

BACKGROUND. Hepatitis C virus (HCV) infects approximately 170 million people worldwide and may lead to cirrhosis and hepatocellular carcinoma in chronically infected individuals. Treatment is rapidly evolving from IFN-α-based therapies to IFN-α-free regimens that consist of directly acting antiviral agents (DAAs), which demonstrate improved efficacy and tolerability in clinical trials. Virologic relapse after DAA therapy is a common cause of treatment failure; however, it is not clear why relapse occurs or whether certain individuals are more prone to recurrent viremia. METHODS. We conducted a clinical trial using the DAA sofosbuvir plus ribavirin (SOF/RBV) and performed detailed mRNA expression analysis in liver and peripheral blood from patients who achieved either a sustained virologic response (SVR) or relapsed. RESULTS. On-treatment viral clearance was accompanied by rapid downregulation of IFN-stimulated genes (ISGs) in liver and blood, regardless of treatment outcome. Analysis of paired pretreatment and end of treatment (EOT) liver biopsies from SVR patients showed that viral clearance was accompanied by decreased expression of type II and III IFNs, but unexpectedly increased expression of the type I IFN IFNA2. mRNA expression of ISGs was higher in EOT liver biopsies of patients who achieved SVR than in patients who later relapsed. CONCLUSION. These results suggest that restoration of type I intrahepatic IFN signaling by EOT may facilitate HCV eradication and prevention of relapse upon withdrawal of SOF/RBV. TRIAL REGISTRATION. ClinicalTrials.gov NCT01441180.

miR-182 and miR-10a are key regulators of Treg specialisation and stability during Schistosome and Leishmania-associated inflammation.

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4(+)Foxp3(+) regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA "regulatory hubs" miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4(+)Foxp3(+) cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function.

IL-17-induced NF-kappaB activation via CIKS/Act1: physiologic significance and signaling mechanisms.

Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases.

Recurrent Granulibacter bethesdensis infections and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by frequent infections, most of which are curable. Granulibacter bethesdensis is an emerging pathogen in patients with CGD that causes fever and necrotizing lymphadenitis. However, unlike typical CGD organisms, this organism can cause relapse after clinical quiescence. To better define whether infections were newly acquired or recrudesced, we use comparative bacterial genomic hybridization to characterize 11 isolates obtained from 5 patients with CGD from North and Central America. Genomic typing showed that 3 patients had recurrent infection months to years after apparent clinical cure. Two patients were infected with the same strain as previously isolated, and 1 was infected with a genetically distinct strain. This organism is multidrug resistant, and therapy required surgery and combination antimicrobial drugs, including long-term ceftriaxone. G. bethesdensis causes necrotizing lymphadenitis in CGD, which may recur or relapse.

Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages.

Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

Analysis of Staphylococcus aureus gene expression during PMN phagocytosis.

Staphylococcus aureus is a leading cause of human infections worldwide and causes a variety of diseases ranging in severity from mild to life-threatening. The ability of S. aureus to cause disease is based in part on its ability to subvert the innate immune system. Advances in genome-wide analysis of host-pathogen interactions have provided the necessary tools to investigate molecular factors that directly contribute to S. aureus pathogenesis. This chapter describes methods to analyze gene expression in S. aureus during interaction with human neutrophils.

Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis.

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with Staphylococcus, certain gram-negative bacteria, and fungi. Granulibacter bethesdensis, a newly described genus and species within the family Acetobacteraceae, was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of Granulibacter bethesdensis, which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related Gluconobacter oxydans genome. Included in the 967 ORFs that are unique to G. bethesdensis are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of G. bethesdensis to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.