Acute West Nile Virus Meningoencephalitis Diagnosed Via Metagenomic Deep Sequencing of Cerebrospinal Fluid in a Renal Transplant Patient.

Solid organ transplant patients are vulnerable to suffering neurologic complications from a wide array of viral infections and can be sentinels in the population who are first to get serious complications from emerging infections like the recent waves of arboviruses, including West Nile virus, Chikungunya virus, Zika virus, and Dengue virus. The diverse and rapidly changing landscape of possible causes of viral encephalitis poses great challenges for traditional candidate-based infectious disease diagnostics that already fail to identify a causative pathogen in approximately 50% of encephalitis cases. We present the case of a 14-year-old girl on immunosuppression for a renal transplant who presented with acute meningoencephalitis. Traditional diagnostics failed to identify an etiology. RNA extracted from her cerebrospinal fluid was subjected to unbiased metagenomic deep sequencing, enhanced with the use of a Cas9-based technique for host depletion. This analysis identified West Nile virus (WNV). Convalescent serum serologies subsequently confirmed WNV seroconversion. These results support a clear clinical role for metagenomic deep sequencing in the setting of suspected viral encephalitis, especially in the context of the high-risk transplant patient population.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Viral co-infection, autoimmunity, and CSF HIV antibody profiles in HIV central nervous system escape.

Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.

Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics.

The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.

Cultivation and serological characterization of a human Theiler's-like cardiovirus associated with diarrheal disease.

Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape.

Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A(2) (PLA(2)s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA(2) activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA(2) activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

Exploring the metabolic and genetic control of gene expression on a genomic scale.

DNA microarrays containing virtually every gene of Saccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes whose expression was affected by deletion of the transcriptional co-repressor TUP1 or overexpression of the transcriptional activator YAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.

Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier.

Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.

The transcriptional program of sporulation in budding yeast.

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

Towards precision quantification of contamination in metagenomic sequencing experiments.

Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from unintentional sequencing of occult contaminants, especially those derived from molecular biology reagents. Recent work suggests that, for any given microbe detected by mNGS, an inverse linear relationship between microbial sequencing reads and sample mass implicates that microbe as a contaminant. By associating sequencing read output with the mass of a spike-in control, we demonstrate that contaminant nucleic acid can be quantified in order to identify the mass contributions of each constituent. In an experiment using a high-resolution (n = 96) dilution series of HeLa RNA spanning 3-logs of RNA mass input, we identified a complex set of contaminants totaling 9.1 ± 2.0 attograms. Given the competition between contamination and the true microbiome in ultra-low biomass samples such as respiratory fluid, quantification of the contamination within a given batch of biological samples can be used to determine a minimum mass input below which sequencing results may be distorted. Rather than completely censoring contaminant taxa from downstream analyses, we propose here a statistical approach that allows separation of the true microbial components from the actual contribution due to contamination. We demonstrate this approach using a batch of n = 97 human serum samples and note that despite E. coli contamination throughout the dataset, we are able to identify a patient sample with significantly more E. coli than expected from contamination alone. Importantly, our method assumes no prior understanding of possible contaminants, does not rely on any prior collection of environmental or reagent-only sequencing samples, and does not censor potentially clinically relevant taxa, thus making it a generalized approach to any kind of metagenomic sequencing, for any purpose, clinical or otherwise.

Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source.

The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.

New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis.

The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae. When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified. The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site. Eight of these genes, PHM1-PHM8, had no previously defined function in phosphate metabolism. The amino acid sequences of PHM1 (YFL004w), PHM2 (YPL019c), PHM3 (YJL012c), and PHM4 (YER072w) are 32-56% identical. The phm3 and phm4 single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and P(i). The phenotype of the phm5 mutant suggests that PHM5 (YDR452w) is essential for normal catabolism of polyP in the yeast vacuole. Taken together, the results reveal important new features of a genetic system that plays a critical role in P(i) acquisition and polyP metabolism in yeast.

Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.

Programmable Microfluidic Synthesis of Over One Thousand Uniquely Identifiable Spectral Codes.

Encoded microparticles have become a powerful tool for a wide array of applications, including high-throughput sample tracking and massively parallel biological multiplexing. Spectral encoding, where particles are encoded with distinct luminescence spectra, provides a particularly appealing encoding strategy because of the ease of reading codes and assay flexibility. To date, spectral encoding has been limited in the number of codes that can be accurately resolved. Here, we demonstrate an automated 5-dimensional spectral encoding scheme using lanthanide nanophosphors that is capable of producing isotropic spherical microparticles with up to 1,100 unique codes, which we term MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding). We further develop a quantitative framework for evaluating global ability to distinguish codes and demonstrate that for six different sets of MRBLEs ranging from 106 to 1,101 codes in size, > 98% of MRBLEs can be assigned to a code with 99.99% confidence. These > 1,000 code sets represent the largest spectral code libraries built to date. We expect that these MRBLEs will enable a wide variety of novel multiplexed assays.

Detection and prevalence of boid inclusion body disease in collections of boas and pythons using immunological assays.

Inclusion body disease (IBD) of boas and pythons is characterized by the intracytoplasmic accumulation of an antigenic 68 kDa viral protein IBDP, more recently known as the nucleoprotein (NP) of the reptarenaviruses. Blood samples of 131 captive boas and pythons (53 boa constrictors, Boa constrictor; 35 rainbow boas, Epicrates cenchria; 22 ball pythons, Python regius; 5 carpet pythons, Morelia spilota; 6 Burmese pythons, Python bivittatus; 4 Jamaican boas, Epicrates subflavus; 5 anacondas, Eunectes spp.; and 1 green tree python, Morelia viridis) were obtained from 28 collections in the USA. Diagnosis of IBD was initially made by the identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (HE) stained blood films and isolated peripheral white blood cells (PWBC). The overall prevalence of IBD in study snakes was 25/131 or 19% (95% CI = 12.4%, 25.8%) with boa constrictors being more commonly infected (22/53 or 41.5%; 95% CI = 28.2%, 54.8%) than other species in this study. Of the 22 IBD positive boa constrictors, 87% were clinically healthy, 13% had various signs of chronic illness, and none showed signs of central nervous system disease. Using a validated monoclonal anti-NP antibody, NP was confirmed within the isolated PWBC by immunohistochemical staining and Western blots. The presence of reptarenaviruses within blood samples of 27 boa constrictors and three rainbow boas was also assessed by PCR. Among boa constrictors, very good agreements were shown between the observation of inclusion bodies (by HE stain) and the presence of NP (by immunohistochemistry, kappa = 0.92; and Western blots, kappa = 0.89), or the presence of reptarenaviruses (by PCR; kappa = 0.92).

Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications.

Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost.

Characterization of three related glucose repressors and genes they regulate in Saccharomyces cerevisiae.

Mig1 and Mig2 are proteins with similar zinc fingers that are required for glucose repression of SUC2 expression. Mig1, but not Mig2, is required for repression of some other glucose-repressed genes, including the GAL genes. A second homolog of Mig1, Yer028, appears to be a glucose-dependent transcriptional repressor that binds to the Mig1-binding sites in the SUC2 promoter, but is not involved in glucose repression of SUC2 expression. Despite their functional redundancy, we found several significant differences between Mig1 and Mig2: (1) in the absence of glucose, Mig1, but not Mig2, is inactivated by the Snf1 protein kinase; (2) nuclear localization of Mig1, but not Mig2, is regulated by glucose; (3) expression of MIG1, but not MIG2, is repressed by glucose; and (4) Mig1 and Mig2 bind to similar sites but with different relative affinities. By two approaches, we have identified many genes regulated by Mig1 and Mig2, and confirmed a role for Mig1 and Mig2 in repression of several of them. We found no genes repressed by Yer028. Also, we identified no genes repressed by only Mig1 or Mig2. Thus, Mig1 and Mig2 are redundant glucose repressors of many genes.

Correction: Programmable microfluidic synthesis of spectrally encoded microspheres.

Correction for 'Programmable microfluidic synthesis of spectrally encoded microspheres' by R. E. Gerver et al., Lab Chip, 2012, 12, 4716-4723.

Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants.

The cDNA from activated mutants of the homologous transcription factors Pdr1p and Pdr3p was used to screen DNA microarrays of the Saccharomyces cerevisiae complete genome. Twenty-six overexpressed targets of the PDR1-3 and/or PDR3-7 mutants were identified. Twenty-one are new targets, the majority of which are of unknown function. In addition to well known ABC transporters, these targets appear to be involved in transport or in membrane lipids and cell wall biosyntheses. Several of the targets seem to contribute to the cell defence against a variety of stresses. Pdr1p and Pdr3p do not act similarly on all targets. Unexpectedly, the expression of 23 other genes appeared to be repressed in the PDR1-3 and/or PDR3-7 mutants. In contrast to the majority of the activated genes, none of the repressed genes contains pleiotropic drug resistance binding sites in their promoter.