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Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

Synchronous endometrial and ovarian (SEO) carcinomas involve endometrioid neoplasms in both the ovary and uterus at the time of diagnosis. Patients were traditionally classified as having independent primary SEO lesions or as having metastatic endometrioid carcinoma. Recent studies have supported that SEO tumors result from the dissemination of cells from one organ site to another. However, whether this can be considered a “metastasis” or “dissemination” remains unclear. In this report, we performed whole-exome sequencing of tumor samples from a woman with well-differentiated endometrioid SEO tumors and a clinical “recurrent” poorly differentiated peritoneal tumor that was diagnosed 8 years after the complete resection of the SEO tumors. Somatic mutation analysis identified 132, 171, and 1214 nonsynonymous mutations in the endometrial, ovarian, and peritoneal carcinomas, respectively. A unique mutation signature associated with mismatch repair deficiency was observed in all three tumors. The SEO carcinomas shared 57 nonsynonymous mutations, whereas the clinically suspected recurrent carcinoma shared only eight nonsynonymous mutations with the SEO tumors. One of the eight shared somatic mutations involved PTEN; these shared mutations represent the earliest genetic alteration in the ancestor cell clone. Based on analysis of the phylogenetic tree, we predicted that the so-called recurrent peritoneal tumor was derived from the same endometrial ancestor clone as the SEO tumors, and that this clone migrated and established benign peritoneal endometriosis where the peritoneal tumor later arose. This case highlights the usefulness of next-generation sequencing in defining the etiology and clonal relationships of synchronous and metachronous tumors from patients, thus providing valuable insight to aid in the clinical management of rare or ambiguous tumors.

Predicting viral evolution remains a major challenge with profound implications for public health. Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics device, the “Evolution Chip”, which propagates millions of independent viral sub-populations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs shape viral evolution.

A family of structurally related protein toxins extracted from bacteria or plants have a similar mechanism of action, namely, blocking protein synthesis by ribosome inactivation. Members of this family of ribosome-inactivating proteins (RIP), classified simultaneously as chemical and biological threat agents, include ricin, abrin, and shiga toxins. We have adapted and streamlined mass spectrometry based functional assays, described by others, to develop a rapid, robust, broad-band and fieldable protocol with the primary goal of RIP toxin detection in unknown powder materials. Complementary to intact protein detection and proteomics methods, this protocol involves monitoring the depurination of selected DNA substrates by mass spectrometry. It is being implemented on the CB-TOF system for rapid detection of bio-threats by MALDI MS. Here we illustrate its performance on the example of a plant (ricin) and a bacterial (shiga) RIP toxin.

Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCE RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

Detection of Plasmodium falciparum malaria during pregnancy is complicated by sequestration of para- sites in the placenta, which reduces peripheral blood microscopic detection. Laser desorption mass spectrometry (LDMS) has previously demonstrated sensitive detection of hemozoin from P. falciparum blood cultures and the ability to track parasitemia in a Plasmodium yoelii malaria mouse model. Here we use a simple, dilution in water, blood sample preparation protocol for LDMS detection of malaria in 45 asymptomatic, pregnant Zambian women. We compare LDMS to microscopy and polymerase chain reaction (PCR) analysis. All women were microscopy negative. LDMS detected P. falciparum hemozoin in 15 out of 45 women, while PCR results were positive in 25 women. Compared with PCR, which analyzed 20-30 L of blood, the sensitivity of LDMS, which analyzed < 1 L of blood, was 52%, with a specificity of 92%. LDMS is a potentially rapid and more sensitive alternate diagnostic method than microscopy.

Rapid diagnosis leading to effective treatment is essential to control escalating infectious diseases such as malaria. Malaria pigment (hemozoin) detection by laser desorption mass spectometry (LDMS) was recently shown to be a sensitive (

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.

A novel method for acquisition and numerical analysis of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectral data is described. The digitized ion current transient from each consecutive laser shot is first acquired and stored independently. Subsequently, statistical correlation parameters between all stored transients are computed. We illustrate the uses of this event-by-event analysis method for studies of sample surface heterogeneity as well as for elucidating the mechanisms of ion formation in MALDI. Other potential applications of the method are also outlined.

New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A′B′ and E′F′ loops. Six mutations in the E′F′ loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.

MALDI-TOF mass spectrometry has been coupled with Internet-based proteome database search algorithms in an approach for direct microorganism identification. This approach is applied here to characterize intact H. pylori (strain 26695) Gram-negative bacteria, the most ubiquitous human pathogen. A procedure for including a specific and common posttranslational modification, N-terminal Met cleavage, in the search algorithm is described. Accounting for posttranslational modifications in putative protein biomarkers improves the identification reliability by at least an order of magnitude. The influence of other factors, such as number of detected biomarker peaks, proteome size, spectral calibration, and mass accuracy, on the microorganism identification success rate is illustrated as well.

Large-scale genomics projects are identifying biomarkers to detect human disease.B. pseudomalleiandB. malleiare two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

Biodefense preparedness begins with the ability to detect and respond to bio-threats, based on accurate interpretation of genetic information with sophisticated, yet easy-to-use bioinformatics tools. Microbial forensics further enables attribution of microbial pathogen samples back to a suspected source. Sample characterization and traceability back to source are dependent on genome identification of specific targets within samples, comprehensive analysis of mixtures of populations’ present, and detection of major/minor variations in the identified genomes and comparison of sample genetic profile against other samples. Commercial Next Generation Sequencing (NGS) platforms offer the promise of dramatically higher detection sensitivity and resolution of forensic DNA samples than is possible with methods in current use. Before applying these technologies for forensic analyses of bacterial samples, however, it is critical to fully elucidate the benefits, caveats and pitfalls of NGS for hypothesis testing in comparative analyses, as ultimately this will be required for NGS use both as an investigative tool and tool for attribution in courts of law. Methods: We developed and evaluated novel probabilistic algorithms to process metagenomic sequence data from direct sample sequencing to identify genomes present in mixtures. Results: We present a pipeline for reference-free sample-to-sample comparisons to improve target characterization beyond one microorganism to characterization of comprehensive sample content. Our tools strengthen statistical confidence to trace the ancestry of samples and attribute samples to source with probabilistic certainties on many targets instead of a single genome. Conclusion: This study developed a novel reference free, bioinformatics strategy to account for and identify genetic diversity in samples. Sequence variants must be non-arbitrarily confirmed in both forward and reverse reads at a rate above the background noise level of sequencer machine error. A similarity distance metric compares genomes within a range of near relationships. Using sequence data from bio-threat agents, we successfully attributed known related strains together, and excluded near relation of known unrelated strains. The major strengths of this forensic method are the non-arbitrary determinations of data validation and relatedness metrics, as well as the ability to compare microbial genomes with or without a reference database of related genomes.

A rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms. Observing such characteristic mass shifts indicates that the microorganism is viable even in the presence of the drug, thus incorporating the isotopic label into characteristic biomarker molecules. The performance of the method is illustrated on the example of intact E. coli, grown in control (unlabeled) and (13)C-labeled media, and analyzed by MALDI TOF MS. Algorithms for data analysis are presented as well.

We describe methods for rapid sequencing of the entire human mitochondrial genome (mtgenome), which involve long-range PCR for specific amplification of the mtgenome, pyrosequencing, quantitative mapping of sequence reads to identify sequence variants and heteroplasmy, as well as de novo sequence assembly. These methods have been used to study 40 publicly available HapMap samples of European (CEU) and African (YRI) ancestry to demonstrate a sequencing error rate, Author Summary This manuscript details a novel algorithm to evaluate high-throughput DNA sequence data from whole mitochondrial genomes purified from genomic DNA, which also contains multiple fragmented nuclear copies of mtgenomes (numts). 40 samples were selected from 2 distinct reference (HapMap) populations of African (YRI) and European (CEU) origin. While previous technologies did not allow the assessment of individual mitochondrial molecules, next-generation sequencing technology is an excellent tool for obtaining the mtgenome sequence and its heteroplasmic sites rapidly and accurately through deep coverage of the genome. The computational techniques presented optimize reference-based alignments and introduce a new de novo assembly method. An important contribution of our study was obtaining high accuracy of the resulting called bases that we accomplished by quantitative filtering of reads that were error prone. In addition, several sites were experimentally validated and our method has a strong correlation (R2 = 0.96) with the NIST standard reference sample for heteroplasmy. Overall, our findings indicate that one can now confidently genotype mtDNA variants using next-generation sequencing data and reveal low levels of heteroplasmy (>10%). Beyond enriching our understanding and pathology of certain diseases, this development could be considered as a prelude to sequence-based individualized medicine for the mtgenome.

The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra, thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation. d-, w-, and y-series ions are prominent in the spectra, complementary to the (a-B)- and w- ions that are typically produced by MALDI post-source decay (PSD). Results are shown for several model DNA and RNA oligonucleotides, including combinations of DAN-induced fragmentation with true tandem TOF MS (MS/MS) for pseudo-MS(3) and "activated-ion PSD."

Detection of Plasmodium falciparum malaria during pregnancy is complicated by sequestration of parasites in the placenta, which reduces peripheral blood microscopic detection. Laser desorption mass spectrometry (LDMS) has previously demonstrated sensitive detection of hemozoin from P. falciparum blood cultures and the ability to track parasitemia in a Plasmodium yoelii malaria mouse model. Here we use a simple, dilution in water, blood sample preparation protocol for LDMS detection of malaria in 45 asymptomatic, pregnant Zambian women. We compare LDMS to microscopy and polymerase chain reaction (PCR) analysis. All women were microscopy negative. LDMS detected P. falciparum hemozoin in 15 out of 45 women, while PCR results were positive in 25 women. Compared with PCR, which analyzed 20-30 microL of blood, the sensitivity of LDMS, which analyzed1 microL of blood, was 52%, with a specificity of 92%. LDMS is a potentially rapid and more sensitive alternate diagnostic method than microscopy.

Rapid diagnosis leading to effective treatment is essential to control escalating infectious diseases such as malaria. Malaria pigment (hemozoin) detection by laser desorption mass spectometry (LDMS) was recently shown to be a sensitive (10 parasites/muL) technique for detecting Plasmodium falciparum parasites cultured in human blood. To examine the use of LDMS in a rapid new malaria screening assay, we followed the time course of P. yoelii infections in mice in parallel with light microscopy and a colorimetric hemozoin assay. Hemozoin was detected by LDMS in 0.3 muL of blood within two days of infection independently of the inoculating dose of 10(6), 10(4), or 10(2) parasite-infected erythrocytes. Microscopy and colorimetric hemozoin determinations lagged the LDMS detection of infections by 2-4 and 3-5 days, respectively, except at the highest inoculation dose. The LDMS detection of hemozoin is a potentially more rapid screen than light microscopy for detecting malaria infection in this mouse model at parasitemias0.1%.

An improved data analysis method is described for rapid identification of intact microorganisms from MALDI-TOF-MS data. The method makes no use of mass spectral fingerprints. Instead, a microorganism database is automatically generated that contains biomarker masses derived from ribosomal protein sequences and a model of N-terminal Met loss. We quantitatively validate the method via a blind study that seeks to identify microorganisms with known ribosomal protein sequences. We also include in the database microorganisms with incompletely known sets of ribosomal proteins to test the specificity of the method. With an optimal MALDI protocol, and at the 95% confidence level, microorganisms represented in the database with 20 or more biomarkers (i.e., those with complete or nearly completely sequenced genomes) are correctly identified from their spectra 100% of the time, with no incorrect identifications. Microorganisms with seven or less biomarkers (i.e., incompletely sequenced genomes) are either not identified or misidentified. Robustness with respect to variations in sample preparation protocol and mass analysis protocol is demonstrated by collecting data with two different matrixes and under two different ion-mode configurations. Statistical analysis suggests that, even without further improvement, the method described here would successfully scale up to microorganism databases with roughly 1000 microorganisms. The results demonstrate that microorganism identification based on proteome data and modeling can perform as well as methods based on mass spectral fingerprinting.

Discontinuities of various types and sizes may be introduced into a material during the manufacturing process and during the service life of the component. Discontinuities generated during the manufacturing process may include stress cracking, slag inclusions, lack of fusion, and gas or shrinkage porosity, whereas typical discontinuities generated during the service life may include fatigue cracks and corrosion [1]. Before the component can be accepted for inventory or continued service, an accurate nondestructive evaluation of the component must be performed.

BackgroundDespite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS).ResultsArchival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype.ConclusionsOur analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.

A successful model of the aerodynamic heat transfer to nose-mounted IR domes on hypersonic missile interceptors is presented. This paper describes a transitional flow heating algorithm, and presents a model for predicting the onset of boundary layer transition on a hemispherical forebody. In addition, this paper discusses the results of temperature predictions for IR domes, including a description of the analytical models and favorable comparisons with experimental data taken at Mach 4.6.

This paper summarizes a test program to verify the Balageas erosion model for Slip Cast Fused Silica in a flight-like erosive environment. The test program is summarized with particular attention paid to documenting the erosive environment. The Balageas model was found to over predict the erosion for these tests and a revised model which gives reasonable agreement with the data is proposed.