Evolution on the Biophysical Fitness Landscape of an RNA Virus.

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.

Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics.

UNLABELLED: 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.

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

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.

Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

UNLABELLED: 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.

Semiquantitative Tissue Perfusion Assessment Using Indocyanine Green in Complex Open Urethral Reconstruction.

OBJECTIVE: Current assessment of critical tissue in genitourinary reconstruction, including graft beds and tissue flaps, primarily relies upon qualitative visual and tactile assessment by experienced surgeons. Here we explore the feasibility of using intravenous indocyanine green (ICG) for semiquantitative assessment of perfusion in complex open urethral reconstruction. METHODS: A standardized protocol for intravenous use of ICG and near-infrared fluorescence was established. Black and white mode was used for qualitative assessment of perfusion based on signal brightness. Quantitative perfusion mode was used to assess relative perfusion to tissue of interest compared to a control area with similar tissue type outside of the studied area. Real-time perfusion was visualized as percentage of perfusion relative to control. RESULTS: In case 1, the graft bed was assessed during dorsal onlay graft substitution urethroplasty. Perfusion to graft bed was compared to that of erectile bodies proximally. A proposed perfusion cutoff of 60% was noted to correlate with clinical judgment of graft bed quality. In case 2, tissue perfusion of Blandy flap in perineal urethrostomy was assessed before and after mobilization. A cutoff of 40% was proposed based on existing flap-based reconstruction literature with the goal to tailor flap and ultimately avoid tissue ischemia and necrosis. In case 3, in a complex staged substitution urethroplasty after hypospadias repair, the use of ICG facilitated a limited excision and shorter graft inlay in this staged reconstruction. CONCLUSION: The application of near-infrared fluorescence tools in open genitourinary reconstruction has the potential to advance quantitative assessment of graft, flaps, and other critical tissue planes, and help establish meaningful perfusion threshold and correlate with clinical outcomes.

Hyperinsulinemia acts via acinar insulin receptors to initiate pancreatic cancer by increasing digestive enzyme production and inflammation.

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown, and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly. Here, we demonstrate that insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are dispensable for glucose homeostasis but necessary for hyperinsulinemia-driven PanIN formation in the context of diet-induced hyperinsulinemia and obesity. Mechanistically, this was attributed to amplified digestive enzyme protein translation, triggering of local inflammation, and PanIN metaplasia in vivo. In vitro, insulin dose-dependently increased acinar-to-ductal metaplasia formation in a trypsin- and Insr-dependent manner. Collectively, our data shed light on the mechanisms connecting obesity-driven hyperinsulinemia and pancreatic cancer development.

Detection of Plasmodium falciparum in pregnancy by laser desorption mass spectrometry.

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 analyzed < 1 microL of blood, was 52%, with a specificity of 92%. LDMS is a potentially rapid and more sensitive alternate diagnostic method than microscopy.

Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics.

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.

A high-throughput drop microfluidic system for virus culture and analysis.

High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 µm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.

Rapid detection of malaria infection in vivo by laser desorption mass spectrometry.

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 parasitemias <0.1%.

Establishing drug resistance in microorganisms by mass spectrometry.

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.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis.

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are 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.

Enhanced in-source fragmentation in MALDI-TOF-MS of oligonucleotides using 1,5-diaminonapthalene.

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."

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

BACKGROUND: Despite 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). RESULTS: Archival 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. CONCLUSIONS: Our 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.

Top-down proteomics for rapid identification of intact microorganisms.

We apply MALDI-TOF/TOF mass spectrometry for the rapid and high-confidence identification of intact Bacillus spore species. In this method, fragment ion spectra of whole (undigested) protein biomarkers are obtained without the need for biomarker prefractionation, digestion, separation, and cleanup. Laser-induced dissociation (unimolecular decay) of higher mass (>5 kDa) precursor ions in the first TOF analyzer is followed by reacceleration and subsequent high-resolution mass analysis of the resulting sequence-specific fragments in a reflectron TOF analyzer. In-house-developed software compares an experimental MS/MS spectrum with in silico-generated tandem mass spectra from all protein sequences, contained in a proteome database, with masses within a preset range around the precursor ion mass. A p-value, the probability that the observed matches between experimental and in silico-generated fragments occur by chance, is computed and used to rank the database proteins to identify the most plausible precursor protein. By inference, the source microorganism is then identified on the basis of the identification of individual, unique protein biomarker(s). As an example, intact Bacillus atrophaeus and Bacillus cereus spores, either pure or in mixtures, were unambiguously identified by this method after fragmenting and identifying individual small, acid-soluble spore proteins that are specific for each species. Factors such as experimental mass accuracy and number of detected fragment ions, precursor ion charge state, and sequence-specific fragmentation have been evaluated with the objective of extending the approach to other microorganisms. MALDI-TOF/TOF-MS in a lab setting is an efficient tool for in situ confirmation/verification of initial microorganism identification.

Covariance mapping in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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.

Microorganism identification by matrix-assisted laser/desorption ionization mass spectrometry and model-derived ribosomal protein biomarkers.

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.