Search

/SummarySARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.Abstract FigureHighlightsS:D614G is ubiquitous among SARS-CoV-2 B-lineage Spikes and is required for infectivity of the main Variants of ConcernIn an example of convergent evolution, SARS-CoV-2 A lineage viruses maintained transmission chains in the absence of S:D614G, but were instead dependent upon S:Q613H or S:H655YS:D614G and S:H655Y are both adaptations to the polybasic S1/S2 cleavage siteIncreased infectivity of S:D614G and S:H655Y is associated with a more open RBD conformation and increased steady-state levels of virion-associated S1

The Human Disease Ontology (DO) (www.disease-ontology.org) database, has significantly expanded the disease content and enhanced our userbase and website since the DO’s 2018 Nucleic Acids Research DATABASE issue paper. Conservatively, based on available resource statistics, terms from the DO have been annotated to over 1.5 million biomedical data elements and citations, a 10× increase in the past 5 years. The DO, funded as a NHGRI Genomic Resource, plays a key role in disease knowledge organization, representation, and standardization, serving as a reference framework for multiscale biomedical data integration and analysis across thousands of clinical, biomedical and computational research projects and genomic resources around the world. This update reports on the addition of 1,793 new disease terms, a 14% increase of textual definitions and the integration of 22 137 new SubClassOf axioms defining disease to disease connections representing the DO’s complex disease classification. The DO’s updated website provides multifaceted etiology searching, enhanced documentation and educational resources., Graphical Abstract Graphical AbstractThe Human Disease Ontology (DO) integrates disease features and etiological factors to describe disease complexity. The DO is extensively cited and integrated into highly cited databases.

Graphical abstract During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03317-4.

Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods permeate specimens with free-radical-chain-growth-polymerized polyacrylate hydrogels, whose network structure limits the local isotropy of expansion as well as the preservation of morphology and shape at the nanoscale. Here we report that ExM is possible using hydrogels that have a more homogeneous network structure, assembled via non-radical terminal linking of tetrahedral monomers. As with earlier forms of ExM, such ‘tetra-gel’-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10× in linear dimension results in a median spatial error of 9.2 nm for localizing the viral envelope layer, rather than 14.3 nm from earlier versions of ExM. Moreover, tetra-gel-based expansion better preserves the virion spherical shape. Thus, tetra-gels may support ExM with reduced spatial errors and improved local isotropy, pointing the way towards single-biomolecule accuracy ExM. Expansion microscopy, or ExM, physically expands biological specimens upon embedding them in swellable polymeric hydrogels, allowing nanoscale imaging using conventional microscopes. This work presents a diamond lattice polymeric hydrogel synthesized via click chemistry, which achieves higher homogeneity than the ones currently in use, allowing a more accurate sample expansion.

Olfactory receptors (ORs), encoded by the largest vertebrate multigene family, enable the detection of thousands of unique odorants in the environment and consequently play a critical role in species survival. Here, we advance our knowledge of OR gene evolution in procellariiform seabirds, an avian group which relies on the sense of olfaction for critical ecological functions. We built a cosmid library of Cory’s Shearwater (Calonectris borealis) genomic DNA, a model species for the study of olfaction-based navigation, and sequence OR gene-positive cosmid clones with a combination of sequencing technologies. We identified 220 OR open reading frames, 20 of which are full length, intact OR genes, and found a large ratio of partial and pseudogenes to intact OR genes (2:1), suggestive of a dynamic mode of evolution. Phylogenetic analyses revealed that while a few genes cluster with those of other sauropsid species in a γ (gamma) clade that predates the divergence of different avian lineages, most genes belong to an avian-specific γ-c clade, within which sequences cluster by species, suggesting frequent duplication and/or gene conversion events. We identified evidence of positive selection on full length γ-c clade genes. These patterns are consistent with a key role of adaptation in the functional diversification of olfactory receptor genes in a bird lineage that relies extensively on olfaction.

The Common Fund Data Ecosystem (CFDE) has created a flexible system of data federation that enables users to discover datasets from across the U.S. National Institutes of Health Common Fund without requiring that data owners move, reformat, or rehost those data. The CFDE’s federation system is centered on a catalog that ingests metadata from individual Common Fund Program’s Data Coordination Centers (DCCs) into a uniform metadata model that can then be indexed and searched from a centralized portal. This uniform Crosscut Metadata Model (C2M2) supports the wide variety of data types and metadata terms used by the individual DCCs and is designed to enable easy expansion to accommodate new data types. We describe its use to ingest and index data from ten DCCs.

During HIV-1 assembly the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5' untranslated region (5'UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5'UTR, promoted by the NC domain of Gag, is predicted during virus maturation. Two 5'UTR dimer conformations, the kissing dimer and the extended dimer, have been identified in vitro, which differ in the extent of intermolecular base pairing. Whether 5'UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy (FCCS) and single-molecule Forster resonance energy transfer (smFRET) imaging to demonstrate that 5'UTRs from two different HIV-1 subtypes form kissing dimers with divergent stabilities. We further show that both 5'UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an extended dimer conformation.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

The Evidence and Conclusion Ontology (ECO) contains terms (classes) that describe types of evidence and assertion methods. ECO terms are used in the process of biocuration to capture the evidence that supports biological assertions (e.g. gene product X has function Y as supported by evidence Z). Capture of this information allows tracking of annotation provenance, establishment of quality control measures and query of evidence. ECO contains over 1500 terms and is in use by many leading biological resources including the Gene Ontology, UniProt and several model organism databases. ECO is continually being expanded and revised based on the needs of the biocuration community. The ontology is freely available for download from GitHub (https://github.com/evidenceontology/) or the project’s website (http://evidenceontology.org/). Users can request new terms or changes to existing terms through the project’s GitHub site. ECO is released into the public domain under CC0 1.0 Universal.

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite., Author summary An estimated 50 million cattle in sub-Saharan Africa are at risk of the deadly livestock disease East coast fever (ECF), caused by the parasite Theileria parva, which imposes tremendous economic hardship on smallholder farmers. An existing ECF vaccine protects against strains circulating among cattle, but not against T. parva derived from African Cape buffalo, its main wildlife carrier. Understanding this difference in protective efficacy requires characterization of the genetic diversity in T. parva strains associated with each mammalian host, a goal that has been hindered by the proliferation of T. parva in nucleated host cells, with much larger genomes. Here we adapted a sequence capture approach to target the whole parasite genome, enabling enrichment of parasite DNA over that of the host. Choices in protocol development resulted in nearly 100% parasite genome specificity and sensitivity, making this approach the most successful yet to generate T. parva genome sequence data in a high-throughput manner. The analyses uncovered a degree of genetic differentiation between cattle- and buffalo-derived genotypes that is akin to levels more commonly seen between species. This approach, which will enable an in-depth T. parva population genomics study from cattle and buffalo in the endemic regions, can easily be adapted to other intracellular pathogens.

SUMMARYThe SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and cells rendered permissive by ectopic expression of various mammalian ACE2 orthologs. Nonetheless, D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts a critical interprotomer contact and that this dramatically shifts the S protein trimer conformation toward an ACE2-binding and fusion-competent state. Consistent with the more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated. These results indicate that D614G adopts conformations that make virion membrane fusion with the target cell membrane more probable but that D614G retains susceptibility to therapies that disrupt interaction of the SARS-CoV-2 S protein with the ACE2 receptor.

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever, a lethal disease endemic to Western Africa. LASV entry is mediated by the viral envelope glycoprotein (GP), a class I membrane fusogen and the sole viral surface antigen. Previous studies have identified components of the LASV entry pathway, including several cellular receptors and the requirement of endosomal acidification for infection. Here, we first demonstrate that incubation at a physiological temperature and pH consistent with the late endosome is sufficient to render pseudovirions, bearing LASV GP, non-infectious. Antibody binding indicates that this loss of infectivity is due to a conformational change in GP. Finally, we developed a single-particle fluorescence assay to directly visualize individual pseudovirions undergoing LASV GP-mediated lipid mixing with a supported planar bilayer. We report that exposure to endosomal pH at a physiologic temperature is sufficient to trigger GP-mediated lipid mixing. Furthermore, while a cellular receptor is not necessary to trigger lipid mixing, the presence of lysosomal-associated membrane protein 1 (LAMP1) increases the kinetics of lipid mixing at an endosomal pH. Furthermore, we find that LAMP1 permits robust lipid mixing under less acidic conditions than in its absence. These findings clarify our understanding of LASV GP-mediated fusion and the role of LAMP1 binding.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca2+ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated., Graphical Abstract, Highlights • The SARS-CoV-2 D614G S protein variant supplanted the ancestral virus in people • D614G increases infectivity on human lung cells or cells with bat or pangolin ACE2 • D614G is potently neutralized by antibodies targeting the receptor-binding domain • D614G shifts S protein conformation toward an ACE2-binding fusion-competent state, Structural and molecular insights into the SARS-CoV-2 spike protein variant D614G reveal the basis of its increased infectivity

Saline migrants into freshwater habitats constitute among the most destructive invaders in aquatic ecosystems throughout the globe. However, the evolutionary and physiological mechanisms underlying such habitat transitions remain poorly understood. To explore the mechanisms of freshwater adaptation and distinguish between adaptive (evolutionary) and acclimatory (plastic) responses to salinity change, we examined genome-wide patterns of gene expression between ancestral saline and derived freshwater populations of the Eurytemora affinis species complex, reared under two different common-garden conditions (0 versus 15 PSU). We found that evolutionary shifts in gene expression (between saline and freshwater inbred lines) showed far greater changes and were more widespread than acclimatory responses to salinity (0 versus 15 PSU). Most notably, 30-40 genes showing evolutionary shifts in gene expression across the salinity boundary were associated with ion transport function, with inorganic cation transmembrane transport forming the largest Gene Ontology category. Of particular interest was the sodium transporter, the Na+ /H+ antiporter (NHA) gene family, which was discovered in animals relatively recently. Thirty key ion regulatory genes, such as NHA paralogue #7, demonstrated concordant evolutionary and plastic shifts in gene expression, suggesting the evolution of ion transporter function and plasticity during rapid invasions into novel salinities. Moreover, freshwater invasions were associated with the evolution of reduced plasticity in the freshwater population, again for the same key ion transporters, consistent with the predicted evolution of canalization following adaptation to stressful conditions. Our results have important implications for understanding evolutionary and physiological mechanisms of range expansions by some of the most widespread invaders in aquatic habitats.

A novel apicomplexan parasite was serendipitously discovered in horses at the United States – Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.

No immunogen has been found that elicits a broadly neutralizing antibody (bNAb) response sufficient for development into an HIV vaccine. In this issue of Cell Host and Microbe, Zhang et al. (2018) rationally design an HIV envelope glycoprotein variant that provides new hope that such an immunogen may be attainable.

Significance Here we have directly visualized conformational changes in the 5′UTR of the HIV-1 genome using single-molecule fluorescence techniques. We find that the monomeric 5′UTR can spontaneously transition between two conformations, which have distinct intramolecular base pairing. One of the observed conformations is competent for dimerization with a second 5′UTR molecule. Our results are consistent with a model in which dimerization initiates by way of localized intermolecular kissing-loop base pairing, which is promoted by tRNA primer annealing. The intermolecular interface then extends, giving rise to the putative extended dimer, which is stabilized by HIV-1 NC. Thus, the 5′UTR is intrinsically dynamic, and both viral and host factors play a role in modulating the RNA conformation and dynamics., The highly conserved 5′ untranslated region (5′UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5′UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5′UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5′UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

Fusion of viral and cellular membranes is an essential step in the entry pathway of all enveloped viruses. This is a dynamic and multistep process, which has been extensively studied, resulting in the endpoints of the reaction being firmly established, and many essential cellular factors identified. What remains is to elucidate the dynamic events that underlie this process, including the order and timing of glycoprotein conformational changes, receptor-binding events, and movement of the glycoprotein on the surface of the virion. Due to the inherently asynchronous nature of these dynamics, there has been an increased focus on the study of single virions and single molecules. These techniques provide researchers the high precision and resolution necessary to bridge the gaps in our understanding of viral membrane fusion. This review highlights the advancement of single-molecule and single-particle fluorescence-based techniques, with a specific focus on how these techniques have been used to study the dynamic nature of the viral fusion pathway.

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also harbor these producers. Thus, we undertook a study to see if these niches also contained omega-3 fatty acid producers. We were successful in isolating and characterizing unique EPA-producing strains of Shewanella from three strictly freshwater native fish species, i.e., lake whitefish ( Coregonus clupeaformis ), lean lake trout ( Salvelinus namaycush ), and walleye ( Sander vitreus ), and from two other freshwater nonnative fish, i.e., coho salmon ( Oncorhynchus kisutch ) and seeforellen brown trout ( Salmo trutta ). We were also able to isolate four unique free-living strains of EPA-producing Shewanella from freshwater habitats. Phylogenetic and phenotypic analyses suggest that one producer is clearly a member of the Shewanella morhuae species and another is sister to members of the marine PUFA-producing Shewanella baltica species. However, the remaining isolates have more ambiguous relationships, sharing a common ancestor with non-PUFA-producing Shewanella putrefaciens isolates rather than marine S. baltica isolates despite having a phenotype more consistent with S. baltica strains.

The ability to detect antibodies that are generated during an immune response is integral to the diagnosis and monitoring of infections. Assays that have been applied to the detection of antibodies are classically referred to as indirect immunoassays, which include three different formats: indirect, double-antigen sandwich, and total IgG capture. Each of these three approaches relies on a unique pair of reagents to capture the target antibody and transduce a detectable signal, which permits assays to be tuned for ideal performance based on the availability and quality of reagents, the resources available to the operator, and the complexity of the sample (e.g., serum vs. saliva). This flexibility, however, can complicate the development of diagnostic tests and increase the complexity of the devices required to perform them. A general platform that can enable the rapid development of inexpensive and simple diagnostic assays would be highly desirable, particularly for those assays intended for use in limited-resource settings. In this manuscript, we describe a paper-based microfluidic device architecture that is capable of supporting these three indirect immunoassay formats by simply changing reagents and without requiring significant alterations to the design or manufacture of the device. We compare the performances of these indirect immunoassays by developing assays for HIV antibodies in human serum. These results suggest that this device format has broad potential for the development of paper-based immunoassays.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2., The Ebola virus envelope glycoprotein is a membrane fusion machine required for the virus to enter into host cells. This study presents direct observation of the conformational changes that the envelope glycoprotein undergoes during the membrane fusion process.

The Human Disease Ontology (DO) (http://www.disease-ontology.org), database has undergone significant expansion in the past three years. The DO disease classification includes specific formal semantic rules to express meaningful disease models and has expanded from a single asserted classification to include multiple-inferred mechanistic disease classifications, thus providing novel perspectives on related diseases. Expansion of disease terms, alternative anatomy, cell type and genetic disease classifications and workflow automation highlight the updates for the DO since 2015. The enhanced breadth and depth of the DO’s knowledgebase has expanded the DO’s utility for exploring the multi-etiology of human disease, thus improving the capture and communication of health-related data across biomedical databases, bioinformatics tools, genomic and cancer resources and demonstrated by a 6.6× growth in DO’s user community since 2015. The DO’s continual integration of human disease knowledge, evidenced by the more than 200 SVN/GitHub releases/revisions, since previously reported in our DO 2015 NAR paper, includes the addition of 2650 new disease terms, a 30% increase of textual definitions, and an expanding suite of disease classification hierarchies constructed through defined logical axioms.

With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method is needed to assign bacterial species designations using genomic data that is consistent with the classically-obtained taxonomy. This is particularly acute for unculturable obligate intracellular bacteria like those in the Rickettsiales, where classical methods like DNA-DNA hybridization cannot be used to define species. Within the Rickettsiales, species designations have been applied inconsistently, often obfuscating the relationship between organisms and the context for experimental results. In this study, we generated core genome alignments for a wide range of genera with classically defined species, includingArcobacter,Caulobacter,Erwinia,Neisseria,Polaribacter,Ralstonia,Thermus, as well as genera within the Rickettsiales includingRickettsia,Orientia,Ehrlichia,Neoehrlichia,Anaplasma,eorickettsia, andWolbachia. A core genome alignment sequence identity (CGASI) threshold of 96.8% was found to maximize the prediction of classically-defined species. Using the CGASI cutoff, theWolbachiagenus can be delineated into species that differ from the currently used supergroup designations, while theRickettsiagenus is delineated into nine species, as opposed to the current 27 species. Additionally, we find that core genome alignments cannot be constructed between genomes belonging to different genera, establishing a bacterial genus cutoff that suggests the need to create new genera from theAnaplasmaandNeorickettsia. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method for bacterial nomenclature that is aligned with classically-obtained results.