Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including the selection of adaptive mutations and the emergence of new variant lineages. Here, we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate the transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ~⅔ were plausibly introduced into these animal species from local human populations, while the remaining ~⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health.

Stereoelectronic Effects on the Binding of Neutral Lewis Bases to CdSe Nanocrystals.

Using 31P nuclear magnetic resonance (NMR) spectroscopy, we monitor the competition between tri- n-butylphosphine (Bu3P) and various amine and phosphine ligands for the surface of chloride terminated CdSe nanocrystals. Distinct 31P NMR signals for free and bound phosphine ligands allow the surface ligand coverage to be measured in phosphine solution. Ligands with a small steric profile achieve higher surface coverages (Bu3P = 0.5 nm-2, Me2P- n-octyl = 2.0 nm-2, NH2Bu = >3 nm-2) and have greater relative binding affinity for the nanocrystal (binding affinity: Me3P > Me2P- n-octyl ∼ Me2P- n-octadecyl > Et3P > Bu3P). Among phosphines, only Bu3P and Me2P- n-octyl support a colloidal dispersion, allowing a relative surface binding affinity ( Krel) to be estimated in that case ( Krel = 3.1). The affinity of the amine ligands is measured by the extent to which they displace Bu3P from the nanocrystals ( Krel: H2NBu ∼ N- n-butylimidazole > 4-ethylpyridine > Bu3P ∼ HNBu2 > Me2NBu > Bu3N). The affinity for the CdSe surface is greatest among soft, basic donors and depends on the number of each ligand that bind. Sterically unencumbered ligands such as imidazole, pyridine, and n-alkylamines can therefore outcompete stronger donors such as alkylphosphines. The influence of repulsive interactions between ligands on the binding affinity is a consequence of the high atom density of binary semiconductor surfaces. The observed behavior is distinct from the self-assembly of straight-chain surfactants on gold and silver where the ligands are commensurate with the underlying lattice and attractive interactions between aliphatic chains strengthen the binding.

Tight Binding of Carboxylate, Phosphonate, and Carbamate Anions to Stoichiometric CdSe Nanocrystals.

To completely displace the carboxylate surface ligands from cadmium selenide nanocrystals, oleic acid impurities are first removed using dimethylcadmium or diethylzinc. In addition to metal carboxylate and methane coproducts, reactions with CdMe2 produce surface bound methyl groups (δ = 0.4 ppm, 0.04-0.22 nm-2) that photolytically dissociate to methyl radicals and n-doped nanocrystals. Without oleic acid impurities, cadmium carboxylate can be completely displaced from the surface using n-alkylamines (NH2R', R' = n-butyl, n-hexyl, n-octyl) (≤0.01 carboxylates nm-2). Colloidal dispersions of amine bound nanocrystals (CdSe-NH2R') are indefinitely stable at amine concentrations of 0.1 M or higher and slowly aggregate at lower concentrations. Dissociation and evaporation of the amine ligands in 4-ethylpyridine, tri-n-butylphosphine, or molten tri-n-octylphosphine oxide solution results in nanocrystal aggregation. CdSe-NH2R' reacts with oleic acid, n-octadecylphosphonic acid, or carbon dioxide to form surface bound n-alkylammonium oleate, phosphonate, and carbamate ion pairs that bind with greater affinity than primary n-alkylamines. The results indicate that nanocrystal dispersions solely stabilized by neutral donor ligands are relatively unstable compared to those stabilized by adsorbed metal carboxylate or phosphonate complexes or by ion pairs. The challenge of differentiating between the neutral ligand bound form and adsorbed ion pairs is discussed.

The advent of genome-wide association studies for bacteria.

Significant advances in sequencing technologies and genome-wide association studies (GWAS) have revealed substantial insight into the genetic architecture of human phenotypes. In recent years, the application of this approach in bacteria has begun to reveal the genetic basis of bacterial host preference, antibiotic resistance, and virulence. Here, we consider relevant differences between bacterial and human genome dynamics, apply GWAS to a global sample of Mycobacterium tuberculosis genomes to highlight the impacts of linkage disequilibrium, population stratification, and natural selection, and finally compare the traditional GWAS against phyC, a contrasting method of mapping genotype to phenotype based upon evolutionary convergence. We discuss strengths and weaknesses of both methods, and make suggestions for factors to be considered in future bacterial GWAS.

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption.

BACKGROUND: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. METHODS: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. RESULTS: Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. CONCLUSIONS: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).

Quantification of four arsenic species in fruit juices by ion-chromatography-inductively coupled plasma-mass spectrometry.

A method using ion chromatography-inductively coupled plasma-mass spectrometry (IC-ICP-MS) for the quantification of arsenic species in fruit juices has been developed and validated. The method is capable of quantifying four anionic arsenic species - arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) - in the presence of unretained species such as arsenobetaine (AsB). Method validation was based on repeatability, analysis of reference materials, recovery of fortified samples, and determination of detection and quantification limits. The method was tested for use with apple, pear, cranberry, grape (red, white and purple) juices, as well as several juice blends. Limits of detection were 0.35, 0.41, 0.45 and 0.70 µg kg⁻¹ for As(III), DMA, MMA and As(V), respectively. Chromatographic recovery was good for most samples (90-107% compared to total arsenic), though recovery for some grape juice samples was lower (67-78%).

Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis.

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

Genetic variation and linkage disequilibrium in Bacillus anthracis.

We performed whole-genome amplification followed by hybridization of custom-designed resequencing arrays to resequence 303 kb of genomic sequence from a worldwide panel of 39 Bacillus anthracis strains. We used an efficient algorithm contained within a custom software program, UniqueMER, to identify and mask repetitive sequences on the resequencing array to reduce false-positive identification of genetic variation, which can arise from cross-hybridization. We discovered a total of 240 single nucleotide variants (SNVs) and showed that B. anthracis strains have an average of 2.25 differences per 10,000 bases in the region we resequenced. Common SNVs in this region are found to be in complete linkage disequilibrium. These patterns of variation suggest there has been little if any historical recombination among B. anthracis strains since the origin of the pathogen. This pattern of common genetic variation suggests a framework for recognizing new or genetically engineered strains.

Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.

BACKGROUND: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. CONCLUSIONS/SIGNIFICANCE: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.

Genomic characterization of the Yersinia genus.

BACKGROUND: New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments. RESULTS: We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogenases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats. CONCLUSIONS: Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.

Population bottlenecks as a potential major shaping force of human genome architecture.

The modern synthetic view of human evolution proposes that the fixation of novel mutations is driven by the balance among selective advantage, selective disadvantage, and genetic drift. When considering the global architecture of the human genome, the same model can be applied to understanding the rapid acquisition and proliferation of exogenous DNA. To explore the evolutionary forces that might have morphed human genome architecture, we investigated the origin, composition, and functional potential of numts (nuclear mitochondrial pseudogenes), partial copies of the mitochondrial genome found abundantly in chromosomal DNA. Our data indicate that these elements are unlikely to be advantageous, since they possess no gross positional, transcriptional, or translational features that might indicate beneficial functionality subsequent to integration. Using sequence analysis and fossil dating, we also show a probable burst of integration of numts in the primate lineage that centers on the prosimian-anthropoid split, mimics closely the temporal distribution of Alu and processed pseudogene acquisition, and coincides with the major climatic change at the Paleocene-Eocene boundary. We therefore propose a model according to which the gross architecture and repeat distribution of the human genome can be largely accounted for by a population bottleneck early in the anthropoid lineage and subsequent effectively neutral fixation of repetitive DNA, rather than positive selection or unusual insertion pressures.