Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

Humoral and T-cell-mediated responses to a pre-clinical Zika vaccine candidate that utilizes a unique insect-specific flavivirus platform.

Vaccination is critical for the control and prevention of viral outbreaks, yet conventional vaccine platforms may involve trade-offs between immunogenicity and safety. Insect-specific viruses have emerged as a novel vaccine platform to overcome this challenge. Detailed studies of humoral and T-cell responses induced by new insect-specific flavivirus (ISFV)-based vaccine platforms are needed to better understand correlates of protection and improve vaccine efficacy. Previously, we used a novel ISFV called Aripo virus (ARPV) to create a Zika virus (ZIKV) vaccine candidate (designated ARPV/ZIKV). ARPV/ZIKV demonstrated exceptional safety and single-dose efficacy, completely protecting mice from a lethal ZIKV challenge. Here, we explore the development of immune responses induced by ARPV/ZIKV immunization and evaluate its correlates of protection. Passive transfer of ARPV/ZIKV-induced immune sera to naïve mice prior to challenge emphasized the importance of neutralizing antibodies as a correlate of protection. Depletion of T-cells in vaccinated mice and adoptive transfer of ARPV/ZIKV-primed T-cells to naïve mice prior to challenge indicated that ARPV/ZIKV-induced CD4 + and CD8 + T-cell responses contribute to the observed protection but may not be essential for protection during ZIKV challenge. However, vaccination of Rag1 KO, Tcra KO, and muMt - mice demonstrated the critical role for ARPV/ZIKV-induced T-cells in developing protective immune responses following vaccination. Overall, both humoral and T-cell-mediated responses induced by ISFV-based vaccines are important for comprehensive immunity, and ISFV platforms continue to be a promising method for future vaccine development.

Coinfection of cattle in Virginia with Theileria orientalis Ikeda genotype and Anaplasma marginale.

Theileria orientalis Ikeda is a newly identified agent of bovine infectious anemia in the United States. Although T. orientalis Ikeda is transmitted by ticks other than the tick that transmits Anaplasma marginale-a bacterial etiology of bovine infectious anemia-the geographic distributions of these 2 infectious organisms overlap, with coinfection reported in some cattle. Only anaplasmosis has an approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex real-time PCR (rtPCR) for A. marginale and T. orientalis. With a cutoff of 38 cycles, the duplex assay has a sensitivity of 97.0% and a specificity of 100% for A. marginale; with a cutoff of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis, compared to existing tests. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, our rtPCR facilitates the study of coinfection of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for T. orientalis, 125 were positive for A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. Hence, coinfection by these 2 agents of bovine infectious anemia does occur within Virginia. It is likely that this pattern of infection will be seen in other regions where T. orientalis and A. marginale infections are endemic, despite the difference in tick vectors.

A draft genome assembly for the eastern fox squirrel, Sciurus niger.

The eastern fox squirrel, Sciurus niger, exhibits marked geographic variation in size and coat color, is a model organism for studies of behavior and ecology, and a potential model for investigating physiological solutions to human porphyrias. We assembled a genome using Illumina HiSeq, PacBio SMRT, and Oxford Nanopore MinION sequencing platforms. Together, the sequencing data resulted in a draft genome of 2.99 Gb, containing 32,830 scaffolds with an average size of 90.9 Kb and N50 of 183.8 Kb. Genome completeness was estimated to be 93.78%. A total of 24,443 protein-encoding genes were predicted from the assembly and 23,079 (94.42%) were annotated. Repeat elements comprised an estimated 38.49% of the genome, with the majority being LINEs (13.92%), SINEs (6.04%), and LTR elements. The topology of the species tree reconstructed using maximum-likelihood phylogenetic analysis was congruent with those of previous studies. This genome assembly can prove useful for comparative studies of genome structure and function in this rapidly diversifying lineage of mammals, for studies of population genomics and adaptation, and for biomedical research. Predicted amino acid sequence alignments for genes affecting heme biosynthesis, color vision, and hibernation showed point mutations and indels that may affect protein function and ecological adaptation.

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.

Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster.

Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. Here, we test for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. We show that winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. We ascribe the observed differences to indirect selection on recombination caused by directional selection on desiccation tolerance. Our findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations).

Ethnically biased microsatellites contribute to differential gene expression and glutathione metabolism in Africans and Europeans.

Approximately three percent of the human genome is occupied by microsatellites: a type of short tandem repeat (STR). Microsatellites have well established effects on (a) the genetic structure of diverse human populations and (b) expression of nearby genes. These lines of inquiry have uncovered 3,984 ethnically biased microsatellite loci (EBML) and 28,375 expression STRs (eSTRs), respectively. We hypothesize that a combination of EBML, eSTRs, and gene expression data (RNA-seq) can be used to show that microsatellites contribute to differential gene expression and phenotype in human populations. In fact, our previous study demonstrated a degree of mutual overlap between EBML and eSTRs but fell short of quantifying effects on gene expression. The present work aims to narrow the gap. First, we identify 313 overlapping EBML/eSTRs and recapitulate their mutual overlap. The 313 EBML/eSTRs are then characterized across ethnicity and tissue type. We use RNA-seq data to pursue validation of 49 regions that affect whole blood gene expression; 32 out of 54 affected genes are differentially expressed in Africans and Europeans. We quantify the relative contribution of these 32 genes to differential expression; fold change tends to be less than other differentially expressed genes. Repeat length correlates with expression for 15 of the 32 genes; two are conspicuously involved in glutathione metabolism. Finally, we repurpose a mathematical model of glutathione metabolism to investigate how a single polymorphic microsatellite affects phenotype. We conclude with a testable prediction that microsatellite polymorphisms affect GPX7 expression and oxidative stress in Africans and Europeans.

DNA sequencing of anatomy lab cadavers to provide hands-on precision medicine introduction to medical students.

BACKGROUND: Medical treatment informed by Precision Medicine is becoming a standard practice for many diseases, and patients are curious about the consequences of genomic variants in their genome. However, most medical students' understanding of Precision Medicine derives from classroom lectures. This format does little to foster an understanding for the potential and limitations of Precision Medicine. To close this gap, we implemented a hands-on Precision Medicine training program utilizing exome sequencing to prepare a clinical genetic report of cadavers studied in the anatomy lab. The program reinforces Precision Medicine related learning objectives for the Genetics curriculum. METHODS: Pre-embalmed blood samples and embalmed tissue were obtained from cadavers (donors) used in the anatomy lab. DNA was isolated and sequenced and illustrative genetic reports provided to the students. The reports were used to facilitate discussion with students on the implications of pathogenic genomic variants and the potential correlation of these variants in each "donor" with any anatomical anomalies identified during cadaver dissection. RESULTS: In 75% of cases, analysis of whole exome sequencing data identified a variant associated with increased risk for a disease/abnormal condition noted in the donor's cause of death or in the students' anatomical findings. This provided students with real-world examples of the potential relationship between genomic variants and disease risk. Our students also noted that diseases associated with 92% of the pathogenic variants identified were not related to the anatomical findings, demonstrating the limitations of Precision Medicine. CONCLUSION: With this study, we have established protocols and classroom procedures incorporating hands-on Precision Medicine training in the medical student curriculum and a template for other medical educators interested in enhancing their Precision Medicine training program. The program engaged students in discovering variants that were associated with the pathophysiology of the cadaver they were studying, which led to more exposure and understanding of the potential risks and benefits of genomic medicine.

A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens.

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.

Analysis of Genomic Sequence Data Reveals the Origin and Evolutionary Separation of Hawaiian Hoary Bat Populations.

We examine the genetic history and population status of Hawaiian hoary bats (Lasiurus semotus), the most isolated bats on Earth, and their relationship to northern hoary bats (Lasiurus cinereus), through whole-genome analysis of single-nucleotide polymorphisms mapped to a de novo-assembled reference genome. Profiles of genomic diversity and divergence indicate that Hawaiian hoary bats are distinct from northern hoary bats, and form a monophyletic group, indicating a single ancestral colonization event 1.34 Ma, followed by substantial divergence between islands beginning 0.51 Ma. Phylogenetic analysis indicates Maui is central to the radiation across the archipelago, with the southward expansion to Hawai'i and westward to O'ahu and Kaua'i. Because this endangered species is of conservation concern, a clearer understanding of the population genetic structure of this bat in the Hawaiian Islands is of timely importance.

Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus.

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BACKGROUND: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. . RESULTS: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple ß-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon ß-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 ß-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. CONCLUSIONS: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.

Desiccation-induced changes in recombination rate and crossover interference in Drosophila melanogaster: evidence for fitness-dependent plasticity.

Meiotic recombination is evolutionarily ambiguous, as being associated with both benefits and costs to its bearers, with the resultant dependent on a variety of conditions. While existing theoretical models explain the emergence and maintenance of recombination, some of its essential features remain underexplored. Here we focus on one such feature, recombination plasticity, and test whether recombination response to stress is fitness-dependent. We compare desiccation stress effects on recombination rate and crossover interference in chromosome 3 between desiccation-sensitive and desiccation-tolerant Drosophila lines. We show that relative to desiccation-tolerant genotypes, desiccation-sensitive genotypes exhibit a significant segment-specific increase in single- and double-crossover frequencies across the pericentromeric region of chromosome 3. Significant changes (relaxation) in crossover interference were found for the interval pairs flanking the centromere and extending to the left arm of the chromosome. These results indicate that desiccation is a recombinogenic factor and that desiccation-induced changes in both recombination rate and crossover interference are fitness-dependent, with a tendency of less fitted individuals to produce more variable progeny. Such dependence may play an important role in the regulation of genetic variation in populations experiencing environmental challenges.

Genomic divergence and adaptive convergence in Drosophila simulans from Evolution Canyon, Israel.

Biodiversity refugia formed by unique features of the Mediterranean arid landscape, such as the dramatic ecological contrast of "Evolution Canyon," provide a natural laboratory in which local adaptations to divergent microclimate conditions can be investigated. Significant insights have been provided by studies of Drosophila melanogaster diversifying along the thermal gradient in Evolution Canyon, but a comparative framework to survey adaptive convergence across sister species at the site has been lacking. To fill this void, we present an analysis of genomic polymorphism and evolutionary divergence of Drosophila simulans, a close relative of Drosophila melanogaster with which it co-occurs on both slopes of the canyon. Our results show even deeper interslope divergence in D. simulans than in D. melanogaster, with extensive signatures of selective sweeps present in flies from both slopes but enhanced in the population from the hotter and drier south-facing slope. Interslope divergence was enriched for genes related to electrochemical balance and transmembrane transport, likely in response to increased selection for dehydration resistance on the hotter slope. Both species shared genomic regions that underwent major selective sweeps, but the overall level of adaptive convergence was low, demonstrating no shortage of alternative genomic solutions to cope with the challenges of the microclimate contrast. Mobile elements were a major source of genetic polymorphism and divergence, affecting all parts of the genome, including coding sequences of mating behavior-related genes.

CAGm: a repository of germline microsatellite variations in the 1000 genomes project.

The human genome harbors an abundance of repetitive DNA; however, its function continues to be debated. Microsatellites-a class of short tandem repeat-are established as an important source of genetic variation. Array length variants are common among microsatellites and affect gene expression; but, efforts to understand the role and diversity of microsatellite variation has been hampered by several challenges. Without adequate depth, both long-read and short-read sequencing may not detect the variants present in a sample; additionally, large sample sizes are needed to reveal the degree of population-level polymorphism. To address these challenges we present the Comparative Analysis of Germline Microsatellites (CAGm): a database of germline microsatellites from 2529 individuals in the 1000 genomes project. A key novelty of CAGm is the ability to aggregate microsatellite variation by population, ethnicity (super population) and gender. The database provides advanced searching for microsatellites embedded in genes and functional elements. All data can be downloaded as Microsoft Excel spreadsheets. Two use-case scenarios are presented to demonstrate its utility: a mononucleotide (A) microsatellite at the BAT-26 locus and a dinucleotide (CA) microsatellite in the coding region of FGFRL1. CAGm is freely available at http://www.cagmdb.org/.

Genomic signatures of experimental adaptive radiation in Drosophila.

Abiotic environmental factors play a fundamental role in determining the distribution, abundance and adaptive diversification of species. Empowered by new technologies enabling rapid and increasingly accurate examination of genomic variation in populations, researchers may gain new insights into the genomic background of adaptive radiation and stress resistance. We investigated genomic variation across generations of large-scale experimental selection regimes originating from a single founder population of Drosophila melanogaster, diverging in response to ecologically relevant environmental stressors: heat shock, heat knock down, cold shock, desiccation and starvation. When compared to the founder population, and to parallel unselected controls, there were more than 100,000 single nucleotide polymorphisms (SNPs) displaying consistent allelic changes in response to selective pressures across generations. These SNPs were found in both coding and noncoding sequences, with the highest density in promoter regions, and involved a broad range of functionalities, including molecular chaperoning by heat-shock proteins. The SNP patterns were highly stressor-specific despite considerable variation among line replicates within each selection regime, as reflected by a principal component analysis, and co-occurred with selective sweep regions. Only ~15% of SNPs with putatively adaptive changes were shared by at least two selective regimes, while less than 1% of SNPs diverged in opposite directions. Divergent stressors driving evolution in the experimental system of adaptive radiation left distinct genomic signatures, most pronounced in starvation and heat-shock selection regimes.

Unique divergence of the breast cancer 2 (BRCA2) gene in Neanderthals.

Unique divergence of the BRCA2, a tumor suppressor gene, in Neanderthals relative to other primates, including modern humans, is highlighted. This divergence with potentially pathogenic consequences raises a question about cancer susceptibility in the archaic species that was replaced by modern humans about 40,000 years ago.