The deterministic force of natural selection and stochastic influence of drift shape RNA virus evolution. New deep-sequencing and microfluidics technologies allow us to quantify the effect of ...mutations and trace the evolution of viral populations with single-genome and single-nucleotide resolution. Such experiments can reveal the topography of the genotype-fitness landscapes that shape the path of viral evolution. By combining historical analyses, like phylogenetic approaches, with high-throughput and high-resolution evolutionary experiments, we can observe parallel patterns of evolution that drive important phenotypic transitions. These developments provide a framework for quantifying and anticipating potential evolutionary events. Here, we examine emerging technologies that can map the selective landscapes of viruses, focusing on their application to pathogenic viruses. We identify areas where these technologies can bolster our ability to study the evolution of viruses and to anticipate and possibly intervene in evolutionary events and prevent viral disease.
Dolan et al. review experimental approaches to the study of evolutionary landscapes of viruses and the forces driving the dynamics of evolving virus populations. Combing various approaches can elucidate evolutionary mechanisms and pathways underlying phenotypic transitions, allowing us to anticipate and possibly intervene in evolutionary events to prevent viral disease.
The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus ...spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) 1, 2. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs 3, 4. EVEs therefore represent a record of past infections and a reservoir of potential immune memory 5. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.
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•Long-read draft assembly of the repeat-rich Aag2 cell line genome•Genome-wide identification of EVEs using the improved assembly•Description of the large-scale organization of the genomic loci harboring EVEs•Evidence supporting antiviral function of piRNAs produced from EVEs
Whitfield et al. perform a genome-wide characterization of the endogenous viral elements (EVEs) present in the Aedes aegypti-derived Aag2 cell line. Using long-read sequencing suited to the highly repetitive mosquito genome, they explore the origin of these sequences and their potential role in mosquito immunity.
The live-attenuated oral poliovirus vaccine (OPV or Sabin vaccine) replicates in gut-associated tissues, eliciting mucosa and systemic immunity. OPV protects from disease and limits poliovirus ...spread. Accordingly, vaccination with OPV is the primary strategy used to end the circulation of all polioviruses. However, the ability of OPV to regain replication fitness and establish new epidemics represents a significant risk of polio re-emergence should immunization cease. Here, we report the development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. We introduced modifications within at the 5′ untranslated region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region to prevent recombination, and 3D polymerase to limit viral adaptability. Prior work established that nOPV2 is immunogenic in preclinical and clinical studies, and thus may enable complete poliovirus eradication.
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•The Sabin vaccine protects from poliomyelitis but can regain neurovirulence•Specific modifications into the Sabin2 genome increase its genetic stability•Slowing down Sabin2 virus evolution prevents reversion and leads to a safer vaccine•The new strain (nOPV2) is safe and immunogenic in preclinical and clinical studies
The live-attenuated oral poliovirus vaccine (OPV; Sabin vaccine) can revert to neurovirulent variants, causing safety concerns. Yeh et al. engineer a poliovirus vaccine strain (nOPV2) that preserves the antigenic and immunogenic characteristics of Sabin2 while stabilizing determinants of attenuation and reducing evolvability. nOPV2 is safe and immunogenic in preclinical studies.
RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major ...contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.
RNA viruses are unique in their evolutionary capacity, exhibiting high mutation rates and frequent recombination. They rapidly adapt to environmental changes, such as shifts in immune pressure or ...pharmacological challenge. The evolution of RNA viruses has been brought into new focus with the recent developments of genetic and experimental tools to explore and manipulate the evolutionary dynamics of viral populations. These studies have uncovered new mechanisms that enable viruses to overcome evolutionary challenges in the environment and have emphasized the intimate relationship of viral populations with evolution. Here, we review some of the emerging viral and host mechanisms that underlie the evolution of RNA viruses. We also discuss new studies that demonstrate that the relationship between evolutionary dynamics and virus biology spans many spatial and temporal scales, affecting transmission dynamics within and between hosts as well as pathogenesis.
IMPORTANCE: American Board of Surgery board certification requires passing both a written qualifying examination and an oral certifying examination. No studies have been conducted assessing the ...effect of sociodemographic variables on board passage rates. OBJECTIVE: To evaluate if trainee sociodemographic factors are associated with board passage rates. DESIGN, SETTING, AND PARTICIPANTS: This national and multi-institutional prospective observational cohort study of 1048 categorical general surgery trainees starting in 2007-2008 were surveyed. Data collection began in June 2007, follow-up was completed on December 31, 2016, and analysis began September 2018. MAIN OUTCOMES AND MEASURES: Survey responses were linked to American Board of Surgery board passage data. RESULTS: Of 662 examinees who had complete survey and follow-up data, 443 (65%) were men and 459 (69%) were white, with an overall board passage rate of 87% (n = 578). In a multinomial regression model, trainees of Hispanic ethnicity were more likely to not attempt the examinations (vs passed both) than non-Hispanic trainees (odds ratio OR, 4.7; 95% CI, 1.5-14). Compared with examinees who were married with children during internship, examinees who were married without children (OR, 0.3; 95% CI, 0.1-0.8) or were single (OR, 0.4; 95% CI, 0.2-0.9) were less likely to fail the examinations. Logistic regression showed white examinees compared with nonwhite examinees (black individuals, Asian individuals, and individuals of other races) (OR, 1.8; 95% CI, 1.03-3.0) and examinees who performed better on their first American Board of Surgery In-Training Examination (OR, 1.03; 95% CI, 1.02-1.05) were more likely to pass the qualifying examination on the first try. White examinees compared with nonwhite examinees (OR, 1.8; 95% CI, 1.1-2.8), non-Hispanic compared with Hispanic examinees (OR, 2.4; 95% CI, 1.2-4.7), and single women compared with women who were married with children during internship (OR, 10.3; 95% CI, 2.1-51) were more likely to pass the certifying examination on the first try. CONCLUSIONS AND RELEVANCE: Resident race, ethnicity, sex, and family status at internship were observed to be associated with board passage rates. There are multiple possible explanations for these worrisome observations that need to be explored. Tracking demographics of trainees to help understand passage rates based on demographics will be important. The American Board of Surgery already has begun addressing the potential for unconscious bias among board examiners by increasing diversity and adding implicit bias training.
transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway ...discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution
genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus,
employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.
Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to ...preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.
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•NPSC differentiation alters proteostasis strategies by rewiring chaperone network•High TRiC/CCT in NSPCs promotes proteome solubility and stress resilience•sHSP induced in neural progeny detoxify misfolded proteins by spatial sequestration•Widespread decline in TRiC/CCT levels and NSPC proteostasis in brain aging
Differentiation of neural stem cells (NSPCs) rewires cellular chaperone networks, fundamentally changing cellular proteostasis strategies. Chaperonin TRiC/CCT, high in NSPCs, promotes stress resilience and cellular fitness. Differentiation lowers TRiC/CCT and induces sHSPs, which promote misfolded protein sequestration into protective inclusions. Aging attenuates TRiC/CCT and NSPC proteostasis, likely contributing to proteinopathies.
Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines ...and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.
Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and ...cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear-vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear-vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear-vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.
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