You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells}
Article:
Abstract:
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.
Introduction:
The evolutionary capacity of RNA viruses allows them to rapidly adapt to their environment and overcome barriers to infection( Alto et al., 2013; Dolan et al., 2018; Domingo and Perales, 2014; Sanjuán, 2016). Quantifying the evolutionary dynamics of virus populations in controlled experimental systems can reveal biological constraints on the viral genome, molecular mechanisms of viral adaptation, and fundamental biophysical and population genetic principles governing molecular evolution in general. Arthropod-borne viruses, or arboviruses, such as Dengue( DENV), Zika( ZIKV), and Chikungunya( CHIKV), are a significant cause of disease globally, with half of the world’s population exposed to arboviral vectors. DENV alone causes approximately 390 million infections and 10, 000 deaths annually( Bhatt et al., 2013; Messina et al., 2019). Arboviruses must cycle between vertebrate and invertebrate hosts, which differ significantly in body temperature, cellular environment, and mode of antiviral immunity, raising questions about the evolutionary strategies they may employ to replicate in these different host environments. Several studies have addressed these alternative landscapes experimentally in vitro and in vivo, identifying mutations that confer increased fitness in each host( Coffey and Vignuzzi, 2011; Filomatori et al., 2017; Forrester et al., 2014; Greene et al., 2005; Pompon et al., 2017; Sessions et al., 2015; Stapleford et al., 2014; Villordo et al., 2015). However, we still lack a comprehensive picture of the alternative genotype-fitness landscapes of any arbovirus defined by the human and insect host environments. Comparing the evolutionary dynamics of viral populations across different host environments could highlight key points of host-specific selection and define the patterns of evolutionary constraint that define the landscape in each host. RNA viruses exist as a dynamic population of co-circulating genotypes surrounding a master sequence( Domingo, 2002; Domingo et al., 2012; Holland et al., 1992; Lauring and Andino, 2010; Wilke, 2005). It is becoming increasingly clear that the distribution and dynamics of minor alleles play important roles in population fitness, adaptation, and disease( Andino and Domingo, 2015; Bordería et al., 2015; Grad et al., 2014; Shirogane et al., 2012; Vignuzzi et al., 2006; Xue et al., 2018, Xue et al., 2016); furthermore, the neighborhood of connected genotypes is thought to be important to population fitness( Moratorio et al., 2017). The genomes of viruses that alternate between hosts, such as arboviruses, are subject to selection in distinct environments, raising the question of how these viruses maintain fitness over alternating hosts. Emerging deep-sequencing techniques allow us to probe the mutational landscape. Library-based methods, such as deep mutational scanning( DMS), screen defined collections of sequences against specific selective pressures. DMS can quantify the fitness effects of individual mutations in viral genomes through intentional diversification of protein sequences( Ashenberg et al., 2017; Setoh et al., 2019; Thyagarajan and Bloom, 2014; Visher et al., 2016). However, these approaches do not capture the evolutionary dynamics of natural populations. The analysis of naturally occurring variation and evolution in experimental virus populations has been limited to allele frequencies greater 1 in 1000, due to the error rates associated with reverse transcriptase used in cDNA synthesis. Recently, high-accuracy sequencing approaches that control for sequencing errors through barcoding, like PrimerID( Jabara et al., 2011), or through template circularization and amplification, like Circular Sequencing( CirSeq)( Acevedo et al., 2014; Acevedo and Andino, 2014), can detect alleles as rare as 1 in 106 in frequency. This sequencing depth enables the observation of the full spectrum of diversity in samples from evolving viral populations. Thus, the ability to globally trace the evolutionary dynamics of individual alleles in viral populations from their genesis at the mutation rate to their eventual fate in a given experiment allows us to describe the viral fitness landscapes that shape adapting populations in unprecedented detail. Importantly, this depth allows observation of common variants that accumulate under positive selection in each experiment, but also reveals rare variants limited to low frequencies by negative selection. This permits quantification of the contribution of genetic constraint on the viral adaptation process. We here use CirSeq to characterize the fitness landscapes of DENV populations adapting to the distinct environments and cellular machineries of human and mosquito cells by tracing individual allele trajectories for almost all possible single nucleotide variants across the DENV genome. This analysis reveals the influence of both positive and negative selection in shaping the evolutionary paths of DENV in these distinct cellular environments. Analysis of the allele repertoire reveals how fitness of the viral population represents a balance between the dynamics of rare beneficial mutations and the significant and constantly replenished load of deleterious alleles during adaptation. We find that adaptation relies on host-specific beneficial mutations that are clustered in specific regions of the DENV genome and enriched in regions of the proteome that exhibit structural flexibility. Of note, these regions are also sites of variation across naturally occurring DENV and ZIKV strains indicating that our analysis provides insights into genetic and biophysical principles of flaviviral evolution and reveals parallels between long- and short-term evolutionary scales.
Discussion:
Here, we used high-resolution sequencing to quantify the contribution of beneficial and deleterious mutation in shaping the evolutionary paths of DENV populations responding to host cell-specific selective pressures. Our analysis shows that DENV populations acquire host-specific population structures defined by distinct genotype-fitness landscapes( Figures 1 and 2), which collectively shift the population in sequence space and result in a concurrent increase in phenotypic fitness, as assessed by focus morphology and absolute titers( Figure 1). Strikingly, we find that simple models of mean population fitness derived from allele frequency measurements alone can predict phenotypic adaptation( Figure 4). This suggests the phenotype and evolutionary dynamics of a virus can be described by the fitness contributions of all alleles in the population. An insight of these analyses is that viral populations carry a large burden of detrimental mutations that imposes a significant fitness cost on the population that persists across passage. Previous studies showed that the high mutation load of RNA viruses is a key determinant on viral evolution and emergence( Agrawal and Whitlock, 2012; Pybus et al., 2007). Our findings quantify the cost of deleterious mutation across the dengue genome, highlighting how negative selection shapes diversity in viral populations. It will be interesting to understand how specific functional and structural features shape these patterns and contribute to overall population fitness. The ability to map evolutionary constraint across the genome allows us to highlight potential vulnerabilities that could be harnessed to develop antiviral drugs and vaccines that are refractory to the emergence of resistance and escape. We find that adaptive mutations, including changes in coding and noncoding regions, cluster in specific regions across the DENV genome. Examination of mosquito specific alterations in a glycosylation site in protein E and in the 3′ UTR suggest that mutations in these clusters can lead to similar phenotypic outcomes. For instance, mutations that cluster in the 3′ UTR and disrupt the structure of stem-loop II, and mutations along loop 150–160 in DENV E protein that disrupt glycosylation in mosquito cells, both sites for gate-keeper mutations for mosquito transmission( Filomatori et al., 2017; Mondotte et al., 2007; Villordo et al., 2015). This suggests that the process of alternating host adaptation relies on maintaining access adaptive phenotypes through highly connected genetic networks made up of phenotypically redundant mutations. We propose that the phenotypic redundancy of such mutations increases the mutational target size associated with key transitions necessary for adaptation, thereby partially relieving possible bottlenecks associated with transmission and early adaptation( Besnard et al., 2020; Girgis et al., 2012). Our study highlights the crucial role of structural constraints in shaping DENV evolution. Adaptive mutations are largely excluded from transmembrane domains and structured regions in DENV proteins. Thus, structural integrity places significant constraints on variation within these regions. It is tempting to speculate that the sequence of these arboviral domains is poised at a compromise that optimizes function in the distinct environments of human and mosquito cells( Bourg et al., 2019; Shoval et al., 2012; Tendler et al., 2015). Of note, our identification of highly constrained DENV regions, where most mutations are lethal, may uncover attractive targets for antivirals. Beneficial mutations are enriched in flexible loops and intrinsically disordered regions of the DENV polyprotein( Figure 6b). The relaxed structural constraints of IDRs allow them to explore more mutational diversity without compromising protein folding or stability, thus enabling access to more extensive sets of adaptive mutations( Charon et al., 2018; Gitlin et al., 2014). Such plasticity may allow viral IDRs to rewire viral protein interactions with host factors, thereby driving adaptation to changing environments( Figure 6f). In the future, it will be informative to characterize how constraint across the genome influences the transmission of arboviruses, by restricting or enabling exploration of the genetic neighborhood and the persistence of specific subpopulations during transmission. Notably, the link between structural properties and fitness effects measured in our study mirrors sequence conservation and variation across natural isolates of DENV and ZIKV( Figure 6c, d). This indicates that the relationships between adaptability, structural flexibility, and phenotypic redundancy uncovered here in a model of DENV adaptation to cultured human and mosquito cells can suggest general principles of flavivirus evolution broadly. While arboviruses that cycle between human and mosquito represent a more extreme case of host switching, most emerging viruses must adapt to changing environments during zoonotic transmission or intra-host spreading. We propose that our simple experimental approach can map the mutational neighborhoods of viral genomes and how selection acts on specific sites of the viral genome and proteome to shape evolutionary outcomes linked to diversification, tropism, and spread for a wide range of RNA viruses and may be particularly useful to study virus without easily accessible animal models, or tools for engineering mutational screens.
Viruses are constantly evolving as a result of mutations in their genetic material and environmental pressures . Viruses switching between insects and mammals face unique evolutionary pressures because they must retain their ability to infect both types of organisms . Yet , the mutations in a virus that may be beneficial in an insect may be different from the ones that may be beneficial in a mammal . Mutations in one host may be even harmful in the other . To learn more about how such viruses thrive as they switch between hosts , Dolan , Taguwa et al . studied the dengue virus , which causes over 390 million infections and over 10 , 000 deaths each year around the globe . They compared the mutations that occurred as the virus multiplied in human and mosquito cells grown in a laboratory . In the experiments , they used a method called ultra-deep RNA sequencing to identify every change that occurred in the genetic material of the virus each time it multiplied . They determined whether the mutations were beneficial or harmful based on whether they became more common – suggesting they helped the virus survive – or whether they did not persist because they were likely harmful or even fatal to the virus . The experiments showed that many harmful mutations constantly occur in the virus , in both human and mosquito cells . Beneficial changes happen rarely , and those that do are usually only helpful in one type of cell . Fatal mutations tended to occur in parts of the genetic material that encodes regions in the viral proteins that must remain the same . These structural elements appear to be essential to the virus’s survival and unable to undergo change , which makes them good targets for antiviral drugs or vaccines . The techniques used in the study may be useful for investigating other viruses and for understanding the evolutionary constraints on viruses more generally . This may help scientists develop antiviral drugs or vaccines that will remain effective even as viruses continue to evolve and mutate .