The mosquito Aedes aegypti is a potent vector of the chikungunya, yellow fever, and dengue viruses, responsible for hundreds of millions of infections and over 50,000 human deaths per year. Mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to provide genomic target sequence specificity. Here, we describe the use of the CRISPR-Cas9 system to generate site-specific mutations in Ae. aegypti. This system relies on RNA-DNA base-pairing to generate targeting specificity, resulting in efficient and flexible genome-editing reagents. We investigate the efficiency of injection mix compositions, demonstrate the ability of CRISPR-Cas9 to generate different types of mutations via disparate repair mechanisms, and report stable germ-line mutations in several genomic loci. This work offers a detailed exploration into the use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.

Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar’s nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

Seasonal coronaviruses (OC43, 229E, NL63, and HKU1) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. It is not known to what extent reinfection by these viruses is due to waning immune memory or antigenic drift of the viruses. Here we address the influence of antigenic drift on immune evasion of seasonal coronaviruses. We provide evidence that at least two of these viruses, OC43 and 229E, are undergoing adaptive evolution in regions of the viral spike protein that are exposed to human humoral immunity. This suggests that reinfection may be due, in part, to positively selected genetic changes in these viruses that enable them to escape recognition by the immune system. It is possible that, as with seasonal influenza, these adaptive changes in antigenic regions of the virus would necessitate continual reformulation of a vaccine made against them.

Seasonal coronaviruses (OC43, 229E, NL63 and HKU1) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. It is not known to what extent reinfection by these viruses is due to waning immune memory or antigenic drift of the viruses. Here, we address the influence of antigenic drift on immune evasion of seasonal coronaviruses. We provide evidence that at least two of these viruses, OC43 and 229E, are undergoing adaptive evolution in regions of the viral spike protein that are exposed to human humoral immunity. This suggests that reinfection may be due, in part, to positively-selected genetic changes in these viruses that enable them to escape recognition by the immune system. It is possible that, as with seasonal influenza, these adaptive changes in antigenic regions of the virus would necessitate continual reformulation of a vaccine made against them.

No abstract

As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.

Despite the appearance of variant SARS-CoV-2 viruses with altered receptor-binding or antigenic phenotypes, traditional methods for detecting adaptive evolution from sequence data do not pick up strong signals of positive selection. Here, we present a new method for identifying adaptive evolution on short evolutionary time scales with densely-sampled populations. We apply this method to SARS-CoV-2 to perform a comprehensive analysis of adaptively-evolving regions of the genome. We find that spike S1 is a focal point of adaptive evolution, but also identify positively-selected mutations in other genes that are sculpting the evolutionary trajectory of SARS-CoV-2. Protein-coding mutations in S1 are temporally-clustered and, in 2021, the ratio of nonsynonymous to synonymous divergence in S1 is more than 4 times greater than in the equivalent influenza HA1 subunit.

As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.