Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

Interferon (IFN) signaling induces the expression of a wide array of genes, collectively referred to as IFN-stimulated genes (ISGs) that generally function to inhibit viral replication. RNA viruses are frequently targeted by ISGs through recognition of viral replicative intermediates and molecular features associated with viral genomes, or the lack of molecular features associated with host mRNAs. The ISGs reviewed here primarily inhibit viral replication in an RNA-centric manner, working to sense, degrade, or repress expression of viral RNA. This review focuses on dissecting how these ISGs exhibit multiple antiviral mechanisms, often through use of varied co-factors, highlighting the complexity of the type I IFN response. Specifically, these ISGs can mediate antiviral effects through viral RNA degradation, viral translation inhibition, or both. While the OAS/RNase L pathway globally degrades RNA and arrests translation, ISG20 and ZAP employ targeted RNA degradation and translation inhibition to block viral replication. Meanwhile, SHFL targets translation by inhibiting -1 ribosomal frameshifting, which is required by many RNA viruses. Finally, a number of E3 ligases inhibit viral transcription, an attractive antiviral target during the lifecycle of negative-sense RNA viruses which must transcribe their genome prior to translation. Through this review, we aim to provide an updated perspective on how these ISGs work together to form a complex network of antiviral arsenals targeting viral RNA processes.

Approximately 800 million people worldwide are infected with one or more species of skin-penetrating nematodes. These parasites persist in the environment as developmentally arrested third-stage infective larvae (iL3s) that navigate toward host-emitted cues, contact host skin, and penetrate the skin. iL3s then reinitiate development inside the host in response to sensory cues, a process called activation. Here, we investigate how chemosensation drives host seeking and activation in skin-penetrating nematodes. We show that the olfactory preferences of iL3s are categorically different from those of free-living adults, which may restrict host seeking to iL3s. The human-parasitic threadworm Strongyloides stercoralis and hookworm Ancylostoma ceylanicum have highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the S. stercoralis tax-4 gene abolishes iL3 attraction to a host-emitted odorant and prevents activation. Our results suggest an important role for chemosensation in iL3 host seeking and infectivity and provide insight into the molecular mechanisms that underlie these processes.

BACKGROUND AND OBJECTIVES: Socioeconomic disparities in cardiovascular health among adults have been documented, but disparities during adolescence are less understood. In this study, we examined secular trends in 7 cardiovascular risk factors and disparities among US adolescents. METHODS: We analyzed NHANES data from 1999 to 2014, including 11 557 (4854 fasting) participants aged 12 to 19 years. To examine trends in cardiovascular risk factors, adolescents were stratified into 3 groups on the basis of family poverty-income ratio: low income (poverty-income ratio, <1.3), middle income (≥1.3 and <3.5), and high income (≥3.5). RESULTS: From 1999 to 2014, the prevalence of obesity increased (16.5%-21.0%, P = .001) but only among low-and middle-income adolescents, with significant disparities in prevalence by income (21.7% vs 14.6% among low-versus high-income adolescents, respectively, in 2011-2014). In addition, there were significant and persistent disparities in the prevalence of smoking (20.8% vs 7.4% among low-versus high-income adolescents, respectively, in 2011-2014), low-quality diet (67.8% vs 49.0%), and physical inactivity (25.6% vs 17.0%). No significant disparities were observed in the prevalence of prediabetes and diabetes, hypertension, or hypercholesterolemia, although the prevalence of prediabetes and diabetes increased among low-income adolescents (21.4%-28.0%, P = .01). Overall, the prevalence of adolescents with 2 or more risk factors declined (48.3% to 37.1%, P<.001), but this decline was only significant for high-and middle-income adolescents. CONCLUSIONS: Recent improvements in cardiovascular health have not been equally shared by US adolescents of varying socioeconomic status.

The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP’s suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25.