Design of antiviral AGO2-dependent short hairpin RNAs.

The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibits significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based model with high predictive power. Using this model, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and transformed them into agshRNAs. We validated the performance of our agshRNA design by evaluating antiviral efficacies of agshRNAs in cells infected with different viruses. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RNA-induced silencing complex (RISC). We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develops a novel antiviral strategy that combines machine learning-based algorithm with agshRNA design to custom design antiviral agshRNAs with high efficiency.

Alphavirus infection triggers antiviral RNAi immunity in mammals.

RNA interference (RNAi) is a well-established antiviral immunity. However, for mammalian somatic cells, antiviral RNAi becomes evident only when viral suppressors of RNAi (VSRs) are disabled by mutations or VSR-targeting drugs, thereby limiting its scope as a mammalian immunity. We find that a wild-type alphavirus, Semliki Forest virus (SFV), triggers the Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs) in both mammalian somatic cells and adult mice. These SFV-vsiRNAs are located at a particular region within the 5' terminus of the SFV genome, Argonaute loaded, and active in conferring effective anti-SFV activity. Sindbis virus, another alphavirus, also induces vsiRNA production in mammalian somatic cells. Moreover, treatment with enoxacin, an RNAi enhancer, inhibits SFV replication dependent on RNAi response in vitro and in vivo and protects mice from SFV-induced neuropathogenesis and lethality. These findings show that alphaviruses trigger the production of active vsiRNA in mammalian somatic cells, highlighting the functional importance and therapeutic potential of antiviral RNAi in mammals.

Nursing Care of an Older Patient With Severe COVID-19 Receiving Prolonged Prone Ventilation: A Case Report.

INTRODUCTION: Prone positioning has been shown to improve ventilation status for patients with severe COVID-19 who are receiving mechanical ventilation. This case report describes the nursing care of a patient with severe COVID-19 who underwent prone ventilation for 72 hours. Relevant nursing management and operational considerations are also discussed. CLINICAL FINDINGS: An 83-year-old woman was admitted to the hospital with fatigue, dizziness, and positive tests for SARS-CoV-2 on nasopharyngeal swab specimens. The patient was intubated. DIAGNOSIS: The patient's positive tests for SARS-CoV-2, chest computed tomography findings, and clinical symptoms were consistent with a diagnosis of severe COVID-19. INTERVENTIONS: When the patient's condition did not improve with mechanical ventilation and intermittent prone positioning, she was placed in the prone position for 72 hours. She received sedation, analgesics, anti-infective medications, and enteral nutrition support in the intensive care unit. Nurses performed dynamic monitoring based on blood gas analysis results to guide lung rehabilitation. OUTCOMES: The patient was weaned from the ventilator on day 20 and successfully discharged home on day 28 of hospitalization. CONCLUSION: During prolonged prone ventilation of a patient with severe COVID-19, nursing strategies included airway management, early lung rehabilitation training guided by pulmonary ultrasonography, skin care, hierarchical management of nurses, hemodynamic support, and enteral nutrition. This report may assist critical care nurses caring for similar patients.

STUB1 regulates antiviral RNAi through inducing ubiquitination and degradation of Dicer and AGO2 in mammals.

RNA interference (RNAi) is an intrinsic antiviral immune mechanism conserved in diverse eukaryotic organisms. However, the mechanism by which antiviral RNAi in mammals is regulated is poorly understood. In this study, we uncovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1) was a new regulator of the RNAi machinery in mammals. We found that STUB1 interacted with and ubiquitinated AGO2, and targeted it for degradation in a chaperon-dependent manner. STUB1 promoted the formation of Lys48 (K48)-linked polyubiquitin chains on AGO2, and facilitated AGO2 degradation through ubiquitin-proteasome system. In addition to AGO2, STUB1 also induced the protein degradation of AGO1, AGO3 and AGO4. Further investigation revealed that STUB1 also regulated Dicer's ubiquitination via K48-linked polyubiquitin and induced the degradation of Dicer as well as its specialized form, termed antiviral Dicer (aviDicer) that expresses in mammalian stem cells. Moreover, we found that STUB1 deficiency up-regulated Dicer and AGO2, thereby enhancing the RNAi response and efficiently inhibiting viral replication in mammalian cells. Using the newborn mouse model of Enterovirus A71 (EV-A71), we confirmed that STUB1 deficiency enhanced the virus-derived siRNAs production and antiviral RNAi, which elicited a potent antiviral effect against EV-A71 infection in vivo. In summary, our findings uncovered that the E3 ubiquitin ligase STUB1 was a general regulator of the RNAi machinery by targeting Dicer, aviDicer and AGO1-4. Moreover, STUB1 regulated the RNAi response through mediating the abundance of Dicer and AGO2 during viral infection, thereby providing novel insights into the regulation of antiviral RNAi in mammals.

Enoxacin Shows Broad-Spectrum Antiviral Activity against Diverse Viruses by Enhancing Antiviral RNA Interference in Insects.

RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we show that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived small interfering RNA (siRNA) into the RNA-induced silencing complex, thereby enhancing the antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited the replication of flaviviruses, including dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrate that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control the vector transmission of arboviruses or viral diseases in insect farming. IMPORTANCE RNAi has been widely recognized as one of the most broadly acting and robust antiviral mechanisms in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that was previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Here, we show that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into the Ago2-incorporated RNA-induced silencing complex and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin displayed effective RNAi-dependent antiviral effects against flaviviruses, such as dengue virus and Zika virus, in mosquito cells. This study is the first to demonstrate that enhancing RNAi by enoxacin elicits potent antiviral effects against diverse viruses in insects.

Plasma Proteomics Identify Biomarkers and Pathogenesis of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is a global public health crisis. However, little is known about the pathogenesis and biomarkers of COVID-19. Here, we profiled host responses to COVID-19 by performing plasma proteomics of a cohort of COVID-19 patients, including non-survivors and survivors recovered from mild or severe symptoms, and uncovered numerous COVID-19-associated alterations of plasma proteins. We developed a machine-learning-based pipeline to identify 11 proteins as biomarkers and a set of biomarker combinations, which were validated by an independent cohort and accurately distinguished and predicted COVID-19 outcomes. Some of the biomarkers were further validated by enzyme-linked immunosorbent assay (ELISA) using a larger cohort. These markedly altered proteins, including the biomarkers, mediate pathophysiological pathways, such as immune or inflammatory responses, platelet degranulation and coagulation, and metabolism, that likely contribute to the pathogenesis. Our findings provide valuable knowledge about COVID-19 biomarkers and shed light on the pathogenesis and potential therapeutic targets of COVID-19.