Rapid detection of intact SARS-CoV-2 using designer DNA nets and a pocket-size smartphone-linked fluorimeter

Rapid, sensitive, and inexpensive point-of-care diagnosis is vital to controlling highly infectious diseases, including COVID-19. Here, we report the design and characterization of a compact fluorimeter called a "Virus Pod” (V-Pod) that enables sensitive self-testing of SARS-CoV-2 viral load in saliva. The rechargeable battery-operated device reads the fluorescence generated by Designer DNA Nanostructures (DDN) when they specifically interact with intact SARS-CoV-2 virions. DDNs are net-shaped self-assembling nucleic acid constructs that provide an array of highly specific aptamer-fluorescent quencher duplexes located at precise positions that match the pattern of spike proteins. The room-temperature assay is performed by mixing the test sample with DNA Net sensor in a conventional PCR tube and placing the tube into the V-Pod. Fluorescent signals are generated when multivalent aptamer-spike binding releases fluorescent quenchers, resulting in rapid (5-min) generation of dose-dependent output. The V-Pod instrument performs laser excitation, fluorescence intensity quantitation, and secure transmission of data to an App via Bluetooth™. We show that the V-Pod and DNA Net assay achieves clinically relevant detection limits of 3.92 × 103 viral-genome-copies/mL for pseudo-typed wild-type SARS-CoV-2 and 1.84 × 104, 9.69 × 104, 6.99 × 104 viral-genome-copies/mL for pathogenic Delta, Omicron, and D614G variants, representing sensitivity similar to laboratory-based PCR. The pocket-sized instrument (∼$294), inexpensive reagent-cost/test ($1.26), single-step, rapid sample-to-answer, and quantitative output represent a capability that is compatible with the needs of frequent self-testing in a consumer-friendly format that can link with medical service systems such as healthcare providers, contact tracing, and infectious disease reporting.

Rapid detection of intact SARS-CoV-2 using designer DNA Nets and a pocket-size smartphone-linked fluorimeter.

Rapid, sensitive, and inexpensive point-of-care diagnosis is vital to controlling highly infectious diseases, including COVID-19. Here, we report the design and characterization of a compact fluorimeter called a "Virus Pod" (V-Pod) that enables sensitive self-testing of SARS-CoV-2 viral load in saliva. The rechargeable battery-operated device reads the fluorescence generated by Designer DNA Nanostructures (DDN) when they specifically interact with intact SARS-CoV-2 virions. DDNs are net-shaped self-assembling nucleic acid constructs that provide an array of highly specific aptamer-fluorescent quencher duplexes located at precise positions that match the pattern of spike proteins. The room-temperature assay is performed by mixing the test sample with DNA Net sensor in a conventional PCR tube and placing the tube into the V-Pod. Fluorescent signals are generated when multivalent aptamer-spike binding releases fluorescent quenchers, resulting in rapid (5-min) generation of dose-dependent output. The V-Pod instrument performs laser excitation, fluorescence intensity quantitation, and secure transmission of data to an App via Bluetooth™. We show that the V-Pod and DNA Net assay achieves clinically relevant detection limits of 3.92 × 103 viral-genome-copies/mL for pseudo-typed wild-type SARS-CoV-2 and 1.84 × 104, 9.69 × 104, 6.99 × 104 viral-genome-copies/mL for pathogenic Delta, Omicron, and D614G variants, representing sensitivity similar to laboratory-based PCR. The pocket-sized instrument (∼$294), inexpensive reagent-cost/test ($1.26), single-step, rapid sample-to-answer, and quantitative output represent a capability that is compatible with the needs of frequent self-testing in a consumer-friendly format that can link with medical service systems such as healthcare providers, contact tracing, and infectious disease reporting.

PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu.

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.

PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.

Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus.

We present a net-shaped DNA nanostructure (called "DNA Net" herein) design strategy for selective recognition and high-affinity capture of intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild-type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers release fluorescence signals upon virus binding that are easily read with a handheld fluorimeter for a rapid (in 10 min), simple (mix-and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive (∼$1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1 × 103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.

Mutations and Evolution of the SARS-CoV-2 Spike Protein.

The SARS-CoV-2 spike protein mediates target recognition, cellular entry, and ultimately the viral infection that leads to various levels of COVID-19 severities. Positive evolutionary selection of mutations within the spike protein has led to the genesis of new SARS-CoV-2 variants with greatly enhanced overall fitness. Given the trend of variants with increased fitness arising from spike protein alterations, it is critical that the scientific community understand the mechanisms by which these mutations alter viral functions. As of March 2022, five SARS-CoV-2 strains were labeled "variants of concern" by the World Health Organization: the Alpha, Beta, Gamma, Delta, and Omicron variants. This review summarizes the potential mechanisms by which the common mutations on the spike protein that occur within these strains enhance the overall fitness of their respective variants. In addressing these mutations within the context of the SARS-CoV-2 spike protein structure, spike/receptor binding interface, spike/antibody binding, and virus neutralization, we summarize the general paradigms that can be used to estimate the effects of future mutations along SARS-CoV-2 evolution.

Gastrointestinal Microenvironment and the Gut-Lung Axis in the Immune Responses of Severe COVID-19.

The global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an unprecedented threat to the human health. A close association of the digestive tract is implied by the high frequency of gastrointestinal syndromes among COVID-19 patients. A better understanding of the role of intestinal microenvironment in COVID-19 immunopathology will be helpful to improve the control of COVID-19 associated morbidity and mortality. This review summarizes the immune responses associated with the severity of COVID-19, the current evidence of SARS-CoV-2 intestinal tropism, and the potential involvement of gut microenvironment in COVID-19 severity. Additionally, we discuss the therapeutic potential of probiotics as an alternative medicine to prevent or alleviate severe COVID-19 outcome.

BTK/ITK dual inhibitors: Modulating immunopathology and lymphopenia for COVID-19 therapy.

Bruton's tyrosine kinase (BTK) signaling is involved in innate immune responses and regulates the production of proinflammatory cytokines that can contribute to COVID-19 immunopathology. Clinical trials with BTK inhibitors in COVID-19 treatment have been proposed, and previous studies have attempted to investigate the therapeutic effects of ibrutinib and underlying mechanisms in treating viral pneumonia. These attempts, however, did not consider potential off target effect of BTK inhibitors on T cell differentiation, function, and survival, which may be beneficial in treatment for COVID-19. Here, we summarize the current knowledge of BTK/IL-2-inducible T-cell kinase (ITK) signaling in immunopathology and lymphopenia and discuss the potential of BTK/ITK dual inhibitors such as ibrutinib in modulating immunopathology and lymphopenia, for COVID-19 therapy.

Cognitive disorders associated with hospitalization of COVID-19: Results from an observational cohort study.

INTRODUCTION: Our understanding of risk factors for COVID­19, including pre-existing medical conditions and genetic variations, is limited. To what extent the pre-existing clinical condition and genetic background have implications for COVID-19 still needs to be explored. METHODS: Our study included 389,620 participants of European descent from the UK Biobank, of whom 3,884 received the COVID-19 test and 1,091 were tested positive for COVID-19. We examined the association of COVID-19 status with an extensive list of 974 medical conditions and 30 blood biomarkers. Additionally, we tested the association of genetic variants in two key genes related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), with COVID-19 or any other phenotypes. RESULTS: The most significant risk factors for COVID-19 include Alzheimer's disease (OR = 2.29, 95% CI: 1.25-4.16), dementia (OR = 2.16, 95% CI: 1.36-3.42), and the overall category of delirium, dementia, amnestic and other cognitive disorders (OR = 1.90, 95% CI: 1.24-2.90). Evidence suggesting associations of genetic variants in SARS-CoV-2 infection-related genes with COVID-19 (rs7282236, OR = 1.33, 95% CI: 1.14-1.54, p = 2.31 × 10-4) and other phenotypes, such as an immune deficiency (p = 5.65 × 10-5) and prostate cancer (p = 1.1 × 10-5), was obtained. CONCLUSIONS: Our unbiased and extensive search identified pre-existing Alzheimer's disease and dementia as top risk factors for hospital admission due to COVID-19, highlighting the importance of providing special protective care for patients with cognitive disorders during this pandemic. We also obtained evidence suggesting a direct association of genetic variants with COVID-19.

Altered Blood Cell Traits Underlie a Major Genetic Locus of Severe COVID-19.

BACKGROUND: The genetic locus 3p21.31 has been associated with severe coronavirus disease 2019 (COVID-19), but the underlying pathophysiological mechanism is unknown. METHODS: To identify intermediate traits associated with the 3p21.31 locus, we first performed a phenome-wide association study (PheWAS) with 923 phenotypes in 310 999 European individuals from the UK Biobank. For genes potentially regulated by the COVID-19 risk variant, we examined associations between their expression and the polygenic score (PGS) of 1263 complex traits in a meta-analysis of 31 684 blood samples. For the prioritized blood cell traits, we tested their associations with age and sex in the same UK Biobank sample. RESULTS: Our PheWAS highlighted multiple blood cell traits to be associated with the COVID-19 risk variant, including monocyte count and percentage (p = 1.07 × 10-8, 4.09 × 10-13), eosinophil count and percentage (p = 5.73 × 10-3, 2.20 × 10-3), and neutrophil percentage (p = 3.23 × 10-3). The PGS analysis revealed positive associations between the expression of candidate genes and genetically predicted counts of specific blood cells: CCR3 with eosinophil and basophil (p = 5.73 × 10-21, 5.08 × 10-19); CCR2 with monocytes (p = 2.40 × 10-10); and CCR1 with monocytes and neutrophil (p = 1.78 × 10-6, 7.17 × 10-5). Additionally, we found that almost all examined white blood cell traits are significantly different across age and sex groups. CONCLUSIONS: Our findings suggest that altered blood cell traits, especially those of monocyte, eosinophil, and neutrophil, may represent the mechanistic links between the genetic locus 3p21.31 and severe COVID-19. They may also underlie the increased risk of severe COVID-19 in older adults and men.

Glycyrrhetinic acid and its derivatives as potential alternative medicine to relieve symptoms in nonhospitalized COVID-19 patients.

SARS-CoV-2 is highly infectious, and infection by this virus results in COVID-19, manifesting predominantly symptoms in the lower respiratory system. Detection of viral genomic materials by RT-PCR is the gold standard for diagnosis. Suspected COVID-19 patients who had a documented history of exposure and exhibited symptoms, but did not have positive PCR test results, were generally self-quarantined with prescriptions aiming to help attenuate their symptoms. These prescriptions are however neither specific nor highly effective for COVID-19 treatment. Given the rapidly growing pandemic and the overwhelmed medical system, the number of self-quarantined patients is increasing. There is an urgent need of alternative medicine to help patients relieve symptoms during self-quarantine, and to potentially help increase their chances of survival and recovery from the infection. We report here a case of severe COVID-19 that never had a positive PCR test result during disease progression but was confirmed with antibody test post recovery. This patient was self-quarantined and received diammonium glycyrrhizinate (DG), a steroid-like molecule, in combination with vitamin C as alternative medicine. This patient went through severe COVID-19 but eventually recovered upon the implementation of this treatment regimen, suggesting potential therapeutic effects of DG as alternative medicine to help relieve COVID-19 symptoms.