Biology subjects

Dewhurst, S.

Publications and source records attributed to Dewhurst, S..

5 recordsLinked to original sources

bioRxiv2023

SARS-CoV-2 induces acute neurological signs while Calcitonin Gene-Related Peptide (CGRP) signaling blockade reduces interleukin 6 (IL-6) release and weight loss in mouse models

COVID-19 can result in neurological symptoms such as fever, headache, dizziness, and nausea. However, neurological signs of SARS-CoV-2 infection have been hardly assessed in mouse models. Here, we infected two commonly used wildtype mice lines (C57BL/6 and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including motion- related dizziness. We then evaluated whether the Calcitonin Gene-Related Peptide (CGRP) receptor antagonist, olcegepant, used in migraine treatment could mitigate acute neuroinflammatory and neurological responses to SARS-COV-2 infection. We infected wildtype C57BL/6J and 129/SvEv mice, and a 129 CGRP-null mouse line with a mouse-adapted SARS- CoV-2 virus, and evaluated the effect of CGRP receptor antagonism on the outcome of that infection. First, we determined that CGRP receptor antagonism provided protection from permanent weight loss in older (>12 m) C57BL/6J and 129 SvEv mice. We also observed acute fever and motion-induced dizziness in all older mice, regardless of treatment. However, in both wildtype mouse lines, CGRP antagonism reduced acute interleukin 6 (IL-6) levels by half, with virtually no IL-6 release in mice lacking CGRP. These findings suggest that migraine inhibitors such as those blocking CGRP signaling protect against acute IL-6 release and subsequent inflammatory events after SARS-CoV-2 infection, which may have repercussions for related pandemic and/or endemic coronaviruses. ImportanceCOVID-19 can cause neurological symptoms such as fever, headache, dizziness, and nausea. However, such neurological symptoms of SARS-CoV-2 infection have been hardly assessed in mouse models. Here, we first infected two commonly used wildtype mice lines (C57BL/6 and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including motion-related dizziness. Further, we showed that migraine treatment drug olcegepant could reduce long-term weight loss and IL-6 release associated with SARS-CoV-2 infection. These findings suggest that a migraine blocker can be protective for at least some acute SARS-CoV-2 infection signs and raise the possibility that it may also impact long-term outcomes of infection.

neuroscience↗

bioRxiv2023

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTSO_LICertain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches. C_LIO_LIUnderstanding Cas13as allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants. C_LIO_LIThese Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/550755v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a3fd63org.highwire.dtl.DTLVardef@1509a82org.highwire.dtl.DTLVardef@cfcd7aorg.highwire.dtl.DTLVardef@bb2f0f_HPS_FORMAT_FIGEXP M_FIG New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a. C_FIG

molecular biology↗

bioRxiv2022

Generation and functional analysis of defective viral genomes during SARS-CoV-2 infection

Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from RNA-seq datasets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hotspots were identified for DVG recombination and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single cell RNA-seq analysis indicated the IFN stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the NGS dataset from a published cohort study and observed significantly higher DVG amount and frequency in symptomatic patients than that in asymptomatic patients. Finally, we observed unusually high DVG frequency in one immunosuppressive patient up to 140 days after admitted to hospital due to COVID-19, first-time suggesting an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. ImportanceDefective viral genomes (DVGs) are ubiquitously generated in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide them the potential for novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex and the recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hotspots for non-homologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide the evidence to harness DVGs immunostimulatory potential in the development of vaccine and antivirals for SARS-CoV-2.

microbiology↗

bioRxiv2022

A single amino acid substitution in PB1 of pandemic H1N1 with A/Ann Arbor/6/60 master donor virus mutations as a novel live-attenuated influenza virus vaccine

Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year old healthy and non-pregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The United States (U.S.) licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain. IMPORTANCEVaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S. licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

microbiology↗

bioRxiv2020

Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro

The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but an effective antiviral therapy has yet to be discovered. To improve treatment options, we conducted a high-throughput drug repurposing screen to uncover compounds that block the viral activity of SARS-CoV-2. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed at 10 dose points (ranging from 20 M to 1 nM). Our screening revealed several FDA-approved agents that act as novel antivirals that block both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 replication by over 50,000-fold without any toxicity and at doses readily achievable in human tissues. Further, both lapatinib and doramapimod could be combined with remdesivir to dramatically improve antiviral activity in cells. These findings reveal novel treatment options for people infected with SARS-CoV-2 that can be readily implemented during the pandemic.

microbiology↗