Mutagenic Compounds as Broad Spectrum Antivirals

Most RNA viruses utilize RNA-dependent RNA polymerases for viral genome replication and synthesis of mRNAs, making them prime targets for mutagenic compounds to be utilized as broad-spectrum antivirals. Primer ID with next-generation sequencing (NGS) allows for accurate and deep characterization of viral populations, which provides for the ideal tool for screening mutagenic compounds and their effects on RNA viruses. In this study, we used this approach to study three potential mutagenic compounds (N4-beta-hydrocytidine, Favipiravir, and Ribavirin) on a panel of RNA viruses, including MERS coronavirus (MERS-CoV), Zika virus (ZIKV), and La Crosse virus (LACV). We hypothesize that these compounds exhibit antiviral effects by inducing lethal mutations in the viral genomes. We used a cell culture model where we grew the viruses in cell culture in the presence and absence of drugs, determined viral titers using plaque assays, and sequenced viruses at different time points to compare sequence differences by using primer ID and NGS. Using this data from the in vitro experiments, we developed a model to predict the percentage of defective viral genomes after treatment with mutagenic compounds. We also used an in vivo mice-MERS coronavirus model to study the antiviral effects and short-term toxicity of NHC. To study the long term toxicity of all three compounds, we used an 8E5 cell model, harboring a single HIV provirus per cell, to monitor the effects of these compounds on DNA dependent RNA polymerase (transcription) and DNA polymerase (DNA replication). The viral titers in the cell culture model showed that 10 μM of NHC had significant inhibition of MERS-CoV and LACV and moderate inhibition of ZIKV, which correlated strongly with an increase of cytosine (C) to uridine (U) transversion mutations, suggesting its mechanism for its antiviral activity is lethal mutagenesis. Both Favipiravir and Ribavirin exhibited no antiviral effects on MERS-CoV or ZIKV but had some antiviral effects on LACV. The In vivo mouse model showed that NHC had great inhibition in MERS-CoV with no significant observed mutations in the mice mRNA. The 8E5 cell model showed that neither Ribavirin or Favipiravir increased the mutation rate, but found that NHC caused an increase in the C to U mutations, suggesting there may be some long term effects of the drug. With the broad-spectrum antiviral effects of these compounds, they could potentially be able to be used to treat a wide variety of viral infections in humans, including newly emerged viruses that lack other forms of treatment.Bachelor of Scienc

β-D-N4-hydroxycytidine (NHC) Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in the genomes of RNA viruses during viral replication. β-D-N 4-hydroxycytidine (NHC, the initial metabolite of molnupiravir) is more than 100-fold more active than ribavirin or favipiravir against SARS-CoV-2, with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate that highly active mutagenic ribonucleosides may hold risk for the host

Reply to Troth et al

We thank Troth et al. for the opportunity to extend the discussion of our data on the mutagenicity of N4-hydroxycytidine (rNHC) [1]. We view our work as providing the proof-of-concept showing that as rNHC is phosphorylated to its active ribonucleoside 5’-triphosphate, the ribonucleoside 5’-diphosphate intermediate that is the immediate precursor to the ribonucleoside 5’-triphosphate also plays the equivalent role of an intermediate precursor for the synthesis of 2’-deoxyribonucleoside 5’-diphosphate (by the activity of ribonucleotide reductase). This is the normal pathway for the synthesis of DNA precursors used from bacteria to humans; thus, it should not be a question of whether the mutagenic form of dNHC as a precursor to DNA is formed, but rather what the impact is. On this point we have unpublished cell-based data supporting conversion of rNHC to dNHC, albeit at low intracellular levels. Also, the near identity of rNHC to cytidine (the addition of a single oxygen atom) makes it likely that rNHC and cytidine undergo similar metabolism in the cell

Can public health reconcile profits and pandemics? An analysis of attitudes to commercial sector engagement in health policy and research

Public health's terms of engagement with unhealthy commodity industries (alcohol, tobacco and ultra-processed food and drinks) have become increasingly contested in policy and research. We sought to identify approaches that could attract consensus support within and across policy domains.Using snowball sampling, we undertook an online survey of 335 health researchers, advocates and policymakers, in 40 countries, assessing responses to stated principles, claims and recommendations for engaging with unhealthy commodity industries in relation to key policy and research initiatives.Most respondents identified a fundamental conflict between industry interests and public health objectives for all three industries, with agreement greatest in relation to tobacco and weakest for food. This pattern was replicated across diverse questions regarding potential forms of engagement, including in rejecting voluntarism and partnership approaches to health policy. While awareness of tobacco industry tactics to influence policy and research was higher than for alcohol and food, most respondents rejected the view that the influence of the latter was less significant for public health. Proposals that health and research organisations should divest their funds attracted less support with respect to food, while restricting publication of industry-funded research in academic journals was the issue that most divided opinion. Respondents reported most difficulty in answering questions about the food industry.The strong consensus around restricting interactions with the tobacco industry supports increased implementation of the WHO Framework Convention on Tobacco Control's conflict of interest provisions. There is strong support for the extension of such practices to the alcohol industry, challenging current norms. More mixed responses indicate a need for greater clarity in defining the food industry, and for research analyzing links, similarities and differences across different types of unhealthy commodity producers. Partnership approaches to addressing non-communicable diseases seem incapable of attracting widespread support across public health, challenging practice in many contexts

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%–100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design

Sensitivity of the IceCube Detector to Astrophysical Sources of High Energy Muon Neutrinos

We present the results of a Monte-Carlo study of the sensitivity of the planned IceCube detector to predicted fluxes of muon neutrinos at TeV to PeV energies. A complete simulation of the detector and data analysis is used to study the detector's capability to search for muon neutrinos from sources such as active galaxies and gamma-ray bursts. We study the effective area and the angular resolution of the detector as a function of muon energy and angle of incidence. We present detailed calculations of the sensitivity of the detector to both diffuse and pointlike neutrino emissions, including an assessment of the sensitivity to neutrinos detected in coincidence with gamma-ray burst observations. After three years of datataking, IceCube will have been able to detect a point source flux of E^2*dN/dE = 7*10^-9 cm^-2s^-1GeV at a 5-sigma significance, or, in the absence of a signal, place a 90% c.l. limit at a level E^2*dN/dE = 2*10^-9 cm^-2s^-1GeV. A diffuse E-2 flux would be detectable at a minimum strength of E^2*dN/dE = 1*10^-8 cm^-2s^-1sr^-1GeV. A gamma-ray burst model following the formulation of Waxman and Bahcall would result in a 5-sigma effect after the observation of 200 bursts in coincidence with satellite observations of the gamma-rays.Comment: 33 pages, 13 figures, 6 table

The Pandora multi-algorithm approach to automated pattern recognition of cosmic-ray muon and neutrino events in the MicroBooNE detector.

The development and operation of liquid-argon time-projection chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens of algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies