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Remdesivir inhibits the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; Nsp12). Here, we conducted viral resistance analyses from the Phase 3 PINETREE trial of remdesivir in nonhospitalized participants at risk of severe COVID-19. Nasopharyngeal swabs (collected at baseline [Day 1], Days 2, 3, 7, and 14) were eligible for analysis if their viral load was above the lower limit of quantification for the RT-qPCR assay (2228 copies/mL). The SARS-CoV-2 genome was sequenced for all remdesivir participants and 50% of placebo participants (baseline, Days 3, 7, and 14) and for participants who progressed to COVID-19-related hospitalization or all-cause death (all time points). Emergent substitutions in Nsp12 and other replication complex proteins were phenotyped using site-directed mutagenesis in a SARS-CoV-2 subgenomic replicon system. Overall, emergent Nsp12 substitutions were detected in 8/115 (7.0%) remdesivir participants and 7/129 (5.4%) placebo participants (1 substitution overlap between groups). Based on a structural analysis, none of the emergent Nsp12 substitutions were in direct contact with the incoming nucleoside triphosphate substrate, the RNA, or the RNA template 5' overhang. One substitution (A376V) showed reduced susceptibility to remdesivir (12.6-fold change in remdesivir half-maximal concentration [EC 50 ]); it also showed reduced fitness when introduced in the SARS-CoV-2 replicon and virus in vitro . Other substitutions had <1.1-fold change in remdesivir EC 50 . None of the emergent substitutions in Nsp8, Nsp10, Nsp13, or Nsp14 (remdesivir, 10/115 [8.7%]; placebo, 10/129 [7.8%]) showed reduced remdesivir susceptibility. In conclusion, emergent substitutions in the SARS-CoV-2 RdRp complex with reduced remdesivir susceptibility were uncommon, indicating a high barrier to remdesivir resistance.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04501952., Competing Interests: L.R., J.L., R.M., D.H., S.X., J.M., N.P., J.K.P., R.H.H., D.P.P., M.A., and C.H. are stockholders and employees of Gilead Sciences, Inc. H.W.L. has nothing to disclose. G.C. is a shareholder and former employee of Gilead Sciences, Inc., and an employee of Vir Biotechnology, Inc. M.G. received funding from Gilead Sciences, Inc., for studies on the mechanism of action of RDV.

As new SARS-CoV-2 variants continue to emerge, it is important to evaluate the potency of antiviral drugs to support their continued use. Remdesivir (RDV; VEKLURY ® ) an approved antiviral treatment for COVID-19, and obeldesivir (ODV) are inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase Nsp12. Here we show these two compounds retain antiviral activity against the Omicron variants BA.2.86, BF.7, BQ.1, CH.1.1, EG.1.2, EG.5.1, EG.5.1.4, FL.22, HK.3, HV.1, JN.1, JN.1.7, JN.1.18, KP.2, KP.3, LB.1, XBB.1.5, XBB.1.5.72, XBB.1.16, XBB.2.3.2, XBC.1.6, and XBF when compared with reference strains. Genomic analysis identified 29 Nsp12 polymorphisms in these and previous Omicron variants. Phenotypic analysis of these polymorphisms confirmed no impact on the antiviral activity of RDV or ODV and suggests Omicron variants containing these Nsp12 polymorphisms remain susceptible to both compounds. These data support the continued use of RDV in the context of circulating SARS-CoV-2 variants and the development of ODV as an antiviral therapeutic.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 ( 2 ) into lung cells, thereby forming the bioactive triphosphate 2-NTP . 2-NTP , an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP . Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3 ) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP , leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2 , which supports development of 3 as a promising COVID-19 treatment.

Enterovirus A71 causes severe disease upon systemic infection, sometimes leading to life-threatening neurological dysfunction. However, in most cases infection is asymptomatic and limited to the gastrointestinal tract, where virus is amplified for transmission. Picornaviruses have previously been shown to exit infected cells via either cell lysis or secretion of vesicles. Here we report that entire Enterovirus A71-infected cells are specifically extruded from the apical surface of differentiated human colon organoids, as observed by confocal microscopy. Differential sensitivity to chemical and peptide inhibitors demonstrated that extrusion of virus-infected cells is dependent on force sensing via mechanosensitive ion channels rather than apoptotic cell death. When isolated and used as inoculum, intact virus-containing extruded cells can initiate new infections. In contrast, when mechanical force sensing is inhibited, large amounts of free virus are released. Thus, extrusion of live, virus-infected cells from intact epithelial tissue is likely to benefit both the integrity of host tissues and the protected spread of this faecal-oral pathogen within and between hosts., (© 2023. The Author(s).)

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium., (© 2023 New York Academy of Sciences.)

Affordable and effective antiviral therapies are needed worldwide, especially against agents such as dengue virus that are endemic in underserved regions. Many antiviral compounds have been studied in cultured cells but are unsuitable for clinical applications due to pharmacokinetic profiles, side effects, or inconsistent efficacy across dengue serotypes. Such tool compounds can, however, aid in identifying clinically useful treatments. Here, computational screening (Rapid Overlay of Chemical Structures) was used to identify entries in an in silico database of safe-in-human compounds (SWEETLEAD) that display high chemical similarities to known inhibitors of dengue virus. Inhibitors of the dengue proteinase NS2B/3, the dengue capsid, and the host autophagy pathway were used as query compounds. Three FDA-approved compounds that resemble the tool molecules structurally, cause little toxicity, and display strong antiviral activity in cultured cells were selected for further analysis. Pyrimethamine (50% inhibitory concentration [IC 50 ] = 1.2 μM), like the dengue proteinase inhibitor ARDP0006 to which it shows structural similarity, inhibited intramolecular NS2B/3 cleavage. Lack of toxicity early in infection allowed testing in mice, in which pyrimethamine also reduced viral loads. Niclosamide (IC 50 = 0.28 μM), like dengue core inhibitor ST-148, affected structural components of the virion and inhibited early processes during infection. Vandetanib (IC 50 = 1.6 μM), like cellular autophagy inhibitor spautin-1, blocked viral exit from cells and could be shown to extend survival in vivo Thus, three FDA-approved compounds with promising utility for repurposing to treat dengue virus infections and their potential mechanisms were identified using computational tools and minimal phenotypic screening. IMPORTANCE No antiviral therapeutics are currently available for dengue virus infections. By computationally overlaying the three-dimensional (3D) chemical structures of compounds known to inhibit dengue virus over those of compounds known to be safe in humans, we identified three FDA-approved compounds that are attractive candidates for repurposing as antivirals. We identified targets for two previously identified antiviral compounds and revealed a previously unknown potential anti-dengue compound, vandetanib. This computational approach to analyze a highly curated library of structures has the benefits of speed and cost efficiency. It also leverages mechanistic work with query compounds used in biomedical research to provide strong hypotheses for the antiviral mechanisms of the safer hit compounds. This workflow to identify compounds with known safety profiles can be expanded to any biological activity for which a small-molecule query compound has been identified, potentially expediting the translation of basic research to clinical interventions., (Copyright © 2020 Moshiri et al.)

Biofilm formation is a common strategy utilized by bacterial pathogens to establish persistence in a host niche. Salmonella enterica serovar Typhi, the etiological agent of Typhoid fever, relies on biofilm formation in the gallbladder to chronically colonize asymptomatic carriers, allowing for transmission to uninfected individuals. S. enterica serovar Typhimurium utilizes biofilms to achieve persistence in human and animal hosts, an issue of both clinical and agricultural importance. Here, we identify a compound that selectively inhibits biofilm formation in both S. Typhi and S. Typhimurium serovars at early stages of biofilm development with an EC 50 of 21.0 and 7.4 μM, respectively. We find that this compound, T315, also reduces biofilm formation in Acinetobacter baumannii , a nosocomial and opportunistic pathogen with rising antibiotic resistance. T315 treatment in conjunction with sub-MIC dosing of ciprofloxacin further reduces S. enterica biofilm formation, demonstrating the potential of such combination therapies for therapeutic development. Through synthesis of two biotin-labeled T315 probes and subsequent pull-down and proteomics analysis, we identified a T315 binding target: WrbA, a flavin mononucleotide-dependent NADH:quinone oxidoreductase. Using a S. Typhimurium strain lacking WrbA we demonstrate that this factor contributes to endogenous S. enterica biofilm formation processes and is required for full T315 anti-biofilm activity. We suggest WrbA as a promising target for further development of anti-biofilm agents in Salmonella , with potential for use against additional bacterial pathogens. The development of anti-biofilm therapeutics will be essential to combat chronic carriage of Typhoid fever and thus accomplish a meaningful reduction of global disease burden.

Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.