In severe COVID-19 patients, excessive inflammation can lead to multiorgan dysfunction. Current anti-inflammatory treatments like glucocorticoids partially improve the outcomes, while immune systems are compromised. We have identified that SARS-CoV-2-infected obese mice were a good model of the cytokine storm seen in COVID-19. Here, we revealed that iguratimod (IGU), an approved agent for rheumatoid arthritis, improved survival by attenuating inflammation with minimal immune suppression. In this study, C57BL/6 mice were fed a high-fat diet (HFD) or a normal-fat diet (NFD) for ten weeks before being infected with a mouse-adapted SARS-CoV-2. IGU significantly improved survival rates and reduced lung inflammation in HFD-fed mice, with minimal impact on interferon-induced genes and viral load. Meanwhile, dexamethasone (DEX) did not improve survival, while it suppressed various immune reactions with different mechanisms to IGU. Interestingly, IGU-treated mice had fewer SARS-CoV-2 positive cells in the lung, although viral replication was comparable to the control mice. Neither IGU nor DEX inhibited the SARS-CoV-2 infection in Vero-E6 cells, unlike the antiviral agent, remdesivir. Of note, IGU was effective prophylactically and therapeutically in HFD mice, and showed beneficial effects in NFD-fed mice with a lethal dose exposure of SARS-CoV-2. We demonstrated that IGU could be a promising treatment for severe COVID-19, especially in obese patients, by fine-tuning inflammation without compromising antiviral immunity. This study supports the possibility of drug repositioning for IGU COVID-19 beyond autoimmune diseases.

Molnupiravir is a medication used to treat COVID-19 by introducing errors into the SARS-CoV-2 virus's genetic code, thereby preventing its replication. Previous studies, both in vitro and in vivo, have yielded conflicting results regarding its mutagenic potential. While most genotoxicity and mutagenicity tests for molnupiravir and its active form, β-d-N4-hydroxycytidine (NHC), were negative, a few in vitro tests showed positive results. Consequently, further investigation is necessary to evaluate various mutagenic endpoints of molnupiravir. In this study, acute toxicity was assessed by measuring the locomotive activity of Caenorhabditis elegans using a WMicroTracker to determine an appropriate dose range for the germline mutagenicity study. The C. elegans worms were treated with different concentrations of molnupiravir and NHC, along with vehicle controls and ethyl methanesulfonate (EMS) as a positive control. To assess germline mutagenicity, P0 worms from a single clone were exposed to selected concentrations of molnupiravir and NHC, as well as vehicle and positive controls, for 4h. Molnupiravir and NHC treatments had no significant effect on the locomotion of C. elegans worms after 1-, 2-, 3-, and 4-h exposures, compared to the vehicle control group. In contrast, EMS significantly reduced the worms' locomotive activity. Subsequent whole-genome sequencing of the F1 progeny from the treated P0 worms revealed that neither molnupiravir nor NHC increased the germline mutation frequency or altered mutation types, compared to the vehicle control. In contrast, EMS treatment significantly increased mutation frequency over the vehicle control, with a specific EMS mutational signature observed. These results suggest that molnupiravir and NHC are not mutagenic in C. elegans germ cells, aligning with previous findings that demonstrate the low mutagenicity of molnupiravir in clinical settings. Additionally, these findings highlight the utility of C. elegans as an alternative animal model for accelerating toxicity assessments and reducing the use of experimental animals.

The global impact of SARS-CoV-2 underscores the need for antiviral treatments beyond vaccines. This study targets Nsp14-MTase, a viral protein essential for replication. Initial quantitative high-throughput screening (qHTS) of ∼15,000 compounds from the selected NCATS in-house libraries identified 135 active hit molecules, reflecting a hit-rate of 1.04%. To enhance the search for promising antiviral agents, we expanded this screening campaign with two rounds of machine learning (ML)-based virtual screening of ∼130,000 compounds. The first iteration yielded 72 active compounds encompassing 27 chemotypes with an IC50 ranging from 1.45 μM to 33.27 μM, increasing the hit-rate 28-fold over the initial qHTS screen. Scaffold clustering of those hits revealed 27 chemotypes. The second iteration added 30 more hits (IC50: 2.18 μM-30.79 μM) across 12 new chemotypes. Initial structure-activity relationship (SAR) exploration around selected chemotypes identified NCGC00606183 (IC50: 0.41 μM) as the most potent hit. Hit-to-lead optimization using scaffold-centric exploration against the ultra large Enamine REAL Space (∼5.6 billion compounds) in HPC clusters identified 78 analogs, with 56 showing potent biochemical activity (IC50: 0.12 μM-18.23 μM) and cellular activity (0.27 μM-23.07 μM) in fully infectious SARS-CoV-2 live virus assays.

4'-Fluorouridine (4'-FU), despite demonstrating potent anti-SFTSV efficacy in vitro and in vivo, faces hindrances in its further development as a promising drug due to its weak chemical stability. Here, we report the discovery and development of VV261, a novel 4'-FU double prodrug with three isobutyryl groups on the ribose moiety and a nicotinoyloxymethyl group linked to the imide-nitrogen on the base moiety, exhibiting notable chemical stability and favorable pharmacokinetic properties. In SFTSV-infected mice, VV261 at 5 mg/kg/d for 7 days demonstrated complete protection against lethal SFTSV infection, prevented weight loss, and even a 2 day treatment significantly reduced both viral RNA copies and infectious virus titers in multiple organs, and notably alleviated splenic tissue lesions. After further preclinical evaluations, VV261, identified as a promising candidate drug for the treatment of SFTS, has entered Phase I clinical trials in China, the first such candidate to reach this stage for SFTS.

Therapeutic options against pathogenic human coronaviruses remain limited. In a recent clinical trial, we demonstrated the therapeutic efficacy of pegylated-IFN-λ in COVID-19 outpatients. However, the emergence of variants that have the potential to evade IFN-mediated antiviral responses raises concerns regarding the continued efficacy of this approach. In this work, we compared the sensitivity of SARS-CoV-2 variants and MERS-CoV to IFN-λ treatment in vitro and explored the potential of combination therapy with other FDA-authorized or approved antiviral agents. We observed that in contrast to the ancestral strain, all other SARS-CoV-2 lineages showed varying, but increased resistance to IFN-λ treatment, from a 5.7-fold increase in EC50 value for the P.1 strain to a 32.7-fold increase for the B.1.1.7 variant. We further show that combination treatment with remdesivir or nirmatrelvir enhanced the antiviral effect of IFN-λ against both SARS-CoV-2 and MERS-CoV. These findings justify the initiation of further in vivo testing that ultimately can help inform the development of more effective therapeutic guidelines against pathogenic coronaviruses.

The coronavirus disease 2019 (COVID-19) pandemic has exerted a catastrophic impact on public health. Meanwhile, the seasonal influenza outbreak overlaps with the current pandemic wave. There is still an urgent need to develop effective therapeutic agents for the treatment of co-infection of multiple respiratory viruses. This study aimed to investigate antiviral effects of EIDD-2801, an orally bioavailable ribonucleoside analog, and its potent therapeutic effects in co-infection of multiple respiratory viruses.

BALB/c mice and hamsters were infected with IFV or SARS-CoV-2, then were dosed orally with EIDD-2801 to measure the antiviral effects of EIDD-2801. Viral replication and mRNA transcription were evaluated by quantitative polymerase chain reaction (qPCR) and protein expression by Western Blot. Influenza viral titer was assessed using EID50 assay.

EIDD-2801 was found to be significantly effective against influenza A virus and influenza B virus. The antiviral activity against SARS-CoV-2 and further co-infection with influenza virus was also distinct. EIDD-2801 had potent antiviral effects against multiple respiratory viruses both in vitro and in vivo.

This study demonstrated that the small-molecule compound EIDD-2801, an orally available broad-spectrum antiviral agent, significantly inhibited the infection of influenza virus and SARS-CoV-2 and effectively protected animals from lethal influenza virus co-infection.

The COVID-19 pandemic highlighted the serious threat that coronaviruses have on public health. Because coronavirus continuously undergoes cross-species transmission, additional therapeutic agents and targets are urgently needed. Here, we show that a 3-deazapurine ribonucleoside, 3-Deazaguanosine (C3Guo, 2), has potent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unexpectedly, C3Guo (2) does not act as an inhibitor of RNA-dependent RNA polymerase (RdRp), which is the therapeutic target of two key nucleoside/nucleotide inhibitors approved for the treatment of COVID-19 (Remdesivir and Molnupiravir); instead, it seems to function by targeting the capping machinery of viral RNA. In hamsters infected with SARS-CoV-2, administration of 2 markedly reduced infectious viral titers, and prevented the development of COVID-19 pneumonia better than Molnupiravir. The potency of 2 against SARS-CoV-2 underscores its potential as an effective therapeutic agent for COVID-19 and future zoonotic coronavirus infections and raises the possibility of antiviral nucleoside analogs with alternative therapeutic targets to RdRp.

To evaluate the clinical applications and treatment outcomes using molnupiravir for the treatment of naturally occurring feline infectious peritonitis virus (FIPv).

Ninety-two client-owned cats with confirmed or presumptive FIP were retrospectively recruited from 35 veterinary practices, primarily in Australia, between February 2023 and March 2024. Cats were categorised based on treatment received: Cohort A: Molnupiravir treatment: monotherapy, maintenance or rescue therapy; Cohort B: Remdesivir and/or GS-441524 treatment. Seventy-eight cats were enrolled. Molnupiravir was administered orally for a median of 84 days, at a median dose of 13.3 mg/kg BID. Remission was defined as the resolution of FIP-related signs with (i) normalisation of serum globulin concentrations and A:G ratio (≥0.6), or (ii) sustained clinical remission for at least 100 days after stopping anti-viral therapy. Cure rate was defined as the percentage of cats achieving sustained remission, without requiring rescue therapy or experiencing a relapsed disease.

Molnupiravir monotherapy resulted in a cure rate of 72% (13/18) while molnupiravir maintenance therapy achieved a cure rate of 86% (25/29), and molnupiravir utilised as a rescue therapy achieved a cure rate of 100% (7/7). Treatment with remdesivir/GS-441524 resulted in a cure rate of 71% (17/24 cats). Survival analysis revealed no difference in outcomes between cats treated with molnupiravir monotherapy and those treated with remdesivir/GS-441524. Adverse events associated with molnupiravir therapy included neutropenia, and transient elevations in hepatic enzymes.

Molnupiravir demonstrated comparable survival outcomes to remdesivir/GS-441524 for treating FIP and serves as an accessible, effective option across various presentations, including ocular and neurologic forms.

The membrane (M) protein of betacoronaviruses is well conserved and has a key role in viral assembly1,2. Here we describe the identification of JNJ-9676, a small-molecule inhibitor targeting the coronavirus M protein. JNJ-9676 demonstrates in vitro nanomolar antiviral activity against SARS-CoV-2, SARS-CoV and sarbecovirus strains from bat and pangolin zoonotic origin. Using cryogenic electron microscopy (cryo-EM), we determined a binding pocket of JNJ-9676 formed by the transmembrane domains of the M protein dimer. Compound binding stabilized the M protein dimer in an altered conformational state between its long and short forms, preventing the release of infectious virus. In a pre-exposure Syrian golden hamster model, JNJ-9676 (25 mg per kg twice per day) showed excellent efficacy, illustrated by a significant reduction in viral load and infectious virus in the lung by 3.5 and 4 log10-transformed RNA copies and 50% tissue culture infective dose (TCID50) per mg lung, respectively. Histopathology scores at this dose were reduced to the baseline. In a post-exposure hamster model, JNJ-9676 was efficacious at 75 mg per kg twice per day even when added at 48 h after infection, when peak viral loads were observed. The M protein is an attractive antiviral target to block coronavirus replication, and JNJ-9676 represents an interesting chemical series towards identifying clinical candidates addressing the current and future coronavirus pandemics.

Coronaviruses (CoVs) are positive, single-stranded RNA viruses that can infect various animal species as well as humans. Particularly relevant for cats is the feline coronavirus (FCoV), which is widespread in cat populations worldwide. Infection with FCoV is usually asymptomatic. However, in multi-cat households, approximately 5-12% of FCoV-infected cats develop feline infectious peritonitis (FIP) due to mutations in the spike gene. FIP is an immune-mediated disease that previously was always fatal. These mutations result in a tropism shift from enterocytes to monocytes and macrophages. The associated change in the virulence of FCoV leads to the characteristic granulomatous vasculitis and perivasculitis observed in FIP. Recently, significant advancements have been made in understanding FIP. Studies show that antiviral drugs used in human medicine, such as the nucleoside analog GS-441524, are effective against FIP and can provide affected cats with a survival chance of up to 100%. Additionally, a novel FCoV variant, FCoV-23, has been identified in cats from Cyprus. According to newest research, this virus arose through a recombination between FCoV and the highly virulent pantropic canine coronavirus; it can be directly transmitted from cat to cat and lead to FIP. Furthermore, increasing evidence suggests that FIP is frequently associated with myocarditis. This article provides an overview of the current knowledge on FIP, including its pathology, clinical signs, effective treatment options, and preventive measures.

Molnupiravir, an orally administered drug for the treatment of mild-to-moderate COVID-19, is a prodrug of the ribonucleoside β-D-N4-hydroxycytidine (NHC). NHC incorporation in the SARS-CoV-2 RNA strand causes an accumulation of deleterious errors in the genome, resulting in reduced viral infectivity and replication. Exposure-response (E-R) analyses for viral RNA mutation rate and virologic outcomes were conducted using data from three phase 2/3 studies of molnupiravir (P006, MOVe-IN, and MOVe-OUT). Three dose levels (200, 400, and 800 mg every 12 hours [Q12H]) and placebo were evaluated. E-R datasets were generated for SARS-CoV-2 RNA mutation and longitudinal SARS-CoV-2 RNA viral load. E-R models were defined for RNA mutation rate and viral load change from baseline at days 5 and 10. The models supported plasma NHC AUC0-12 as the appropriate pharmacokinetic driver for assessing E-R relationships. The highest percentage of participants with > 20 low-frequency nucleotide substitutions (LNS) per 10,000 bases, a measure of likely meaningful drug effect, was predicted in the 800 mg Q12H treatment group. A strong drug effect on the reduction of viral load was observed on days 5 and 10. E-R relationships were best represented by an Emax structural model with reasonable consistency in the estimated AUC50s (~2.3-fold), across the models, of 10,260 and 4390 nM*hr. for day 5 viral load change from baseline and LNS error rate, respectively. These biomarker E-R curves support the choice of 800 mg Q12H as providing near-maximal drug effect, consistent with findings from the previously published molnupiravir E-R model of clinical outcomes.

The efficacy of molnupiravir for COVID-19 treatment remains controversial due to substantial heterogeneity in dosage and study settings across randomised controlled trials (RCTs).

We systematically searched Medline, PubMed, Embase, and the Cochrane Register of Clinical Trials up to February 3, 2025, for RCTs and real-world studies evaluating molnupiravir 800 mg twice daily as an early treatment for COVID-19 to prevent mortality and hospitalisation in high-risk adult outpatients. The primary outcomes were all-cause mortality and all-cause hospitalisation. Random-effects models were used to estimate pooled effect sizes.

Thirty-four studies were included, comprising 30,345 participants from 11 RCTs and 1,581,737 participants from 23 cohort studies. Molnupiravir reduced mortality risk by 55 %-65 % at 28 days (RCTs: risk ratio [RR] 0.35; 95 % CI 0.12-0.98, I2 0 %; cohort studies: RR 0.45; 95 % CI 0.27-0.73, I2 91 %). This benefit persisted at 3 months (RR 0.47; 95 % CI 0.23-0.95, I2 93 %) and 6 months (RR 0.62; 95 % CI 0.52-0.74, I2 0 %). The effectiveness in preventing 28-day hospitalisation varied by participants' mean age in both RCTs (35-45 vs. 45-57 years: RR 0.55; 95 % CI 0.36-0.84 vs. 1.06; 95 % CI 0.81-1.39, subgroup difference P = 0.01) and cohort studies (62-74 vs. 75-85 years: RR 0.88; 95 % CI 0.77-1.01 vs. 0.56; 95 % CI 0.44-0.72, subgroup difference P < 0.01).

Molnupiravir significantly reduces the risk of mortality. It also lowers the risk of hospitalisation in the oldest group (mean age ≥75 years) but not in younger groups (mean age 45-74 years).

Directed evolution in mammalian cells offers a powerful approach for advancing synthetic biology applications. However, existing mammalian-based directed evolution methods face substantial bottlenecks, including host genome interference, small library size and uncontrolled mutagenesis. Here we engineered an orthogonal alphaviral RNA replication system to evolve RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells. This system generates a large, continuously diversified library of replicative RNAs through replicase-limited mode of replication and inducible mutagenesis. Using REPLACE, we engineered fluorescent proteins and transcription factors. Notably, cells equipped with REPLACE can undergo Darwinian adaptation, allowing them to evolve in response to both cell-extrinsic and cell-intrinsic challenges. Collectively, this work establishes a powerful platform for advancing mammalian synthetic biology and cell engineering applications through directed evolution.

The global healthcare and economic challenges caused by the pandemic of COVID-19 reinforced the urgent demand for quick and effective therapeutic and preventative interventions. While vaccines served as the frontline of defense, antivirals emerged as adjunctive countermeasures, especially for people who developed infection, were immunocompromised, or were reluctant to be vaccinated. Beyond the serious complications of SARS-CoV-2 infection, the threats of long-COVID and the potential for zoonotic spillover continue to be significant health concerns that cannot be overlooked. Moreover, the incessant viral evolution, clinical safety issues, waning immune responses, and the emergence of drug-resistant variants pinpoint towards more severe viral threats in the future and call for broad-spectrum innovative therapies as a pre-pandemic preparedness measure. The present review provides a comprehensive up-to-date overview of the strategies utilized in the development of classical and next-generation vaccines against SARS-CoV-2, the clinical and experimental data obtained from clinical trials, while addressing safety risks that may arise. Besides vaccines, the review also covers recent breakthroughs in anti-SARS-CoV-2 drug discovery, emphasizing druggable viral and host targets, virus- and host-targeting antivirals, and highlighting mechanistically representative molecules that are either approved or are under clinical investigation. In conclusion, the integration of both vaccines and antiviral therapies, along with swift innovative strategies to address viral evolution and drug resistance is crucial to strengthen our preparedness against future viral outbreaks.

Molnupiravir is a prodrug of the antiviral ribonucleoside analogue N4-hydroxycytidine (NHC), for use in the treatment of coronavirus disease 2019 (COVID-19). However, it is generally considered that NHC-triphosphate is incorporated into the host genome to induce mutations. In our previous preliminary report, we proposed oxidative DNA damage by NHC via cytidine deaminase (CDA)-mediated ROS formation. In the present study, we investigated cell viability using the HL-60 human leukemia cell line and its H2O2-resistant clone, HP100 cells. The survival rate was significantly reduced in HL-60 cells treated with NHC, but not in HP100 cells. LC-MS analysis revealed that uridine formation occurred from CDA-treated NHC, suggesting that CDA metabolizes NHC to uridine and hydroxylamine. We clarified mechanisms of CDA-mediated reactive oxygen species (ROS) generation and DNA damage by NHC using isolated DNA. CDA-treated NHC induced DNA damage in the presence of Cu(II). The DNA damage was enhanced by NADH addition and piperidine treatment. CDA-treated NHC and Cu(II) caused piperidine-labile sites at thymine, cytosine, and guanine, and the DNA cleavage pattern was similar to that of hydroxylamine. Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H2O2 and Cu(I). An indicator of oxidative DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanosine formation by CDA-treated NHC, was lower under hypoxic conditions than under normal conditions. Therefore, hydroxylamine, possibly produced from NHC treated with CDA, could induce metal-dependent H2O2 generation during the redox reactions, suggesting that oxidative DNA damage induced by ROS plays an important role in molnupiravir-related cytotoxicity and mutagenicity.

Coronaviruses (CoVs) encode non-structural proteins (nsp's) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3'-5' exoribonuclease in non-structural protein 14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14-ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-nsp10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between wild type-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-nsp10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-nsp10 interface for viral inhibition and attenuation.IMPORTANCECoronavirus replication requires assembly of a replication transcription complex composed of nsp's, including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic cofactor nsp10, but the determinants and importance of the nsp14-nsp10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-nsp10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non-active-site targets for virus inhibition and attenuation.

Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.IMPORTANCEEmerging coronaviruses like SARS-CoV, SARS-CoV-2, and MERS-CoV cause a range of disease outcomes in humans from an asymptomatic, moderate, and severe respiratory disease that can progress to death but the factors causing these disparate outcomes remain unclear. Understanding host responses to mild and life-threatening infections provides insight into virus-host networks within and across organ systems that contribute to disease outcomes. We used multi-omics approaches to comprehensively define the host response to moderate and severe MERS-CoV infection. Severe respiratory disease was associated with dysregulation of the immune response. Key lung epithelial cell populations that are essential for lung function get infected and die. Mice lacking key immune cell populations experienced greater virus replication but decreased disease severity implicating the immune system in both protective and pathogenic roles in response to MERS-CoV. These data could be utilized to design new therapeutic strategies targeting specific pathways that contribute to severe disease.

The orally administered antiviral drug Lagevrio or molnupiravir (MOV) and the combination antiviral drug nirmatrelvir/ritonavir or Paxlovid (PAX) have been shown to reduce the likelihood of hospitalization and death for high-risk patients with COVID-19. Clinical studies, including those comparing PAX and MOV, were reviewed; both drugs are effective in reducing morbidity and mortality in COVID patients, although PAX generally appears to be more efficacious. Both drugs received Emergency Use Authorization in the United States for mild to moderate COVID-19 infection, while only PAX has subsequently been given full FDA approval. The principal disadvantage of PAX is that it interacts with many commonly used drugs, while MOV does not. The purpose of this review is to summarize current information and knowledge about these two drugs. The two drugs have completely different mechanisms of action. PAX inhibits viral replication while MOV induces viral replication errors that are expected to lead to viral inactivation. There is, however, the potential that MOV also could mutate host DNA and cause the virus to mutate into variants with new features. The package insert for MOV states that patients should be notified of relevant toxicity issues before administration. Sensitive mutation detection/analysis studies, such as error corrected Next Generation Sequencing (ecNGS) or HPRT mutation detection assays, in MOV-treated patients are needed to establish the safety of MOV.

Currently, the trials found that the clinical efficacy of molnupiravir is lower than ritonavir-boosted nirmatrelvir. An explanation for these different efficacies in clinical treatments is still limited. The analysis method was developed and validated to simultaneously quantify nirmatrelvir, ritonavir, and beta-D-N4-hydroxycytidine (NHC) in human plasma and bronchoalveolar lavage fluid (BALF) by electrospray ionization mass spectrometry. Our method was validated over a linear range of 30-10000 ng/mL for both matrices, with precision and accuracy within 15 % across four concentrations. Recovery rates for both analytes from plasma and BALF were between 90.7-102.2 % and 90.5-107.7 %, respectively. The analytical method was then applied to monitor therapeutic drug concentrations in 59 plasma samples from 23 patients receiving ritonavir-boosted nirmatrelvir or molnupiravir. By setting target plasma concentrations of 292 ng/mL for nirmatrelvir and 1205 ng/mL for NHC, based on in vitro antiviral 90 % virus inhibitory concentrations (EC90), the drug's molecular weight and its binding to human plasma proteins, we observed that ritonavir-boosted nirmatrelvir had substantially greater rates of achieving target plasma concentrations. Additionally, we monitored epithelial lining fluid in 4 BALF samples from 4 patients and observed that NHC exhibited higher permeability in lung tissue (approximately 20 % higher than nirmatrelvir). However, subtherapeutic antiviral concentrations of NHC were also present in epithelial lining fluid. These findings highlight the importance of considering these factors in determining the efficacy of these drugs in treating coronavirus disease 2019 (COVID-19).

The impact of remdesivir on SARS-CoV-2 diversity and evolution in vivo has remained unclear. In this single-center, retrospective cohort study, we assessed SARS-CoV-2 diversification and diversity over time in a cohort of hospitalized patients who did or did not receive remdesivir. Whole-genome sequencing was performed on 98 paired specimens collected from 49 patients before and after remdesivir administration. The genetic divergence between paired specimens was not significantly different in this cohort compared with that in a control group of patients who did not receive the drug. However, when we focused on minority variants, several positions showed preferential diversification after remdesivir treatment, some of which were associated with specific variants of concern. Most notably, remdesivir administration resulted in strong selection for a nonsynonymous mutation in nsp12, G671S, previously associated with enhanced viral fitness. This same mutation was found to be enriched in a second cohort of 143 inpatients with specimens collected after remdesivir administration compared with controls. Only one other mutation previously implicated in remdesivir resistance (nsp12:V792I) was found to be preferentially selected for after remdesivir administration. These data suggest that SARS-CoV-2 variants with enhanced replicative fitness may be selected for in the presence of antiviral therapy as an indirect means to overcome this selective pressure.

A class of tetrahydropyrazino[2,1-a:5,4-a']diisoquinoline derivatives were synthesized under environmentally friendly conditions using water as the solvent. The 3-D structures of some synthesized compounds were determined by X-ray diffraction. Since naturally occurring isoquinoline alkaloids have significant antiviral activities against a wide range of viruses, including coronaviruses, the synthesized compounds were assayed for their inhibitory activities against SARS-CoV-2. Our results showed that the active compounds 50 and 96 blocked the delta SARS-CoV-2 entry into VeroE6 cells to display EC50 of 26.5 ± 6.9 and 17.0 ± 3.7 μM, respectively, by inhibiting the interaction between SARS-CoV-2 Spike's receptor binding domain (RBD) and human receptor angiotensin-converting enzyme 2 (ACE2), and CC50 greater than 100 μM. This study provides a green synthesis method of tetrahydropyrazinodiisoquinoline for antiviral or other applications.

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral RNA. We identified potent lipophilic small interfering RNA (siRNA) conjugates targeting highly conserved regions of SARS-CoV-2 outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single siRNA approach. Viral resistance to single siRNA treatment occurred due to emergence of point mutations at critical positions required for siRNA-mediated target binding and cleavage, which led to a loss of siRNA efficacy. With a two-siRNA combination, emergence of mutations within the siRNA binding site was abolished. When delivered intranasally, two-siRNA combination protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach with high resistance barrier to counteract SARS-CoV-2 emergent variants and complement vaccination. Most importantly, given that the siRNAs can be rapidly developed from a new pathogen sequence, this strategy has implications as a new type of preventive medicine that may protect against future coronavirus pandemics.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that has re-emerged to cause large outbreaks of human infections worldwide. There are currently no approved antivirals for treatment of CHIKV infection. Recently, we reported that the ribonucleoside analog 4'-fluorouridine (4'-FlU) is a highly potent inhibitor of CHIKV replication, and targets the viral nsP4 RNA dependent RNA polymerase. In mouse models, oral therapy with 4'-FlU diminished viral tissue burdens and virus-induced disease signs. To provide critical evidence for the potential of 4'-FlU as a CHIKV antiviral, here we selected for CHIKV variants with decreased 4'-FlU sensitivity, identifying two pairs of mutations in nsP2 and nsP4. The nsP4 mutations Q192L and C483Y were predominantly responsible for reduced sensitivity. These variants were still inhibited by higher concentrations of 4'-FlU, and the mutations did not change nsP4 fidelity or provide a virus fitness advantage in vitro or in vivo. Pathogenesis studies in mice showed that the nsP4-C483Y variant caused similar disease and viral tissue burden as WT CHIKV, while the nsP4-Q192L variant was strongly attenuated. Together these results support the potential of 4'-FlU to be an important antiviral against CHIKV.

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.

RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4'-Fluorouridine (4'-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4'-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4'-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4'-FlU-resistant mutant influenza A virus was re-sensitized towards 4'-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4'-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4'-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy.

The COVID-19 pandemic, driven by the rapid evolution of the SARS-CoV-2 virus, presents ongoing challenges to global public health. SARS-CoV-2 is characterized by rapidly evolving mutations, especially in (but not limited to) the spike protein, complicating predictions about its evolutionary trajectory. These mutations have significantly affected transmissibility, immune evasion, and vaccine efficacy, leading to multiple pandemic waves with over half a billion cases and seven million deaths globally. Despite several strategies, from rapid vaccine development and administration to the design and availability of antivirals, including monoclonal antibodies, already having been employed, the persistent circulation of the virus and the emergence of new variants continue to result in high case numbers and fatalities. In the past four years, immense research efforts have contributed much to our understanding of the viral pathogenesis mechanism, the COVID-19 syndrome, and the host-microbe interactions, leading to the development of effective vaccines, diagnostic tools, and treatments. The focus of this review is to provide a comprehensive analysis of the functional impact of mutations on diagnosis, treatments, and vaccine effectiveness. We further discuss vaccine safety in pregnancy and the implications of hybrid immunity on long-term protection against infection, as well as the latest developments on a pan-coronavirus vaccine and nasal formulations, emphasizing the need for continued surveillance, research, and adaptive public health strategies in response to the ongoing SARS-CoV-2 evolution race.

We investigated the mutation profiles of severe acute respiratory syndrome coronavirus 2 in samples collected from a molnupiravir and nirmatrelvir/ritonavir combination therapy in macaques. We found that molnupiravir induced several nirmatrelvir resistance mutations at low abundance that were not further selected in combination therapy. Coadministration of nirmatrelvir/ritonavir lowered the magnitude of the mutagenetic effect of molnupiravir.

Drug repurposing can serve an important role in rapidly discovering medicament options for emerging microbial pandemics. In this study, a pragmatic approach is demonstrated for screening and testing drug combinations as potential broad-spectrum therapies against SARS-CoV-2 and other betacoronaviruses. Rapid cell-based phenotypic small molecule screens were executed using related common-cold-causing HCoV-OC43 betacoronavirus to identify replication inhibitors from a library of drugs approved by regulatory agencies for other indications. Given the best inhibitors, an expedient checkerboard strategy then served to identify synergistic drug combinations. These combinations were then validated using more challenging assays involving SARS-CoV-2 and variants. Promising drug combinations against multiple viral variants were discovered and involved Tilorone with Nelfinavir or Molnupiravir.

The SARS-CoV-2 pandemic has significantly challenged global public health, highlighting the need for effective therapeutic options. This study focuses on the papain-like protease (PLpro) of SARS-CoV-2, which is a critical enzyme for viral polyprotein processing, maturation, and immune evasion. We employed a combined approach that began with computational models in a virtual screening campaign, prioritizing compounds from our in-house chemical library against PLpro. Out of 81 virtual hits evaluated through enzymatic and biophysical assays, we identified a modest inhibitor featuring a naphthyridine core with an IC50 of 73.61 μM and a K i of 22 μM. Expanding our exploration, we synthesized and assessed 30 naphthyridine analogues, three of which emerged as promising noncovalent, nonpeptidomimetic inhibitors with IC50 values between 15.06 and 51.81 μM. Furthermore, in vitro ADMET assays revealed these compounds to possess moderate aqueous solubility, low cytotoxicity, and high microsomal stability, making them excellent candidates for further development targeting SARS-CoV-2 PLpro.