Chikungunya virus (CHIKV) and other alphaviruses in the Togaviridae family are positive-sense RNA viruses and major human pathogens, causing millions of infections worldwide. In humans, alphaviruses such as CHIKV, Mayaro and Ross River viruses typically cause arthritogenic disease characterized by debilitating arthralgia, joint inflammation, fever, and rash. Although a vaccine was recently approved for use against CHIKV, no vaccines are licensed against other alphaviruses. No antiviral treatments are available to prevent or treat infections by any alphavirus. To address this unmet need, we used a CHIKV nanoluciferase reporter virus to develop a high-throughput screening assay for novel small-molecule inhibitors. From this campaign, we identified several unique inhibitors of CHIKV replication. Mechanistic characterization of two inhibitors revealed that both target the nsP4 RNA-dependent RNA polymerase, while susceptibility profiling pinpointed unique nsP4 mutations that specifically confer resistance. In silico docking analyses indicated potential binding poses of the inhibitors near the polymerase active site. Collectively, these results define multiple chemotypes for further development and highlight novel molecular targets within nsP4 for CHIKV inhibition. IMPORTANCE:Chikungunya virus is a mosquito-borne pathogen that has caused millions of human infections worldwide, producing severe fever, rash, and long-lasting joint pain that can persist for months. Related viruses such as Mayaro and Ross River viruses also cause debilitating disease, yet no antiviral drugs are available to treat any infection caused by this family of viruses. In this study, we developed a high-throughput assay that allowed us to rapidly identify compounds capable of blocking chikungunya virus replication. We discovered new hit compounds that inhibit virus growth. In addition, we determined that two of the most promising hit candidates target the viral nsP4 polymerase. By identifying these novel inhibitors and characterizing both their mechanisms of action and resistance profiles, we have established the groundwork for future efforts to develop much needed therapies against chikungunya virus and related pathogens.

Urban green spaces provide many benefits, including to human wellbeing, ecosystem services, and urban wildlife. Thus, there are many reasons to green up urban spaces, especially by using native species. Furthermore, urban green spaces are suited to enhancing biodiversity without negatively impacting food or fiber production. Municipalities and private landowners invest substantially in landscaping and its maintenance. However, much of that outlay supports non-native plants that may be less adapted to local conditions such as rainfall patterns and temperature ranges, thus having greater resource requirements, as well as being less supportive of native wildlife and possessing a greater potential of becoming invasive. Here, we explore ways to increase the use of native plant species in urban settings to reduce the need for watering or chemical application, enhance the support of native species, and reduce the risk of invasion to urban and ex-urban habitats. We identify three main impediments: the perception of native species as less aesthetically pleasing, the availability of native plants in the nursery industry, and the willingness of policymakers to take supportive measures. We propose methods to address all three, providing successful examples from a number of US localities and a case study that demonstrates what drivers might exist and what actions remain to be taken.

Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global vaccination campaigns. MeV belongs to the mononegavirus order of viral pathogens that store their genetic information in non-segmented negative polarity RNA genomes. Genome replication and viral gene expression are carried out by a virus-encoded RNA-dependent RNA polymerase (RdRP) complex that has no immediate host cell analog. To better understand the organization and regulation of the viral RdRP and mechanistically characterize antiviral candidates, biochemical RdRP assays have been developed that employ purified recombinant polymerase complexes and synthetic RNA templates to monitor the initiation of RNA synthesis and RNA elongation in vitro. In this article, we will discuss strategies for the efficient expression and preparation of mononegavirus polymerase complexes, provide detailed protocols for the execution and optimization of RdRP assays, evaluate alternative options for the choice of template and detection system, and describe the application of the assay for the characterization of inhibitor candidates. Although MeV RdRP assays are the focus of this article, the general strategies and experimental approaches are readily transferable to related viruses in the mononegavirus order.

Small-molecule antivirals can be used as chemical probes to stabilize transitory conformational stages of viral target proteins, facilitating structural analyses. Here, we evaluate allosteric pneumo- and paramyxovirus polymerase inhibitors that have the potential to serve as chemical probes and aid the structural characterization of short-lived intermediate conformations of the polymerase complex. Of multiple inhibitor classes evaluated, we discuss in-depth distinct scaffolds that were selected based on well-understood structure-activity relationships, insight into resistance profiles, biochemical characterization of the mechanism of action, and photoaffinity-based target mapping. Each class is thought to block structural rearrangements of polymerase domains albeit target sites and docking poses are distinct. This review highlights validated druggable targets in the paramyxo- and pneumovirus polymerase proteins and discusses discrete structural stages of the polymerase complexes required for bioactivity.

FMRI data are noisy, complicated to acquire, and typically go through many steps of processing before they are used in a study or clinical practice. Being able to visualize and understand the data from the start through the completion of processing, while being confident that each intermediate step was successful, is challenging. AFNI's afni_proc.py is a tool to create and run a processing pipeline for FMRI data. With its flexible features, afni_proc.py allows users to both control and evaluate their processing at a detailed level. It has been designed to keep users informed about all processing steps: it does not just process the data, but first outputs a fully commented processing script that the users can read, query, interpret and refer back to. Having this full provenance is important for being able to understand each step of processing; it also promotes transparency and reproducibility by keeping the record of individual-level processing and modeling specifics in a single, shareable place. Additionally, afni_proc.py creates pipelines that contain several automatic self-checks for potential problems during runtime. The output directory contains a dictionary of relevant quantities that can be programmatically queried for potential issues and a systematic, interactive quality control (QC) HTML. All of these features help users evaluate and understand their data and processing in detail. We describe these and other aspects of afni_proc.py here using a set of task-based and resting state FMRI example commands.

Quality control (QC) assessment is a vital part of FMRI processing and analysis, and a typically under-discussed aspect of reproducibility. This includes checking datasets at their very earliest stages (acquisition and conversion) through their processing steps (e.g., alignment and motion correction) to regression modeling (correct stimuli, no collinearity, valid fits, enough degrees of freedom, etc.) for each subject. There are a wide variety of features to verify throughout any single subject processing pipeline, both quantitatively and qualitatively. We present several FMRI preprocessing QC features available in the AFNI toolbox, many of which are automatically generated by the pipeline-creation tool, afni_proc.py. These items include: a modular HTML document that covers full single subject processing from the raw data through statistical modeling; several review scripts in the results directory of processed data; and command line tools for identifying subjects with one or more quantitative properties across a group (such as triaging warnings, making exclusion criteria or creating informational tables). The HTML itself contains several buttons that efficiently facilitate interactive investigations into the data, when deeper checks are needed beyond the systematic images. The pages are linkable, so that users can evaluate individual items across a group, for increased sensitivity to differences (e.g., in alignment or regression modeling images). Finally, the QC document contains rating buttons for each "QC block", as well as comment fields for each, to facilitate both saving and sharing the evaluations. This increases the specificity of QC, as well as its shareability, as these files can be shared with others and potentially uploaded into repositories, promoting transparency and open science. We describe the features and applications of these QC tools for FMRI.

Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease.

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Hormones can induce trait development in one species yet have no effect on the same trait in a closely related species, but the mechanisms underlying these differences are unclear. Here, we compare two closely related lizard species to explore the cellular mechanisms associated with the evolutionary loss of hormonally mediated ventral coloration. The eastern fence lizard (Sceloporus undulatus) has sexually dimorphic blue and black ventral coloration that develops when maturational increases in androgens induce melanin synthesis in males. The closely related striped plateau lizard (Sceloporus virgatus) has sexually monomorphic white ventral skin that does not produce melanin in response to the same signal. We used immunohistochemistry to localize the androgen receptor (AR) in the skin of both species and to test whether the loss of ventral coloration in S. virgatus corresponds to the loss of AR in the skin. We found that the ventral skin of S. virgatus displays little or no AR staining in the pigment cell layer, potentially explaining the loss of androgen sensitivity in this tissue, relative to the robust AR staining in the same layer of S. undulatus. Based on the location of three markers for melanophores (microphthalmia-associated transcription factor, dopachrome tautomerase, and tyrosinase), AR appears to be present in melanophores in S. undulatus. However, we could not detect these melanophore markers in the skin of S. virgatus. Therefore, the evolutionary loss of ventral coloration may have occurred via the loss of the AR-producing melanophore in mature ventral skin, preventing the development of a male-typical trait and sexual dimorphism in this tissue.

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread in the population. We recently reported the production of bovine colostrum-derived antibodies that can neutralize the virus. These have been formulated into a nasal spray. The immunoglobulin preparation is capable of blocking interaction of the trimeric spike protein (Tri S) of SARS-CoV-2 with the cellular receptor angiotensin-converting enzyme 2 (ACE2), entry of a pseudovirus carrying the Tri S into ACE2 over-expressing human embryonic kidney (HEK) cells, and entry of the virus into live Vero E6 cells. Using an ELISA assay, we demonstrate here that this holds true for different SARS-CoV-2 variants of concern. Using the ferret transmission model, we show that the nasal spray formulation of anti-SARS-CoV-2 immunoglobulins efficiently blocks transmission of SARS-CoV-2 from infected to uninfected ferrets. The results indicate that the use of the nasal spray in humans can add an effective additional layer of protection against the virus, and might be applicable for other viruses of the upper respiratory tract.

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission. In this study, the authors report the small molecule inhibitor EDP-235 as a potent inhibitor of SARS-CoV-2 and show that it is effective against a range of variants and other coronaviruses and that it suppresses virus replication, reduces lung damage, and prevents transmission in small animal models.

Abstract Background Remdesivir (RDV, Veklury) is the first FDA-approved direct-acting antiviral treatment for COVID-19. While RDV requires IV administration, obeldesivir (ODV, GS-5245) is an oral prodrug of GS-441524, the parent nucleoside of RDV, designed for effective oral delivery. ODV is being tested in two Phase 3 clinical studies for outpatient treatment of COVID-19. Methods In vitro ODV activity against SARS-CoV-2 was assessed in A549-hACE2 cells. In vivo therapeutic efficacy of oral ODV was evaluated in mouse, ferret, and African Green Monkey (AGM) SARS-CoV-2 models. GS-441524 pharmacokinetics (PK) following ODV oral administration was assessed in animals and in a Phase 1 study in humans. Results ODV is a potent inhibitor of SARS-CoV-2 in vitro (EC50 = 1.9 µM) and showed 2- to 7-fold increased GS-441524 oral bioavailability in multiple animal models compared to parent GS-441524. Oral ODV reduced infectious lung viral loads by 2.8 log10 plaque forming units (pfu)/g tissue and ameliorated viral pathophysiological effects in a pathogenic mouse model of SARS-CoV-2 when dosed at 10 mg/kg twice daily beginning 12 hours post infection (hpi). In a SARS-CoV-2 asymptomatic upper airway infection ferret model, oral ODV dosed once daily at 20 mg/kg starting 12 hpi reduced nasal infectious viral titers by >3.3 log10 pfu/mL. Viral transmission among co-housed ferrets was also prevented by ODV. Once daily oral administration of 60 mg/kg ODV in SARS-CoV-2 infected AGMs initiated 8 hpi significantly reduced viral loads in bronchoalveolar lavage and multiple respiratory tissues. Efficacious oral doses of ODV in the mouse, ferret and AGM models were achieved at estimated plasma GS-441524 exposures (AUC0-24h) of 36, 98 and 111 µM·h, respectively. Based on PK data from a Phase 1 dose escalation study in healthy volunteers, an ODV dose of 350 mg twice daily (BID) is expected to achieve plasma GS-441524 exposures (77-103 µM·h) in the efficacious range derived from these animal models. Conclusion ODV is a promising investigational oral COVID-19 treatment based on its preclinical in vitro and in vivo SARS-CoV-2 efficacy, its oral bioavailability in preclinical species, and its oral PK and safety profile in a Phase 1 trial. Collectively, these data support the 350 mg BID dose for the ongoing Phase 3 clinical trials. Disclosures Jared Pitts, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Darius Babusis, BA, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Rita Humeniuk, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Nicholas C. Riola, BS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Joy Feng, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Venice Du Pont, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Olena Anoshchenko, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Meghan Vermillion, DVM, PhD, Gilead Sciences, Inc.: Employee Mazin Abdelghany, MD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Robert H. Hyland, DPhil, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Sandhya Girish, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Tomas Cihlar, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Joe Llewellyn, PharmD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Helen Winter, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Roy Bannister, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Raju Subramanian, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Richard L. Mackman, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds John P. Bilello, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds

The development of Canopeo as a close-range remote sensor for measuring ground cover fraction (GCF) offered farmers and scientists an accurate, simple, low-cost tool for monitoring health and development throughout the plant lifecycle. However, a significant obstacle to image-based monitoring of plant performance is the difficulty of object distinction between plant and background sharing similar colors. The overall goal of this research was to test Canopeo's sensitivity for detecting GCF when plants were imaged on different colored backgrounds in a greenhouse environment. We therefore tested Canopeo's ability to detect plant versus non-plant pixels in each image (resolution 72 × 72) using ten complex flat backgrounds. Multicolored backgrounds resembling flooring which may be found in a greenhouse setting (concrete, brick painted white, natural wood plank, and wood painted white with scuffs) resulted in least amount of deviation (<0.46) when analyzed with the control (flat black) background. Canopeo overestimated GCF of Viburnum sp. and E. pinnatum cv. on a green background which resulted in the greatest amount of deviation (>20). Canopeo demonstrated greatest underestimation GCF for Viburnum sp. and E. pinnatum cv. on a red background. When GCF of the green background was omitted, the r2 value of 0.75, or goodness of fit, suggest approximately 75% of the sampling variation can be described by the background color and not experimental error. Canopeo is an easily accessible tool for researchers and farmers to monitor plant growth and development on a diversity of backgrounds beyond soil and field settings.

Summary After years of the COVID-19 pandemic, over 40 million children worldwide are at risk of measles due to delayed vaccination 1 and temporary SARS-CoV-2 viral dominance 2 . Acute measles has a case-fatality rate of ∼1%, but most morbidity and mortality arise post-measles due to destruction of pre-existing immune memory by lymphotropic measles virus (MeV) 3,4 , a paramyxovirus of the Morbillivirus genus. MeV-induced immune amnesia is not mitigated by post-exposure vaccination and the impact of unrelated respiratory virus disease history on measles severity has not been defined. We used a lethal canine distemper virus (CDV)-ferret model as surrogate for human morbillivirus disease 5 and employed the orally efficacious broad-spectrum paramyxovirus polymerase inhibitor GHP-88309 6 to establish measles treatment paradigms. Applying a receptor tropism-intact recombinant CDV with low lethality, we provide in vivo confirmation of the morbillivirus immune amnesia hypothesis and reveal an 8-day advantage of antiviral treatment versus therapeutic vaccination in preserving immune memory. Infection of ferrets with non-lethal influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks prior to CDV caused exacerbated CDV disease that rapidly advanced to fatal hemorrhagic pneumonia associated with lung onslaught by commensal bacteria. RNAseq of BAL samples and lung tissue identified CDV-induced expression of trefoil factor (TFF) peptides, which was absent in animals pre-infected with IAV, thus highlighting that immune priming by unrelated respiratory viruses influences morbillivirus infection outcome. Non-invasive pulmonary ferret MRI revealed that severe outcomes of consecutive IAV/CDV infections were prevented by oral GHP-88309 treatment even when initiated after peak clinical signs of CDV. These findings validate the morbillivirus immune amnesia hypothesis, define treatment paradigms for measles, identify prior disease history as risk factor for exacerbated morbillivirus disease, and demonstrate that treating morbillivirus infection with direct-acting oral antivirals provides therapeutic benefit regardless of whether the time window to mitigate primary clinical signs of infection has closed.

Therapeutic options against SARS-CoV-2 are underutilized. Two oral drugs, molnupiravir and paxlovid (nirmatrelvir/ritonavir), have received emergency use authorization. Initial trials suggested greater efficacy of paxlovid, but recent studies indicated comparable potency in older adults. Here, we compare both drugs in two animal models; the Roborovski dwarf hamster model for severe COVID-19-like lung infection and the ferret SARS-CoV-2 transmission model. Dwarf hamsters treated with either drug survive VOC omicron infection with equivalent lung titer reduction. Viral RNA copies in the upper respiratory tract of female ferrets receiving 1.25 mg/kg molnupiravir twice-daily are not significantly reduced, but infectious titers are lowered by >2 log orders and direct-contact transmission is stopped. Female ferrets dosed with 20 or 100 mg/kg nirmatrelvir/ritonavir twice-daily show 1–2 log order reduction of viral RNA copies and infectious titers, which correlates with low nirmatrelvir exposure in nasal turbinates. Virus replication resurges towards nirmatrelvir/ritonavir treatment end and virus transmits efficiently (20 mg/kg group) or partially (100 mg/kg group). Prophylactic treatment with 20 mg/kg nirmatrelvir/ritonavir does not prevent spread from infected ferrets, but prophylactic 5 mg/kg molnupiravir or 100 mg/kg nirmatrelvir/ritonavir block productive transmission. These data confirm reports of similar efficacy in older adults and inform on possible epidemiologic benefit of antiviral treatment.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles.

AbstractPre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4’-fluorouridine (4’-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4’-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09).In vitroviral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4’-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4’-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4’-FlU.Author SummaryReduced sensitivity to FDA-approved influenza drugs is a major obstacle to effective antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate drug, 4’-FlU, against seasonal, pandemic, and highly pathogenic avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4’-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the drug. Testing of resistant variants against two chemically unrelated nucleoside analog inhibitors of influenza virus, conditionally approved favipiravir and the broad-spectrum SARS-CoV-2 drug molnupiravir, revealed cross-resistance of one cluster with favipiravir, whereas no viral escape from molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4’-FlU. These results demonstrate that partial viral escape from 4’-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.