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Path to another drug against COVID-19 The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. Owen et al . report the discovery and characterization of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protects against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armory against future viral threats. —VV

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50 10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shownin vitroactivity against Ebola virus and demonstrated activity against SARS-CoV-2in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activityin vitroand efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virusin vitroin HeLa cells and of mouse adapted Ebola virus in mousein vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50values of 180 nM and IC50198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kdvalues of 339 nM and 647 nM, respectively. Human Cmaxfor pyronaridine and quinacrine is greater than the IC50hence justifyingin vivoevaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

Background Inhaled budesonide benefits patients with COVID-19. ProLung™-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung™-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it's effect on SARS-CoV-2 replication is unknown. Objective To determine the efficacy of ProLung™-budesonide against SARS-CoV-2-infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation. Methods SARS-CoV-2-infected Vero 76 cells were treated with ProLung™-budesonide ([0.03-100 µg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung™-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung™ carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL). Results ProLung™-budesonide showed significant inhibition of viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value >24. Weekly ProLung™-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung™-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challenge. Conclusions ProLung™-budesonide significantly inhibited viral replication in SARS-CoV-2-infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).

Traditional bolus vaccine administration leads to rapid clearance of vaccine from lymphoid tissue. However, there is increasing evidence suggesting that the kinetics of antigen delivery can impact immune responses to vaccines, particularly when tailored to mimic natural infections. Here, we present the specific enhancements sustained release immunization confers to seasonal influenza vaccine, including the magnitude, durability, and breadth of humoral responses. To achieve sustained vaccine delivery kinetics, we have developed a microneedle array patch (MIMIX), with silk fibroin-formulated vaccine tips designed to embed in the dermis after a short application to the skin and release antigen over 1-2 weeks, mimicking the time course of a natural influenza infection. In a preclinical murine model, a single influenza vaccine administration via MIMIX led to faster seroconversion, response-equivalence to prime-boost bolus immunization, higher HAI titers against drifted influenza strains, and improved protective efficacy upon lethal influenza challenge when compared with intramuscular injection. These results highlight infection mimicry, achieved through sustained release silk microneedles, as a powerful approach to improve existing seasonal influenza vaccines, while also suggesting the broader potential of this platform technology to enable more efficacious next-generation vaccines and vaccine combinations.

Enterovirus D68 (EV-D68) is a non-polio enterovirus that affects the respiratory system and can cause serious complications, especially in children and older people with weakened immune systems. As an emerging virus, there are no current antiviral therapies or vaccines available. Our goal was to develop a mouse model of human EV-D68 infection that mimicked the disease observed in humans and could be used for evaluation of experimental therapeutics. This is the first report of a respiratory disease model for EV-D68 infection in mice. We adapted the virus by 30 serial passages in AG129 mice, which are deficient in IFN- α/β and –γ receptors. Despite a lack of weight loss or mortality in mice, lung function measured by plethysmography, showed an increase in enhanced pause (Penh) on days 6 and 7 post-infection. In addition, as virus adapted to mice, virus titer in the lungs increased 320-fold, and the pro-inflammatory cytokines MCP-1 and RANTES increased 15-fold and 2-fold in the lung, respectively. In addition, a time course of mouse-adapted EV-D68 infection was determined in lung, blood, liver, kidney, spleen, leg muscle, spinal cord and brain. Virus in the lung replicated rapidly after intranasal inoculation of adapted virus, 10(6) CCID(50)/mL by 4 hours and 10(8.3) CCID(50)/mL by 24 hours. Virus then spread to the blood and other tissues, including spinal cord and brain. This mouse model for EV-D68 infection includes enhanced pause (Penh) as an indicator of morbidity, and viremia, virus titers and proinflammatory cytokines in the lung, and lung histopathology as indicators of disease. Our mouse-adapted virus has a similar antiviral profile to the original isolate as well as another respiratory picornavirus, rhinovirus-14. This model will be valuable in evaluating experimental therapies in the future.

The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse- adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. ClinicalTrials.gov Identifier: NCT04756531 One-Sentence Summary PF-07321332 is disclosed as a novel, orally active, investigational small-molecule inhibitor of the SARS-CoV-2 main protease, which is being evaluated in clinical trials for the treatment of COVID-19.

BackgroundInhaled budesonide benefits patients with COVID-19. ProLung™-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung™-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it’s effect on SARS-CoV-2 replication is unknown.ObjectiveTo determine the efficacy of ProLung™-budesonide against SARS-CoV-2 infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation.MethodsSARS-CoV-2-infected Vero 76 cells were treated with ProLung™-budesonide ([0.03– 100 μg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung™-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung™-carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL).ResultsProLung™-budesonide showed significant inhibition on viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value > 24. Weekly ProLung™-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung™-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challengeConclusionsProLung™-budesonide significantly inhibited viral replication in SARS-CoV-2 infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.

As the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mpro compared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs., Graphical Abstract

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 μM range in vitro. Furthermore, Exebryl-1, a β-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 μM, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

As the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mprocompared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs.

The coronavirus main protease, Mpro, is a key protein in the virus life cycle and a major drug target. Based on crystal structures of SARSCoV2 Mpro complexed with peptidomimetic inhibitors, we recognized a binding characteristic shared with proline-containing inhibitors of hepatitis C virus protease. Initial tests showed that this subclass of HCV protease inhibitors indeed exhibited activity against Mpro. Postulating a benefit for a preorganized backbone conformation, we designed new ketoamide-based Mpro inhibitors based on central proline rings. One of the designed compounds, ML1000, inhibits Mpro with low-nanomolar affinity and suppresses SARSCoV2 viral replication in human cells at sub-micromolar concentrations. Our findings identify ML1000 as a promising new pre-organized scaffold for the development of anti-coronavirus drugs.

X-ray crystal structures of SARS-CoV-2 Mpro with dual inhibitors provide a new direction for the designing of COVID-19 antivirals., The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of Mpro in complex with calpain inhibitors II and XII and three analogs of GC-376. The structure of Mpro with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Enterovirus D68 (EV-D68) is unique among enteroviruses because of the ability to cause severe respiratory disease as well as neurological disease. We developed separate models of respiratory and neurological disease following EV-D68 infection in AG129 mice that respond to antiviral treatment with guanidine. In four-week-old mice infected intranasally, EV-D68 replicates to high titers in lung tissue increasing the proinflammatory cytokines MCP-1 and IL-6. The respiratory infection also produces an acute viremia. In 10-day-old mice infected intraperitoneally, EV-D68 causes a neurological disease with weight-loss, paralysis, and mortality. In our respiratory model, treatment with guanidine provides a two-log reduction in lung virus titers, reduces MCP-1 and IL-6, and prevents histological lesions in the lungs. Importantly, viremia is prevented by early treatment with guanidine. In our neurological model, guanidine treatment protects mice from weight-loss, paralysis, and mortality. These results demonstrate the utility of these models for evaluation of antiviral therapies for EV-D68 infection.

To identify potential therapeutic stop-gaps for SARS-CoV-2, we evaluated a library of 1,670 approved and reference compounds in an unbiased, cellular image-based screen for their ability to suppress the broad impacts of the SARS-CoV-2 virus on phenomic profiles of human renal cortical epithelial cells using deep learning. In our assay, remdesivir is the only antiviral tested with strong efficacy, neither chloroquine nor hydroxychloroquine have any beneficial effect in this human cell model, and a small number of compounds not currently being pursued clinically for SARS-CoV-2 have efficacy. We observed weak but beneficial class effects of β-blockers, mTOR/PI3K inhibitors and Vitamin D analogues and a mild amplification of the viral phenotype with β-agonists.

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F (P

The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.

Enterovirus D68 (EV-D68) causes outbreaks of respiratory illness, and there is increasing evidence that it causes outbreaks of acute flaccid myelitis (AFM). There are no licensed therapies to prevent or treat EV-D68 infection or AFM disease. We isolated a panel of EV-D68-reactive human monoclonal antibodies that recognize diverse antigenic variants from participants with prior infection. One potently neutralizing cross-reactive antibody, EV68-228, protected mice from respiratory and neurologic disease when given either before or after infection. Cryo-electron microscopy studies revealed that EV68-228 and another potently neutralizing antibody (EV68-159) bound around the fivefold or threefold axes of symmetry on virion particles, respectively. The structures suggest diverse mechanisms of action by these antibodies. The high potency and effectiveness observed in vivo suggest that antibodies are a mechanistic correlate of protection against AFM disease and are candidates for clinical use in humans with EV-D68 infection.

SARS-CoV-2 infection of host cells is driven by binding of the SARS-CoV-2 spike-(S)-protein to lung type II pneumocytes, followed by virus replication. Surfactant protein SP-D, member of the front-line immune defense of the lungs, binds glycosylated structures on invading pathogens such as viruses to induce their clearance from the lungs. The objective of this study is to measure the pulmonary SP-D levels in COVID-19 patients and demonstrate the activity of SP-D against SARS-CoV-2, opening the possibility of using SP-D as potential therapy for COVID-19 patients. Pulmonary SP-D concentrations were measured in bronchoalveolar lavage samples from patients with corona virus disease 2019 (COVID-19) by anti-SP-D ELISA. Binding assays were performed by ELISAs. Protein bridge and aggregation assays were performed by gel electrophoresis followed by silver staining and band densitometry. Viral replication was evaluated in vitro using epithelial Caco-2 cells. Results indicate that COVID-19 patients (n = 12) show decreased pulmonary levels of SP-D (median = 68.9 ng/mL) when compared to levels reported for healthy controls in literature. Binding assays demonstrate that SP-D binds the SARS-CoV-2 glycosylated spike-(S)-protein of different emerging clinical variants. Binding induces the formation of protein bridges, the critical step of viral aggregation to facilitate its clearance. SP-D inhibits SARS-CoV-2 replication in Caco-2 cells (EC90 = 3.7 μg/mL). Therefore, SP-D recognizes and binds to the spike-(S)-protein of SARS-CoV-2 in vitro, initiates the aggregation, and inhibits viral replication in cells. Combined with the low levels of SP-D observed in COVID-19 patients, these results suggest that SP-D is important in the immune response to SARS-CoV-2 and that rhSP-D supplementation has the potential to be a novel class of anti-viral that will target SARS-CoV-2 infection.

Graphical abstract, Highlights • An in-house library of (N)-methanocarba nucleosides was screened against Enteroviruses. • A hit against Enterovirus A71, adenosine derivative 17, was structurally expanded providing an initial SAR. • The rigid bicyclo[3.1.0]hexane substitution of ribose was needed for antiviral activity. • A narrow SAR was found, not resembling other EV-A71 antiviral nucleosides. • Several novel nucleosides, including a 4′-fluoromethyl analogue MRS7704 48, had selectivity index >10 at EV-A71., Various (North)-methanocarba adenosine derivatives, containing rigid bicyclo[3.1.0]hexane ribose substitution, were screened for activity against representative viruses, and inhibition was observed after treatment of Enterovirus A71 with a 2-chloro-N6-1-cyclopropyl-2-methylpropan-1-yl derivative (17). µM activity was also seen when testing 17 against other enteroviruses in the Picornaviridae family. Based on this hit, structural congeners of 17, containing other N6-alkyl groups and 5′ modifications, were synthesized and tested. The structure activity relationship is relatively narrow, with most modifications of the adenine or the methanocarba ring reducing or abolishing the inhibitory potency. 4′-Truncated 31 (MRS5474), 4′-fluoromethyl 48 (MRS7704) and 4′-chloromethyl 49 nucleosides displayed EC50 ~3–4 µM, and 31 and 48 achieved SI ≥10. However, methanocarba analogues of ribavirin and N6-benzyladenosine, shown previously to have anti-EV-A71 activity, were inactive. Thus, we identified methanocarba nucleosides as a new scaffold for enterovirus inhibition with a narrow structure activity relationship and no similarity to previously published anti-enteroviral nucleosides.

Enterovirus D68 (EV-D68) caused a large outbreak in the summer and fall of 2014 in the United States. It causes serious respiratory disease, but causation of associated paralysis is controversial, because the virus is not routinely identified in cerebrospinal fluid. To establish clinical correlates with human disease, we evaluated EV-D68 infection in non-lethal paralysis mouse models. Ten-day-old mice lacking interferon responses were injected intraperitoneally with the virus. Paralysis developed in hindlimbs. After six weeks of paralysis, the motor neurons were depleted due to viral infection. Hindlimb muscles were also infected and degenerating. Even at the earliest stage of paralysis, muscles were still infected and were degenerating, in addition to presence of virus in the spinal cord. To model natural respiratory infection, five-day-old mice were infected intranasally with EV-D68. Two of the four infected mice developed forelimb paralysis. The affected limbs had muscle disease, but no spinal cord infection was detected. The unique contributions of this study are that EV-D68 causes paralysis in mice, and that causation by muscle disease, with or without spinal cord disease, may help to resolve the controversy that the virus can cause paralysis, even if it cannot be identified in cerebrospinal fluid.

The emergence of pandemic influenza strains, particularly the reemergence of the swine-derived influenza A (H1N1) in 2009, is reaffirmation that influenza viruses are very adaptable and influenza remains as a significant global public health treat. As recommended by the World Health Organization (WHO), the use of adjuvants is an attractive approach to improve vaccine efficacy and allow dose-sparing during an influenza emergency. In this study, we utilized CaPtivate Pharmaceutical’s proprietary calcium phosphate nanoparticles (CaPNP) vaccine adjuvant and delivery platform to formulate an inactivated whole virus influenza A/CA/04/2009 (H1N1pdm) vaccine as a potential dose-sparing strategy. We evaluated the relative immunogenicity and the efficacy of the formulation in BALB/c mice following single intramuscularly administration of three different doses (0.3, 1, or 3 μg based on HA content) of the vaccine in comparison to non-adjuvanted or alum-adjuvant vaccines. We showed that, addition of CaPNP in vaccine elicited significantly higher hemagglutination inhibition (HAI), virus neutralization (VN), and IgG antibody titers, at all dose levels, relative to the non-adjuvanted vaccine. In addition, the vaccine containing CaPNP provided equal protection with 1/3rd of the antigen dose as compared to the non-adjuvanted or alum-adjuvanted vaccines. Our data provided support to earlier studies indicating that CaPNP is an attractive vaccine adjuvant and delivery system and should play an important role in the development of safe and efficacious dose-sparing vaccines. Our findings also warrant further investigation to validate CaPNP’s capacity as an alternative adjuvant to the ones currently licensed for influenza/pandemic influenza vaccination.

Received 11 November 2013; returned 21 January 2014; revised 19 March 2014; accepted 21 March 2014 Objectives: Emerging drug resistance to antiviral therapies is an increasing challenge for the treatment of influenza virus infections. One new antiviral compound, BTA938, a dimeric derivative of the viral neuraminidase inhibitor zanamivir, contains a 14-carbon linker bridging two zanamivir moieties. In these studies, we evaluated antiviral efficacy in cell cultures infected with influenza virus and in mouse models of lethal influenza using H1N1pdm09, H3N2 and oseltamivir-resistant (H275Y) viruses. Methods: In vitro activity was evaluated against 22 strains of influenza virus. Additionally, in vivo studies compared the efficacy of BTA938 or zanamivir after intranasal treatment. We also tested the hypothesis of a dual mode of action for BTA938 using scanning electron microscopy (SEM). Results: BTA938 inhibited the viruses at nanomolar concentrations in vitro with a median 50% effective concentration value of 0.5 nM. In mouse models, the dimer provided � 10-fold greater protection than zanamivir. The data also showed that a single low dose (3 mg/kg) protected 100% of mice from an otherwise lethal oseltamivirresistant (H275Y) influenzavirus infection. Remarkably, a single prophylactic treatment (10 mg/kg) administered 7 days before the challenge protected 70% of mice and when administered 1 or 3 days before the challenge it protected 90% of mice. Additionally, SEM provides evidence that the increased antiviral potency may be mediated by an enhanced aggregation of virus on the cell surface. Conclusions: In vitro and in vivo experiments showed the high antiviral activity of BTA938 for the treatment of influenza virus infections. Moreover, we demonstrated that a single dose of BTA938 is sufficient for prophylactic and therapeutic protection in mouse models.

Few anti-influenza drugs are licensed in the United States for the prevention and therapy of influenza A and B virus infections. This shortage, coupled with continuously emerging drug resistance, as detected through a global surveillance network, seriously limits our anti-influenza armamentarium. Combination therapy appears to offer several advantages over traditional monotherapy in not only delaying development of resistance but also potentially enhancing single antiviral activity. In the present study, we evaluated the antiviral drug susceptibilities of fourteen pandemic influenza A (H1N1) virus isolates in MDCK cells. In addition, we evaluated favipiravir (T-705), an investigational drug with a broad antiviral spectrum and a unique mode of action, alone and in dual combination with the neuraminidase inhibitors (NAIs) oseltamivir, peramivir, or zanamivir, against oseltamivir-sensitive pandemic influenza A/California/07/2009 (H1N1) and oseltamivir-resistant A/Hong Kong/2369/2009 (H1N1) virus. Mean inhibitory values showed that the tested virus isolates remained sensitive to commonly used antiviral drugs, with the exception of the Hong Kong virus isolate. Drug dose-response curves confirmed complete drug resistance to oseltamivir, partial sensitivity to peramivir, and retained susceptibility to zanamivir and favipiravir against the A/Hong Kong/2369/2009 virus. Three-dimensional analysis of drug interactions using the MacSynergy(TM) II program indicated an overall synergistic interaction when favipiravir was combined with the NAIs against the oseltamivir-sensitive influenza virus, and an additive effect against the oseltamivir-resistant virus. Although the clinical relevance of these drug combinations remains to be evaluated, results obtained from this study support the use of combination therapy with favipiravir and NAIs for treatment of human influenza virus infections.

Background: The discovery of novel influenza virus inhibitors remains an important priority in light of the emergence of drug-resistant viruses. Toward this end, a library of over 6,000 compounds was tested for antiviral activity. Methods: Strains of influenza virus were evaluated by cytopathic effect (CPE) inhibition and virus yield reduction assays. Intracellular nucleoside triphosphate pools were analysed by strong anion exchange HPLC. Dihydroorotate dehydrogenase inhibition assays were conducted. Influenza virus-infected mice were treated for 5 days with D282. Results: A non-nucleoside, 4-[(4-butylphenyl)amino]-2-methylene-4-oxo-butanoic acid (D282), was discovered that inhibited influenza A and B virus CPE by 50% at 6–31 μM (giving selectivity indices of >13 to >67, based on cytotoxicity of >400 μM in stationary cell cultures). Ribavirin (positive control) was active at 14–44 μM (yielding selectivity indices of >9 to >29, with >400 μM toxicity). D282 and ribavirin inhibited virus yield by 90% at 9.5 ±3.3 and 10.8 ±3.2 μM, respectively. The antiviral activity of D282 in vitro was reversed by addition of uridine, cytidine and orotic acid. D282 exhibited an uncompetitive inhibition of mouse liver dihydroorotate dehydrogenase (inhibitor constant [Ki] of 2.3 ±0.9 μM, Michaelis constant [Km] of 150 ±16 μM). Because cellular pyrimidine biosynthesis was inhibited, D282-treated cells had decreased uridine triphosphate and cytidine triphosphate levels. D282 (≤100 mg/kg/day) failed to prevent death of mice infected with influenza. Conclusions: D282 was active against influenza A and B viruses by inhibiting de novo pyrimidine biosynthesis. Although effective in vitro, the compound, like others in its class, was devoid of antiviral activity in infected mice.

Background: Limited in vivo studies in the scientific literature suggest that components of green tea and elderberry may be beneficial in treating influenza virus infections. They are thought to act by blocking virus adsorption to cells. TheraMax® is a proprietary medication administered by nasal spray that contains both green tea and elderberry extracts that was evaluated for antiviral activity. Methods: TheraMax was tested by dilution in Madin–Darby canine kidney cell cultures in standard viral cytopathic effect (CPE) inhibition and virucidal assays against eight influenza A and B strains. It was also administered intranasally to mice to determine protective activity compared to oral oseltamivir against an influenza A/NWS/33 (H1N1) infection. Results: In cell culture assays, TheraMax was found to inhibit viral CPE by 50% at a 1:20 dilution for seven of the eight virus strains, with no virucidal activity at 1:2 dilution. The undiluted product was administered to anaesthetized mice twice daily for 4 days starting 2 h before or 12 h after infection. Alternatively, TheraMax and virus were combined for treatment and infection. Oseltamivir was given orally twice daily for 5 days at 10 mg/kg/day starting at −2 h. TheraMax (combined directly with virus) and oseltamivir each prevented death and curtailed weight loss during the infection, and improved lung haemorrhage scores on day 6. TheraMax starting at −2 h or 12 h significantly delayed death by >2 days and reduced lung haemorrhage scores, but most animals died. Conclusions: These combined results indicate that TheraMax delayed symptoms during animal infections, likely through blocking of virus adsorption.

An amantadine-resistant influenza A/Duck/MN/1525/81 (H5N1) virus was developed from the low-pathogenic North American wild-type (amantadine-sensitive) virus for studying treatment of infections in cell culture and in mice. Double combinations of amantadine, oseltamivir (or the cell culture-active form, oseltamivir carboxylate), and ribavirin were used. Amantadine-oseltamivir carboxylate and amantadine-ribavirin combinations showed synergistic interactions over a range of doses against wild-type virus in Madin-Darby canine kidney (MDCK) cell culture, but oseltamivir carboxylate-ribavirin combinations did not. Primarily additive interactions were seen with oseltamivir carboxylate-ribavirin combinations against amantadine-resistant virus. The presence of amantadine in drug combinations against the resistant virus did not improve activity. The wild-type and amantadine-resistant viruses were lethal to mice by intranasal instillation. The resistant virus infection could not be treated with amantadine up to 100 mg/kg body weight/day, whereas the wild-type virus infection was treatable with oral doses of 10 (weakly effective) to 100 mg/kg/day administered twice a day for 5 days starting 4 h prior to virus exposure. Drug combination studies showed that treatment of the amantadine-resistant virus infection with amantadine-oseltamivir or amantadine-ribavirin combinations was not significantly better than using oseltamivir or ribavirin alone. In contrast, the oseltamivir-ribavirin (25- and 75-mg/kg/day combination) treatments produced significant reductions in mortality. The wild-type virus infection was markedly reduced in severity by all three combinations (amantadine, 10 mg/kg/day combined with the other compounds at 20 or 40 mg/kg/day) compared to monotherapy with the three compounds. Results indicate a lack of benefit of amantadine in combinations against amantadine-resistant virus, but positive benefits in combinations against amantadine-sensitive virus.

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal cowpox (Brighton strain) and vaccinia (WR strain) virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI), upper respiratory infections (URI), and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term) or 56d (long-term) prior to virus challenge. Single nasal sinus treatments of 10(6) and 10(7) PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201). Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7) mDEF201 treatments, but a 10(6) dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7) PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5) to 10(7) PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32-100 μM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01-10 nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations. Infected mice were treated twice-daily for 5 days starting 2 hours prior to virus challenge using drug doses of 0.05-0.4 mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4 mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2 mg/kg/day) compared to single drug treatments The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.

Background: The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. New classes of inhibitors are essential to combat swine influenza viral infection. Methods: A series of isatine-sulfadimidine derivatives were screened for antiviral activity against swine influenza A/California/07/2009 (H1N1) virus in Madin–Darby canine kidney (MDCK) cell culture. Cytotoxicity of the synthesized compounds was also tested in uninfected MDCK cells. Results: All the compounds inhibit the influenza A (H1N1) in MDCK cells. The most active compounds, SPIII-5Br and SPIII-5H, inhibited virus-induced cytopathology by 50% at 27 and 30 μM, respectively, with 50% cytotoxicity occurring at a much higher dose (975–1,000 μM). The positive control compound ribavirin inhibits the replication of the virus at 18 μM and cytotoxic concentration was found to be >1,000 μM. Conclusions: SPIII-5Br and SPIII-5H exhibited potency in the same range as ribavirin and are suitable candidate molecules for further investigation.

Favipiravir (T-705 [6-fluoro-3-hydroxy-2-pyrazinecarboxamide]) and oseltamivir were combined to treat influenza virus A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1) infections. T-705 alone inhibited viruses in cell culture at 1.4 to 4.3 μM. Oseltamivir inhibited these three viruses in cells at 3.7, 0.02, and 0.16 μM and in neuraminidase assays at 0.94, 0.46, and 2.31 nM, respectively. Oral treatments were given twice daily to mice for 5 to 7 days starting, generally, 24 h after infection. Survival resulting from 5 days of oseltamivir treatment (0.1 and 0.3 mg/kg/day) was significantly better in combination with 20 mg/kg of body weight/day of T-705 against the H1N1 infection. Treatment of the H3N2 infection required 50 mg/kg/day of oseltamivir for 7 days to achieve 60% protection; 25 mg/kg/day was ineffective. T-705 was ≥70% protective at 50 to 100 mg/kg/day but inactive at 25 mg/kg/day. The combination of inhibitors (25 mg/kg/day each) increased survival to 90%. The H5N1 infection was not benefited by treatment with oseltamivir (≤100 mg/kg/day for 7 days). T-705 was 30 to 70% protective at 25 to 100 mg/kg/day. Survival improved slightly with combination treatments. Increased activity was seen against H5N1 infection by starting treatments 2 h before infection. Oseltamivir was ineffective at ≤40 mg/kg/day. T-705 was 100% protective at 40 and 80 mg/kg/day and inactive at 20 mg/kg/day. Combining ineffective doses (20 mg/kg/day of T-705 and 10 to 40 mg/kg/day of oseltamivir) afforded 60 to 80% protection and improved body weights during infection. Thus, synergistic responses were achieved with low doses of T-705 combined with oseltamivir. These compounds may be viable candidates for combination treatment of human influenza infections.

OBJECTIVES To determine the metabolism of favipiravir (T-705, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) to its ribosylated, triphosphorylated form (T-705 RTP) in uninfected and influenza A/Duck/MN/1525/81 (H5N1) virus-infected cells. Effects of treatment on intracellular guanosine triphosphate (GTP) pools and influenza virus-inhibitory activity were also assessed. METHODS A strong anion exchange HPLC separation method with UV detection was used to quantify T-705 RTP and GTP levels in Madin-Darby canine kidney cells. Antiviral activity was determined by virus yield reduction assay. RESULTS Accumulation of T-705 RTP in uninfected cells increased linearly from 3 to 320 pmol/10(6) cells in cells exposed to 1-1000 microM extracellular T-705 for 24 h, approaching maximum levels by 9 h. Virus infection did not result in greater T-705 RTP accumulation compared with uninfected cells. Catabolism of T-705 RTP occurred after removal of T-705 from the extracellular medium, with a half-life of decay of 5.6 +/- 0.6 h. Based upon these results, short-term incubation of T-705 with H5N1 virus-infected cells was predicted to provide an antiviral benefit. Indeed, 4-8 h 10-100 microM T-705 treatment of cells resulted in virus yield reductions, but less than continuous exposure. A 100-fold higher extracellular concentration of T-705 was required to inhibit intracellular GTP levels compared with ribavirin, which helps explain ribavirin's greater toxicity. CONCLUSIONS The favourable intracellular metabolic properties of T-705 combined with its reduced cell-inhibitory properties make this compound an attractive candidate for treating human influenza virus infections.

Neurological respiratory deficits are serious outcomes of West Nile virus (WNV) disease. WNV patients requiring intubation have a poor prognosis. We previously reported that WNV-infected rodents also appear to have respiratory deficits when assessed by whole-body plethysmography and diaphragmatic electromyography. The purpose of this study was to determine if the nature of the respiratory deficits in WNV-infected rodents is neurological and if deficits are due to a disorder of brainstem respiratory centers, cervical spinal cord (CSC) phrenic motor neuron (PMN) circuitry, or both. We recorded phrenic nerve (PN) activity and found that in WNV-infected mice, PN amplitude is reduced, corroborating a neurological basis for respiratory deficits. These results were associated with a reduction in CSC motor neuron number. We found no dramatic deficits, however, in brainstem-mediated breathing rhythm generation or responses to hypercapnia. PN frequency and pattern parameters were normal, and all PN parameters changed appropriately upon a CO2 challenge. Histological analysis revealed generalized microglia activation, astrocyte reactivity, T cell and neutrophil infiltration, and mild histopathologic lesions in both the brainstem and CSC, but none of these were tightly correlated with PN function. Similar results in PN activity, brainstem function, motor neuron number, and histopathology were seen in WNV-infected hamsters, except that histopathologic lesions were more severe. Taken together, the results suggest that respiratory deficits in acute WNV infection are primarily due to a lower motor neuron disorder affecting PMNs and the PN rather than a brainstem disorder. Future efforts should focus on markers of neuronal dysfunction, axonal degeneration, and myelination.

Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. Methods To gain insights into the pathogenesis of TCRV infection in AG129 mice, we assessed hematologic and cytokine responses during the course of infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute infection, and measured serum and tissue viral titers out to 40 days post-infection to determine whether the virus is ultimately cleared in recovering mice. Results We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2–3 weeks post-infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. Conclusions Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.