Identification of a Macrocyclic Compound Targeting the Lassa Virus Polymerase.

There are no approved vaccines or therapeutics for Lassa virus (LASV) infections. To identify compounds with anti-LASV activity, we conducted a cell-based screening campaign at biosafety level 4 and tested almost 60,000 compounds for activity against an infectious reporter LASV. Hits from this screen included several structurally related macrocycles. The most potent, Mac128, had a sub-micromolar EC50 against the reporter virus, inhibited wild-type clade IV LASV, and reduced viral titers by 4 orders of magnitude. Mechanistic studies suggested that Mac128 inhibited viral replication at the level of the polymerase.

Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.

Development of reverse genetic tools to study Chapare and Machupo viruses.

Arenaviruses are highly pathogenic viruses that pose a serious public health threat. Chapare virus (CHAV) and Machupo virus (MACV), two New World arenaviruses, cause hemorrhagic fevers with case fatality rates of up to 45%. Research on therapeutic drug targets and vaccines for these viruses is limited because biosafety level 4 containment is required for handling them. In this study, we developed reverse genetics systems, including minigenomes and recombinant viruses, that will facilitate the study of these pathogens. The minigenome system is based on the S segment of CHAV or MACV genomes expressing the fluorescent reporter gene ZsGreen (ZsG). We also generated recombinant CHAV and MACV with and without the ZsG reporter gene. As a proof-of-concept study, we used both minigenomes and recombinant viruses to test the inhibitory effects of previously reported antiviral compounds. The new reverse genetics system described here will facilitate future therapeutic studies for these two life-threatening arenaviruses.

Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics.

Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ∼2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. To test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics.

One Health Investigation of SARS-CoV-2 in People and Animals on Multiple Mink Farms in Utah.

From July−November 2020, mink (Neogale vison) on 12 Utah farms experienced an increase in mortality rates due to confirmed SARS-CoV-2 infection. We conducted epidemiologic investigations on six farms to identify the source of virus introduction, track cross-species transmission, and assess viral evolution. Interviews were conducted and specimens were collected from persons living or working on participating farms and from multiple animal species. Swabs and sera were tested by SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) and serological assays, respectively. Whole genome sequencing was attempted for specimens with cycle threshold values <30. Evidence of SARS-CoV-2 infection was detected by rRT-PCR or serology in ≥1 person, farmed mink, dog, and/or feral cat on each farm. Sequence analysis showed high similarity between mink and human sequences on corresponding farms. On farms sampled at multiple time points, mink tested rRT-PCR positive up to 16 weeks post-onset of increased mortality. Workers likely introduced SARS-CoV-2 to mink, and mink transmitted SARS-CoV-2 to other animal species; mink-to-human transmission was not identified. Our findings provide critical evidence to support interventions to prevent and manage SARS-CoV-2 in people and animals on mink farms and emphasizes the importance of a One Health approach to address emerging zoonoses.

Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524).

The necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn. IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

Broad-spectrum in vitro antiviral activity of ODBG-P-RVn: an orally-available, lipid-modified monophosphate prodrug of remdesivir parent nucleoside (GS-441524).

The intravenous administration of remdesivir for COVID-19 confines its utility to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524) against viruses that cause diseases of human public health concern, including SARS-CoV-2. ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had near-equivalent activity to remdesivir in primary-like human small airway epithelial cells. Our results warrant investigation of ODBG-P-RVn efficacy in vivo.

Screening and Identification of Lujo Virus Inhibitors Using a Recombinant Reporter Virus Platform.

Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Design, synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles as inhibitors of ebola virus infection.

Novel 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1H-benzimidazoles were designed and synthesized as Ebola virus inhibitors. The proposed structures of the new prepared benzimidazole-piperidine hybrids were confirmed based on their spectral data and CHN analyses. The target compounds were screened in vitro for their anti-Ebola activity. Among tested molecules, compounds 26a (EC50=0.93 µM, SI = 10) and 25a (EC50=0.64 µM, SI = 20) were as potent as and more selective than Toremifene reference drug (EC50 = 0.38 µM, SI = 7) against cell line. Data suggests that the mechanism by which 25a and 26a block EBOV infection is through the inhibition of viral entry at the level of NPC1. Furthermore, a docking study revealed that several of the NPC1 amino acids that participate in binding to GP are involved in the binding of the most active compounds 25a and 26a. Finally, in silico ADME prediction indicates that 26a is an idealy drug-like candidate. Our results could enable the development of small molecule drug capable of inhibiting Ebola virus, especially at the viral entry step.

Hantavirus Infection Is Inhibited by Griffithsin in Cell Culture.

Andes virus (ANDV) and Sin Nombre virus (SNV), highly pathogenic hantaviruses, cause hantavirus pulmonary syndrome in the Americas. Currently no therapeutics are approved for use against these infections. Griffithsin (GRFT) is a high-mannose oligosaccharide-binding lectin currently being evaluated in phase I clinical trials as a topical microbicide for the prevention of human immunodeficiency virus (HIV-1) infection (ClinicalTrials.gov Identifiers: NCT04032717, NCT02875119) and has shown broad-spectrum in vivo activity against other viruses, including severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, and Nipah virus. In this study, we evaluated the in vitro antiviral activity of GRFT and its synthetic trimeric tandemer 3mGRFT against ANDV and SNV. Our results demonstrate that GRFT is a potent inhibitor of ANDV infection. GRFT inhibited entry of pseudo-particles typed with ANDV envelope glycoprotein into host cells, suggesting that it inhibits viral envelope protein function during entry. 3mGRFT is more potent than GRFT against ANDV and SNV infection. Our results warrant the testing of GRFT and 3mGRFT against ANDV infection in animal models.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

Potent in vitro activity of ß-D-4'-chloromethyl-2'-deoxy-2'-fluorocytidine against Nipah virus.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that continues to cause outbreaks in humans characterized by high mortality and significant clinical sequelae in survivors. Currently, no therapeutics are approved for use in humans against NiV infection. Here, we report that 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (ALS-8112) inhibits NiV. ALS-8112 is the parent nucleoside of lumicitabine, which has been evaluated in phase I and II clinical trials to treat pediatric and adult respiratory syncytial virus infection. In this study, we tested ALS-8112 against NiV and other major human respiratory pneumo- and paramyxoviruses in 2 human lung epithelial cell lines, and demonstrated the ability of ALS-8112 to reduce infectious wild-type NiV yield by over 6 orders of magnitude with no apparent cytotoxicity. However, further cytotoxicity testing in primary cells and bone marrow progenitor cells indicated cytotoxicity at higher concentrations of ALS-8112. Our results warrant the evaluation of lumicitabine against NiV infection in relevant animal models.

Probing the Effects of Pyrimidine Functional Group Switches on Acyclic Fleximer Analogues for Antiviral Activity.

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as "fleximers" have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

Characterisation of infectious Ebola virus from the ongoing outbreak to guide response activities in the Democratic Republic of the Congo: a phylogenetic and in vitro analysis.

BACKGROUND: The ongoing Ebola virus outbreak in the Ituri and North Kivu Provinces of the Democratic Republic of the Congo, which began in July, 2018, is the second largest ever recorded. Despite civil unrest, outbreak control measures and the administration of experimental therapies and a vaccine have been initiated. The aim of this study was to test the efficacy of candidate therapies and diagnostic tests with the outbreak strain Ituri Ebola virus. Lacking a virus isolate from this outbreak, a recombinant Ituri Ebola virus was compared with a similarly engineered Makona virus from the 2013-16 outbreak. METHODS: Using Ebola virus sequences provided by organisations in DR Congo and a reverse genetics system, we generated an authentic Ebola virus from the ongoing outbreak in Ituri and North Kivu provinces. To relate this virus to other Ebola viruses in DR Congo, we did a phylogenetic analysis of representative complete Ebola virus genome sequences from previous outbreaks. We evaluated experimental therapies being tested in clinical trials in DR Congo, including remdesivir and ZMapp monoclonal antibodies, for their ability to inhibit the growth of infectious Ituri Ebola virus in cell culture. We also tested diagnostic assays for detection of the Ituri Ebola virus sequence. FINDINGS: The phylogenetic analysis of whole-genome sequences from each Ebola virus outbreak suggests there are at least two Ebola virus strains in DR Congo, which have independently crossed into the human population. The Ituri Ebola strain initially grew slower than the Makona strain, yet reached similar mean yields of 3 × 107 50% tissue culture infectious dose by 72 h infection in Huh-7 cells. Ituri Ebola virus was similar to Makona in its susceptibility to inhibition by remdesivir and to neutralisation by monoclonal antibodies from ZMapp and other monoclonal antibodies. Remdesivir inhibited Ituri Ebola virus at a 50% effective concentration (EC50) of 12nM (with a selectivity index of 303) and Makona Ebola virus at 13nM (with a selectivity index of 279). The Zmapp monoclonal antibodies 2G4 and 4G7 neutralised Ituri Ebola virus with a mean EC50 of 0·24 µg/mL and 0·48 µg/mL, and Makona Ebola virus with a mean EC50 of 0·45 µg/mL and 0·2 µg/mL. The Xpert Ebola and US Centers for Disease Control and Prevention real-time RT-qPCR diagnostic assays detected Ituri and Makona Ebola virus sequences with similar sensitivities and efficiencies, despite primer site binding mismatches in the Ituri Ebola virus. INTERPRETATION: Our findings provide a rationale for the continued testing of investigational therapies, confirm the effectiveness of the diagnostic assays used in the region, and establish a paradigm for the use of reverse genetics to inform response activities in an outbreak. FUNDING: US Centers for Disease Control and Prevention.

Aneurysmal bone cyst treated with percutaneous doxycycline: is a single treatment sufficient?

OBJECTIVE: The purpose of this case series is to report on the effectiveness of a single percutaneous injection of doxycycline as a primary treatment for aneurysmal bone cyst (ABC). MATERIALS AND METHODS: A retrospective cohort study was conducted on seven patients diagnosed with ABC at various anatomical sites, with the intention to treat by a single percutaneous injection of doxycycline. Mean patient age was 14 years. RESULTS: Signs of treatment response were seen in six of seven patients after one injection. Three of the seven received a second treatment, despite signs of response. Another had expansion of the lesion after treatment, requiring excision. In total, three patients had a single injection of doxycycline as their sole treatment and another three showed signs of response after a single injection. CONCLUSIONS: A single percutaneous injection of doxycycline should be considered a viable primary treatment option for ABC.

A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus.

There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, which encodes the α and ß subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.

Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD.IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune system are characteristic features of EVD, statins could be explored as part of EVD therapeutics.

Identification of 2'-deoxy-2'-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne orthonairovirus, causes a severe hemorrhagic disease in humans (Crimean-Congo hemorrhagic fever, CCHF). Currently, no vaccines are approved to prevent CCHF; treatment is limited to supportive care and the use of ribavirin, the therapeutic benefits of which remain unclear. CCHF is part of WHO's priority list of infectious diseases warranting further research and development. To aid in the identification of new antiviral compounds, we generated a recombinant CCHFV expressing a reporter protein, allowing us to quantify virus inhibition by measuring the reduction in fluorescence in infected cells treated with candidate compounds. The screening assay was readily adaptable to high-throughput screening (HTS) of compounds using Huh7 cells, with a signal-to-noise ratio of 50:1, and Z'-factors > 0.6 in both 96- and 384-well formats. A screen of candidate nucleoside analog compounds identified 2'-deoxy-2'-fluorocytidine (EC50 = 61 ± 18 nM) as having 200 × the potency of ribavirin (EC50 = 12.5 ± 2.6 µM), as well as 17 × the potency of T-705 (favipiravir), another compound with reported anti-CCHFV activity (EC50 = 1.03 ± 0.16 µM). Furthermore, we also determined that 2'-deoxy-2'-fluorocytidine acts synergistically with T-705 to inhibit CCHFV replication without causing cytotoxicity. The incorporation of this reporter virus into the high-throughput screening assay described here will allow more rapid identification of effective therapeutic options to combat this emerging human pathogen.

Flex-nucleoside analogues - Novel therapeutics against filoviruses.

Fleximers, a novel type of flexible nucleoside that have garnered attention due to their unprecedented activity against human coronaviruses, have now exhibited highly promising levels of activity against filoviruses. The Flex-nucleoside was the most potent against recombinant Ebola virus in Huh7 cells with an EC50=2µM, while the McGuigan prodrug was most active against Sudan virus-infected HeLa cells with an EC50 of 7µM.