Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

CNP blocks mitochondrial depolarization and inhibits SARS-CoV-2 replication in vitro and in vivo.

The COVID-19 pandemic has claimed over 6.5 million lives worldwide and continues to have lasting impacts on the world's healthcare and economic systems. Several approved and emergency authorized therapeutics that inhibit early stages of the virus replication cycle have been developed however, effective late-stage therapeutical targets have yet to be identified. To that end, our lab identified that 2',3' cyclic-nucleotide 3'-phosphodiesterase (CNP) inhibits SARS-CoV-2 virion assembly. We show that CNP inhibits the generation of new SARS-CoV-2 virions, reducing intracellular titers without inhibiting viral structural protein translation. Additionally, we show that targeting of CNP to mitochondria is necessary for inhibition, blocking mitochondrial depolarization and implicating CNP's proposed role as an inhibitor of the mitochondrial permeabilization transition pore (mPTP) as the mechanism of virion assembly inhibition. We also demonstrate that an adenovirus expressing virus expressing both human ACE2 and CNP inhibits SARS-CoV-2 titers to undetectable levels in lungs of mice. Collectively, this work shows the potential of CNP to be a new SARS-CoV-2 antiviral target.

Diet-induced obesity and diabetes enhance mortality and reduce vaccine efficacy for SARS-CoV-2.

IMPORTANCE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a wide spectrum of diseases in the human population, from asymptomatic infections to death. It is important to study the host differences that may alter the pathogenesis of this virus. One clinical finding in coronavirus disease 2019 (COVID-19) patients is that people with obesity or diabetes are at increased risk of severe illness from SARS-CoV-2 infection. We used a high-fat diet model in mice to study the effects of obesity and type 2 diabetes on SARS-CoV-2 infection as well as how these comorbidities alter the response to vaccination. We find that diabetic/obese mice have increased disease after SARS-CoV-2 infection and they have slower clearance of the virus. We find that the lungs of these mice have increased neutrophils and that removing these neutrophils protects diabetic/obese mice from disease. This demonstrates why these diseases have increased risk of severe disease and suggests specific interventions upon infection.

Target-agnostic drug prediction integrated with medical record analysis uncovers differential associations of statins with increased survival in COVID-19 patients.

Drug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. A Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. Simvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

Placental SARS-CoV-2 Infection and Its Implications for Increased Risk of Adverse Pregnancy Outcomes.

OBJECTIVE: Pregnant women are at increased risk of coronavirus disease 2019 (COVID-19). This could be explained through the prism of physiologic and immunologic changes in pregnancy. In addition, certain immunological reactions originate in the placenta in response to viral infections.This study aimed to investigate whether severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can infect the human placenta and discuss its implications in the pathogenesis of adverse pregnancy outcomes. STUDY DESIGN: We conducted a retrospective cohort study in which we collected placental specimens from pregnant women who had a laboratory-confirmed SARS-CoV-2 infection. We performed RNA in situ hybridization assay on formalin-fixed paraffin-embedded tissues to establish the in vivo evidence for placental infectivity by this corona virus. In addition, we infected trophoblast isolated from uninfected term human placenta with SARS-CoV-2 variants to further provide in vitro evidence for such an infectivity. RESULTS: There was a total of 21 cases enrolled, which included 5 cases of spontaneous preterm birth (SPTB) and 2 intrauterine fetal demises (IUFDs). Positive staining of positive-sense strand of SARS-CoV-2 virions was detected in 15 placentas including 4 SPTB and both IUFDs. In vitro infection assay demonstrated that SARS-CoV-2 virions were highly capable of infecting both cytotrophoblast and syncytiotrophoblast. CONCLUSION: This study implies that placental SARS-CoV-2 infection may be associated with an increased risk of adverse obstetrical outcomes. KEY POINTS: · SARS-CoV-2 can effectively infect human placenta.. · Such infectivity is confirmed by in vitro experiments.. · Placental SARS-CoV-2 corelates with adverse obstetrical outcomes..

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine.

BACKGROUND: NVX-CoV2373 is a recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) nanoparticle vaccine composed of trimeric full-length SARS-CoV-2 spike glycoproteins and Matrix-M1 adjuvant. METHODS: We initiated a randomized, placebo-controlled, phase 1-2 trial to evaluate the safety and immunogenicity of the rSARS-CoV-2 vaccine (in 5-µg and 25-µg doses, with or without Matrix-M1 adjuvant, and with observers unaware of trial-group assignments) in 131 healthy adults. In phase 1, vaccination comprised two intramuscular injections, 21 days apart. The primary outcomes were reactogenicity; laboratory values (serum chemistry and hematology), according to Food and Drug Administration toxicity scoring, to assess safety; and IgG anti-spike protein response (in enzyme-linked immunosorbent assay [ELISA] units). Secondary outcomes included unsolicited adverse events, wild-type virus neutralization (microneutralization assay), and T-cell responses (cytokine staining). IgG and microneutralization assay results were compared with 32 (IgG) and 29 (neutralization) convalescent serum samples from patients with Covid-19, most of whom were symptomatic. We performed a primary analysis at day 35. RESULTS: After randomization, 83 participants were assigned to receive the vaccine with adjuvant and 25 without adjuvant, and 23 participants were assigned to receive placebo. No serious adverse events were noted. Reactogenicity was absent or mild in the majority of participants, more common with adjuvant, and of short duration (mean, ≤2 days). One participant had mild fever that lasted 1 day. Unsolicited adverse events were mild in most participants; there were no severe adverse events. The addition of adjuvant resulted in enhanced immune responses, was antigen dose-sparing, and induced a T helper 1 (Th1) response. The two-dose 5-µg adjuvanted regimen induced geometric mean anti-spike IgG (63,160 ELISA units) and neutralization (3906) responses that exceeded geometric mean responses in convalescent serum from mostly symptomatic Covid-19 patients (8344 and 983, respectively). CONCLUSIONS: At 35 days, NVX-CoV2373 appeared to be safe, and it elicited immune responses that exceeded levels in Covid-19 convalescent serum. The Matrix-M1 adjuvant induced CD4+ T-cell responses that were biased toward a Th1 phenotype. (Funded by the Coalition for Epidemic Preparedness Innovations; ClinicalTrials.gov number, NCT04368988).

Expanded Histopathology and Tropism of Ebola Virus in the Rhesus Macaque Model: Potential for Sexual Transmission, Altered Adrenomedullary Hormone Production, and Early Viral Replication in Liver.

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.

The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses.

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.

A SARS-CoV-2 Delta Variant Case Manifesting as Extensive Placental Infection and Fetal Transmission.

INTRODUCTION: Studies indicate a very low rate of SARS-CoV-2 detection in the placenta or occasionally a low rate of vertical transmission in COVID-19 pregnancy. SARS-CoV-2 Delta variant has become a dominant strain over the world and possesses higher infectivity due to mutations in its spike receptor-binding motif. CASE PRESENTATION: To determine whether SARS-CoV-2 Delta variant has increased potential for placenta infection and vertical transmission, we analyzed SARS-CoV-2 infection in the placenta, umbilical cord, and fetal membrane from a case where an unvaccinated mother and her neonate were COVID-19 positive. A 35-year-old primigravida with COVID-19 underwent an emergent cesarean delivery due to placental abruption in the setting of premature rupture of membranes. The neonate tested positive for SARS-CoV-2 within the first 24 h, and then again on days of life 2, 6, 13, and 21. The placenta exhibited intervillositis, increased fibrin deposition, and syncytiotrophoblast necrosis. Sequencing of viral RNA from fixed placental tissue revealed SAR-CoV-2 B.1.167.2 (Delta) variant. Both spike protein and viral RNA were abundantly present in syncytiotrophoblasts, cytotrophoblasts, umbilical cord vascular endothelium, and fetal membranes. CONCLUSION: We report with strong probability the first SARS-CoV-2 Delta variant transplacental transmission. Placental cells exhibited extensive apoptosis, senescence, and ferroptosis after SARS-CoV-2 Delta infection.

Broad Anti-coronavirus Activity of Food and Drug Administration-Approved Drugs against SARS-CoV-2 In Vitro and SARS-CoV In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease.IMPORTANCE There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV in vitro We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine in vivo using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.

Quantification of Viral and Host Biomarkers in the Liver of Rhesus Macaques: A Longitudinal Study of Zaire Ebolavirus Strain Kikwit (EBOV/Kik).

Zaire ebolavirus (EBOV) causes Ebola virus disease (EVD), which carries a fatality rate between 25% and 90% in humans. Liver pathology is a hallmark of terminal EVD; however, little is known about temporal disease progression. We used multiplexed fluorescent immunohistochemistry and in situ hybridization in combination with whole slide imaging and image analysis (IA) to quantitatively characterize temporospatial signatures of viral and host factors as related to EBOV pathogenesis. Eighteen rhesus monkeys euthanized between 3 and 8 days post-infection, and 3 uninfected controls were enrolled in this study. Compared with semiquantitative histomorphologic ordinal scoring, quantitative IA detected subtle and progressive features of early and terminal EVD that was not feasible with routine approaches. Sinusoidal macrophages were the earliest cells to respond to infection, expressing proinflammatory cytokine interleukin 6 (IL6) mRNA, which was subsequently also observed in fibrovascular compartments. The mRNA of interferon-stimulated gene-15 (ISG-15), also known as ISG15 ubiquitin like modifier (ISG15), was observed early, with a progressive and ubiquitous hybridization signature involving mesenchymal and epithelial compartments. ISG-15 mRNA was prominent near infected cells, but not in infected cells, supporting the hypothesis that bystander cells produce a robust interferon gene response. This study contributes to our current understanding of early EVD progression and illustrates the value that digital pathology and quantitative IA serve in infectious disease research.

Filoviruses Infect Rhesus Macaque Synoviocytes in Vivo and Primary Human Synoviocytes in Vitro.

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.

Phase 2 Placebo-Controlled Trial of Two Vaccines to Prevent Ebola in Liberia.

BACKGROUND: The safety and efficacy of vaccines to prevent Ebola virus disease (EVD) were unknown when the incidence of EVD was peaking in Liberia. METHODS: We initiated a randomized, placebo-controlled, phase 3 trial of the chimpanzee adenovirus 3 vaccine (ChAd3-EBO-Z) and the recombinant vesicular stomatitis virus vaccine (rVSV∆G-ZEBOV-GP) in Liberia. A phase 2 subtrial was embedded to evaluate safety and immunogenicity. Because the incidence of EVD declined in Liberia, the phase 2 component was expanded and the phase 3 component was eliminated. RESULTS: A total of 1500 adults underwent randomization and were followed for 12 months. The median age of the participants was 30 years; 36.6% of the participants were women. During the week after the administration of vaccine or placebo, adverse events occurred significantly more often with the active vaccines than with placebo; these events included injection-site reactions (in 28.5% of the patients in the ChAd3-EBO-Z group and 30.9% of those in the rVSV∆G-ZEBOV-GP group, as compared with 6.8% of those in the placebo group), headache (in 25.1% and 31.9%, vs. 16.9%), muscle pain (in 22.3% and 26.9%, vs. 13.3%), feverishness (in 23.9% and 30.5%, vs. 9.0%), and fatigue (in 14.0% and 15.4%, vs. 8.8%) (P<0.001 for all comparisons); these differences were not seen at 1 month. Serious adverse events within 12 months after injection were seen in 40 participants (8.0%) in the ChAd3-EBO-Z group, in 47 (9.4%) in the rVSV∆G-ZEBOV-GP group, and in 59 (11.8%) in the placebo group. By 1 month, an antibody response developed in 70.8% of the participants in the ChAd3-EBO-Z group and in 83.7% of those in the rVSV∆G-ZEBOV-GP group, as compared with 2.8% of those in the placebo group (P<0.001 for both comparisons). At 12 months, antibody responses in participants in the ChAd3-EBO-Z group (63.5%) and in those in the rVSV∆G-ZEBOV-GP group (79.5%) remained significantly greater than in those in the placebo group (6.8%, P<0.001 for both comparisons). CONCLUSIONS: A randomized, placebo-controlled phase 2 trial of two vaccines that was rapidly initiated and completed in Liberia showed the capability of conducting rigorous research during an outbreak. By 1 month after vaccination, the vaccines had elicited immune responses that were largely maintained through 12 months. (Funded by the National Institutes of Allergy and Infectious Diseases and the Liberian Ministry of Health; PREVAIL I ClinicalTrials.gov number, NCT02344407 .).

Pan-Filovirus Serum Neutralizing Antibodies in a Subset of Congolese Ebolavirus Infection Survivors.

One year after a Zaire ebolavirus (EBOV) outbreak occurred in the Boende Health Zone of the Democratic Republic of the Congo during 2014, we sought to determine the breadth of immune response against diverse filoviruses including EBOV, Bundibugyo (BDBV), Sudan (SUDV), and Marburg (MARV) viruses. After assessing the 15 survivors, 5 individuals demonstrated some degree of reactivity to multiple ebolavirus species and, in some instances, Marburg virus. All 5 of these survivors had immunoreactivity to EBOV glycoprotein (GP) and EBOV VP40, and 4 had reactivity to EBOV nucleoprotein (NP). Three of these survivors showed serologic responses to the 3 species of ebolavirus GPs tested (EBOV, BDBV, SUDV). All 5 samples also exhibited ability to neutralize EBOV using live virus, in a plaque reduction neutralization test. Remarkably, 3 of these EBOV survivors had plasma antibody responses to MARV GP. In pseudovirus neutralization assays, serum antibodies from a subset of these survivors also neutralized EBOV, BDBV, SUDV, and Taï Forest virus as well as MARV. Collectively, these findings suggest that some survivors of naturally acquired ebolavirus infection mount not only a pan-ebolavirus response, but also in less frequent cases, a pan-filovirus neutralizing response.

Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures.

Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

Ebola Virus Neutralizing Antibodies Detectable in Survivors of theYambuku, Zaire Outbreak 40 Years after Infection.

The first reported outbreak of Ebola virus disease occurred in 1976 in Yambuku, Democratic Republic of Congo. Antibody responses in survivors 11 years after infection have been documented. However, this report is the first characterization of anti-Ebola virus antibody persistence and neutralization capacity 40 years after infection. Using ELISAs we measured survivor's immunological response to Ebola virus Zaire (EBOV) glycoprotein and nucleoprotein, and assessed VP40 reactivity. Neutralization of EBOV was measured using a pseudovirus approach and plaque reduction neutralization test with live EBOV. Some survivors from the original EBOV outbreak still harbor antibodies against all 3 measures. Interestingly, a subset of these survivors' serum antibodies could still neutralize live virus 40 years postinitial infection. These data provide the longest documentation of both anti-Ebola serological response and neutralization capacity within any survivor cohort, extending the known duration of response from 11 years postinfection to at least 40 years after symptomatic infection.

Assessment and Optimization of the GeneXpert Diagnostic Platform for Detection of Ebola Virus RNA in Seminal Fluid.

Recent studies have suggested that Ebola virus (EBOV) ribonucleic acid (RNA) potentially present in the semen of a large number of survivors of Ebola virus disease (EVD) in Western Africa may contribute to sexual transmission of EVD and generate new clusters of cases in regions previously declared EVD-free. These findings drive the immediate need for a reliable, rapid, user-friendly assay for detection of EBOV RNA in semen that is deployable to multiple sites across Western Africa. In this study, we optimized the Xpert EBOV assay for semen samples by adding dithiothreitol. Compared to the assays currently in use in Liberia (including Ebola Zaire Target 1, major groove binder real-time-polymerase chain reaction assays, and original Xpert EBOV assay), the modified Xpert EBOV assay demonstrated greater sensitivity than the comparator assays. Thus, the modified Xpert EBOV assay is optimal for large-scale monitoring of EBOV RNA persistence in male survivors.

CNP blocks mitochondrial depolarization and inhibits SARS-CoV-2 replication in vitro and in vivo.

The COVID-19 pandemic has claimed over 6.5 million lives worldwide and continues to have lasting impacts on the world's healthcare and economic systems. Several approved and emergency authorized therapeutics that inhibit early stages of the virus replication cycle have been developed however, effective late-stage therapeutical targets have yet to be identified. To that end, our lab identified that 2',3' cyclic-nucleotide 3'-phosphodiesterase (CNP) inhibits SARS-CoV-2 virion assembly. We show that CNP inhibits the generation of new SARS-CoV-2 virions, reducing intracellular titers without inhibiting viral structural protein translation. Additionally, we show that targeting of CNP to mitochondria is necessary for inhibition, blocking mitochondrial depolarization and implicating CNP's proposed role as an inhibitor of the mitochondrial permeabilization transition pore (mPTP) as the mechanism of virion assembly inhibition. We also demonstrate that an adenovirus expressing virus expressing both human ACE2 and CNP inhibits SARS-CoV-2 titers to undetectable levels in lungs of mice. Collectively, this work shows the potential of CNP to be a new SARS-CoV-2 antiviral target.

SIRT-1 is required for release of enveloped enteroviruses.

Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.