Prevention and control of Ebola virus transmission: mathematical modelling and data fitting.

The Ebola virus disease (EVD) has been endemic since 1976, and the case fatality rate is extremely high. EVD is spread by infected animals, symptomatic individuals, dead bodies, and contaminated environment. In this paper, we formulate an EVD model with four transmission modes and a time delay describing the incubation period. Through dynamical analysis, we verify the importance of blocking the infection source of infected animals. We get the basic reproduction number without considering the infection source of infected animals. And, it is proven that the model has a globally attractive disease-free equilibrium when the basic reproduction number is less than unity; the disease eventually becomes endemic when the basic reproduction number is greater than unity. Taking the EVD epidemic in Sierra Leone in 2014-2016 as an example, we complete the data fitting by combining the effect of the media to obtain the unknown parameters, the basic reproduction number and its time-varying reproduction number. It is shown by parameter sensitivity analysis that the contact rate and the removal rate of infected group have the greatest influence on the prevalence of the disease. And, the disease-controlling thresholds of these two parameters are obtained. In addition, according to the existing vaccination strategy, only the inoculation ratio in high-risk areas is greater than 0.4, the effective reproduction number can be less than unity. And, the earlier the vaccination time, the greater the inoculation ratio, and the faster the disease can be controlled.

Dysglycaemia in Ebola virus disease: a retrospective analysis from the 2018 to 2020 outbreak.

BACKGROUND: Ebola virus disease (EVD) is associated with multisystem organ failure and high mortality. Severe hypoglycaemia is common, life-threatening, and correctable in critically ill patients, but glucose monitoring may be limited in EVD treatment units. METHODS: We conducted a retrospective review of patients admitted to EVD treatment units in Butembo and Katwa, Eastern DRC. Glucose measurements were done using a handheld glucometer at the bedside or using the Piccolo xpress Chemistry Analyzer on venous samples. FINDINGS: 384 patients (median age 30 years (interquartile range, IQR, 20-45), 57% female) and 6422 glucose measurements (median 11 per patient, IQR 4-22) were included in the analysis. Severe hypoglycaemia (≤2.2 mmol/L) and hyperglycaemia (>10 mmol/L) were recorded at least once during the ETU admission in 97 (25%) and 225 (59%) patients, respectively. A total of 2004 infusions of glucose-containing intravenous solutions were administered to 302 patients (79%) with a median cumulative dose of 175g (IQR 100-411). The overall case fatality rate was 157/384 (41%) and was 2.2-fold higher (95% CI 1.3-3.8) in patients with severe hypoglycaemia than those without hypoglycaemia (p = 0.0042). In a multivariable Cox proportional hazards model, periods of severe hypoglycaemia (adjusted hazard ratio (aHR) 6.2, 95% CI 3.2-12, p < 0.0001) and moderate hypoglycaemia (aHR 3.0, 95% CI 1.9-4.8, p < 0.0001) were associated with elevated mortality. INTERPRETATION: Hypoglycaemia is common in EVD, requires repeated correction with intravenous dextrose solutions, and is associated with mortality. FUNDING: This study was not supported by any specific funding.

"If I die for touching him, let me die": a rapid ethnographic assessment of cultural practices and Ebola transmission in high-risk border regions of Tanzania.

BACKGROUND: Ebola Virus Disease (EVD) is a rare but contagious disease caused by Ebola Virus (EBOV). The first Ebola outbreaks were reported in the Democratic Republic of Congo (DRC) before subsequent reported cases in Western and East African countries, including Uganda, which borders Tanzania. Proximity to EVD-infected countries raises the prospect of cross-border transmission, raising alarm in Tanzania. This study aimed to explore the cultural practices likely to prevent or escalate EVD transmission in the event of its outbreak in the country. METHODS: This rapid ethnographic assessment employed observation, interviews, and focus group discussions to collect data from people with diverse characteristics in five regions of Tanzania Mainland namely, Kagera, Kigoma, Mwanza and Songwe regions and Zanzibar Island. The qualitative data was then subjected to thematic analysis. FINDINGS: Cultural practices may escalate the transmission of EVD and hinder its prevention and control. These cultural practices include caring sick people at home, confirmation of death, mourning, and body preparation for burial. Communal life, ceremonies, and social gatherings were other aspects observed to have the potential for compounding EVD transmission and hindering its containment in case of an outbreak. CONCLUSION: Cultural practices may escalate EVD transmission as identified in the study settings. As such, Risk Communication and Community Engagement (RCCE) activities should be interventionist in transforming cultural practices that may escalate the spread of EVD as part of preparedness, prevention, and control efforts in the event of an outbreak.

Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Psychological distress among healthcare professionals in Mbarara, following the 2022 Ebola Virus Disease outbreak in Uganda: a mixed methods study.

BACKGROUND: The 2022 Ebola Virus Disease (EVD) outbreak occurred at a time when Uganda was still battling the social and psychological challenges of the COVID-19 pandemic; placing health care professionals (HCPs) at a much higher risk of developing psychological distress. Psychological distress among HCPs can cause decreased workplace productivity and ineffective management of their patients. The current study aimed to investigate and understand psychological distress among HCPS in Mbarara city in Southwestern Uganda following the 2022 EVD outbreak. METHOD: We enrolled 200 HCPs through convenient sampling from one private and one public health facility in Mbarara city in Southwestern Uganda, in a cross-sectional convergent parallel mixed method approach where qualitative and quantitative data were collected concurrently. Quantitative data, utilizing the Kessler Psychological Distress (K10) Scale, provided us with a quantitative measure of the prevalence of psychological distress among HCPs, and were analyzed using STATA version 16. Qualitative data, on the other hand, offered deeper insights into the nature, perceptions, and contextual factors influencing this distress, and were analyzed using emergent theme analysis. RESULTS: The prevalence of psychological distress was 59.5% and it was higher among females (63.9%) compared to males (36.1%). HCPs vividly expressed distress and anxiety, with heightened suspicion that every patient might be an EVD carrier, creating a pervasive sense of unsafety in the workplace. However, the outbreak had an educational affect where concerns about the announcement of another EVD outbreak were diverse, with HCPs expressing anxiety, despair, and dissatisfaction with the country's management of potential outbreaks. CONCLUSION: High levels of psychological distress were experienced by HCPs in Southwestern Uganda as a result of the 2022 EVD pandemic. HCPs express a wide range of feelings, such as dread, anxiety, despair, pessimism, and discontent with the way the outbreaks are handled throughout the nation. We recommend implementation of comprehensive psychosocial support programs tailored to the unique needs of HCPs, including counseling services, stress management workshops, and peer support networks.

Identification of Prospective Ebola Virus VP35 and VP40 Protein Inhibitors from Myxobacterial Natural Products.

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.

Prevalence of somatic symptoms among Ebola Virus Disease (EVD) survivors in Africa: a systematic review and meta-analysis.

BACKGROUND: Many Ebola virus disease (EVD) survivors have reported somatic and neuropsychological symptoms after discharge from the Ebola Treatment Unit (ETU). Since the 2014-2016 Ebola epidemic in West Africa, various studies have investigated and identified these symptoms. Evidence on somatic symptoms is widely available in the literature, however, there is no concise overview of the prevalence across different time intervals. METHODS: This meta-analysis was conducted following the (PRISMA) guidelines. A database search was conducted to identify original studies that reported the prevalence of symptoms. The primary outcome measure was the prevalence rate of several somatic symptoms. Results were pooled, and prevalence rates were assessed over time, to elucidate any particular trends. RESULTS: We included 23 studies (5,714 participants). The pooled prevalence was: arthralgia 50% (95% CI: 41%-59%); headache 44% (95% CI: 36%-52%); myalgia 32% (95% CI: 26%-38%); abdominal pain 27% (95% CI: 15%-39%); fatigue 25% (95% CI: 19%-31%); numbness of feet 16% (95% CI: 14%-18%); numbness of hands 12% (95% CI: 10%-14%) and hearing loss 9% (95% CI: 5%-12%). Prevalence across different time intervals revealed significant patterns. All the symptoms persisted for more than 2 years after discharge except for abdominal pain. CONCLUSION: The pooled prevalence rates of somatic symptoms are notably high. Arthralgia and headache are the most prevalent of the symptoms, with hearing loss and numbness in hands and feet being the least. We found that arthralgia, myalgia, and abdominal pain decreased over time. However, headache, fatigue, numbness of hands and feet, and hearing loss increased over time.

Evaluation of potency and metabolic stability of diphyllin-derived Vacuolar-ATPase inhibitors.

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.

(+)-fenchol and (-)-isopinocampheol derivatives targeting the entry process of filoviruses.

The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (-)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC50) 1.4-20 µÐœ against Lenti-EboV-GP infection and 11.3-47 µÐœ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration.

Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics.

BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Ebola virus circulation in a non-epidemic Guinean rural area: A mixed-method approach to assessing endemicity.

OBJECTIVES: This study aimed to investigate the prevalence of orthoebolavirus antibodies in Madina Oula, a non-epidemic rural area in Guinea, in 2022. METHODS: A cross-sectional study was conducted from March 14 to April 3, 2022 involving recording household and socio-demographic characteristics, lifestyle data, and collecting dried blood spots from 878 individuals in 235 households. Dried blood spots were tested using multiplex serology to detect antibodies to different orthoebolaviruses: Ebola virus, Bundibugyo virus, Sudan virus, Reston virus, and Bombali virus. Seroprevalence was estimated with a 95% confidence interval and a Z-test was performed to compare the seropositivity between children aged under 15 years and those over 15 years. Household and participant characteristics were analyzed using descriptive statistic, and socio-historical conditions were discussed. RESULTS: The serological analysis conducted in 2022 on 878 participants revealed varying reactivity to orthoebolavirus antigens, notably, with glycoprotein antigens, particularly, glycoprotein Sudan virus (16%). A total of 21 samples exhibited reactivity with at least two antigens, with a median age of 27 years (interquartile range 10.00-35.00), ranging from 2 to 80 years. There is no significant difference between seropositivity in children aged under 15 (2.86%) years and those over 15 (2.14%) years. The antibody presence varied per village, with the highest prevalence observed in Ouassou and Dar-es-Salam. CONCLUSIONS: Serological data in a region unaffected by recent Ebola outbreaks indicate possible orthoebolavirus endemicity, emphasizing the need for preparedness against known or novel orthoebolaviruses with potential cross-reactivity.

Assessment of health care workers preparedness to epidemics: A case of Ebola virus disease preparedness in private hospitals in Kampala, Uganda.

INTRODUCTION: Unrecognized Ebola Virus Disease (EVD) can lead to multiple chains of transmissions if the first caretakers are not trained and prepared. This study aimed to assess healthcare workers (HCWs) preparedness in private hospitals located in Kampala, to detect, respond and prevent EVD. METHODOLOGY: A descriptive cross-sectional study was carried out among HCWs in direct clinical care provision in four private hospitals, and in one Ebola Treatment Unit (ETU) using a self-administered questionnaire from March to June 2020. RESULTS: 222 HCWs agreed to participate aged from 19 to 64 years and with 6 months to 38 years of practice where most were nurses (44%). 3/5 hospitals did not have written protocols on EVD case management, and only one (ETU) had an exclusive emergency team. 59% were not sure whether contact tracing was taking place. Private hospitals were not included in EVD trainings organized by the Ministry of Health (MoH). In addition, HCWs in private hospitals were not empowered by the MoH to take part in EVD case management. Despite these shortcomings, only 66% of HCWs showed an interest to be immunized. Knowledge about potential Ebola vaccines was generally poor. CONCLUSIONS: In Kampala, Uganda, establishment of a more comprehensive preparedness and response strategy for EVD outbreaks is imperative for HCWs in private facilities, including a wide vaccination educational program on Ebola vaccination. The findings from this study if addressed will likely improve the preparedness and management of future Ebola outbreaks in Uganda.

Safety and Immunogenicity of an Accelerated Ebola Vaccination Schedule in People with and without Human Immunodeficiency Virus: A Randomized Clinical Trial.

The safety and immunogenicity of the two-dose Ebola vaccine regimen MVA-BN-Filo, Ad26.ZEBOV, 14 days apart, was evaluated in people without HIV (PWOH) and living with HIV (PLWH). In this observer-blind, placebo-controlled, phase 2 trial, healthy adults were randomized (4:1) to receive MVA-BN-Filo (dose 1) and Ad26.ZEBOV (dose 2), or two doses of saline/placebo, administered intramuscularly 14 days apart. The primary endpoints were safety (adverse events (AEs)) and immunogenicity (Ebola virus (EBOV) glycoprotein-specific binding antibody responses). Among 75 participants (n = 50 PWOH; n = 25 PLWH), 37% were female, the mean age was 44 years, and 56% were Black/African American. AEs were generally mild/moderate, with no vaccine-related serious AEs. At 21 days post-dose 2, EBOV glycoprotein-specific binding antibody responder rates were 100% among PWOH and 95% among PLWH; geometric mean antibody concentrations were 6286 EU/mL (n = 36) and 2005 EU/mL (n = 19), respectively. A total of 45 neutralizing and other functional antibody responses were frequently observed. Ebola-specific CD4+ and CD8+ T-cell responses were polyfunctional and durable to at least 12 months post-dose 2. The regimen was well tolerated and generated robust, durable immune responses in PWOH and PLWH. Findings support continued evaluation of accelerated vaccine schedules for rapid deployment in populations at immediate risk. Trial registration: NCT02598388 (submitted 14 November 2015).

Ebola Virus-Specific Neutralizing Antibody Persists at High Levels in Survivors 2 Years After Resolution of Disease in a Sierra Leonean Cohort.

Ebola virus (EBOV) infection results in Ebola virus disease (EVD), an often severe disease with a nonspecific presentation. Since its recognition, periodic outbreaks of EVD continue to occur in sub-Saharan Africa. The 2013-2016 West African EVD outbreak was the largest recorded, resulting in a substantial cohort of EVD survivors with persistent health complaints and variable immune responses. In this study, we characterize humoral immune responses in EVD survivors and their contacts in Eastern Sierra Leone. We found high levels of EBOV IgG in EVD survivors and lower yet substantial antibody levels in household contacts, suggesting subclinical transmission. Neutralizing antibody function was prevalent but variable in EVD survivors, raising questions about the durability of immune responses from natural infection with EBOV. Additionally, we found that certain discrete symptoms-ophthalmologic and auditory-are associated with EBOV IgG seropositivity, while an array of symptoms are associated with the presence of neutralizing antibody.

A scoping review of ethics review processes during public health emergencies in Africa.

BACKGROUND: The COVID-19 pandemic forced governments, multilateral public health organisations and research institutions to undertake research quickly to inform their responses to the pandemic. Most COVID-19-related studies required swift approval, creating ethical and practical challenges for regulatory authorities and researchers. In this paper, we examine the landscape of ethics review processes in Africa during public health emergencies (PHEs). METHODS: We searched four electronic databases (Web of Science, PUBMED, MEDLINE Complete, and CINAHL) to identify articles describing ethics review processes during public health emergencies and/or pandemics. We selected and reviewed those articles that were focused on Africa. We charted the data from the retrieved articles including the authors and year of publication, title, country and disease(s) reference, broad areas of (ethical) consideration, paper type, and approach. RESULTS: Of an initial 4536 records retrieved, we screened the titles and abstracts of 1491 articles, and identified 72 articles for full review. Nine articles were selected for inclusion. Of these nine articles, five referenced West African countries including Liberia, Guinea and Sierra Leone, and experiences linked to the Ebola virus disease. Two articles focused on South Africa and Kenya, while the other two articles discussed more general experiences and pitfalls of ethics review during PHEs in Africa more broadly. We found no articles published on ethics review processes in Africa before the 2014 Ebola outbreak, and only a few before the COVID-19 outbreak. Although guidelines on protocol review and approval processes for PHEs were more frequently discussed after the 2014 Ebola outbreak, these did not focus on Africa specifically. CONCLUSIONS: There is a gap in the literature about ethics review processes and preparedness within Africa during PHEs. This paper underscores the importance of these processes to inform practices that facilitate timely, context-relevant research that adequately recognises and reinforces human dignity within the quest to advance scientific knowledge about diseases. This is important to improve fast responses to PHEs, reduce mortality and morbidity, and enhance the quality of care before, during, and after pandemics.

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses.

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Improved protein splicing through viral passaging.

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEIntervening proteins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.