Current updates on the epidemiology,pathogenesis and development of small molecule therapeutics for the treatment of Ebola virus infections

Ebola virus disease(EVD)is a rare,highly contagious and a deadly disease with a variable fatality rate ranging from 30%to 90%.Over the past two decades,Ebola pandemic has severely affected the sub-Sahara region including Democratic Republic of the Congo(DRC),and Uganda.The causative agents of the most EVD cases are three distinct species out of six Ebolaviruses namely Zaire Ebolavirus(ZEBOV),Sudan Ebolavirus(SUDV)and Bundibugyo Ebolavirus(BDBV).In recent years,significant strides have been made in therapeutic interventions.Notably,the US Food and Drug Administration has approved two monoclonal antibodies:InmazebTM(REGN-EB3)and Ansuvimab or EbangaTM.Additionally,many small molecules are currently in the developmental stage,promising further progress in medical treatment.Addressing the critical need for preventive measures,this review provides an in-depth analysis of the licensed Ebola vaccines-Ervebo and the combination of Zabdeno(Ad26.ZEBOV)and Mvabea(MVA-BN-Filo)as well as the vaccines which are currently being tested for their efficacy and safety in clinical studies.These vaccines might play an important role in curbing the spread and mitigating the impact of this lethal disease.The current treatment landscape for EVD encompasses both nutritional(supportive)and drug therapies.The review comprehensively details the origin,pathogenesis,and epidemiology of EVD,shedding light on the ongoing efforts to combat this devastating disease.It explores small molecules in various stages of the development,discusses patents filed or granted,and delves into the clinical and supportive therapies that form the cornerstone of EVD management.This review aims to provide the recent developments made in the design and synthesis of small molecules for scientific community to facilitate a deeper understanding of the disease and fostering the development of effective strategies for prevention,treatment,and control of EVD.

Global Epidemic of Ebola Virus Disease and the Importation Risk into China: An Assessment Based on the Risk Matrix Method

Objective To analyze the global epidemic status of the Ebola virus disease(EVD) and assess the importation risk into China.Methods Data from World Health Organization reports were used. We described the global epidemic status of EVD from 1976–2021, and assessed and ranked the importation risk of EVD from the diseaseoutbreaking countries into China using the risk matrix and Borda count methods, respectively.Results From 1976–2021, EVD mainly occurred in western and central Africa, with the highest cumulative number of cases(14,124 cases) in Sierra Leone, and the highest cumulative fatality rate(85%) in the Congo. Outbreaks of EVD have occurred in the Democratic Republic of the Congo and Guinea since 2018. The importation risk into China varies across countries with outbreaks of disease.The Democratic Republic of the Congo had an extremely high risk(23 Borda points), followed by Guinea and Liberia. Countries with a moderate importation risk were Nigeria, Uganda, Congo, Sierra Leone,Mali, and Gabon, while countries with a low importation risk included Sudan, Senegal, and Co te d’Ivoire.Conclusion China is under the risk of EVD importation with the globalization and severe epidemic status of EVD. Key attention need to be paid to the Democratic Republic of the Congo, Guinea, and Liberia. Therefore, it is necessary to prevent and prepare in advance for importation risk in China.

Ebola virus disease: past, present and future

Ebola virus disease is one of the most deadly ailments known to mankind due to its high mortality rate(up to 90%) accompanying with the disease. Ebola haemorrhagic fever(EHF) is an infectious disease of animal that can be transmitted to both human and non-human primates. The first epidemic of EHF occurred in 1976 in the Democratic Republic of the Congo. The incubation period of ebola is less than 21 days. Ebola virus infections are depicted by immune suppression and a systemic inflammatory response that leads to damage of the vascular, coagulation and immune systems, causing multi-organ failure and shock. Five genetically distinct members of the Filoviridae family responsible for EHF are as follows: Zaire ebolavirus, Sudan ebolavirus, C?te d'Ivoire ebolavirus, Bundibugyo ebolavirus and Reston ebolavirus. The ongoing 2014 West Africa ebola epidemic has been considered as the most serious panic in the medical field with respect to both the number of human cases and death toll. The natural host for ebola virus is unknown, thus it is not possible to carry out programs to regulate or abolish virus from transmission to people. The ebola virus infection provides little chance to develop acquired immunity causing rapid progression of the disease. It is pertinent to mention that at present, there is no antiviral therapy or vaccine that is helpful against ebola virus infection in humans. The impediment of EHF necessitates much better understanding of the epidemiology of the disease, particularly the role of wildlife, as well as bats, in the spread of ebola virus to humans.

Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

Ebola Virus Disease: General Characteristics, Thoughts, and Perspectives

In December 2013, a new round of Ebola virus disease （EVD） first occurred in a remote countryside of Guinea and then spread in Guinea, Liberia, Sierra Leone, and Nigeria of West Africa. EVD, caused by Ebolavirus and previously known as Ebola hemorrhagic fever, is an acute infectious disease with fatality rates up to 90%. As of August 22, 2014, the number of suspected and confirmed cases was 2615, causing 1427 deaths[I]. On August 8, 2014, World Health Organization announced the current outbreak in West Africa as an international public health emergency. The global epidemic tendency remains ambiguous to date. In recent years, China closely collaborates with West Africa in labor, business, overseas education, and also sends aid medical team there. Thus, the risk of importing the disease cannot be ignored. We conduct this literature review of epidemiology, pathogen, prophylaxis, and treatment to provide evidence for controlling the risk and carrying out effective interventions.

Ebola virus disease: From epidemiology to prophylaxis

The outbreak of Ebola virus disease(EVD) continues to spread through West Africa. Since the first reported EVD in March 2014, the number of cases has increased rapidly, with the fatality rate of >50%. The most prevalent Ebola virus belongs to the species of Zaire ebolavirus, with a mortality rate as high as 90%. Although there were introduced cases in other continents, Africa is the endemic area where fruit bats and apes are suspected to be Ebola virus carriers. The virus might be transmitted from the host animals to humans if humans consume relative raw and contaminated meats; however, human-to-human transmission via close contact is the major route of current outbreaks. EVD happens at any seasons and affected people of any race in any age groups. Direct contact with body fluids of EVD patients and living in the contaminated environment greatly increase the risk of being infected. Transmission viaaerosol is less possible but the transmission via droplet is possible in humans. Thus, health care providers are facing danger of getting Ebola virus infection. So far, there are limited vaccines, drugs and/or therapies to prevent Ebola virus infection or treat EVD. Medical workers should follow the current standard prophylactic procedures. Military forces can orchestrate efficient care to mass EVD casualties. Although it is necessary to speed up the pace of developing effective vaccine and therapeutics for the prevention and treatment of EVD, public health prophylaxis is the most important issue at present to control the spread of this disease cost-effectively.

The Ebola virus:a review of progress and development in research

The Ebola virus was identified in the year 1976 and has caused periodic outbreaks in West African countries.The disease has a case fatality rate up to 90%.Ebola has been classified as a biosafety level four pathogen and there is no currently approved vaccine or treatment for the virus.However,remarkable progress has been demonstrated by researchers in understanding the pathogenicity of the Ebola virus.Several animal models have been cultivated to develop diagnostics,vaccines and therapeutic drugs.

Detection and Analysis of Ebola Virus in Sierra Leone-China Friendship Biosafety Laboratory from March 11 to April 20, 2015

Ebola virus disease reemerged in Western Africa in 2014.Chinese Center for Disease Control and Prevention dispatched the first Ebola virus（EBOV）detection team to run newly established Sierra Leone-China Friendship Biological Safety Laboratory.The aims of study were to understand epidemiology,clinical manifestations and survival time of EBOV in patient＇s blood.A total of 913specimens were tested between March 11 and April20, 2015. EBOV positivity occurred in 7.37% of the blood and 0.53% in throat swabs.

Ebola virus disease: Recent advances in diagnostics and therapeutics

Ebola virus disease(EVD)is associated with haemorrhagic fever in humans and nonhuman primates,with a high rate of fatality(up to 90%).Some outbreaks in human history have proven the lethality of EVD.The recent epidemic of 2014 and 2015 in West Africa was the deadliest of all time(11 284 deaths).To understand the transmission dynamics,we have reviewed the epidemiology of EVD to date.The absence of any licensed vaccines or approved drugs against Ebola virus(EBOV)further highlights the severity and crisis level of EVD.Some organizations(public and private)are making considerable efforts to develop novel therapeutic approaches or vaccines to contain the outbreak of EBOV shortly.Here,we summarized the various potential drugs and vaccines(undergoing multiple phases of clinical trials)that have arisen as an alternative against EBOV,and we highlighted the numerous issues and limitations hindering this process.Alternatively,an increasing focus on strengthening the medical and civic health structure could provide speedy benefits in containing the spread of EVD,as well as offer a resilient foundation for the deployment of novel drugs and vaccines to the affected countries,once such drugs and vaccines become available.

Genetic diversity in Ebola virus:Phylogenetic and in silico structural studies of Ebola viral proteins

Objective:To explore the genetic diversity and the modification of antibody response in the recent outbreak of Ebola Virus.Methods:Sequences retrieved from public databases,the selective pressure analysis and the homology modelling based on the all protein(nucleoprotein.VP35,VP40.soluble glycoprotein,small soluble glycoprotein.VP30,VP24 and polymerase) were used.Results:Structural proteins VP24.VP30.VP35 and VP40 showed relative conserved sequences making them suitable target candidates for antiviral treatment.On the contrary,nucleoprotein.polymerase and soluble glycoprotein have high mutation frequency.Conclusions:Data from this study point out important aspects of Ebola virus sequence variability that for epitope and vaccine design should be considered for appropriate targeting of conserved protein regions.

Phosphorylation sites within Ebola virus nucleoprotein

To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

Obstetrical Surgery in the Context of Ebola Virus Disease (Ebola) in Guinea: Lessons Learned from 2 Cases

In this manuscript, the authors have studied obstetrical surgery in the context of Ebola virus disease in Guinea. No protocol recommends childbirth outside of Ebola treatment center, although it has no technical platform and no qualified providers in this area. These were unknown MVE cases in pregnant women/parturient women aged 25 and 40 years, with no education, who were confirmed in the RT-PCR test after surgical management. To fight Ebola virus transmission, traditional protection protocols must be strengthened. Training, supervision and monitoring of providers are key elements for the protection of staff in the event of an EVD outbreak. Improving working conditions and strengthening hand washing, usage of PPE/EPP, decontamination of equipment with 0.5% chlorine solution, hygiene of premises, immunization of personnel involved, are effective measures to combat EVD.

Knowledge, Attitudes and Preventive Practices on Ebola Virus Disease in the Kintampo Districts of Ghana

This study describes community members’ knowledge of Ebola Virus Disease (EVD), their attitudes and preventive practices. A mixed methods approach was used. A random sample of 1028 community members aged 15 - 65 years was interviewed in a quantitative survey. This was complemented with a qualitative study involving 24 opinion leaders who were carefully selected. The study was conducted in Kintampo North and South districts of Ghana from August 2014 to October 2014. 83% of respondents had heard of EVD, but 62.5% did not know the duration between the time of infection and onset of clinical symptoms. The most popular symptom mentioned spontaneously was bleeding through body orifices (48.6%). Majority of respondents mentioned handshake or skin contact as a mode of transmission (57.3%) and reduced contact with bats as a means to prevent the spread of EVD (58.1%). Knowledge of transmission of body fluids such as faeces, blood or urine was low (<10%), though this varied significantly by socio-demographic group. Majority (94%) of respondents acknowledged that EVD was a serious disease, however, only 58% saw themselves at risk. Current preventive behaviours included: improved hand hygiene (83%) and avoidance of handshakes and physical contact with people (81%). Community members in the Kintampo districts have high level of awareness of EVD, but important gaps in knowledge of EVD still exist, especially concerning body fluids as a mode of transmission. There is the need to intensify educational messages as part of Ghana’s preparedness towards a potential EVD outbreak.

Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present.Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus.The in-depth analysis of the viral protein to find the binding site,active pocket is needed.Here,the authors analyzed the envelope glycoprotein GP2 from Ebola virus.Identification of active pocket and protein draggability within envelope glycoprotein GP2 from Ebola virus was done.According to this assessment,7 active pockets with varied draggability could be identified.

Synthetic Biology Construct of Ebola Virus in Bacteria Surrogate Is Stable and Safe for Rapid Detection Studies in a BSL-2 Laboratory Setting

Rapid detection of virulent pathogens during an outbreak is critical for public health advisories and control of the disease in a population. While many molecular techniques for point of care and clinical diagnosis abound, the US experience with the COVID-19 testing in the early stages of the pandemic underscores the critical importance of determining the appropriate target gene(s) with in-built controls that reliably detect pathogens with high sensitivity and specificity. Assays and research for diagnostics and therapy could be slowed during an epidemic because access to the required BSL-3 and BSL-4 laboratories are limited. So, during the 2014 West Africa Ebola outbreak, we tested the hypothesis that using synthetic cDNA of Ebolavirus in a bacteria surrogate (fit for all lab settings), would remain unmutated and safe after several generations, serving as an effective positive control in research settings, self test and point-of-care detection platforms. Primers were designed for the detection and quantification of the nucleoprotein (NP) gene of the 2014 Makona Ebola strain (KR781608.1, 733 - 1332 bp). To test the stability of artificially inserted translation arrest in the Orf of the model gene, it was edited to include three STOP codons in the RNA transcript using SNAP GENE. The segment was then spliced into a high copy number plasmid, cloned into One ShotTM TOP10 Escherichia coli (Invitrogen), and tested for stability and safety by periodic subculture, extraction and sequencing. Unlike COVID-19, rapid detection of blood-borne etiologies like Ebola requires optimized protocols for blood matrix. Using real-time PCR and newly designed primer pairs, the EBOV surrogate was detected and enumerated in human blood and regular broth and buffers. Based on aligned sequence analysis, the EBOV synthetic NP gene was stable (>99.9999% similarity coefficient) for at least 3 months. Detection sensitivity in broth and blood was at least 100 cells/ml or about 5.8 × 103 to 7.3 × 103 virion equivalents per ml. While the developments of transcription-and-replication-competent virus like particles (trVLP) have made it possible to study the infection and replication cycles of virulent pathogens in BSL-2 laboratories, the simplicity of our model and the reproducibility of detection and enumeration show the utility of synthetic bio-components as positive controls for point of care diagnostic tools. The inserted stop codons remained intact after many generations, suggesting that expressed virulent proteins can be easily silenced in synthetic biology models for research in BSL-1 and 2 and a wide range of pathogens. Synthetic bio-components can thereby aid further research by reducing costs and improving safety for workers and stakeholders.

Laboratory Detection and Diagnosis of Filoviruses

Ebola virus(EBOV) and Marburg virus(MARV),belonging to the Filoviridae family,emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates.As high as 50-90% mortality,filoviruses can cause significant threats to public health.However,so far no specific and efficient vaccine has been available,nor have other treatment methods proved to be effective.It is of great importance to detect these pathogens specific,rapidly and sensitively in order to control future filovirus outbreaks.Here,recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.

Establishment and application of a surrogate model for human Ebola virus disease in BSL-2 laboratory

The Ebola virus(EBOV)is a member of the Orthoebolavirus genus,Filoviridae family,which causes severe hemorrhagic diseases in humans and non-human primates(NHPs),with a case fatality rate of up to 90%.The development of countermeasures against EBOV has been hindered by the lack of ideal animal models,as EBOV requires handling in biosafety level(BSL)-4 facilities.Therefore,accessible and convenient animal models are urgently needed to promote prophylactic and therapeutic approaches against EBOV.In this study,a recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein(VSV-EBOV/GP)was constructed and applied as a surrogate virus,establishing a lethal infection in hamsters.Following infection with VSV-EBOV/GP,3-week-old female Syrian hamsters exhibited disease signs such as weight loss,multi-organ failure,severe uveitis,high viral loads,and developed severe systemic diseases similar to those observed in human EBOV patients.All animals succumbed at 2–3 days post-infection(dpi).Histopathological changes indicated that VSV-EBOV/GP targeted liver cells,suggesting that the tissue tropism of VSV-EBOV/GP was comparable to wild-type EBOV(WT EBOV).Notably,the pathogenicity of the VSV-EBOV/GP was found to be species-specific,age-related,gender-associated,and challenge route-dependent.Subsequently,equine anti-EBOV immunoglobulins and a subunit vaccine were validated using this model.Overall,this surrogate model represents a safe,effective,and economical tool for rapid preclinical evaluation of medical countermeasures against EBOV under BSL-2 conditions,which would accelerate technological advances and breakthroughs in confronting Ebola virus disease.

Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays

Ebola virus （EBOV） and Marburg virus （MARV） are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China, while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus （ZEBOV） and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 101 RNA copies per milliliter of virus culture supernatant, equivalent to 10,000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable

Animal models for filovirus infections

The family Filoviridae, which includes the genera Marburgvirus and Ebolavirus, contains some of the most pathogenic viruses in humans and non-human primates （NHPs）, causing severe hemorrhagic fevers with high fatality rates. Small animal models against filoviruses using mice, guinea pigs, hamsters, and ferrets have been developed with the goal of screening candidate vaccines and antivirals, before testing in the gold standard NHP models. In this review, we summarize the different animal models used to understand filovirus pathogenesis, and discuss the advantages and disadvantages of each model with respect to filovirus disease research.

Niemann-Pick C1蛋白在埃博拉病毒感染中的作用及其靶向药物研究进展

埃博拉病毒属丝状病毒科,具有高传染性,能引起人类和灵长类动物出现严重出血热等症状,病死率高达90%。Niemann-Pick C1(NPC1)蛋白是埃博拉病毒感染过程中表达于宿主细胞内体膜上的一个重要受体,其与埃博拉病毒被组织蛋白酶裂解的糖蛋白(GP)的相互作用是病毒感染宿主的关键环节,介导病毒囊膜与内体膜的融合,进而将病毒基因组释放到宿主细胞。近年来,将NPC1蛋白作为广谱抗丝状病毒药物靶点研发的小分子抑制剂、单克隆抗体和基因治疗药物均有突破性进展。本文介绍了NPC1的结构及其在埃博拉病毒感染中的作用,并对靶向NPC1的小分子抑制剂、单克隆抗体药物和基因治疗药物的研究现状进行总结。