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How does sGP of Ebola virus help it to evade host humoral immunity?

How does sGP of Ebola virus help it to evade host humoral immunity?


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During Ebola infection, the viruses secret a lot of sGP. What's its function? Since anti GP antibodies are effective at inhibiting Ebola infection, wouldn't sGP stimulate host immune system to produce more anti GP antibodies? Why does Ebola virus expose its weakness deliberately?


Molecular mechanisms of Ebola virus pathogenesis: focus on cell death

Ebola virus (EBOV) belongs to the Filoviridae family and is responsible for a severe disease characterized by the sudden onset of fever and malaise accompanied by other non-specific signs and symptoms in 30–50% of cases hemorrhagic symptoms are present. Multiorgan dysfunction occurs in severe forms with a mortality up to 90%. The EBOV first attacks macrophages and dendritic immune cells. The innate immune reaction is characterized by a cytokine storm, with secretion of numerous pro-inflammatory cytokines, which induces a huge number of contradictory signals and hurts the immune cells, as well as other tissues. Other highly pathogenic viruses also trigger cytokine storms, but Filoviruses are thought to be particularly lethal because they affect a wide array of tissues. In addition to the immune system, EBOV attacks the spleen and kidneys, where it kills cells that help the body to regulate its fluid and chemical balance and that make proteins that help the blood to clot. In addition, EBOV causes liver, lungs and kidneys to shut down their functions and the blood vessels to leak fluid into surrounding tissues. In this review, we analyze the molecular mechanisms at the basis of Ebola pathogenesis with a particular focus on the cell death pathways induced by the virus. We also discuss how the treatment of the infection can benefit from the recent experience of blocking/modulating cell death in human degenerative diseases.


Abstract

Virus infection presents a significant challenge to host survival. The capacity of the virus to replicate and persist in the host is dependent on the status of the host antiviral defense mechanisms. The study of antiviral immunity has revealed efective antiviral host immune responses and enhanced our knowledge of the diversity of viral immunomodulatory strategies that undermine these defences. This review describes the diverse approaches that are used by RNA viruses to trick or evade immune detection and response systems. Some of these approaches include the specific targeting of the major histocompatibility complex-restricted antigen presentation pathways, apoptosis, disruption of cytokine function and signaling, exploitation of the chemokine system, and interference with humoral immune responses. A detailed insight into interactions of viruses with the immune system may provide direction in the development of new vaccine strategies and novel antiviral compounds.


Ebolavirus evolves in human to minimize the detection by immune cells by accumulating adaptive mutations

The current outbreak of Zaire ebolavirus (EBOV) lasted longer than the previous outbreaks and there is as yet no proven treatment or vaccine available. Understanding host immune pressure and associated EBOV immune evasion that drive the evolution of EBOV is vital for diagnosis as well as designing a highly effective vaccine. The aim of this study was to deduce adaptive selection pressure acting on each amino acid sites of EBOV responsible for the recent 2014 outbreak. Multiple statistical methods employed in the study include SLAC, FEL, REL, IFEL, FUBAR and MEME. Results show that a total of 11 amino acid sites from sGP and ssGP, and 14 sites from NP, VP40, VP24 and L proteins were inferred as positively and negatively selected, respectively. Overall, the function of 11 out of 25 amino acid sites under selection pressure exactly found to be involved in T cell and B-cell epitopes. We identified that the EBOV had evolved through purifying selection pressure, which is a predictor that is known to aid the virus to adapt better to the human host and subsequently reduce the efficiency of existing immunity. Furthermore, computational RNA structure prediction showed that the three synonymous nucleotide mutations in NP gene altered the RNA secondary structure and optimal base-pairing energy, implicating a possible effect on genome replication. Here, we have provided evidence that the EBOV strains involved in the recent 2014 outbreak have evolved to minimize the detection by T and B cells by accumulating adaptive mutations to increase the survival fitness.

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3 A prospective strategy

In the symptomatic stage, the supportive care is mainly toward aggressive prevention of intravascular volume depletion, correction of profound electrolyte-abnormalities, and prevention of the shock complications (Fowler et al., 2014). There is neither precaution for the incubation period nor effective medication for the terminal stage. We propose that a preventative antiviral intervention for the incubation period may lower the consequent virus burden. Furthermore, we propose that an immunomodulatory strategy for the terminal stage may reduce the damages caused by the cytokine storm, thus prolonging the survival time of the patients, making it possible for the patients’ adoptive immunity to recover and beat the infection.

3.1 Type I IFN intervention during the incubation period may render a beneficial outcome

Type I IFNs are cytokines that are secreted by infected cells. They induce cell-intrinsic antiviral states in infected and neighbor cells, they also modulate innate immune responses in a balanced manner and activate the adaptive immune system (le Bon and Tough, 2002 Ivashkiv and Donlin, 2014) (Fig. 3).

Type I IFN controls innate and adaptive immunity and intracellular antiviral programs

This figure was created in reference to Ivashkiv and Donlin (2014) in combination with our own considerations

Type I IFNs have a broad spectrum of antiviral capability, which is able to fight most virus infections. To give an example, the recombinant IFN-α2b has been shown to have a significant nonspecific inhibition of herpes simplex virus, influenza virus, and severe acute respiratory syndrome (SARS) coronary virus replication (Cao et al., 2011).

The Ebola virus blocks the production of type I IFN by APCs including DCs and macrophages it also blocks the IFN-mediated antiviral response by virus proteins. It is a crucial mechanism whereby the viruses evade attacks from the host immune system directly. However, recombinant IFN-α2b (200 IU/ml) can suppress Ebola replication by 100-folds in Vero cells in vitro, early treatment of Ebola-infected cynomolgus with recombinant IFN-α2b delayed onset of viremia and death by several days (Jahrling et al., 1999). In addition, IFN-β treatment was associated with reducing the plasma and tissue viral burden it thus significantly increased survival time in macaques infected with the Ebola virus in vivo (Smith et al., 2013).

Collectively, these results indicate that type I IFN may have therapeutic potential: it is reasonable to postulate direct effects on viral replication as well as adaptive immune response. The observation that the virus titers were not detectable in the incubation period indicates that the vast majority of cells were not infected by the Ebola virus. Based on this fact, we suggest that exogenous administration of type I IFN may induce uninfected cells into an antiviral state. Type I IFN might limit the spread of the Ebola virus and prolong survival if administered immediately after exposure to Ebola viruses.

3.2 Mesenchymal stromal cell therapy during the terminal stage may prevent cytokine storm, massive cell apoptosis, and septic shock

Some patients indeed recovered from the Ebola infection without receiving specific interventions despite Ebola infection’s high mortality rate. This fact suggests that appropriate immune responses can help the body heal itself. The outcome highly depends on two cellular-level competing mechanisms: the apoptosis of endothelial cells executed by autoimmune attacks and vascular regeneration by stem cells. Interestingly, the patients under the age of 21 years had a lower fatality rate of 57%, whereas the fatality rate was up to 94% in those over the age of 45 years, between these two age groups, the fatality rate was 74% (Schieffelin et al., 2014). Stem cells are the essential for the regenerative processes of almost all tissues and organs. The significant differences indicate that patients’ cellular-level healing capacity in different age groups may be relevant to the consumption of age-related changes in the stem cell reservoir. We suggest that treatment with mesenchymal stromal cells (MSCs) during the terminal stage of EVD should be considered.

MSCs are mesoderm-origin, multipotent cells that exist in many tissues and are capable of differentiating into several different cell types, and the numbers or potential of MSC populations in adult organs decline during aging (Stolzing et al., 2008 Toledano et al., 2012). After exogenous administration, MSCs migrate to injured tissue sites where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. MSCs operate through a variety of effector mechanisms on key cells of the innate and adaptive immune systems, mostly through manipulating the cell cycle or inducing maturation arrest without apoptosis (Tyndall and Pistoia, 2009). The therapeutic effects of MSCs may depend largely on the capacity of MSCs to regulate inflammation and tissue homeostasis via an array of immunosuppressive factors, cytokines, growth factors, and differentiation factors. MSCs reduce inflammation by shutting down the TNF-α pathway for immune cell activation and prevent a cytokine storm by inhibiting or disabling T-cell response. MSCs reprogram macrophages, neutrophils, NK cells, DCs, T lymphocytes, and B lymphocytes, all of which counteract sepsis (Plock et al., 2013) (Fig. 4).

Immunological function of MSCs on different cell types of the innate/adaptive immunosystem while MSCs diminish damage and induce repair

This figure was created in reference to Plock et al. (2013) in combination with our own considerations. NK: natural killer IL: interleukin PGE2: prostaglandin E2 IDO: indoleamine 2,3-dioxygenase sHLA-G5: soluble human leukocyte antigen-G5 TNFR: tumor necrosis factor receptor IFN: interferon EGF: epidermal growth factor

MSCs can specifically communicate with the inflammatory microenvironment and this immunoregulatory function of MSCs is highly plastic (Wang et al., 2014). While stimulating tissue repair by mitogenic and angiogenic effects, MSCs inhibit ongoing inflammation, apoptosis, and later fibrosis of injured tissue, and support endothelial cell growth and blood vessel repair this strategy can help avoid the abuse of steroid hormones and various sequelae. It has been reported that graft-versus-host disease (GVHD), systemic lupus erythematosus (SLE), and sepsis can be successfully treated by MSCs (Kebriaei et al., 2009 Sun et al., 2009 Wannemuehler et al., 2012 Pedrazza et al., 2014). It would be interesting to use the available nonhuman primate models of EVD to test such a therapeutic hypothesis.

3.3 Non-specific treatment as an essential strategy against viral diseases in the foreseeable future

It has been almost 40 years since the first Ebola outbreak in 1976. To date, there are no effective therapeutic or prophylactic interventions available to prevent this infection. Several experimental interventions are in early stages of development, and their availability is limited and intermittent (Zhang and Wang, 2014). Positive results were observed in several cases where ZMapp and TKM-Ebola were administrated, but it was unclear whether the positive outcome was due to the drugs or due to better supportive care in Western countries than that of Africa. Clinical trial on an Ebola vaccine developed by Merck and NewLink has been suspended due to unexpected side effects recently. Meanwhile, Médecins Sans Frontières (MSF) has selected three existing interventions for clinical trials, they are Favipiravir approved in Japan for treating influenza, Brincidofivir approved in the USA for virus treatment, and Ebola convalescent serum, and they might all be present less of a supply challenge or are already approved for other purposes. However, preliminary data show that their potency against the Ebola virus is limited. The outlook for the development of Ebola therapeutics is not optimistic.

There are many different kinds of viruses. These viruses mutate their DNA/RNA signatures and evolve rapidly. There has been no specific therapy for even the most common human viral infection such as HBV, HCV, HIV, HPV, and avian influenza. Realistically, the non-specific treatment is an essential strategy against viral diseases in the foreseeable future. When our immune system is given sufficient time for intentional activation when we are exposed to deadly viruses, there is a good chance that it can gear up and eliminate the viruses by itself.


Ebola-GP DNA Prime rAd5-GP Boost: Influence of Prime Frequency and Prime/Boost Time Interval on the Immune Response in Non-human Primates

  • Hadar Marcus
  • , Emily Thompson
  • , Yan Zhou
  • , Michael Bailey
  • , Mitzi M. Donaldson
  • , Daphne A. Stanley
  • , Clement Asiedu
  • , Kathryn E. Foulds
  • , Mario Roederer
  • , Juan I. Moliva
  • & Nancy J. Sullivan

Frontiers in Immunology (2021)

Mining of Ebola virus genome for the construction of multi-epitope vaccine to combat its infection

  • Uma Shankar
  • , Neha Jain
  • , Subodh Kumar Mishra
  • , Md Fulbabu Sk
  • , Parimal Kar
  • & Amit Kumar

Journal of Biomolecular Structure and Dynamics (2021)

Investigating the Interaction between Negative Strand RNA Viruses and Their Hosts for Enhanced Vaccine Development and Production

  • Kostlend Mara
  • , Meiling Dai
  • , Aaron M. Brice
  • , Marina R. Alexander
  • , Leon Tribolet
  • , Daniel S. Layton
  • & Andrew G. D. Bean

Ebolavirus: Comparison of Survivor Immunology and Animal Models in the Search for a Correlate of Protection

Frontiers in Immunology (2021)

Application of Nanotechnology in the COVID-19 Pandemic

International Journal of Nanomedicine (2021)


Contents

EBOV carries a negative-sense RNA genome in virions that are cylindrical/tubular, and contain viral envelope, matrix, and nucleocapsid components. The overall cylinders are generally approximately 80 nm in diameter, and have a virally encoded glycoprotein (GP) projecting as 7–10 nm long spikes from its lipid bilayer surface. [13] The cylinders are of variable length, typically 800 nm, but sometimes up to 1000 nm long. The outer viral envelope of the virion is derived by budding from domains of host cell membrane into which the GP spikes have been inserted during their biosynthesis. Individual GP molecules appear with spacings of about 10 nm. Viral proteins VP40 and VP24 are located between the envelope and the nucleocapsid (see following), in the matrix space. [14] At the center of the virion structure is the nucleocapsid, which is composed of a series of viral proteins attached to an 18–19 kb linear, negative-sense RNA without 3′-polyadenylation or 5′-capping (see following) the RNA is helically wound and complexed with the NP, VP35, VP30, and L proteins this helix has a diameter of 80 nm. [15] [16] [17]

The overall shape of the virions after purification and visualization (e.g., by ultracentrifugation and electron microscopy, respectively) varies considerably simple cylinders are far less prevalent than structures showing reversed direction, branches, and loops (e.g., U-, shepherd's crook-, 9-, or eye bolt-shapes, or other or circular/coiled appearances), the origin of which may be in the laboratory techniques applied. [18] [19] The characteristic "threadlike" structure is, however, a more general morphologic characteristic of filoviruses (alongside their GP-decorated viral envelope, RNA nucleocapsid, etc.). [18]

Each virion contains one molecule of linear, single-stranded, negative-sense RNA, 18,959 to 18,961 nucleotides in length. [20] The 3′ terminus is not polyadenylated and the 5′ end is not capped. This viral genome codes for seven structural proteins and one non-structural protein. The gene order is 3′ – leader – NP – VP35 – VP40 – GP/sGP – VP30 – VP24 – L – trailer – 5′ with the leader and trailer being non-transcribed regions, which carry important signals to control transcription, replication, and packaging of the viral genomes into new virions. Sections of the NP, VP35 and the L genes from filoviruses have been identified as endogenous in the genomes of several groups of small mammals. [21] [22] [23]

It was found that 472 nucleotides from the 3' end and 731 nucleotides from the 5' end are sufficient for replication of a viral "minigenome", though not sufficient for infection. [18] Virus sequencing from 78 patients with confirmed Ebola virus disease, representing more than 70% of cases diagnosed in Sierra Leone from late May to mid-June 2014, [24] [25] provided evidence that the 2014 outbreak was no longer being fed by new contacts with its natural reservoir. Using third-generation sequencing technology, investigators were able to sequence samples as quickly as 48 hours. [26] Like other RNA viruses, [24] Ebola virus mutates rapidly, both within a person during the progression of disease and in the reservoir among the local human population. [25] The observed mutation rate of 2.0 x 10 −3 substitutions per site per year is as fast as that of seasonal influenza. [27]

Proteins encoded by Zaire ebolavirus
Symbol Name UniProt Function
NP Nucleoprotein P18272 Wraps genome for protection from nucleases and innate immunity.
VP35 Polymerase cofactor VP35 Q05127 Polymerase cofactor suppresses innate immunity by binding RNA.
VP40 Matrix protein VP40 Q05128 Matrix.
GP Envelope glycoprotein Q05320 Cleaved by host furin into GP1/2 to form envelope with spikes. Also makes shed GP as a decoy.
sGP Pre-small/secreted glycoprotein P60170 Shares ORF with GP. Cleaved by host furin into sGP (anti-inflammatory) and delta-peptide (viroporin).
ssGP Super small secreted glycoprotein Q9YMG2 Shares ORF with GP created by mRNA editing. Unknown function.
VP30 Hexameric zinc-finger protein VP30 Q05323 Transcriptional activator.
VP24 Membrane-associated protein VP24 Q05322 Blocks IFN-alpha/beta and IFN-gamma signaling.
L RNA-directed RNA polymerase L Q05318 RNA replicase.

There are two candidates for host cell entry proteins. The first is a cholesterol transporter protein, the host-encoded Niemann–Pick C1 (NPC1), which appears to be essential for entry of Ebola virions into the host cell and for its ultimate replication. [28] [29] In one study, mice with one copy of the NPC1 gene removed showed an 80 percent survival rate fifteen days after exposure to mouse-adapted Ebola virus, while only 10 percent of unmodified mice survived this long. [28] In another study, small molecules were shown to inhibit Ebola virus infection by preventing viral envelope glycoprotein (GP) from binding to NPC1. [29] [30] Hence, NPC1 was shown to be critical to entry of this filovirus, because it mediates infection by binding directly to viral GP. [29]

When cells from Niemann–Pick Type C individuals lacking this transporter were exposed to Ebola virus in the laboratory, the cells survived and appeared impervious to the virus, further indicating that Ebola relies on NPC1 to enter cells [28] mutations in the NPC1 gene in humans were conjectured as a possible mode to make some individuals resistant to this deadly viral disease. The same studies described similar results regarding NPC1's role in virus entry for Marburg virus, a related filovirus. [28] A further study has also presented evidence that NPC1 is the critical receptor mediating Ebola infection via its direct binding to the viral GP, and that it is the second "lysosomal" domain of NPC1 that mediates this binding. [31]

The second candidate is TIM-1 (a.k.a. HAVCR1). [32] TIM-1 was shown to bind to the receptor binding domain of the EBOV glycoprotein, to increase the receptivity of Vero cells. Silencing its effect with siRNA prevented infection of Vero cells. TIM1 is expressed in tissues known to be seriously impacted by EBOV lysis (trachea, cornea, and conjunctiva). A monoclonal antibody against the IgV domain of TIM-1, ARD5, blocked EBOV binding and infection. Together, these studies suggest NPC1 and TIM-1 may be potential therapeutic targets for an Ebola anti-viral drug and as a basis for a rapid field diagnostic assay. [ citation needed ]

Being acellular, viruses such as Ebola do not replicate through any type of cell division rather, they use a combination of host- and virally encoded enzymes, alongside host cell structures, to produce multiple copies of themselves. These then self-assemble into viral macromolecular structures in the host cell. [33] The virus completes a set of steps when infecting each individual cell. The virus begins its attack by attaching to host receptors through the glycoprotein (GP) surface peplomer and is endocytosed into macropinosomes in the host cell. [34] To penetrate the cell, the viral membrane fuses with vesicle membrane, and the nucleocapsid is released into the cytoplasm. Encapsidated, negative-sense genomic ssRNA is used as a template for the synthesis (3'-5') of polyadenylated, monocistronic mRNAs and, using the host cell's ribosomes, tRNA molecules, etc., the mRNA is translated into individual viral proteins. [35] [36] [37]

These viral proteins are processed: a glycoprotein precursor (GP0) is cleaved to GP1 and GP2, which are then heavily glycosylated using cellular enzymes and substrates. These two molecules assemble, first into heterodimers, and then into trimers to give the surface peplomers. Secreted glycoprotein (sGP) precursor is cleaved to sGP and delta peptide, both of which are released from the cell. As viral protein levels rise, a switch occurs from translation to replication. Using the negative-sense genomic RNA as a template, a complementary +ssRNA is synthesized this is then used as a template for the synthesis of new genomic (-)ssRNA, which is rapidly encapsidated. The newly formed nucleocapsids and envelope proteins associate at the host cell's plasma membrane budding occurs, destroying the cell. [ citation needed ]

Ebola virus is a zoonotic pathogen. Intermediary hosts have been reported to be "various species of fruit bats . throughout central and sub-Saharan Africa". Evidence of infection in bats has been detected through molecular and serologic means. However, ebolaviruses have not been isolated in bats. [8] [38] End hosts are humans and great apes, infected through bat contact or through other end hosts. Pigs in the Philippines have been reported to be infected with Reston virus, so other interim or amplifying hosts may exist. [38] Ebola virus outbreaks tend to occur when temperatures are lower and humidity is higher than usual for Africa. [39] Even after a person recovers from the acute phase of the disease, Ebola virus survives for months in certain organs such as the eyes and testes. [40]

Zaire ebolavirus is one of the four ebolaviruses known to cause disease in humans. It has the highest case-fatality rate of these ebolaviruses, averaging 83 percent since the first outbreaks in 1976, although fatality rates up to 90 percent have been recorded in one outbreak (2002–03). There have also been more outbreaks of Zaire ebolavirus than of any other ebolavirus. The first outbreak occurred on 26 August 1976 in Yambuku. [41] The first recorded case was Mabalo Lokela, a 44‑year-old schoolteacher. The symptoms resembled malaria, and subsequent patients received quinine. Transmission has been attributed to reuse of unsterilized needles and close personal contact, body fluids and places where the person has touched. During the 1976 Ebola outbreak in Zaire, Ngoy Mushola travelled from Bumba to Yambuku, where he recorded the first clinical description of the disease in his daily log: [42]

The illness is characterized with a high temperature of about 39°C, hematemesis, diarrhea with blood, retrosternal abdominal pain, prostration with "heavy" articulations, and rapid evolution death after a mean of three days.

Since the first recorded clinical description of the disease during 1976 in Zaire, the recent Ebola outbreak that started in March 2014, in addition, reached epidemic proportions and has killed more than 8000 people as of January 2015. This outbreak was centered in West Africa, an area that had not previously been affected by the disease. The toll was particularly grave in three countries: Guinea, Liberia, and Sierra Leone. A few cases were also reported in countries outside of West Africa, all related to international travelers who were exposed in the most affected regions and later showed symptoms of Ebola fever after reaching their destinations. [43]

The severity of the disease in humans varies widely, from rapid fatality to mild illness or even asymptomatic response. [44] Studies of outbreaks in the late twentieth century failed to find a correlation between the disease severity and the genetic nature of the virus. Hence the variability in the severity of illness was suspected to correlate with genetic differences in the victims. This has been difficult to study in animal models that respond to the virus with hemorrhagic fever in a similar manner as humans, because typical mouse models do not so respond, and the required large numbers of appropriate test subjects are not easily available. In late October 2014, a publication reported a study of the response to a mouse-adapted strain of Zaire ebolavirus presented by a genetically diverse population of mice that was bred to have a range of responses to the virus that includes fatality from hemorrhagic fever. [45]


Ebola Virus Disease (EVD)

Ebola virus (EBOV) VP40 is a major driving force of nascent virion production and a negative regulator of genome replication/transcription. Here, we showed that the YIGL sequence at the C-terminus of EBOV VP40 is important for virus-like particle (VLP) production and the regulation of genome replication/transcription. Accordingly, a mutation in the YIGL sequence caused defects in VLP production and genome replication/transcription. The residues I293 and L295 in the YIGL sequence were particularly critical for VLP production. Furthermore, an in silico analysis indicated that the amino acids surrounding the YIGL sequence contribute to intramolecular interactions within VP40. Among those surrounding residues, F209 was shown to be critical for VLP production. These results suggested that the VP40 YIGL sequence regulates two different viral replication steps, VLP production and genome replication/transcription, and the nearby residue F209 influences VLP production.

ICTV Virus Taxonomy Profile: Filoviridae

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15–19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.

Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein–protein interactions on a functional level.

Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays and ELISA

The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck hindering research efforts. A safe alternative to working with native viruses are pseudotyped viruses (PV) which are non-replicating particles bearing surface glycoprotein(s) that can be used for antibody detection. The aim of this study was to create a diagnostic tool to distinguish between genera and species of pathogenic filoviruses (e.g. neutralization tests and ELISA), avoiding the cross reactivity currently seen. High titre PVs bearing the receptor glycoprotein (GP) of different filovirus species, plus specific epitope chimeras, were successfully generated. Next, lyophilisation studies to assess particle stability/degradation transportation and long-term storage were conducted. Filoviruses maintained their titres for at least 1.5 years after lyophilisation when kept in temperatures of up to 4 °C, with all filovirus genera following a similar trend. At higher temperatures, PVs degraded to unworkable titres. Reconstituted PVs also performed well in neutralisation assays. A chimeric cuevavirus GP bearing ebolavirus (Zaire sp.) epitopes KZ52 and 1 H3 retained infectivity, with average titres of approximately 1×10 7 RLU ml −1 , similar to wild type, indicating its structure was not compromised. These chimeras are now being assessed in neutralisation tests using specific monoclonal antibodies and incorporated into ELISA with PVs as antigens. The data suggests lyophilised PVs are amenable to long-term storage, and their GPs can be modified to create artificial antigens for diagnostics and serosurveillance.

How myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases

Up to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014–2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host’s innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.

Ebola returns to its Congo Basin heartland

Immunogenicity of propagation-restricted vesicular stomatitis virus encoding Ebola virus glycoprotein in guinea pigs

Vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (GP) in place of the VSV glycoprotein G (VSV/EBOV-GP) is a promising EBOV vaccine candidate which has already entered clinical phase 3 studies. Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and in vitro virus-spreading inhibition test that employed recombinant VSV/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. However, VSV/EBOV-GP(F88A) rapidly reverted to a fully propagation-competent virus indicating that a single-point mutation is not sufficient to maintain the propagation-restricted phenotype.

Different effects of two mutations on the infectivity of Ebola virus glycoprotein in nine mammalian species

Ebola virus (EBOV), which belongs to the genus Ebolavirus , causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann–Pick C1.

The serology of Ebolavirus – a wider geographical range, a wider genus of viruses or a wider range of virulence?

Viruses of the genus Ebolavirus are the causative agents of Ebola virus disease (EVD), of which there have been only 25 recorded outbreaks since the discovery of Zaire and Sudan ebolaviruses in the late 1970s. Until the west African outbreak commencing in late 2013, EVD was confined to an area of central Africa stretching from the coast of Gabon through the Congo river basin and eastward to the Great Lakes. Nevertheless, population serological studies since 1976, most of which were carried out in the first two decades after that date, have suggested a wider distribution and more frequent occurrence across tropical Africa. We review this body of work, discussing the various methods employed over the years and the degree to which they can currently be regarded as reliable. We conclude that there is adequate evidence for a wider geographical range of exposure to Ebolavirus or related filoviruses and discuss three possibilities that could account for this: (a) EVD outbreaks have been misidentified as other diseases in the past (b) unidentified, and clinically milder, species of the genus Ebolavirus circulate over a wider range than the most pathogenic species and (c) EVD may be subclinical with a frequency high enough that smaller outbreaks may be unidentified. We conclude that the second option is the most likely and therefore predict the future discovery of other, less virulent, members of the genus Ebolavirus .

Chloroquine inhibited Ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model

Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. Owing to the lack of approved therapies, screening programmes of potentially efficacious drugs have been undertaken. One of these studies has demonstrated the possible utility of chloroquine against EBOV using pseudotyped assays. In mouse models of EBOV disease there are conflicting reports of the therapeutic effects of chloroquine. There are currently no reports of its efficacy using the larger and more stringent guinea pig model of infection. In this study we have shown that replication of live EBOV is impaired by chloroquine in vitro . However, no protective effects were observed in vivo when EBOV-infected guinea pigs were treated with chloroquine. These results advocate that chloroquine should not be considered as a treatment strategy for EBOV.

The 2014 Ebola virus disease outbreak in West Africa

On 23 March 2014, the World Health Organization issued its first communiqué on a new outbreak of Ebola virus disease (EVD), which began in December 2013 in Guinée Forestière (Forested Guinea), the eastern sector of the Republic of Guinea. Located on the Atlantic coast of West Africa, Guinea is the first country in this geographical region in which an outbreak of EVD has occurred, leaving aside the single case reported in Ivory Coast in 1994. Cases have now also been confirmed across Guinea as well as in the neighbouring Republic of Liberia. The appearance of cases in the Guinean capital, Conakry, and the transit of another case through the Liberian capital, Monrovia, presents the first large urban setting for EVD transmission. By 20 April 2014, 242 suspected cases had resulted in a total of 147 deaths in Guinea and Liberia. The causative agent has now been identified as an outlier strain of Zaire Ebola virus. The full geographical extent and degree of severity of the outbreak, its zoonotic origins and its possible spread to other continents are sure to be subjects of intensive discussion over the next months.

Coronaviruses in bats from Mexico

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (β)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs four were β-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance and that a single species/genus of bat can contain multiple CoVs. A β-CoV with 96.5 % amino acid identity to the β-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure

Marburg virus (MARV) and Ebola virus, members of the family Filoviridae , cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

Lethality and pathogenesis of airborne infection with filoviruses in A129 α/β −/− interferon receptor-deficient mice

Normal immunocompetent mice are not susceptible to non-adapted filoviruses. There are therefore two strategies available to establish a murine model of filovirus infection: adaptation of the virus to the host or the use of genetically modified mice that are susceptible to the virus. A number of knockout (KO) strains of mice with defects in either their adaptive or innate immunity are susceptible to non-adapted filoviruses. In this study, A129 α/β −/− interferon receptor-deficient KO mice, strain A129 IFN-α/β −/−, were used to determine the lethality of a range of filoviruses, including Lake Victoria marburgvirus (MARV), Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV) and Côte d’Ivoire ebolavirus (CIEBOV), administered by using intraperitoneal (IP) or aerosol routes of infection. One hundred percent mortality was observed in all groups of KO mice that were administered with a range of challenge doses of MARV and ZEBOV by either IP or aerosol routes. Mean time to death for both routes was dose-dependent and ranged from 5.4 to 7.4 days in the IP injection challenge, and from 10.2 to 13 days in the aerosol challenge. The lethal dose (50 % tissue culture infective dose, TCID 50 ) of ZEBOV for KO mice was 50 ml −1 when administered by either the IP or aerosol route of infection for MARV the lethal dose was 50 ml −1 by the IP route of infection and 50 ml −1 by the aerosol route. In contrast, there was no mortality after infection with SEBOV or REBOV by either IP or aerosol routes of infection all the mice lost weight (

15 % loss of group mean body weight with SEBOV and

7 % with REBOV) but recovered to their original weights by day 14 post-challenge. There was no mortality in mice administered with CIEBOV via the IP route of infection and no clinical signs of infection were observed. The progression of disease was faster following infection with ZEBOV than with MARV but ultimately both viruses caused widespread infection with high titres of the infectious viruses in multiple organs. Histopathological observations were consistent with other animal models and showed widespread organ damage. This study suggests that MARV and ZEBOV are more virulent when administered via the IP route rather than by aerosol infection, although both are highly virulent by either route. The KO mouse may provide a useful model to test potential antiviral therapeutics against wild-type filoviruses.

Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP 1,2 ) and the ribonucleoprotein (RNP) complex components however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP 1,2 , the RNP components and VP40, but not GP 1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.


VASCULAR PERMEABILITY AND COAGULATION DEFECTS

In addition to inducing apoptosis within lymphocytes, the large release of TNF-α from infected monocytes/macrophages can increase endothelial permeability, resulting in vascular leakage [48]. In vitro studies show that increased endothelial permeability is temporally associated with the release of TNF-α from MARV-infected human monocytes/macrophages [93]. Similarly, the release of nitric oxide, which is an important effector molecule in the homeostasis of the cardiovascular system, can result in the loss of vascular smooth-muscle tone and hypotension [53, 94]. In addition, ZEBOV infection of macrophages leads to the up-regulation of surface TF as well as the release of membrane microparticles containing TF, resulting in the overactivation of the extrinsic pathway of coagulation and the development of disseminated intravascular coagulation [58]. Expression of TF is further up-regulated by proinflammatory cytokines, notably IL-6, which are abundant during acute ZEBOV infection, exacerbating the intravascular coagulation phenotype [95].

In addition, ZEBOV-induced paralysis of the host response facilitates viral dissemination to hepatocytes, adrenal cortical cells, and endothelial cells of connective tissue in cynomolgus macaques [45] (Fig. 2). Hepatocellular necrosis results in decreased synthesis of coagulation proteins, whereas infection and necrosis of adrenocortical cells may negatively affect blood pressure homeostasis, leading to hemorrhage [45]. Coagulation abnormalities are initiated early during ZEBOV infection in cynomolgus macaques. Specifically, a dramatic decrease in plasma levels of anticoagulant protein C occurs as early as 2 d postinfection. This is followed by an increase of both tissue plasminogen activator, which is involved in dissolving blood clots, and fibrin-degradation products (d-dimers) at d 5 postinfection [58]. Thrombocytopenia and prolonged prothrombin time are indicators of dysregulated blood coagulation and fibrinolysis during ZEBOV infection and may manifest as petechiae, ecchymoses, mucosal hemorrhages, and congestion [39, 58]. Toward the terminal stage of the infection, and after the onset of hemorrhagic abnormalities, ZEBOV replicates in endothelial cells [46]. However, although infection of endothelial cells is thought to have a role in the pathogenesis, the molecular mechanisms of endothelial damage are not yet fully understood.


History of Ebola Virus Disease

Ebola virus disease (EVD), one of the deadliest viral diseases, was discovered in 1976 when two consecutive outbreaks of fatal hemorrhagic fever occurred in different parts of Central Africa. The first outbreak occurred in the Democratic Republic of Congo (formerly Zaire) in a village near the Ebola River, which gave the virus its name. The second outbreak occurred in what is now South Sudan, approximately 500 miles (850 km) away.

Initially, public health officials assumed these outbreaks were a single event associated with an infected person who traveled between the two locations. However, scientists later discovered that the two outbreaks were caused by two genetically distinct viruses: Zaire ebolavirus and Sudan ebolavirus. After this discovery, scientists concluded that the virus came from two different sources and spread independently to people in each of the affected areas.

Viral and epidemiologic data suggest that Ebola virus existed long before these recorded outbreaks occurred. Factors like population growth, encroachment into forested areas, and direct interaction with wildlife (such as bushmeat consumption) may have contributed to the spread of the Ebola virus.

Since its discovery in 1976, the majority of cases and outbreaks of Ebola Virus Disease have occurred in Africa. The 2014-2016 Ebola outbreak in West Africa began in a rural setting of southeastern Guinea, spread to urban areas and across borders within weeks, and became a global epidemic within months.

Identifying a Host

Following the discovery of the virus, scientists studied thousands of animals, insects, and plants in search of its source (called reservoir among virologists, people who study viruses). Gorillas, chimpanzees, and other mammals may be implicated when the first cases of an EVD outbreak in people occur. However, they &ndash like people &ndash are &ldquodead-end&rdquo hosts, meaning the organism dies following the infection and does not survive and spread the virus to other animals. Like other viruses of its kind, it is possible that the reservoir host animal of Ebola virus does not experience acute illness despite the virus being present in its organs, tissues, and blood. Thus, the virus is likely maintained in the environment by spreading from host to host or through intermediate hosts or vectors.

African fruit bats are likely involved in the spread of Ebola virus and may even be the source animal (reservoir host). Scientists continue to search for conclusive evidence of the bat&rsquos role in transmission of Ebola. 1 The most recent Ebola virus to be detected, Bombali virus, was identified in samples from bats collected in Sierra Leone. 2

Understanding Pathways of Transmission

The use of contaminated needles and syringes during the earliest outbreaks enabled transmission and amplification of Ebola virus. During the first outbreak in Zaire (now Democratic Republic of Congo &ndash DRC), nurses in the Yambuku mission hospital reportedly used five syringes for 300 to 600 patients a day. Close contact with infected blood, reuse of contaminated needles, and improper nursing techniques were the source for much of the human-to-human transmission during early Ebola outbreaks. 3

In 1989, Reston ebolavirus was discovered in research monkeys imported from the Philippines into the U.S. Later, scientists confirmed that the virus spread throughout the monkey population through droplets in the air (aerosolized transmission) in the facility. However, such airborne transmission is not proven to be a significant factor in human outbreaks of Ebola. 4 The discovery of the Reston virus in these monkeys from the Philippines revealed that Ebola was no longer confined to African settings, but was present in Asia as well.

By the 1994 Cote d&rsquoIvoire outbreak, scientists and public health officials had a better understanding of how Ebola virus spreads and progress was made to reduce transmission through the use of face masks, gloves and gowns for healthcare personnel. In addition, the use of disposable equipment, such as needles, was introduced.

During the 1995 Kikwit, Zaire (now DRC) outbreak, the international public health community played a strong role, as it was now widely agreed that containment and control of Ebola virus were paramount in ending outbreaks. The local community was educated on how the disease spreads the hospital was properly staffed and stocked with necessary equipment and healthcare personnel was trained on disease reporting, patient case identification, and methods for reducing transmission in the healthcare setting. 5

In the 2014-2015 Ebola outbreak in West Africa, healthcare workers represented only 3.9% of all confirmed and probable cases of EVD in Sierra Leone, Liberia, and Guinea combined. 6 In comparison, healthcare workers accounted for 25% of all infections during the 1995 outbreak in Kikwit. 7 During the 2014-2015 West Africa outbreak, the majority of transmission events were between family members (74%). Direct contact with the bodies of those who died from EVD proved to be one of the most dangerous &ndash and effective &ndash methods of transmission. Changes in behaviors related to mourning and burial, along with the adoption of safe burial practices, were critical in controlling that epidemic. 8

1 Baseler L., Chertow D, et. Al. The Pathogenesis of Ebola Virus Disease. Annu. Rev. Pathol. Mech. Dis. 2017. 12:387&ndash418.

3 Amundsen, S. Historical Analysis of the Ebola Virus: Prospective Implications for Primary Care Nursing Today. Clinical Excellence for Nurse Practitioners. Vol 2. No 6. 1998. 343-351.

4 Baseler L., Chertow D, et. Al. The Pathogenesis of Ebola Virus Disease. Annu. Rev. Pathol. Mech. Dis. 2017. 12:387&ndash418.

5 Amundsen, S. Historical Analysis of the Ebola Virus: Prospective Implications for Primary Care Nursing Today. Clinical Excellence for Nurse Practitioners. Vol 2. No 6. 1998. 343-351.

6 WHO. Health worker Ebola infections in Guinea, Liberia and Sierra Leone: A Preliminary Report 21 May 2015. Accessed June 20, 2017. http://www.who.int/hrh/documents/21may2015_web_final.pdf pdf icon [917 &ndash KB] external icon

7 Khan A. et al. The Reemergence of Ebola Hemorrhagic Fever, Democratic Republic of the Congo, 1995. J Infect Dis (1999) 179 (Suppl 1): S76-86.

8 Baseler L., Chertow D, et. Al. The Pathogenesis of Ebola Virus Disease. Annu. Rev. Pathol. Mech. Dis. 2017. 12:387&ndash418.



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