Ebola, the Negative Stranded RNA Virus

Ebola virus (EBV) is a deadly virus that has resulted in a number of deaths during its outbreaks in Africa in 2014–2016 and 2018–2019. This virus causes a hemorrhagic fever like other pathogenic viruses of the Filoviridae family with high mortality rate. The exact reservoir of the ebola virus is not known, but different mammal groups are the source from which it is transferred to the human population. The transmission among the human population is through body fluids of patients and also through aersol droplets in the air. The role of different glycoproteins in the budding formation has helped a lot in understanding the physiology of the ebola virus. Most of these viral glycoproteins synthesis and the replication enzymes offer a good inhibitory target for drug design against the ebola virus. Recently, different groups have claimed the development of a successful vaccine for the ebola virus. However, the availability of the vaccines to the poor population of Africa and other parts of the world is still not practical.


Introduction and background
The Ebola virus was first discovered back in 1976 when its breakout occurred simultaneously in South Sudan and in the area of Ebola River in Yambuku city of Democratic Republic of Congo (Figure 1) [1]. It reemerges in 1990s, 2000s, peaked in 2014 and 2018-2019 (Figure 1). This RNA viral disease caused hemorrhagic fever in humans and non-human primates [2]. The African originated species of Ebola virus fatality rate in humans is nearly 90% [2]. Ebola, Marburg, and Cueva virus are the three infectious viruses with a common ancestor of the family Filoviridae [2]. The word "filo" in Filoviridae is derived from the Latin word film, which means thread like, as these virions appear thread-like under the electron microscope [3]. The Filoviridae family is part of the order Mononegavirales, class Monjiviricetes and phylum Negarnaviricota in the virus taxonomic classification [4,5]. Ebola, also known as Ebola virus disease (EVD/EBOV) and Ebola hemorrhagic fever (EHF) in the past, is a filamentous, enveloped, non-segmented, single stranded negative sense ribonucleic acid (RNA) virus [4]. The replication of Filoviridae family of viruses takes place in the cytoplasm of the host cell [6,7]. The genomic RNA is non-contagious alone because it is not able to serve as a template for protein synthesis [6,7]. To start the transcription of positive-sense messenger RNA (mRNA) the viral protein must connect with the genomic RNA [6,7]. The

History
The Ebola virus is named after a small river, Ebola in Zaire and this viral infection first appeared in 1976 in both South Sudan and Zaire in the African continent [18][19][20]. The international teams of WHO reached the affected area but were unsuccessful in saving more lives and collecting vital information due to lack of medical facilities and illiteracy in rural areas [21]. This virus family becomes genetically more advanced and spread into other areas, and appeared in the united states in 1989 [22]. EBOV did not appear in Africa for 15 years but as the natural reservoir  was not completely eliminated and due to this reason the virus showed itself again in 1994-1996 [23]. During 1994-1996, the new subtype of Ebola Coted'Ivoire was discovered, while the old subtypes were also present. The people infected were living near the tropical forest area. The ethnologist's discovered that the fifth subtype was found in the chimpanzee group with a high fatality rate. They were transferred to Switzerland for suitable facilities and medical care by a group of scientists [24]. The outbreak in 2014 has the highest fatality rates in the history of EBOV [25].

Transmission, symptoms, and treatment
EBOV is a rare and deadly disease caused by infection with one of the Ebola virus strains. Humans and non-human primates are the primary hosts of Ebola [26]. The different types of insectivorous bat genus, especially Mops condylurus and frugivorous bat species are also the host of EBOV [27][28][29]. The fruits eating bat is also a common reservoir of ebola virus [30,31]. The virus is transmitted through contaminated blood and bodily fluids of infected person or animal. Fever, muscle ache, loss of appetite, and fatigue are the common symptoms after onset of EBOV, while in fatal cases, severe vomiting, diarrhea, hemorrhage, septic shock, and multi-organ failure may occur that may lead to death in 30-90% patients [32]. The patients can be treated as the symptoms of EBOV appear in them. Currently, there is no proper Federal Drug Authority (FDA) approved medicines or vaccines. The following are some of the interventions and precautions for survival against EBOV; a. Intravenous fluids (IV) and body salts should be regularly provided to the EBOV infected patients.
b. The blood pressure and oxygen status of patients should be monitored and maintained to the normal level.
c. The blood lost during the illness should be replaced and medicines should be provided to control the blood loss.
d. The patient should increase the intake of fluids.
e. The other infections which develop during treatment should also be treated simultaneously [33].

EBOV genomic organizations
The EBOV genome has nucleotide sequences which are extragenic. The sequences combine to produce secondary structures that contain 3′ and 5′ ends. The genome serves to initiate transcription and genome replication [34,35]. The negative-stranded RNA genome of EBOV contains seven important genes that are arranged in a linear fashion and has a total length of approximately 19 kilobases and it translated into eight proteins [36]. Its sequence is as follows 3′-NP, VP35, VP40, GP, VP30, VP24, L-5′as shown in Figure 2. The VP (35) is RNA-dependent RNA polymerase cofactor, VP(40) is a matrix protein, GP(1,2) is spike glycoprotein, VP(30) is a transcriptional activator, VP (24) is the second protein matrix and (L) is RNA polymerase enzyme [6]. The RNA genome of the virus is coated by a complex of ribonuclear protein (RNP). It is also known as nucleocapsid. The nucleocapsid is composed of major nucleoprotein (NP), minor nucleoproteins that include VP (30), VP (35), and polymerase (L) protein as shown in Figure 3 [37,38]. The above complex is then enveloped in a layer of matrix. The matrix consists of the major and minor matrix protein VP40, VP24, respectively. Then, the matrix proteins are encapsulated by a double layer of lipid which is then peppered with the viral glycoprotein (GP) that help in binding to the host cell [39,40]. All five EBOV species discovered so far has the same GP gene organization which is divided into sGP, GP1, GP2, and ssGP [41,42].

Structure of glycoproteins
It has been observed that most of the viral components play an active role in the pathogenesis and infection of this viral disease, but the major role is played by the glycoproteins of the EBOV [45]. Thus, they possess a significant importance in the virulence of EBOV and targeting its synthesis is one of important step for controlling this infection [45]. EBOV includes different glycoproteins each of which plays their roles in different aspects of viral life cycle. Each gene product has distinct biochemical and biological properties [46,47]. The fourth gene, GP   is formed by a process known as glycosylation in which carbohydrate are linked covalently to the polypeptide chain [48].

Production of glycoproteins
The biosynthesis of glycoproteins occurs by transcription and expression strategies. The precursor of the secreted glycoproteins and full length glycoproteins are the initial products of EBOV glycoprotein. Pre-sGP is transcripted and translated to soluble products sGP and ∆-peptide while glycoprotein GP1, 2 is cleaved to GP1 and GP2. These two fragments are linked together by disulfide-bonding (S▬S). The GP1 helps in binding to the host cell and has a crucial role of EBOV entry across the host cell membrane [49]. The flowchart is shown in Figure 4 [45].

Classification and functions of EBOV glycoproteins
The Ebola virus actually produces the following soluble glycoproteins during infection:

Small secreted glycoprotein of EBOV
Small secreted glycoprotein (ssGP) is translated from part of mRNA. It is nonstructural and generated when two adenosines are combined or one is deleted during transcription [50]. The ssGP has monomeric structure which has identical 295 amino acids from starting point with secreted GP and full length GP but they vary at the C-terminus of the full length glycoprotein [51]. The host ADAM17 metalloprotease enzyme is responsible to generate ssGP. The antibodies that neutralize the viral glycoproteins are quickly blocked by the secreted complex GP [14]. This complex plays a key function in the virus pathogenesis. In spite of extensive necrosis and massive virus production, it therefore contributes to less inflammatory reaction seen during the infection [14]. When the EBOV enters the host cell, the small secretary glycoprotein neutralizes the host cell neutrophils through CD16b and resulting in lymphocyte death and further vascular dysregulation [52].

Secreted or soluble glycoprotein and delta peptide of EBOV
The soluble glycoprotein (sGP) is a non-structural protein with a single frame and has total 364-372 residues. At the N-terminal, 295 residues which also include the signal sequence are similar with full length GP. It differs in the length of C-terminal sequences [46,50]. The formation of sGP is from Precursor (pre-sGP). Unedited mRNA is frequently produced from infected cells which results in the formation of precursor (pre-sGP). Precursor (pre-sGP) is cleaved by the furin protein to produce sGP and delta peptide [46,50]. The orientation of sGP is antiparallel and linked together by disulfide bonding. It possesses dimerization between amino and carboxy groups as shown in Figure 5. The sGP plays a role in the evasion of humoral immune response by absorbing elicited antibodies. It also predicted to be involved in interaction with neutrophils of the host cells and their neutralization [53].

Envelope or full length glycoprotein of EBOV
It is generated on the surface of mature infectious virions and formed when RNA is edited by the process of transcription. The GP protein has 676 amino acids and is a structural polypeptide chain. It is encoded in two frames and functions in attachment and entry of virus into the defenseless cells of the host [48]. The GP is sub-divided into two glycoproteins; GP1 and GP2. They are membrane associated proteins and are linked with each other through disulfide bonding as shown in Figure 6. The GP1 is 130 kDa and GP2 is 24 kDa in size [47]. The GP1 helps in attachment to the receptors which are present on target cells [54]. The interactions allow the binding of viral particles to the dendritic cells and thus facilitates virus into cell entry [48]. GP2 is known as a class I viral fusion protein. Some characteristics of GP2 subunit protein of EBOV are as follows: towards the C-termini is the transmembrane helix while the "fusion-peptide" is found at the N-terminus [55]. The formation of this structure drives to combine the cell membranes of target cells and EBOV. It also helps the particles to enter into the cytoplasm of a healthy cell. The penetration of virus is mediated by the fusion of the membranes of endosomes with the endocytosed virus particle [56].
Survey of the literature showed that currently there are no crystal structures of both secreted and envelope glycoproteins available which are important for designing novel drugs that can inhibit the attachment of these glycoprotein to the host cells receptors and also to halt the activity of the secretary and soluble glycoproteins. In one of our study, we have constructed 3D structures through homology modeling [57]. The molecular dynamics simulation study of the obtained homology models and several drug docking results showed that these proteins can be targeted with small  ligand molecules [57]. Further computation studies and experimental work will prove the usefulness of these small molecules as inhibitor of the EBOV important proteins [57]. The current advancement in cryo-electron microscopy should be utilized for the elucidation of EBOV structure like that of influenza, dengue, Zika and other arboviral diseases [57][58][59][60]. The availability of protein structure will help in molecular dynamics simulation and docking of these important viral proteins with suitable ligands and it will enhance the exploration of novel antiviral drugs [59,60].

Progress in vaccines development for EBOV
A number of researchers have developed vaccines in their laboratories which are in initial stages and have some minor side effects [61]. A recent investigation showed the development of a vaccine when the EBOV glycoproteins like GP and VP40 were injected in the guinea pigs and a protective immune response was observed [62]. The constructed vaccine recovers the infected rhesus macaque in a single dose [62]. Similarly the glycoproteins along with the matrix proteins VP24 and VP40 and in their absence were also tested in guinea pigs [63]. The antibodies produced against these antigens completely cure the tested subjects from EBOV [63]. Several other vaccine of EBOV was developed recently and they showed promising results in murine models with high success rate [64]. Thus, a success in the development of vaccine has been achieved but passing the clinical trials and its availability in the market will take time.

Prevention and control
A number of steps can be taken for the control and prevention of EBOV breakout and its spread. It is always better to avoid traveling to areas of EBOV outbreak and strict quarantine should be imposed on the people of that particular towns. Only medical teams with proper safety masks, cloths should be allowed for the care of patients infected with EBOV [65]. The damaged fruits and vegetables eaten by bat should not be consumed. Bats, monkeys, pangolins, and other wild animals' meat and their utilization in food, medicine, and soaps should be avoided [66]. Contacting these animals should be discouraged. Proper washing of hands and use of sanitizers should be encouraged in schools, airports, shopping malls, trains, and all public areas. It is always necessary for hospitals and health working organizations to share and spread information related to viral diseases outbreak to the general public on time and awareness campaigns should be launched at regular intervals of time. The advancement in cryo-electron microscopy should be utilized for structural resolution of the important enzymes of the EBOV and other fatal viruses so that novel drugs can be synthesized for their inhibition and control [67].

Conclusions
With the 2018 outbreak of EBOV and high probability of future outbreaks and spread, it is highly important to expediate the production of effective vaccine and immunization of vulnerable population across different countries in Africa, that will help in the control of this viral disease. Joint efforts are also needed by the local public health departments and scientific community across the globe for information sharing on different viral outbreaks, vaccine development, and easy access of immunization and medicines.