Ebola Hemorrhagic Fever: Recent Update On Disease Status, Current Therapies And Advances In Treatment

Swiftly growing viruses are a major intimidation to human health. Such viruses are extremely pathogenic like Ebola virus, influenza virus, HIV virus, Zika virus etc . Ebola virus, a type of Filovirus, is an extremely infectious, single-stranded ribonucleic acid virus that infects both humans and apes, prompting acute fever with hemorrhagic syndrome. The high infectivity, severity and mortality of Ebola has plagued the world for the past fifty years with its first outbreak in 1976 in Marburg, Germany, and Frankfurt along with Belgrade and Serbia. The world has perceived about 28,000 cases and over 11,000 losses. The high lethality of Ebola makes it a candidate for use in bioterrorism thereby arising more concern. New guidelines have been framed for providing best possible care to the patients suffering from Ebola virus i.e Grading of Recommendation Assessment, Development And Evaluation (GRADE) methodology to develop evidence-based strategy for the treatment in future outbreak of Ebola virus. No drugs have been approved, while many potent drugs like rVSV-EBOV, Favipiravir, ZMapp are on clinical test for human safety. In this review we will discover and discuss perspective aspects that lead to the evolution of different Ebola variants as well as advances in various drugs and vaccines for treatment of the disease.


INTRODUCTION
The Ebola viruses (EBOV) belonging to family Filoviridae are non-segmented, negative-sense and single-stranded RNA viruses, with a size approximately 19kbp. Firstly discovered in 1976, near to the Ebola River and prevalent regions of central, eastern and western Africa. Ebola is the cause for more than twenty lethal outbreaks of EVD (Ebola Virus Disease) having been investigated in Africa since 1976. A new strain known as Makona was responsible for outbreak in 2014. Ebola virus has been responsible for more than ten thousand deaths.
Ebola haemorrhagic fever (Ebola HF) is known globally as a fatal disease in humans and non-human primates (monkeys, chimpanzees and gorillas).

EBOLA VIRUS DISEASE
EBOV leads to severe hemorrhagic fever resulting in lethal outcome in humans, and several species of non-human primates. Human Ebola outbreaks usually occur suddenly from a indefinite source, that spreads rapidly from person to person. EBOV were earlier categorized as "hemorrhagic fever viruses", based on the clinical appearances, that includes coagulation defects, bleeding, and shock. But it's no longer categorized as such because not all patients affected by Ebola developed substantial hemorrhagic symptoms, that frequently arises at terminal phase of fatal illness (Leroy et al., 2011).

RISK FACTORS AND MODE OF TRANSMISSION
Ebola Virus is transmitted to humans via close contact with the blood, secretions, organs or other bodily fluids of diseased animals. In Africa, infection spread through handling of ill or dead infected chimpanzees, gorillas, fruit bats, monkeys, forest antelope and porcupines. There is no possibility of infection with asymptomatic persons as well as there is very low risk of infection during the incubation period and also during the first week of symptomatic illness.
Higher risk of transmission arises in funerals due to contact with infected corpse (Brainard et al., 2015). Threats of infection are also high among health care workers who take care of the infected, via unprotected contact with bodily fluids. (Francesconi et al., 2003).

ACUTE NEUROLOGICAL MANIFESTATIONS
Throughout acute phase, EVD patients have number of neurological signs and symptoms, while serious neurological manifestations are proportionately unusual. Most commonly, patients will have nonspecific headache, which often presents as an early symptom. Altered mental status, which may range from mild confusion to delirium with hallucinations, may also occur, but may be secondary to a host of variables, including electrolyte abnormalities and shock. Severe cases may lead to coma (West & von saint, 2014). Meningitis and encephalitis associated to EVD have been recognised in recent outbreak, also in prior outbreaks, while the prevalence is not well noted (

DIAGNOSIS
Diagnosis through laboratory testing show low levels of white blood cell and platelet along with elevated liver enzyme levels (Dallatomasina et al., 2015).

PATHOPHYSIOLOGY OF THE DISEASE
Ebola virus invades the tissue via infected fluid that associates with mucosal or skin breaks, where they effectively reproduce in the monocytes, macrophages, and dendritic cells (Beeching et al., 2014). In vitro studies revealed that virus envelope made of glycoprotein is liable for both receptor binding and fusion with host cell membrane. The host immune attack fails to defend as heavily glycosylated viral envelope includes both N-and O-linked glycan providing protection to the virus (Ansari, 2014). The white blood cells carry the virus within the entire body to tissues and organs such as liver, lymph nodes, lungs and spleen. Presence of viral particles within the body and cell injuries caused by viruses promotes release of chemical signals (TNF-a, IL-6 and IL-8) responsible for fever and inflammation. Ebola infection damages human cells by causing infection to the endothelial cells that reduces reliability of the blood vessels and cell adhesion molecules leading to liver damage and improper clotting (Hensley et al., 2005). When a cell is infected with EBOV, receptors located in the cytosol recognizes the infectious molecule associated with virus that leads to activation of protein together with interferon regulatory factor 3 and factor 7 that ultimately triggers signaling cascade and releases type 1 interferon. Type 1 interferon then binds to the receptors IFNAR1 and IFNAR2 of the neighboring cell that further activates STAT1 and STAT2 signaling proteins that moves into the nucleus and triggers gene expression coding antiviral proteins (Leung et al., 2006). EBOV's V24 proteins interferes with the production of antiviral proteins by constraining STAT1 signaling protein entry into neighboring cell nucleus, thus with the inhibition of these immune responses EBOV quickly spreads throughout the body   (Fig.1).

MANAGEMENT
No specific treatment for Ebola hemorrhagic fever has been approved by FDA yet though prospective candidates are present, being in phase 3 or waiting approval. Patients identified to be at risk of infection are immediately isolated and attended by health care personnel that have been trained in bio-safety and outfitted with protective equipment. All equipment utilized must be disinfected due the high risk of contamination from infected bodily fluids and biowaste should be disposed properly. Disinfection can be carried using bleach, detergents etc. Boiling equipment in water for 5 minutes or providing heat of 60°C for about 1 Hour can also eliminate the virus (Cook et al., 2015). Primarily, supportive treatment is provided to increase survival chance of patient involving fluid balance maintenance, treatment of any other infections that may occur and symptomatic treatment (Clark et al., 2012). Supportive care guidelines for care of Ebola patients have been developed utilizing Grading of Recommendations Assessment, Development And Evaluation (GRADE) methods.
Due to gastrointestinal fluid losses, fluid balance must be maintained by utilizing oral re-hydration solutions or intravenous fluids depending on the status of the patient (Hunt et al., 2015;MacDermott & Herberg, 2017). Loperamide may be utilized to decrease the fluid losses in the patient (Chertow et al., 2015). Electrolyte balance must be maintained by replacement and regular monitoring (Hunt et al., 2015). Symptomatic treatment is provided for various complications. Paracetamol is utilized for management of fever and pain, although opioid analgesics may be utilized for severe pain. Ondansetron, metoclopramide are utilized for nausea and vomiting. Antacids are given in case of dysphagia/acid reflux. Anticonvulsants (e.g., phenobarbital) used for management of seizures which may rarely occur. In case of agitation, sedatives are recommended. Hemorrhage usually occurs, thereby clotting factors and blood products are regularly administered. Broad spectrum antibiotics are utilized in case of sepsis (Clark et al., 2012).
Also, contact tracing is performed to control the outbreak by observing all persons that have been in contact with the afflicted person for 21 days for development of signs and symptoms of Ebola Virus (EBOV) infection. In case,

DRUGS ON CLINICAL TESTING
Many therapies recently developed have not been fully tested until now for safety and efficacy (Bishop, 2015). The biggest challenge being that the virus needs bio-safety level 4 facilities for handling (Kortepeter et al., 2008).

Monoclonal Antibodies(mAbs) :
MB-003 is a mixture of three monoclonal antibodies c13C6, h13F6 and c6D8 (Qiu et al., 2014). Treatment with MB-003 increased survival in animals infected with EBOV as it targets the surface glycoprotein of EBOV (Davidson et al., 2015). Another mAB, ZMAb, a combination of m1H3, m2G4 and m4G7 monoclonal antibodies, has been used to produce protection from EBOV for more than 10 weeks in primates (Qiu et al., 2014). After testing the six monoclonal antibodies, ZMapp was created which has m2G4 and m4G7 from ZMAb combined with c13C6 from MB-003 (Davidson et al., 2015). It was found to be highly efficacious in primates but in a trial on patients in Liberia, Guinea, results were found to be lacking efficacy hence further research is required (Prevail et al., 2016). Also, a cocktail of three monoclonal antibodies isolated from mammalian cells, MIL-77 has been sanctioned for use on compassionate basis with an IND for phase-1 trial having been filed (Qiu et al., 2016).

Blood and blood products:
Convalescent Blood and plasma have been shown to have high efficacy due to the fact that patients surviving Ebola disease have been known to produce antibodies against it. Trials done on guinea pig regarding convalescent plasma have shown no outstanding results but no adverse effects were noted deeming the treatment safe (Van Griensven et al., 2016).

Vaccines:
No vaccines have been approved by FDA for treatment of humans till date. But various promising candidates exist and are being tested in clinical trials. rVSV-EBOV, the first proven vaccine to be highly effective against EBOV ChAd3-ZEBOV, developed by National Institute of Allergy and Infectious Diseases and GlaxoSmithKline, has been derived from chimp adenovirus type 3 (ChAd3) and expresses glycoproteins of two Ebola virus species, Zaire and Sudan. This when administered produces antibodies against EBOV. Genetic modifications prevent the virus to replicate in humans. Single dose administration has been proven to be effective with only mild adverse effects (Ledgerwood et al., 2017). MVA-Bn-Filo has been used a booster vaccine along with ChAd3-ZEBOV to elongate immunity (Tapia et al., 2016).
Ad26.ZEBOV, a vaccine developed by Janssen Pharmaceutica, Johnson and Johnson, obtained from human adenovirus serotype 26, expresses the Mayinga Ebola variant glycoprotein. This is given along with a boost vaccine MVA-Bn-Filo. The combination causes elongated immunity up to 8 months (Milligan et al., 2016).
MVA-Bn-Filo (multivalent modified vaccinia Ankara) is a vaccine developed by Bavarian Nordic. It encodes multiple filovirus glycoproteins, thereby, elucidating its use along with other vaccines as a booster vaccine (Sridhar, 2015;Milligan et al., 2016) Ad5.ZEBOV, developed in china, is a relatively newer vaccine utilizing the adenovirus type 5 vector of the more recent 2014 Zaire Guinea strain. Although at an early clinical stage, Phase I and II trials proved good safety although efficacy in humans is still not proven. GamEvac

Interferons:
Interferons have a major role in immune response against viruses. Infected cells release interferons which then activate immune cells and halt viral replication by inhibiting viral gene expression. Use of modified Interferon alpha prolonged survival in Non Human Primates (NHP) (Bradfute, 2017). Conducted studies proved that interferons are moderately effective and treatment is beneficial only in early stages (Dyall et al., 2017).

Antivirals:
Favipiravir, formerly T-705, is a broad spectrum antiviral drug that acts by forming an active metabolite which prevents viral RNA replication by inhibiting RNA dependent RNA polymerase. Oral doses have proven effective against EBOV in animals even after a week post infection. In humans, however, highest effect was noticed in patients with moderate viral spread, but it was ineffective against higher level of viremia. Oral use, good tolerability and availability make it a viable candidate for further studies (Haque et al., 2015;Sissoko et al., 2016).
Brincidofovir, an experimental drug, inhibits viral DNA polymerase, thereby, halting viral replication. Brincidofovir showed anti EBOV activity but the Phase 2 clinical trials were halted due to lack of new cases thereby withdrawn from investigational use (Florescu & Keck, 2014).
Galidesivir (BCX4430) is a broad spectrum antiviral drug developed by BioCryst Pharmaceuticals. This adenosine analogue, prevents viral RNA replication by inhibiting RNA Polymerase. It has shown efficacy against EBOV and Marbug Virus in animals while not effecting human RNA or DNA, thereby, Phase I study were initiated and are still ongoing. The drug is administered intramuscularly, but administration through oral route is also possible. (Warren et al., 2014;Kilgore et al., 2015).
TKM-Ebola was developed by Arbutus Biopharma as an experimental antiviral which was a combination of siRNAs that targeted EBOV proteins, thereby, inhibiting replication. Effect was proven in non-human primates (NHP) but the drug was not able to prove efficacy in humans. After Phase II studies were halted due to no proof of efficacy, the company suspended production (Haque et al., 2015;Thi et al., 2015;Dunning et al., 2016).
AVI-7537, an antiviral developed by Sarepta Therapeutics, consists of phosphorodiamidate morpholino oligomers, which targets EBOV gene. The drug improved survival chance in NHPs that were infected. Phase 1 studies offered evidence of safety and pharmacokinetics but later Phase 1 studies were stopped due to funding issues (Heald et al., 2014;Haque et al., 2015).
JK-05, an antiviral compound developed in china, is reportedly similar to Favipiravir and acts by inhibiting RNA polymerase, thereby, halting replication. The compound has been sanctioned for emergency use and preclinical studies have been reportedly performed. Although no clinical data is available, China has been reported to have sent the drug to west Africa during the outbreak in 2014 (Kilgore et al., 2015).
GS-5734, a novel antiviral drug developed by Gilead Sciences is a nucleoside pro drug that is highly potent against Filo viruses. It metabolizes into an active component which then constrains viral RNA replication. Treatment in NHP produced high efficacy and also presence of active metabolite in various organs suggests its potential in decreasing virion presence in bodily fluids. Its been used in some cases of EBOV infections in humans though the most notable would be its effectiveness in a newborn where GS-5734, along with ZMapp and covalescent blood transfusion decreased viral presence without producing any developmental defects (Warren et

Other Drugs:
FX06, developed by MChE-F4Pharma is a human fibrin derived synthetic peptide currently being tested for vascular leak syndrome. The drug has been administered to two patients, during late stages of EBOV infection. One patient reportedly recovered. However no concrete data regarding efficacy is available (Wolf et al., 2015). rNAPc2 (Recombinant nematode anticoagulant protein c2), an anticoagulant that inhibits tissue factor developed from saliva of hookworm by Arca Biopharma, USA has been evaluated for prevention of thrombosis. The drug has been used to increase survival in EBOV infected NHP but still no data on tolerance or efficacy in humans is available (Geisbert et al., 2003).
During screening of green fluorescent protein of EBOV, various compounds with activity were identified out of which FGI-103, FGI-104 and FGI-106 produced protection through prophylactic treatment in Ebola mouse model ( Azithromycin, an antibiotic was tested for efficacy against EBOV. Increased survival was noted in mouse models however the drug was ineffective when given to guinea pigs (Madrid et al., 2015;Sweiti et al., 2017).
Various FDA approved drugs have been utilized in treatment of Ebola. Amiodarone, an antiarrythmic drug was used in one study involving Ebola patients in Sierra Leone which reported decreased mortality although effectiveness was not established. The use was justified due to inhibition of filo virus cell entry in preclinical studies (Turone, 2014;Sweiti et al., 2017).
Clomiphene and Toremiphene which are FDA approved Selective estrogen receptor modulators (SERM) used for infertility and breast cancer respectively, have been studied for anti-Ebola activity in mouse. These SERMs inhibited EBOV entry, thereby, increasing survival in mouse models (Johansen et al., 2013).
A combination therapy utilizing Atorvastatin, Irbesartan and in some cases Clomiphene, was reportedly given to 100 Ebola patients in Sierra Leone out of which only 2 deaths were reported rest survived (Fedson & Rordam, 2015).
Chloroquine, an antimalarial drug, has been indicated to have some anti-Ebola activity by barring EBOV entry into host cells. Tests in animals produced increased survival (Madrid et al., 2015;Sweiti et al., 2017). Another antimalarial drug, Amodiaquine was used in a comparative study against lumefantrine in Ebola patients. The study found that patients treated with Amodiaquine had a better survival rate than those treated with lumefantrine, although no data on effectiveness of Amodiaquine was given (Gignoux et al., 2016).

CONCLUSION
Although the recent outbreak in Africa (2014) has been declared as an emergency no more by WHO, the fear of future outbreaks prompt researchers throughout the world to take action. Also high lethality and infectivity of Ebola Virus put it as a candidate for use in Bio-terrorism. This along with the emergence of new strains like Makona (west Africa 2014 outbreak) forced development of new therapeutics against the virus, through which many promising drugs like ZMapp, rVSV vaccine etc, have emerged. Though many new therapeutics have been developed or are in development, performing clinical trials to measure their safety and efficacy remains a huge hurdle due to the bio-safety level 4 status of the disease and unavailability of subjects. Even if future outbreaks may occur, it is needless to say that the world is much better equipped to handle Ebola then it was back in 2014. With Better diagnostics, better management and therapeutics future outbreaks will be much better contained and have better survival rates.