key: cord-0074944-uieywrg4 authors: Ahad, Abdul; Mahnoor, Sabahat; Zaid, Muhammad; Ali, Muhammad; Afzal, Muhammad Sohail title: Ebolavirus: Infection, Vaccination and Control date: 2022-02-21 journal: Mol Gen Microbiol Virol DOI: 10.3103/s0891416821050037 sha: 36a12c2a9fce5121a6445ec2604089c95b290c98 doc_id: 74944 cord_uid: uieywrg4 Members of the genus Ebolavirus (family Filoviridae) are among the deadliest viral pathogens spread throughout the world with severe rate of mortality, at least 90% in some outbreaks. Their virions are filamentous and enveloped with enclosed negative-sense single-stranded RNA genome. The genome potentially expresses seven structural and nonstructural proteins. The replication cycle is complex consisting of multiple molecular processes and interactions with human-host factors and proteins. Due to high mortality rate of infection, the studies regarding cure is still infancy. This review covers the current understanding of the virus replication cycle and vaccine development, and herbal treatments to control Ebola covering the available literature on the subject. Ebolavirus disease (EVD) is among the world's lifethreatening infections repeatedly re-emerging specifically in Africa from the last 50 yr [1] . EVD named after Ebola River that passes close to the Yambuku town, the place where first outbreak has occurred. Ebola outbreak in 1976 caused the death of 151 patients (out of 281) in Sudan. They were the vaccine workers dealing with the specimen of African green monkey. In Yambuku, 280 individuals died in 318 infected individuals [2] . In December 2013, the world's largest Ebola outbreak occurred in Guinea, Liberia, and Sierra Leone [3] . It was the first outbreak outside the Central Africa where Zaire ebolavirus was the causal virus [4] . The World Health Organization (WHO), in August 2014, nominated the West African flare-up as a Public Health Emergency of International Concern [5] . Whereas, WHO reported 10 141 cases and 4922 deaths in October 2014 [6] . Recently, an epidemic has been reported from Congo with some 2000 deaths (Table 1) . Ebola hemorrhagic fever is zoonotic viral disease that initially happened in 1976 in the provincial territories of Central Africa [7] . The non-specific symptoms that can be observed in an Ebola infected individual may be fever, abdominal pain, vomiting, diarrhea, sore throat, muscle pain, cough, headache, nausea, bleeding, rashes, organ failure, and arthralgia [8] . Upon contamination, the infection moves through direct contact with body liquids like blood etc. Along with this, the virus was detected in individual's body fluids like tears, sweat, saliva, breast milk, semen, urine [9] . Amazingly, the infected dead-body may also transmit the disease. Ebola is among those pathogens that may replicate in a dead body and may release with the infectious fluid [10] . This review is an effort to encompass EVD in context with the literature and knowledge available on the viral physiology, pathology, pathogenesis and the host immune responses. Despite the fact that there is no cure or commercially approved vaccine for Ebolavirus, the recombinant vesicular stomatitis virus-Zaire ebolavirus (VSV-EBOV) immunization was first utilized in Guinea in 2016, and the Democratic Republic of Congo in 2017-2018 [11] . Some other treatments and vaccines are monoclonal antibody cocktail, "Zmapp", and a chimpanzee adenovirus-derived vaccine, "cAd3-ZEBOV", respectively [12] . Clinical trials are summarized in Table 2 . Natural reservoir hosts for EBOV is yet to be confirmed. Studies showed that the fruit bats may be associated with it. EBOV outbreaks among humans is associated with their direct contact to fruit bats. Epidemiological studies also indicated that fruit bat could be the intermediate host. Studies also showed that EBOV outbreaks could link drastic shift from dry to wet condition. Further studies indicated that EBOV outbreaks could be linked with higher humidity and lower temperature. EBOV infection transmission is possible from human to human through air/space, touching/bath and sex/blood. The replication cycle and pathogenesis of Ebolavirus is complex that involve binding of viral proteins with the host factors. Ebolavirus spike glycoprotein (GP) accomplishes entry into the host cell for replication that might also trigger micropinocytosis pathway. The host cathepsin L and cathepsin B process GP into GP1. However, cathepsin L and B are not always required for EBOV replication. The GP1 then interacts with Niemann-pick C-1 (NPC-1) in the late endosome followed by its fusion with the vesicle-membrane. As a result, the virion contents (including the virus-encoded polymerase) are released in the cytoplasm. Viral mRNAs are transcribed using the polymerase and translated using host translation machinery. Viral genomic-RNA replication commences once enough nucleoprotein are expressed to encapsulate the newly synthesized negative-sense (-ve) single-stranded (ss) RNA genome. Finally, VP40 helps in the assembly of virion particles and in budding. The VP24 protein suppresses host immune system thereby inhibiting interferon signaling ( Fig. 1 Fig. 2 ). These proteins are transcribed in an order of 3'-NP-VP35-VP40-GP-VP30-VP24-L-5' (Fig. 1) Even though there is no exact treatment for Ebolavirus disease, some precautionary and control measures must considered. They include, (1) administer intravenous fluid to avoid dehydration, (2) maintain normal blood pressure, (3) blood clotting through medications, (4) maintain the cleanliness around the patient, (5) use of breathing devices and drugs to control fever, (6) avoid contact with healthy individuals, however, if necessary, personal protective equipment must be worn [13] . DNA vaccines or DNA immunization in comparison with live/attenuated vaccines has a number of advantages, where plasmids can be used to induce antigenic response. They are non-infectious and easily adaptable for evolving pathogens. They can easily be produced in large amounts. However, booster doses are required for immunity. They can induce cellmediated response as well as humoral response. One major advantage is that the existing immunity may not be a problem with DNA vaccines [14] . First successful vaccination was done in 1998 on mice in which four doses having plasmids encoding Zaire ebolavirus Glycoprotein (ZEBOV-GP) and Zaire Ebolavirus Nucleoprotein (ZEBOV-NP) were used in the mice [15] . Unfortunately, there is no data available for DNA vaccine for Non-Human Primates alone. Studies have shown that DNA vaccine combined with recombinant vectors like adenovirus encoding GP protected the cynomolgus macaques from lethal EBOV challenge [16] . A clinical trial was conduct in which 3 doses of DNA vaccine were given to the 20 participants encoding ZEBOV-GP, NP and SEBOV-GP. No side effects were found in participants and induction of antibodies and CD4* T-cells were observed. CD8* T-cells were also detected in 8 of the participants [17] . Another DNA vaccine was developed having two plasmids encoding the wild type glycoproteins of EBOV and SEBOV. It was experimented on 10 healthy persons in the US. A dose of 4 mg of dose was administered thrice during 4-weeks period followed by a booster dose after 32 weeks. As the T-cell response was NP poor, a booster dose was therefore, needed for the longevity of vaccination [18] . Combination of different plasmids encoding ZEBOV-GP, SEBOV-GP and Marburgvirus glycoprotein (MARV-GP) was proved effective to protect guinea pigs from lethal ZEBOV challenge. Studies have shown that induction of antibodies and T-cells were observed in mouse model and was 100% protected from lethal ZEBOV challenge [19] . Further experimentation required to analyses the effectiveness of this vaccine in the NHP. Main requirement of DNA vaccine is the need of multiple/booster doses for the longevity of vaccination. The use of replication deficient EBV was one of the earliest strategies used to create vaccine. The mutant EBOVAVP30 (deletion of VP30 gene) make the virus unable to replicate. VP30 is necessary for the reasonability of the infection and for replication along these lines encoding viral polymerase cofactors. It was considered a key step to use a whole virus for vaccination as it could provide a better protection against Ebolavirus by triggering the immune system using the complete viral protein and the genetic material. To check the safety assessment EBOVAVP30 was introduced in immunocompromised mice (STAT-1 double knockout mice) and surprisingly mice showed no clinical signs or viremia. On further investigation, it was found that EBOVAVP30 is completely safe for mice and guinea pigs. EBOVAVP30 proved to be very effective because it is genetically stable even after serial passage in Vero cells expressing VP30 [20] . Later on EBOVAVP30 proved 100% safe and protective in NHP when one or two doses of 10 7 infectious unit was given to them. The subjects were fully protected against lethal EBOV infection. Some of them had shown little signs of viremia but they eventually clear the virus. To make vaccine more protective, it was treated with hydrogen peroxide but the immune response of vaccine that was treated with hydrogen peroxide was slightly lower as compared to nontreated vaccine but it was considered sufficient [21] . Adenovirus family based vaccines have been proven effective against many infections. AdHu5 (adenovirus serotype (5) is an attractive choice as vaccine vector due to its potential to induce effective immune response. Deletion of E1 gene makes them unable to replicate and further E3 and E4 deletion increased the size of vector [22] . A single dose of AdHU5 expressing EBOV GP and NP presented a total protection in NHP against ZEBOV challenge. The issues with AdHu5 is the pre-existing immunity, it is estimated that 30 to 50% of the population in North America and more than 90% of the population in developing countries have pre-existing immunity to this vector, making it less applicable. Another adenovirus vector rAd5 with EBOV GP provide 100% protection against EBOV lethal challenge in NHP. The higher amount of rAd5 may be toxic but the non-replicating behavior make them safe to use. The vector Ad5 having codon optimized ZEBOV GP that was tested on rodent models with much lower dose successfully survived EBOV lethal challenge [23] . Again the main problem associated with Ad5 is the pre-existing immunity in certain population, varying 60 to 90% but high dose of rAd5 may overcome the problem. The dose required is about 1.6 × 10 11 vp. To avoid preexisting immunity adenovirus serotypes having a very low seroprevalence such as Ad26, chimpanzee CHAd3 and Ad35 were used to substitute Ad5. However, there is not much data available of EBOV challenges for these vaccines but studies have shown that they can induce antigen-specific antibodies and T cell response in NHP. Ad26-EBOV that was tested on NHP provided only partial protection. One month later after providing heterologous Ad35-EBOV complete protection was witnessed [24] . Vesiculovirus stomatitis virus (VSV) is a negative stranded RNA infection belonging to Rhabdoviridae family that can infect cattle and in humans it can cause little illness. VSV can be utilized in replication compe-tent form so it can influence a high humoral and cellular response in humans. VSV expressing Ebolavirus glycoproteins conferred a complete protection in mice against ZEBOV challenge [25] . After successful vaccination in mice, it was tested on NHP thereby conferring complete protection against ZEBOV challenge. VSV also can give cross protection against different species as well. Recombinant VSV expressing SEBOV GP, ZEBOV GP, ICEBOV GP in equal amount administered to NHP showed 100% survival and protection against viral challenges. In another studies to measure the multivalent protective ability of this vaccine rVSV encoding MARV GP, TEBOV GP, SEBOV GP, EBOV GP was tested and the results showed that cross protection is possible against all these viruses. Another study has shown that this vaccine can be safe for immunocompromised people as well [26] . In 2014 initial trials were setup to analyze the safety and side effects of the VSV base vaccines. Phase 1 clinical trials were conduct in which 158 adult healthy volunteers were given a single IM injection of VSV-EBOV at different doses in escalating manner (3 × 10 5 , 3 × 10 6 , 1 × 10 7 , 2 × 10 7 and 5 × 10 7 pfu). Viremia was noted in all the individuals as confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Approximately 90% vaccine recipient's experiences different adverse effects such as pain at the site of injection, fatigue and myalgia. Some participants developed arthritis in Geneva, Kenya and Germany but no viremia was detected. After immunization within 24 to 36 h all the symptoms were resolved. It was observed that higher (3 × 10 6 ) dose of VSV-EBOV induce a strong immune response than the lower dose (3 × 10 5 ) but antibodies response was not significantly different [27] . In 2016, a clinical trial was conducted in Guinea to analyze the efficiency of heterozygous based vaccine combining two components-the recombinant vesicular stomatitis virus (VSV) and the adenovirus serotype-5 (Ad-5). Both components were genetically altered expressing Ebola virus glycoproteins. Seroconversion was observed in all patients with minimal adverse effects. The most common adverse effect observed was pain at the site of injection. Other than this, no significant adverse reactions was found. Two different doses were given to the subjects. The first group was given half doses of VSV and Ad-5 and the other group was given full doses. On day 42, all participants (including both groups) were detected with glycoprotein specific antibodies. This study demonstrates that the vaccine was completely safe and could induce high cellular and humoral response [28] . Vaccination providing protection may be classified into two different categories. Mechanistic that provide main protection against the virus and non-mechanistic that may participate in the protection but is not much effective. Pre-clinical studies on NHP showed that VSV-EBOV could induce GP-specific antibodies with levels correlating protection. Further studies showed that cell-mediated immunity does not play important role in VSV-EBOV vaccine mediated protection because lethal challenge was not affected by the depletion of CD8 + T cells or CD4 + T cells. It is evident that CD8 + T cells play regulatory role in VSV-EBOV mediated protection. CD8 + T cells play important role in the protection given by Venezuelan equine encephalitis virus (VEEV) based replicon vaccine. Protection conferred by VLP vaccine requires GP and specific CD8 + T cells. EBOV vaccine platforms elicit distinct immune profiles thereby considering different strategies of protection (Table 4) . HERBAL AND OTHER REMEDIES Many diseases like Alzheimer, diabetes mellitus, cardiovascular disorder have reached to endemic pro-portion. Whereas, the emergence of some deadly diseases like HIV, bird flu, rabies, EBOV have increased the mortality rate. The origin of the virus plays a key role to control epidemics and mortality rate [13] . Still there is no proven treatment for Ebola. However, some clinical-, herbal-and immune-therapies are used to suppress the adverse effects. Some natural cures have enlisted in (Table 5) . Since centuries, plants are used as therapeutic agents treating human and animal diseases. Plants produce secondary metabolites, which have a diverse range of pharmacological activities to fight against certain diseases. These plant-derived remedies show some significant treatment of Ebola. Some chief drugs like Belladonna, Arsenic, Bryonia, Aconite and Gelcemium have shown symptomatic treatment of Ebola. Belladonna herb also known as deadly nightshade leaf. It is originated and cultivated in England and European countries. The herb is also found in India and in the forests of Jammu. Belladonna showed significant cure in fever, brain infections, cough, measles, menstrual irregularity, pregnancy defects, whooping cough, uterine infections and abdominal related issues. Aconite herb was used as a strong poison in the past some decades. Now a days, it is also used to fight against many infections like vertigo, dropsy, chicken pox, asthma, fever and headache disorders. However, it causes many adverse and intense effects (pupil contraction, anxiety attacks and short of breath) in patients. It is, therefore, not recommended to the patients who suffer from some cardiovascular and nervous sensitivity disorders. Bryonia is originated and cultivated in the western Eurasia, North Africa, South Asia and Canary Island. It is found to be effective in the cure of breast infection, respiratory disorders, whooping cough, fever, constipation, gastric disorders and meningitis. Gelcemium, poisonous flowering plant family, initially was originated and cultivated in China, North America and Southeast Asia. The plant shows some significant effects in the treatment of many diseases like cerebral disorders, dengue fever, locomotors Ataxia, fever, cardiovascular disorders and eye infections. It was found that Belladonna, Aconite and Bryonia treats all the infections of Ebola except blindness, red and itchy eyes and blood vomiting respectively. While Gelcemium treats all the significant symptoms of Ebola (Table 5) . Berries are powerful antioxidants and found in red, blue and black colors. They are originated and cultivated in Europe. They provide an effective coverage from the bacterial and viral infections. Green tea is native to China, where it is cultivated and exported to other countries. It contains flavonoid known as epigallocatechin gallate, which acts as a protective shield in body against infections and involves in the boosting of immune system. Phyllanthus amarus is a naturally occurring herb cultivated in America. It also is used to treat cold and other bacterial infections. Chili pepper originated in Mexico contains capsaicin, which helps in the boosting of immune system. A necessary vitamin, beta-carotene also found in chili pepper, which helps in the maintenance of immune system. Ginger originated and cultivated in Island Southeast Asia. It acts as a strong antioxidant, antimicrobial, antiinflammatory, antibiotic and antiseptic. Mushrooms originated and cultivated in Paris. They are rich source of antioxidant and minerals (selenium & copper) assist in activating the immune system. Brazil nuts, originated from Brazil, eastern Colombia, eastern Peru and eastern Bolivia, are rich source of selenium. Two Brazil nuts daily help preventing from cold and influenza. Turmeric, originated and cultivated in India, has an anti-microbial activity. It helps to prevent cold and influenza. Garlic, originated and cultivated in Central Asia, South Asia or Southwestern Siberia, contains antifungal, antiviral and antibiotic activity to fight against the infections. Eucalyptus oil helps to relieve headaches and in soothing of throat (Table 5) . Arsenic is a semi-metallic element found in the earth crust with atomic weight of 74.9 g. Its pure form exists in three forms alpha (yellow), beta (black) and gamma (grey). It is found to be effective in the treatment of alcoholism, cancer infection, cholera, kidney infection, vomiting, pleurisy and rickets. Another drug known as TCM (Traditional Chinese Medicine), widely used in China to cure the deadly diseases. TCM approach rely on the belief that virus-induced fever causes a deficiency of energy in the body. Soups of cheap and effective herbs are, therefore, used to recover the depleted energy. Master Zhang include "Ma-Huang Soup," "Da-Qing-Long Soup," "Gui-Zhi Soup," and "Ge-Geng Soup." The effective symptomatic control of TCM has been observed against the Zika virus infection. In Ebola infection, hemorrhage is a lethal issue, while the herbs (sanqi, chuanxiong, danshen) present in TCM options have the potential to regulate the circulatory system (Table 5) . Ebolavirus (EBOV) has the ability to invade the adaptive and innate immunity and can inhibit both type-I IFNs synthesis and response. Some more facts are still needed to explore the replication mechanism of EBOV in the host. Vaccination against EBOV is still under clinical trials. Vesicular stomatitis virus (VSV)based vaccines showed some real protection against EBOV but still more clinical trials are needed to analyze the protection and effectiveness of the vaccine. The main problem associated with VSV-based vaccine is the adverse effects that need to be addressed. Future research will unravel the mechanisms associated with the infection cycle and the associated high mortality rate. The protection approaches like the use of herbs, specifically the TCM has the potential to be used as cure against EBOV disease. 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Vaccines Immunother Conflict of interests. The authors declare that they have no conflicts of interest.Statement on the welfare of humans. All applicable international, national, and/or institutional guidelines were followed.