key: cord-300133-yc2wxgid
authors: Martínez, Miguel J.; Salim, Abdulbaset M.; Hurtado, Juan C.; Kilgore, Paul E.
title: Ebola Virus Infection: Overview and Update on Prevention and Treatment
date: 2015-09-12
journal: Infect Dis Ther
DOI: 10.1007/s40121-015-0079-5
sha: 
doc_id: 300133
cord_uid: yc2wxgid

In 2014 and 2015, the largest Ebola virus disease (EVD) outbreak in history affected large populations across West Africa. The goal of this report is to provide an update on the epidemic and review current progress in the development, evaluation and deployment of prevention and treatment strategies for EVD. Relevant information was identified through a comprehensive literature search using Medline, PubMed and CINAHL Complete and using the search terms Ebola, Ebola virus disease, Ebola hemorrhagic fever, West Africa outbreak, Ebola transmission, Ebola symptoms and signs, Ebola diagnosis, Ebola treatment, vaccines for Ebola and clinical trials on Ebola. Through 22 July 2015, a total of 27,741 EVD cases and 11,284 deaths were reported from all affected countries. Several therapeutic agents and novel vaccines for EVD have been developed and are now undergoing evaluation. Concurrent with active case investigation, contact tracing, surveillance and supportive care to patients and communities, there has been rapid progress in the development of new therapies and vaccines against EVD. Continued focus on strengthening clinical and public health infrastructure will have direct benefits in controlling the spread of EVD and will provide a strong foundation for deployment of new drugs and vaccines to affected countries when they become available. The unprecedented West Africa Ebola outbreak, response measures, and ensuing drug and vaccine development suggest that new tools for Ebola control may be available in the near future. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s40121-015-0079-5) contains supplementary material, which is available to authorized users.

. The scope and severity of the EVD outbreak underscore the urgent need for development and evaluation of affordable therapeutic and prophylactic agents that can be made available for at-risk populations across Africa. Over the past 17 months, the West Africa EVD outbreak has provided an important opportunity to consider use of and evaluate several therapeutic and prophylactic agents (e.g., vaccines) to determine their safety and efficacy [5, 6] .

For this review, we considered published and [9] [10] [11] . In 2011, a novel third genus of filovirus named Cuevavirus was reported from post-mortem tissues of bats collected in 2002 in Northern Spain [12] . Cuevavirus has not been grown in cell culture, and its pathogenic potential for humans remains unknown. To date, a single species (Lloviu cuevavirus) has been approved by the International Committee on Taxonomy of Viruses (ICTV) [9] . The current West Africa outbreak is caused by Zaire ebolavirus, which shows 97% identity to EBOV strains from the DRC and Gabon [13] . The genome of EBOV contains seven genes named nucleoprotein (NP), virion protein (VP) 24, VP30, VP35, VP40, glycoprotein (GP) and L protein [14] . Each one of these genes encodes a corresponding structural protein. part of the nucleocapsid and, together with VP24, interferes with innate host immunity.

The surface GP is responsible for the attachment to the cellular receptor and viral entry. L protein is the RNA-dependent RNA polymerase [15] [16] [17] [18] [19] [20] .

Early reports suggest that the EBOV variant of the 2014-2015 West Africa outbreak accumulated mutations that may have an impact on the performance of certain diagnostic tests or even on the efficacy of several experimental treatments. Gire et al.

analyzed the genetic sequence of 99 EBOV genomes from 78 patients in the four most affected countries of the West African region [16] . They found significant rates of genomic variation in EBOV in the current outbreak when compared with the EBOV genomic sequence in the 2004 Ebola outbreak in the DRC. Although the impact of these mutations on the diagnostic tests and experimental therapeutics has not yet been proven, some mutations exist in viral genes that are targeted by primers of some reverse transcription-polymerase chain reaction (RT-PCR) protocols [21] , as well as mutations in the binding sites of target proteins of some experimental treatments such as anti-GP monoclonal antibodies [22] . In 2015, Hoenen and colleagues studied full-length sequences of two clusters of EBOV imported from Mali and found that the gene sequence of EBOV has remained stable during the current Ebola outbreak [23] .

Ebola viruses have been responsible for 33 outbreaks in six African countries [2] . Historically, the outbreaks have affected hundreds of individuals where effective control of outbreaks was achieved primarily through isolation of cases and contact tracing.

However, from 2000, EVD outbreaks have been recognized almost every year with substantial variation in morbidity and case-fatality rates ranging from 24% to 81% [24] . High case-fatality rates have been associated with the Zaire and Sudan subtypes [25] . In the ongoing Ebola outbreak, the overall case-fatality rate has been estimated to be approximately 41% for West Africa and other affected countries [26] . Most recently, although the number of cases has declined substantially ( Fig. 2) , EVD cases continue to be reported (please see the supplemental table for details) in Guinea, Liberia and Sierra Leone with WHO Ebola situation reports noting weekly cases in July 2015 [27] . In July 2015, situation report statistics from the WHO suggest that the greatest current burden of EVD is found in Guinea (n = 43) and Sierra Leone (n = 31).

Liberia reported the lowest EVD case number (n = 3) in the week of 29 June through 5 July [27] .

The search for the natural reservoir host of EBOV has been a matter of investigation during the last decades. There is mounting evidence that a number of mammal species may harbor and transmit the virus. Several bat species (i.e., Epomops franqueti, Hypsignathus monstrosus and Myonycteris torquata) have been found to carry filoviruses [28] [29] [30] [31] [32] [33] [34] [35] [44, 45] . Patients often present to health care providers within 1 week of symptom onset [21, 46] . In the early clinical phase of EVD, patients manifest signs and symptoms that mimic common tropical illnesses (e.g., dengue, malaria, typhoid fever and other viral infections) [16, 47, 48] . patients with EVD. These investigators found hemorrhage, shortness of breath and myalgia were independently associated with death [52] . Clinical deterioration may progress rapidly resulting in death within 7 to 10 days.

Vulnerable populations include children under the age of 5 years, pregnant women and the elderly [49] . EVD in these groups also include unspecific symptoms in the clinical presentation. Qin et al. did not find differences related to mortality between patients less than 10 years of age and others between 11 to 20 years old, but they found that patients aged \30 years had a much lower case fatality rate than those aged [30 years [22/38 (57.9% ) and 20/23 (87.0%), respectively, with p = 0.0175] and that survivors attended Ebola Treatment Centers earlier after the onset of symptoms [53] . No evidence suggests that pregnant women are more susceptible to EBOV infection than the general population. However, they might be at increased risk of severe illness and fetal loss. Although no large series are available, the fetal outcome is generally fatal.

Immune suppression and a systemic inflammatory response due to the release of cytokines and other proinflammatory mediators lead to the impairment of vascular, coagulation and immune systems [54] . This can result in multiorgan failure and shock resembling a septic shock syndrome. Massive fluid losses due to intense vomiting and profuse diarrhea can result in dehydration and hypovolemic shock [49] . Severe lymphopenia as well as significant deterioration of renal and liver functions, which may be reflected in high blood urea nitrogen, serum creatinine and hepatic enzymes (i.e., aminotransferases and alkaline phosphatase), can occur [21, 55, 56] .

Since EBOV is a contagious pathogen, the WHO and Centers for Disease Control (CDC) have issued recommendations for proper handling of biological specimens from suspected cases of EVD [57, 58] . Extreme caution should take place at all stages (i.e., specimen acquisition, transport, processing and testing) of specimen processing, and appropriate biosafety laboratory procedures must be used when handling biological specimens from patients with suspected EVD. tests such as loop-mediated isothermal amplification (LAMP) assays [60, 61] .

Prior to 2000, antigen detection methods [e.g., enzyme-linked immunosorbent assay (ELISA)] were the gold standard for EBOV detection in some outbreaks [62] . In the acute phase of EVD, ELISA has a relatively high sensitivity (93%), but EBOV antigen levels decline as disease progresses, rendering lower sensitivity for antigen detection 1-2 weeks following symptom onset [41, 63] . Several other antigen detections tests are currently under evaluation and may be deployed in the near future to complement RT-PCR testing [60] . ELISA testing has been largely replaced by RT-PCR, which permits more rapid detection and can now be deployed in mobile (portable) testing platforms in outbreak settings [64] .

Detection of IgM antibodies against EBOV is performed by ELISA in the first week after the onset of symptoms with a peak of IgM levels occurring in the 2nd week of illness [41, 48, 62] .

IgM antibodies are cleared at variable rates from 1 to 6 months after illness onset [41] . Data showed that serology can be highly specific for the EVD diagnosis but less sensitive in the intensive care unit setting. Hence, antibody testing may be less clinically useful in the diagnosis and management of critically ill EVD patients [49] . Although IgG antibodies appear soon after the IgM and may persist for years

[41], a substantial number of EVD patients have died before they develop an IgG antibody response [48] .

Nucleic acid tests (NATs), particularly RT-PCR, are regarded as the gold standard for EVD diagnosis, in part because of their high sensitivity and specificity in detecting the Ebola viral genome. This is generally accomplished by international mobile teams deployed in institutions such as the European

Mobile Laboratory or CDC. RT-PCR is a rapid and highly sensitive nucleic acid amplification test to detect EBOV nucleic acid [65] . The sensitivity and specificity of RT-PCR are approximately 100% and 97%, respectively [63] . Within the first 3 days of illness, molecular assays may not detect the viral genome, which may lead to false-negative results. Therefore, RT-PCR should be repeated in subsequent samples [49, 66] . To minimize false-negative results, proper sampling, collection, storage or transportation, and a proper RT-PCR technique have to be implemented to avoid cross-contamination [49, 54] . Quantitative RT-PCR has been developed and could possibly be used to monitor the viral load since data suggest high viremia might be associated with unfavorable outcomes and death [21, 46] . For those patients receiving experimental treatments, EBOV viral load monitoring could be useful to assess treatment response [48] .

The 

The provision of clinical supportive care is now a cornerstone of EVD patient management, which includes rehydration, nutrition, analgesics and blood transfusion when appropriate, though no clear evidence proves their effectiveness [21] . A key aspect of supportive care is the maintenance of intravascular volume with oral rehydration solution (ORS) or intravenous fluids that provide appropriate electrolyte replacement. The use of antiemetics and antidiarrheal agents may also be important for patients with persistent vomiting and diarrhea [21, 49, 50] 

Favipirarvir is a nucleotide analog and viral RNA polymerase inhibitor with a wide range of antiviral effects against numerous negative-or positive-strand RNA viruses [83] [84] [85] [86] [87] [88] . Initially, favipiravir was developed to treat influenza viruses, and a phase III clinical trial was completed in which favipiravir was tested on several thousands of people and proven to be safe and effective [84] . Recently, favipiravir has also shown efficacy against EBOV in vitro and in vivo in a mouse model [89] been established, although early treatment in high-risk or potentially EBOV-exposed individuals may be an option [96] . Oral administration of BCX4430 may be feasible, although the pharmacokinetic data suggest that the intramuscular route may provide more favorable therapeutic levels [95] .

Brincidofovir is a prodrug of cidofovir and a fairly recent oral nucleotide analog that prevents viral replication by inhibiting DNA polymerase [97] . Brincidofovir has shown broad-spectrum antiviral activity against DNA viruses such as herpes viruses and adenovirus and is currently in a phase III clinical trial against cytomegalovirus and adenovirus [98, 99] . Although the exact mechanism of action for brincidofovir in EVD is not yet well understood, brincidofovir may interfere with RNA polymerase of EBOV. The US FDA has put brincidofovir on fast-track approval for treatment of EVD based on in vitro data alone [99] . A phase II open-label multicenter study to assess the safety and efficacy of brincidofovir against EBOV in humans has been withdrawn prior recruitment (ClinicalTrials.gov Identifier:

NCT02271347). As a result of the dramatic decline in the number of new cases in Liberia in the month of January 2015 where the trial was first initiated, Chimerix, Inc., decided to discontinue the trial with no further discussions [100] .

There are a number of known agents and newly identified compounds that have shown anti-EBOV activity. For example, Compound 7 is a benzodiazepine derivative that also prevents EBOV entry into the host cells [101] .

Preliminary analysis suggests that Compound 7 binds near a hydrophobic pocket near the EBOV GP1 and GP2 interface. Analysis of this compound suggests that it may have activity against several filoviruses including EBOV [101] .

No animal or human trials have been reported to date.

NSC 62914 is a small molecule, which has an antioxidant property, and was found to inhibit many viruses, including EBOV, Lassa virus, Venezuelan equine encephalitis virus and Rift Valley fever virus [102] . This compound, a reactive oxygen species, has shown in vitro activity against EBOV as well as some evidence in the EBOV mouse model for protection against lethal EBOV infection at a treatment dose of 2 mg/kg/injection (higher doses did not improve survival in the mouse model). Further studies of this or related compounds in mouse and other animal models may be warranted to elucidate their role in the treatment of EBOV and other filovirus infections.

Of additional interest are compounds that have been proven to be effective in preventing the entry of filoviruses, including EBOV, into host cells [103] . LJ-001 binds to lipid membranes and prevents virus-cell fusion across a wide range of viruses. Additional research will be needed to understand how such compounds can be optimally formulated to maximize both the safety and pharmacologic activity in vivo. FGI-103, FGI-104, and FGI-106 are a group of broad-spectrum antiviral agents that inhibit viral replication in a dose-dependent manner among multiple and genetically distinct viruses including EBOV, bunyaviruses, dengue virus,

HIV and hepatitis C virus [104] . Using a mouse model, Aman et al. found that FGI-106 yields a protection after a challenge with a lethal dose of EBOV. Aman and colleagues showed that protection was also found when FGI-106 was administered in a prophylactic fashion. In related studies, FGI-103 and FGI-104 are also small molecules that inhibit filovirus infection and are found to protect EBOV-or Marburg-infected mice, although their mechanism of actions are unclear [105, 106] . [117] . For use of CP or CWB, the WHO has provided guidance to improve the safety of product development as well as safety for patients who receive these products [118] [119] [120] . Convalescent sera-based therapy may cause some toxicity related problems, such as transmission of undetected pathogen(s) and transfusion reactions. A recent case report from treating a Spanish nurse who had contracted EBOV during her care to a patient with EVD in Spain shed some light on the issue of using experimental therapeutics including CP [121] .

The infected nurse had received convalescent sera from two survivors of EVD, high-dose favipiravir and other supportive care treatment.

On the 10th day of clinical disease, the nurse developed clinical signs and symptoms suggestive of post-transfusion acute lung injury, which was managed conservatively without the need of supportive mechanical ventilation.

Although purified IgG can lower these risks, lot-to-lot variation remains a potential problem.

Previous experience also highlights the risk of antibody-dependent enhancement of EBOV infection [122] . could overcome future ZMapp shortages. Using magnifection, 0.5 g of ZMapp can be extracted and purified from 1 kg N. benthamiana leaf biomass [134] . While this product holds promise, a major hurdle in its future utility is to manufacture large quantities of each monoclonal antibody from plants in a way that ensures sustained high yield of monoclonal antibodies at reasonable cost [135] . To overcome potential rate-limiting steps in large-scale production, ZMapp can be manufactured using Chinese hamster ovary (CHO) cells.

In Despite the high safety profile compared with other newly discovered antivirals, these agents cannot be used alone because of their low efficacy, but they can be used in combination with other treatments available including supportive care measures.

Interferon: Since its discovery in 1950, interferon has not been widely used because of the associated adverse events [123] . The potential use of interferons in the treatment of EVD is rooted in the evidence that EBOV interferes with functions of type I interferons [137] [138] [139] . Previous pre-clinical research showed that exogenous interferon-a or interferon-b could delay the occurrence of viremia or prolong survival time, but could not save animals from death [140, 141] NHPs (usually rhesus and cynomolgus macaque) can be infected with non-adapted strains and best mimic disease progression in humans, and therefore they are considered the ''reference'' animal model for vaccine studies [147] . Differences can also be found in the EBOV antigens included in the vaccines. promising in providing more timely and accurate EBOV detection, there will be a need for additional research to study optimal strategies for deploying these diagnostics to locations where testing is most needed. In the near future, there is a substantial likelihood that one or more drugs and vaccines will be approved for use in the treatment and prevention of EVD. The availability of new agents for prevention, acute therapy and post-exposure prophylaxis will require additional research to identify and reduce 

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