Ebola virus disease: A review on virological characteris

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Ebola virus disease: A review on virological characteris- tics, clinical profile, diagnosis and treatment

Bui Tien Sy*, Phan Quoc Hoan*, Nguyen Hong Thang**,

Nguyen Thai Son**, Hoang Xuan Su*

* 108 Military Central Hospital

" 103 MiUtary Hospital, Vietnam Military Medical University '" Medical Microbiology Department Bio-medical and Pharmaceuti-

cal Applied Research Center, ViePiam Military Medical University

Summary

Objective: To describe the current Ebola virus epidemic and the potential options for management of Ebola virus disease. Data sources: A PubMed literature search (1976 through January, 2014} was conducted using the search term Ebola. Study selection and data extraction: Epidemiology, clinical trial studies, CDC and WHO reports published in English were selected. Studies published within the past 5 years were the primary focus of this review. Data synthesis: The current Ebola virus epidemic has primarily been contained in West Africa though it has subsequently spread to other areas, including the United States. Treatment options for patients infected with Ebola virus are limited. Supportive therapy is cen- tered on fluid resuscitation, electrolyte imbalance correction, treating complicating infections, and preventing complications of shock. Experimental therapies (ZMapp, brincidofovir, TKM-Ebola, and favipi- ravir) have been used during this current outbreak. Different vaccine therapies are also in early-stage de- velopment. Conclusion: Ebola virus is highly virulent and fatal, and treatment options are limited. Several experimental and existing therapies may be options for preventing and treating Ebola virus disease.

Keywords: Ebola virus, Filoviridae, ZMapp.

Background

Ebola virus disease (EVD) discriminated in West Africa since late 2013 was currently the largest and longest EVD outbreak ever recorded w i t h a highest case fatality in t h e nearly four-decade history of this disease, it was t h e first time the EVD expanded out o f t h e original area in Guinea's rainforest t o affect West Africa. Up t o 21/1/2015, there has been in ex- cess of 21.000 confirmed, probable, and suspected cases of EVD w i t h more than 8600 deaths in eight different countries, but majority in Guinea, Liberia, and Sierra Leone [1]. Due t o t h e fast speed of viral transmission and hight mortality, WHO declared in 08/08/2014 that "the current outbreak of the Ebola

Correspondence to: Bui Tien Sy - Department of Mo- lecular Biology, 108 Military Central Hospital;

Email: [email protected]

is a public health emergency o f international concern" and urged international c o m m u n i t y t o reduce t h e spread o f Ebola virus.

Ebola virus classification

Ebola virus as well as Marburg virus found in Eastern Africa belong t o Filoviridae family. The last- est member o f this family was Llovlucuevavirus idenfied in bats in Spain, which has been not determined t o cause disease among humans. Most of Ebola virus and Marburg virus, however, may cause severe hemorrhagic fever a m o n g humans.

Five different antigenically Ebola viruses are identified and named by t h e location in which they were first detected. Three Ebola viruses isolated f r o m EVD endemics in Central Africa containing:

Ebola Sudan and Zaire were first determined in Su- dan and Democratic Republic of Congo (DRC) In

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1976, and recently Ebola Bundibugyo virus in Ugan- da in 2007 [2], The f o u r t h viral sub-type is Ebola Tal Forest virus (formly k n o w n as Ebola Cote d'lvote v i - rus) w h i c h was first detected f r o m an ethnologist in 1994 [3]. The last one, Reston Ebola virus, was isolated f r o m monkeys in Reston, USA. These monkeys were i m p o r t e d t o USA f r o m t h e Philippines. This sub-type virus is k n o w n not t o cause h u m a n disease.

Ebola virus organization

The virions are enveloped, filamentous particle with 80 n m diameter and greatly variable length up to 1400 n m . The viral g e n o m e is a linear, negative, single stranded RNA approximately 19 kb in length [4], including a 3 ' conserved, non-coding region, following by seven genes encoding for structural proteins and ending by a 5' conserved non-coding region. In addition, regions containing promoters for replication and transformation are interposed between structural genes. Using full genome of Ebola virus and IVlarburg virus retrieved f r o m GenBank, Baize and et al reported t h a t t h e most genetic diversity among Filoviridae family is between Ebola Tai Forest virus and Ebola Reston virus (08-C strains) with 36.3% of nucleotide diversity [5]. This diver- gence is rarely more t h a n 4 % in a virus genus [6].

Within Ebola genus w h i c h currently causing t h e outbreak in Guinea, t h e greatest nucleotide diver- gence is 3% between t h e "1994 Gabon" strain and the present "Gueckedou-C05" train [5].

There are seven Ebola proteins including: a nucleoprotein (NP), a glycoprotein (GP), an RNA de- pendent RNA polymerase and 4 structural proteins VP24, VP30, VP35 and VP40 [7]. VP40 is as a matrix protein which play a role in virus b u d d i n g and re- leasing from t h e host cell [8]. Viral V24, also a matrix protein, involves in nucleocapsid f o r m a t i o n and virion assembly [9]. The envelope surface glycoprotein (GP) o f t h e virus mediates for virus attachment, membrane fusion and entering into t h e host cell [101. GP is main factor of virus in pathogenesis. W i t h - in 3 days virions reach t o t h e endothelial system and stimulate t o secrete interferons, w h i c h help for spreading t h e virus in t h e host. The virus contains a phospholipids bilayer coat t o protect t h e viral genome as well as accelerate virus entering into t h e host cell.

Due t o the mRNA edditing function shown only in Ebola virus but not in Marburg virus, t w o isoforms of GP are translated only in this species containing full-length GP (ftGP) and truncated GP or soluble GP (sGP) but not in Marburg virus. The sGP is shorter b u t more abundantly synthesized. It was reported t o have antagonistic effects on TNF-a induced endothelial barrier function and severe cytotoxicity t o t h e vascular cell, which are characteristics of Filovirus.

Meanwhile ftGP is less synthesized and its concen- tration strongly correlating w i t h t h e severe cytotoxicity of the vascular cell [11-13]. Renard and e t a l reported that high level expression of ftGP was deter- m i n e d t o hide specialized antigenic epitopes itself which misleads t h e virus into disappearing f r o m t h e plasma membrane. In addition, they showed that t h e over expression of ftGP also masked t h e cellular surface containing major histocompatibility c o m - plex classl. These t w o mechanisms help t h e virus evading i m m u n e system [14].

The methods of Ebola virus detection Identificaion of EVD at the outbreak onset has still been a challenge due t o non-specific clinical symptoms of t h e disease. When EVD endemic occur- ring, all the suspective cases should be considered as high risk of infection for better managing t h e endemic.

All techniques for detecting t h e virus are only performed in modern equipment laboratories containing microscopy, ELiSA, PCR and culture. Viral culture may be conducted by injecting the infectious samples into mouse, rat or primate mammals. The Vero and Vero E6 cell lines are most c o m m o n l y used to isolate the Ebola virus. Viral culture is a basic me- t h o d and a gold standard for specifically identifying Filovirus. However, isolating virus requires the bio- safety level-4 laboratory due to high infectivity and dissemination of the virus.

Filovirus might be directly detected under elec- tron microscopy basing on the viral morphological structure. This method can differentiate Marburg virus and Ebola virus, but it impossibly identifies specific sub-type of Ebola virus genus [15]. Al- t h o u g h , It is easy and quick m e t h o d but its limita-

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tions are: inability t o quickly and stimultaneously examine a large a m o u n t o f samples, expensive cost, t h e complexity of microscopy maintenance, and re- quire a high skill personnel. In addition, only samples w i t h high viral load can be detected by this m e - t h o d [15].

Some potential methods c o m m o n l y using for identifying Ebola virus are Elisa and PCR. The viral antigens including g e n o m e as well as specific antibody against Ebola virus m i g h t be detected several days after t h e onset of clinical symptoms [16]. IgM and IgG antibody were c o m m o n l y used t o develop assays for viral detection. While IgM can be eady d e - termined even only t w o days and disappeared f r o m 30-168 days, IgG m i g h t be detected 6-18 days after t h e presence of t h e first clinical symptoms and exist many years [16,17].

There has not been any evidence of t h e presence of Ebola and Marburg viruses in blood w i t h i n EVD incubation period yet. However, the viruses were identified in blood at t h e first day of clinical symptoms by genome-based assays [18]. Conven- tional RT-PCR t o detectect Ebola virus were first developed by Sanchez [19], and improved by Leroy [20]. Multiplex RT-PCR and realtime RT-PCR were used t o detect and differentiate viruses in Filovirus family. Dorsten et al employed the primer sets de- signed by Sanchez t o amplify a fragment DNA in L gene t o determine Ebola virus and Marburg virus and discriminate those from Dengue, Rift Valley Fe- ver viruses [19, 21]. Another method, nested RT-PCR, was developed t o diagnose infection of the Ebola virus sub-type Zaire and Sudan basing on t h e specific primers targeting coding regions of nucleopro- t e i n [ 1 8 ] .

Human outbreaks and viral epidemiology Two EVD outbreaks were first recorded at t h e same time in Central Africa in 1976, one in Sudan and another in Congo (formally Zaire) [22, 23]. These t w o viruses were subsequently named as Ebola Su- dan and Ebola Zaire, respectively. Up t o now, many haemorrhagic fever outbreaks were identified in Af- rica including Ebola virus haemorrhagic fever in 1995 and 2000 taken place in Congo and Uganda, respectively; Marburg virus haemorrhagic fever in Congo in 1998-1999 and in Angola in 2004-2005.

The outbreaks causing by Filovirus have been a great challenge t o t h e public health. This concern has gradually risen in recent years and w i t h the pin- nacle when t h e current Ebola virus hemorrhagic fe- ^ ver emerged in late 2013. This outbreak quickly spread t o neighboring areas, even f r o m Africa to Europe and America w i t h unprecedented infection patients.

a) 1976-1979: The first recorded Ebola outbreak in Congo and Sudan

The first Ebola outbreaks had 53% fatality with 150 deaths of t h e 284 confirmed Ebola patients in Sundan. The second one t o o k place in Congo in the same year f r o m August t o November, 1976. Its epi- center was in Yambuku about 800km far from the area of t h e first outbreak [23]. Subsequently, this previously u n k n o w n epidemic was named for the river Ebola w h i c h f l o w i n g past Yambuku and the virus isolate was also k n o w n Ebola Zaire causing 89% mortality (284 deaths of 318 Ebola infection confirmed patients).

b) 1994-1997: Ebola re-emergence There have not been identified any EVD cases during 14 years f r o m 1980 t o 1993. The first human EVD case indicated t h e resurgence of EVD was a Swiss female ethnologist. She g o t sick after autopsy- ing a dead chimpanzee in Ivory Coast in June, 1994 [3]. Both t h e w o m a n and chimpanzee were later confirmed t o be infected by an Ebola virus strain, which lately d e n o m i n a t e d Ivory Coast Ebola.

However, t h e first Ebola outbreak in this period occurred in July, 1995 w h i c h caused 256 deaths out of 315 patients ( 8 1 % mortality), and t h e Ebola Zaire virus was identified as t h e culprit o f this outbreak.

Three consequent outbreaks were recorded in Ga- bon causing Ebola Zaire w i t h more than 60% of mortality rate [24,25].

c) 2000-2004: Geographical appearance of Ebola Zaire and resurgence of Ebola Sudan

This period had a number o f outbreaks caused by t h e Ebola Zaire strain in a relatively limited area (border between Gabon and Republic of Congo) and emerged by t h e revival of Ebola Sudan in Sudan and Uganda. There were 4 outbreaks were recorded by Ebola Zaire and one of those had a very high fatality rate of 90.2% [26]. Meanwhile, 02 outbreaks caused

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by Ebola Sudan in Uganda and Sudan had nearly equal mortality of about 41%; however, their scales of discrimination were completely contradictory (27, 28).

d) Outbreak of Ebola in West Africa in 2014 On 23 March 2014, WoHd Health Organization (WHO) issued the first press release of new out- breaks caused by Ebola virus which initially rose in the Republic of Guinea, Guekedou province in the eastern tropical forest area. The disease quickly spread to Macenta province, 80 km far from the east [6, 291. Centers for Disease Control and Prevention (CDC) confirmed the third case of Ebola virus dis- ease, Mr. Thomas Eric Duncan, who was a medical staff caring for the first Ebola patient in the United State of America in 17/10/2014 [30, 31]. Moreover, Ebola patients were also identified in Spain. This was the first time ever the Ebola endemic has passed over the original area in central Africa to cause a huge outbreak in West Africa, America and Europe.

Up to date, more than 21.000 were considered to infect by Ebola virus and nearly a half of those were death.

To deal with the Ebola epidemic unprecedented in the history, the first-ever United Nations emer- gency health mission, the UN Mission for Ebola Emergency Response (UNMEER) has been estab- lished. Its strategic priorities is to prevent the disease spread, treat patients, provide essential services, maintain stability, and prevent the disease spread to the countries unaffected by Ebola epidemic.

Clinical features of EVD

The Ebola virus is transmitted through direct contact with patients or indirect contact with Ebola infected surfaces, especially intact skin, mucosal ex- posure, or mother-to-child transmission, or contact with body fluids of Ebola virus disease patients. The incubation period is 2-21 days, and an average of 7- 10 days. The outbreak is considered as ending when the last case has been recorded at least twice as much as the maximum incubation period; for exam- ple, 42 days after the last deaths or cases causing by the virus.

The disease typically has an abrupt onset after 5-15 days exposure with clinical symptoms like flu such as fever, headache, stomachache, muscle and

joint pains. Therefore, it might be easy to confuse with malaria or dengue diseases in tropical areas.

About half of patients have symptoms of cough, sore throat and dysphasia. Some patients appear digestive disorders such as diarrhea, nausea and vomiting. Haemorrhage occurs with 30%-80% of patients, most in the final stage of the disease, mani- fested by purpura, epistaxis, dental bleeding, gastro- intestinal bleeding, or bleeding in other body parts, and haemorrhage seems to be related to the sever- ity of disease. Seriously-ill patients have very early symptoms and often die within 6-16 days due to hypovolaemic shock and multi-organ failure. In non- serious cases, patients will have fever a few days and improve quickly around 6 to 11 days that is sufficient for producing antibodies against the virus [32].

Frequency of symptoms, especially haemor- rhage, is related to the strain of virus. The most viru- lent strain is Ebola Zaire with the mortality rate of 60-90%, followed by Ebola Sudan with the mortality rate of 40-60%. Meanwhile, the mortality rate re- corded in Bundibugyo Ebola virus is about 25% and only 01 patient who survived was reported to infect Tai Forest virus [17]. The Ebola Reston virus is seemed not to cause serious disease for human al- though the studies in laboratory show that this strain can infect hosts [32].

Blood analysis shows the evidences of neutro- penia {related to the increase of self-die process of lymphocyte), thrombocytopenia, increase of liver enzyme concentrations, thrombin and throm- boplastin time together with detection of fibrin deg- radation products imply appearance of dissemi- nated intravascular coagulation.

Recent reports indicated that pig was consid- ered as a new host of Ebola virus [33-35]. Ebola transmission in pigs due to fruit bats may become a serious pathogen to the host in Filoviridae group.

These fruit bats with an extensive geographical

range can rapidly and widely spread the disease. In

addition, another great concern for highly conta-

gious Ebola virus in pigs is Reston strain which cause

respiratory tract infection instead of hemorrhagic

fever [36). Therefore it can be transmitted from pigs

to pigs and from pigs to humans via respiratory se-

cretion. Although currently there is no evidence

proving that Reston virus strains cause human dis-

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ease, b u t after mutations, it may become patho- genic t o humans. The mortality rate is high in several recent outbreaks and especially t h e present one in West Africa, and t h e absence o f preventive methods and specific-disease treatment, t h e geographic range of potential reservoir species, possibility o f aerosol transmission and being possibly used as a biological weapon caused t h e m t o classify by t h e National Institute o f Allergy and Infectious Diseases Category A priority pathogens and CDC category A agents of bioterrorism. The increasing natural infec- t i o n rate of Filoviridae virus, expansion into new hosts, and high transmission of occupational exposure t o these viruses in laboratories and in outbreak locations, requires t o immediately develope thera-

pies for pre-exposure and post-exposure t r e a t m e n t References

EVD Treatment ^ There has been neither a licensed vaccine nor a

specific therapy t o be used for the treatment of t h e 2.

human Ebola virus infection up-to-date [37]. How- ever, due t o t h e quick discrimination and high mortality o f recent EVD outbreaks, t h e World Health Or- ganization (WHO) has ethically allowed to engage unproven interventions that have shown promising results in laboratory and animal models, but not yet been evaluated for safety and efficacy in humans as potential sources of treatment or prevention [38]

including monoclonal antibody (mAbs)-based therapies (e.g. ZMapp), anti-sense phosphorodiamidate morpholino oligomers (PMO AVl-6002), lipid nano- ^ particle small interfering RNA (LNP-slRNA: TKM- Ebola), and an EBOV glycoprotein-based vaccine using live-attenuated recombinant vesicular stoma- ~ titis virus (rVSV-EBOGP) or a chimpanzee adenovirus (rChAd-EBOGP)-based vector. Partners will begin clinical trials o f t h e s e products in West Africa shortly.

Human trial results of these agents w o u l d not be available until next year. Therefore, t h e WHO issued a consensus statement that the use of whole blood therapies and convalescent blood serum needs t o be considered as a matter of priority in t h e recent EBOV outbreak in West African countries like ZMapp, ZMAb, MB-003... While no licensed available drugs and vaccines, g o o d outbreak control bases on ap- 6.

plying package of interventions, namely case management, surveillance and contact tracing, a g o o d laboratory service, safe burials and social m o -

bilisation t o be very i m p o r t a n t and useful ap- proaches for reducing t h e transmission o f t h e endemic. C o m m u n i t y engagement plays a key role to successfully controlling outbreaks. Raising aware- ness of risk factors for Ebola infection and protective measures that individuals can take is an effective way t o reduce h u m a n transmission.

Conclusion

Ebola virus is highly virulent and fatal, and treatment options are limited. Several experimental and existing therapies may be options for preventing and treating Ebola virus disease.

WHO (2015) Ebola Situation report.

http://www.who.int/csr/disease/ebola/situation- reports/en/.

Towner, J. S., T. K. Sealy, M. L. Khristova, C. G.

Albarino, S. Conlan, S. A. Reeder, P. L Quan, W. I.

Lipkin, R. Downing, J. W. Tappero, S. Okware, J.

Lutwama, B. Bakamutumaho, J. Kayiwa, J. A.

Comer, P. E. Rollin, T. G. Ksiazek, and S. T. Nichol (2008) Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS pathogens 4: e l 000212.

Le Guenno, B., P. Formenty, M. Wyers, P.

Gounon, F. Walker, and C. Boesch (1995}

Isolation and partial characterisation of a new strain of Ebola virus. Lancet 345:1271 -1274.

Regnery, R. L, K. M. Johnson, and M. P. Kiley (1980) Virion nucleic acid of Ebola virus. Journal of virology 36:465-469.

Baize, S., D. Pannetier, L. Oestereich, T. Rieger, L Koivogul, N. Magassouba, B. Soropogui, M. S.

Sow, S. Keita, H. De Clerck, A. Tiffany, G.

Dominguez, M. Loua, A. Traore, M. Kolie, E. R.

Malano, E. Heleze, A. Bocquin, S. Mely, H. Raoul, V. Caro, D. Cadar, M. Gabriel, M. Pahlmann, D.

Tappe, J. Schmidt-Chanasit, B. Impouma, A. K.

Diallo, P. Formenty, M. Van Herp, and S. Gunther (2014) Emergence of Zaire Ebola virus disease in Guinea. The New England journal of medicine 371:1418-1425.

Gatherer, D (2014) The 2014 Ebola virus disease outbreak in West Africa. The Journal of general virology 95:1619-1624.

(6)

JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY VollO-Apr/2015

7. Muhlberger, E (2007) Filovirus replication and transcription. Future virology 2:205-215.

8. Urata, S., T. Noda, Y. Kawaoka, S. Morikawa, H.

Yokosawa, and J. Yasuda (2007) Interaction of TsglOl with Marburg virus VP40 depends on the 17.

PPPy motif, but not the PT/SAP motif as in the case of Ebola virus, and TsgiOl plays a critical role in 18.

the budding of Marburg virus-like particles induced by VP40, NP and GP. Journal of virology 81:4895- 4899.

9. Noda, T., P. Halfmann, H. Sagara, and Y. Kawaoka (2007) Regions in Ebola virus VP24 that are important for nucleocapsid formation. The Journal of infectious diseases 196 Suppi 2; S247-

250. 19-

10. Licata, J. M., R. F. Johnson, Z. Han, and R. N. Harty (2004) Contribution of ebola virus glycoprotein, nucleoprotein and VP24 to budding ofVP40 virus- like particles, ioutna] of vito\ogy 7S:7M4-735^.

11. Falzarano, D., O. Krokhin, V. Wahl-Jensen, J.

Seebach, K. Wolf, H. J. Schnittler, and H. 20.

Feldmann (2006) Structure-function analysis of the soluble glycoprotein, sGP, of Ebola virus.

Chembiochem : a European journal of chemical biology 7; 1605-1611.

12. Sanchez, A , S. G. Trappier, B. W. Mahy C. J.

Peters, and S. T. Nichol (1996) The virion 21.

glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. Proceedings of the National Academy of Sciences of the United States of America 93:3602-3607.

13. Volchkov, V. E., S. Becker, V. A. Volchkova, V. A.

Ternovoj, A. N. Kotov, S. V. Netesov, and H. D.

Klenk (1995) GP mRNA of Ebola virus is edited by 22.

the Ebola virus polymerase and by T7 and vaccinia virus polymerases. Virology 214:421-430.

14. Reynard, O , M. Borowiak, V. A. Volchkova, S. 23 Delpeut, M. Mateo, and V. E. Volchkov (2009) Ebolavirus glycoprotein CP masks both its own epitopes and the presence of cellular surface proteins. Journal of virology 83:9596-9601. 24 15. Wang, Y. P., X. E. Zhang, and H. P. Wei (2011)

Laboratory detection and diagnosis of Filoviruses.

Virologica Sinica 26:73-80.

15. Rowe, A. K., J. Bertolli, A. S. Khan, R. Mukunu, J. J.

Muyembe-Tamfum, D. Bressler, A. J. Williams, C.

J Peters, L Rodriguez, H. Feldmann, S. T. Nichol, P. E. Rollin, and T. G. Ksiazek (1999) CImical, virologic, and immunologic follow-up of convalescent Ebola hemorrhagic fever patients

and their household contacts, Kikwit, Democratic Republic of the Congo. Commission de Lutte contre les Epidemics a Kikwit. The Journal of infectious diseases 179 SuppI l:S28-35.

Feldmann, H., and T W. Geisbert (2011) Ebola haemorrhagic fever. Lancet 377:849-862.

Towner, J. S., P. E. Rollin, D. G. Bausch, A.

Sanchez, 5. M. Crary, M. Vincent, W. F. Lee, C. F.

Spiropoulou, T G. Ksiazek, M. Lukwiya, F.

Kaducu, R. Downing, and S. T. Nichol (2004) Rapid diagnosis of Ebola hemorrhagic fever by reverse transcriptton-PCR In an outbreak setting and assessment of patient viral load as a predictor of outcome. Journal of virology 78:4330-4341.

Sanchez, A , T. G. Ksiazek, P. E. Rollin, M. E.

Miranda, S. G. Trappier, A. S. Khan, C. J. Peters, and 5. T. Nichol (1999) Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates. The Journal ofinfectious diseases 179 Supp11:5164-169.

Leroy E. M., S. Baize, C. Y. Lu, J. B. McCormick, A.

J. Georges, M. C. Georges-Courbot, J. Lansoud- Soukate, and S. P. Fisher-Hoch (2000) Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting. Journal of medical virology 60:

463-467.

Drosten, C, S. Gottig, S. Schilling, M. Asper, M.

Panning, H. Schmitz, and 5. Gunther (2002) Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus. Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. Journal of clinical microbiology 40; 2323-2330.

WHO (1978) Ebola haemorrhagic fever in Zaire, 1976. Bulletin of the World Health Organization 56:271-293.

WHO (1978) Ebola haemorrhagic fever in Sudan, 1976. Report o f a WHO/lnternationat Study Team.

Bulletin of the World Health Organization 56:

247-270.

Georges, A. J., E. M. Leroy, A. A. Renaut. C. T.

Benissan, R. J. Nabias, M. T. Ngoc, P. I Obiang, J.

P. Lepage, E. J. Bertherat, D. D. Benoni, E. J.

Wickings, J. P. Amblard, J M. Lansoud-Soukate, J.

M. Milleliri, S. Baize, and M. C. Georges-Courbot (1999) Ebola hemorrhagic fever outbreaks in Gabon, 1994-1997: epidemiologic and health control issues. The Journal of infectious diseases 179 SuppI 1:565-75.

(7)

JOURNAL OF 108-C ,^1. rVlFUICINE AND PHARMACY V o l l 0 - A p r / 2 0 l l '

Amblard, J, P. Obiang, 5. Edzang, C. Prehaud, M.

Bouloy and B. L Guenno (1997) Identification of the Ebola virus in Gabon In 1994. Lancet 349:181 -182.

Formenty, P., F. Libama, A. Epelboin, Y.

Ailarangar, E. Leroy, H. Moudzeo, P. Tarangonia, A. Molamou, M. Lenzi, K. Ait-lkhlef, B. Hewlett, C.

Roth, and T. Grein (2003) [Outbreak o f Ebola hemorrhagic fever in the Republic of the Congo, 2003: a new strategy?]. Medecine tropicale: revue du Corps de sante colonial 63:291 -295.

WHO (2001) Outbreak of Ebola haemorrhagic fever, Uganda, August 2000-January 2001. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations Weekly epidemiological record / Health Section of the Secretariat of the League of Nations 76:41-46.

WHO (2004) Ebola haemorrhagic fever in south Sudan-update. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations = Weekly epidemiological record / Health Section of the Secretariat of the League of Nations 79.

WHO (2014) Ebola haemorrhagic fever In Guinea.

Disease Outbreak News.

CDC (2014) Texas Reports Positive Test for Ebola in One Additional Healthcare Worker.

WHO (2014) The outbreak of Ebola virus disease in Senegal is over.

Sanchez, A., T. Geisbert, and H. Feldmann (2006) Filoviridae: Marburg and Ebola viruses. Lippincott Williams & Wilkins, Philadelphia.

Barrette, R. W., S. A. Metwally, J. M. Rowland, L Xu, 5. R. Zaki, 5. T. Nichol, P. E. Rollin, J. S. Towner, W. J. Shieh, B. Batten, T. K. Sealy, C. Carrillo, K. E.

Moran, A. J. Bracht, G. A. Mayr, M. Slrios-Cruz, D.

P. Catbagan, E. A. Lautner, T. G. Ksiazek, W. R.

White, and M. T. Mcintosh. (2009) Discovery of swine as a host for the Reston ebolavirus. Science 325: 204-206.

WeingartI, H. M., C Embury-Hyatt, C. Nfon, A.

Leung, G. Smith, and G. Koblnger (2012) Transmission of Ebola virus from pigs to non- human primates. Scientific reports 2:811.

Pan, v., W. Zhang, L Cui, X. Hua, M. Wang, and Q.

Zeng (2014) Reston virus In domestic pigs in China. Archives of virology 159:1129-1132.

Koblnger, G. P., A. Leung, J. Neufeld, J, 5.

Richardson, D. Falzarano, G. Smith, K. Tierney, A.

Patel, and H. M. WeingartI. (2011) Replication, pathogenicity, shedding, and transmission of Zaire ebolavirus in pigs. The Journal of infectious diseases 204:200-208.

WHO (2014) World Health Organization Statement on the WHO Consultation on potential Ebola therapies and vaccines.

WHO (2014) World Health Organization: Ethical considerations for use o f unregistered interventions for Ebola viral disease.