Ebola virus – epidemiology, diagnosis, and control: threat to humans, lessons learnt, and preparedness plans – an update on its 40 year's journey

Figures & data

Figure 1. Structure of Ebola virus and its genome. Ebola virus possesses negative-sense RNA genome with exceptionally 14000 nm length with 3' nucleoprotein and 5' RNA polymerase end.

Figure 2. Evolutionary relationships of Ebola virus complete genomes. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987). The optimal tree with the sum of branch length = 1.06917633 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (Nei & Kumar 2000) and are in the units of the number of base differences per site. The analysis involved 56 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 17,999 positions in the final data-set. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013).

Table 1. Outbreaks of Ebolavirus genus between 1976 and March 2016.

Ebola subtype	Number of transmission events	Country	Reported number of human cases	Reported number (%) of deaths among cases	Remarks
Recent outbreaks (since 2014)
Ebolavirus	–	Multiple countries mainly three West African countries, namely Guinea, Liberia, Sierra Leone	28,652	11,325 (40)	Data between March 2014 and 27 March 2016. Seven countries (Italy, Mali, Nigeria, Senegal, Spain, the United Kingdom, and the United States of America) have previously reported a case or cases imported from a country with widespread and intense transmission.
Ebolavirus	1	DRC	66	49 (74)	August–November 2014; the outbreak was unrelated to the outbreak of Ebola in West Africa.
Outbreaks between 1976 and 2013
Bundibugyo virus	2	DRC, Uganda	185	50 (27)
Reston virus	7	Philippines, USA, Italy	13	0 (0)	Four outbreaks in monkeys only; one outbreak in pigs with antibody detection in six workers; two out breaks in moneys with seven people developed antibodies.
Sudan virus	8	South Sudan, Uganda, England	779	412 (53)
Taï Forest virus	1	Côte d'Ivoire (Ivory Coast)	1	0 (0)
Zaire virus	15	Republic of Congo, DRC, Russia, Gabon, South Africa	1383	1086 (79)
Total			31079	12922

Source: http://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html; http://apps.who.int/ebola/current-situation/ebola-situation-report-30-march-2016

Figure 3. Ebola-affected countries on the world map. The coloured area depicts Ebola presence from 1976 to 2016.

Figure 4. Historical timeline of Ebola virus. The number of outbreaks in Sudan, the Democratic Republic of Congo, Uganda, and Tai Forest are dipcted.

Table 2. Chronological data of Ebola virus cases and fatality since its first incidence.

Years	Country	Cases reported	Deaths	Case fatality rate
1976	Zaire	318	280	88%
	Sudan	284	151	53.1%
	England	1	0	0%
1977	Zaire	1	1	100%
1979	Sudan	34	22	64.7%
1989–1990	Philippines	3	0	0%
1990	USA	4	0	0%
1994	Gabon	52	31	59.6%
	Côte d'Ivoire Ivory Coast	1	0	0%
1995	Democratic Republic of Congo	315	250	71.4%
1996	Gabon	37	21	56.7%
1996–1997	Gabon	60	45	75%
1996	South Africa	2	1	50%
2000–2001	Uganda	425	224	52.7%
2001–2002	Gabon	65	53	81.5%
	Republic of Congo	57	43	75.4%
2002–2003	Republic of Congo	143	128	89.5%
2003	Republic of Congo	35	29	82.8%
2004	South Sudan	17	7	41.1%
	Russia	1	1	100%
2007	Democratic Republic of Congo	264	187	70.8%
2007–2008	Uganda	149	37	24.8%
2008	Philippines	6	0	0%
2008–2009	Democratic Republic of Congo	32	15	46.8%
2011	Uganda	1	1	100%
2012	Uganda	11	4	36.3%
	Democratic Republic of Congo	36	13	36.1%
2012–2013	Uganda	6	3	50%
2014–2016	United States of America	4	1	25%
	Spain	1	0	0%
	United Kingdom	1	0	0%
	Italy	1	0	0%
	Guinea	3814	2544	66.7%
	Sierra Leone	14124	3956	28%
	Senegal	1	0	0%
	Mali	9	6	66.6%
	Liberia	10678	4810	45%
	Nigeria	20	8	40%
Total		31013	12872	41.5%

Note: Data compiled from WHO situation reports apps.who.int/ebola/ebola-situation-reports?m = 20141121 and CDC updates on Ebola virus https://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/previous-updates.html

Figure 5. Transmission, pathogenesis, and clinical signs of Ebola virus.

Table 3. Chronology of Ebola diagnosis efforts.

S. No.	Technique(s)	Input molecule (starting material)	Target to be used for detection	Advantage(s)	Reference/year
(1)	Formalin-fixed tissue-specimens examination after enzyme treatment.	Formalin-fixed tissue slides	–	Advantages are minimized due to arrival of sophisticated and efficient diagnostic techniques.	Kurata et al. (1983)
(2)	Immunofluorescence focus assay.	Serum	Ebola antigen		Truant et al. (1983)
(3)	Enzyme immunosorbent assay for primates.				Ksiazek et al. (1992)
(4)	Immuno-enzyme test system.				Borisevich et al. (1996)
(5)	Transmission electron microscopic study of the hemorrhagic spots.	Squamous epithelial cells and the capillary endothelial cells on mucous membrane of mucous membrane of soft palate	Direct virus visualization	Visualization of virus in the enlarged Golgi apparatus.	Wang et al. (1997)
(6)	Immuno-histochemical and electron microscopic examination techniques.	Tissue	Viral antigens and particles directly visualized	For safe, sensitive, and specific detection of Ebola virus in formalin-fixed skin.	Zaki et al. (1999)
(7)	Diagnosis by RT-PCR in an epidemic setting.	Peripheral blood mononuclear cells (PBMC)	Nucleic acid	PCR-based diagnostic assay.	Leroy et al. (2000)
(8)	Development and evaluation of a fluorogenic 5' nuclease assay.	Cultured virus in Vero cells	Glycoprotein (GP) antigens	Detect and differentiate between Zaire and Sudan subtypes.	Gibb et al. (2001)
(9)	5' nuclease probe detection by real-time reverse transcription-PCR.	Serum	Nucleic acid	Detection limit: 9–16 RNA copies per reaction.	Drosten et al. (2002)
(10)	Antigen capture ELISA directed against the envelope glycoprotein of Ebola virus.	Serum	Glycoprotein (GP) antigens	Sensitivity up to ∼10^3 pfu/ml. Able to detect even SDS-inactivated virus.	Lucht et al. (2004)
(11)	Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome.	Serum, blood, or plasma	Nucleoprotein (NP)	One-step fluorogenic Q-RT-PCR assay	Towner et al. (2004)
(12)	Immune-filtration based antigen-detection assay for rapid detection (only Zaire and Sudan strain-specific).	Serum	Matrix protein (VP40)	Sensitivity similar to ELISA and rapid assay without need of the higher technical skill.	Lucht et al. (2007)
(13)	Reverse transcription-loop-mediated isothermal amplification assay (Zaire strain-specific).	Vero cells cultured virus	NP	Portable amplification-based platform. Negative for other febrile disease viruses such as Lassa virus, Rift Valley fever virus, Yellow fever virus.	Kurosaki et al. (2007)
(14)	Quantitative 5’ nuclease assay for NP/-	Vero cells cultured virus	NP	Evaluation of commercial mastermixes for sensitivity and reaction time.	Stephens et al. (2010)
(15)	SYBR Green I real-time RT-PCR.	Cell cultured virus	NP	No cross reactivity with Marburg, Dengue, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome viruses.	Liu et al. (2012, 2014)
(16)	Multiplex RT-PCR for simultaneous detection of Marburg virus and Ebola virus.	Cell cultured virus	Nucleic acid	Sensitive with ability to test two hemorrhagic viruses in a single assay.	Yang et al. (2012)
(17)	Indirect ELISA for detecting Abs specific to Ebola and Marburg viruses.	Cell culture supernatant	NP	Very sensitive assay.	Huang et al. (2014)
(18)	Novel biomarker soluble CD40 ligand (sCD40L) correlates of EVD clinical outcome.	Serum	Different 26 cytokines	Simultaneous detection of 26 proinflammatory cytokines. Increased soluble CD40 ligand (sCD40L) level is indicator of diseases severity.	McElroy et al. (2014)
(19)	Comprehensive multiplex one-step TaqMan qRT-PCR assay.	Cell cultured virus	Nucleic acid	In a single qRT-PCR assay, 28 hemorrhagic viruses may be detected.	Pang et al. (2014)
(20)	Aptameric sensing technology for rapid pandemic monitoring.	Serum	Protein	Aptamers are bio-affinity probes with improved pathogen detection ability.	Acquah et al. (2015)
(21)	GeoSentinel surveillance network. Differential diagnosis of illness in travelers arriving from Sierra Leone, Liberia, or Guinea.	General health surveillance of physical illness	Patients	Based on clinical data collected on GeoSentinel global surveillance network platform (57 specialized travel and tropical medicine clinics).	Boggild et al. (2015)
(22)	Antigen capture ELISA and lateral flow immunodiagnostic.	Serum	Matrix VP40 and glycoprotein (GP) antigens	Sensitivity sufficient for clinical diagnosis.	Boisen et al. (2015)
(23)	Optofluidic analysis system for amplification-free, direct detection of Ebola.	Serum	Nucleic acid	Chip-scale approach Single nucleic acid detection in 10 minutes.	Cai et al. (2015)
(24)	Fe3O4 magnetic nanoparticle-based immuno-chromatographic strip (Nanozyme-strip)	Serum	Gycoprotein (GP) antigens	Comparable to ELISA, but faster and simpler.	Duan et al. (2015)
(25)	Rapid recombinase polymerase amplification Ebola virus detection assay.	Serum and swab	Nucleic acid	Assay can combine extraction and detection in a single step.	Faye et al. (2015)
(26)	Real-time EBOV-L-RT-qPCR for detection of Zaire Ebola virus.	Serum or blood	L gene	No false positives with serum or EDTA bloods samples.	Jääskeläinen et al. (2015)
(27)	The Ebola Spatial Care Path™ (SCP): accelerating point-of-care testing (POCT).	Whole blood	Platelet count, white blood cell count, granulocyte count and percentage, and lymphocyte/monocyte count and percentage, prothrombin time, virus-specific IgM and IgG, hemoglobin and hematocrit	Ebola molecular diagnostics kits for rapid POCT. The reader/processor/cartridges in sealed, disposable pouches containing the biological and chemical reagents.	Kost et al. (2015)
(28)	Ultrafast, sensitive and large-volume on-chip real-time PCR.	(General pathogen detection)		Ultrafast RT-PCR system using microfluidic thermalization can detect Ebola in 7.5 minutes.	Houssin et al. (2016)
(29)	Prototype Cepheid GeneXpert Ebola Assay.	Stored serum and plasma samples	GP and NP gene	No cross-reactivity. Less input sample volume.	Pinsky et al. (2015); Jensen et al. (2016)
(30)	RT-loop-mediated isothermal amplification (RT-LAMP) assay	Cultured virus	Untranscribed trailer region or nucleoprotein coding region of the viral RNA	Higher sensitivity to nanoscale. No cross-reactivity to other hemorrhagic fever viruses and arboviruses.	Kurosaki et al. 2016
(31)	Upconversion nanoprobe/nanoporous membrane system.	Inactivated Ebola virus samples	Ultrasensitive detection of Ebola virus oligonucleotide	Detection up to pico-mole level. Low cost, rapid, and ultrasensitive detection.	Tsang et al. (2016)
(32)	Biofire Film-Array BioThreat-E Test (v2.5).	EDTA-blood, serum or plasma	–	Whole blood may be used.	Leski et al. (2015); Weller et al. (2016)
(33)	Zaire Ebola virus (ZEBOV)-specific enzyme-linked immunosorbent assay (ELISA) using inactivated ZEBOV isolate Makona	Serum	Glycoprotein (GP) antigens	Simple and cost effective.	Krähling et al. (2016)
(34)	RT-LAMP	Blood	Untranscribed trailer region or nucleoprotein coding region of the viral RNA.	Whole blood used to detect GP gene of EBOV high sensitivity Result within 40 minutes	Benzine et al. (2016); Kurosaki et al. (2016)
(35)	ReEBOV Antigen Rapid Test (ReEBOV RDT)	Whole blood or serum	VP antigen (anti-EBOV VP40)	Highly specific Point of care test for EVD	Cross et al. (2016)

Table 4. Diagnostic kits developed for Ebola virus (EBOV) detection.

S. No.	Kit name	Type of the test	Input material(s)	Company name, country
(1)	–	Antigen detection: LAMP	Serum, plasma, amniotic, or synovial fluid	Lucigen, USA
(2)	ABICAP-rapid-Ebola test	Antigen detection – antibody immuno-column for analytical purpose	Whole blood, serum, urine, stool, tears, and saliva as well as in environmental samples such as water, air, and soil	Senova GmbH, Germany
(3)	AccuPower EBOV Quantitative RT-PCR Kit (EBO-1111A)	Nucleic acid detection: RT- PCR	Serum or plasma	Bioneer, Korea
(4)	eZYSCREEN kit	Antigen detection	Blood or serum	French Atomic Energy Commission (CEA), France
(5)	LiferiverTM Ebola Virus (EBOV) Real Time RT-PCR Kit	Real-time reverse transcription PCR	Blood, serum, plasma (non-heparin anticoagulant)	Shanghai ZJ BioTech Co., Ltd., China
(6)	LightMix Modular Ebola virus Zaire	Real-time reverse transcription PCR	Whole Blood	Tib-molbiol/Roche, Germany
(7)	Lipsgene SEBOV/ZEBOV Kit	Real-time reverse transcription PCR	Blood, serum, or plasma	Lipsdiag, Germany
(8)	OraQuick® Ebola Rapid Antigen Test Kit	Antigen detection	Cadaveric oral fluid/whole blood	OraSure Technologies, Inc., USA
(9)	Rapid Response Ebola Virus Test Cassette	Antigen-detection chromatographic immunoassay	Whole blood/serum/plasma	BTNX Inc., Canada
(10)	RealStar Filovirus Screen RT-PCR Kit 1.0	Real-time reverse transcription PCR	EDTA plasma	Altona Diagnostics, Germany
(11)	ReEBOV Antigen Rapid Test Kit	Antigen detection: immuno-chromatographic dipstick immunoassay	Blood/serum/plasma	Corgenix, USA
(12)	SD Q Line Ebola Zaire Ag	Antigen detection – lateral flow chromatographic immunoassay	Blood or serum	SD Biosensor Inc., GyeongGi-do, Republic of Korea
(13)	Xpert® Ebola Test	Real-time reverse transcription PCR in self-contained cartridge system -	Whole blood EDTA or buccal swab specimens	Cepheid AB, Sweden

Table 5. Different available therapies for Ebola virus treatment.

Type of therapy	Name of molecule/modality	Modus operandis	Model organism (onto which the test has been conducted)	Company	References
siRNA therapy	TKM-Ebola	Cocktail of three siRNAs targeting L, VP24, and VP35 genes	Monkeys	Tekmira, Canada	Geisbert et al. (2010)
Antiviral drug	AVI-7537	Through gene silencing	Mice and monkeys	Sarepta Therapeutics	Warren et al. (2010); Iversen et al. (2012); Heald et al. (2014)
	Favipiravir (T-705), a pyrazinecarboxamide derivative	Inhibit viral RNA-dependent RNA polymerase activity and induce lethal RNA mutations	Mice	Toyama Chemical, Japan	Smither et al. (2014); Oestereich et al. (2014)
	Brincidofovir (CMX001)	Improved bioavalability and activity against dsDNA viruses	Human (but not for ebola-specific)	Chimerix of Durham, North Carolina	Florescu et al. (2015)
ZMapp	Combination of humanized monoclonal antibodies (c13C6 + c2G4 and C4G7	Conformational epitopes of GP2 or GP trimer stem region are targeted	Macaques	Mapp Biopharmaceutical, San Diego, California	Haque et al. (2015); Kilgore et al. (2015)
Nucleotide analogue	BCX4430	Nucleoside RNA polymerase inhibitor, incorporate into growing RNA and lead to premature termination	Mice	BioCryst Pharmaceuticals, Inc., Durham, North Carolina	Warren et al. (2014)
Induction of Niemann-Pick C1 mutant phenotype	U18666A/clomiphene/toremiphene induce cholesterol accumulation in lysosomes and inhibit Ebola virus infection	Membrane fusion and virus escape is prevented	In vitro	A chemical supplied by several vendors across the world	Carette et al. (2011)
Repurposed drug	FTY720 (FDA-approved drug for multiple sclerosis)	Prevent cytokine storm and sepsis, reduce inflammation	Human	Sigma/Novartis	Japtok and Kleuser (2009)
	FX06 (anticoagulant therapy)	Fibrin-derived peptide help in vascular leakage	Human	MChE-F4Pharma, Vienna, Austria	Wolf et al. (2015)
	Rosuvastatin, atorvastatin, and pravastatin (lipid lowering drugs)	Reduction of cholesterol content affect the biosynthesis machinery in Ebola virus	Human	By several vendors across the world	Calza et al. (2012); Haque et al. (2015)
	Chloroquine, hydroxychloroquine (antimalarial/lupus erythematous /rheumatoid arthritis treatment with its anti-inflammatory properties	Prevent endosome acidification and Ebola virus is trapped inside endosome	Human	By several vendors across the world	Madrid et al. (2013); Olsen et al. (2013); Kuehne et al. (2016)
	rNAPc2 (recombinant nematode anticoagulant protein c2)	Modulation of coagulation factors	Primates	ARCA biopharma, Colorado	Geisbert et al. (2003)
	Suramin (anti-trypanosomal agent)	Anti-heparin activity, interfere with viral replication and viral entry into the cell	In vitro	Germanin, Bayer	Henß et al. (2016)
Convalescent plasma (CP) therapy	Plasma for Ebola survivors	Providing highly specific antibodies and replenish lost serum components	Rhesus macaques	NA	Jahrling et al. (2007); Dye et al. (2012); Mendoza et al. (2015)
Viral-vectored vaccines	Recombinant chimpanzee adenovirus serotype 3 vector	GP gene	Human	NA	Choi et al. (2015)
	Recombinanat Vesicular stomatitis virus		Cynomolgus macaques	NA	Jones et al. (2005); Qiu et al. (2009); Falzarano et al. (2011)
	Venezuelan equine encephalitis virus (VEEV) replicons	GP/NP	Mice and guinea pigs	NA	Pushko et al. (2003)
DNA vaccine	Inserted NP/GP	Antigen presentation by both MHC-I and II	Guinea pigs or chimpanzees	NA	Sullivan et al. (2009); Hoenen et al. (2012)
Virus-like particles (VLPs)	DNA vectors expressing GP and VP40 proteins in human 293T cells	Morphologically similar to virus and provoke protective immune response	Mice	NA	Ye and Yang (2015); Carra et al. (2015)
Trojan-horse antibodies	Bispecific antibody	Specific for NCP1 or GP receptor binding site coupled to mAb against conserved surface exposed GP epitope	Mice	NA	Wec et al. (2016)
Human transbodies	Cell penetrable human ScFv against VP40	Inhibit egress of EBOV VLPs from hepatocytes	In vitro	NA	Teimoori et al. (2016)
TmPyP4	Cationic porphyrin	Inhibition of L gene of EBOV	In vitro	NA	Wang et al. (2016)

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