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The Ebola virus: what we know, what we don’t know and why we don’t know. Aisha Belgore
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Page 1: The Ebola virus: what we know, what we don’t know and why we don’t know. · The Ebola Virus: What we know, what we don't know and why we don’t know. By Aisha Belgore Table of

The Ebola virus: what we know, what we don’t know and why we don’t know.

Aisha Belgore

Page 2: The Ebola virus: what we know, what we don’t know and why we don’t know. · The Ebola Virus: What we know, what we don't know and why we don’t know. By Aisha Belgore Table of

The Ebola Virus: What we know, what we don't know and why we don’t know.

By Aisha Belgore

Table of Contents ABSTRACT 2 ............................................................................................................................................................

INTRODUCTION 2 ...................................................................................................................................................

STUCTURE AND REPLICATION 4 .............................................................................................................................

Nucleocapsid Proteins 5 .....................................................................................................................................

Envelope Proteins 6 ............................................................................................................................................

What we don’t know about the proteins: The sGP Protein 7 .............................................................................

Viral Protein Synthesis and ReplicaOon Within Cells 8 .......................................................................................

TRANMISSION AND RESERVIOR HOSTS 10 .............................................................................................................

Animal to Human Transmission 10 .....................................................................................................................

Human to Human Transmission 11 ....................................................................................................................

What we don’t know about transmission: Droplet transmission 12 ..................................................................

INFECTION AND PATHOGENESIS 15 ........................................................................................................................

Receptor AWachment and Entry and Membrane Fusion Mechanisms 15 ..........................................................

Immune System DeregulaOon 16 .......................................................................................................................

“Impairment of the vascular system” 17 ............................................................................................................

CoagulaOon 18 ...................................................................................................................................................

Organ infecOon 19 ..............................................................................................................................................

Death 20 .............................................................................................................................................................

What InfecOon Routes do we not know 20 ........................................................................................................

TREATMENT AND VACCINE 22 ................................................................................................................................

Treatment Methods 22 .......................................................................................................................................

Vaccine Developments 23 .................................................................................................................................

CONCLUSION: WHY WE DON’T KNOW? 24 ............................................................................................................

Social Issues 25 ...................................................................................................................................................

Geographic challenges 25 ..................................................................................................................................

Economic and poliOcal Challenges 25 ................................................................................................................

ScienOfic Challenges 25 ......................................................................................................................................

BIBLIOGRAPHY 27...................................................................................................................................................

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ABSTRACT Ebola virus is a long filamentous virus which is the sister virus to Marburg virus and together they make up the Filovirdea family. There are 5 strains of Ebola virus: Sudan (SUDV), Reston (RESTV), Bundibugyo (BDBV), Tai forest (TAFV) and Ebola (EBOV- which was formally known Zaire Ebola). This report focuses on EBOV and the lethal Ebola Virus Disease (EVD) it causes in a human host. It also gives a detailed and in depth explanation of what is known about the virus i.e. the method used to infect its hosts, the function of its proteins, the mechanisms used to cause EVD in hosts and the effort being made by the scientific community to combat the disease. It is made evident in the report that a lot of the information available on Ebola is yet to be fully proven. Part of the purpose of writing the report are highlighting these ‘grey areas’ and explain why further investigation is a challenge.

INTRODUCTION Ebola viruses are the causative agents of Ebola Virus Disease in mammalian organisms. There are 5 species of the Ebola viruses. These are: Sudan (SUDV), Reston (RESTV), Bundibugyo (BDBV), Tai forest (TAFV) and Ebola (EBOV- which was formally known Zaire Ebola) . They all vary slightly in the sequence of their 1

ribonucleic acid (RNA) and this causes them to all produce different versions of the same proteins. Because different proteins are produced they behave differently. For example, each strain of the virus can infect primates and cause the Ebola Virus Disease, however RESTV can only infect non-human primates . Of the 2 3

four other strains which can infect humans, EBOV is the most lethal strain with a mortality rate varying in the range 25-90% . For this essay, EBOV will be the only strain considered. 4

The Ebola Virus (EBOV), is a virus of order Mononegavirales, family Filovirdea, genus Ebolavirus and species Ebola . The Belgian virologist Peter Piot, who visited the area to carry out an epidemiological 5 6

evaluation of the villages infected, named the genus after the closest river, the Ebola River; and the species after the country it was first discovered in, The Republic of Zaire (what is now Democratic Republic of Congo) . 7

EBOV was first discovered in a small village called Yambuku. Its first known host was a primary school teacher who had previously been in contact with monkey and antelope meat. After a number of days he visited the hospital complaining to have symptoms identical to that of malaria which is a common illness in Zaire. The nurses at the hospital injected the malaria medicine Chloroquine into his bloodstream. As was common practice then, the needle used to administer his injection was later used for other patients that day. A fortnight later, he broke out with Ebola; his family, the people who attended his funeral and the people who also visited

InternaOonal CommiWee on taxonomy of Viruses, 'Virus taxonomy: 2015 Release', Interna'onal Commi.ee on 1

taxonomy of Viruses (ICTV), <hWp://ictvonline.org/virustaxonomy.asp>, [accessed 30 July, 2016].

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 2

1994), page 115.

Jens Kunn, 'PROPOSAL FOR A REVISED TAXONOMYOF THE FILOVIRDEA: CLASSIFICATION, NAMES OF TAXA AND VIRUSES 3

ANA VIRUS ABBREVIATIONS', Achieves of Virology, Vol.155, (December 2010), pp. 2083- 2103.

Amanda Shaffer, 'Key Protein May Give Ebola Virus It is Opening', New York Times, 16 January 2012, pp.1-2.4

InternaOonal CommiWee on taxonomy of Viruses, 'Virus taxonomy: 2015 Release', Interna'onal Commi.ee on 5

taxonomy of Viruses (ICTV), <hWp://ictvonline.org/virustaxonomy.asp>, [accessed 30 July, 2016].

Jens Kunn, 'PROPOSAL FOR A REVISED TAXONOMYOF THE FILOVIRDEA: CLASSIFICATION, NAMES OF TAXA AND VIRUSES 6

ANA VIRUS ABBREVIATIONS', Achieves of Virology, Vol.155, (December 2010), pp. 2083- 2103.

Carl Zimmer, 'A PLANET OF VIRUSES’ (Chicago and London: University of Chicago Press, 2015) pp.80.7

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the hospital the same day as him- two weeks earlier- all contracted the virus soon after. This led to the first known breakout of Ebola in 1976 . 8 9 10

EBOV is also responsible for most recent outbreak in 2014 in West Africa which concluded on the January 14th 2016 with its final casualty in Liberia. Liberia was declared Ebola free by the Centre for Disease Control (CDC) and the World health Organisation (WHO) after going 42 days without a reported case which is double the maximum incubation time of the virus (21 days) . The 2014 outbreak originated from Meliandou, a small 11

Guinea village. This was the first time this region of Africa was infected with the virus . During the outbreak 12

EBOV infected 28,652 people globally (as of April 4th 2016), of this 28616 were in Sierra Leone, Liberia and Guinea which were the most affected countries. Other countries such as The United States of America, Nigeria, United Kingdom, Spain, Italy, Mali and Senegal had fewer casualties totalling to 36 cases in total . 13

Sierra Leone, Guinea and Liberia- the heavily infected countries received help from global institutions and charities such as World Health Organisation and Red Cross . 14 15

Throughout the rest of this report each section will include a descriptive subsection, explaining what we do know about the virus then a subsequent subsection describing what is unknown about that virus in the context of the essay. In the conclusion section of the report, the ‘why we don’t know’ is fully explained.

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 8

1994), page 115.

InternaOonal CommiWee on taxonomy of Viruses, 'Virus taxonomy: 2015 Release', Interna'onal Commi.ee on 9

taxonomy of Viruses (ICTV), <hWp://ictvonline.org/virustaxonomy.asp>, [accessed 30 July, 2016].

Jens Kunn, 'PROPOSAL FOR A REVISED TAXONOMYOF THE FILOVIRDEA: CLASSIFICATION, NAMES OF TAXA AND 10

VIRUSES ANA VIRUS ABBREVIATIONS', Achieves of Virology, Vol.155, (December 2010), pp. 2083- 2103.

Unknown Author, '2014 Ebola Outbreak in West Africa- Case Counts', Centres for disease control and preven'on, 11

<hWp://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-count.html>, [accessed 29 July, 2016].

World Health OrganisaOon, 'Latest Ebola Outbreak Over in Liberia; West Africa is at zero, but new flare ups likely to 12

occur,' World Health OrganisaOon, <hWps://www.who.int/mediacentre/news/releases/2016/ebola-zero-liberia/en>, [accessed 02 August, 2016].

Unknown Author, '2014 Ebola Outbreak in West Africa- Case Counts', Centres for disease control and preven'on, 13

<hWp://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-count.html>, [accessed 29 July, 2016].

American Red Cross, ‘Ebola Outbreak In Africa’, American Red Cross hWp://www.redcross.org/about-us/our-work/14

internaOonal-services/ebola#Overview [accessed 04 November, 2016].

The World Bank, ‘World Bank Ebola Response Fact Sheet’, The World Bank hWp://www.worldbank.org/en/topic/15

health/brief/world-bank-group-ebola-fact-sheet [Accessed 04 November 16].

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16

STUCTURE AND REPLICATION EBOV is a member of the family Filovirus and the Latin derivative of filovirus are the words 'filum ‘and 'virdea' these translates to 'thread' and 'virus' . Hence, Ebola has a long and tubular structure. The length of 17

individual virions vary in the range of 860 - 1200 nm (10-9 m) long which is significantly larger than the average virus size, this is another feature common to filoviruses, they're larger than other virus families. However, the width of virions is fixed as all are 80 nm wide. It is described as having a "6 shape" or "U- shape" due to the bending and looping in the viruses’ structure as seen in Figure 1 . 18 19 20

The EBOV contains a single-stranded nonsegmented negative sense (running from 3’ to 5’) ribonucleic Acid (SS-RNA) genome. This Genome comprises 7 genes arranged on one long linear strand, coding for 8 proteins and is 18957 bases long. This makes up for approximately 1% of an individual virions mass. The seven genes are: NP, VP35, VP40, GP, VP30, VP24 and L. As shown in figure 2 the genome is capped with leader and trainer regions of nucleotides. In these regions, there are origins for replication and promoters for transcription initiation, and encapsidation signals. Also, shown in figure 2 by the purple arrows there are overlaps between VP35 and VP40, GP and VP30 and the last overlap between VP24 and L. These overlaps mean that during

Jeffrey DelVisco, ‘A Witness to Ebola’s Discovery’ The New York Times, 9 August 2014.16

DicOonary.com, 'Filovirus', Dictonary.com, hWps://dicOonary.com/browse/filovirus 17

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 18

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Centre for Biosecurity, 'Ebolavirus', Public Health Agency of Canada, <hWps://wwww.phac-aspc.gcca/lab-bio/rews/19

psds-xss/eblola-eng.php>, [03 November, 2016].

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 20

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26

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Figure 1: A transition electron microscope (TEM) micrograph of the Ebola Virus. Taken by Fredrick A. Murphy for Centre for Disease Control.

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transcription combinations of genes are transcribed by RNA polymerase into an mRNA however the different proteins have their start codon positioned at different locations along the mRNA . 21 22 23

!

Each gene produces a corresponding protein, except for the GP gene which also produces both the GP and sGP proteins . The proteins are categorized into two groups, those that contribute to the envelope structure 24

and those involved in formation of the nucleocapsid . 25

Nucleocapsid Proteins The four proteins NP, VP35, VP30 and L are all nucleocapsid associated proteins. NP and VP30 are the major and minor nucleocapsid proteins. NP binds to the RNA forming NP-RNA this process is called encapsidation. When combined, the two macromolecules form coil complex of 40nm diameters. During virion replication, the NP protein must be present to encapsidate the new viral RNA produced as a result of transcription 11. VP30 has two roles, one is being included to NP, effectively joining the nucleocapsid; the other is as a transcription activator, promoting the attachment of the polymerase protein to the viral genome to produce mRNA. The rate at which the protein performs its roles is based on the amount of phosphorylation it has undergone. When highly phosphorylated it will fully bind to NP however it will not act efficiently as an activator. On the other hand, when lightly phosphorylated it will not be able to bind as effectively to NP but will carry out its role as an activator increasing the transcription rate by 160 folds . The polymerase enzyme 26

is a complex of the proteins L and VP35. The L protein provides the main structure of the RNA dependent RNA polymerase, the main function of the polypeptide chain is to catalyse the polymerisation, polyadenylation and methylation reactions during replication and transcription. While the VP35 protein is a cofactor for the polymerase protein; it affects the method of RNA synthesis during transcription or replication of the genome. VP35 directs the polymerase complex to a specific encapsidated region on the genome. The three proteins (NP, L and VP35) then form a triple complex whilst the enzyme catalyses the replication or

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 21

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 22

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Winifred Weissenhorn, STUCTURE OF VIRAL PROTEIN, EBOLA AND MARBURG VIRUSES: MOLECULAR AND CELLULAR 23

BIOLOGY, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Mark-Antoine de La Vega, Gary Wang, Gary P. Kobinger and Xiangguo Qui, ‘THE MULTIPLE ROLES OF SGP IN EBOLA 24

PATHOGENESIS,’ Viral Immunology, vol. 28 (01 February,2015), pp.3-9.

Centre for Biosecurity, 'Ebolavirus', Public Health Agency of Canada, hWps://wwww.phac-aspc.gcca/lab-bio/rews/psds-25

xss/eblola-eng.php , [03 November 2016].

Miguel J. MarOnez, Nadine Biedenkopf, ValenOna Volchkova, Bezna Hartlieb, Nathalie Alazard-Dany , Olivier 26

Reynard , Stephan Becker , Viktor Volchkov, ‘ROLE OF EBOLA VIRUS VP20 IN TRANSCRIPTION REINITIATION’, Journal of Virology, Vol. 82, (01 October 2008), pp. 12569-12573.

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Figure 2: Arrangement of the genes in the Ebola Virus nonsegmented sequence.

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transcription process. VP35 also contributes to the virulence of the virus, it is an inhibitor which disallows the host’s innate immune system from reacting effectively (explained further in the section on infection) . 27 28 29

30

Envelope Proteins The proteins involved in the envelope structure are GP, VP40 and VP24 . The Glycoproteins (GP) are the 31

insoluble surface proteins shown in Figure 2 that resemble small ‘buds’ off the viral external membrane. These ‘buds’ are called peplomers and are between 7 and 10 nm high. As the name implies this protein is heavily glycosylated, the glycosylation is what permits its behaviour as a peripheral protein. The main function of the GP protein peplomer is as the receptor that gains the virion entry into the host cell. The GP binds with cell receptors (described in more detail in the infection section) and then fuses the cell membrane of the virus with the cell membrane of the host’s cell, therefore entering the cell via endocytosis. Because of its roles this protein contributes heavily to the pathogenicity of the virus, also it determines the antigens produced by the host’s immune system. The GP peplomers can be subdivided into two different types of GP proteins performing different structural functions: GP1 and GP2. GP1 is the polypeptide that contains the receptor binding region, it is the longer part that protrudes away from the viral envelope. The GP2 is a trimer

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 27

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 28

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 29

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Grace Redberry, Gene silencing: new research (New York: Nova Science Publishers, 2006) pp. 1-530

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 31

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26

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Figure 3: shows the location of all virion proteins in the virion structure (all except sGP protein)

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(three polypeptide chains) forming rod like molecules that embed the whole GP into the phospholipid bilayer membrane of the virus to ensure that protein isn’t loosely lost off the virions surface . 32 33

The proteins VP40 and VP24 are matrix proteins. Due to the high attraction between the membrane and the matrix proteins both lie immediately below the membrane, between the membrane and the nucleocapsid forming the matrix. Most the matrix comprises of VP40 which also happens to be the most abundant protein, by mass, in the virion. Both proteins are known to be largely associated with replication of the virion particles. The main role of VP40 in replication is as the moderator of budding of new virus particles off the cells membrane. A second function of VP40 is RNA silencing, which is the process by which certain genes in the genome are suppressed prevent the transcription of one or more genes . Another function is assisting in 34 35

morphogenesis, which is the process of maintaining the typical tubular shape of virus particles as new virions are produced. VP40 binds the phospholipid bilayer and the bonds formed between the two compounds promote the bending and wrapping of the membrane around the predeveloped virion particle . 36

Much less is known about the role of VP24. The protein is found in very small amount in individual particles, it makes up a small part of the matrix, hence it is commonly known as the secondary matrix protein. One of its known functions is as an interferon antagonist. Therefore, it inhibits and weakens the host’s immune response and contributes to pathogenicity, a role it shares with VP35 . This immune response will be explained in 37

better detail in the ‘Pathogenesis’ section of the essay.

What we don’t know about the proteins: The sGP Protein The final protein produced by EBOV is the secreted glycoprotein (sGP) protein which is the main product of the GP gene as it less modified than the GP protein (less glycosylation). It is unique because it is the only EBOV protein secreted, all others remain within the structure of an individual virion. As already mentioned, sGP is secreted from infected host cells therefore it is detected, in large amounts, in the host’s bloodstream. Very little research is done on the role that sGP plays to benefit the virus in vivo. Many theories have been researched and were later disproved. For instance:

• It was observed that during an Ebola infection host leukocyte cells (B and T cells) undergo apoptosis. Because sGP is found in large amounts in host’s blood serum it was hypothesized that sGP is the cause of the apoptosis as leukocytes are not cells prone to infection. Also, the leukocytes most likely encounter sGP in bloodstream of infected patients. However, this theory was rejected due to an

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 32

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 33

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 34

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Swiss InsOtute of bioinformaOcs, ‘Ebolavirus’, Viral Zone <hWp://viralzone.expasy.org/all_by_species/207.html> 35

[accessed 03/11/16].

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 36

MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 37

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

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experiment where recombinant sGP was place in vitro with Jurkat cells and after fixed period, no apoptosis occurred.

• Vascular dysregulation is another symptom of EVD. As with the previous example because sGP is found in the blood stream it is easily assumable that it would influence the endothelial vascular cells. However, after infection endothelial cells remain inactivated. Therefore, it was then theorized that sGP performs an anti-inflammatory role. This was proven false because, sGP was added to activate endothelial cell s from an umbilical cord and cells remained swollen.

The only known function of sGP is as a decoy antigen . This role of sGP is explained in greater detail further 38

on in the infection section.

39

Viral Protein Synthesis and Replication Within Cells Once the nucleocapsid (NC) of the virion is released into the cytoplasm of host’s cell the first viral process is the transcription of viral proteins . The polymerase complex stored in the NC with the negative strand RNA 40

initiates this process assisted by the transcription factor protein VP30. Using the RNA genome as a template, the process starts at the 3’ leader with the NP gene and continues to the 5’ end finishing with the L gene

Mark-Antoine de La Vega, Gary Wang, Gary P. Kobinger and Xiangguo Qui, ‘THE MULTIPLE ROLES OF SGP IN EBOLA 38

PATHOGENESIS,’ Viral Immunology, vol. 28 (01 February,2015), pp.3-9.

Gina BaWaaglia ‘Ebola: New Technology for Diagnosis, Treatment and PrevenOon’, BiorediaOons hWp://39

www.bioradiaOons.com/ebola-new-techniques-for-diagnosis-treatment-and-prevenOon/ [accessed 12 December 2016].

Elke Muhlberger, ‘FILOVIRUS REPLICATION AND TRANSCRIPTION’, FUTURE VIROLOGY, vol. 2 (March 2007), pp. 40

205-215.

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Figure 4: Shows the viral activity within an individual infected host cell.

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(Figure 2). Seven monocistronic messenger RNA (mRNA) molecules are produced . Transcription starts and 41

stops as the polymerase encounters state and stop sites. The transcription start sites for all the genes are identical with the nucleotide sequence 3’CUUCUAAUU and the stop site are all also all identical as they are polyadenylated.

After transcription, the polymerase is released from the genomic RNA. The polymerase can reattach to the promoter same gene it has just previously copied. This property of the polymerase causes the genes further down the genome to be transcribed less as RNA polymerase can continuously transcribe the early genes. Due to this, a large amounts of NP mRNA can be detected in infected cells just 7 hours after infection hence it is the most produced viral protein in a host cell whilst L mRNA is not detected by northern hybridisation, which is used to study (qualitatively and quantitatively) mRNA levels in cells . 42

Following transcription viral mRNA is transported to the rough endoplasmic reticulum (RER) where it is translated in an attached ribosome. Translation produces unmodified protein sequences which are folded and modified in the RER or (in the case of GP/sGP) in the Golgi apparatus. The concentration of all proteins in the cytoplasm build up over time and inclusion bodies are developed. These inclusion bodies can grow large enough to be visible using a light microscope.

The build-up of viral proteins in the cytoplasm prompts the polymerase to switch from transcription to RNA replication. Using an alternative mechanism to transcription the RNA polymerase will attach to the same 3’ promoter as in transcription however doesn’t terminate at the intrinsic stop sites till the 5’ end. The complementary positive-sense (5’ to 3’) anti genome copy produced is also stored in the NC with the original genome. After the antigenome is formed it is then used as a template to produce multiple copies of the original Viral RNA. Once the new viral RNA has been produced surrounding NP protein encapsidate the RNA. Low levels of NP in the cytoplasm then induces the relaunch of transcription. This cycle continually occurs till the cell is saturated with NC complexes.

Following transcription, the proteins move to various regions with in the cell : 43

• sGP is secreted out of cell

• VP24 and VP40 resides below the plasma membrane

• GP peplomers stick out of cell membrane

• NP rests in inclusion bodies in the cytoplasm

• VP35, VP30 and L are all involved in further production of protein during transcription.

At the saturation point the NP will move to membrane where its packaged, by VP40 into viral particles. The VP40 and VP24 on the membrane form the matrix around the NC at the cells inner surface. The viral particles

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 41

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945

Elke Muhlberger, ‘GENOME ORGANISATION, REPLICATION, AND TRANSCRIPTION’, EBOLA AND MARBURG VIRUSES: 42

MOLECULAR AND CELLULAR BIOLOGY’, ed. by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26

Khadija Khataby et al. ‘EBOLA VIRUS’S GLYCOPROTEIN AND ENTRY MECHANISM’, EBOLA, ed. Crtomir Podlipnik (Intech: 43

10 September 2016) pp. 119-138.

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evacuate the cell by budding off the plasma membrane, in this process the plasma membrane -and included GP peplomers- convolute the matrix forming a viral particle . 44 45

TRANMISSION AND RESERVIOR HOSTS Animal to Human Transmission Reservoir host are the long- term host of a pathogen, because of this they do not develop the disease caused by the pathogen . Second to humans, EBOV is highly pathogenic in non-human primates . However, viral 46 47

particles or RNA traces of EBOV are found in other mammals, including: pigs, dogs , guinea pigs , mice 48 49

, fruit bats and antelopes. Many of the animals listed also develop Ebola virus disease although, it affects 50 51

each of them differently. For example, in pigs, viral particles are found in high concentration in the lung so it is transmissible though droplets but in non-human primates it is only infectious through direct contact . The 52

other animals are asymptomatic when infected, this implies that they could potentially be reservoir hosts, however it has not been confirmed.

Many scientists suggest that three specific fruit bats are the natural reservoir hosts for Ebola. The three-bat species are the hammer-headed bat (Hypsignathus monstrosus), Franquet's epauletted bat (Epomops franqueti) and the little collared bat (Myonycteris torquata) . These bats are identified this way because they are the 53

only known species to be asymptomatic and contain specific Immunoglobulin G (antibodies) for the antigen of the EBOV (GP peplomers).

Ilana Kelsey, ‘Ebola Virus: How it infects people, and how scienOsts are working to cure it’, Science In The News: 44

University of Harvard The Graduate School of Arts and Science hWp://sitn.hms.harvard.edu/flash/2014/ebola-virus-how-it-infects-people-and-how-scienOsts-are-working-to-cure-it/# [accessed 20 November 2016].

Elke Muhlberger, ‘FILOVIRUS REPLICATION AND TRANSCRIPTION’, Future Virology, vol. 2 (March 2007), pp. 205-21545

Medical DicOonary for the Health Professions and Nursing, ‘Reservoir Host’, Medical DicOonary for the Health 46

Professions and Nursing <hWp://medicaldicOonary.thefreedicOonary.com/reservoir+host> [accessed November 22 2016].

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 47

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

Weingartl H.M., ‘REVIEW OF EBOLA VIRUS INFECTIONS IN DOMESTIC ANIMALS’, InternaOonal Symposium, vol. 135 48

(2015), pp. 211-218.

Elena Ryabichikova et al.‘EBOLA VIRUSINFECTION I THE GUINEA PIG’, EBOLA AND MARBURG VIRUSES: MOLECULAR 49

AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 239-251

Mike Bray, ‘PATHOGENESIS OF FILOVIRUS INFECTON IN MICE’ EBOLA AND MARBURG VIRUSES: MOLECULAR AND 50

CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 255-272

Gerado Chowell, Hiroshi Nishiura, ‘TRANSMISSION DYNAMICS AND CONTROL OF EBOLA VIRUS DISEASE (EVD): A 51

REVIEW’, VOL. 12 (10/10/2014), PP. 196.

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 52

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45

Eric M Leroy, ‘FRUIT BATS AS RESERVOIRS OF EBOLA VIRUS’, Nature, vol. 438 (01 December 2005), p. 575.53

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In any outbreak, the primary case is the first human to contract the disease . All the primary cases of all the 54

outbreaks between 1995 and 2007 were caused by spillover events. A spillover event occurs when viral particles are transmitted between species . Therefore, one or more of the animals above provides the 55

causative agent in each outbreak. Note that it is not only the reservoir host species that can be the source of infection, symptomatic animals can also infect humans. Due to the location of the outbreaks (rural villages in Central Africa) it is probable that spillover events are responsible for primary cases. This is because, many of the local indigenes hunt and eat bats, non-human primates and other mammals as “bush meat” . This can be 56

viewed as an advantage because, at the moment, many of these tribes are unaware of the effect of eating these meats however if they are educated on the risk involved the chance of spill over events could reduce.

57

Human to Human Transmission Following the infection of patient zero (animal to human transmission), the outbreak is then driven by transmission between subsequent human hosts. An infectious dose of as little as 10 Ebola virions are a sufficient to cause infection in humans and a single teaspoon of infected blood can contain up to 10 billion viral particles packed within it so an millilitre of blood can have up to 5.9 x 1010 virions in it . This 58 59 60

allows the EVD to be highly transmissible between individuals. Fortunately, Ebola cannot be transmitted to others whilst host is asymptomatic during incubation phase which lasts between 2 to 21 days. The infection is only transmitted when the hosts begins to fall ill. In some cases, it could start very early in the infection cycle,

Johan Glesecke, ‘PRIMARY AND INDEX CASES, The Lancet, vol. 384 (6 December 2014) p.202454

Suresh Rewar, ‘TRANSMISSION OF EBOLA VIRUS DISEASE: AN OVERVEIW’, Annals of Global Health, vol.88 (2014), pp. 55

444-451

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 56

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

World FederaOon of ScienOfic Journalists, ‘Ebola: The Basic Facts’, World FederaOon of ScienOfic Journalists hWp://57

wfsj.org/ebola/basics-of-ebola/ [accessed 12 December 2016].

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 58

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 59

1994), page 115.

Denise Grady, ‘Questions Rise on Preparations at Hospitals to Deal with Ebola’, The New York Times, 13 October 60

2014, p. A1.

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Figure 5: Shows the various known methods of transmission.

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where it is very difficult for doctors to immediately suspect Ebola, the symptoms usually displayed shown are very similar to other infections such as malaria which is also prevalent in Africa . Human to human 61

transmission can occur via two routes; direct contact and indirect contact . 62

Indirect contact occurs when fomites contaminated with infected bodily fluids come in contact with an orifice of another individual. Ebola viral particle are easily destroyed using domestic bleaching agents. Although there are more precautious measures that can be taken to ensure every virion is rendered inactive such as gamma radiation, heating to 60 degrees for an hour or boiling for 10 minutes . On the other hand, if 63

contaminated surfaces are left disinfected, the virions can survive on dry surfaces for up to 6 hours and if held under blood at room temperature the particles remain active for several days . 64

Direct contact occurs when the bodily fluids of an infected individual (alive or deceased) come in contact with another individual’s mouth, eyes, nose (mucosal membranes) or open wound or cut (bloodstream) . Bodily 65

fluids that contain viral particles include: blood, saliva, mucus, vomit, urine, faeces, breast milk, tears, and sweat . In fatal cases, large amounts of viral particles are found on skin and in sweat. In these situations, 66

Ebola can be contracted from merely touching an infected person . The fluids that contain the most Ebola 67

viral particles are the faeces, vomit and blood of human host. In support of this, 67.6% cases suffer from emesis, 65.6% suffer high volumes of diarrhoea and 18% suffer instantons tears in the skin causing external bleeding . The virus is engineered to cause the development of these symptoms in the hosts for further 68 69

propagation of the outbreak . 70

What we don’t know about transmission: Droplet transmission Droplet transmission is the transfer of viral particles via respiratory droplets (small mixtures of mucus and virions) from an infected patient to the mucosal membrane of the recipient, this usually occurs over very short distances (less than 2m). Currently no study has confirmed that Ebola is spread by droplet transmission

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 61

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

Gerado Chowell, Hiroshi Nishiura, ‘TRANSMISSION DYNAMICS AND CONTROL OF EBOLA VIRUS DISEASE (EVD): A 62

REVIEW’, BMC Medicine, VOL. 12 (10 October 2014), PP. 196.

Suresh Rewar, ‘TRANSMISSION OF EBOLA VIRUS DISEASE: AN OVERVEIW’, Annals of Global Health, vol.88 (2014), pp. 63

444-451.

Centre for Disease Control, Q and A on Transmission, Centre for Disease Control hWp://www.cdc.gov/vhf/ebola/64

transmission/qas.html [16/11/2016].

Centre for Disease Control, ‘Ebola (Ebola Virus Disease) - Transmission’, Centres for Disease Control and PrevenOon 65

hWps://www.cdc.gov/vhf/ebola/transmission/#searchTarget, [16 Nov. 16].

Centre for Disease Control, Q and A on Transmission, Centre for Disease Control hWp://www.cdc.gov/vhf/ebola/66

transmission/qas.html [16/11/2016].

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 67

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

Donald G Mc Neil Jr., ‘Ask Well: How does Ebola spread? How long can the virus survive?’, New York Times, 03 68

October 2014.

Centre for Disease Control, ‘Review of Human-to-Human Transmission of Ebola Virus’, Centres for Disease Control and 69

PrevenOon < hWp://www.cdc.gov/vhf/ebola/transmission/human-transmission.html>, [16 Nov. 16].

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 70

1994), page 115.

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however, it is highly speculated that it can spread through large droplets in coughs and sneezes when an infected individual is very ill so virion density is high and couching is a symptom of EVD occurring in 49% of patients . Also, Ebola virions have been found in the cells of the respiratory tract such as alveolar epithelial 71

cells and macrophages.

The role of droplets remains unclear because of the lack of epidemiological data. Information is limited because:

• There have been few outbreaks: As shown in Figure 6, since 1976 when Ebola made its debut in Zaire there have been 13 outbreaks. These outbreaks never lasted for more than two years and the cases were never more than 500. Due to these reasons, there has been ample time for epidemiological experts to carry out studies and observe patterns . 72 73

• Few Studies: Scientists are not encouraged to carry out evaluations of outbreaks because most project have inherent recall bias which weakens the credibility of their work. This bias is introduced for two main reasons. Either, the hosts died from the lethal agent so the scientist used surrogates (family member), who often misrepresents the host, as their source of information. Or, the few people who survive had amnesia of the whole experience so the information they provide is not certainly true . 74

!

Centre for Disease Control, ‘How is Ebola Spread’, Centre for Disease Control and PrevenOon hWp://www.cdc.gov/vhf/71

ebola/pdf/infecOon-spread-by-air-or-droplets.pdf [accessed 15/11/16].

Centre for Disease Control, ‘Outbreaks Chronology: Ebola Virus Disease’, Centres for Disease Control and PrevenOon < 72

hWp://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html>, [16 Nov. 16].

Jean- Phillipe Chippaux, ‘OUTBREAKS OF EBOLA VIRUS DIEASE IN AFRICA: THE BEGINNINGS OF A TRAGIC SAGA’ 73

JOURNAL OF VENOMOUS ANIMALS AND TOXINS INCLUDING TROPICAL DISEASES, vol. treat20 (December 2014)pp.1-14.

Michael T. Osterholm, ‘TRANSMISSION OF EBOLA VIRUSES: WHAT WE KNOW AND WHAT WE DON’T KNOW’, 74

American Society for Microbiology, vol. 6 (19 February 2015), pp 137-45.

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Figure 6: A timeline of the EBOV outbreaks since 1976 also showing location country of outbreak.

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INFECTION AND PATHOGENESIS In this section, the path taken by the virus in the human body that causes EVD is explained. Along with this, all symptoms observed during the viral infection are explained.

Receptor Attachment and Entry and Membrane Fusion Mechanisms Infection doesn’t start until the virion(s) makes it past the plasma membrane cell barrier of host’s cell. This process of entering the cell is carried out using an entry mechanism. The GP proteins (GP-1 and GP-2) are the only proteins expressed on the outer surface therefore it is critical for viral entry. The GP-1 trimer is responsible for the attachment of the virion to host cell proteins in the plasma membrane, whilst the GP-2 oversees fusion of the virions envelope with the membrane of the endosome releasing the nucleocapsid complex and its constituent proteins . 75

For the virus to enter cells the GP-1 protein must bind with a receptor, via the mucin like region, which will trigger an endocytosis mechanism used by the virus to enter the cell. The receptors that the GP protein can potential bind to are: DC-SIGN, l-SIGN, hMGL, LSECtin, Folate receptor 1 (FOLR1), Beta- Integrins and Tryo3 receptors. Although it can bind to all these receptors, none of them are nesessary for Ebola to infect a cell . However at least one must be present for Ebola to enter the cell. 76 77 78 79

As implied by the name, glycoprotein (GP), the viral peplomers contain glycosylation sites where oligosaccharides are attached to the protein at the Asparagine (amino acid) residues. It also contains a mucin like region. These sugars allow the virion to evade the host immune system as the glycoprotein antigen is not recognized by the host’s defence system and it also blocks the more recognizable epitopes on the protein . 80

After attachment, the membrane is triggered to initiate the entry mechanism. Like other viruses, Ebola uses micropinocytosis to enter the host’s cell . Micropinocytosis is a form of endocytosis in which the cell non-81

selectively internalizes the solute molecules in the surrounding fluid . In the cell, the proteases cathepsin B 82

(CatB) and cathepsin L (CatL) diffuse into the endosome with virion particles. The enzymes catalyse the hydrolysis of the GP protein, removing the mucin like domain and the glycan cap, leaving behind a much smaller and more exposed GP-1 which is still attached to GP-2 . The cleaved protein is now described as 83

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 75

PATHOLOGY, vol. 11 (2015) e1004731

Jeffrey E. Lee, Erica Ollmann Saphire, ‘EBOLAVIRUS GLYCOPROTEIN STRUCTURE AND MECHANISM OF ENTRY’, FUTURE 76

VIROLOGY, vol. 4 (2009), pp. 621-635

Khadija Khataby et al. ‘EBOLA VIRUS’S GLYCOPROTEIN AND ENTRY MECHANISM’, EBOLA, ed. Crtomir Podlipnik (Intech: 77

10/09/16) pp. 119-138.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 78

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 79

PATHOLOGY, vol. 11 (2015) e1004731

Joseph r. Fancica et al. ‘STERIC SHEILDING OF SURFACE EPITOPES AND IMPAIRED IMMUNE RECOGNITION INDUCED BY 80

THE EBOLA VIRUS GLYCOPROTEIN’, PLOS PATHOLOGY, vol. 6 (9 September 2010).

J. Mercer, A. Helenius, ‘VIRUS ENTRY BY MACROPINOCYTOSIS’, NATURE: CELL VIROLOGY, vol. 11 (2009), pp. 510-520481

Jet Phey Lim, Paul A Gleeson, ‘MACROPINOCYTOSIS: AN ENDOCYTIC PATHWAY FOR INTERNALISING LARGE GULPS’, 82

IMMUNOLOGY AND CELL BOILOGY, vol.89 (2011), pp.836-843.

Jeffrey E. Lee, Erica Ollmann Saphire, ‘EBOLAVIRUS GLYCOPROTEIN STRUCTURE AND MECHANISM OF ENTRY’, FUTURE 83

VIROLOGY, vol. 4 (2009), pp. 621-635.

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“Activated GP” because the receptor binding site (RBS) is now exposed . The activated GP peplomers then 84

go on to bind with NPC1 receptors in the membrane to initiate the fusion process.

The NPC1-GP (activated) complex triggers the fusion by revealing GP-2. The lysosome must be acidic for this to occur. The acidic conditions cause a conformational change in the tertiary structure of GP-2 allowing it to form a fusion loop at the apex. Following the attachment of the fusion loop, GP-2 unwinds into 6 bundles, this allows the fusion loop and the GP transmembrane domain to meet, creating a gap thereby attaching it to the membrane, and releasing the Nucleocapsid . After release of nucleocapsid into the cell cytoplasm, viral 85

replication is initiated with the infect cell. Soon after virion particles begin to be released into interstitial fluid where it can then infect other cells.

Immune System Deregulation EBOV initially infects dendritic cells (DC), tissue macrophages, and monocytes all of which are peripheral blood mononuclear cells (PBMCs) . Infection causes macrophages and monocytes to synthesize and 86 87

release large amounts of cytokines (mainly chemokines) which are immune cell signalling proteins, and other soluble factors. The release of cytokines conscripts more macrophages and monocytes to the infected area. Following the introduction of the immune cells to the area, virion particles are released from the plasma membrane of the infected immune cells. This provides more target cells for the virus to infect . 88

Among the cytokines released by the PBMC’s, type I Interferons (IFN) are released. The function of this subgroup of interferons is to regulate the activity of the immune system by stimulating the activity of NK cells and macrophages to start an anti-viral response. Within host cells (macrophages), prior to virion assembly, free floating VP35 is able to supress the production IFN- B mRNA production and cause a change in the tertiary structure of the IFN receptors (IFNAR1 and IFNAR2) preventing it from binding with external IFN 89

. 90

Although these 3 PBMCs are heavily infected they do not show large amounts of cell death as viral particles bud off the surface leaving all cellular structures intact. This contributes to their role as the disseminator of virions, as the cells need to be alive to travel around the body. The cells transport the virions to other organs via the lymphatic vessels and the bloodstream . 91

To avoid the adaptive immune system, the viral particles release large volumes of the sGP protein into the blood stream. The sGP protein has the same N- terminus as GP for up to the first 300 amino acids. So, after

Stephen Y. Chan, Mark A. Goldsmith, MOLECULA MECHANISMS OF FILOVIRUS ENTRY, EBOLA AND MARBURG 84

VIRUSES: MOLECULAR AND CELLULAR BIOLOGY, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 1-26

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 85

PATHOLOGY, vol. 11 (2015) e1004731

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 86

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Parameshwran Ramanan et al. ‘FILOVIRAL IMMUNE EVASION MECHANISMS’, VIRUSES, VOL. 3 (2011), PP. 1634-1649. 87

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 88

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945

Pestka S., ‘THE INTERFERON RECEPTORS’, SEMINARS IN ONCOLOGY, vol. 24 (June 1997), p.1989

Raymond M. Walsh et al, ‘TYPE 1 INTERFERINS AND ANTIVIRAL CD8 T CELL RESPONCES’, PLOS PATHOGENS, vol. 8 90

(January 2012), p. 1

L Falasca et al. ‘MOLECULAR MECHANISMS OF EBOLA VIRUS PATHOGENESIS: FOCUS ON CELL DEATH’ CELL DEATH AND 91

DIFFERENTIATION, vol.22(2015), pp. 1250-1259

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secretion, the humoral immune response makes antibodies with complementary paratopes to the epitopes found on the sGP. Therefore, the immune system recognizes the presence of the sGP and antibody complex. Meanwhile the Ebola virions are successfully entering new cells unnoticed by the immune system. This the second known misdirection mechanism of the virus.

Ebola is a unique virus in that it has a broad cell tropism, it can infect almost all cell types. This is because of the large number of receptors that the GP peplomer complex can bind with during the entry process. One cell type it cannot infect are lymphocytes. However, during infection there is a significant drop in lymphocyte cell count resulting in lymphopenia . The loss of lymphocytes is caused by: 92

• Soluble factors such as chemokines and nitric oxide are released which are cell apoptosis factors. NO regulates the “on and off” switch of apoptotic pathways in lymphocytes, especially T cells and Natural Killer Cells (NK cells) . 93 94

• Loss of dendritic cell function. Dendritic cells and helper T cells (Th cells: a type of lymphocyte) usually form a complex following an infection that activates Th cells, permitting them to perform their immune function of encouraging clonal expansion of cytotoxic T cells (Tc cells) and sustaining antibody production in B cells. However, the complex of infected DC cells with Th cells will instead induce apoptosis in the Th cell . 95

• It is suspected that when lymphocytes encounter antigen of Ebola or the sGP protein. They bind to receptors involved in cell apoptosis. The interaction between the receptors and the viral protein triggers the apoptosis mechanism and the cell dies . 96

The combination of all the immunological activity, overwhelms and weakens the immune system of the host organism. This allows Ebola unrestricted access to cells and uninterrupted viral activity.

“Impairment of the vascular system” 97

Within humans, endothelial cells (lining vascular tissue) perform a barrier function; separating the blood from the interstitial fluid. These cells also monitor homeostasis by regulating the blood pressure, coagulation and organ perfusion. As the compromised PBMCs travel in the blood stream, viral particles are released into the blood, increasing viremia. The virions in the blood are then able to infect endothelial cells, the secondary target cells.

Infected endothelial cells undergo the normal reactions (protein synthesis and replication) as in other infected cells, however the GP proteins (GP1 and 2), stuck on the outer membrane of the endothelial cells, prevent the expression of cellular adhesion molecules. This further increases the porosity of the endothelia, as the cells are

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 92

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 93

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Bernhard Brune, ‘NITROGEN OXIDE: NO APOPTOSIS OR TURNING IT ON?’, CELL DEATH AND DIFFERENTIATION, vol. 10 94

(2003), pp. 864-869.

L Falasca et al. ‘MOLECULAR MECHANISMS OF EBOLA VIRUS PATHOGENESIS: FOCUS ON CELL DEATH’ CELL DEATH AND 95

DIFFERENTIATION, vol.22(2015), pp. 1250-1259

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 96

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 97

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

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not adhering to each other with the same strength prior to infection. This is observed in vitro as short as 12 hours after infection . 98 99

Proinflammatory mediators are among the soluble factors secreted by PBMCs. The mediators activate endothelial cells altering their shape and size. Following this, the cells lose their flat shape and become more rounded. This shape change leads to weakened barrier function this in turn makes the vessels more leaky, decreases the integrity of endothelium and depletes the intravascular volume therefore making host hypovolemic . 100 101

(81)

Coagulation Another effect caused by the secretion of cytokines is the expression of tissue factor in the plasma membrane. This protein initiates the coagulation cascade (reactions that result in blood clotting). When the protein is over-expressed the host develops disseminated intravascular coagulation (DIC), a form of coagulopathy . 102

Coagulation of blood takes place within the blood cells of hosts. In fatal cases, the clots formed are big enough to block the blood supply to or away from the organ(s). Also, in other areas of the body excess bleeding will occur as all coagulating proteins are at the location of where DIC is occurring. DIC induces the beginning of thrombocytopenia (lowered platelet count) and increased levels of fibrin (coagulator enzyme)

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 98

PATHOLOGY, vol. 11 (2015) e1004731

Hans-Joachim SchniWler et al., ‘THE ROLE OF ENDOTHELLIAL CELLS IN FILOVIRUS HEAMORRHAGIC FEVER’ EBOLA AND 99

MARBURG VIRUSES: MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 279-294

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 100

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 101

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

US Department of Health and Human Services, ‘What Is Disseminated Intravascular CoagulaOon?’, NaOonal Heart, 102

Lung and Blood insOtute <hWps://www.nhlbi.nih.gov/health/health-topics/topics/dic>, [accessed 06/12/16].

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Figure 7: Shows the sequenOal path taken by the virus in its human host.

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hydrolysis which further decreases the chance of coagulation at sites not suffering from DIC. The virus can 103

take advantage of this because along with no coagulation in the endothelium, the endothelial cells gaps make it very easy for blood to escape from blood vessels which allows it to freely infect organ parenchymal cells. This is the main cause of the “haemorrhage” in the former name of the disease “Ebola Haemorrhagic Fever”

. 104

This bleeding is manifested as emesis (vomiting), petechia, ecchymosis, mucosal haemorrhages and internal vascular congestion. Note that emesis and faeces are the most common forms of external haemorrhage. And even these symptoms aren’t observed till the very late stages of the infection and not enough blood is lost by these methods to cause death. This is what prompted the medical society to change the name of the disease caused by EBOV from Ebola haemorrhage fever (EHF) to Ebola virus disease (EVD). Both substances fill up the gastrointestinal tract causing chronic abdominal pain for host. The digestive product/substrate material is filled with blood containing vast amounts of viral particles. In patients far along in their infection, the emesis is synonymized as “black vomit” because there are black patches which consist of large clumps . Mucosal 105

haemorrhages cause the coughing and sneezing of blood although this does not occur often. The virus has evolved to carry out this mechanism to further infect other people.

Organ infection In contrast to vascular endothelium, the endothelial cells of other organs such as the live kidneys spleen lymph nodes, and adrenal glands are discontinuous. There are large intercellular spaces between the endothelial cells. Therefore, after the leukocyte extravasation, and blood loss from endothelium, Ebola virions can easily enter the organs via these intercellular spaces to infect cells . 106

Hepatocytes (liver cells) are the most commonly infected. The infection of hepatocytes causes large scale cell necrosis however not large enough to be causative of death. Therefore, there is a dysregulation of coagulation, most coagulator factors are normally synthesized in hepatocytes. In place of the necrosed cells is “karyorrhectic debris” which is the chromatin previous stored within the cells . A large volume of viral 107

particles are mixed in with this debris. Adrenal cells also undergo necrosis, this has a strong impact on the blood pressure of the patient. Without adrenocortical cells, steroid- synthesizing enzymes aren’t produced. This will drastically lower the blood pressure of the patient. This is the reason why the patients experience extreme tiredness and weakness.

Ebola does not significantly infect the brain in the way it does to the liver and immune cells due to the presence of the blood brain barrier that prevents the entry of large molecules (such as proteins). However large brain defects are observed. This is because the brain has the highest oxygen demand of all organs within the brain. Due to hypovolemic loss throughout the body the lowered partial pressure of oxygen the oxygen delivered to the brain is not enough for it to sustain normal activity. Therefore, the hypothalamus is unable to

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 103

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 104

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 105

1994), page 115.

Elena Ryabichikova et al. ‘EBOLA VIRUSINFECTION I THE GUINEA PIG’, EBOLA AND MARBURG VIRUSES: MOLECULAR 106

AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 239-251.

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 107

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

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regulate the core body temperature. Meanwhile, the cells within the body are carrying out respiration at high rates (releasing a lot of heat energy) to sustain protein synthesis within infected cells to produce viral proteins. This is what causes the fever observed in patients. The remaining symptoms observed (headache, brain swelling and confusion) are all caused by the absence of oxygen which prevents efficient respiration.

Death Death in fatal cases usually occurs between 6 to 16 days after the onset of symptoms . Death due to EVD is 108

commonly misconstrued to be caused by excess internal bleeding, although this is contributory it is not the primary reason. The most common cause of death of EVD patients is due to hypovolemic shock and 109

multiorgan failure. The intravascular content of the blood is too low and the body goes into shock as most organs are not receiving enough oxygen to sustain aerobic respiration. Although all body cells shut down, the deceased body is still heavily infectious.

This was not known in the earliest outbreaks so many of those who came in contact with the corpse often soon showed symptoms. Now cadavers are handled very carefully by health care workers wearing personal protective equipment (PPE) who clean the deceased patient, then wrap them up in a mortuary sac before transporting them to their place of burial as promptly as possible.

What Infection Routes do we not know When the virus was first detected in 1976 there was very little room for further research as it was such a dangerous virus. Since then new scientific techniques have then been devised to allow the safe study of the virus. All the information discussed in this report have been obtained from five different methods or models, these are:

1. Pseudotyped virus. The gene coding the attachment protein of the Vesicular Stomatitis Virus(VSV) is replaced by the EBOV GP/sGP gene to code for GP on the outside of the virions. These virion particles are used to infect cells in vitro. This method permits the researchers to observe the activity of the GP protein in the attachment, entry and fusion processes. Also, it allows them to carry out these experiments in Biosafety Level 2 facilities while the remaining four methods require biosafety level 4 as whole viral particles are used in experimentation. At my work experience at Professor Alain Townsend’s lab at the Weatherall Institute of Molecular Medicine virus they have used a recombinant form of H5N1 influenza that codes for EBOV GP to the efficacy of the vaccine produced by the Oxford University . 110

2. Testing cells in vitro. EBOV can be added to real human cells in vitro to observe the effect of Ebola infection on specific target cells. This is mainly used for the testing of very crucial infection target cells such as PBMCs and endothelial cells. The most common cells used are macrophages and human umbilical vein endothelial cell (HUVEC).

3. Animal models. Scientists have developed models in which animals (most commonly guinea pigs, non-human primates and mice) where organisms are intentionally infected with the virus to critically monitor and observe the effect of the viral disease on the animals. This has been used to show

16. PriOsh K.Tosh et al. ‘WHAT CLINICIANS SHOULD KNOW ABOUT THE 2013 EBOLA OUTBREAK’ MAYO CLINC, VOL.108

89 (DECEMBER 2014), PP. 1710-1717.

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 109

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 110

PATHOLOGY, vol. 11 (2015) e1004731.

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methods of transmission, internal degradation such as the reduction in integrity of the vascular endothelium.

4. Observations during an outbreak. This is data is obtained from infected patients during an outbreak in Africa where the outbreak takes place. The patient’s physical conditions are monitored by care taker, such as pupil dilation, blood pressure, vomiting. Conversely other internal factors are observed by taking internal sample such as a blood sample. The blood sample can be tested for the presence of relevant antibodies, viral proteins (sGP and VP35), viral particles, steroid synthesising enzymes and coagulation factors. The blood sample can also indicate the platelet count, lymphocyte count, oxygen partial pressure and concentration of antibodies.

5. Deceased patients. After death, relatives of the disease are asked for permission to carry out an autopsy of the corpse. From the autopsy, internal organ samples are obtained and dead cells can be observed. This is the main method used to evaluate the state of hepatocytes prior to organism death.

Although the testing methods listed above have led to vast discoveries about the mechanisms used by the virus that leads to degradation of its human hosts, there are still so many viral mechanisms that aren’t understood.

The mechanism the virus uses to trigger micropinocytosis on the cell surface of the potential host cells is still a mystery. This is because micropinocytosis, in most enveloped viruses, is triggered by the attachment to the phosphatidylserine (PtdSer) receptors. However, EBOV GP has never been shown to attach to this receptor. The difficultly scientist are faced with when they try to understand this phenomenon, is that the receptor that must be present for Ebola entry has not yet been determined. Once this obstacle has been overcome, scientist can then begin to investigate how this receptor initiates the micropinocytosis mechanism.

This issue that lies with further investigating this property is the initially mystery to discovered which receptor on the member must always be present for EBOV infection. When this has been determined, research can than advance into discovering how this required protein initiates micropinocytosis . 111 112

One study has shown that during an infection, GP is suspected to have a role other than an apoptosis initiator. GP is hypothesised to cause an upregulation in the amount of cellular adhesion molecules (CAMs) in the peripheral blood mononuclear cells (PBMCs) infected. This causes the cells to agglutinate in the blood. This has three disadvantages to the host. Firstly, the clumps of cells promote coagulation at sites of clump formation. Secondly, if the clumps grow large enough to block blood vessels this increases the amount of blood restriction to critical parts of the body. And thirdly, if the clumps attach to endothelial cells, large scale extravasation will occur allowing more viral cells come in contact with host cells. However, this hasn’t been confirmed because a contradictory study has been published showing the rather than GP up regulating CAMs, the cytokine released cause the downregulation of CAMs in some PBMCs. There is still a lot of debate on this third function of GP . 113

There are reports of EBOV infection in which the infected patients were asymptomatic. Scientists theorise that at the start of the infection there is the regular spike in cytokine levels in the blood however, the high cytokine levels aren’t maintained for very long, they soon return to the baseline level. This has led scientists to believe

Sven Moller-Tank, Wendy Maury, ‘EBOLA VIRUS ENTRY: A CURIOUS AND COMPLEX SERIES OF EVENTS’, PLOS: 111

PATHOLOGY, vol. 11 (2015) e1004731

25. Sven Moller-Tank, Wendy Maury, ‘PHOSPHATIDYLSERINE RECEPTORS: ENHANCERS OF ENVELOPED VIRUS 112

ENTRY AND INFECTION’, VIROLOGY, vol. 0 (November 2014), pp. 565-580.

Mark-Antoine de La Vega, Gary Wang, Gary P. Kobinger and Xiangguo Qui, ‘THE MULTIPLE ROLES OF SGP IN EBOLA 113

PATHOGENESIS,’ Viral Immunology, vol. 28 (01 February,2015), pp.3-9

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that the best method for post infection survival is an “early and robust” cytokine release at the start of the infection however this is still yet to be confirmed . 114

TREATMENT AND VACCINE Treatment Methods At the moment, there is no curative therapy for the disease. During the 2014 west African epidemic, only experimental therapies were given to a small population of the infected.

The experimental drug that was used in the 2014 outbreak was called ZMapp™, produced by Mapp biopharmaceuticals . It was administered to 6 healthcare workers during the epidemic and 2 died . 115 116 117

ZMapp™ is an antibody cocktail composed of three monoclonal antibodies(mAbs) with complimentary shapes for three different Ebola GP epitopes . The three mAbs are components of other cocktails, c2G4 and 118

c4G7 make up the ZMAb component cocktail and c13C6 makes up the MB-003 component. The antibodies are produced in an Australian species of Tobacco which has been genetically modified to specifically produce the antibody cocktail. Tobacco was the desired species of synthesis because it can be grown to produce an enormous amount of antibodies. The process of generating the antibodies in the plant is that, human antibody genes are fused with that of the tobacco plant then scientists infect the plant with the virus in which the plants undergoes an immune response producing antibodies with human constant regions due to the recombinant human gene fused with the plants genes . The way the ZMapp™ drug works is that; the antibodies are 119

neutralising antibodies that stick to Ebola and act as marker for the immune system cells to easily spot therefore producing an artificial immune response to the virus.

The most common animal models used to test drugs for Ebola are mice, guinea pigs and non-human primates. This is because the EVD observed in these animals resembles EVD in humans . Prior to its administration 120

humans in 2014 ZMapp™ was tested in rhesus monkeys. It showed promising results, 100% of the monkeys

Heinz Feldmann, Anthony Sanchez, and Thomas W. Geizsbert, FILOVIRDEA: MARBURG AND EBOLA VIRUS, FEILD 114

VIROLOGY, edited by David Knipe and Peter M. Howly (Philadelphia: LipcoW Williams & Wilkins, 2013), pp. 923 – 945.

Mapp BiopharmaceuOcals, ‘LeafBio Announces Conclusion of ZMapp™ Clinical Trial’, Mapp BiopharmaceuOcals 115

<hWp://mappbio.com/lea�io-announces-conclusion-of-zmapp-clinical-trial/> [accessed 07/12/2016].

John W King, ‘Ebola Virus InfecOon Treatment & Management’, Medscape hWp://emedicine.medscape.com/arOcle/116

216288-treatment [accessed 07/12/2016].

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 117

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

Vincent Racaniello, ‘How ZMapp anObodies bind to Ebola virus’, Virology blog hWp://www.virology.ws/2014/11/25/118

how-zmapp-anObodies-bind-to-ebola-virus/ [accessed 07/12/2016]

Treye Green, ‘ZMapp Ebola Treatment: What To Know About The Experimental Drug Made From Tobacco’ 119

InternaOonal Business Times, 08/06/2014.

Gary J Nabel et al., ‘CELLULAR AND MOLECULAR MECHANISM OF EBOLA PATHOGENICITY AND APPROACHES TO 120

VACCINE DEVELOPMENT’ EBOLA AND MARBURG VIRUSES: MOLECULAR AND CELLULAR BIOLOGY’, edited by Hanz- Dieter Klenk and Heinz Feldmann (Trowbridge: The Cromwell Press, 2004) pp. 301-315.

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survived with protection from 5 days after infection compared with only 10% of the control group surviving . 121 122

In an Ebola outbreak in 1995 in Kikwit DRC human convalescent blood serum was administered to 8 patients to test the effect of passive immunity on infection. Of the 8 patents tested, 7 survived the infection. Doctors then believed that this method of treatment could potentially be curative, however subsequent studies did not show promising results, there was no improvement for many patients in the study so the effectiveness of this approach is still undefined . 123

Patients that are not given an experimental drug or convalescent blood serum are treated with “Supportive care”. To elaborate, the patient is treated by the appearance of symptoms. Once a patient develops EVD after infection healthcare workers try to manage the haemodynamic and haemostasis by intravenous restoration of electrolyte (body salt) balance and water content of the blood and a source of increased partial pressure oxygen as hypovolemia, hypotension and diarrhoea are enhanced . When the patients develop DIC they 124 125

are provided with a replacement of the coagulation factors lost in clotting and heparin, a blood thinner . 126

Vaccine Developments Currently there are no licensed vaccines providing protection against Ebola. But there are 8 potential vaccines in clinical trials ongoing. Five of the 8 vaccines all use recombinant Vesicular Stomatitis Virus- Ebolavirus (rVSV-EBOV) as a vector. This vaccine was developed by the Public Health Agency of Canada but was distributed to different groups for trial at the end of 2014 . These vaccines are the ones showing the most 127

promising result and are closest to being licenced for public use . 128

The rVSV-EBOV based vaccines contain genetically modified samples of Vesicular Stomatitis Virus. The most virulent VSV protein gene is removed from the genome of VSV and replaced with the GP gene of EBOV. This way when the VSV virions replicate, they are producing virions with the GP peplomers on their

John W King, ‘Ebola Virus InfecOon Treatment & Management’, Medscape hWp://emedicine.medscape.com/arOcle/121

216288-treatment [accessed 07/12/2016].

Nancy Kass, ‘EBOLA, ETHICS AND PUBLIC HEALTH: WHAT NEXT?’ ANNALS OF INTERNAL MEDICINE, vol. 161 (2014) 122

pp. 744-745.

John W King, ‘Ebola Virus InfecOon Treatment & Management’, Medscape hWp://emedicine.medscape.com/arOcle/123

216288-treatment [accessed 07/12/2016].

Duane J. Funk, Anand Kumar, ‘EBOLA VIRUS DIESEASE: AN UPDATE FOR ANESTHESIOLOGISTS AND INTENSIVISTS’, 124

CANADIAN JOURNAL OF ANEASTHESIA, vol. 62 (2015), pp.80-91.

M. Goeijenbier et al. ‘EBOLA VIRUS DISEASE: A REVIEW ON EPIDEMIOLOGY. SYMPTOMS TREATMENT AND 125

PATHOLOGENESIS’, THE NETHERLANDS JOURNAL OF MEDICINE, vol. 72 (November 2014), pp. 442 – 448.

Centre for Disease Control, ‘Treatment’, Centre for Disease Control < hWp://www.cdc.gov/vhf/ebola/treatment/> 126

[accessed on 07/12/2016].

World Health OrganisaOon, ‘ World on the verge of an effecOve vaccine’, World Health OrganisaOon hWp://127

www.who.int/mediacentre/news/releases/2015/effecOve-ebola-vaccine/en/ [accessed on 07/12/2016].

World Health OrganisaOon, ‘Ebola vaccines, therapies, and diagnosOcs’, World Health OrganisaOon hWp://128

www.who.int/medicines/emp_ebola_q_as/en/ [accessed on 07/12/2016].

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outer envelope, mimicking EBOV . This allows the individuals given the vaccine to produce neutralising 129 130

antibodies as a response to the invading antigen.

Vaccine clinical trials have two main targets, ensuring the vaccine administered is safe and effective. Safety in terms of how the vaccinated individuals react to the vaccine to ensure there aren’t any severely detrimental side effects and ensure patient is not susceptible to other infections. The side effects associated with rVSV-EBOV based vaccines are all minor and are common to many other vaccine which indicates that they’re usually caused by the solvent and adjuvant used. The side effects are:

• 1 in 2 people have a sore arm, headaches, or muscle aches that are relieved after the first 24 hours.

• 1 in 8 people feel nausea that stops after the first two days.

• 1 in 7 people experience swollen joints that return to normal within a week or two.

• 1 in 20 people developed skin rashes that cleared after a fortnight . 131

Many of these vaccines didn’t start human administration till late 2015, towards the end of the epidemic so there was not much evidence showing the efficacy of the virus . However, at the end of 2015, the trial run 132

by the Médecins Sans Frontiers in Guinea showed 100% efficacy with the sample group of 7651 used as the participants were in direct contact with Ebola infected patients . Approval of this vaccine is suspected to be 133

approved by the American Federal Drug Administration in early 2017 . In the UK the vaccine at university 134

of oxford haven’t concluded their stage three research so, their data hasn’t yet been approved by the Medicines & Healthcare products Regulatory Agency (MHRA) for use overseas.

CONCLUSION: WHY WE DON’T KNOW? Globally, the reason why Ebola is feared is because it is detonated as the world’s second most lethal virus, losing the race to its sister virus Marburg Virus . However, this fear is amplified by the fact that Ebola 135

outbreaks are occurring more frequently and with many more casualties. However, the real fear that should be associated with Ebola lies with the fact there is such little information available about the virus. The lack of research is caused by a variety of factors ranging from economic, political, geographic, scientific and social factors.

Centre for Disease Control, ‘Sierra Leone Trial to Introduce a Vaccine against Ebola (STRIVE) Q&A’, Centre for Disease 129

Control hWp://www.cdc.gov/vhf/ebola/strive/qa.html [accessed 07/12/2016].

World Health OrganisaOon, ‘Ebola vaccines, therapies, and diagnosOcs’, World Health OrganisaOon hWp://130

www.who.int/medicines/emp_ebola_q_as/en/ [accessed on 07/12/2016].

Centre for Disease Control, ‘Sierra Leone Trial to Introduce a Vaccine against Ebola (STRIVE) Q&A’, Centre for Disease 131

Control hWp://www.cdc.gov/vhf/ebola/strive/qa.html [accessed 07/12/2016].

World Health OrganisaOon, ‘Ebola vaccines, therapies, and diagnosOcs’, World Health OrganisaOon hWp://132

www.who.int/medicines/emp_ebola_q_as/en/ [accessed on 07/12/2016].

NHS Choices, ‘ScienOsts hail ‘100% effecOve’ Ebola vaccine’, NaOonal Health Service < hWp://www.nhs.uk/news/133

2015/08August/Pages/scienOsts-who-hail-100-percent-effecOve-ebola-vaccine.aspx > [accessed on 07/12/2016].

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2016/05/160503131401.htm > [accessed on 07/12/2016].

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ten-most-dangerous-viruses-in-the-world/a-17846283> [accessed07/12/16 ] (1)

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Social Issues Many of the scientists involved in Ebola research could be sceptical about working on viruses as their health is at risk with the probability of contracting the virus from handling samples in the lab. So only very keen scientists are usually involved. Although this is positive as all employees have invested interest in what is being researched, such personnel is limited.

The scientists who work with real viral particles must work in Biosafety Level four (BSL4) conditions where they would have to don and doff their PPE often which can be very challenging. This process included taking long chemical showers which is a very inconvenient process that provides uncomfortable working conditions that can discourage people from participating in research.

Before the 2014 epidemic there was no significant outbreak for 19 years (Kikwit 1995). So scientists weren’t motivated to study the virus as it occurs so infrequently unlike other virus like influenza which make an annual debut. Researchers are unsure of when/ if the next epidemic is going to occur. As stated in the treatment section ZMapp was the only drug (even though it was legally approved) available. This shows the lack of interest and investment in the virus when there’s no outbreak.

Geographic challenges There are only 49 BSL4 labs globally. Only two of these are in Africa: one in south Africa and the other in Gabon, while the virus is most prevalent in the Democratic Republic of Congo, Liberia, Sierra Leone and Guinea. So, researchers will have to be imported from these countries into Gabon (the closer of the two) to perform their work which is discouraging as they may be separated from their families. This is also disadvantageous because viral particles that are stored in a deceased infected person’s blood are transported across borders to other countries for foreign scientists to experiment with . This is potentially harmful to the 136

citizens of the importing country as if the viral particles escape their transport medium, this could lead to an outbreak in another country that was previously not at risk.

Economic and political Challenges Prior to the 2014 outbreak, governments and private funding organisations were not encouraged to fund EBOV research as it occurs infrequently and there is very little room in the government’s financial budget for preventative measures as there are more urgent and pressing needs of the citizens. Another reason why finding funding was problematic for researchers is that the government and funding organisation poured their resources into other areas such as HIV.

Another danger of researching Ebola is that it is a potential bio-terrorism agent. So it must be kept in BSL4 to prevent the viral particles from falling into the wrong hands to cause harm.

Scientific Challenges Theoretically speaking, if all the issues mentioned above are resolved there’s is still a significant issue for human in vivo studies: the number of patients in the study will be limited. The death window of Ebola lies between the 8th - 37th day post infection, which is very narrow. So, the availability of infected patients in a study is usually limited as previously mentioned (in the analysis of sGP subsection).

Another issue is the challenge faced with the confirmation of observations. This can be split in two, statistical relevance and contradictory published research. As just explained there is usually a limited study set of living EBOV sufferers so any observation noticed may be statistically irrelevant because sample size is too small which was observed (as previously mentioned) during the 2014 West African outbreak with ZMapp™ testing.

Richard Preston, THE HOT ZONE: THE CHLLING TRUE STORY OF AN EBOLA OUTBREAK (London: Transworld Publisher, 136

1994), page 115.

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Secondly, contradiction, which is an issue faced in all aspects of scientific research; however, in EBOV testing this happens to very high. The cause of this is that scientist don’t know a lot about the virus and how it behaves so they come up with the right concepts however go about experimentation the wrong way. For example, a study was carried out testing the contribution of sGP in the coagulation dysfunction mechanism of the virus. However, they investigated the wrong protein, the protein that affects coagulation most is the GP protein. It took many subsequent tests (with focus on each protein) discover this . During their process of 137

elimination, they wasted resources that could be allocated to do other things.

To conclude, research alternatives have shown positive results such as using pseudotyped viruses in vaccine development and determining the function of individual viral proteins or in animal models which allows scientists some leeway to study in safer and more convenient environments. The issue with these alternative methods is that they do not completely replicate the activity of EBOV in a human, the virus may use alternative mechanisms in different host species or may require certain proteins to perform a function which the pseudotyped virus does not have so a key mechanism could not be observed. Overall, EBOV is a very complex, “intelligent” and unique agent that scientist hope to conquer.

16. Mark-Antoine de La Vega, Gary Wang, Gary P. Kobinger and Xiangguo Qui, ‘THE MULTIPLE ROLES OF SGP IN 137

EBOLA PATHOGENESIS,’ Viral Immunology, vol. 28 (01 February,2015), pp.3-9

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