The what and why of the Ebola virus disease

 Electron micrograph of Ebola virus (Cynthia Goldsmith, CDC (wikimedia commons)

With the death toll surpassing one thousand, this is the largest Ebola outbreak since its emergence in 1976. The outbreak is unusual in its occurrence on the west coast of Africa rather than central and also in its spread to urban areas. The outbreak has received much news coverage in recent weeks as the death toll rises and two Americans infected were flown home to receive “experimental therapy.” Why does the outbreak capture the international attention so much more than diseases such as malaria, which kills three times that number…in one day? Perhaps one reason is that malaria is perceived as a chronic condition in developing countries, with eradication a far-fetched goal. The horrific effects of Ebola, hemorrhagic fever, internal bleeding and death resulting from multiple organ failure are probably even more of a contributor. This, combined with a mortality rate of up to 90% (though the numbers are closer to 60% in the current outbreak), makes for a terrifying disease. With the announcement by the WHO this week that experimental drugs can be used in Africa, it seemed like a good time to write a post about the biology behind the Ebola disease and how these much-discussed experimental drugs work. I apologize in advance if this information seems basic to readers in the scientific field, especially virologists, but this post is aimed at those less familiar with science.

The pathogen that causes Ebola is a virus. Viruses are different from bonafide living organisms in that they lack cellular structure and cannot make their own proteins. Proteins are the workhorses of the cell, molecular machines that replicate the genome, allow cells to divide, secrete, move, etc. But viruses are just genetic material, either RNA or DNA, enclosed in a protein coat and possibly a lipid envelope (depending on the type of virus). In order to make more of themselves, the virus must invade cells, be it cells of bacteria, plants, or mammals, and hijack the host machinery. The Ebola virus is a single-stranded negative sense RNA virus. This refers to what has to happen inside the host cell in order to replicate. Its genome is encoded in RNA, which, although very similar to DNA (used by most organisms to encode their genome), is chemically less stable than DNA. The virus has a helical protein coat that protects its genome, giving it a striking long filamentous appearance.

Journey of the Ebola virus:
The virus can spread to a new person when blood or body secretions containing the virus come in contact with mucus membranes or broken skin. A glycoprotein (made of a chain of amino acids and sugar) forms spikes on the virus surface and attach to the host cells. The viral proteins bind receptors on the host cell and trick the host cell into engulfing the virus, bringing it inside the cell (Saeed et al., 2010). The virus then needs to make a version of its genome that the cell can read like a blueprint to make more RNA genome, as well as the structural elements of the virus. Luckily for the virus, it has brought a protein, made during its previous infection to convert the genome into a proper blueprint. The newly copied and packaged viruses then must get out of the cell and invade new ones. The Ebola virus “buds” from the host cell, enclosing itself in lipids from the plasma membrane of the host cell, which is the barrier that separates cells from their environment. One protein in particular on the virus envelope is central to this step and is therefore being investigated as a potential drug target (Jasenosky et al., 2001, Timmons et al., 2001). However this is not what is in the ZMapp drug given to three Ebola patients.

Why is Ebola so deadly?
While some viruses infect only certain types of cells, the Ebola virus spreads through the blood system to infect cells in many organs. Patients often suffer liver, kidney, and other organ failure. A major contributor of the mortality of the disease is “immune paralysis” where patients are unable to mount a defense. This is because many of the proteins the virus makes are specialized to interfere with our defense systems allowing the pathogen to wreak havoc unchecked (Lubaki et al., 2013). For example, while our immune system is designed to sense viral RNA (double stranded RNA), a protein made by the Ebola virus covers and hides the RNA from the host. And any amount of interferon signaling activation that is achieved is inhibited later on by the same and other viral proteins (Leung et al., 2010). Additionally one of the first cells infected by the virus are macrophages, immune cells required early on to recruit other immune cells.  This both prevents them from doing their job and causes them to secrete substances that leads to the cell death of other immune cells and makes capillaries leaky, causing internal bleeding (Geisbert et al, 2003).

What’s in this experimental drug?
When infected by bacteria or viruses, our immune systems produce antibodies, “Y”-shaped proteins that bind a protein on the foreign invader similarly to a lock and key mechanism, using the two tips of the “Y”. This neutralizes the pathogen and recruits other parts of the immune system. Therefore antibodies against Ebola virus injected into a patient could help fight the infection.

But where can we get these antibodies?
Mice similarly produce antibodies so scientists at Mapp Biopharmaceutical injected mice with the virus and harvested the antibodies the mice produced. After identifying the antibody reactive against Ebola, they modified the genetic blueprint encoding the antibody so it looked more human-like. This is to prevent an immune reaction in humans, in which the immune system would see the injected antibody as a foreign invader rather than a helping hand.

How to make large amounts of this antibody? 
Since all organisms use the same genetic code, scientists can make all kinds of cells produce the protein of their choice. In graduate school, I made E. coli bacteria produce proteins from the malaria parasite so that I could study the protein more easily (hence my comment about the deadliness of malaria in the introduction). However since bacteria aren’t very good at making complex antibodies, mammalian cell lines are often used instead. Mapp Biopharmaceutical uses tobacco plants because production is faster and cheaper. The DNA is inserted into the plant cells and the plant’s machinery makes the recombinant protein, which is then harvested. ZMapp, the serum that was used on the two Americans, is a combination of antibodies made in this way. It is important to keep in mind that although the two American patients appear to be responding positively to ZMapp, it is impossible to determine from this that the drug works. The only way to determine the effectiveness of a drug is through clinical trials in which a control group receives a placebo. However with both the ethical questions of testing drugs in developing countries and the extremely short supply of serum, clinical trials are unlikely to occur in the near future.

References:
Geisbert, T., et al. (2003). Pathogenesis of Ebola Hemorrhagic Fever in Cynomolgus Macaques. Am J Pathol. 163, 2347-2370.

Feldmann H., et al. (2001). Biosynthesis and role of filoviral glycoproteins. J Gen Virol. 82, 2839-48.

Jasenosky, L. D., Neumann, G., Lukashevich, I., and Kawaoka, Y. (2001). Ebola virus VP40-induced particle formation and association with the lipid bilayer. J Virol. 75, 5205–5214.

Lubaki, N.M., et al. (2013). The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains. J Virol. 87, 12506.

Leung, D.W., et al. (2010). Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Nat Struct and Mol Biol. 17, 165–172.

Timmins, J., Scianimanico, S., Schoehn, G., and Weissenhorn, W. (2001). Vesicular release of ebola virus matrix protein VP40. Virol. 283, 1–6.

Saeed, M. F., et al. (2010). Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes. Plos Path. 6, 1-15.

Wahl-Jensen, V., et al. (2005). Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function. J Virol. 79, 10442-10450.