Thursday, December 11, 2014

Ebola Virus

Although the Ebola epidemic isn't in the forefront of the news in the United States, its still a significant issue in west Africa. In Guinea, Liberia, and Sierra Leone, there have been 20,416 cases and 8,004 deaths (as of December 31, 2014 as reported at the CDC website) since the epidemic began in March. Only one African country was able to control the Ebola outbreak and as of October 19, 2014, Nigeria has been declared free of Ebola.

Many people, and even may scientists, do not know much about Ebola virus, nor how it causes disease. It causes the deadly disease hemorrhagic fever and mortality rates can be as high as 90%. As such, a biosafety level 4 (BSL-4) facility is needed to study the intact infectious virus. There aren't many of these labs and it requires much equipment and training to be able to work in these labs:

Scientists in a BSL-4 facility at US Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Md. Photo from:

The first known outbreak of Ebola occurred in 1976 in northern Zaire, now known as the Democratic Republic of the Congo. Since then there have been sporadic outbreaks with the current epidemic being the worst. Ebola is a zoonotic disease as it enters a human population from an infected animal. It is still not known which animal species (the reservoir) contains the virus for spread to other animals and humans but studies indicate that it is likely a species of bat.

Ebola virus is a member of the Filoviridae family of viruses. Filoviruses have an RNA genome, a helical capsid, and a lipid envelope with glycoprotein spikes. Viruses in this family include the Ebola virus, Marburg virus, and the recently identified Cueva virus. Ebola and Marburg viruses cause hemorrhagic fever. The Cueva virus was recently identified in bats in a cave in Spain and has not been shown to infect humans.There are five species of Ebola virus, three of which cause disease in humans: Zaire, Sudan, and Bundibugyo viruses. The Zaire Ebola virus is the one that is causing the current epidemic. Reston Ebola virus infects primates and pigs in the Philippines and does not cause disease in human.

Ebola virus virion. From

The Ebola virus genome has seven different genes which code for proteins that allow the virus to attach to and enter cells, replicate the virus chromosome, make more viruses, and cause disease. Some of these proteins pull double duty: they are needed for replication of the virus but they are also involved in the viruses ability to cause disease. These multi-tasking virus proteins are glycoprotein (gp), VP24, and VP35.

Ebola virus genome. From:

As with all viruses, Ebola virus needs to be able to enter cells and take over the cell to be able to make more viruses. The viral glycoprotein binds to a specific cell surface protein (the receptor) and gets the viral nucleocapsid (the protein coat & viral genome) into the cell to begin the replication cycle. The Ebola virus genome is a negative-sense RNA genome. What this means is that it can't be used immediately to direct synthesis of viral proteins. The genome needs to first be copied to a positive-sense RNA. When Ebola virus enters cells it brings with it extra proteins needed for this first copying step - L (viral RNA polymerase), VP30, and VP35. Once a positive-sense RNA is made, the cell's ribosomes will then translate that RNA to make all of the viral proteins. The L/VP30/VP35 complex will also copy the positive-sense RNA to make many copies of the virus chromosome. The various proteins that make up new Ebola viruses will then assemble with the negative-sense RNA copies to make nucleocapsids inside the cell. Ebola virus acquires its lipid envelope from the plasma membrane of infected cells via a process known as budding. The viral glycoproteins are incorporated into the plasma membrane during synthesis, VP40 and VP24 attach to the glycoproteins, then the nucleocapsid with VP35 and L attach to VP40 initiating a process by which the virus forces itself out of the cell (budding).

Ebola virus life cycle. From:

The goal for a virus is to make more viruses and get to the next host. To be able to do this, a virus will often express proteins that counteract the immune response. The goal of the host's immune response is to stop viruses from replicating and to get rid of the viruses already made. As a result, many symptoms, especially early symptoms of hemorrhagic fever, are actually your body's way of trying to get rid of viruses  (or anything foreign entering your body). So in essence, a virus infection is an arms race and a detente is reached - the virus wins by spreading to other people and you usually don't get killed - which is why vaccination is important (get your vaccines!). Three Ebola virus proteins have been shown to upset this arms race: glycoprotein (GP), VP35, and VP24. So how do these three viral proteins affect the immune response?

There are actually three forms of glycoprotein that are expressed. Some of these are due to RNA editing whereby extra adenines are added during copying of the negative-sense RNA and some are due to processing of the expressed glycoprotein. These three glycoproteins (GP) are the full-length glycoprotein that becomes part of the virus, a small secreted GP (ssGP)  and a secreted GP (sGP) that forms a dimer. sGP acts as a decoy antigen (an antigen is something that is specifically recognized by cells and proteins of the immune system). It will bind to antibodies that are directed against GP preventing those antibodies from binding GP and preventing virus infection, which means the antibody response against Ebola will not be as effective resulting in more virus infection. sGP is also expressed in much higher quantities than GP; therefore, more B cells are activated and more antibodies produced against sGP than against GP. This is called antigen subversion as the specific immune response against the virus ends up being weaker than it should. Again, the result is that more virus will be able to infect cells and produce even more viruses.

VP35 and VP24 both target what is known as the interferon pathway. Interferons (IFN) are a group of proteins whose function is to activate an anti-viral response in cells. IFN acts as a warning signal for uninfected cells. When a cell becomes infected with a virus, the cell responds by producing IFN, which is then released from the cell. IFN then binds to uninfected cells and activates an anti-viral response in uninfected cells so that cell will be primed to fight off the virus if the virus happens to get into the cell. Many viruses have evolved ways to stop this process so that more viruses can be produced.

One of the activators of the IFN response is double-stranded RNA, which is produced when the Ebola virus L/VP30/VP35 protein complex makes copies of the RNA genome. The cell expresses proteins, RIG-1/MDA-5, that bind to double-stranded RNA to activate the IFN response. VP35 of Ebola virus also binds to double-stranded RNA preventing RIG-1/MDA-5 from binding. Not all the double-stranded RNA will get bound by VP35 so the IFN response may still proceed. VP35 can also bind to proteins that are down-stream of the pathway (IRF-3/IRF-7) preventing these proteins from becoming activated. The end result is that very little to no IFN will be produced in cells in which VP35 is expressed.

Some IFN is produced in cells in response to Ebola virus infection, which will activate the anti-viral response in other cells. In these cells that are then infected by Ebola virus, VP24 will block activation of the anti-viral response. Upon binding of IFN to a cell surface receptor, STAT1, a transcription factor, becomes activated. It is transported to the nucleus via a carrier protein called karyopherin a1 to turn on genes needed for the anti-viral response. VP24 binds to karyopherin a1 to prevent it from bringing STAT1 into the nucleus, which means that the cell will be less effective at stopping virus infection. VP24 also binds to STAT1 directly but the effects of this are not currently known. The end result is more viruses being produced!

Ebola VP24 & VP35 inhibits IFN pathway. From:APP Zhang et al. Virulence 5:440, 2012.
Many of these proteins also have other effects on the tissues and organs of someone who is infected with Ebola virus resulting in many of the symptoms of hemorrhagic fever. Initial symptoms include: high fever, fatigue, dizziness, muscle, bone or joint aches, and weakness (similar to many other viral infections). As the disease progresses, bleeding may occur as well as some of the following symptoms: shock, nervous system malfunctions, coma, delirium, kidney failure, and liver failure. The hemorrhagic manifestations are a maculopapular rash (flat, red area on the skin that is covered with small confluent bumps) and mucosal bleeding (especially in the gastrointestinal tract).

On-Line Resources:
PLOS Ebola Collection:
UC Santa Cruz Ebola Genome Portal:
Science special collection:
Centers for Disease Control and Prevention: