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: http://northshorejournal.org/us-military-assisting-in-ebola-outbreak

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 http://viralzone.expasy.org/all_by_species/207.html

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: http://viralzone.expasy.org/all_by_species/207.html

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: http://viralzone.expasy.org/all_by_species/5016.html

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: https://flipboard.com/section/plos-ebola-collection-bRVgYj
UC Santa Cruz Ebola Genome Portal: http://genome.ucsc.edu/ebolaPortal/
Science special collection: http://www.sciencemag.org/site/extra/ebola/?intcmp=HP-COLLECTION-PROMO-EBOLA
Centers for Disease Control and Prevention: http://www.cdc.gov/vhf/ebola/index.html

How to Write a Research Paper.

So you have to write a research paper for the science class that you had to take. Now what!?

First, what is a research paper? A research paper is a way of synthesizing information about a topic from a variety of sources in a coherent fashion. You will want to have enough information written in an organized manner to demonstrate to your professor that you learned something about the topic. You will want the paper to be well written and organized so you get a good grade and you don't bore your professor (this may not seem important but remember that she will be determining your grade and may have a lot of papers to grade. So make the paper stand out in a good way!)

1. Pick a topic that's interesting to you. Sometimes your professor will give you a list of topics; other times you will be given some general guidelines and have to come up with your own topic. Since you will be spending a fair amount of time doing research and writing for this assignment you should find something that may be of interest to you. Many students have no clue what to write about. You could start with your text book and see what jumps out at you. Perhaps you have a family member that was recently diagnosed with a disease that you could learn more about. Or you can look over the short articles at ScienceDaily where recent research papers are summarized.

2. Make sure the topic is appropriate to the assignment. Your topic should be within the parameters of what your professor wants. Read the instructions (you will see this statement many times because far too many students fail this part of the assignment!) or check with your professor. Listen to what she says during class about the requirements of the paper and write it down! You will want to make sure that your topic is not too broad so that you can include details and real information in your paper. Glossing over topics and writing a superficial paper will make it a boring paper for your professor to read and will likely result in you earning fewer points.

3. Use appropriate sources for the information for your paper. There is a lot of information on the web that can be used to write a research paper. Many of it will not be appropriate, some will. Your search for information should always begin at the library. Your library will have subscribed to a variety of databases that can be searched. Think of databases as a smaller version of Google. The database will be a collection of research papers or books that can be searched. Searching databases is more focused than doing a Google search. Google should not be your main way of looking for information for your research paper!

Some good sources of information:

  - an encyclopedia. Some good ones are Encylopedia Britannica, Tree of Life

  - articles in Scientific American

  - books

  - review articles in scientific journals. These have summarized the research results from a large number of labs. They are written for other scientists but many non-scientists should be able to read and understand it.

   - research papers in scientific journals.

   - some information from organizations such as American Cancer Society or similar organizations is ok but they should not be the main source of your information.

What is not appropriate?

  - Blogs

  - Wikipedia!

  - Videos or news pieces. Some professors may allow documentaries but you should always check first.

  - News articles. A single news article can be used as a starting point for a research paper but should not be a main source of information.

*** Be wary of web sites and organizations that claim there is a conspiracy or scientists are bought by companies to keep information hidden. Most of your information for a research paper should be coming from scientific articles and books. Do not rely only on web-based information from sources that are not familiar. If you are getting information from an organization that is not recognizable make sure to check the validity of the information presented! 

4. Write the paper in your own words! A research paper is not a collection of quotes connected together by a few words or sentences of your own. Once you've started gathering your sources, you should first read the source without taking notes. This will give you a good idea of what information is there and what is likely to be relevant to your paper. Then you can take your notes. Note at the top of your paper, the source, include all the information that you will need for your bibliography/end notes/work cited. Do not write in complete sentences and write the notes in your own words. You will find that there are some things that can't be rewritten in your own words. This does not mean you should quote the material. For example, many papers on AIDS and the virus that causes it has this sentence: Acquired immunodeficiency syndrome is caused by human immunodeficiency virus. There really isn't a way to write this any differently so you don't need to quote it. If you take notes in your own words then you will have to worry less about plagiarism.

When using information from a research paper, you do not need to write many details about how the experiment was done. What you should include is the relevant results and conclusions. Do not fill up your paper with extraneous words and information.

5. Organizing your notes will result in an organized paper. After writing notes of all the information from different sources, you should organize them so that the various pieces of information fit together. Often your professor will suggest that you make an outline. Here is a good explanation of an outline and how to put it together: What is an Outline? When writing your own outline you don't have to be so formal with Roman numerals, etc. but you should organize your thoughts before attempting to do any writing. Make sure you include your sources in your outline. You don't need the full reference but enough so you know where the information came from. This will help you to properly cite the sources when writing your paper (see #7).

6. Use transitions. When you are writing your paper, remember that the various sections should be linked together so that the paper flows. The last sentence of a paragraph should lead from that paragraph and into the topic of the next paragraph.

7. Properly cite your sources. Only listing your sources at the end of the paper is insufficient for a science research paper. You need to use in text citations. Make sure you use the proper format for your citations. Your professor will let you know what is required. For a science paper this will often be APA or AMA format. Here's a good resource of How and When to cite in a paper. And make sure to include your list of references at the end of your paper and in the proper format! Make sure you have the correct information. LOOK for the list of authors. If the source is a research paper in a journal you should have authors, title of the paper, year published (the month and day should not be included), journal name, volume, and page numbers.

8. Use correct spelling and proper grammar. This is a critical skill to learn. When you are out in the workforce, you want to be able to demonstrate that you do know what you are talking about! A poorly written letter/email/report will suggest that you do not. If you struggle with this, you should consult the tutors in the writing center. Also, read the paper out loud. You will often find your own mistakes when you actually hear them yourself!
      a. Avoid use of vernacular language. This is every day type of language. Words such as lots, a lot, got, gotten, etc. should be avoided. And it goes without saying that using texting type shorthand should NEVER be used!
      b. Make sure your tenses match. If you use the past tense make sure to use it throughout the paper. Generally when talking about research that was done in the past, the past tense should be used.
      c. Your subject and verb should match. This means that if you have more than one subject, the verb should be also for multiple subjects. For example: Chimpanzees have opposable thumbs. But also remember that inanimate subjects can't do anything. For example, when referencing a source, authors can state or conclude. The institutions in which the research was performed or the journal in which the paper was published cannot state or conclude.
       d. 15 words you should refrain from using! http://mashable.com/2015/05/03/words-eliminate-vocabulary/?utm_cid=mash-com-fb-main-link

9. Proofread your paper! Once you've written your paper you should set it aside for a few days. Then go back and reread the paper. You may even want to read the paper out loud. If it sounds weird or not right, correct it!