Virology: The Ebola Virus

NICK TSAKMAKLIS '08

The Ebola virus is named after a small river in Sudan, the site of the first cases. It is one of the world’s most dangerous agents of death. Since the first major outbreak in 1976, similar outbreaks occurred in 1979, 1984, 1995, 2000, and 2001. According to the Centers for Disease Control and Prevention and the World Health Organization, 54.5 percent of individuals who contracted the Ebola virus Sudan (EBO-S) passed away. Additionally, 81.3 percent of people who became infected with Ebola virus Zaire (EBO-Z), perished. The Ebola virus belongs to the Filoviridae family of viruses. There are four distinct types of this virus: Zaire, Sudan, Cote d’Ivoire, and Reston. Out of these four versions of the virus, only the first three are responsible for causing humans illness. The fourth subtype, Reston, has not caused “clinical illness” in the few individuals who contracted it . As evident, there are multiple types of Ebola virus, with the majority of them being fatal to those who become infected.

The Ebola Virus was first discovered in a western province of Sudan and in a region of what is now known as the Republic of Congo (formerly Zaire) in 1976. In this outbreak, 284 people were infected. Of them, 151 succumbed to the deadly virus. At the same time, in the Republic of Congo, the virus proved fatal in 280 of the 318 reported cases. Another outbreak occurred in 1979 in Sudan which was responsible for 22 deaths (of the 33 individuals who contracted the virus). In 1989, the Ebola – Reston virus was isolated in monkeys in Reston, Virginia. Through the next seven years, a small number of cases occurred in monkeys and humans (though none were fatal). In 1994, the Ebola virus hit Gabon for the first time, killing 9 of the 19 people it infected. Subsequent breakouts occurred in February of that same year (killing 21 of 37 individuals), and in July of 1996 (killing 45 of 60 individuals). In 1995, the virus struck the Republic of Congo again. This time, 250 of the 315 infected individuals died. Between September of 2000 and January of 2001, the Ebola virus hit Sudan for the first time since 1979. This time, 224 of the 425 infected patients passed away, marking this as the largest Ebola epidemic ever. More recently, from October of 2001 to December of 2003, the Zaire subtype of the virus struck Gabon and the Republic of Congo, totaling 254 deaths (out of 302 reported cases). The total number of casualties due to the virus is staggering: out of the roughly 1,850 confirmed cases since its discovery, 1,200 did not survive.

The Ebola virus contains a single-stranded, negative –sense RNA molecule. This means that the RNA molecule must be translated to positive-sense RNA (meaning that it can be translated directly into proteins) before translation . Ebola’s genome consists of 18,959 base pairs. The virus consists of seven genes. Of these, four are structural proteins (VP30, VP35, nucleoprotein, and polymerase protein). The remaining three genes code for membrane-associated proteins (VP40, glycoprotein, and VP24). The length of the virus varies, but the optimum size for infectivity is 970 nanometers. The virus can take the shape of a long filamentous rod, U-shaped, or ring-shaped figures. Despite the variations in length and shape, the diameter of the virion is consistently eight nanometers in diameter. Not much is known about the receptor that is involved with Ebola.

Little is known about the Ebola virus’ life cycle and reproductive tendencies, though there are some theories and ideas that attempt to decipher the puzzle. It is assumed that the Ebola virus replicates similar to other negative-sense viruses, such as the influenza virus. The replication process is believed to happen in the cytoplasm of the host cell. Likely, the viral polymerase makes mRNA transcripts and ultimately begins the replication process. First, positive RNA strands are made, followed by negative ones. The negative-sense RNA then goes through another round of mRNA transcription and replication. In time, as enough viral proteins and negative-sense RNA strands accumulate, the virus buds. How the Ebola virus replicates and buds is yet to be determined, however. During this process, an interesting event occurs. One of its genes can only be expressed in a process known as transcriptional editing. Here, the viral polymerase actually adds an extra Adenosine base into the growing mRNA in order to create the proper protein sequence for the GP protein. The non-edited mRNA sequences produce SGP, which is a small, non-structural protein that is removed by the cell in large quantities. It is inferred that SGP plays some kind of role in confusing the immune system of the infected individual, thus throwing off any kind of immune response.

The incubation period of the virus is anywhere from two to twenty-one days. It is highly contagious, and infected individuals remain infected, even after death. Ebola virus can be spread through direct contact of blood, secretions, or bodily fluid of infected individuals. Additionally, contact with the deceased helped pass along the contagious virus. Commonly, unprepared workers, nurses, and doctors at hospitals contracted the virus from the infected patients. Individuals also became infected at funerals of the deceased, when proper burial protocol was not followed. As a result, many mourners received the virus. Additionally, the virus can be spread through family interactions. For example, an infected individual might unknowingly pass on the virus to his whole family during one of their meals.

To protect against this type of senseless spreading of the virus, stronger measures were taken. Patients suspected of containing the virus were isolated and barricaded from the rest of the hospital. Individuals associated with the suspected patients were also tested to make sure that they were not infected. All hospital’s personnel were briefed on the contagious nature of the virus. The importance of being careful while executing intravenous processes such as exacting blood, or handling other bodily secretions such as urine or saliva, was emphasized. Disinfection of soiled clothing and bed linens was also required due to the contagiousness of the virus and the high risk of transmission . Nurses and doctors were asked to wear masks, gloves, gowns, and goggles to prevent any spreading. All equipment was to be sterilized or thrown out . Lastly, it was the responsibility of the community where the outbreak occurred to educate its inhabitants on the seriousness of the virus and the ways in which it can be spread.

Though it is known that the virus can affect both humans and primates and that is can be transmitted rapidly between the two species, there is still a large piece of missing information. Little is still known about the origins of the virus, or about the original host. The “natural reservoir” of the virus is still unknown, but it is believed that it lives somewhere in the rain forests of Africa and in Western Asia. Although primates have been known to be able to contract and transmit the virus, they are not believed to be the original source. Instead, it is believed that they, like humans, contracted the virus from the mysterious “natural reservoir”. The virus is not exclusive to just humans and primates, however. Ebola infections have been linked to contact with forest antelope and porcupines. This indicates that other organisms might also be able to contract the virus and pass it to others, though they might not be the original source. One of the better hypotheses is one which speculates that bats might be the source of the “natural reservoir”. The reasoning behind this is that in laboratory tests and experiments, bats infected with the Ebola virus did not die. This proves that they might be the carriers of the virus, and perhaps the original source as well.

The most common way to diagnosis a possibly infected individual is through an indirect immunofluorescence test. One of the problems associated with diagnosing potentially infected patients is the risk of further spreading the disease through contact with the patient’s blood or other fluids. Another problem is that many of the preliminary symptoms, such as itchy eyes and skin rash, are not exclusive to the Ebola virus and are quite frequent in many other diseases. Currently, definitive diagnosis of the virus is based on its isolation in tissue cultures. Isolating the virus, however, poses a problem. Due to the contagiousness of the virus and its rapid transmission, the utmost caution must be taken when handling it. As a result, few laboratories in the world can handle it properly and safely. Another means of detection involves an immunoglobulin M and immunoglobulin G enzyme linked immunosorbent assay (ELISA) using Ebola Zaire antigens. Other commonly used tests include the Western Blot Analysis and radioimmunoprecipitation assays (uses radioisotope labeled antigens) . Immunoprecipitation assays involve attempting to remove an antigen from a solution by using an antibody that attaches to the antibody . A Western Blot Analysis is when an antibody is used to detect a protein in a mixture of many other proteins . Survivors of the virus can be tested for IgM and IgG antibodies (immunoglobulin M and G, respectively). Deceased individuals can be tested after death by polymerase chain reaction (PCR) or virus isolation to determine if they had succumbed to the virus. Though there are a few methods used in diagnosing patients, the large risk is in attaining samples and handling them.

Individuals infected with the Ebola virus can be subjected to a plethora of symptoms. Early symptoms might include fatigue, headaches, sore throats, vomiting/nausea, diarrhea or arthritis. As the virus begins to replicate and proliferate, more serious symptoms arise. These symptoms might include inflammation of the eyes (conjunctivitis), rashes, swelling of the genitals, internal bleeding, bleeding of the eyes, throat and mouth, and/or seizures and coma . The more severe symptoms involve internal bleeding, such as bloody noses (epistaxis), bleeding of the lungs, vomiting of blood (hematemesis), and bloody eyes (conjunctival hemorrhages). Death usually occurs within a week of infection, usually due to shock or internal bleeding.

Currently, there is no cure to the Ebola virus. Since the source of the virus is still unknown, it is impossible to control or eliminate the natural reservoir. The speed at which the virus works has also proved to be a problem, as few people have been able to develop a proper immune response. Additionally, obtaining samples and studying them is extremely difficult to do, especially in the areas of outbreak. Few places in the world have the kind of equipment and laboratories needed to study the lethal virus safely. Supportive therapy is provided to infected patients. This includes balancing their fluids and electrolytes, and maintaining healthy oxygen and blood pressure levels. Also, patients are treated for their headaches, fatigue, sore throats and/or any other curable symptom they display. Though there is no current cure for the more devastating effects of Ebola, one of the best ways to contain the virus is by isolating those individuals who are presumed to be infected. By isolating them, the virus is not allowed to spread. This might seem like a passive way of eliminating a virus (as opposed to finding an actual cure), it is absolutely necessary in preventing more deaths.

While there is no vaccine out there for the time being, one is being developed and is in the Phase I Clinical trial. A three-plasmid DNA vaccine was developed that encoded the envelope glycoprotein from the Ebola virus Zaire and Sudan subtypes. The plasmids were put together in a way so that three Ebola virus proteins would be constructed. These proteins include a nucleoprotein taken from the Ebola Zaire virus, and two glycoproteins (taken from the Zaire and Sudan subtypes). The glycoproteins contained deletions that were meant to eliminate toxicity. The three plasmids cannot replicate in animals, and would not be able to produce a potentially infectious virion even if some sort of genetic recombination were to occur.
This was injected into twenty healthy adults, ranging from 18 to 44 years old. They received the vaccine three times, at two milligrams, 4 milligrams, 8 milligrams, or placebo. The subjects were then tested by ELISA, Immunoprecipitation, and Western Blot analysis. The testing went well, with no adverse effects or coagulation abnormalities. All twenty of the subjects developed a specific antibody response to at least one of the three antigens. Though the experiment was fairly basic, it did prove that vaccination through DNA plasmids might be possible, and deserves to be further studied.

Placed in the wrong hands, the Ebola virus can become a major agent of bioterrorism. Ebola is a strong candidate for bioterrorism because of its high mortality rate, infectious nature, and lack of treatment. Among all hemorrhagic fevers, Ebola has the highest mortality rate. The speed and ease at which it infects is unprecedented. Uninformed clinics or hospitals could further spread the virus during a bioterrorist attack. The fact that the virus currently has no cure also puts it at the top of the list. With no cure, treatment or vaccine in the immediate future, the chances of defending against such an attack is slim. For this reason, the virus is listed as a category A bioweapon agent. Unfortunately for bioterrorists, obtaining enough samples to make an impact is next to impossible outside of the world’s top laboratories . This has not stopped groups in the past, however. Attempts to use the virus have been made. The former Soviet Union amassed significant amounts of Marburg (a similar virus, in the same family as Ebola) and Ebola. Japan’s Aum Shinrikyo cult tried to obtain samples of Ebola during outbreaks in the mid-nineties from Kikwit and Zaire.

The Ebola virus is among the world’s most dangerous viruses. Its catastrophic nature was put on display in the Cote d’Ivoire, the Republic of Congo, and Sudan beginning in the late seventies. The virus belongs to the Filoviridae family. Only three of the four subtypes have affected humans: Zaire, Sudan, Cote d’Ivoire, and Reston. Ebola has been responsible for at least 1,200 deaths (out of approximately 1,850 cases). Symptoms of the virus include: headaches, conjunctivitis, rashes, and internal bleeding. There is still no cure or vaccine to the virus, though quite a few are being tested at the moment. The need to find a treatment for the Ebola virus is crucial, as the virus’ ease of transmission and high mortality rate can be used in an attack against innocent individuals in bioterrorist attacks.

Bibliography
A, Polesky, and Bhatia G. "Ebola Hemorrhagic Fever in the Era of Bioterrorism." NCBI. 14 Jan. 2008.
"Definition of Fever, Ebola Virus." MedicineNet.Com. 04 Nov. 2001. 12 Jan. 2008.
"Ebola Hemorrhagic Fever." World Health Organization. Sept. 2007. 11 Jan. 2008.
"Ebola Hemorrhagic Fever." Medicine Plus. 06 Mar. 2006. 13 Jan. 2008.
Graham, Barney S. "A DNA Vaccine for Ebola Virus is Safe and Immunogenic in a Phase I Clinical Trial." PubMed Central. 20 Aug. 2006. 13 Jan. 2008.
"Immunoprecipitation." Wikipedia. 6 Jan. 2008. 12 Jan. 2008.
Khalsa, Guruatma. "Western Blotting." Mama Ji's Molecular Kitchen. Arizona State University. 12 Jan. 2008.
King, John W., and Anurag Markanday. "Ebola Virus." EMedicine. 31 Jan. 2007. 11 Jan. 2008.
Paustian, Timothy. "Ebola Virus." Microbiology and Bacteriology. 2006. University of Wisconsin-Madison. 11 Jan. 2008.
"Questions and Answers About Ebola Hemorrhagic Fever." CDC. 18 Nov. 2005. 13 Jan. 2008.
"Sense (Molecular Biology)." Wikipedia. 9 Jan. 2008. 12 Jan. 2008.
Sullivan, Nancy, Zhi-Yong Yang, and Gary J. Nabel. "Ebola Virus Pathogenesis: Implications for Vaccines and Therapies." PubMed Central. Sept. 2003. 13 Jan. 2008
"Viral Hemorrhagic Fevers Summary." Bioterrorism and Emerging Infections Education. University of Alabama At Birmingham. 15 Jan. 2008.
Wattam, Rebecca. "Ebola Virus." 11 May 2004. Virginia Tech. 12 Jan. 2008.