Death Called a River
Ebola hemorrhagic fever is one of the most virulent diseases known to humankind. Very few pathogens are more dangerous than Ebola virus once a person is infected. There is no cure, and with a case-fatality rate of between 50 and 90 percent, depending on which strain in involved, it is one of the deadliest viruses on the planet.
But Ebola virus is also one of the rarest viral infections in humans. Despite the tremendous attention it has received in recent years, of all the pathogens that have plagued mankind throughout history, Ebola virus is little more than a bit player. It has sustained but nine major outbreaks since it was first recognized some 25 years ago, claiming at most a few hundred lives each time. By comparison, five times as many people die in workplace accidents in the United States each year than all those who have ever died of Ebola hemorrhagic fever.
"People say to me, 'Why do you work on Ebola virus when it has still killed less than 1,000 people that we know of?'" says Immunology Professor Dennis Burton.
"My answer," he says, "is that we don't know where the virus is headedwhether sooner or later [it] could become more dangerous to a wider population."
The scariest thing about Ebola virus is not what it has done but what it might do.
A Virus Emerges
In the summer of 1976, Ngoy Mushola, a doctor from Bumba, Zaire, traveled to Yambuku, a town on the shores of the Ebola river.
There, at a local hospital, Mushola recorded the first clinical description of a new disease that was killing almost all of the patients who contracted it. "The illness is characterized with a high temperature of about 39°C, hematemesis [the vomiting of blood], diarrhea with blood, retrosternal abdominal pain, prostration with "heavy" articulations, and rapid evolution death after a mean of three days," he wrote in his daily log.
The illness, which was later named Ebola hemorrhagic fever after the nearby river, was successfully contained in Zaire over the course of a few months, but not before 318 people contracted the virus. Nearly 90 percent of the victims died within a few days of becoming infected.
Hundreds of miles away, in Maridi and Nzara, two cities in the southern tip of Sudan, doctors were witnessing an outbreak, describing patients with high fevers, aches, nausea, bleeding, delirium, and what they termed a "mask-like" or "ghost-like" face. Two hundred and eighty-four were infected and over half died.
One of the main risk-factors associated with Ebola virus in the Sudan outbreak was caring for the sick. The disease was spread within hospitals, and many medical care personnel were infected. In several of the Ebola hemorrhagic fever outbreaks that have followed, health care workers have been at risk, and there have been many documented cases of doctors and nurses contracting Ebola virus from the patients they were tending.
Scientists and laboratory personnel working with the live virus are also at risk, and a few months after the Sudan outbreak, a scientist working with the virus in England became infected after he accidentally stuck himself with an infected needle.
Virulent and Rare
Burton, who does not work with live virus, began to study Ebola virus in the mid-1990s, expanding his successful studies on the interplay of viruses and antibodies in humans. In particular, he had been looking at several "slow" virusesincluding human immunodeficiency virus and herpes simplex virusand wanted to raise antibodies against a virus that was very different. He chose Ebola.
"The virus is intriguing because it acts so quickly," says Burton. "It kills people in two weeks or less."
As deadly as Ebola virus is, it has never sustained a large outbreak, probably due to its speed of action and how powerfully sick it makes people. Even as case-fatality can approach 90 percent, infected patients become bed-ridden while they are most infectious, and infection is spread only through direct contact with bodily fluids. Thus, patients are easily quarantined and outbreaks contained.
"Humans are the unlikely target," says Neuropharmacology Professor Michael Buchmeier, who has studied Ebola virus and related viruses for a number of years. Humans are not the natural reservoir for Ebola virus, but merely incidental or accidental hosts.
Until recently, Buchmeier and Burton were co-investigators on a National Institutes of Health project to research immune therapies against Lassa virus and Ebola virus. Now Buchmeier is concentrating his research on the virus that causes Lassa Fever, which is believed to infect over 100,000 people a year, cause 3,000 to 5,000 deaths, and be the leading cause of fetal death in some West African countries.
Ebola and Lassa are both non-human viruses. They are persistent in animal populations in the wild, and remain in this animal "reservoir" population because they are not deadly enough to kill the infected animalsan evolutionary advantage for a virus to remain endemic in its host species population.
In the case of Lassa fever virus, the animal host is the multimammate rat, a rodent common to many parts of Africa. Humans become infected when they come into direct contact with virus particles in rat waste. Scientists suspect a similar host for the Ebola virus.
"There has to be something out there that harbors Ebola virus," says Buchmeier.
Though much work has gone into identifying the source of this virus, none has yet been found. Ebola virus and the closely related Marburg virus have both been found to infect humans and monkeyssome strains infect one or the other, and some strains infect bothbut neither human nor monkey populations harbor the virus in between outbreaks.
Some believe that the natural source of the Ebola and Marburg viruses might be bats, because of the association of some outbreaks with people who had visited caves and mines containing many bats. Given the source of Lassa virus, rodents are another good candidate. However, despite repeated attempts to culture Ebola virus from animals in the wild, the source of the virus has never been found.
Burton thinks an antibody he has made might provide a technology that would help.
From the Marrow of a Survivor
Antibodies to Ebola virus appear 10 days to two weeks after the infection, which is bad timing for the infected person as the virus has more often than not run its lethal course by then.
"We got bone marrow from two survivors [of the 1995 Ebola hemorrhagic fever epidemic in Kikwit, Democratic Republic of Congo]," says Burton. Case workers for the U.S. Centers for Disease Control and Prevention (CDC) provided the marrow. "We made phage display libraries from that bone marrow."
Phage display is a method for selecting from billions of protein variants those that bind to a particular target. In the technique, libraries of antibodies are fused to the viral coat protein of the phagea filamentous virus that infects bacteria. Then the virus is allowed to reproduce in culture, where it copiously makes new copies of itself and the antibody library.
"In effect, we reconstituted the antibody response [the survivors] made in Africa six months later in the laboratory," says Burton.
Since the phage virus displays these proteins on the surface of the virions, a scientist can easily select for antibodies to them in vitro by passing the viral stew over a stationary phase containing the target substratein this case, irradiated, inactivated Ebola virions. Those that can bind do, and the best antibodies are those that bind the tightest and resist being washed off the stationary phase.
"You are left with the ones you are interested in," explains Burton. Then these antibodies of interest can be sequenced and placed into an expression system where they can be mass producedBurton has produced gram quantities of one such antibody in the past few years.
This antibody reacted particularly strongly against the viral coat glycoprotein on inactivated Ebola virus. Subsequent tests carried out by Burton's collaborators in BioSafety Level 4 laboratories have shown the antibody to be reactive against live Ebola virus in cell culture and in live models. Promising results so far.
Burton and his colleagues are interested in looking at the possibility of using the antibody derived from this patient as a serum that might be used to treat patients, particularly as a first-line defense for laboratory workers who accidentally receive a needle prick injury.
He also is developing a colorimetric assay to test samples taken from animals in the wild to look for evidence of Ebola virus. This assay might then be made into a field-ready kit so health care workers have better odds of identifying Ebola virus's animal reservoir.
"If the animal has antibodies against Ebola virus in its serum, then you can see that in this color test," says Burton.
Such a detection method would also prove invaluable for safeguarding against the accidental import of Ebola virus into the United States or other countries through monkeys, as has happened on several occasions. In 1989, for instance, an outbreak of Ebola hemorrhagic fever in Reston, Virginia killed several monkeys that had been imported from the Philippines.
And Burton uses his antibodies as probes to study the basic science of Ebola, an important advance, because much about the virus is unknown.
There is much about the Ebola virus that is still a mystery. Replication strategies are poorly understood. The mechanism for Ebola entry into a cell is not known. We do know that once Ebola virus is inside cells, it goes about replicating itself, and we know that the virus requires the recognition of a receptor on the surface of a cell to enter that cell. But we do not know for certain what that receptor is.
Ebola forms long filamentous virions inside infected cells. When a virion is made, the structural proteins associate with the RNA strand, packaging it in a capsid that then associates with viral proteins that insert into the cell membrane, which allows the whole package to bud off from the infected cell and form a new virion. The genetic material is a single strand of antisense (-) RNA of about 20,000 nucleotides. When transcribed by its own polymerase enzyme, the viral RNA codes for a nucleoprotein, a few structural proteins, the polymerase, and the glycoprotein target of Burton's antibody.
"We're interested in the function of the glycoprotein," says Buchmeier, though he adds that he works mostly with the related family of arenaviruses, which like the filovirus family to which Ebola belongs, cause hemmoragic fever in humans.
The glycoprotein forms spikes, approximately seven nanometers long, on the virion surface. These glycoproteins define the receptor specificity, mediate the cell fusion and cell entry, and may have certain domains that interfere with other cell functions. Like all viruses, Ebola has a certain cell specificityit targets endothelial cells and macrophages. Ebola may even use its spikes to spread from cell to cell, thus evading the immune system and increasing its virulence.
"That," says Burton, "probably has something to do with its extreme pathogenicity and the fact that the immune response to it is so slow."
Once inside a cell, the virion uncoats and the polymerase transcribes the viral (-) RNA into a (+) sense strand inside a host cell's cytoplasm. There, the sense strand, and at some point, the polymerase switch into replication mode and copy the (+) sense strand into an anti-(-) sense strand. These are packaged with other virus components and released, along with components of infected cells.
"[Infected cells] release a storm of early cytokines, like TNF-alpha, interleukin-6, and the interferons alpha and beta," says Buchmeier. "These cytokines are very toxic and cause shock and damage to the body."
Death comes from a combination of dehydration, massive hemmoraging, and shock, which results from this massive release of cytokines.
Though there are vaccines in trial, there is currently no cure for Ebola hemorrhagic fever. The best treatment consists of administering fluids and taking protective measures to ensure containment, like isolating the patient and washing sheets with bleach.
The Once and Future Virus?
The timing of the appearance of Ebola hemorrhagic fever in Africa 25 years ago was a case of epidemiological irony.
Even as this new threat was emerging, another deadly virus was being cornered there. In 1976, the World Health Organization was monitoring progress on its global smallpox eradication effort started a decade earlier. This effort was to be successful within a yearthe last case of smallpox on earth occurred in Somalia in 1977.
Today it is Ebola virus that looms large, though perhaps not in numbers. Ebola hemorrhagic fever has killed hundreds. Smallpox hundreds of millions. Still, what will eventually become of Ebola virus is impossible to say. In the latest outbreak, the World Health Organization was reporting, as of last week, 33 confirmed cases of Ebola and 24 deaths in the countries of Gabon and the Democratic Republic of Congo.
For his part, Burton is interested in how the antibody he has isolated might be used as a possible treatment.
"You cannot be complacent about something like Ebola virus," says Burton. "You have to watch out for [it]."