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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 phage—a 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 substrate—in 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 produced—Burton 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.

Dangerous Mystery

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 specificity—it 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 year—the 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]."


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Transmission electron micrograph of Ebola virus. Photo courtesy of the Centers for Disease Control and Prevention.