Chapter 14. Filovirus
Four days ago, the tissue and blood samples, air-shipped by a medical overnight delivery service from Tanzania, had arrived at the canary-colored building on the Via Portuense in Rome called the Instituto Nazionale per le Malattie Infettive Lazzarro Spallanzani. Matteo Graciano was the Level 4 Biohazard Lab Manager at the Instituto Nationale. The doctor at the hospital in Kigoma who had called had been most adamant when he expressed his concern that they might be dealing with a very serious viral hemorrhagic fever. The symptoms were consistent with a VHF: fever, dizziness, head ache, bleeding under the skin, bleeding from orifices, bright red eyes, an expressionless face with a mass of subcutaneous bruises, hot black vomit. Graciano had heard this before at the Institute, of course. In his twelve years working at the Instituto Nationale, he had only seen one of these scares turn out to be the real thing. However, in dealing with potential biohazards, every sample had to be treated as if it were a Level 4 pathogen. Four days ago, Graciano had gone to work immediately, unwrapping the specimens from the biohazard container.
Graciano had first performed a series of simple tests for a host of more common diseases like malaria and typhoid fever. These had come back negative. Then Graciano used a testing methodology developed at Columbia University called the Greene Mass Tag Panel VHF, which could simultaneously test for viral hemorrhagic fevers like Ebola Zaire, Ebola Sudan, Ebola Cote d'Ivoire, Ebola Bundibugyo, Marburg Virus, Lassa Virus, Rift Valley Fever, Crimean-Congo hemorrhagic fever, Hantaan, Seuol virus, yellow fever, and Kyasanur Forest disease. The Greene Mass Tag Panel used a technology called "real-time" RT-PCR. This test involved taking a tiny DNA sequence and amplifying it into billions of copies. The process was called "real time," because it used to take days and now could be done in "real time," or just hours. Once the sequence of DNA was copied billions of times, the analyzing machine could then chart whether specific gene sequences found in existing forms of Viral Hemorrhagic Fevers were present in the sample. Graciano removed a 64-well yellow plastic microtiter plate which contained sixty-four tubes with snap-on lids. With a six-inch long pipette, he plunged into each of the sixty-four tubes some serum taken from the boy in Tanzania. Then he followed a painstaking recipe, which involved placing various chemicals into the tubes and incubating them at various temperatures.
This was painstaking work, but Graciano was up to the task. Ever since he was a young boy, he and his two brothers had been interested in viruses. It was an agricultural virus which had ruined his father’s business. Graciano always felt that he was getting a little vengeance for his father every time he saved the world from one of these dangerous little bugs.
Graciano inserted into the mixture a fluorescent "probe." The probe was a tiny piece of DNA, which contained a fluorescent marker. After billions of copies of the molecule were made, the lights could be detected, like an airplane pilot viewing the thousands of candles at Lady Diana's night vigil. A computer detected the light and registered it on a read out.
Graciano was not surprised when the results of this initial real-time RT-PCR test were negative for any form of known Viral Hemorrhagic Fever. In all likelihood, the African doctor's diagnosis was incorrect. This could even be something as simple as the flu. After all, it would not be the first time that someone in a third world country died of the flu. However, the existence or non-existence of a Level 4 pathogen was not determined with only one test. Graciano had several more tests to complete. Because this new sample did not appear to be a VHF, Graciano put the remaining tests off for a while, so that he could complete more pressing work in the lab.
Graciano was working on a report for his boss, who was scheduled to speak at the World Health Organization in a few days. The subject was potential cures for some of the world’s most dangerous diseases. Graciano had been in touch with research labs all over the world to find the most up-to-date answers on curing these dangerous viruses. The Powerpoint was nearly complete. Graciano enjoyed working later hours, in the deathly quiet of the Italian lab. It gave him time to think. He had a lot of personal demons, and the quiet gave him comfort. He pulled out a recent study from Belgium and summarized the contents on his Powerpoint slide. After the presentation was completed, he returned to testing the African samples.
Graciano had removed the Tanzanian samples and proceeded to test them with the Ebola virus and Marburg virus "antigen capture test." The antigen was the unique and distinctive part of the foreign invading target which had entered the body. The antigen capture assay test was a simple test in which the bottoms of the wells of a multi-well microtiter plate were coated with specific Ebola virus and Marburg virus antibodies. Then the blood of the infected boy was inserted into the coated wells, and Graciano would observe whether any of the specific Ebola and Marburg antibodies would "bind" to any antigens in the blood samples. If the result was positive, the well would be colored. If the test was negative, the result would be clear. When Graciano finished this test several hours later, all the wells were clear.
Graciano completed the third test on the samples just before dinner. The third test was the IgM and IgG ELISA assay test. "Ig" stood for immunoglobin, one of the main types of antibodies comprising the body's immune system. The antibodies were the body's police force. When a foreign substance harmful to the body enters, these Y-shaped structures latch on to the invading criminal and put him behind bars. If these young Tanzanian boys had been infected with a viral hemorrhagic fever, the body's white cells should have been in overdrive trying to produce more anti-Ebola or anti-Marburg police officers. Graciano expected this final test to be negative as well. When Graciano ran this test, and plunged the chemicals from the IgG and IgM assay test into the wells of the boys' blood, he was shocked by the result. Although the wells for the antibodies of Marburg virus, Ebola Sudan, Ebola Reston, Ebola Ivory Coast, and Ebola Bundibugyo were clear, every well with Ebola Zaire antibodies was colored! That did not make any sense. He repeated the test again and got the same results.
Now he was stumped. How could the body of this boy be producing Ebola Zaire antibodies, but the antigens for Ebola Zaire were not present, and the real time RT-PCR test showed no sign of Ebola Zaire? Unless…. His next thought concerned him greatly. Could this be an entirely new strain of Ebola that was, perhaps, similar to Ebola Zaire but with a different chemical structure? Graciano recalled something similar with the CDC's investigation of the newly-discovered Ebola Bundibugyo Virus in Uganda in 2007. Graciano went down to his office and opened a black filing cabinet. Pulling out a file marked "Ebola Buggy," Graciano removed from the file a November 2008 article from the PLoS Pathogens Journal. He spent twenty minutes scanning the article. As he finished the article, he was sure he was on to something. There, as here, the initial RT-PCR test had been negative, but the IgG and IgG tests had been positive. This must be a new strain of Ebola!
However, Graciano could not make a firm diagnosis with such equivocal results. He would ultimately need to get this blood gene-sequenced. But first he needed to look at this "bugger" up close and personal. Graciano could not go to sleep now. He went back to the biohazard container, and removed the small tissue samples from the Tanzanian boy's spleen. He crushed them on a table with a mortar and pestle, and dropped the bloody mess into a flask with human kidney cells. He did the same for the boy's blood cells, repeating the process until he had a rack of flasks. Then he put the flasks into an incubator, letting them "cook." While the flasks were in the oven, he pulled out some more articles on Ebola Zaire from his filing cabinet and read them. He fell asleep at about 2:00 a.m. at his desk.