Ebola virus: Questions, answers, and more questions
ABSTRACTEbola virus causes a hemorrhagic fever with a high case-fatality rate. Treatment remains supportive although a variety of specific treatments are still in the early stages of investigation. This report reviews the clinical virology of Ebola virus, the reported proposed treatments, and the current outbreak.
KEY POINTS
- Ebola virus is spread by contact with body fluids, with no evidence to date that it is airborne.
- Ebola virus is likely maintained in a reservoir of small animals, possibly bats.
- The incubation period is about 5 to 7 days, during which the patient is not infectious.
- Symptoms begin abruptly, with fever, chills, and general malaise, which in some patients leads to weakness, severe headache, myalgia, nausea, vomiting, diarrhea, and abdominal pain.
- Once the disease is symptomatic, patients have high levels of virus in the blood and other body fluids and are therefore infectious.
- Survivors show improvement in the second week of illness, during which viremia resolves and virus-specific antibodies appear.
EBOLA IS AN IMMUNODEFICIENCY VIRUS
The main targets of infection are endothelial cells, mononuclear phagocytes, and hepatocytes. Ebola virus replicates at an unusually high rate. Macrophages infected with Zaire ebolavirus produce tumor necrosis factor alpha, interleukin (IL) 1 beta, IL-6, macrophage chemotactic protein 1, and nitric oxide. Virus-infected macrophages synthesize cell-surface tissue factor, triggering the extrinsic coagulation pathway.
Ebola is an immunodeficiency virus. Dendritic cells, which initiate adaptive immune responses, are a major site of filoviral replication. Infected cells cannot present antigens to naïve lymphocytes. Patients who die of Ebola virus disease do not develop antibodies to the virus. Lymphocytes remain uninfected, but undergo “bystander” apoptosis induced by inflammatory mediators.
CLINICAL MANIFESTATIONS
The incubation period is generally 5 to 7 days (range 2 to 28 days), during which the patient is not infectious. Symptoms begin abruptly, with fever, chills, general malaise, weakness, severe headache, and myalgia. By the time of case detection in West Africa, most patients also had nausea, vomiting, diarrhea, and abdominal pain. Once symptoms arise, patients have high levels of the virus in their blood and fluids and are infectious. Hemorrhagic symptoms have apparently been uncommon in West Africa, occurring in 1.0% to 5.7%, but “unexplained bleeding” has been documented in 18% of cases.4 Among those in whom the disease enters its hemorrhagic terminal phase, there is characteristic internal and subcutaneous bleeding, vomiting of blood, and subconjunctival hemorrhage.4
Laboratory findings include lymphocytopenia (often with counts as low as 1.0 × 109/L), thrombocytopenia (with counts in the range of 50 to 100 × 109/L), elevated aminotransferase levels (including aspartate aminotransferase levels 7 to 12 times higher than alanine aminotransferase in fatal cases), low total protein (due to capillary leak), and disseminated intravascular coagulation. Those who survive begin to improve in the second week, during which viremia resolves in association with the appearance of virus-specific antibodies.4
DIAGNOSIS
In symptomatic patients, Ebola virus infection is diagnosed by detection in blood or body fluids of viral antigens by enzyme-linked immunosorbent assay, or RNA sequences by reverse transcriptase polymerase chain reaction. The diagnosis is confirmed with cell culture (in a BSL-4 containment laboratory) showing characteristic viral particles by electron microscopy.
CARING FOR PATIENTS
The most detailed descriptions of the care of patients with Ebola virus disease have come from Dr. Bruce Ribner, of Emory University Hospital, in an October 2014 report of his experience caring for Ebola-infected patients at Emory University Hospital in Atlanta, GA.5 He described fluid losses of 5 to 10 L/day, profound hyponatremia, hypokalemia, and hypocalcemia, which were associated with cardiac arrhythmias and the need for intravenous and oral electrolyte repletion and hemodialysis. Intensive one-to-one nursing was critical, as was the coordination of many medical subspecialties. The Emory team arranged point-of-care testing near the unit and generally kept laboratory testing to a minimum. The team was surprised to learn that commercial carriers refused to transport specimens even when they were licensed for category A agents. Difficulties with the local water authority and waste disposal contractor required the hospital to dedicate an autoclave to process all materials used in clinical care.
TREATMENT: SUPPORTIVE AND EXPERIMENTAL
Treatment is supportive to maintain circulatory function and blood pressure and to correct coagulopathy. However, a variety of vaccines, antibodies, small-molecule agents, and antiviral agents are undergoing testing, mostly in animals at this point.
Vaccines. A therapeutic vaccine that worked only slightly was a live-attenuated recombinant vesicular stomatitis virus expressing Ebola virus transmembrane glycoproteins, which was tested in mice, guinea pigs, and rhesus macaques who had been exposed to Ebola virus.6
A preventive vaccine worked better. Stanley et al7 evaluated a replication-defective chimpanzee adenovirus 3-vectored vaccine that also contained Ebola virus glycoprotein. They gave macaques a single injection of this vaccine, and then 5 weeks later gave them a lethal dose of Ebola virus. All the vaccinated animals survived the infection, and half (2 of 4) survived when challenged 10 months later. With a prime-boost strategy (modified vaccinia virus Ankara, a poxvirus), all survived when challenged 10 months later.
KZ52, a neutralizing antibody, did not work. Oswald et al8 gave a human IgG monoclonal antibody against Zaire Ebola virus, designated KZ52, to four rhesus macaques, challenged them with the virus 24 hours later, and administered a second shot of KZ52 on day 4. All of them died.
ZMAb is a combination of three murine monoclonal antibodies, designated 1H3, 2G4, and 4G7. Ad-IFN is a human adenovirus, serotype 5, that expresses human interferon alpha. Qui et al9 gave ZMAb and Ad-IFN to macaques in several experiments. In experiment 1, eight macaques were infected and then were given ZMAb and Ad-IFN 3 days later, and ZMAb again on days 6 and 9. Seven of the eight survived. In a second experiment, Ad-IFN was given first, when the viral load was still less than the limit of detection of known assays, and then ZMAb was given upon detection of viremia and fever. Two of four macaques survived. Control animals had undetectable levels of IgG, whereas Ebola virus GP–specific IgG levels were detected in all survivors. IFN-gamma ELISpots showed high EBOV-GP–specific T-cell response in all survivors.
ZMapp is another cocktail of monoclonal antibodies, containing two from ZMab (2G4 and 4G7), plus a third, c13C6. In experiments in rhesus macaques, three groups of six animals each received three doses of ZMapp at varying times after being infected with Ebola virus: at 3, 6, and 9 days; at 4, 7, and 10 days, and at 5, 8, and 11 days. All 18 macaques treated with ZMapp survived. Thus, Zmapp extended the treatment window to 5 days postexposure.10 One of the American health care workers who contracted Ebola virus in Liberia received this medication.
HSPA5-PMO. Endoplasmic reticulum chaperone heat shock 70 kDa protein 5 (HSPA5) is instrumental in the maturation of envelope proteins in hepatitis C and influenza A virus. It plays a role in viral entry for coxsackievirus A9 and dengue virus serotype 2, and it may be involved in Ebola viral budding. Phosphorodiamidate morpholino oligomers (PMOs) are a class of antisense DNA nucleotide analogs.
Reid et al11 reported that mice treated with HSPA5–PMO were completely protected from lethal Ebola challenge. Therefore, HSPA5 appears to be a promising target for the development of antifilovirus countermeasures.
Favipiravir, an antiviral agent also known as T-705, is a pyrazinecarboxamide derivative. Invented in 2002 by Toyama Chemicals as an inhibitor of influenza virus replication, it acts as a nucleotide analog, selectively inhibiting the viral RNA-dependent RNA polymerase, or causes lethal mutagenesis upon incorporation into the virus RNA. Favipiravir suppresses Ebola virus replication by 4 log10 units in cell culture.12
Mice were challenged with intranasal inoculation of 1,000 focus-forming units of Ebola virus diluted in phosphate-buffered saline. Until the first day of treatment (postinfection day 6), all mice in the T-705 group lost weight similarly to control mice, developed viremia, and showed elevated serum levels of aspartate aminotransferase and alanine aminotransferase. Within 4 days of T-705 treatment (post-infection day 10), the animals had cleared the virus from blood. Surviving mice developed Ebola virus-specific antibodies and CD8+ T cells specific for the viral nucleoprotein.12
The authors hypothesized that suppression of virus replication by T-705 allowed the host to mount a virus-specific adaptive immune response, and concluded that T-705 was 100% effective in the treatment of Zaire Ebola virus infection up to postinfection day 6 but was hardly beneficial at the terminal stage of disease.12 Of note, favipiravir is undergoing phase 2 and phase 3 trials as an anti-influenza agent in Japan.
