Both Marburg virus and Ebola virus cause an acute febrile illness associated with a high mortality rate. This illness is characterized by multisystem involvement that begins with the abrupt onset of headache, myalgias, and fever and proceeds to prostration, rash, and shock and often to bleeding manifestations. Epidemics usually begin with a single case acquired from an unknown reservoir in nature (bats are suspected; see “Epidemiology,” below) and spread mainly through close contact with sick persons or their body fluids, either at home or in the hospital.
The family Filoviridae (Fig. 197-1) comprises two antigenically and genetically distinct genera: Marburgvirus and Ebolavirus. Ebolavirus has five readily distinguishable species named for their original sites of recognition: Zaire, Sudan, Côte d′Ivoire, Bundibugyo, and Reston. Except for the Reston virus, all the Filoviridae are African viruses that cause severe and often fatal disease in humans (Figs. 197-2 and 197-3). The Reston virus, which has been exported from the Philippines on several occasions, has caused fatal infections in monkeys but only subclinical infections in humans. Different strains of the five Ebola species, isolated over time and space, exhibit remarkable sequence conservation, indicating marked genetic stability in their selective niche.
Phylogenetic tree of filoviruses. Marburgvirus and Ebolavirus are seen to be two different genera. The genus Ebolavirus includes five distinct species. Note that the Yambuku and Kikwit Zaire viruses are virtually identical even though the epidemics for which they were responsible are separated by two decades and hundreds of kilometers. Virtually every virus sequenced from each of those two epidemics is identical over the part of the genome examined. This pattern is typical of that seen with single introductions followed by human-to-human passage via needle or close contact in an African hospital. In the Marburgvirus branch of the tree, there is one major clade with a slightly divergent group characterized by the Ravn 1987 Kenya isolate. All the viruses from the major Angola 2005 outbreak are represented by a single virus because the sequences in this human-to-human epidemic are virtually identical. However, in the outbreak occurring in the Democratic Republic of the Congo (DRC) in 1999 and resulting from multiple independent infections after cave entry, two viruses with slightly different phylogenies are represented within the major group, and there is even another virus within the Ravn subgroup. These sequences were selected from hundreds determined at the U.S. Centers for Disease Control and Prevention and elsewhere. (Adapted from Peters, 2010.)
Left: Geographic sites of Ebolavirus species identification, as represented by dots (yellow, Zaire; green, Sudan; red, Côte d'Ivoire; black, Bundibugyo), in or adjacent to the Central African primary or secondary forest. Even Ebolavirus Côte d'Ivoire was isolated in the Tai forest reserve. Right: Amplified map of Uganda shows the zone along the border of the Democratic Republic of the Congo (DRC, formerly Zaire) where the newest Ebolavirus species, Bundibugyo, was identified. Bundibugyo and the nearby town of Kikyo, which was also affected by this epidemic, are tourist destinations close to the Ugandan capital of Kampala. (Adapted from Peters, 2010.)
Maps of the African continent and the country of Gabon (with adjacent Republic of the Congo) show the geographic distribution of Marburgvirus identification. Red dots indicate a case or an epidemic. Uige, Angola, is the site of the largest Marburg epidemic (252 cases, 90% mortality rate). The Angolan strains differ by only 0–0.07% at the nucleotide level (Fig. 197-1). The Durba outbreak lasted 3 years and was characterized by multiple introductions of virus into men entering a subterranean mine. Nine distinct lineages were detected, of which one was in the rather distant (21%) Ravn lineage. Red dots on the Gabon map indicate detection of virus in bats by PCR. (Adapted from Peters, 2010.)
Typical filovirus particles contain a single linear, negative-sense, single-stranded RNA arranged in a helical nucleocapsid. The virions are 790–970 nm in length; they may also appear in elongated, contorted forms (Fig. 197-4). The lipid envelope confers sensitivity to lipid solvents and common detergents. The viruses are largely destroyed by heat (60°C, 30 min) and by acidity but may persist for weeks in blood at room temperature. The glycoprotein self-associates to form the virion surface spikes, which presumably mediate attachment to cells and fusion. The glycoprotein's high sugar content may contribute to its low capacity to elicit effective neutralizing antibodies. A smaller form of the glycoprotein, bearing many of its antigenic determinants, is produced by in vitro–infected cells and is found in the circulation in human disease; it has been speculated that this circulating soluble protein may suppress the immune response to the virion surface protein or block antiviral effector mechanisms. Both Marburg virus and Ebola virus are biosafety level 4 pathogens because of their high associated mortality rate and aerosol infectivity.
Ebola virions: diagnostic specimen from the first passage in Vero cells of a blood sample from a patient. Some of the filamentous (negatively stained) virions were fused together, end-to-end, giving the appearance of a "bowl of spaghetti." This image was from the first isolation and visualization of Ebola virus in 1970. (Courtesy of Fredrick A. Murphy, MD, University of Texas Medical Branch, Galveston, Texas; with permission.)
Marburg virus was first identified in Germany in 1967, when infected African green monkeys (Cercopithecus aethiops) imported from Uganda transmitted the agent to workers in a vaccine laboratory. Of the 25 human cases acquired from monkeys, seven ended in death. The six secondary cases were associated with close contact or parenteral exposure. Secondary spread to the wife of one patient was documented, and virus was isolated from the husband's semen despite the presence of circulating serum antibodies. Isolated cases of Marburg virus infection were reported from eastern and southern Africa, with limited spread. Then, in 1999, repeated transmission of Marburg virus to workers in a gold mine in eastern Democratic Republic of the Congo (DRC; formerly Zaire) was studied. The secondary spread of the virus among patients′ families was more extensive than previously noted, resembling that of Ebola virus and suggesting the importance of hygiene and proper barrier nursing in the epidemiology of these viruses in Africa. Finally, in 2004–2005, an alarming, massive Marburg virus epidemic, with >250 cases, occurred in Angola. The epidemiologic features resembled those of the Ebola virus epidemics described below, and the case-fatality rate was 90%. This high figure may have been due in part to poor conditions in African hospitals; however, the virus isolated in this epidemic was slightly different phylogenetically from other known strains and exhibited increased virulence in nonhuman primates.
Ebola virus first appeared in 1976, causing simultaneous epidemics of severe hemorrhagic fever (550 human cases) in Zaire and Sudan. Later, it was shown that different species of virus (with associated mortality rates of 90% and 50%, respectively) had caused the two epidemics. Both epidemics were associated with interhuman spread (particularly in the hospital setting) and the use of unsterilized needles and syringes—a common practice in developing-country hospitals. The epidemics dwindled as the clinics were closed and as persons in the endemic area increasingly shunned affected persons and avoided traditional burial practices.
After an interval of apparent inactivity of almost 20 years, the Zaire Ebola virus recurred in a major epidemic (317 cases) in the DRC in 1995 and in smaller epidemics in Gabon in 1994–1996. Mortality rates were high (88% in the DRC), transmission to caregivers and others who had direct contact with body fluids was common, and poor hygiene in hospitals exacerbated spread. In the DRC epidemic, an index case was infected in Kikwit in January 1995. The epidemic smoldered until April, when intense nosocomial transmission forced closure of the hospitals; samples were finally sent to the laboratory for Ebola testing, which yielded positive results within a few hours. International assistance, with barrier nursing instruction and materials, was provided; nosocomial transmission ceased, hospitals reopened, and patients were segregated to prevent intrafamilial spread. The last case was reported in June 1995.
Separate emergences of Ebola virus (Zaire) were detected in Gabon in 1994–2003, usually in association with deep-forest exposure and subsequent familial and nosocomial transmission. Die-offs of nonhuman primates were sometimes documented, and Ebola infection was confirmed in at least some animals. In a 1996 episode, a physician exposed to Ebola-infected patients traveled to South Africa with a fever; a nurse who assisted in a cutdown on the physician developed Ebola hemorrhagic fever and died despite intensive care. The index patient was identified retrospectively on the basis of serum antibodies and virus isolation from semen. No additional cases arising from care of the primary or secondary case were detected, nor did any secondary cases follow care of an unsuspected Côte d′Ivoire Ebola case in Switzerland. Thus, distant transport of Ebola virus is an established risk, but limited nosocomial spread occurs under proper hygienic conditions.
After its first documented activity in 1976, the Sudan Ebola species returned in epidemic form to cause an indolent outbreak in Uganda in 2000–2001. This outbreak claimed the lives of 224 (53%) of 425 patients.
Reston Ebola virus was first seen in the United States in 1989, when it caused a fatal, highly transmissible disease among cynomolgus macaques imported from the Philippines and quarantined in Reston, Virginia, pending distribution to biomedical researchers. This and other appearances of the Reston virus have been traced to a single export facility in the Philippines, but no source in nature had been established until the discovery of this viral species in Philippine pigs. Occasional serologic evidence of human infection was found, but no cases of human disease were identified.
Epidemiologic studies (including a specific search in the Kikwit epidemic) have failed to yield evidence for an important role of airborne particles in human disease. This lack of epidemiologic evidence is surprising and seems to conflict with the viruses′ classification as biosafety level 4 pathogens (which is based in large part on aerosol infectivity) and with formal laboratory assessments showing a high degree of aerosol infectivity for monkeys. Sick humans apparently do not usually generate sufficient amounts of infectious aerosols to pose a significant hazard to those around them.
Although numerous die-offs have been reported among chimpanzees and gorillas (some even threatening the viability of these endangered species), these animals (like humans) appear to be sentinels for virus activity. Speculation about the true reservoirs has centered on bats, and preliminary evidence indicates that bats may indeed be the reservoirs of filoviruses. This evidence includes the detection of antibodies and reverse-transcriptase polymerase chain reaction (RT-PCR) products in bats, the epidemiologic findings in subterranean gold mines in Durba (DRC) where Marburg transmission has occurred, and reported associations of human antibody production with the handling of bats. Recent isolation of Marburg virus from Egyptian fruit bats (Rousettus aegyptiacus) captured in Uganda in proximity to cases of human disease further supports bats as reservoirs, but the exact biologic relation and the natural cycle remain to be elucidated.
Pathology and Pathogenesis
In humans and in animal models, Ebola and Marburg viruses replicate well in virtually all cell types, including endothelial cells, macrophages, and parenchymal cells of multiple organs. In macaques, the earliest involvement—that of the mononuclear phagocyte system—is responsible for initiation of the disease process. In human disease and macaque models, upregulation of tissue factor and disseminated intravascular coagulation (DIC) are the inciting mechanisms. Viral replication is associated with cellular necrosis both in vivo and in vitro. Significant findings at the light-microscopic level include liver necrosis with Councilman bodies, intracellular inclusions that correlate with extensive collections of viral nucleocapsids, interstitial pneumonitis, cerebral glial nodules, and small infarcts. Antigen and virions are abundant in fibroblasts, interstitium, and (to a lesser extent) the appendages of the subcutaneous tissues in fatal cases; escape through small breaks in the skin or possibly through sweat glands may occur and, if so, may be correlated with the established epidemiologic risk of close contact with patients and the touching of the deceased. Inflammatory cells are not prominent, even in necrotic areas.
In addition to sustaining direct damage from viral infection, patients infected with Ebola virus (Zaire) have high circulating levels of proinflammatory cytokines, which presumably contribute to the severity of the illness. In fact, the virus interacts intimately with the cellular cytokine system. It is resistant to the antiviral effects of interferon α, although this mediator is amply induced. Viral infection of endothelial cells selectively inhibits the expression of major histocompatibility complex class I molecules and blocks the induction of several genes by the interferons. In addition, glycoprotein expression inhibits αV integrin expression, an effect that leads to detachment and subsequent death of endothelial cells in vitro and that correlates with the limited inflammatory response evident in lesions.
Acute infection is associated with high levels of circulating virus and viral antigen. Clinical improvement takes place when viral titers decrease concomitant with the onset of a virus-specific immune response, as detected by enzyme-linked immunosorbent assay (ELISA) or fluorescent antibody testing. In fatal cases, there is usually little evidence of an antibody response, and there is extensive depletion of spleen and lymph nodes. Ebola Sudan virus amplification by PCR shows a correlation between serum viral RNA concentration and the likelihood of death. Recovery is apparently mediated by the cellular immune response: convalescent-phase plasma has little in vitro virus-neutralizing capacity and is not protective in humans or in passive transfer experiments in monkey and guinea pig models.
After an incubation period of ∼7–10 days (range, 3–16 days), the patient abruptly develops fever, severe headache, malaise, myalgia, nausea, and vomiting. Continued fever is joined by diarrhea (often severe), chest pain (accompanied by cough), prostration, and depressed mentation. In light-skinned patients (and less often in dark-skinned individuals), a maculopapular rash appears around day 5–7 and is followed by desquamation. Bleeding may begin about this time and is apparent from any mucosal site and into the skin. In some epidemics, fewer than half of patients have had overt bleeding, and this manifestation has been absent even in some fatal cases. Additional findings include edema of the face, neck, and/or scrotum; hepatomegaly; flushing; conjunctival injection; and pharyngitis. Around 10–12 days after the onset of disease, the sustained fever may break, with improvement and eventual recovery of the patient. Recrudescence of fever may be associated with secondary bacterial infections or possibly with localized virus persistence. Late hepatitis, uveitis, and orchitis have been reported, with isolation of virus from semen or detection of PCR products in vaginal secretions for several weeks.
Leukopenia is common early on; neutrophilia has its onset later. Platelet counts fall below (sometimes much below) 50,000/μL. Laboratory evidence of DIC is found, but its clinical significance and the need for therapy are controversial. Serum levels of alanine and aspartate aminotransferases (particularly the latter) rise progressively, and jaundice develops in some cases. The serum amylase level may be elevated, and this elevation may be associated with abdominal pain, suggesting pancreatitis. Proteinuria is usual; decreased kidney function is proportional to shock.
Most patients acutely ill as a result of infection with Ebola or Marburg virus have high concentrations of virus in blood. Antigen-detection ELISA is a sensitive, robust diagnostic modality. Virus isolation and reverse-transcription PCR are also effective and provide additional sensitivity needed in some cases. Recovering patients develop IgM and IgG antibodies that are readily detected by ELISA. The indirect fluorescent antibody test with paired sera is an effective diagnostic tool in most acute cases but is extremely misleading in population-based serologic surveys for Ebola virus activity. Real-time PCR is extremely useful in detecting the need for quarantine or geographic spread. Skin biopsies are an extremely useful adjunct in postmortem diagnosis of infection with Ebola virus (and, to a lesser extent, Marburg virus) because of the presence of large amounts of viral antigen, the relatively low risk posed by sample collection, and the lack of cold-chain requirements for formalin-fixed tissues.
Treatment: Marburg and Ebola Virus Infections
No virus-specific therapy is available, and—given the extensive viral involvement in fatal cases—supportive treatment may not be as useful as was once hoped. However, studies in rhesus monkeys have shown improved survival among animals treated with an inhibitor of factor VIIa/tissue factor or with activated protein C; this effect demonstrates the importance of DIC in pathogenesis. In addition, direct intervention against viral replication with small interfering RNA (siRNA) is effective in postexposure prophylaxis against the highly virulent Zairespecies in macaques. Vigorous treatment of shock should take into account the likelihood of vascular leak in the pulmonary and systemic circulation and of myocardial functional compromise. The membrane fusion mechanism of Ebola virus resembles that of retroviruses, and the identification of “fusogenic” sequences suggests that inhibitors of cell entry may be developed. Despite the poor neutralizing capacity of polyclonal convalescent-phase sera, phage display of immunoglobulin mRNA from convalescent-phase bone marrow has yielded monoclonal antibodies that have in vitro neutralizing capacity and mediate protection in guinea pig models (but, unfortunately, not in the more sensitive monkey models).
No vaccine or antiviral drug is currently available, but barrier nursing precautions in African hospitals can greatly decrease the spread of filoviruses beyond the index case and thus prevent epidemics of infection with these viruses and other agents as well. An adenovirus-vectored Ebola glycoprotein gene has proved protective in nonhuman primates and is undergoing phase 1 trials in humans. An experimental vesicular stomatitis virus–based vaccine has protected macaques when given both before and after infection with the Zaire Ebola virus.