Filovirus Research: Knowledge Expands to Meet a Growing Threat
- 1 Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
- 2 Department of Pathology, University of Texas Medical Branch, Galveston
- Reprints or correspondence: Dr. Mike Bray, Integrated Research Facility, NIAID/ NIH, 6700A Rockledge Dr., Rm. 5128, Bethesda, MD 20892 (mbray{at}niaid.nih.gov).
This article concludes the special supplement of the Journal of Infectious Diseases (JID): “Filoviruses: Recent Advances and Future Challenges.” The importance of the filoviruses as uniquely virulent agents of human disease is underscored by the fact that this is the second time that they have been the subject of a JID supplement. The first, entitled “Ebola: The Virus and the Disease,” appeared in 1999 and was inspired by the occurrence of a large epidemic of Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo (DRC), in 1995 and a subsequent scientific colloquium held in Antwerp, Belgium [1]. Eight years later, the preparation of this new supplement was again prompted by events in Africa— in this case, epidemics of both Ebola and Marburg hemorrhagic fever—and by another international symposium, this time held in Winnipeg, Canada.
In bringing this special issue of the Journal to an end, it seems appropriate for us to compare the current status of the filovirus field to its condition when the first supplement appeared 8 years ago. The more than 40 papers published in this issue testify to the vast expansion in knowledge of filovirus biology that has been achieved over that time, ranging from the elucidation of viral replication mechanisms to the development of new vaccines and therapies that protect laboratory primates against Marburg virus (MARV) or Ebola virus (EBOV) challenge. At the same time, numerous articles published here and elsewhere have documented the growing burden of filoviral hemorrhagic fever in central Africa, where epidemics have taken place with increasing frequency, and the spread of Zaire EBOV (ZEBOV) now poses a threat to the survival of great apes [2]. Of the nearly 2300 human infections that have been documented since filoviruses were first identified 40 years ago, one-half have occurred in the past 8 years. Not once but twice, MARV has caused large outbreaks in Africa, one of which occurred in a country where filoviral hemorrhagic fever had never before been seen [3, 4]. The fact that case-fatality rates noted in association with recent epidemics have been as high as those observed in the 1970s shows how much remains to be done before progress in the laboratory will have an impact in the field.
Resurgence of Marburg Hemorrhagic Fever
Of all the developments in the filovirus field in the past 8 years, none has been more striking than the reemergence of MARV as a major pathogen. In 1999, it was appropriate for a JID supplement to focus solely on EBOV, because its Sudan and Zaire species (SEBOV and ZEBOV, respectively) had, by that time, caused 7 epidemics in Africa, and the threat posed by the recently discovered Reston EBOV and Ivory Coast EBOV species was still not known. By contrast, Marburg hemorrhagic fever seemed to be a minor problem, because the only recorded outbreak, caused by the inadvertent introduction of infected monkeys into Europe in 1967, had been associated with a relatively low mortality rate, and because, in ensuing decades, just 6 additional cases were detected at scattered sites in east Africa. However, even as the first Ebola supplement was coming off the press in 1999, an epidemic was under way in a remote gold-mining area in Durba/Watsa, DRC, that would eventually claim the lives of 83% of the 154 infected persons. In 2005, an even larger outbreak of Marburg hemorrhagic fever occurred near the coast of Angola, far to the west of the site of occurrence of any previously known case, and had a similarly high associated mortality rate.
Not only did the emergence of MARV in the Durba/Watsa area surprise the research community because of the agent's unexpectedly high lethality, it also revealed a novel epidemiologic pattern. Earlier studies indicated that outbreaks of filoviral hemorrhagic fever had resulted from a single introduction of virus into a community, followed by person-to-person transmission through contact with body fluids; large epidemics occurred when a lack of proper infection control in hospitals amplified the spread of virus [5]. In Durba/Watsa, however, sequencing revealed significant differences among viruses isolated from individual patients, indicating that repeated introductions of the virus had taken place, and retrospective case tracking found comparatively little secondary transmission [3]. Speculation as to the source of infection focused on the daily exposure of gold miners to large number of bats, reviving an idea first proposed in 1980, when a person who had visited a bat-infested cave in Kenya developed fatal Marburg hemorrhagic fever [6].
The Angola outbreak also had a unique epidemiologic feature, in that the disease was first recognized in the pediatrics ward of the Uige provincial hospital, and most of the initial patients were younger than 5 years of age [7]. It otherwise largely recapitulated the pattern of Ebola hemorrhagic fever epidemics in Yambuku and Kikwit, in that an initial introduction of virus was followed by the rapid spread of infection through medical procedures—in this instance, apparently through the reuse of contaminated transfusion equipment. The near-uniform lethality of illness in both Durba/Watsa and Angola and the remarkable pathogenicity of the Angola agent in laboratory primates have changed the image of MARV, leaving no doubt that it poses as great a threat as EBOV to populations of central Africa [8].
EBOV in Africa: An Increasingly Complex Picture
Despite the progress that has been made in the laboratory, the means by which ZEBOV and SEBOV are maintained in central Africa and transmitted to humans are still as mysterious as they were in 1999. In fact, the situation has been made even more complicated—and more threatening—by the newly recognized spread of ZEBOV among wild primates. A first glimpse of a connection between infections of humans and apes was provided by an article in the first filovirus supplement in the Journal, which described a point-source outbreak of Ebola hemorrhagic fever that occurred in 1996, when villagers in Gabon butchered and ate a chimp found dead in the forest [9]. Since then, the spread of the virus among gorillas and chimpanzees in the border region between Gabon and the Republic of the Congo (RC) has caused both an extremely high number of deaths among those animals and a series of outbreaks when hunters have come across infected animals [2, 10]. As the epicenter of ZEBOV transmission in Africa, the Gabon-RC region may prove to be the site where the elusive maintenance host of the filoviruses is finally discovered, and it may also serve as a future testing ground for educating local populations, improving case detection, and developing a rapid, coordinated outbreak response.
While ZEBOV was spreading in Gabon and RC, SEBOV reappeared far to the northeast after an absence of more than 2 decades, marking the first year of the new millennium by causing the largest epidemic of filoviral hemorrhagic fever yet recorded, with 425 cases and 224 deaths occurring in Gulu, Uganda [11]. In 2004, the virus reemerged near its original outbreak site in Sudan, this time fortunately claiming a much smaller number of victims [12]. The only positive aspect of these tragic events is that the ⋃50% case-fatality rate associated with SEBOV hemorrhagic fever has enabled researchers to identify prognostic markers of survival, including genetic determinants of resistance to lethal infection, that may prove critical for vaccine development and as guides to therapy [13–16]. With regard to the other 2 Ebola species, Ivory Coast EBOV, which was first described in the 1999 supplement, has not been seen since, and the Reston EBOV has not ventured from its hiding place in The Philippines during the past 8 years [17, 18].
The Hunt for the Reservoir Host
The filoviruses are now the only causative agents of viral hemorrhagic fever for which a natural maintenance cycle has not been identified. Great apes obviously cannot serve as the viral reservoir, because their numbers are too small, they die of infection too quickly, and they are not present at many sites where human outbreaks have occurred. Instead, it appears likely that MARV and EBOV are maintained in 1 or more species of small animals that are widely distributed across central Africa and that they serve as the source of infection for both humans and wild primates [6, 19]. Evidence is accumulating that incriminates fruit bats, which are ubiquitous in central Africa, but the supporting data still remain limited to the detection of anti-EBOV antibodies in some animals and the recovery of short viral sequences from tissues by means of nested reverse transcription polymerase chain reaction [20, 21]. The “Holy Grail” of virus isolation has not yet been attained.
Once the reservoir host has finally been identified, studies using captive animals in the laboratory could help to define the biological basis of filoviral maintenance and transmission, perhaps shedding light on why epidemics have been so unpredictable in their timing and location. Studies of filoviral infection in the maintenance host may also help to resolve such pathogenesis-related puzzles as the function of the EBOV-secreted glycoprotein, which cannot be determined by studying primates, which are only accidental hosts for these agents.
Filoviruses and the Host Cell
While the search for the maintenance host has been under way, teams of investigators thousands of miles from the African rain forest have made remarkable advances in elucidating the mechanisms of filoviral replication at the molecular level. As is often the case, progress has been greatly facilitated by the development of new experimental methods. Reverse-genetics techniques have been especially valuable in enabling researchers in biosafety level 4 laboratories to investigate determinants of pathogenicity by constructing a variety of live, recombinant filoviruses with specific sequence modifications [22, 23]. At the same time, the creation of noninfectious minigenome systems and the generation of pseudotyped or chimeric viruses have permitted a wide range of studies to be performed under lowlevel containment [24, 25]. As indicated by articles appearing in this supplement and elsewhere, these efforts have been especially productive in explaining how filoviruses exploit normal cellular processes to support their own replication. The use of recombinant systems to elucidate the function of individual viral proteins has also been important in demonstrating that VP24 and VP35 play a triple role as virion structural proteins, components of the replication complex, and antagonists of type I interferon (IFN) responses [26, 27]. The fact that filoviruses encode 2 different IFN inhibitors that block both the production of IFN-b and the response to exogenous IFN-α and IFNγ by infected cells helps to explain how these pathogens disseminate so quickly from an initial site of infection.
Filoviruses and the Immune System
Progress in elucidating replication mechanisms within host cells has been paralleled by advances in understanding how MARV and EBOV interact with the human immune system to cause rapidly overwhelming disease. Although data from patients in Africa are still limited, the understanding of pathogenesis has been greatly aided by time-course studies of pathologic changes in rodents and macaques lethally infected with ZEBOV [28–30]. This work has shown that filoviral hemorrhagic fever represents a catastrophic failure of innate immunity, in which macrophages, the ubiquitous sentinels that should defend the host against invading pathogens, instead serve as the principal site of viral replication, and the proinflammatory mediators that these cells produce, instead of helping to restrict the spread of virus, induce a fulminant systemic inflammatory syndrome [31–33]. This new picture of filovirus pathogenesis is not merely of academic interest; by revealing the underlying mechanisms shared with the far more common syndrome of septic shock, it is leading to the successful application of sepsis therapies to macaques infected with lethal doses of EBOV [34, 35].
Not only have recent laboratory and clinical studies helped to explain how humans die of filoviral hemorrhagic fever, they have also provided important insights into how some patients manage to survive infection. The first data were presented in an article in the JID Ebola supplement published in 1999, which reported that only those patients in the Kikwit outbreak who produced anti-EBOV IgM or IgG during the course of illness were likely to survive infection [36]. The notion that a failure of adaptive immune function might be partly responsible for a fatal outcome of filoviral hemorrhagic fever is consistent with studies showing that EBOV-infected dendritic cells are unable to present antigens to T cells, and that lethal filoviral infections in mice and nonhuman primates (and, probably, in humans) are accompanied by a massive loss of lymphocytes through “bystander” apoptosis [37–40]. Current research suggests that the latter process is a feature of many severe infections, and that it could be at least partially prevented through specific countermeasures, helping to preserve immune function [41]. Further progress in identifying determinants of survival could benefit from the development of new animal models with lessthan- uniform lethality, mirroring the 50% mortality rate associated with SEBOV infection.
Progress in Vaccine Development
Readers of the 1999 JID filovirus supplement who noted the lack of any vaccines that could protect nonhuman primates against MARV or EBOV might have concluded that no effective prophylaxis would ever be developed against such highly virulent pathogens. Eight years later, the situation has changed dramatically. A DNA/adenovirus prime-boost vaccine that solidly protects macaques against subsequent challenge with ZEBOV is now undergoing safety testing of its individual components in human volunteers [42], and several other products have also proved to be protective in laboratory primates but have not yet advanced to a phase 1 trial. The candidate vaccines described in this issue include a noninfectious preparation of ZEBOV virus-like particles [43], a replication-defective recombinant adenovirus encoding the EBOV surface glycoprotein (GP) [44], and a live chimeric vesicular stomatitis virus (VSV) encoding the MARV or EBOV GP [45]. Remarkably, the VSV vaccine has also protected macaques against MARV when inoculated 30 min after an otherwise lethal challenge, and it was partially protective against ZEBOV under similar circumstances [46, 47]. No cross-protection was observed, indicating that protection was based on the rapid induction of specific immunity to the MARV or EBOV GP—a response that fails to occur in the untreated infection. If it is found to be safe in humans, the VSV vaccine could be used both as a conventional vaccine and as an immediate treatment for persons infected in a laboratory accident or during a disease outbreak.
Progress in Experimental Therapy
At the time of publication of the first filovirus supplement to the Journal, several types of therapy had been devised that could protect mice or guinea pigs against Ebola or Marburg hemorrhagic fever, but none had proved to be effective in laboratory primates. Eight years later, several different interventions have been shown to be beneficial in macaques, so long as treatment is begun before or soon after virus challenge. Interestingly, no medication has yet been developed that has a mechanism of action similar to the antiviral drug ribavirin, a nucleoside analogue that inhibits many RNA viruses but is inactive against the filoviruses. Instead, direct inhibition of viral replication has been achieved using antisense or siRNA molecules that block transcription by targeting specific sequences on viral mRNA [48, 49].
Other successful approaches have acted indirectly, by blocking critical host responses identified in pathogenesis studies. The discovery that filovirus-infected macrophages trigger disseminated intravascular coagulation by synthesizing cell-surface tissue factor has led to the therapeutic use of recombinant nematode anticoagulant protein c2, an anticoagulant that blocks the interaction of tissue factor and factor VIIa, to treat both EBOV- and MARV-infected macaques [8, 50]. In the former animals, the effect of treatment was remarkably wide-ranging, including marked reductions in coagulopathy, inflammatory responses,and viral replication, revealing that what previously were considered to be separate disease markers are actually tightly interlinked [51]. The modulation of coagulopathy through intravenous administration of activated protein C, in the same manner in which it is now used to treat patients with sepsis, has also proved to be beneficial in EBOV-infected macaques [35]. If one includes the VSV vaccines cited above as postexposure “drugs,” this means that 3 different therapeutic approaches have proved beneficial the first time that they were tested in nonhuman primates. Because they differ in their mechanism of action, there is reason to expect that their use in combination would produce an additive or a synergistic benefit.
Even though none of these experimental approaches has been shown to be effective in nonhuman primates when initiated after the early incubation period, one should not minimize the value of effective postexposure prophylaxis for Marburg or Ebola hemorrhagic fever. Such treatment could be lifesaving in the event of a laboratory accident, and it could also be used to treat local residents and health care workers exposed during the course of an African epidemic. Because filoviral hemorrhagic fever is more slowly progressive in humans than in macaques, and because it is associated with a mortality rate of less than 100%, it is reasonable to hope that treatments that are beneficial in nonhuman primates will be even more effective in humans.
Unfortunately, despite these significant advances in the laboratory, the past 8 years have seen little or no progress in improving the treatment of patients in African outbreaks. The new millennium began on a positive note, when the response to the epidemic in Gulu demonstrated that local and expatriate medical personnel could work together to create a well-managed isolation ward, supported by a field laboratory with rapid diagnostic capability [13]. However, efforts by local health workers and international responders to use traditional case finding and isolation to deal with the series of Ebola hemorrhagic fever outbreaks in Gabon and RC and with the Marburg epidemic in Angola were often met with so much suspicion and hostility from local populations as to prevent the provision of even basic medical care [52–54].
The continuing failure to reduce the case-fatality rate associated with filoviral hemorrhagic fever has convinced many clinicians and researchers that no matter how well organized and technologically proficient the response to a filovirus epidemic may be, it cannot be considered a success unless its primary aim is the delivery of effective medical treatment [52]. Turning this concept into reality will require close collaboration among laboratory scientists, international organizations, and local physicians and health workers in central Africa. A near-term goal for such a network might be the development of standard protocols for basic medical care that can be explained to affected populations, so that their response to outside assistance will be based on hope, rather than fear. The introduction of new, experimental therapies will be a far more challenging issue and may require the development of both a field research infrastructure and a system of scientific and ethical review.
The Road Ahead
Compared with malaria, tuberculosis, and other plagues that affect millions of people, Marburg and Ebola hemorrhagic fever are minor public health problems. Can the risk to investigators and the costly containment laboratories required to study these “exotic” illnesses be justified? There is no doubt that the answer is yes. Filoviral infections stand out from all other diseases in revealing an alarming gap in our defense against zoonotic agents: the human immune system, product of a billion years of evolution, is helpless when confronted by a few particles— possibly, a single virion—of MARV or EBOV. Our future security against emerging pathogens demands that we understand and repair this striking vulnerability.
The need for filovirus research becomes even more apparent when it is realized that the same host responses that enable MARV and EBOV to cause fatal hemorrhagic fever also contribute to morbidity and death associated with a wide variety of other conditions, ranging from gram-negative bacterial sepsis to smallpox and avian influenza. Just as the chronic disease induced by HIV inspired the scientific community to study cell-mediated immunity and its defects, so the rapidly overwhelming illness induced by the filoviruses should stimulate investigators to learn how unregulated innate responses may lead to fatal inflammatory syndromes and to apply that knowledge to all areas of infectious diseases research.
The efforts and courage of a global network of scientists and health workers have brought about a vast increase in our understanding of Marburg and Ebola hemorrhagic fever. In 1999, the first filovirus supplement in the Journal provided a detailed characterization of the threat, but it had little to offer in the way of counter measures. Eight years later, this new special issue presents a variety of vaccines and therapies that have protected nonhuman primates against otherwise lethal filoviral challenge, suggesting that they will be efficacious in humans. The greatest challenge lies ahead—to move these new capabilities forward through clinical trials and use them to reduce the death toll from Marburg and Ebola hemorrhagic fever in Africa. Let us hope that a future JID supplement will report success in this endeavor.
Acknowledgments
Supplement sponsorship. This article was published as part of a supplement entitled “Filoviruses: Recent Advances and Future Challenges,” sponsored by the Public Health Agency of Canada, the National Institutes of Health, the Canadian Institutes of Health Research, Cangene, CUH2A, Smith Carter, Hemisphere Engineering, Crucell, and the International Centre for Infectious Diseases.
Footnotes
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Potential conflicts of interest: none reported.
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Financial support: supplement sponsorship is detailed in the Acknowledgments.
- © 2007 by the Infectious Diseases Society of America
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