Bats as Reservoir Hosts for Filoviruses

Learning where Ebola and Marburg viruses reside in nature and how they are transmitted from these natural reservoirs to humans will lead to better understanding of how outbreaks start, Towner explained. A reservoir host species' natural history must support viral persistence at the population level, he observed, as evidenced by the following characteristics: consistent infection in host populations; maintenance of viral circulation through adequate viral loads and rates of viral shedding; and host populations that are, overall, clinically healthy.

Results of two studies (Amman et al., 2012; Towner et al., 2009) conducted during an outbreak in miners in Kitaka, Uganda, and the occurrence of unrelated cases in foreign tourists in nearby Python Cave suggested that Marburg virus is horizontally transmitted among bat populations, Towner said. Infection rates generally increased as bats aged, with the exception of a spike among older juvenile bats—a phenomenon that has been noticed in other studies of viral host populations, he noted. Further analysis suggested that periods of increased active infection in juvenile bats coincided with incidences of spillover to humans (Amman et al., 2012). He speculated that older animals inhabit preferred sites deep in the cave, but younger bats, being lower in the hierarchy, stay closer to the outside, where they are likelier to encounter humans or other primates.

Based on experimental infectious studies (Amman et al., 2015; Paweska et al., 2012) Towner stated, “We suspect that bat-to-bat transmission [of Marburg virus] is probably through biting. For bat-to-human transmission, we suspect it's probably through infected feces, or urine containing infectious virus.” This scenario is consistent with the experience of tourists infected with the virus at Python Cave who did not actually enter the caverns, and yet were probably exposed to bat excrement.

Genomic Surveillance of Ebola Transmission

Recent advances in high-throughput genetic sequencing allow researchers to track viruses as they spread through host populations, evolving as they go. Stephen Gire of Broad Institute of the Massachusetts Institute of Technology and Harvard University described the application of these methods to reveal how and when the Ebola virus spilled over to humans in West Africa, how many such events occurred, and how the virus evolved as the epidemic unfolded (Gire et al., 2014).

The medical team at Kenema Government Hospital in Sierra Leone, where that country's first laboratory-confirmed Ebola case was diagnosed, acted quickly to identify more cases through contact tracing, all of whom had attended the funeral of a traditional healer who had treated Guinean Ebola patients, Gire reported. Samples from 12 of the Sierra Leonean patients were sent immediately to Gire's team in Boston, who sequenced and analyzed them within 10 days, using a strategy they had created to track Lassa fever transmission. As the epidemic continued, the researchers continued to sequence patient samples from that hospital, publishing their initial findings (on 114 viral isolates from 78 patients) in August 2014. It was important to make this information available to the scientific community as soon as possible, Gire said, in order to quickly bring as much diverse expertise to its analysis, and thereby, to benefit efforts to quell the epidemic.

The key questions of where and how the epidemic began were quickly answered, he noted. Based on viral phylogeny, Gire and coworkers traced the origin of the West African strain to the Ebola-Zaire strain that caused the 1976 outbreak. Comparing genetic differences between the West African isolates and the sequences of other Ebola viruses, and taking into account the rate by which the virus typically undergoes sequence change—its substitution rate—they were able to determine that the specific variant of Ebola-Zaire that sparked the West African epidemic had emerged within the past 10 years in Central Africa, he noted. Further, he said, “We were able to show that it was actually one individual introduction from the animal reservoir into the human population that then was sustained in the population by human-to-human contact afterward.”

Detecting Ebola

An acute infection, Ebola symptoms commence after an incubation period of variable length, during which the pathogen replicates. These symptoms can vary widely from case to case, thus the controversy as to whether or not the disease should be denoted a hemorrhagic fever. By the time the virus is readily detectable, the patient typically is at the tipping point between death and recovery, Feldmann observed. “Often, particularly with less sensitive assays, you do not see a very strong or any immune response in people that succumb to infection very suddenly and early during disease progression,” he said. Thus, pathogen detection assays, rather than those that target host immune response, are key to diagnosing Ebola in outbreak situations.

Standard Ebola diagnostic tests detect either the nonsegmented, negative-stranded RNA genome of the virus or its protein components, Feldmann explained; both methods were used to diagnose Ebola in West Africa, as was the presence of antibody (IgM and IgG) in survivors of the disease.⁴ Ebola and other filoviruses are enclosed by an envelope studded with glycoprotein trimers. The majority of the protective immune response is directed against these molecules; however, immune response to the virus's other six structural proteins is important to its diagnosis, he noted.

The list of commercially produced Ebola diagnostic tests has grown significantly over the course of the epidemic and continues to expand, Feldmann observed; however, the majority of field laboratories still use their own assays, many of which lack positive controls because those require biosafe containment. “I think we have to do much, much more . . . to make sure that the laboratories . . . that are doing diagnostics in certain countries actually evaluate their tests,” he urged—and better yet, to fulfill the World Health Organization's (WHO's) demand for rapid, sensitive, safe, and simple Ebola diagnostics. “I think we are on the way to achieving that,” he observed, but as discussed below, some obstacles remain to meeting that goal.

Putting the present in perspective, Feldmann noted that until around 2000, Ebola diagnosis took weeks to months, as samples were shipped to reference centers such as the CDC. The construction of in-country reference centers reduced that to days; with current on-site mobile support, diagnosis takes about 2 to 6 hours, he said. “The future is minutes,” he predicted, but he cautioned that methods currently capable of such speed are not sufficiently sensitive.

The View from Monrovia

At the time of the workshop, Feldmann's team from the NIH and another from the CDC had been staffing the laboratory of a Médecins Sans Frontières (MSF) ETU in Monrovia, Liberia, since August 2014. “We started out in a tent structure, as almost all mobile laboratories nowadays do,” he recalled. MSF can construct such facilities within a day, allowing laboratory operations to begin in less than 48 hours, he said.

Performing diagnostic tests in the tents was challenging, according to Feldmann; nevertheless, despite incomplete physical separation of different stages of the process, there were few reports of contamination. Later the laboratory moved to a fixed structure, which improved workflow and also afforded better protection from rain and heat. A mobile plastic BSL-3 laboratory under negative pressure was also available but was not used because sample loads did not reach critical levels, he said.

The ELWA 3, an MSF Ebola management center, operated a laboratory during the West African epidemic, and employed quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to diagnose Ebola. This method permitted detection of several different pathogens within about an hour, but it requires sophisticated equipment and trained personnel, Feldmann noted. Reverse transcription loop-mediated isothermal amplification (RT-LAMP), a faster, less-demanding method, is not as well established, but it is moving into field use, he added; however, as currently performed, it is vulnerable to detecting false negatives. An even faster method, isothermal recombinase polymerase amplification, has potential, but it has not been appropriately evaluated for field use.

Extracting viral RNA from patient samples for analysis requires an additional 30 to 60 minutes, but it can be performed easily using kits from several sources, Feldmann noted. The most common are based on chaotropic agents and can be either performed manually or automated. Although some of these processes must be adjusted in order to completely inactivate the samples, this approach has been used for more than a decade in the field and in the laboratories without incident, he said. “If you do the test in the laboratory, you have breakthrough with rest infectivity unless you add . . . ethanol. That completely inactivates the sample. Be careful, and test your procedures,” he advised.

The ELWA 3 team used a two-target approach to diagnose Ebola, performing dual PCR assays for separate genomic regions. “The positive is only a positive if both are consistent and agree on the result,” Feldman said. “Not every mobile lab does that. Some labs operate on a single target system, which, of course, is more prone to potential false negatives if you have sequence variation.” If the results of their PCR assays disagreed, they ran a third assay on a different genomic region as a tie-breaker, and used clinical parameters as well to determine their conclusion, he said. The assays, conducted in tandem by the NIH and the CDC teams, were nearly perfectly concordant, he noted—evidence of their successful collaboration.

Under optimal conditions, patients at ELWA 3 were tested as soon as they entered the facility and if positive, were isolated, Feldman reported. Surviving patients who were asymptomatic for 3 days then required two consecutive negative tests, between one and three days apart, for discharge. However, he noted, that algorithm was frequently compromised in August and September 2014, when the facility became overwhelmed with cases. “You had to make the decision between someone that you may send home who may be a false negative versus someone who is extremely ill with Ebola knocking on your door, and you can't take that patient into your clinic,” he recalled.

Beginning in October 2014, as the crisis began to abate, the laboratory also began testing for malaria. A frequent coinfection with Ebola, malaria needs to be studied as to its possible effect on survival, Feldmann remarked. The researchers also began performing blood tests for Ebola using bedside instruments; however, he said, antigen-based “dipstick” tests (as noted by Heymann) are better prospects for future analyses, and capable of providing results within 15 minutes. Although such tests are not as sensitive as qRT-PCR, they can potentially be used successfully in an appropriately adjusted diagnostic algorithm, Feldmann suggested. “For an incoming patient, a negative [result from a rapid test] does not exclude an infection,” he emphasized. “For a survivor, a negative test does not exclude infectivity or rest infectivity.” Laboratory-based amplification approaches will still be needed to measure viral load, he added.

While unsuited to the diagnostic demands of the epidemic at its height, serology—which had been employed in previous outbreaks—may still be used to analyze samples to better understand what happened, and be developed as a basis for rapid tests, Feldmann observed. Here too, inactivating the virus is problematic, he noted: heat alone is not sufficient and must be combined with a chemotropic agent.

The Science of Containment Care

Compared with other infectious diseases, Ebola “is pretty hard to catch,” Kortepeter observed; transmission could be said to occur only in the right place at the wrong time, which unfortunately describes the fate of many health care workers.

Hospitals—the “right place”—have historically served as epicenters of Ebola spread through such unsafe practices as the reuse of contaminated needles or syringes on patients, accidental needle sticks of health care workers, exposures during surgical procedures, rubbing eyes and other mucous membranes with soiled gloves, and the splashing of infected water or body fluids, Kortepeter stated. Within households, transmission results primarily from direct physical contact with blood or body fluids of an individual who has been ill, sleeping in the same hut or mat, sharing a needle, sharing a hospital bed, or having close contact with a cadaver, he said.

The Ebola virus concentrates in the blood but is also present in other bodily fluids, Kortepeter pointed out. Yet, the extent to which such nonblood fluids pose an infection risk remains largely to be determined (Osterholm et al., 2015). In general, increasing viremia over the course of the illness means that any exposure at its peak—around 7 to 10 days after symptom onset—confers maximum risk, he noted. During this period, moreover, patients are most likely to experience high-volume diarrhea, vomiting, bleeding, and coughing. It is also unknown whether or under what circumstances asymptomatic or mildly ill patients can transmit Ebola, he added.

Evolution of Containment Care

In a 2006 consensus article (Smith et al., 2006), Ribner and colleagues defined a biocontainment patient care unit as a clinical facility designed specifically for the care of patients to prevent nosocomial transmission of highly hazardous and contagious diseases, and incorporating those engineering or safety measures that are often used in containment laboratories and have been tested over time in laboratories. Kortepeter, who worked in this type of facility at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), noted that their biocontainment unit was established in 1967, the same year Marburg virus was identified, and 2 years before the discovery of Lassa fever.

Additional advances in biocontainment occurred on the occasion of the first lunar landing, which—fueled in part by Michael Crichton's best-selling novel, The Andromeda Strain—raised public concern about extraplanetary contamination. In response, the National Aeronautics and Space Administration developed a specialized negative-pressure Airstream trailer to isolate the astronauts for a period of postflight quarantine, Kortepeter said; it included a patient isolator that was never used, except on the set of the film E.T.: The Extra-Terrestrial.

These designs informed USAMRIID's next high-level containment care unit, dubbed “The Slammer,” Kortepeter observed. It incorporated a host of features, including an external direct pass-thru entry, positive pressure suits, air intake and exhaust filtration, and a steam-sterilized sewer system. Between 1972 and 2004, 22 patients with potentially dangerous infections were admitted to The Slammer, he reported. Quoting Ribner in a recent Time magazine story (von Drehle and Baker, 2014), Kortepeter recalled that The Slammer was once viewed as a sort of “Noah's Ark.” Today, it serves as an early example of a BSL-4 facility, he said.

Additional improvements were necessary to complete the transition from laboratory biosafety to patient care, Kortepeter explained. When experience with outbreak management demonstrated that incorporating isolation and barrier precautions into patient triage procedures significantly reduced the spread of infectious diseases to health care providers, PPE precautions changed accordingly to incorporate anterooms for donning and doffing, negative pressure where possible, and the mainstay gloves, gowns, masks, shoe covers, and protective eyewear.

However, even with those precautions in place, laboratory transmission of Ebola occurred among nonhuman primates in Reston, Kortepeter recounted. That, along with demonstrations of nonhuman primate infection by aerosol and other routes and of seroconversion to Ebola-Reston among the primates' human caregivers, resulted in new personal protective equipment requirements for negative pressure and high-efficiency particulate air filter (HEPA) respirators that could be used by those treating patients with prominent cough, vomiting, diarrhea, or hemorrhage, he reported. In 2005, further updates in isolation recommendations stated that hospitals may choose to institute airborne precautions when caring for patients with severe pulmonary involvement or who are undergoing potentially aerosol-generating procedures, he noted.

These recommendations guided the treatment of the initial Ebola patients in the United States in 2014, Kortepeter reported. Then, after two nurses in Dallas were infected with Ebola (as described by Ribner), caregivers were instructed to wear a respirator and to cover all of their skin, Kortepeter stated.

What Next?

The aforementioned advances in containment care have not prevented shocking numbers of health care workers from becoming infected with Ebola in West Africa, Kortepeter acknowledged. Continued uncertainty about the circumstances of transmission is a problem, he noted, but the primary challenge is to protect caregivers from high concentrations of virus in bodily fluids during peak infection. There is no room for mistakes under these circumstances; therefore, as in BSL-4 laboratories, redundancy in protective measures is essential, he insisted. Such measures are best taken in designated containment care facilities capable of handling rare, dangerous infections with which typical hospital staff and clinicians are potentially unfamiliar, he insisted (Kortepeter et al., 2015). At the same time, every hospital should evaluate its capacity to address infectious diseases, and should be competent to perform basic tasks, such as triage, specimen collection, and isolation, for patients that are best treated in containment care facilities, he said.

Even those critical of the specialized facility model can agree on basic principles of containment care, Kortepeter argued. These include the provision of deliberate care, the need to coordinate movement in treatment and laboratory facilities to prevent contamination, the importance of assessing the risk versus benefit of every procedure, the necessity of practicing key procedures, and the requirement for vigilance by care team members, who will monitor each others' actions and point out inappropriate actions before harm is done.

Reflecting on the evolution of containment care, Kortepeter concluded that the field has come of age. That being the case, he said, addressing Ribner: if The Slammer was indeed Noah's Ark, then perhaps it can be said that your ship has either come in, or it has set sail.

Vaccines

Several promising Ebola vaccine candidates existed years prior to the West African epidemic, Geisbert noted. However, because of the limited market for such products and lack of financial incentives for their development, all had stalled in animal trials; moreover, he asserted, progress toward a human vaccine was slowed by the constraints of the FDA's Animal Rule.⁷ This legislation, spurred by biodefense concerns and intended to permit the licensure of drugs for which human efficacy trials would be unethical, nevertheless had impeded potential responses to emerging diseases (Cohen, 2014). As the epidemic spread, those barriers were overcome, and several vaccine candidates were rapidly advanced.

Geisbert briefly characterized four “fast-tracked” Ebola vaccines known to have entered clinical trials at the time of the workshop. Two had already completed phase I safety trials in humans: ChAd3, a chimpanzee adenovirus vector bearing surface glycoprotein genes from two Ebola strains, developed by GlaxoSmithKline (GSK) in collaboration with NIAID and, VSV-delta G, an attenuated vesicular stomatitis virus (a common infection of livestock) engineered to express the Ebola surface glycoprotein, produced by NewLink Genetics and Merck (Geisbert and Feldmann, 2011). These were followed by a more-attenuated “next-generation” VSV-delta G vaccine (Cooper et al., 2008), developed by Profectus Biosciences, and also by a two-step protocol that employs a modified smallpox virus to boost the efficacy of ChAd3, produced by Johnson and Johnson in collaboration with Bavarian Nordic.⁸

Kurilla reported that a ring vaccination trial in Guinea is under way for one of the candidates. These achievements represent a decade of focused effort, Kurilla emphasized. Nevertheless, he observed, researchers have yet to gain a firm understanding of successful immune response to the Ebola virus, in part because so few people had survived the disease. A December 2014 workshop NIAID cosponsored with the CDC, the FDA, the U.S. Department of Defense, and the Biomedical Advanced Research and Development Authority (BARDA) examined this problem, he noted.

Geisbert agreed that, despite the impressive progress achieved, important questions remain. He noted three areas with key gaps in knowledge. The first concerns the durability of the vaccine candidates. Nearly all filovirus vaccines have been evaluated in virus-challenged nonhuman primates within 8 weeks of their final vaccination—too early to predict their long-term potency. A study found that the ChAd3 vaccine alone at two different doses fails to protect monkeys adequately from Ebola as few as 10 months later (Stanley et al., 2014). “You have to boost with MVA [modified smallpox virus] to get long-term protection,” he reported—but the MVA vaccine has produced adverse effects at the subcutaneous injection site, he added.

While live attenuated viruses such as VSV have historically been durable, and a Marburg-VSV vaccine has proven sturdy (Mire et al., 2014), the Ebola construct has not been sufficiently tested to determine if it follows suit, according to Geisbert.

The potency of the virus used to create the current group of Ebola vaccines has also been called into question. New data suggest that these Ebola-Zaire virus seed stocks represented a less-pathogenic genetic variant of the wild-type virus, Geisbert remarked (Kugelman et al., 2012; Volchkova et al., 2011). While this makes a high percentage of the soluble glycoprotein that enables antigenic subversion,⁹ as described by Kurilla in the next section, the laboratory stock has largely lost that ability, Geisbert explained (Mohan et al., 2012). Accordingly, he added, recent evidence demonstrates that the wild-type Ebola-Zaire virus kills nonhuman primate hosts faster than the laboratory strain.

Given the possible implications of these results for Ebola vaccine efficacy, Geisbert's group conducted a test of candidate vaccines in macaques that subsequently were infected with wild-type Ebola-Zaire virus. Unexpectedly, Geisbert reported, all the monkeys that received either of the VSV-based vaccines were completely protected and asymptomatic, while all but one monkey that received adenovirus-based vaccines became ill, and one died. “This really tells us that this is something that we have to pay attention to as we develop medical countermeasures, both vaccines and treatments for Ebola, particularly in the context of the current outbreak,” he concluded. However, he added, two promising postexposure therapeutics, ZMapp and TKM-Ebola (described below), had been tested and found effective against the wild-type virus.

In subsequent discussion, Feldmann pointed out that almost all of the laboratory-stain viruses had been grown in Vero cells, rather than in target cells derived from host animals, in which the virus retains its wild-type form. “Primary target cells in animals have a selective pressure to that wild-type virus,” he explained. “Vero cells, unfortunately, do not. That is where most of the variance is generated.” Switching to primary target cell lines would go a long way toward solving this problem, he concluded.

The third area Geisbert discussed as needing further study is the possibility of protecting against multiple species and strains of filoviruses with a single vaccine. “Long-term, we really want to make sure that we have vaccines and treatments that protect against multiple species and strains even within one species of Ebola or Marburg,” he urged. As the small sequence change underlying the capacity for antigenic subversion suggests, even strains with near-perfect sequence homology can vary in their response to the same vaccine.

Postexposure Treatments

Geisbert and Kurilla provided an overview of several approaches that have been taken to mitigate the effects of Ebola infection. One prominent strategy, antibody therapy to neutralize the virus, was the focus of a workshop presentation by Erica Ollmann Saphire, described in the next section. Attempts have included the treatment of patients with whole blood, serum, or plasma donated by Ebola survivors, as was first attempted during the 1995 outbreak in Kikwit. Researches have also tried to transfer whole blood among nonhuman primates that survived Ebola, but in most cases it has not worked well, Geisbert noted (Jahrling et al., 2007; Sullivan et al., 2011).

Inhibiting transcription and replication of the Ebola virus is theoretically possible, but it has not been demonstrated. The antiviral drug Favipiravir (also known as T-705 or Avigan), known to be active against several viruses, including West Nile and yellow fever, was recently used to treat Ebola patients, according to Geisbert. Considered a broad spectrum antiviral, its mechanism of action is thought to involve the selective inhibition of the RNA-dependent RNA polymerase, but that has yet to be definitively proven. At the time of the workshop, several studies of this drug were under way in nonhuman primate models of Ebola, he reported. Similarly, the antiviral drug Brincidofovir appears to inhibit replication of a variety of DNA viruses through an unknown mechanism and, surprisingly, Ebola—an RNA virus—in cell culture. Brincidofovir was used to treat the locally acquired Ebola patient in Dallas, he said; it was also intended for studies in Africa that were abruptly halted by its manufacturer, Chimerix. The results of those attempts should be instructive, he observed.

Antisense technology, in the form of small interfering RNAs (siRNAs), provides another means to inhibit Ebola virus activity by suppressing gene expression. These powerful, sequence-specific reagents naturally defend eukaryotic cells against viral pathogens, Geisbert explained. They have been used in cell-culture systems to inhibit the replication of human viruses, including HIV, hepatitis C, poliovirus, severe acute respiratory syndrome, and Marburg; however, effective delivery of siRNAs in vivo remains elusive (Xie et al., 2006).

Geisbert and co-workers attempted to use siRNAs to target three essential Ebola-Zaire virus proteins, he said. In their initial postexposure studies of guinea pigs, only about one in five animals were protected. However, when they repeated the study using a novel lipid-based delivery system for siRNA from Tekmira, every animal survived (Geisbert et al., 2010). This formulation, called TKM-Ebola, was produced by Tekmira and cleared by the FDA under compassionate use to treat several patients who contracted Ebola while serving in Liberia. Geisbert noted that a phase II trial of TKM-Ebola was planned for Africa, if enough patients could be enrolled.

In general, Geisbert observed, repatriated U.S. Ebola patients, many of whom received experimental or compassionate use drugs, fared well compared with their West African counterparts. “I don't think we can point to any one postexposure treatment or intervention that is the reason for that,” he concluded; rather, single drugs, combinations of drugs, and 24/7 monitoring by highly trained staff likely all supported superior outcomes.

Convening a Global Conversation

At the urging of World Bank president Jim Kim, Dzau agreed to guide the NAM to lead the development of a global health risk framework—and to do it as the West African crisis was still being addressed, in order to take advantage of the “real-time learning” under way. At a December 2014 meeting at the NAM, chaired by the WHO Director-General Margaret Chan and including major stakeholders in the Ebola epidemic, sentiment was overwhelming to proceed as a group to create such a framework, Dzau recalled. On March 5, 2015, participants in a similar meeting at the NAM decided to convene an independent commission to create a detailed report that hoped to inform future United Nations (UN) and WHO policy.

Dzau reported that the Rockefeller, Gates, and Ford Foundations; the Wellcome Trust; and USAID, among others, had already provided funding to support this effort. The report will be prepared by a global commission convened by the NAM, but it will not be an NAM or a U.S. product itself; the NAM's role is limited to that of secretariat.

For the report to have the hoped-for impact, it has to be issued from an independent international commission, Dzau explained; thus, 17 commissioners, reflecting a range of expertise and geopolitical representation, will oversee the report's preparation. As shown in the organizational diagram depicted in Figure 4-1, four workstreams will address each of the previously identified gap areas, based on information gathered at four workshops scheduled between August and September 2015 and organized by the National Academies of Sciences, Engineering, and Medicine.¹⁰

FIGURE 4-1

Development process for a global health risk framework. SOURCE: Dzau presentation, March 25, 2015.

Strategic dissemination will also be necessary to ensure that this report is effective and its recommendations implemented, Dzau acknowledged. “It has to start with the UN and the WHO, because they are going to be the recipients of many of the suggestions,” he observed. A financing mechanism to support distribution was under discussion with potential donors, he said; however, much more work will be needed to ensure that “it's not just going to be a report sitting on the shelf.”¹¹