I spoke with virologist Ian Goodfellow, whose laboratory works on noroviruses, about why he went to Sierra Leone to establish an Ebolavirus diagnostic and sequencing laboratory. The obstacles he encountered were considerable, but the results were very useful. Recorded at the Emerging Infectious Diseases A to Z (EIDA2Z) conference hosted by the National Emerging Infectious Diseases Laboratories (NEIDL).
Jeremy Luban, Aaron Lin, and Ted Diehl join the TWiV team to discuss their work on identifying a single amino acid change in the Ebola virus glycoprotein from the West African outbreak that increases infectivity in human cells.
You can find TWiV #415 at microbe.tv/twiv, or listen below.
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When viruses cross species, serial transmission may lead to the selection for mutations that confer improved replication or transmission in the new host. Identifying such mutations in human viruses is extremely difficult: we cannot conduct the appropriate experiments in humans, and often do not have viral isolates spanning the time from spillover through prolonged circulation. The 2013-2016 outbreak of Ebola virus in West Africa is unique because viral genome sequences were obtained early and throughout the epidemic. The results of two new studies (link to paper one, link to paper two) suggest that some of the observed mutations increase infectivity for human cells. The impact of these mutations on infection of humans, and their role in the West African outbreak, remain unknown.
Many mutations have been identified among the many hundreds of genome sequences obtained during the recent Ebola virus epidemic. One stands out: a mutation that leads to a single amino acid change in the viral glycoprotein, from alanine to valine at position 82 (A82V). This change arose early in the outbreak (it was first observed in Guinea in March 2014) and was subsequently found in most of the isolates. It has never been observed in previous Ebolavirus outbreaks.
The effect of the A82V change on viral infectivity was determined by building pseudotyped viral particles – in this case, HIV particles with the Ebola virus glycoprotein. Human cells in culture were infected with pseudotyped viruses with the Ebola virus glycoprotein with either alanine or valine at position 82. Infectivity was measured by quantifying the production of a protein from the HIV genome. The results show that A82V increases infectivity by twofold. The effect is also observed in cells from non-human primates, but not from rodents, dogs, or cats. However, the A82V change decreased infectivity in bat cells.
The A82V change is located at the binding site of the Ebola virus glycoprotein with the cell fusion receptor, NPC1. It appears to increase the fusion activity of the viral glycoprotein.
Other amino acid changes in the Ebola virus glycoprotein were also observed to increase infectivity in human cells, and decrease infectivity in bat cells.
The pattern of increased infectivity in primate cells, and decreased infectivity in bats, is consistent with the hypothesis that the outbreak virus came from bats, and after circulation of the virus in humans, it lost some ability to infect bat cells while becoming better at infecting human cells. However there is still no solid proof that bats are a reservoir of Ebolaviruses.
What does increased infectivity have to do with infection of humans? The idea is that the mutation increases the efficiency of virus entry into cells, and hence increased viral gene expression is observed. Fewer viruses needed to infect a cell, the better chance of initiating an infection. But is the two-fold increase observed in cells enough to impact infection in humans?
The assays used in these papers measure protein production from an HIV genome. The experiments need to be repeated using bona fide Ebola virus, to make sure that the mutations have the same effect. The changes might have impacts on other stages of viral replication. Furthermore, the impact of the changes in the viral glycoprotein should be assessed in animal models, to determine if improved infectivity has any impact on pathogenesis and transmission. Ultimately, we can’t prove that these mutations have any effect in humans – the needed experiments cannot be done.
I’m curious about why the A82V change was not seen in previous Ebola virus outbreaks. Those were in different parts of Africa – could the changes be driven by population genetics, ecology, or other factors? It will be important to determine if the same change is selected in future outbreaks.
The authors are sufficiently cautious in their conclusions. From paper #2:
Despite the experimental data provided here, it is impossible to clearly establish whether the adaptive mutations observed were in part responsible for the extended duration of the 2013–2016 epidemic. Indeed, it seems likely that the prolonged nature of the outbreak in West Africa was primarily due to epide- miological factors, such as an increased circulation in urban areas that in turn led to larger chains of transmission.
From paper #1:
Our findings raise the possibility that this mutation contributed directly to greater transmission and thus to the severity of the outbreak. It is difficult to draw any conclusions from this hypothesis, though…
As I feared, press coverage of these findings has been inaccurate. For example, a BBC headline proclaims “Ebola adapted to easily infect people”. Even the journal Cell, which published both papers, made an incorrect conlcusion: see the screen capture below from the journal website.Both Cell and the BBC might have taken too literally the unfortunate title of one of the papers, “Human adaptation of Ebola virus during the West African Outbreak.” The results suggest adaptation to human cells, not to humans. The title of the second paper is sufficiently careful: “Ebola virus glycoprotein with increased infectivity dominated the 2013-2016 epidemic”. But that’s not a BBC headline.
Four years after filming ‘Threading the NEIDL’, Vincent and Alan return to the National Emerging Infectious Diseases Laboratory BSL4 facility at Boston University where they speak with science writer David Quammen.
You can find TWiV #408 at microbe.tv/twiv, or watch/listen here.
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On episode #318 of the science show This Week in Virology, the TWiV gang reviews ten fascinating, compelling, and riveting virology stories from 2014.
You can find TWiV #318 at www.microbe.tv/twiv.
On episode #314 of the science show This Week in Virology, Vincent travels to Albert Einstein College of Medicine where he speaks with Kartik, Ganjam, and Margaret about their work on Ebolavirus entry, a tumor suppressor that binds the HIV-1 integrase, and the entry of togaviruses and flaviviruses into cells.
You can find TWiV #314 at www.microbe.tv/twiv.
ZMapp, a mixture of three antibodies against Ebola virus, became a household name after it was used to treat two Americans who were infected while working in Liberia. The structure of these antibodies bound to the Ebola virus glycoprotein suggest how they inhibit infection and ways to improve ZMapp.
The three monoclonal antibodies that comprise ZMapp (called c13C6, c2G4, and c4G7) were produced by immunizing mice with a recombinant vesicular stomatitis virus in which the glycoprotein was replaced with that from Ebola virus. Antibodies that bound the viral glycoprotein and protected mice from infection were identified, and three were made to resemble human antibodies and produced in tobacco plants. Ecco Zmapp!
Embedded in the membrane of the filamentous Ebola virus particle are many copies of the glycoprotein, seen as club-shaped spikes in the image to the right (image credit: ViralZone). The viral glycoprotein is essential for entry of the virus into cells. The antibodies in ZMapp are directed against the viral glycoprotein.
To determine how the antibodies bind the virus particle, they were individually mixed with purified Ebola virus glycoprotein, and the structures were determined by electron microscopy and image reconstruction. The results, shown in the illustration, indicate precisely where each antibody binds to the Ebola virus glycoprotein. The individual antibodies colored red (c2G4), yellow (c4G7), and purple (c13C6) are bound to a single Ebola virus glycoprotein in white, with the viral membrane below (Image credit).
The structures reveal that c13C6 (purple) binds at the tip of the viral glycoprotein, perpendicular to the plane of the viral membrane. The other two antibodies (red, yellow) bind at the base of the viral glycoprotein. Their binding sites overlap but are not identical (the Ebola virus glycoprotein is a trimer, and in the image, the yellow and red antibodies are shown binding to different subunits for clarity). Two other antibodies that block Ebola virus infection also bind at the base of the glycoprotein.
Antibody c13C6, which binds to the tip of the viral glycoprotein, does not neutralize viral infectivity. Nevertheless, it can protect animals from Ebola virus infection. This observation suggests that the c13C6 antibody may work in concert with complement, a collection of serum proteins, to block virus infection. It is not known why c13C6 antibody is non-neutralizing, but one possibility is that it binds to a part of the viral glycoprotein that is removed by an endosomal protease, cathepsin, before receptor binding in late endosomes.
Antibodies c2G4 and c4G7, which bind to the membrane-proximal part of the viral glycoprotein, neutralize viral infectivity. How they do so is not known, but one possibility is that they prevent structural changes of the viral protein that are essential for the fusion with the endosomal membrane, a process that delivers the viral nucleic acid into the cell cytoplasm.
These studies reveal two general areas of the Ebola virus glycoprotein that are important targets for antibodies that protect animals from Ebola virus infection. Those directed at the base of the glycoprotein neutralize infectivity while those that bind the tip do not. This information can now be used to isolate additional antibodies that bind either site. These can be used in animal protection studies to design mixtures that are even more potent than ZMapp.
After the governors of New York and New Jersey decided that health workers who have returned from the Ebola virus outbreak in West Africa should be subject to a 21-day quarantine, two Nobel laureates entered the fray. Bruce Beutler feels that the quarantine is the right thing to do, while Peter Doherty says it’s wrong. Which laureate is right?
The key issue in this debate is the assumption that someone who has been infected with Ebola virus, and does not display symptoms, is not contagious. Beutler doesn’t believe that there is enough evidence for this assumption: “People may have said that without symptoms you can’t transmit Ebola. I’m not sure about that being 100 percent true. There’s a lot of variation with viruses.” (source: NJ.com). On the other hand, Doherty says “the evidence-based consensus among the professionals seems to be that this is not necessary” (source: NJ.com).
As I’ve written before, our knowledge of the non-contagious nature of Ebola virus infected patients is based on experience with previous outbreaks. To make sure I wasn’t missing anything that Beutler had noted, I examined the published summaries of the previous outbreaks of Ebolaviruses. There have been 24 outbreaks caused by these viruses, summarized in a table by CDC. In poring over the outbreak data, I looked for information on how the virus is transmitted. These data are typically obtained by interviewing Ebola virus patients or their families, and constructing chains of transmission – who infected who, and how the infection was transmitted. Because it is not always possible to determine exactly how transmission took place, the interviewers attempt to determine what kinds of activities are most associated with acquiring infection. These activities are called ‘risk factors’.
I was particularly interested in determining if the following was a risk factor in any of the 23 previous outbreaks: some type of contact with a healthy person who subsequently developed Ebola hemorrhagic fever (EHF). I didn’t find any. All transmissions that could be assessed involved an obviously sick individual, and never from anyone who was healthy.
The first two recognized outbreaks of infection were in 1976 in Zaire (Ebola virus) and in Sudan (Sudan virus). The former comprised 318 cases, and infection was spread by close contact with patients and by using contaminated needles. The Sudan oubreak of 284 cases started in workers of a cotton factory, and was amplified by transmission in a hospital. Transmission required close contact with an acute case, usually the act of nursing a patient.
Sudan, 1979 (Sudan virus, 34 cases) The index case worked in a textile factory. Virus was then introduced into 4 families from the local hospital. Every case, except the index patient, could be traced to a human source of infection. Twenty-nine cases occurred in chains of secondary spread in 4 families, all from direct physical contact. No illness was observed among persons who were exposed to cases in confined spaces, but without physical contact.
Gabon, 1994 (Ebola virus, 52 cases) This outbreak began in three different gold mining camps. A second wave of infections outside the camp was initiated by contact with a hospitalized patient from first wave.
DRC, 1995 (Ebola virus, 315 cases) The index case was a charcoal worker, with no known contacts with other EHF patients, probably infected in a charcoal pit or on his farm. He infected 3 members of his family and 10 secondary cases occurred among his family members. Some of these individuals introduced virus into a maternity hospital, and then a general hospital. Prior contact with a suspected patient was reported by 93.5% (159/170) of case-patients for whom data were available. Eleven patients reported receiving an injection within 3 weeks of symptom onset. No role for airborne transmission was observed in human-to-human transmission. Sleeping in the same room with patient not a risk factor.
Gabon, 1996 (Ebola virus, 37 cases) A chimpanzee found dead in the forest was eaten by people hunting for food. Eighteen people who were involved in butchering the animal became ill. Ten other cases occured in their family members. Of 190 individuals who contacted sick people, none developed EHF.
Gabon, 1996-97 (Ebola virus, 60 cases) The index case was a hunter who lived in a forest camp. A dead chimpanzee found in the forest at the time was determined to be infected with Ebola virus. Disease was spread by close contact with infected persons. This outbreak was not well documented.
South Africa, 1996 (Ebola virus, 2 cases) A nurse in South Africa developed EHF; she had been previously exposed to the blood of a sick doctor brought from Gabon. This doctor had treated EV infected patients.
Uganda, 2000-2001 (Sudan virus, 425 cases) The three most important risk factors identified: attending funerals of EHF patients; interfamilial or nosocomial transmission. The source of infection of primary patients could not be identified. The transmission risk factors among 83 contacts were determined. The most important risk factor was direct repeated contact with a sick person’s body fluids, as occurs during care. Risk of infection was higher during late stage of disease. Simple physical contact with sick person was neither necessary nor sufficient for developing EHF. Transmission through contaminated fomites is possible – such as sleeping on the same mat, or sharing meals with a sick person. Having washed the clothing of a sick person was not a risk factor. There was no evidence for airborne transmission during this outbreak – sleeping in the same hut as an infected person was not a risk factor.
Gabon, 2001-2002 and Republic of Congo (Ebola virus, 65 and 67 cases, respectively) All but two cases were linked to recognized chains of transmission. There were six different introductions of virus into humans during the outbreak, each related to a hunting episode. An unusually high number of animals were found dead in the rainforest at this time, mainly gorillas, chimps, and monkeys. Ebola virus was detected in the carcass of a gorilla that had been butchered by one of the index cases shortly before onset of illness. The vast majority of secondary cases were related to community based transmission, and health care workers caring for infected patients included.
Republic of Congo 2002-03 (Ebola virus, 143 cases) Risk factors in this outbreak were determined to be participating in the funeral/burial ritual of a patient, and providing nursing care to an individual with EHF. Quote from the paper: ‘There is no contamination by air or just handshake’.
Republic of Congo 2003 (Ebola virus, 35 cases) Risk factors in this outbreak were determined to be participating in the funeral/burial ritual of a patient, and providing nursing care to an individual with EHF.
Sudan, 2004 (Sudan virus, 17 cases) Risk factors in this outbreak were determined to be participating in the funeral/burial ritual of a patient, and providing nursing care to an individual with EHF. There was one introduction of virus into the index case-patient, who had been hunting baboons in forest, and was in contact with fresh monkey meat 5 days before onset of symptoms. All cases were epidemiologically linked, with 4 generations of transmission observed.
Democratic Republic of Congo, 2007 (Ebola virus, 264 cases) Index case-patient bought freshly killed fruit bats from hunters for consumption. She infected 11 family members who provided care, who in turn passed infection to others in a chain of transmission.
Uganda 2007-08 (Bundibugyo virus, 149 cases) Identified a chain of transmission from a prominent individual in the community to 27 others; they had contact with him before or after his death.
I did not find risk factor analysis for outbreaks in Democratic Republic of Congo, 2008-2009 (Ebola virus, 32 cases) ; Uganda, 2012 (Sudan virus, 11 cases; and Democratic Republic of Congo, 2012 (Bundibugyo virus, 36 cases). Transmission studies on the recent Ebola virus outbreak in West Africa has not yet been published. However the recent outbreak of Ebola virus in Nigeria was traced from an index case who traveled there from Liberia, where he had cared for a sibling who died of EHD. Upon receiving medical attention in Nigeria, he infected health care workers and the disease spread further. Few of the 898 contacts linked to this index case became infected.
From this analysis it is clear that contact with an asymptomatic individual, who subsequently developed EHV, is not a risk factor for disease. All risk factors involve close contact with sick individuals, such as would occur in a health care facility, among family members caring for a sick individual, and during funeral proceedings.
I am aware that not all of the cases in these outbreaks could be traced and assigned risk factors for infection. One could argue that among these cases, some might have been acquired by contact with an individual in the incubation period. However it makes no sense that these individuals would have been missed in all of the outbreaks. The more likely conclusion is that during the incubation period, patients infected with Ebola virus are not contagious.
There is one more piece of information to support the conclusion that there is no transmission during the Ebola virus incubation period. During the Ebola virus outbreak of 2000-2001 in Uganda, blood samples were collected from health care workers who became symptomatic with EHF; of these, 27 died and 18 survived. Ebola virus RNA could be detected in the blood on the first day that symptoms were observed, at levels just above the detection threshold (illustrated; image credit). Each day thereafter, the virus load increased, much more substantially in patients with a fatal outcome. While no viral loads were determined before the onset of symptoms, it is reasonable to predict that viral loads would be even lower. This conclusion is consistent with the observation that infected patients do not transmit infection before the onset of symptoms, probably because the levels of virus that they produce are too low. The results of this study are also consistent with observations from some outbreaks that the risk of transmission is increased later in disease, when more virus is present in the blood.
These data lead to the conclusion that Ebola virus transmission does not occur during the incubation period. Therefore it is not necessary to quarantine those who might have had contact with Ebola virus infected individuals. Daily reporting of temperature and other vital signs should be sufficient to allow detection of the onset of disease.
In other words, I disagree with Dr. Beutler’s statement that there are no good data supporting lack of Ebola virus transmission during the incubation period. The fact that Dr. Doherty disagrees with Dr. Beutler just goes to show that having a Nobel Prize doesn’t mean you are always right.
On episode #309 of the science show This Week in Virology, the TWiVocytes answer questions about Ebola virus, including mode of transmission, quarantine, incubation period, immunity, and much more.
You can find TWiV #309 at www.microbe.tv/twiv.
I usually don’t post TWiM episodes here, but #90 has a lot of virology. In this episode, recorded in La Jolla, CA at the annual meeting of the Southern California Branch of the American Society for Microbiology, I first speak with Laurene Mascola, Chief of Acute Communicable Diseases at the Los Angeles County Department of Public Health. Dr. Mascola talks about how Los Angeles county has prepared for an outbreak of Ebola virus. Next up is David Persing, Executive Vice President and Chief Medical and Technology Officer at Cepheid. His company has developed an amazing, modular PCR machine that is brining rapid diagnosis everywhere, including the United States Post Office. And it might even be available on your refrigerator one day.