To catch a killer

As the body count continues to rise – do we have any defence against Ebola virus? It’s the mid-seventies – and in a small village in the Republic of Zaire the headmaster of the local school busies himself with the everyday challenges of education in deepest rural West Africa. And what challenges they must have been, not least of which convincing the mainly subsistence community of the virtues of education at all. Still, he has a school room and desks and pupils – and life proceeds in much the same way as you might imagine life to proceed in villages across West Africa. This emblematic existence however, wasn’t to last. By the end of August of 1976 Mabalo Lokela had been taken ill; a relapse, it was thought, of a previous malaria infection. Less than two weeks later it became clear this was something else – something dangerously different. He started to vomit uncontrollably, developed acute diarrhea, headaches and found it difficult to breathe. Shortly afterwards his nose, eyes and gums began bleeding. On September 8, approximately two weeks after presenting the first symptoms of the illness, Lokela was dead. Within a week other cases began to erupt. The small hospital where he was treated closed after 11 of its 17 staff members died – and in all, 318 cases were identified of which 280 perished. Analysis at the Pasteur Institute in Paris soon confirmed the disease was indeed different; a virus entirely new to science had not only found its way into Lokela’s body, but also into our imaginations – a new and deadly threat. Yambuku, a village hitherto inconspicuous, goes down in medical history as the location of the index case of Ebola virus disease, a disease which nearly 40 years later plagues West Africa once again with the biggest outbreak on record. Named after a stream in the vicinity of Yambuku from which it was thought to originate, Ebola has no vaccines and only experimental therapies – the effectiveness of which remains to be seen. Little wonder then the media reaction to the recent outbreak has reached almost fever pitch levels – all the ingredients are there for an unmatched ability to grab headlines: The macabre symptoms, the virulence, the awful images of suffering – all set in the deepest Congo. Perhaps it is unwarranted attention – as Dr Seth Berkley of the GAVI alliance, an organisation promoting vaccination in developing nations, points out: “Ebola is without doubt a truly horrible disease, but then there are many other bad ones that kill far more.” Yet however true that fact may be, it is of little consolation for the countries of Guinea, Liberia, Nigeria and Sierra Leone. It is here that the virus is working with its deadly efficiency to crippling effect; it is here that quarantine zones are set up and movement is restricted; it is here that barriers set up to stop transmission of disease inevitably hinder delivery of supplies and the pangs of hunger and thirst begin to dig in. So what, if anything, can be done? In the short term – which of course for the sufferers of the disease is the only timescale that matters – desperation has crept into the response. A week after the WHO officially declared the outbreak as a public health emergency their own consultants agreed that unproven experimental interventions could be ‘ethically’ used. Putting aside the inherent risk of adverse effects and poor efficacy, the main problem with this strategy is one of supply and demand – given these drugs are experimental, not one of them benefits from the speed and efficiency of full-on production. The Ebola virus has sickened at least 2127 people and killed 1145 of them, yet as sobering as they are, those numbers “vastly underestimate the magnitude of the outbreak” the WHO warns. A little left over in a lab here, or a small amount set aside for preclinical work there just won’t cut it. As a prime example of this Mapp Biopharmaceutical, a company developing an experimental Ebola drug called ZMapp, announced last month the last of the medication had been sent to West Africa after receiving a request in early August. “The available supply of ZMapp,” they say “…is exhausted”. If not experimental drugs then what? Back to basics say the WHO, as they consider using the blood of people who have recovered from an infection to treat those still fighting the virus. Convalescent serum has been used in this way before, and relatively recently too – it formed part of the response in China to the SARS outbreak in 2002 – but doubts have been raised about its use against Ebola. As of going to print there are no official plans to administer convalescent serum to those currently affected, but the WHO said it will assess if the treatment approach was “safe and feasible”. All correct and proper of course, but being correct and proper takes time. Something Ebola-infected patients don’t have. “There is not a lot of extra time to experiment with unproven therapies,” writes Armand Sprecher, an epidemiologist and public health specialist at Doctors Without Borders, in New Republic. “We cannot subject our patients to all of the possible things that might work. We have to choose wisely.” And so to the longer term. Is there a way to expose the virus; to break its adeptness? Of course there are the experimental interventions currently mooted, however the vast majority under consideration appear to be cocktails of antibodies (such as ZMapp) or drugs designed to work on other diseases which may, or may not, confer an advantage to sufferers of Ebola. Then there are reports that Ebola victims in the Nigerian city of Lagos are to receive Nano Silver in an attempt to treat the infection, but the basis of this approach is at best unclear, seemingly stemming from a letter written to the Nigerian government by a doctor who represents an organisation who just happen to sell – you guessed it – colloidal silver. Joanne Liu, international president of Doctors without Borders, estimates it will take officials around six months to contain the outbreak – it seems likely therefore that the natural epidemiological ebb and flow of this outbreak will reach maturity before a robust therapeutic approach can be developed. So what of the future? Where will the medicinal ‘big guns’ come from for the next outbreak? Dr Gaya Amarasinghe from the Washington University School of Medicine thinks he might know. And, as is so often the case, the key to his approach is to understand the enemy. In doing so he suggests the chink in the formidable armour of Ebola virus resides in the very thing it uses to expose the chink in our own. “We’ve known for a long time that infection with Ebola obstructs an important immune compound called interferon,” says Amarasinghe. “Now we know how Ebola does this, and that can guide the development of new treatments.” The reason Ebola is such an effective pathogen resides in one of the great biological swindles of all time; a confidence trick of incredible ambition. As a virus, of course Ebola must avail itself of its host’s cellular equipment required to produce the proteins it needs to replicate, yet at the same time it manages to remain invisible to the immune system – preventing the cell’s innate antiviral immune response by some fiendish molecular sleight of hand. Ordinarily, when a virus infects a host cell the innate immune system – that evolutionary throw back which remains our first line of defence – pulses into action. As with most pathogen/immune system interplays there are a large number of cast members – each with a very specific role to play. A starring role, as always, must however go to interferon – the great immune conductor.It controls a variety of responses to viral infection, including the self-destruction of infected cells and the blockage of supplies necessary for viral reproduction. A vital component of this interferon pathway is the transcription factor STAT1. It is the lone messenger carrying interferon’s antiviral message to the nucleus hidden deep in the cellular interior. Once there it activates the genes for hundreds of proteins involved in antiviral responses. Evolution has of course worked on the selective pressure of temporality here – when viral infection strikes, time is most certainly of the essence. As such, an important part of innate immunity is that STAT1 is afforded a ‘rapid access lane’ as cellular transporter proteins whisk the vital signal from membrane to nucleus. And it is this rapid access lane which Ebola seems to switch off. But how does it do this? How does Ebola avoid this ancient barricade against infection? Well, building on previous work(1,2) Amarasinghe and his team have uncovered the molecular basis behind this ability to shoot the cellular messenger. Together with researchers at the Icahn School of Medicine at Mount Sinai, the University of Texas Southwestern Medical Center, Howard University and biopharmaceutical company Microbiotix, Amarasinghe’s team developed a detailed map of how an Ebola protein – VP24 – binds to one of these transporter proteins. Think back here to the old lock-and-key analogy of agonist/antagonist receptor binding and you’ll begin to see how VP24 can do this. It binds, say the team, not just in a competitive way but in a preferential way – and once it has done so the rapid transport of STAT1 becomes impossible. The line of communication between the virally ravaged cell and its isolated – but all important – nucleus goes dead. Yet of course this is one very specific transport pathway that has been shut down – Amarasinghe’s proposed model also predicts that all other nucleocytoplasmic trafficking will be largely unaffected by the binding of VP24. This is the devilishly ‘clever’ part of mechanism, because it means other proteins that travel in and out of the cell nucleus – so important to viral replication – are likely to be unaffected. As Amarasinghe et al write in their paper published in Cell Host & Microbe(3): “By targeting the binding site that is critical for PY-STAT1 recognition and nuclear transport, EBOV disables cell-intrinsic antiviral signalling in order to facilitate virus replication without impacting normal cellular cargo transport.” Ebola can now go about the deadly business of highjacking the cells proteomic machinery to replicate itself, whilst the immune system remains none-the-wiser. It can’t pick up any cellular red flags, because the cell hasn’t the ability to fly them. Interesting? Undoubtedly. It is a testament – if one were needed – to the ever resourceful evolutionary processes at work that something as ‘simple’ as a virus can act in such a sophisticated way. But is this just academic? Can we derive some medicinal insight from this? The team certainly think so. “Now that our map of the combined structure of these two proteins has revealed one critical way Ebola works, the information it provides will guide the development of new treatments,” says Amarasinghe. It is hoped that the structural insights from the study could provide the framework for targeting the VP24/transporter interface pharmacologically to resensitise Ebola virus to interferons. But there have been concerns raised because, as always with the development of therapeutics, money talks. Ebola is rare, and rare diseases make for particularly unenticing targets for big pharma. The truth – as awful as it may be to consider as people continue to die in West Africa – is that any new drug would probably be used only occasionally in relatively small numbers of people. The molecular target is there, at least potentially, but to really make headway against this disease we need to look to investment from international aid agencies rather than the pockets of big pharma. [caption id="attachment_39587" align="alignleft" width="600"] Life cycles of the Ebola virus. Fruit bats of the Pteropodidae family are considered to be the natural host of the Ebola virus. Credit CDC[/caption] Deadly simple The current outbreak is killing between 50% and 60% of people infected. It is not known which factors allow some people to recover while most succumb. Genus Ebolavirus is 1 of 3 members of the Filoviridae family (filovirus), along with genus Marburgvirus and genus Cuevavirus. Genus Ebolavirus comprises 5 distinct species. Each Ebola virion contains one molecule of linear, single-stranded, negative-sense RNA, 18,959 to 18,961 nucleotides in length. It codes for just seven structural proteins and one non-structural protein. The gene order is 3? – leader – NP – VP35 – VP40 – GP/sGP – VP30 – VP24 – L – trailer – 5?; The leader and trailer are non-transcribed regions, which carry important signals to control transcription, replication, and packaging of the viral genomes into new virions. The Treatment Gap The current outbreak is killing between 50% and 60% of people infected. It is not known which factors allow some people to recover while most succumb. No specific medicine has been proven to be effective against Ebola. There are experimental treatments, including: - ZMapp, being developed by Mapp Biopharmaceutical. A combination of three different monoclonal antibodies that bind to the protein of the Ebola virus. - Tekmira Pharmaceuticals have a drug (TKM-Ebola) based on small interfering RNA (siRNA) which has shown efficacy when given to monkeys infected with the Marburg virus - a related species to Ebola. - DNA vaccines and adenovirus-based vaccines have entered clinical trials, however no vaccine is currently available for humans. References

1. Bray, M. and Murphy, F.A.Filovirus research: knowledge expands to meet a growing threat.J. Infect. Dis. 2007; 196:S438–S443
2. Geisbert, T.W., Young, H.A., Jahrling, P.B., Davis, K.J., Larsen, T., Kagan, E., and Hensley, L.E.Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells.Am. J. Pathol. 2003; 163:2371–2382
3. 3.Wei Xu, Megan R. Edwards, Dominika M. Borek, Alicia R. Feagins, Anuradha Mittal, Joshua B. Alinger, Kayla N. Berry, Benjamin Yen, Jennifer Hamilton, Tom J. Brett, Rohit V. Pappu, Daisy W. Leung, Christopher F. Basler, Gaya K. Amarasinghe. Ebola Virus VP24 Targets a Unique NLS Binding Site on Karyopherin Alpha 5 to Selectively Compete with Nuclear Import of Phosphorylated STAT1. Cell Host & Microbe Volume 16, Issue 2, p187–200, 13 August 2014