Why is Ebola virus so deadly?

In December 2013 Guinea saw the first death from an outbreak of Ebola virus. This, still on going, Ebola epidemic is now the largest in recorded history. I’ve therefore put together this blog post that will tell you about the virus species and what can make it so deadly.

The first recorded outbreaks of Ebola virus were in 1976 in Zaire and Sudan, with the name being derived from the Ebola River in Zaire (now the Democratic Republic of Congo). These outbreaks were caused by two different species of the Ebola virus family, with the Zaire strain being more deadly. In Sudan, there were a total of 151 deaths from 284 confirmed cases, whereas Zaire had 280 deaths from 318 cases.

The Ebola virus structure - false colouring of an electron microscope image

Zaire is still the most deadly of the five currently defined Ebola species. However, its case fatality rate is often misconstrued in my opinion. There have been a total of 14 ‘outbreaks’ of Zaire Ebola virus with case fatality ranging from 44-100%. But two of those ‘outbreaks’ have been of a single person, who died, giving two 100% case fatality ‘outbreaks.’ If you remove those two, the average case fatality from all recorded Zaire Ebola virus outbreaks is 75%. It’s still very high, but not the often-reported 90%.

With that said, Ebola viruses are undoubtedly extremely deadly. For a point of comparison, the worst influenza outbreak, 1918 Spanish Flu, had a case fatality rate of around 2.5%. But what is it that can make Ebola virus deadly?

Like all other viruses, Ebola virus must enter and infect cells. Different viruses have different cells they infect. For example, influenza preferentially infects lung cells, while HIV preferentially infects white blood cells. Ebola targets a lot of cells, but it is thought the earliest to be infected are white blood cells. Similarly to HIV, Ebola infection kills these cells that constitute our immune system. However, while HIV takes years, Ebola can kill in a matter of weeks.

As Ebola viruses infect cells of the immune system, our bodies try to fight back by releasing chemicals known as cytokines. Cytokines attract immune cells to the sites of infection and help to activate the immune response – they are essential to combating infection. Cytokine release causes the general symptoms associated with viral infections such as headaches, fever, nausea and inflammation. In addition to infection triggering release of cytokines, cell death is also a major cause. With Ebola killing cells and the immune response, there is a huge release of cytokines.

While the cytokine response is essential to fighting infection, it is a response balanced on a knife-edge. Cytokines act through a positive feedback mechanism; infected cells release cytokines to activate immune cells, these activated cells then release more cytokines and so on. If this isn’t regulated, there can be an uncontrolled release of cytokines throughout the body, known as a cytokine storm. Ebola infection can cause this uncontrolled release of cytokines resulting in a massively exaggerated response and extreme fever and vomiting, to name just two symptoms.

So Ebola viruses can cause cytokine storm, while also, somewhat paradoxically, decimating the immune system. But it doesn’t stop there; Ebola can also damage and infect endothelial cells that line the blood vessels through our bodies, producing holes in these vessels and allowing fluids to escape. Combine this with the dilation of blood vessels caused by the inflammatory response from cytokines and you have a situation where Ebola virus causes huge fluid loss and drop in blood pressure (potentially resulting in shock).

Leaky blood vessels, combined with the cytokine-induced fever, give the symptoms Ebola is best known for, ‘haemorrhagic fever’. The haemorrhaging causes massive blood blisters around the body and release of blood from the gums and into the eyes (causing them to turn red). This is just the striking exterior; there is also internal bleeding to exacerbate the situation.

With all the fluids lost due to leaking of blood vessels through the body, combined with further loss from vomiting and diarrhoea, hydration becomes one of the biggest issues for patients infected with Ebola. Indeed, rehydration therapy is one of the few treatments available to patients with Ebola, but needs to be given early, before the late stages of haemorrhaging and fever.

With all of these different symptoms combined, hopefully it’s clear why Ebola virus can cause such a deadly infection.

Ebola virus is spread through close contact with bodily fluids from an infected person (or animal); hence the vomiting and diarrhoea are highly advantageous to the virus. This tends to put close family members and medical staff at the highest risk of contracting the virus. However, there can also be sexual spread for weeks after a patient has apparently recovered from infection.

The current West Africa outbreak is suspected to have killed 826 people from 1440 cases (as of 4th August 2014 – this site is regularly updated). This gives the current outbreak of the Zaire species of Ebola a case fatality rate of around 57%, much lower than the average for this species. The reasons for why some outbreaks of the same Ebola species are so deadly compared to others are unclear, but I’d speculate one major factor is the work of health care personnel. Limiting spread by isolation of patients, giving rapid rehydration therapy and tracking spread as much as possible are unboundedly having a huge impact in controlling this outbreak. In the past, outbreaks had occurred in areas of huge diplomatic unrest and poverty making the necessary responses much harder. The current outbreak is in areas of huge poverty, but the international response is undoubtedly having a major impact on limiting deaths. 

A health care worked in an isolation unit

While Ebola virus is unquestionably a dangerous and deadly virus, the need for close contact spread would suggest to me that it will never really take off as a pandemic. I would speculate that developed parts of the world would see nowhere near as much death from the virus, even if an outbreak were to occur, because of being able to limit spread with isolation measures and the speed with which this could be done in richer parts of the world. Hopefully with the continued work of health care professionals in West Africa, this current outbreak will soon be curtailed. 


Amendment
Clearly my hope that the Ebola outbreak would "soon be curtailed" hasn't come to fruition. It is now a little over a year since the first death from this Ebola outbreak and the death toll has reached 8,414 (15th Jan 2015) from 21,261 cases (13,427 being laboratory confirmed). However, it has been reported today that the number of new cases may be declining. Hopefully this trend will continue and 2015 will see the end of this Ebola epidemic. 

A breakthrough in the treatment of hepatitis C virus

A staggering 130-150 million people are estimated to have chronic hepatitis C virus (HCV) infection worldwide, that’s approximately 5 times the number of people that are infected with HIV. HCV can be divided into 11 major genotypes, based on divergence of the genetic material that makes up the virus. Of the global total for HCV infections, genotype 1 constitutes 60% of these infections.

Even though HCV is a global problem, treatment for infection is sorely lacking, especially for genotype 1. Until recently, up to 48 weeks worth of ribavirin and interferon injections were the only option. Neither of these treatments specifically targets the virus, and both cause significant side effects. Interferons are proteins produced by our bodies in response to infection and are responsible for the associated general symptoms; fever, chills, headache, malaise, nausea and so on. Ribavirin, on the other hand, is a drug that partially mimics a building block of RNA. This incomplete mimicry means ribavirin can interfere with production of RNA; a process essential for replication of the RNA genome of HCV. This is how ribavirin is thought to work, however, it has never been proved, and its full function isn’t know. Similarly to interferon, ribavirin can cause fever, chills etc., but can also cause many more serious side effects such as depression and problems with vision. 

 

Hepatitis C virus particles under an electron microscope

Due to the problems associated with HCV therapy, many patients opt out, choosing to live with chronic infection. Indeed, in the UK, only 3% of patients receive treatment. Opting out may seem rational since the vast majority of chronic infections are completely asymptomatic, why take treatment that will make you feel appalling, when you feel fine without it? Combine that with the knowledge that only 50-60% of patients infected with genotype 1 viruses will achieve a sustained virologic response (defined as having no virus in the blood for 24 weeks following the treatment, essentially a cure), and you can appreciate why many people opt out. However, chronic HCV infection will inevitably lead to complications in the liver, including cirrhosis, failure and cancer. HCV is the most common cause of liver transplantation in the US, for example, and is responsible for 300,000-500,000 deaths per year globally.

With all that said, things are starting to improve for HCV patients with many new, HCV specific, drugs starting to enter the market. HCV was only classified in 1989 and, following much research, the essential proteins the virus makes were identified and their structures defined just before the turn of the millennium. Knowing these structures allowed for drugs to be designed that could specifically interfere with their functions, and now, finally, we are starting to see the outcome of these research programs. Currently, four HCV specific drugs have been approved for clinical use, while many others are in phase 2 and 3 clinical trials. Phase 3 clinical trials are essentially the final stage of testing any new drug and where its efficacy is tested in a large number of patients, if it proves to be effective, and safe, it can then be approved for general use.

This leads me into the topic of this blog post, the results of a phase 3 trial published earlier this month in the New England Journal of Medicine showing remarkable promise for the treatment of HCV genotype 1. The treatment is a single, orally administered, pill combining two drugs named Ledipasvir and Sofosbuvir. Ledipasvir interferes with the function of the viral protein NS5A, which plays many key functions for HCV infection, the most well characterized being modulation of the immune response against the virus. While Sofosbuvir inhibits the function of the viral RNA polymerase, blocking the production of new HCV genomes, and by extension, virus particles.

A schematic of the hepatitis C virus genome,
and the proteins it encodes

The study treated 865 patients, chronically infected with HCV genotype 1. These individuals were evenly divided into four groups. Two groups received Ledipasvir and Sofosbuvir for 12 or 24 weeks, while the remaining two groups received Ledipasvir and Sofosbuvir along with ribavirin for the same time periods. In all four groups, the percentage of sustained virologic response (essentially a cure) was over 95%. This is an incredible increase from the current efficacy of HCV genotype 1 treatment, and was reported in a wonderfully understated manner, a direct quote from the manuscript; “the rates of sustained virologic response in all four treatment groups were superior to the historical rate of 60%. The rates of sustained virologic response 12 weeks after the end of treatment were as follows… 99%… 97%… 95%… 99%.” Modest reporting of a fantastic result.

Furthermore, the inclusion of ribavirin had no impact on the percentage of patients that attained sustained viriologic response, suggesting it can be dropped from the therapy, along with it’s associated side effects. The difference between 12 and 24 weeks was also negligible, with both being around 98% effective. The treatment with Ledipasvir and Sofosbuvir proved to be extremely effective, and also proved to be safe, with only mild side effects being reported. Indeed, the majority of reported adverse reactions were from the group receiving ribavirin, and most of those reactions were known to be associated with ribavirin itself.

It’s worth noting three cases of virologic failure, patients who cleared the virus but then had detectable levels within 12 weeks of cessation of treatment. In one case, the patient had undetectable levels of metabolites from the drugs, suggesting non-adherence to the regimen. The other two cases were patients who had relapse of the virus, even though they had adhered to the treatment regimen. Importantly, both of these patients were found to have viruses with mutations to NS5A that made the protein resistant to Ledipasvir, before the trial. This was concluded to be the cause of the relapse and in future clinical situations it would be easy to remove Ledipasvir and use an alternative drug that targets another protein of the virus.

Another point to note is that this study was not designed to look at any long-term effect. Chronic HCV infection causes damage of the liver that can result in complications such as cirrhosis and cancer. It is not known if the clearance of the virus with Ledipasvir and Sofosbuvir will have any effect on reducing the occurance of these issues. However, I would speculate that the liver, an organ highly adept at repairing itself (as anyone who has ever had a hangover will indirectly know), would become healthy again once the virus has been removed.

Hepatitis C virus infection has been historically difficult to treat, particularly genotype 1. Until recently, the only available treatments were a combination of ribavirin and interferon, both of which come with substantial side effects and only work about 50-60% of the time. However, following a huge amount of research on HCV in labs around the world, numerous drugs have been developed to specifically stop HCV replication. Many of these drugs are finally starting to reach the point of being clinically available. Ledipasvir and Sofosbuvir seem to be incredibly promising for the treatment of HCV having a nearly perfect rate of sustained virologic response and only minimal side effects. I imagine it won’t be long before the single pill of these two drugs reaches patients around the world.

Shutting the door to HIV? (part 2 of 2)

This is post two of a blog on HIV gene therapy; I’d highly recommend post one to help with understanding of what is to come, that being the discussion of a study in which CCR5 is deleted from cells of HIV infected individuals.

Over recent years ‘gene editing’ has started to take off, with new technologies being developed and made safe. One such approach uses artificial proteins known as zinc finger nucleases (ZFNs). These proteins are a combination of two naturally occurring proteins, a zinc finger protein and a nuclease (known as Fok1). Zinc fingers bind to specific stretches of DNA, while a nuclease is an enzyme capable of cutting through double stranded DNA. Combining the two produces a protein that can cut through DNA at specific locations.

A diagram demonstrating the principle of
ZFN cleavage of DNA

When breaks are made in both strands of DNA, a cell attempts to repair the damage – with one of three outcomes. The cell dies, the DNA is stitched back together and the original sequence is preserved, or the DNA is repaired with the random insertion or deletion of parts of the original base sequence. The third of these outcomes leads to the deletion of a gene. Using ZFNs to damage DNA at specific sites therefore allows for deletion of a specific gene – such as CCR5.

This leads me to the aforementioned study. In the work, published in the New England Journal of Medicine, HIV infected patients had blood taken, CD4 T-cells collected and then treated with a ZFN to specifically delete CCR5. Following the procedure, these cells were then infused back into the patient.

Twelve individuals received the ZFN treatment, in a study designed primarily to determine if this was safe. To that end they mostly achieved their aim. The majority of the patients in the study developed only mild reactions with 130 reported side effects such as, fever, chills, headaches etc. Out of these, only 32 of the side effects were linked directly to the modified cells, with the remaining number being attributed to the infusion process. Sadly one of the twelve did develop a severe adverse reaction and needed hospitalisation, but this was a response to the infusion, rather than the modified cells themselves. It would be nice to have none of these issues, but, as with any medical procedure, there is a likelihood of side effects, and the ones caused by infusion of the CCR5 deleted cells are largely acceptable.

So what impact did these cells have? As you may expect, the infusion of 10 billion or so cells caused an increase in the number of CD4 T-cells (the loss of CD4 T-cells is a hallmark feature of HIV infection). The mutant cells were detected at a reasonably high level, making up 13.9% of the circulating CD4 T-cells at any given time. Furthermore, the mutant cells were estimated to stay in an individual for close to 96 weeks. At this stage it’s clear that the infusion of CCR5 deleted cells is largely safe, and that these cells can survive in the blood for a fairly long period of time.

The main aim of this study was to assess the safety of CCR5 ZFN treatment, but there was also some investigation into the impact these CCR5 deleted cells had on HIV levels. All of the patients were on antiretroviral therapy at the start of the trial, and, as a result, had undetectable levels of HIV. However, six of the twelve patients were taken off their medication for twelve weeks, four weeks after the infusion of CCR5 deleted cells. Initially these patients showed virologic rebound, the phenomenon where HIV levels start to rise as soon as therapy is stopped. A peak in this was seen at six to eight weeks following cessation of therapy. However, at that point the levels began to decline – perhaps, optimistically, suggesting a protective effect from the CCR5 deleted cells. Moreover, as CD4 T-cell death occurred from the increased level of HIV, it was noted that the CCR5 deleted cells died at a slower rate than normal CD4 T-cells, further suggesting these cells may be protected from HIV infection.

At this stage no real conclusions can be drawn about the efficacy of infusing CCR5 deleted cells for treating HIV. This study achieved its aim to demonstrate safety of the procedure, but little can be learned about any protective effect at this stage. However, that is not to say this isn’t a promising avenue for HIV therapy in the near future.

An artistic representation of HIV showing the outer layer being
peeled away to show the inner core

A few aspects still need to be ironed out. For instance, of the approximately 10 billion cells infused to the patient, only 20% of these had the CCR5 deletion, meaning there are still a large number of cells for the virus to infect. Furthermore, the patients will continue to make their own immune cells from their own stem cells, which won’t have the CCR5 deletion, and will therefore, effectively, dilute the impact of the CCR5 deleted cells. Continual infusions may therefore be necessary for any protective effect. Alternatively, it may be possible to use a similar technique to engineer a CCR5 deletion into stem cells, allowing the patients to develop modified cells for the rest of their life.

Another issue may arise from the fact that HIV is a rapidly evolving virus, capable of becoming resistant to any treatment given in isolation. I would speculate that if a patient, infected with HIV were to be taken off all medication and given CCR5 deleted cells, then HIV viruses that bind CXCR4 would become more prevalent, or perhaps some other work around would evolve. What impact this may have is unknown at this stage.

No doubt a large scale, phase 2 clinical trial is, soon to be, or already underway to assess the protective effects of the CCR5 deletion against HIV over a longer time period of time, with a larger number of patients. It will be interesting to see how much clinical impact this procedure can truly have. And what ways the virus may find to escape the intervention.

Shutting the door to HIV? (part 1 of 2)

Earlier this month a study was published that suggested a potential new approach to treat HIV infection using genetic modification of the T-cells HIV infects. This study rightly generated media interest, so I thought I’d give my take on it and outline some of the details. I’ve divided the post into two parts, allowing for an ample amount of background to aid understand of the details of the study, without scaring you off with one long post.

To begin with, a bit of cell biology. A protective layer of lipid molecules, in a double sheet, surrounds our cells. This ‘lipid bilayer’ provides separation between the inside and outside of the cell. However, cells needs to interact with the environment and transport molecules across the lipid bilayer. One of the major mechanisms to have evolved are cell surface receptor proteins. These proteins reside in the lipid bilayer, exposed to the environment, and can bind molecules, in some cases these are then transported into the cell. These proteins are essential for the normal functioning of our cells.

A schematic of a lipid bilayer with inserted proteins
spanning it (transmembrane proteins)

Like other viruses, HIV must enter cells to replicate. Without wanting to overly anthropomorphise the situation, viruses are devious in the ways they do this. In the case of HIV, binding to two cell surface receptors drives a shape change in a protein on the virus that causes it to fuse with the lipid membrane of the cell. This fusion event allows entry of the viral genetic material, which can then undergo replication.

CD4 is the essential receptor for HIV; it is the first receptor the virus interacts with. CD4 is a protein expressed by cells of the immune system, such as T-cells and macrophage. Once bound to CD4, the virus must bind to one of two co-receptors, either CCR5 or CXCR4. When this second binding event has occurred, the shape change will happen, and the virus will fuse with the cell and enter.

HIV fusion

The vast majority of viruses that are transmitted between individuals, and many of the viruses within an individual are CCR5 tropic (they bind CCR5 and not CXCR4). You could therefore speculate that without CCR5, HIV transmission would be blocked, and infection levels reduced. Without CCR5 on cells, could we shut the door to HIV infection? However, an important question is whether this would be safe? Can people survive without CCR5?

Interestingly, around 4-16% of people with European descent have a mutation in at least one copy of the CCR5 gene (CCR5 – italics denote a gene) known as CCR5 delta 32 (there is deletion of 32 base pairs from the gene). People with this mutation in one gene copy, 'heterozygotes', express roughly half the level of the protein compared to a person with no mutation. It is also possible to have the delta 32 mutation in both copies of the gene, 'homozygotes', leading to no CCR5 protein being present on any cells – and these people are perfectly healthy. What’s more, people carrying CCR5 delta 32 mutations have resistance to HIV infection, and slower progression to AIDS if they are infected, particularly the homozygotes.

The hypothesis that disruption of CCR5 could be protective was further strengthened in 2008 with the ‘Berlin patient.’ This individual, later disclosed as being named Timothy Rae Brown, has been functionally cured of HIV. Brown was diagnosed with HIV in 1995, and was subsequently diagnosed with a leukaemia in 2006. The leukaemia could be treated with a bone marrow transplant. Brown’s doctor, Gero Hütter, decided to acquire the bone marrow from a donor homozygous for CCR5 delta 32. Stem cells of the immune system reside in bone marrow; so receiving bone marrow from a homozygous CCR5 delta 32 individual meant Brown developed an immune system populated by cells containing the mutation. In 2009, Brown had been off his antiretroviral therapy, used to treat HIV, for over a year and had had no detectable virus. He remains, to this day, free from antiretroviral therapy and still has no detectable HIV.

As amazing as the story of the Berlin patient is, bone marrow transplants are not a realistic option to treat HIV infection because of cost and the risks associated with the procedure. However, what if there were other, less drastic, ways of introducing mutations to CCR5?

This is where I’ll leave the first part of this post. Tomorrow I’ll post part two looking in detail at a study assessing the safety of genetically modifying cells to cause deletion of CCR5, and to see the impact this has on HIV infection. So come back tomorrow for more…

Pithovirus - More than just a large storage container

I have a fascination with giant viruses; they have made two previous appearances on my blog (here/here and here). The reason for this fascination is two-fold; firstly, their discovery shattered the notion of viruses as tiny, sub-microscope, particles and secondly, because they have resulted in some intriguing ideas surrounding the beginning of life itself and even the suggestion of a fourth domain of life (see the two previous posts for more details). It has been over 10 years since the first of these giant viruses, Mimivirus, was characterised. Following that discovery, multiple related viruses were identified, and the Megaviridae family was defined. It was thought that these viruses were the only family of giant viruses, one (literally) huge exception amongst a plethora of viral families. That was until the discovery of Pandoravirus, a virus that was even larger than any members of the Megaviridae, and, moreover, completely different. This opened the door for there to be more families of giant viruses, the Megaviridae were no longer an exception. A third viral family has now entered the furor, sitting somewhere between the two previously defined families - this is Pithovirus.

The name derives from the Greek pithos meaning ‘large storage container,’ and was chosen for the fact that a pithos was given to Pandora (Pandora's box) - hinting towards the link with Pandoravirus. The two viruses look very similar as you can see in the images below. Additionally, the naming, either deliberately or fortuitously, hints at another feature of Pithovirus - that being its size. It is even larger than Pandoravirus (the previous giant virus record holder) measuring 1.5 micrometers in length and 0.5 micrometers wide - making it a ‘large storage container’ indeed (as viruses go anyway). To put that size in context, influenza virus is roughly spherical, with diameter of approximately 0.1 micrometers.

Pandoravirus (false colouring for display)

Pithovirus (false colouring for display)

What's even more intriguing about the size of Pithovirus is the fact the genome is comparatively tiny. Pandoravirus measures at 1 x 0.5 micrometers, and weights in with a genome of 2.8 million base pairs of double stranded DNA (the same genetic building blocks we use). However, the physically larger Pithovirus has a genome of only 610,033 base pairs - about 4.5 times smaller than Pandoravirus. Additionally, while the two viruses may look similar from the outside, their genomes are completely different. DNA is made up of bases, of which there are 4: A, T, C and G. The genome of Pithovirus is 64% AT, whereas Pandoravirus is 61% GC. Furthermore, there are only 5 proteins made by Pithovirus that have any similarity those of Pandoravirus.

It seems strange that Pithovirus has such a large shell to encase, comparatively, such little genetic material. The virus Phaeocystis globosa has a similar sized genome, but this is encased in an icosahedron of only 0.15 micrometers in diameter.

One possible explanation for the discrepancy between the viral particle size and genome size could be due to gene loss - an idea suggesting that viruses originate from independent cellular organisms that lose genes and become dependent on a host. Perhaps Pithovirus has lost genes and become parasitic, but has not evolved into a small particle. (See the Pandoravirus blog for more details about the idea of gene loss).

An alternative explanation for the size of Pithovirus could be that this feature is essential for its lifecycle. Pithovirus is a virus that infects amoeba, and indeed, it's ability to infect Acanthamoeba castellani was how it was discovered. This is something in common between all the giant viruses - they all prey on amoeba. Amoeba are single celled organisms that obtain nutrients (eat) through a process known as phagocytosis - the same process our macrophages use to engulf foreign organisms and protect us from infection. Could it be that these viruses need to be giant to ensure that amoeba confuse them for nutrients and phagocytose them? Without being phagocytosed, the virus would not be able to enter the amoeba and could not replicate. Viruses are incredibly adept at subverting essential mechanisms of a cell to their own end, so perhaps the sheer size of these viruses is another example of this. It will be interesting to see, as more giant viruses are discovered, whether any can infect organisms that do not rely so heavily on phagocytosis. 

Not only is the size of Pithovirus fascinating, but also how it was discovered. The team, working from the labs of Jean-Michel Claverie and Chantal Abergel, obtained samples of Siberian permafrost dating back approximately 30,000 years. Using these permafrost samples the teams then inoculated cultures of Acanthamoeba castellani and found that Pithovirus was able to grow from these cultures. What is truly remarkable about this is that Pithovirus had been frozen in Siberian permafrost for over 30,000 years and could be thawed out and become infectious. This makes it the oldest infectious organism ever discovered.

The discovery of Pithovirus in 30,000-year-old permafrost does beg the question about what else might be lurking in sediments hidden away in the Siberian ice. Climate change in the Russian Arctic is more evident than many other parts of the world, having seen a 3°C temperature increase in the last 100 years, compared to a global average increase of 0. 7°C. As the ice thaws, it may be possible that other ancient infectious organisms could be released. While this study does not say that this will happen for sure, it makes it clear that surveying permafrost regions may be an important scientific expedition, not just to detect anything dangerous, but also to potentially uncover new and exciting organisms.

The discovery of Pithovirus is fascinating as it adds yet another layer of complexity to our understanding of the giant viruses. It seems to sit somewhere in between the two previously describe families, looking like Pandoravirus, but being genetically more closely related to members of the Megaviridae. It was a pithos that Pandora received from the Gods containing all the worlds’ evils. While Pithovirus is not going to bring any evils to anything other than amoeba, the fact that a virus can be preserved for that long in Siberian permafrost does come with a warning that more exploration may be important. And not only important, but also exciting, who knows what we may find!