The Zombie Virus

The frozen ground

Often confused with ice, permafrost is permanently frozen ground (with temperatures hovering around -10 °C) in which all metabolic activity is suspended due to a lack of water. These cold environments with neutral pH and sheltered from light and oxygen are perfect for housing microorganisms buried deep within the permafrost. With a hardness comparable to concrete, some water-rich permafrosts, such as Yedoma, exhibit high stratigraphical stability for easy and reliable dating. 

Studying microbes in permafrost has been a long research tradition in Russia, with the first viable microorganism finding dating back to 1911 (1). Between the 1950s and present day, a veritable discipline has developed from realization of global warming consequences on microbial flora. After several controversial studies with possible contamination by modern microbes, there’s now a consensus that bacteria can survive permafrost samples up to half a million years in age (2) – a timeline that lies closely with the emergence of the Homo sapien species.

From plants to viruses

My team’s interest in permafrost was sparked by a study undertaken by David Gilinchinsky’s group to resuscitate a small flowering plant (Silene stenophylla) whose tissues had been frozen for more than 30,000 years (3). This research suggested that alongside bacteria, multicellular organisms could also remain alive once trapped in ancient permafrost. This finding has since extended to animals with a nematode species revival after 46,000 years of cryptobiosis in Siberian permafrost (4). 

After reading this study, I was sure that, if a plant could be revived, then a virus would surely survive in permafrost. I contacted the team behind the research and they were happy to provide me with a few grams of their permafrost sample, and our project took off rapidly. 

A key part of our research involved isolating virus particles, which may seem risky when you take into account that these viruses were unknown at the time of study. However, this risk can be strictly controlled thanks to the ways in which viruses replicate. To multiply, virus particles must infect a cellular host. We used this fundamental property to detect and amplify particles that remained infectious in a given sample by providing “bait sample cells” evolutionarily distant from us. Offering this alternative cell type to infect (specifically an Amoeba that diverged from Homo sapiens a billion years ago), ensured our safety throughout without needing to conduct research in a high security BSL4 laboratory. 

Our first attempts at reviving the Amoeba-infecting virus was quickly successful. Within 12 months, we could identify and describe two different viruses from the 30,000 year old sample provided by our Russian collaborators. Moreover, these first zombie viruses (called Pithovirus and Mollivirus (5,6)) were representatives of two new viral families never seen before – supporting us in dispelling a putative laboratory contamination. 

In retrospect, our research protocol was quite effective. The process involved placing a small amount of permafrost onto a laboratory culture of Amoebae (genus Acanthamoeba). Soil samples are typically full of bacteria, which would usually multiply rapidly and overwhelm the culture medium. However, amoebae feed on bacteria, and with some antibiotic help, they can clean their own culture. Another advantage is that most viruses infecting Acanthamoeba produce large particles visible under a light microscope. This makes it easy to detect when a virus is multiplying in the amoeba cultures and rule out cultures dying from other causes, such as fungal infections.

Additionally, Acanthamoeba is found in various soils and aquatic environments, making them an ideal probe and positive control. If no amoeba-infecting viruses are found, there are likely no other infectious eukaryotic viruses present either. Finally, Acanthamoeba cells are easy to cultivate in a simple medium and are only mild human pathogens, posing a minimal risk. This allows virology experiments to be performed on a BSL2 level bench, making the setup cheap and straightforward.

At the same time, the viruses infecting amoebae are very similar to those infecting animals and humans – using similar replication mechanisms as pathogens like asfarviruses (which cause lethal swine fever) and poxviruses (which cause smallpox). This suggests that these pathogens could also retain infectivity for thousands of years in Siberian permafrost. Therefore, our studies on amoeba viruses can be used as a risk-free approach to estimate the potential for other viruses, including harmful human pathogens, to be resuscitated after permafrost thaws. 

Introducing metagenomics

But what about the presence of other viruses in ancient permafrost? Thanks to advances in sequencing technology, it’s now possible to analyze all living organism DNA present in a sample. The result is a mixture of several billion fragments of genomic sequences (including viruses, bacteria, plants, and animals) that modern bioinformatic techniques sort and identify through comparison with gigantic databases. This metagenomic approach (7) allows for identification of viruses in permafrost without the risk of reactivation. This means that if zombie viruses (such as those capable of causing smallpox) are present in ancient permafrost and remain infectious, they cannot escape from the laboratory. 

Our follow-up study conducted last year (8) highlighted two points. Firstly, finding infectious viruses trapped in prehistoric permafrost is a common occurrence, and secondly, the general time limit for age of these viruses is 48,500 years. However, this limit isn’t set in stone – it simply corresponds with technical limitations of radiocarbon dating. Attempts are currently taking place to revive viruses that originate from a time before Homo sapiens, which could expose us to viruses that we know nothing about and our immune systems have never come into contact with.

Climate change consequences

Permafrost is greatly impacted by the weather, meaning that global warming plays a large part in how concerned we should be about the release of zombie viruses. During the Siberian summer, when outside temperatures can exceed 30 °C, huge quantities of viral particles are defrosted each day and dumped into local rivers. So far we’re unable to estimate how long viruses released from prehistoric permafrost can remain infectious when exposed to UV light, oxygen, and heat. However, it’s clear that the associated risk is bound to increase alongside global warming due to an acceleration of thawing permafrost and increased population in the Arctic in the wake of industrial ventures.

Preventing a zombie virus pandemic

The probability of a pandemic triggered by a completely new virus is extremely low. However, if a virus of this nature spreads through the human population, it would have drastic consequences on a far greater scale than the COVID-19 pandemic. So is it possible to prevent a zombie virus pandemic? Our team proposes focusing surveillance on sick patients who are frequently exposed to environmental hazards (miners) or zoonotic risks (animal handlers). This way, we only need to study new viruses that have already infected humans or jumped from animals to humans. It would also reduce the data size and help detect emerging viruses early, before they can spread from person to person.

With this approach in mind, I’ve proposed the creation of a circumpolar surveillance network under the auspices of UARCTIC – a large international consortium of universities with a long-standing interest in Arctic research and a unique network of relationships with indigenous populations. This network will take form in a community-based scientific project, giving locals the ability to report suspected diseases and isolate atypical patients before infectious disease specialists arrive on site to assess the situation. Only time will tell if this approach is effective, but one thing is for certain: surveillance will play a central role in managing and preventing ancient viruses from re-emerging.