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How do we know that Viruses exist?

The SARS-CoV-2 virus has a huge negative impact on our lives, a strong contrast to the incredible small size of the virus. This elusiveness poses major challenges to our understanding and ability to fight it. In times of fake news and politicians who are abusing the pandemic for their own agenda by claiming that the virus is a hoax, there is a valid question to ask: ‘How can we be sure that the virus exists if they are even too small to be seen under the microscope?’

This question describes a scientific problem that can be traced back to the 19th century, when the true nature of viruses was a mystery even to microbiologists and doctors.

Here, we will go on a hunt for the proof of virus’s virulent existence. We will discuss the two main indications scientists look out for in matters like this, and how the scientific community solved this mystery during the past two centuries:

Indication 1: Evidence by Causality

On the first step of our hunt let us have a look at the subject: Viruses! One infamous virus is the one that is responsible for rabies. Rabies has 100% lethality after the first symptoms occur. These symptoms include fever, intense pain and a digestive tract that goes haywire. Later symptoms include hyperactivity as well as hydrophobia. This last symptom leads to the ill person developing an aversion to swallow their own saliva and feeling uncomfortable around any type of water. A terrible disease that made doctors and scientists desperately searching for the cause. But even long after bacteria had been found and identified as the origin of certain diseases (culprits for diseases are also called pathogens), no such bacteria that could be held responsible for causing rabies were ever found.

Thus the scientists of the time were confronted with a question: What exactly was responsible for diseases like rabies? Was the pathogen comparable to bacteria, or just so very alien in nature that it would not compare to anything known until then? Was it even physical in nature?

How do we know that Viruses exist? 1
Luis Pasteur. Image by Ernest Board licensed under CC BY 4.0.

One of the persons who came one step closer to the answer of these questions was the French microbiologist Luis Pasteur, who lived in the 19th century. Previous successes with anthrax and fowl cholera established him as an icon in medicine and microbiology. But his meticulous search for the pathogen responsible for rabies was fated to be unsuccesful from day one, since none of the responsible pathogens could ever have been seen with the microscopy techniques of the time.

Given the disease’s severe nature and lethality, Pasteur tried to develop a vaccine against rabies, despite the fact that there was no hint on a pathogen’s existence except the symptoms it caused. Drawing on his experience with bacterial pathogens he eventually succeeded to develop an effective vaccine. He circumvented the need to isolate the pathogen by using infected nerve tissue to pass the disease to different mammal species like rabbits and dogs, reducing its virulence with every cycle. Just a few years later after succesfully testing it on dogs - in 1885 - the vaccine safed the life of the 9-year-old French boy Joseph Meister, making him the first person ever to be immune to rabies.

How do we know that Viruses exist? 2
Dogs, loyal companions to us humans since the stone age. And apparently not only helping us with hunting and cuddling… Image by Pixabay.

So, even when the true nature of the rabies pathogen remained a mystery, Pasteur was still able to prevent it from infecting people (and dogs), just by carefully observing the matter at hand, and learning from the way bacteria work. The fact that this technique worked implied that this pathogen was interacting with our bodies in a similar way to bacteria, proving that it must be physical in nature and hence we must be able to find it.

The development of the Chamberland filter, by Charles Chamberland, one of Pasteur’s assistants at the time, narrowed the physical properties of the pathogen down further. The filter basically consists of a porcelain tube with an opening on one side through which a liquid can be filtered, provided sufficient pressure.

This filter was shown to separate observable microorganisms from the liquid, including pathogenic bacteria. In 1892, Russian biologist Dimitry Iwanowski, who was investigating the mosaic disease of certain plants, filtered juice from infected leaves with the Chamberland filter and documented that the extracted juice was still infectious to healthy tobacco plants. He also observed that, unlike disease-inducing bacteria which would lose their ability to survive after a short time without nutrients, the filtrate remained infectious for many months after the filtration process. This not only added further evidence to the true nature of viruses but incidentally foreshadowed one of viruses' most annoying traits: The ability to survive without any kind of metabolism, akin to plants, animals or bacteria.

How do we know that Viruses exist? 3
Components of a Pasteur-Chamberland filter, as displayed in the Science History Institute. The white cylinder is a porcelain tube with one opening for incoming water on the top. Tightly anchored and sealed in the larger cylinder on the left, it filters the water if the incoming pressure is high enough. The filtrate leaves the setup on the other side. Image by Science history Institute licensed under CC BY-SA 3.0.

This experiment was repeated with many other diseases in the following years, including rabies. Dutch microbiologist Martinus Beijerinck first introduced the word “virus” for the filtered infectious liquid in 1898 (From Latin virus: "poison, slime, venom"). And although scientist were still uncertain if viruses were liquid or composed of particles, these experiments demonstrated that the responsible pathogens must be soluble in water and small enough to pass the Chamberland filter. This is a prime example of how scientists can demonstrate the existence of something from its effects on its environment under controlled conditions, which may even permit educated guesses about its nature.

But as they say: “Seeing is Believing”, (not "Educated Guessing is Believing") so let’s proceed our hunt for specifically visible proof with:

Indication 2: Evidence by Visualization

In the years after the discovery of the infectious liquids called “viruses” microscopy techniques improved more and more, to the point where clumps of multiple virus particles could be shown. Some scientists supposedly saw these clumped viruses without knowing it to be viruses; but the resolution and magnification abilities of light microscopes were nowhere near high enough to determine any kind of structure. There is a physical limit to the magnification a microscope can achieve. To discuss this limit, we must digress a bit and take a closer look on how waves work, and how light and matter interact:

Light can be seen as a wave. Reflection, diffraction and refraction alongside things like absorption and emission are the main interaction mechanisms between this wave and matter and they allow us to see. However, all these mechanisms depend on some level on the compared size between the waves legth and the object. Smaller objects would just be “ignored” by the wave. Since waves of any kind share some of their basic principles, a comparison with water waves can come in handy to visualize this property: Imagine boulders disrupting the waves on a beach. One could deduce the boulder's existence and make assumptions on their size by the way the waves move past these boulders and arrive on the beach. Now imagine a 20 m high super wave, which would just sweep over them and not show any sign of their existence. Similarly, electromagnetic waves ignore obstacles that are much smaller than the wavelength.​*​ This is the reason why viruses are being “ignored” by the light waves used in light microscopy. To be precise: The spectrum of visible light (which is most commonly used in light microscopy) covers wavelengths from roughly 700 nm down to ca. 400 nm. Considering the just discussed dependence of light-matter interaction on size and the way a microscope guides its light, adding the application of some math, we find the highest theoretical resolution in light microscopy to be calculated around 250 nm. Even today, however, a great expense of resources and effort must be spent to come close to this level of resolution. Most viruses are much smaller. The SARS-CoV-2 virus measures in at roughly 120 nm diameter, less than half the theoretical limit!

Now, since light microscopy cannot let us see viruses, what other way might there be to make them visible to us, on our hunt for the visible proof of viruses existence?

Enter the Electron Microscope:

How do we know that Viruses exist? 4
First electron microscope with resolving power higher than that of a light microscope. By Ernst Ruska, 1980. Currently displayed in the “Deutsches Museum” in Munich. Image by J. Brew licensed under DFDL.

This revolutionary device was first built by German scientists Max Knoll and Ernst Ruska in 1931. This was shortly after French physicists Lois de Broglie’s hypothesis that particles can also behave like waves went ‘viral’ in 1923. The electron microscope takes this concept and circumvents the electromagnetic wavelength problem by waving electromagnetic waves goodbye and using the wave behaviour of accelerated electrons instead. Here, the lenses which focus the light were replaced with magnetic lenses in order to focus electrons (which are charged negatively). These coil arrays produce magnetic fields that bend the pathway of the electrons. After having passed the sample, and another magnetic lens, the electrons are detected by a fluorescent screen, which translates the signals of the incoming electrons into signals we can see. Since one could theoretically accelerate the electrons to velocities very close to the speed of light - thereby shortening their wavelength more and more - the theoretical resolution limit for electron microscopes is basically unlimited.

However, relativistic effects, exponentially growing energy costs for acceleration close to the speed of light, and technical aspects of magnetic lenses limit the practical resolution limit of modern electron microscopes to some still mind blowing 0.1 - 0.2 nm.

The electron microscope opened doors to deeper and smaller levels of the microcosmos for the scientific community, and as fate would have it, the brother of Ernst Ruska, Helmut Ruska, happened to be a physician and biologist. Very soon, he realized his brother’s inventions potential for microbiology, and together with their colleague Bodo von Borries they published the first microscopic picture of a virus in 1938.

How do we know that Viruses exist? 5
EM picture of the Ectromeliavirus, a virus that causes Mousepox in mice. It was discovered in 1930. This picture was taken and published in 1938 as the first micrograph of a virus. Image from "Klinische Wochenschrift 17.27 (1938): 921-925.".

What followed was a golden age for virology as the electron microscope revealed the true nature of different viruses, allowing scientists to understand how viruses operated and how to classify them.

And the new Coronavirus?

Fast forward to 2020: electron microscopy developers were not sleeping. In the 80 years that have passed since the brothers Ruska, many new methods have been developed and existing techniques were refined. And so, after the emergence of a new Coronavirus in the beginning of 2020, it was just a matter of time until EM pictures of SARS-CoV-2 were published. With this we can both see and believe SARS-CoV-2 exists!

How do we know that Viruses exist? 6
Transmission electron micrograph of a SARS-CoV-2 virus particle. Image by NIAID-RML.

Further reading and sources:

Von Borries, B., E. Ruska, and H. Ruska. "Bakterien und Virus in übermikroskopischer Aufnahme." Klinische Wochenschrift 17.27 (1938): 921-925.

Ackermann, Hans-W., and Hans-W. Ackermann. "The first phage electron micrographs." Bacteriophage 1.4 (2011): 225-227.

Knoll, Max, and Ernst Ruska. "Das elektronenmikroskop." Zeitschrift für physik 78.5-6 (1932): 318-339.

Kruger, D. H., Peter Schneck, and H. R. Gelderblom. "Helmut Ruska and the visualisation of viruses." The Lancet 355.9216 (2000): 1713-1717.

Horzinek, Marian C. "The birth of virology." Antonie van Leeuwenhoek 71.1-2 (1997): 15-20.


  1. ​*​
    Note that this analogy is not 100% accurate, but for our case this is sufficient.

Author:

Erik Nebelung

Nanoscience M.Sc. Student @ Institute for Nanostructure and Solid-State Physics, Hamburg University
Erik is studying nanoscience with a focus on biochemical methods and applications. From August 2020 till January 2021 he pursued his studies at the iNano institute in Aarhus, before starting his master's thesis back in Hamburg. He had his first taste of protein crystallization during his bachelor's thesis work and this sparked his interest in […]

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