Occam’s Razor advises us not to accept the theory that diseases like influenza, the common cold, and COVID-19 are caused by pathogenic viruses until, at the very least, the existence of such pathogens has been established beyond all reasonable doubt. But has this ever been done for any pathogenic virus? That is what we will be looking at in the next few articles, beginning with a brief look at the early history of the science of virology.
Etymology
The word virus was borrowed into English from the Latin language, where its meaning had little if anything to do with biology:
- Latin: vīrus, slimy liquid, slime : poison : offensive odour or taste
In English, the word’s original meaning introduced an unmistakably biological association:
- Oxford English Dictionary (OED): virus, venom, such as is emitted by a poisonous animal (Craigie et al 243)
The earliest appearance of the word according to the OED was in 1599, when the theologian Hugh Broughton used it in Master Broughton’s Letters:
Wherein you have spent all the vires and power you have for the defence of a vain paradox, and spit out all the virus and poison you could conceive, in the abuse of his reverend person. (Broughton 14)
Other uses of the word with the same meaning cited by the OED refer to the poison of Cleopatra’s asp, the poison of the viper, and the sting of a bee (Craigie et al 243).
The use of the word as a technical term in the science and practice of pathology dates from the early 18th century:
- Pathology: virus, A morbid principle or poisonous substance produced in the body as the result of some disease, esp. one capable of being introduced into other persons or animals by inoculation or otherwise and of developing the same disease in them. (Craigie et al 243)
This usage was introduced by Ephraim Chambers in his Cyclopædia of 1728:
virulent, a Term apply’d to any thing that yields a Virus; that is, a corrosive or contagious Pus. (Chambers 312)
Edward Jenner’s theory of vaccination popularized this use of the term, especially when referring to the pus found in the blisters or pustules of a patient suffering from smallpox or cowpox:
Morbid matter of various kinds, when absorbed into the system, may produce effects in some degree similar; but what renders the Cow-pox virus so extremely singular, is, that the person who has been thus affected is for ever after secure from the infection of the Small Pox; neither exposure to the variolous effluvia, nor the insertion of the matter into the skin, producing this distemper. (Jenner 6)
For Jenner, the pus from which he made his smallpox vaccine simply consisted of morbid matter of various kinds. Beyond this he was not willing to speculate.
In 1928, when the first edition of Volume 10, Part 2, of the OED appeared, the term virus meant no more than this. None of the definitions or citations in that volume refer to anything more than poisonous matter that either causes disease or is itself a by-product of disease. The actual agent that made such pus pathogenic was still unknown, though various hypotheses had been put forward by the end of the 19th century. For example, several pathologists had surmised that smallpox was actually caused by a protozoan, or single-celled organism:
Guarnieri ... L Pfeiffer ... E Pfeiffer ... and others ... consider the small, easily stained bodies, surrounded by a clear zone, which are found in the epithelium in the early stages of variola [smallpox virus] and vaccinia [cowpox virus], to be protozoa. Guarnieri has designated the supposed parasite as Cytoryctes vaccina. The parasitic nature of these bodies has not yet been demonstrated. After Salmon ... had spoken against such a view, A. Hückel [Armand Hückel, father of Erich] ... proved, through exact and carefully conducted investigations, that in vaccinia at the point of inoculation into the cornea certain portions of the epithelial cells undergo especial disease changes, and that from their protoplasm there arise those peculiar structures which have been mistaken for parasites. (Ziegler 690)
Giuseppe Guarnieri’s hypothetical pathogens Cytoryctes vaccina [cell-destroyers of smallpox] are now identified as Guarnieri Bodies, aggregate masses of protein allegedly produced by viruses in the nuclei of infected cells—in other words, products of the so-called cytopathic effect.
Germ Theory
The word virus ultimately acquired its modern acceptation as a result of the germ theory of disease, which had been widely embraced by the scientific community by the end of the 19th century. This theory has its roots, however, in the ancient world. The concept of contagion—the spread of a disease from person to person—can be found in the Torah and the ancient Indian text known as the Sushruta Samhita, both of which may date back to the middle of the first millennium BCE, if not earlier These early theories, however, had little to say about the nature of the pathogenic agent that was supposedly being transmitted from person to person.
The first to attempt to identify these vectors was, perhaps, the Roman poet and philosopher Titus Lucretius Carus, who suggested in Book 6 of his didactic poem De Rerum Natura [The Nature of Things] that contagious diseases were spread by certain seeds (semina), which could enter a person’s body through inhalation or ingestion.
A few decades later, another Roman writer, the prolific polymath Marcus Terentius Varro, ascribed some contagious diseases to animals too small to be seen with the naked eye. In Book 1, Chapter 12, of his De Re Rustica [On Agriculture] we read:
Note also if there be any swampy ground, both for the reasons given above, and because certain minute animals, invisible to the eye, breed there, and, borne by the air, reach the inside of the body by way of the mouth and nose, and cause diseases which are difficult to be rid of. (Storr-Best 39)
Similar ideas to both of these can be found throughout the Middle Ages, alongside the so-called miasma theory, which attributed contagious plagues to noxious air—whatever that means. It was only in the middle of the 17th century, however, that German polymath Athanasius Kircher first demonstrated the existence of living organisms too small to be seen with the naked eye. In his Scrutinium Physico-Medicum Contagiosae Luis, Quae Pestis Dicitur [A Physico-Medical Examination of the Contagious Pestilence Called the Plague] of 1658 Kircher proposed that bubonic plague was caused by a microscopic organism. Kircher’s discoveries were replicated twenty-seven years later by the Dutch microscopist Anton van Leeuvenhoek.
By the beginning of the 18th century, some physicians were attributing many other diseases, including smallpox, to such micro-organisms. Others, however, restricted this new theory to diseases associated with marshes and other sources of noxious air. It was believed that by extending the theory to diseases in general, one was only bringing it into ridicule.
The germ theory gradually came to be accepted by the wider medical community in the course of the 19th century. An important step in this process was taken in 1835 by the Italian entomologist Agostino Bassi, who demonstrated that muscardine, a silkworm disease that was ravaging the French silk industry at the time, was caused by a fungal mould now known as Beauveria bassiana.
In 1854 the English physician John Snow curtailed an outbreak of cholera in London by applying the principals of his 1849 paper On the Mode of Communication of Cholera. Snow believed that cholera was caused by a unicellular organism too small to be seen with the microscope, which could reproduce within the body of an infected person (Snow 15). Today cholera is attributed to various strains of bacteria.
The final victory of the germ theory over its rivals was largely due to the work of French chemist Louis Pasteur. In the 1850s and ’60s Pasteur investigated the causes of souring in beverages such as wine and milk. His experiments led him to conclude that micro-organisms in these beverages were feeding on the sugars in them and producing lactic acid as a by-product, which soured the taste. These results not only led to the introduction of pasteurization—the heating of beverages to kill these micro-organisms—but also inspired Pasteur with the idea that infectious micro-organisms could cause diseases. Over a period of several decades he investigated the causes of disease, and became a champion of vaccination as an effective means of protecting susceptible populations from such diseases as rabies, anthrax, and chicken cholera.
Meanwhile in Germany, the physician Robert Koch was also promoting the germ theory of disease. His name is now inextricably linked to four fundamental criteria—Koch’s Postulates—for demonstrating in a scientifically sound manner that a particular disease is caused by a particular pathogenic organism. This is a curious state of affairs, as Koch himself never actually formulated any such postulates or considered them binding on his research. They were deduced from what Koch wrote in two papers on the cause of tuberculosis in 1882 and 1884. Thomas Brock’s Milestones in Microbiology (1962) has English translations of the relevant passages from both papers:
Koch and other epidemiologists were happy to relax these requirements if they contradicted the germ theory. For example, Koch came to believe that cholera and typhoid fever could be spread by asymptomatic carriers of their respective pathogens, though the very idea of an asymptomatic carrier violates the First Postulate.
But the commonly held belief that Koch ever considered himself bound by such principles in the first place may have to be revisited:
It would seem that the uncritical enthusiasm of later commentators has saddled Koch with expressions that he did not intend. Koch’s postulates as ordinarily interpreted are not logical or cogent, and there is no adequate evidence that Koch so intended them. The determination of etiology is a considerably more complex subject than is indicated by the postulates. Perhaps textbooks might dispense with consideration of the postulates and substitute therefor a discussion of the canons of induction and the validity of inference, that is, an analysis of what we mean by “cause.” (King 361)
Contagium vivum fluidum
In the course of the 18th and 19th centuries, advances in microscopy led to the discovery of numerous varieties of micro-organisms which were believed to be the infectious agents of different diseases. These animalcules fell into three main groups of unicellular organisms:
- Fungi and fungal spores, including yeast (2 to 50 μm in size)
- Bacteria (0.5-5 μm)
- Protozoa (1 μm to several cm)
Mycology, the systematic study of fungi, began with the publication of the Italian botanist Pier Antonio Micheli’s Nova plantarum genera [New Types of Plants] in 1729. Bacteria were first observed by Anton van Leeuwenhoek in 1676, but the systematic science of bacteriology dates from the work of Ferdinand Cohn in the middle of the 19th century. Anton van Leeuwenhoek was also the first person to observe Protozoa, but the classification and systematic study of these unicellular animals dates from 1818, when the German zoologist Georg Goldfuss coined the term.
According to the germ theory of disease, each disease was a contagium vivum—living contagion—because it was spread by a living organism, such as a bacterium or a protozoan. In the 19th century what we now call the Germ Theory of Disease was generally referred to as the Doctrine of Contagium Vivum (Roberts 307 ff).
Proponents of this germ theory, however, soon discovered that there were some diseases for which no such pathogen could be found. The key figure in this discovery was the Dutch botanist Martinus Willem Beijerinck. In 1898 he published the results of his research into the plant disease known as the tobacco mosaic disease. Twelve years earlier the German agricultural chemist Adolf Mayer had first described and named this disease of the tobacco plant, and had discovered that, like bacterial infections, it could be transferred from plant to plant. Beijerinck, however, found that the fluid from a tobacco plant suffering from the disease could still infect healthy plants after being passed through the finest filter then available—Chamberland’s porcelain filter, developed in 1884 by a French colleague of Pasteur’s, Charles Chamberland. Beijerinck concluded that the pathogen which caused the tobacco mosaic disease was even smaller than bacteria, which were typically a few micrometres across (1 μm = 10–6 m).
In 1892 the Russian botanist Dmitri Iosifovich Ivanovsky had made a similar discovery, but he suspected that either his filters were faulty or the infectious agent was a chemical toxin produced by bacteria, and did not follow up his discovery (Wilson & Topley 3). Beijerinck referred to his filterable infectious fluid as a contagium vivum fluidum, Latin for contagious living fluid. Some scientists thought that the pathogenic agent must be a soluble chemical toxin, but Beijerinck and others noted that the contagium vivum fluidum of an infected plant could be transferred successively from healthy plant to healthy plant without any apparent diminution in its virulence. This seemed to imply that the pathogenic agent was replicating within each infected plant, which in turn implied that the agent was a living organism. It is to Beijerinck that we owe the belief that viruses cannot replicate on their own: they can only replicate inside living tissue. Nevertheless, Beijerinck’s terminology reflects his belief that the pathogenic agent was of a liquid nature, which allowed it to pass through even the finest filters.
In the same year as Beijerinck carried out his studies into the transmission of tobacco mosaic disease, two other researchers, Friedrich Loeffler & Paul Frosch, came to similar conclusions with regard to foot-and-mouth disease.
For their work, Ivanovsky and Beijerinck are now recognized as the co-discoverers of viruses, even though neither actually observed any such organisms. Ivanovsky never hypothesized their existence, while Beijerinck conceived of the pathogen as a living fluid—hardly a virus as we understand the term today. In 1900, in fact, in a paper on the subject, he wrote of:
Autres maladies infectieuses des plantes, provoquées par un principe contagieux fluide, et non par des parasites.
[Other infectious diseases of plants, caused by a fluid contagious principle and not by parasites.] (Beijerinck 308)
Curiously, what are believed to be viruses had already been observed by the Scottish bacteriologist John Brown Buist. In 1886 Buist developed a method of staining and fixing the infectious fluid from a pustule of an animal suffering from cowpox. Upon observing the resulting slide, Buist noticed tiny bodies, which he identified as fungal spores. Today, these are identified as virions of the cowpox virus, vaccinia, which is said to be one of the largest viruses in existence—about 360 nm long, which is close to the limit of the resolving power of the best optical microscopes in 1886. Buist, who used crystal violet to stain the bodies, estimated their length at 100–500 nm (Wilson & Topley 3).
It was around the beginning of the 20th century that epidemiologists and microbiologists began to use the term virus to refer to the hypothetical agent in Beijerinck’s contagium vivum fluidum. Curiously, the Eleventh Edition of _The Encyclopædia Britannica_does not include any articles for Virus or Virology. It was published in 1911, when the existence of these organisms was still unproven. Almost a quarter of a century would pass before the word virology was coined.
Tissue Culture
The current practice of culturing viruses in the laboratory using tissue cultures was developed in the first decade of the 20th century—decades before the existence of viruses had been confirmed. The pioneer in this field of research was the American biologist Ross Granville Harrison. In 1906-07, Harrison successfully grew embryonic nerve cells from a frog in vitro, using a medium of clotted lymph to nurture the cells.
In 1913, three microbiologists, Edna Steinhardt, C Israeli, and Robert A Lambert, adapted Harrison’s techniques in an attempt to culture viruses. They embedded fragments of corneal tissue from guinea pigs and rabbits in clotted plasma to create a nurturing medium for the cowpox virus, vaccinia. Although they could not detect any vaccine bodies (ie virions of the hypothetical vaccinia virus) in any of their incubated samples, they did observe an increase in infectivity when they applied the samples to the shaven skin of test rabbits (Steinhardt et al 297-298). From this they concluded that whatever the pathogenic agent was, it was replicating in the tissue culture. Control samples, on the other hand, in which the virus was incubated in paraffin instead of tissue, showed a decrease in infectivity after one week and a loss of infectivity after three weeks. (Steinhardt, Israeli & Lambert 299-300).
The success of these experiments ensured that the in vitro culturing of viruses in tissue cultures would eventually become recognized as the gold standard for virus isolation. It must be remembered that these experiments were carried out when the existence of viruses was still hypothetical and their nature, if they did exist, a complete mystery. It should also be noted that the results of similar experiments with the rabies virus were not as clear cut:
The three viruses studied thus far with the new method have been those of rabies, vaccinia, and syphilis. In the study of the former we attempted, first, to determine whether the Negri bodies, the nature of which is still in dispute, would show any development or multiplication in vitro when fragments of brain from rabid animals were incubated in blood plasma, and, second, to see if the characteristic bodies could be produced in vitro by combining normal brain and virus. we found that no development or multiplication took place in vitro, and that, altho structures indistinguishable from certain forms of Negri bodies developed in the virus-normal brain preparations, the same bodies were found in control preparations in which were pieces of normal brain without virus. In a single instance the virus remained alive after 8 days’ incubation; animal inoculations of preparations incubated for a longer time gave in all case negative results. (Steinhardt & Lambert 87)
The results of these rabies experiments seem to contradict those of the vaccinia experiments.
Electron Microscopy
In the spectrum of electromagnetic radiation, visible light comprises wavelengths of approximately 400–900 nm. Objects smaller than this cannot be resolved by visible light. If the hypothetical viruses of Beijerinck’s contagium vivum fluidum were smaller than this, an instrument with greater resolving power would be needed to image them.
In 1924, the French physicist Louis de Broglie pointed the way to such an instrument when he proposed his theory of Wave-Particle Duality. According to this theory, wavelike phenomena such as light also exhibit particle-like properties, and particle-like phenomena such as electrons and atoms also exhibit wavelike properties. In other words, the electron is both a particle and a wave. This opened the door to the possibility of replacing visible light with cathode rays (ie electrons as waves), which could be generated with wavelengths much smaller than those of visible light. Because electrons are electrically charged, the cathode rays are focused using electromagnetic fields rather than glass lenses.
The electron microscope was invented in 1931 at the Berlin Technische Hochschule (Technical University of Berlin) by the physicist Ernst Ruska and the electrical engineer Max Knoll. Within a few years electron microscopes that exceeded the resolution of optical microscopes were commercially available. Two types of electron microscopes, the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM), are used to image viruses.
Helmut Ruska, brother of Ernst, developed the electron microscope for biological and medical applications. He was one of the first scientists to study viruses with the electron microscope:
Until 1939, next to nothing was known about the structure of viruses because of the constraints imposed by the limited resolving power of light, even of short wavelengths in the ultraviolet range. In that year Kausche, Pfannkuch and Ruska visualized a virus with the newly invented electron microscope (EM), in which beams of electrons travelling in a vacuum are focused on the object with electromagnetic fields. Given the then current interest in crystalline tobacco mosaic virus (TMV) ... it is not surprising that this was the first virus to be examined. Kausche and his colleagues employed the technique of shadow-casting, whereby objects on the specimen grid were coated with gold particles generated by heating the metal in a vacuum; they defined the size of the crystals as about 25 x 300 nm. (Topley & Wilson 5)
After the Second World War, new techniques were developed to exploit the electron microscope’s superior resolving power. Before an object can be imaged in an electron microscope using these methods, however, extensive processing and preparation may be required. One of the most widely used techniques of imaging viruses is called negative staining, in which the background—not the virus—is stained with an electron-dense salt of tungsten, uranium, molybdenum, ruthenium, osmium, and other suitable elements. This increases the contrast between the viruses and the background, allowing the former to be distinguished:
Negative stain—suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably coated EM grid, blotted, then allowed to dry. Viewing of this preparation in the TEM should be carried out without delay for best results. The method is important in microbiology for fast but crude morphological identification, but can also be used as the basis for high-resolution 3D reconstruction using EM tomography methodology when carbon films are used for support. (Wikipedia)
This technique of negative staining was only developed in the late 1950s (Brenner & Horne 1959). Positive staining, in which the virus particles are coated with the stain, is not as useful, as fine detail is obscured.
In the 1940s, it was discovered that virus particles could by crystallized, allowing their structures to be examined in detail using the technique of X-Ray Diffraction. The tobacco mosaic virus was the first to be examined using this technique. The first X-ray diffraction images of the crystallised virus were obtained by Bernal and Fankuchen in 1941. Based on her X-ray crystallographic pictures, Rosalind Franklin is alleged to have discovered the full structure of the TMV in 1955.
Detecting Viruses
Four principal methods are used today to detect the presence of a virus:
Electron Microscopy The virus is imaged directly using an electron microscope.
Cytopathic Effect The virus is grown in a cell culture and the cytopathic effect which the virus has on the host cells is observed.
Serology The presence of a virus in an infected organism is detected by monitoring the production of antibodies as a reaction to the presence of the virus (ie antigen).
PCR The presence of a virus is detected by replicating a gene unique to the the virus’s genome.
We have already looked at PCR and seen that the discovery of the virus and the sequencing of its genome must have already taken place before a suitable PCR test can be developed. This method, therefore, cannot be cited as a method of proving that a virus exists in the first place. But what of the other three methods? Do they prove the existence of pathogenic viruses?
In the next article, we will review the history of electron microscopy and its application to the science of virology.
And that’s a good place to stop.
References
- Martinus Beijerinck, De l’existence d’un principe contagieux vivant fluide, agent de la nielle des feuilles de tabac, Verzamelde geschriften van M. W. Beijerinck, Volume 3, Pages 296-312, Delft (1921)
- Thomas Brock (translator & editor), Milestones in Microbiology, Prentice-Hall, Inc, Englewood Cliffs, New Jersey (1962)
- Hugh Broughton, Master Broughton’s Letters, John Wolfe, London (1599)
- Ephraim Chambers, Cyclopædia: or, An Universal Dictionary of Arts and Sciences, Volume 2, James & John Knapton et al, London (1728)
- W A Craigie (editor) et al, A New English Dictionary on Historical Principles, Volume 10, Part 2, The Clarendon Press, Oxford (1928)
- Edward Jenner, An Inquiry into the Causes and Effects of the Variolæ Vaccinæ, a Disease Discovered in some of the Western Counties of England, Particularly Gloucestershire, and Known by the Name of the Cow Pox, Sampson Low, London (1798)
- Lester S King, Dr. Koch’s Postulates, Journal of the History of Medicine and Allied Sciences, Volume 7, Number 4, Pages 350-361, Oxford University Press, Oxford (1952)
- Robert Koch, Die Aetiologie der Tuberculose, Berliner Klinische Wochenschrift, Volume 19, Number 15, Pages 221–230, (1882)
- Robert Koch, Die Aetiologie der Tuberkulose, Mittheilungen aus dem Kaiserlichen Gesundheitsamte, Volume 2, Pages 1–88, (1884)
- William Roberts, The Doctrine of Contagium Vivum and its Application to Medicine, Journal of Cell Science, New Series, Volume 17, Issue 68, Pages 307-329, London (1877)
- John Snow, On the Mode of Communication of Cholera, Second Edition, John Churchill, London (1855)
- Edna Steinhardt, C Israeli, R A Lambert, Studies on the Cultivation of the Virus of Vaccinia, The Journal of Infectious Diseases, Volume 13, Issue 2, Pages 294-300, Oxford University Press, Oxford (1913)
- Edna Steinhardt & Robert A Lambert, Studies on the Cultivation of the Virus of Vaccinia. II, The Journal of Infectious Diseases, Volume 14, Number 1, Pages 87-92, Oxford University Press, Oxford (1914)
- Lloyd Storr-Best (translator), Varro on Farming, G Bell and Sons, Ltd, London (1912)
- William Whiteman Carlton Topley & Graham Selby Wilson et al, Topley & Wilson’s Microbiology and Microbial Infections, Ninth Edition, Volume 1, Virology, Arnold, London (1998)
- Ernst Ziegler, General Pathology, or The Science of the Causes, Nature, and Course of the Processes of Disease, Translated from the Tenth Revised German Edition and edited by Aldred Scott Warthin, William Wood and Company, New York (1903)
Image Credits
- COVID-19 Poster: © 2021 Dublin Region Homeless Executive, Fair Use
- The Early History of Virology: © R&D Systems, Inc, Fair Use
- Edward Jenner: Anonymous Portrait, Wellcome Library, Public Domain
- Giuseppe Guarnieri: Anonymous Photograph, Public Domain
- Titus Lucretius Carus: Anonymous Illustration of a Seal Bearing the Head of Titus Lucretius Carus, H A J Munro (translator), T Lucreti Cari: De Rerum Natura Libri Sex, George Bell and Sons, London (1898), Public Domain
- John Snow: Anonymous Autotype of an Anonymous Presentation Portrait (1856), Public Domain
- Robert Koch: Anonymous Photograph, Public Domain
- A Microscopic View of the Different Animalcules: D Dodd (artist), John Pass (engraver), J Wilkes (publisher), Public Domain
- Martinus Willem Beijerinck: Anonymous Photograph (1921), Public Domain
- Dmitri Iosifovich Ivanovsky: Anonymous Photograph, Public Domain
- Tobacco Mosaic Disease: R J Reynolds Tobacco Company (photographers), USDA Forest Service, Public Domain
- Edna Steinhardt: © Rebekah Honce (designer), Anonymous Photograph, Fair Use
- Ernst Ruska & Max Knoll’s Electron Microscope: Deutsches Museum, Munich, J Brew (photographer), Creative Commons License
- Ernst Ruska: Anonymous Photograph, Copyright Unknown, Fair Use
- Helmut Ruska: © Erdman A Ruska, Fair Use
- Alleged MERS-CoV Virions Imaged in a TEM by Negative Staining: © Cynthia Goldsmith and Azaibi Tamin (electron microscopists), CDC, Fair Use
Online Resources
