We will be beginning by looking at the history of virology. Observations had been made, and are still being made, about infections in various organisms. We will trace how these symptoms came to be attributed to viral infections. Saying that an infection is caused by a virus required the development of the concept of a virus as an infectious agent - an agent that is different than other disease-causing entities. Coming up with the concept of a virus as "different" was dependent on advances in bacteriology, genetics, biochemistry, and molecular biology. An integral part of defining a virus was demonstrating that its infectivity and the characteristics of the infection were caused by nucleic acid.
Viruses have probably been around as long as there have been single-celled organisms. People knew the effects of viruses long before they knew anything about their characteristics. This is an Egyptian bas-relief dating from 1500 BC. (Fig. 2-1). What do you think caused the priest depicted to have the withered leg? A more appealing example of the effects of a virus is shown here (Fig. 2-2). These tulips are striped ('broken', variegated) because of infection with what we now call tulip mosaic virus. Bulbs of this type became collectors items - they were originally imported to Europe from the East - and very valuable. In Holland in 1625, the following items were traded for ONE variegated bulb!4 tons wheat, 8 tons, rye, 4 fat oxen, 8 fat pigs, 2 hogsheds wine, 4 barrels beer, 2 barrels butter, 1,000 pounds of cheese, 1 full-dress suite, 1 silver goblet and 1 bed with accessories (unnamed)The disabled man and and the striped tulip are examples of infection. Other symptoms of viral infection that we as humans have experienced include lesions (as in chicken pox or herpes), fever, aches, pains, runny nose, cough, upset stomach, diarrhea, and others. Can you think of additional ones?
As you can see, mammals are not the only organisms that are infected by viruses. For example, bacteria can be infected by viruses called bacteriophage (phage = eat). Bacteriophage infection causes cells to die and fall apart, a process call lysis - a liquid culture of bacteria before infection can be turbid, while after infection it will be clear. If bacteriophage are mixed with bacteria which are then plated on agar, each infected cell will be killed and break apart (lyse). The viruses that are released from the first cell that was infected will then infect the surrounding cells and cause clearing of an area that is called a "plaque" (Fig. 2-3).
If the initial infection is set up properly, the number of infectious virus particles in the preparation of virus can be estimated.
Plants also can be infected by viruses. Many viruses are transmitted from plant to plant by insects; alternatively, plants can be infected through contaminated farm instruments. One problem with infecting plants or their cells is that the virus must get across the plant cell wall, which is much tougher than the cell˜s membrane. Both of the methods of transmission described above involve some mechanical means of broaching the cell wall. The insect will transfer the virus through its proboscis, which acts like a little syringe; the instruments will actually break the plant cell wall. The economic effects of virus infection can be devastating and plants are often bred to resist infection. A typical symptom of viral infection is the appearance of a lesion (Fig. 2-4).
Other symptoms in plants include stunting of growth and loss of chlorophyll.
In mammals, there are 'silent', non-visible responses to virus infection. The most important of these is the production of proteins called antibodies by cells of the immune system, These antibodies are found in blood serum and defend the organism against infection. Any individual antibody molecule has a particular amino acid sequence and shape that allows it to recognize that proteins of viruses (called antigens) are foreign to the body and therefore a threat to its integrity. Once these antibodies are produced by cells of the immune system, cells capable of producing the specific antibody are retained in the body. They will quickly produce the specific antibody against the viral antigen if the organisms is infected with that virus for a second time. That's why no one gets chicken pox twice.
Additionally, some viruses can hang around in an organism for a long time after the initial or "acute" infection when symptoms are seen. These are called "latent" infections. Latent viral infections may damage tissue even without gross symptoms, as in the case of the human hepatitis viruses (causing either chirrosis or liver cancer), or they may reactivate and cause visible symptoms, as with herpes virus or shingles (zoster), which is the late onset version of chicken pox (varicella) (Fig. 2-5).
Bacteriophage l can 'hide' inside cells for many generations, during which time it is not at all obvious that the cell is infected, a phenomenon called lysogeny. An environmental stimulus, typically ultraviolet light, causes the virus to become activated and lyse the cells.
This brief description of some of the characteristics of virus infection holds many hidden assumptions about the nature of viruses. The most basic is that there is a infectious agent, a virus, that has certain properties that differentiate it from other infectious agents, such as bacteria, fungi, prions, etc.
If there are two agents that can cause similar symptoms, it is necessary to be able to separate one from the other and to know something about each before an investigator can ascribe particular properties to each.
In the case of defining a virus, it was important to differentiate viruses from bacteria. Not until something was known about bacteria was it possible to say that an infection was NOT caused by a bacterium, and that it was therefore necessary to look for another 'organism'. In other words, the advances in microbiology were a necessary predecessor to the development of the science of virology. Of these advances, the most important were the concept of sterility (the absence of bacteria), the ability to grow bacteria on defined medium, and at least one of Koch's postulates (1880), the one that says that a pure culture of an organism will produce the disease when injected into in susceptible animal.
In the 1870's and 1880's. bacteriologists developed the operational concept of sterility. If liquid were forced by air pressure through a clay or porcelain filter, now called a Chamberland candle, the liquid that came through was considered to be sterile, since the bacteria were held back in the filter. As these filters became more refined, it was possible to regulate the size of their pores, so that an idea could be obtained about the dimensions of the particles that were retained. Some filters held back entities as small as 4 microns (Fig. 2-6).
The operational definition of sterilty, of course, required the ability to grow bacteria in suitable ways, so that the lack of bacterial growth using the filtrate could be monitored. Robert Koch had observed the growth of bacteria as colonies on the cut surfaces of potatoes, and by 1887, Robert Petri had developed the technique still in use today of growing bacteria on dishes containing agar. The use of agar, which had the advantage of remaining solid at 37 C, had been suggested by Fannie Hesse, the wife of an associate of Koch.
The history of virology is really based in plants, not animals. In 1886, a German investigator, Adolf Mayer, showed that the symptoms of tobacco mosaic disease (see Fig. 2-4 above) could be passed to a healthy plant. He ground up the leaf of a diseased plant with some water, and inoculated the healthy plants with a fine capillary tube. Nine of ten plants so inoculated developed the same symptoms as the original infected plant. (Koch's postulate mentioned above still is applicable, even though no one had a pure culture of a virus.)
In 1892, Dmitri Iwanovski of St. Petersburg, Russia, showed that tobacco mosaic disease could be transferred to a healthy plant using the material that came through a Chamberland candle when ground up infected leaves were put into the candle. I have found that the sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filter-candles. Iwanovski interpreted his findings as showing that the disease was caused by a toxin produced in the plants by a bacterium. There is a sophisticated argument that rules out the possibility that the agent in question is simply a toxin. Can you come up with it? Assume that a toxin is a protein molecule, and that there are 1000 molecules of toxin in plant 1. The infection is passed from plant 1 to plant 2 .... to plant n, with filtration between each passage. What do you think will happen and does this rule out Iwanowski's conjecture?
The idea of the infectivity of filtrates was boosted by the discovery, by Loeffler and Frosch in 1897, that filtrates of lesion of cattle with hoof and mouth disease could cause disease symptoms. Within the scientific community, the demonstration of the same phenomenon in more that one organism increases the probability that the observation is 'true'. Therefore, the same observation in plants and in animals boosted its believability.
In 1898, the Dutch scientist, Martinius Beijerinck (BYE-YER-RINK), repeated Iwanovsky's experiment, but came to a startlingly different conclusion. He called the material that passed through the filter 'contagium fluidum vivum' (living contagious fluid), a 'water soluble molecule able to replicate only when incorporated in the living protoplasm of the cells, into whose reproduction it is, in a manner of speaking, passively drawn.'
Beijerinck's description is remarkably prescient and eventually came to be believed, simply because filtrates that could cause infection when inoculated onto healthy organisms gave no growth under conditions under which bacteria would grow. The agents in the filtrates were remarkably small - they couldn't be seen with the microscope.
The advent of World War I effectively caused a halt to the dissemination of research in virology, although some important discoveries were made. The effects of bacteriophage were found independently by a Canadian, Twort, in 1915, and by the Frenchman, d'Herelle, in 1917.
The history of virology after World War I must be looked at in the context of the advances in genetics that had taken place during the first two decades of the 20th Century. This work was carried out using the fruit fly Drosophila, by Thomas Hunt Morgan and his colleagues at Columbia University. Their main achievement was the realization that visible traits were determined by genes and that these genes were located on chromosomes.
One of Morgan's co-workers, H.J. Mueller (who later won a Nobel prize for the effects of radiation on genetic material) wrote the following about viruses in 1921:This paragraph refers to bacteriophage; a similar thought was expressed about the agent of tobacco mosaic disease by Duggar and Joanne Karrer Armstrong
If these d'Herelle bodies (bacteriophage) were really genes, fundamentally like our chromosome genes, they would give us an entirely new angle from which to attack the gene problem. They are filterable, to some extent isolable (isolatable?) can be handled in test tube and the properties, as shown by their effects on the bacteria, can then be studied after treatment. It would be rash to call them genes, and yet at present we must confess that there is no distinction between genes and them.
The analysis of the nature of viruses also benefited from post-World War I developments in protein chemistry. By 1935 Wendell Stanley had crystallized tobacco mosaic virus. The paper describing this result was titled 'Isolation of a crystalline protein possessing the properties of TMV.' This title implies that the properties of TMV are ascribable solely to a protein(s). In my mind this goes to show that in science, sometimes you get what you look for.
That the causal agency in mosaic disease may be, in any particular case, a sometime product of the host cell; not a simple product, such as an enzyme, but a particle of chromatin or of some structure with a definite heredity, a gene perhaps, that has, so to speak, revolted from the shackles of co-ordination, and being endowed with a capacity to reproduce itself, continues to produce disturbance in its path and 'stimulation',but its path is only the living cell.'
But - this wasn't the end of the story. In 1936, the British workers Bawden and Pirie reported that they had found the element phosphorus in TMV in a form 'that could be isolated as a nucleic acid of the ribose type from the protein denatured by heat.'
So - where are we in terms of what portion of the virus causes its infectivity? The difficulty with assigning the 'active principle' to the nucleic acid was based on the fact that what was known about nucleic acids was not helpful. At this time, it was known that nucleic acids were composed of four different constituents, but the current concept that the linear sequence of these compounds could have biological meaning had not yet evolved. Nucleic acids, in the 1930s, were thought to be stupid.
The answer to the question of which part of the virus is reponsible for its heritable characteristics was not obtained until after World War II. As with World War I, there was a major disruption of biological research during this period. The experiment that gave this information was carried by Alfred Hershey and Martha Chase using bacteriophage T. Its design was based on two previously obtained pieces of data. The first was the information that both protein and nucleic acid were components of viruses (work of Bawden and Pirie, above). The other was the development of the electron microscope. One of the first uses of the electron microscope was to 'see' not only what a virus looked like, but also to look at how it infected bacteria. The virus used was one of the so-called 'T-even' series (T-2, T-4, T-6, etc.) Can you imagine what a thrill it was to scientists to not only see the shape of a virus but also to see that the viruses had more than one shape? above that suggested that the virus was like a syringe - that upon the shortening of the rod, something in the head was injected into the cell.
It also could not have been done without the development of the biological application use of radioactive isotopes. By the time the Hershey-Chase experiment was carried out, it was clear that a radioactive isotope of phosphorus, e.g., P32, or of sulfur, S35, would be 'seen' by biological systems exactly like the non-radioactive element.
These investigators realized that the electron micrographs suggested that some portion of the virus was 'injected' into the cells and that some portion of the virus remained outside. The hypothesis was that whatever went inside was responsible for the production of progeny virus. Did the nucleic acid go inside or did the protein? How would you design an experiment to find this out, if radioactive markers, one which was essentially specific for nucleic acid (P32) and a different one, specific for protein (S35) were available? Again, try to think about what 'tricks' were part of the experimental design that allowed a (relatively) clear answer. The graphic outline of the Hershey-Chase experiment is shown in Fig. 2-7.
From the current perspective, the results of this experiment are really rather sloppy. However, the interpretation was influenced by previous work of Avery, McLeod, and McCarty that showed that nucleic acid was responsible for the phenotype of bacteria.
A more direct demonstration of the role of nucleic acid in viral replication was obtained using our old friend TMV (Fig. 2-8).
Fraenkel-Conrat and Singer took advantage of the fact that the nucleic acid and protein of this virus could be separated by use of high concentrations (8 M) of a denaturing agent called urea. The took two strains of TMV (let's call them A and B), each one of which caused different symptoms on a plant. They separated the nucleic acid and the protein of A and B and reconstituted infectious virus by mixing and matching. They produced a virus that was nucleic acid(A) with protein(B), another with nucleic acid(B) and protein(A). They used these recombinant viruses to infect plants and scored the phenotype of the infection. What do you think the result was and how was it interpreted? They also reconstituted nucleic acid(A) with protein(A) and nucleic acid (B) with protein(B) and infected plants with these viruses. Why do you think they did this part of the experiment?
Historically, viruses were first identified by the types of effects they caused on organisms. They were first characterized as 'filterable agents', entities that could cause infection even if the medium containing them were considered sterile by the standards of bacteriology. An important part of the definition of a virus was that developed by Beijerinck, that a virus was dependent upon its host to make more of itself. Once genetics was established as a discipline, viruses were hypothesized to be essentailly genetic elements, This contrasted with the ability to isolate a virus using the tools of protein chemistry, that implied that viruses were proteinaceous in nature. However, the finding of nucleic acid in viruses confirmed that they might indeed contain genetic information. Nucleic acid was confirmed to be responsible for the production of viruses as well as the characteristic of virus infection.