Advances in Understanding Viruses

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Advances in Understanding Viruses

Overview

At the beginning of the twentieth century, the term "virus" commonly referred to infectious agents that could not be seen under the microscope, trapped by filters, or grown in laboratory cultures. Today the term refers to a minute entity composed of an inner core of nucleic acid and an envelope of protein. The fundamental difference between a virus and other microbes is that a virus can only reproduce by entering a living host cell and taking over its metabolic apparatus. Studies of tobacco mosaic disease by Adolf Mayer, Martinus Beijerinck, and Dimitri Ivanovski led to the discovery of the tobacco mosaic virus, which was crystallized by Wendell M. Stanley. The discovery of bacterial viruses, or bacteriophages, by Frederick Twort and Félix-Hubert d'Hérelle provided one of the most important experimental tools in the development of molecular biology.

Background

The modern definition of a virus refers to a minute entity composed of an inner core of nucleic acid and an envelope of protein; a virus particle can only reproduce by entering a living host cell and taking over its metabolic apparatus. The meaning of the word "virus" has undergone many changes since its original usage in Latin for "slime," an unpleasant substance, but not necessarily dangerous. Eventually, "virus" assumed the connotation of a dangerous poison or venom or of a mysterious, unknown infectious agent. Eighteenth-century medical writers applied the term to the contagion that transmitted an infectious disease, for example, the "cowpox virus" or the "variolous virus." After the establishment of germ theory in the late nineteenth century, "virus" was generally used in reference to obscure entities with infectious properties. The infectious agents of some diseases displayed complex life cycles and could not be cultured by conventional techniques. Obviously, the failure of some infectious agents to grow in the laboratory might simply mean that they required special growth conditions.

Bacteriologists discovered that the causative agents of many diseases could be identified under the microscope, grown in the laboratory, and removed from growth media by using special filters, such as the filtration device developed by Charles Chamberland (1851-1908). The Chamberland filter was used to separate visible microorganisms from their culture medium and to prepare bacteria-free liquids. Chamberland was also instrumental in the development of the autoclave, a device for sterilizing materials that uses steam heat under pressure. Rigorous use of these techniques made possible the establishment of a new category of infectious agents, which were generally called "invisible-filterable viruses."

"Invisible viruses" were thought to cause many important diseases of humans and animals, but their nature remained obscure. Therefore, in the early twentieth century such infectious agents were defined operationally as "filterable" and "invisible" microbes. In other words, the viruses were defined operationally in terms of their ability to pass through filters that trapped bacteria and their ability to remain invisible under a light microscope. Criteria based on specific techniques, however, provided little insight into the biochemical nature of viruses. Moreover, further studies of the invisible-filterable viruses indicated that these two operational criteria were not necessarily linked. Eventually, some microbiologists began to consider the possibility that viruses might be a special category of microparasites that could only reproduce in the cells of other organisms.

Investigations of plant viruses, especially the tobacco mosaic virus, provided the basis for the new science of virology. Adolf Eduard Mayer (1843-1942), Martinus Beijerinck (1851-1931), and Dimitri Ivanovski (1864-1920) are generally regarded as the founders of virology. Plant virology can be traced back to 1886, when Mayer discovered that tobacco mosaic disease could be transmitted to healthy plants by inoculating them with extracts of sap from the leaves of diseased plants. Mayer could not culture the causative agent on artificial media, but he was able to prove that filtered sap from infected plants could transmit the disease.

In 1892 Ivanovski demonstrated that the infectious agent for tobacco mosaic disease could pass through the finest filters available. Using Chamberland's filter method, Ivanovski found that a filtered extract of infected tobacco leaves produced the same disease in healthy plants as an unfiltered extract. He read a paper on his discovery to the Academy of Sciences of St. Petersburg that same year. Ivanovski thought his observations might be explained by the presence of a toxin in the filtered sap, but he did not discount the possibility that unusual bacteria had passed through the pores of the filter. Unable to isolate a toxin or culture the "tobacco microbe," Ivanovski turned to research on alcoholic fermentations. Nevertheless, Russian historians consider Ivanovski the founder of virology.

Apparently unaware of Ivanovski's work, Beijerinck began research on tobacco mosaic disease as a graduate student under Mayer at the University of Leiden. In 1895 Beijerinck became professor of microbiology at the Polytechnical School in Delft. His research interests included botany, microbiology, chemistry, and genetics as well as tobacco mosaic disease. Like his predecessors, Beijerinck found that the sap of plants with tobacco mosaic disease remained infectious after passage through a filter. He was particularly intrigued by the ability of filtered sap to transmit the disease after passing through a series of a large number of plants; this fact indicated that the causative agent was an entity that could reproduce and multiply itself and was not a poison or toxin. Based on reports in the botanical literature, Beijerinck thought that many other plant diseases were caused by agents like the tobacco mosaic disease virus.

The fundamental difference between bacteria and virus was probably not size or filterability. In 1900 Beijerinck published an extensive review of his work and his theory. Unlike bacteria, the virus might be an obligate intracellular parasite, an agent that can only reproduce within living cells. In thinking about the way in which filtered plant sap could be used to transmit tobacco mosaic disease to a large series of plants, Beijerinck concluded that the disease must be caused by an agent that needed the living tissues of the plant in order to reproduce itself. In other words, the virus could be thought of as some kind of "contagium vivum fluidum," or contagious living fluid. The crucial difference between a virus and other microbes might not be simply size. Some microbes might be obligate parasites of living organisms that could not be cultured in vitro on any cell-free culture medium. Beijerinck, therefore, concluded that tobacco mosaic disease must be caused by an agent that had to incorporate itself into a living cell in order to reproduce. Beijerinck's theory of the "contagium vivum fluidum" was a first step towards a new way of thinking about the special nature of the virus.

One of the first detailed studies of an animal disease caused by a filterable virus was conducted by Friedrich Loeffler (1852-1915) and Paul Frosch (1860-1928) in 1898. Foot-and-mouth disease was an important disease of cattle, but all efforts to culture bacteria from fluid taken from lesions in the mouths and udders of sick animals were unsuccessful. Loeffler and Frosch were unable to isolate the causative agent, but they did prove that it could be transmitted to cattle and pigs with minute amounts of filtered, apparently bacteria-free, fluids from the vesicles of infected animals. These experimentally infected animals could transmit the disease to other animals. Using mixtures of blood and lymph from the vesicles of animals with the disease, Loeffler and Frosch were able to immunize healthy animals. These experiments suggested that a living agent, rather than a toxin, must have been in the filtrate. They concluded that the disease might be caused by a living agent small enough to pass through the pores of their filters. Their experiments suggested that only a living agent, one capable of reproducing itself, could continue to cause the disease after passage through a series of animals. Loeffler and Frosch suggested that other infectious diseases, such as smallpox, cowpox, and cattle plague, might be caused by similar organisms. Nevertheless, they continued to think of the infectious agent as a very small and unusual microbe that had not yet been cultured in vitro, rather than a novel entity that could only multiply within the cells of its host.

Within a few years of these studies, filterable viruses were suspected of being the cause of various plant, animal, and human diseases. These predictions were confirmed in 1911, when Francis Peyton Rous (1879-1970) injected chicken with a cell-free filtrate from tumor cells and discovered a virus that could cause cancer. A farmer had noticed that one of his Plymouth Rock hens had developed a breast tumor and brought it to the Rockefeller Institute in New York. After performing an autopsy, Rous prepared an extract of the malignant tumor cells and injected it into healthy chickens. Even when the extract was filtered to remove whole cells, hens of the same purebred stock developed tumors. The virus became known as the Rous sarcoma virus. Rous was awarded a Nobel Prize in physiology or medicine in 1966 for his demonstration that a malignant tumor could be transmitted by a virus.

Impact

Demonstrations of the existence of plant and animal viruses were followed by the discovery of viruses acting as parasites of bacteria. In 1915 Frederick William Twort (1877-1950) discovered that even bacteria could be attacked by invisible viruses. While trying to grow viruses on artificial medium in agar plates, Twort noted that certain bacterial colonies contaminating the plates became glassy and transparent. If pure colonies of this micrococcus were touched by a tiny portion of material from the glassy colonies, they too became transparent. Twort later became obsessed with speculative work on the possibility that bacteria evolved from viruses that had developed from even more primitive forms. These viruses became known as "Twort particles. " Twort demonstrated that these microparasites were filterable, like the infectious agent of many mysterious plant and animal diseases. His landmark paper on the nature of ultra-microscopic viruses was published in the British medical journal Lancet in 1915. Unfortunately, Twort's research was interrupted by World War I and his paper had little immediate impact on microbiology.

In 1917, while working on the dysentery bacillus at the Pasteur Institute, Félix d'Hérelle (1873-1949) discovered what he called "an invisible microbe that is antagonistic to the dysentery bacillus." d'Hérelle obtained the invisible microbe from the stools of patients recovering from bacillary dysentery. When an active filtrate was added to a culture of Shiga bacilli, bacterial growth soon ceased and bacterial death and lysis (dissolution) followed. A trace of the lysate produced the same effect on a fresh Shiga culture. More than 50 such transfers gave the same results, indicating that a living agent was responsible for bacterial lysis. The invisible microbe could not grow on laboratory media or heat-killed bacilli but grew well in a suspension of washed bacteria in a simple salt solution. d'Hérelle concluded that the invisible anti-dysentery microbe must be an obligate parasite of the Shiga bacillus; therefore, he called it a "bacteriophage," that is, an entity that eats bacteria. Speculating on the implications of the phenomenon he had discovered, d'Hérelle predicted that bacteriophages for other bacteria would be found. He hoped that bacteriophages might be modified in the laboratory and used to destroy pathogenic bacteria. Unfortunately, d'Hérelle's hope that the bacteriophage would become the "microbe of immunity" did not come true. In honor of the scientists who had discovered them, bacterial viruses were sometimes called "Twort-d'Hérelle particles." Bacterial viruses remained mere laboratory curiosities until molecular biologists adopted bacteriophages as their primary experimental system, at which point the bacteriophage became known as the Rosetta stone of molecular genetics.

During the 1930s and 1940s researchers began to examine the biochemistry of viruses. By the 1940s improved biochemical techniques were making it possible to understand the nature of complex biological macromolecules. Biochemical studies supported the belief that the virus lay on the borderline between cells and molecules. At about the same time, x-ray crystallography and the electron microscope provided a new picture of the previously invisible viruses.

In 1935 Wendell M. Stanley (1904-1971) reported that he had isolated and crystallized a protein having the infectious properties of the tobacco mosaic virus. The title of his landmark paper in the journal Science was "Isolation of a Crystalline Protein Possessing the Properties of Tobacco Mosaic Virus." Stanley had seen that investigating the chemical nature of viruses was closely related to the problem of purifying an enzyme. Isolating a virus, however, was more difficult than purifying an enzyme because the chemical reaction catalyzed by an enzyme could be measured more easily than the biological activity of a virus. Nevertheless, Stanley attempted to purify the virus by methods similar to those that James Sumner (1887-1955) and John Northrop (1891-1987) had employed to isolate enzymes. Starting with literally a ton of infected tobacco leaves, Stanley obtained a spoonful of crystalline material that essentially consisted of protein but was still capable of producing tobacco mosaic disease. In 1945 he produced small amounts of crystals that were extremely active as infectious agents. He referred to the purified protein as a "molecular virus." Stanley found that even after several recrystallizations, tobacco mosaic virus retained its physical, chemical, and biological properties. He concluded that tobacco mosaic virus was an auto-catalytic protein that could only reproduce and multiply in the presence of living cells. Further work showed that the purified virus contained nucleic acid as well as protein, but until the 1950s it was unclear whether protein, nucleic acid, or nucleoprotein was the infectious component. Stanley's work made possible the modern definition of viruses as particles composed of an inner core of nucleic acid enclosed in a protein overcoat. Stanley won the Nobel Prize in chemistry in 1946 for his demonstration that an apparently pure chemical substance could reproduce itself and multiply as if it were a living organism. Appropriately, he shared the prize with Northrop and Sumner. Northrop was awarded the prize for developing methods for the crystallization of proteins and Sumner was honored for providing the first convincing proof that enzymes are proteins.

Only a few years after Stanley characterized it, scientists obtained the first portrait of the tobacco mosaic virus by electron microscopy. A review of microbiology that set forth the fundamental differences between bacteria and viruses was published by Thomas Rivers (1888-1962) in 1927. Rivers helped to establish virology as a distinct field of study. During the 1930s the electron microscope made it possible to see viruses as well as bacteria. In 1934 Ladislaus Laszlo Marton published the first electron micrographs of biological materials. Three years later, he published the first electron micrographs of bacteria. In 1940 Helmuth Ruska prepared the first electron micrographs of a virus. In 1949 John Franklin Enders (1897-1985), Thomas H. Weller (1915- ), and Frederick Chapman Robbins (1916- ) developed a technique that made it possible to grow the polio virus in cultures of human tissue. Their methods provided a valuable tool for the isolation and study of viruses, which have been described as "living molecules." By the end of the twentieth century, however, scientists had discovered that some diseases were caused by other, previously unknown submicroscopic creatures, such as "slow" viruses, viroids, and prions.

LOIS N. MAGNER