THE HISTORY OF VIROLOGY (Lecture 2) STM 326

STM 326          THE HISTORY OF VIROLOGY Lecture 2

The history of virology — the scientific study of viruses and the infections they cause – began in the closing years of the 19th century. The first evidence of the existence of viruses came from experiments with filters that had pores small enough to retain bacteria. 

Louis Pasteur (1822–1895) was unable to find a causative agent for rabies and speculated about a pathogen too small to be detected using a microscope. By the early 1870s, Pasteur had already established himself as a renowned leader in research, and in 1877 Pasteur began to fully immerse himself in the study of disease. At the time, Pasteur was studying chicken cholera (Pasteurella multocida), a diarrhoeal disease that was destroying the breeding chicken population. In 1879, Pasteur observed, by chance, that old bacterial cultures lost their virulence. He had instructed an assistant to inject the chickens with a fresh culture of the viral bacteria before a holiday. The assistant, however, forgot and went on vacation. When he returned a month later, he performed the procedure using the old cultures. Unexpectedly, the chickens only showed mild signs of the disease and survived. When they were healthy again, Pasteur, intrigued by the results, injected them with fresh bacteria. The chickens did not become ill. The discovery of the chicken cholera vaccine by Louis Pasteur revolutionized work in infectious diseases and can be considered the birth of immunology. The notion of using a weakened form of the disease to provide immunity was not new, but Pasteur was the first to take the process to the laboratory, impacting all virologists who followed after him.

The microbe, weakened in the lab, had taught the chicken immune system to fight the infection without causing any serious harm to the chicken. This type of vaccine is called a live, attenuated vaccine. Louis Pasteur continued to explore illnesses in the pursuit of new vaccines.

Then, in 1885, Rabies had presented a new obstacle for Pasteur in the development of a successful vaccine. Unlike chicken cholera and anthrax, both caused by bacterium, the microorganism causing the disease could not be specifically identified, meaning Pasteur would not be able to develop the vaccine in vitro (in the laboratory). Pasteur did not know this at the time, but the reason he could not find the microorganism is because rabies is a viral disease. Viruses are small infectious agents that replicate quickly and have a high mutation rate. These rapid mutations can be used to the benefit of researchers in the development of an attenuated vaccine. By serial passage of a virus through a different species, the virus becomes more adapted to that species, and less adapted to its original host, deceasing virulence with respect to the original host (e.g. it is “attenuated”). By passing the virus through rabbits, Pasteur made the virus less dangerous to human hosts, while still giving the body enough information to recognize the antigen and develop immunity to the “wild” version of the disease.

After successfully protecting dogs from the disease, Pasteur agreed to treat his first human patient, a nine-year-old boy who had been so severely attacked by feral dogs there was little doubt he would die if nothing was done. Pasteur injected the boy with a daily series of progressively more virulent doses of the vaccine from the rabies-infected rabbits. The boy never developed symptoms and Pasteur became an international hero. 

His procedure was originally called “Pasteur’s treatment”, but Pasteur decided to honor the 18th century virology pioneer Edward Jenner, who publicized the cure for smallpox, and give these artificially weakened diseases the generic name of “vaccines”. Thus, we largely have Pasteur to thank for today’s definition of a vaccine as a “suspension of live (usually attenuated) or inactivated microorganisms (e.g., bacteria or viruses) or fractions thereof administered to induce immunity and prevent infectious disease. Louis Pasteur’s work advanced the nascent field of virology and served to spur vaccine research all over the globe. 

Charles Chamberland (1851–1931) The invention that allowed viruses to be discovered at all was the ChamberlandPasteur filter. This was developed in 1884 in Paris by Charles Chamberland, the French microbiologist who worked with Louis Pasteur. It consisted of unglazed porcelain “candles”, with pore sizes of 0.1 – 1 micron (100 - 1000 nm), which could be used to completely remove all bacteria or other cells known at the time from a liquid suspension. Though this simple invention essentially enabled the establishment of a whole new science – virology - the continued development of the discipline required a string of technical developments.   

Martinus Beijerinck (1851–1931) In 1876, Adolf Mayer, who directed the Agricultural Experimental Station in Wageningen was the first to show that what he called "Tobacco Mosaic Disease" was infectious, he thought that it was caused by either a toxin or a very small bacterium.

 Later, in 1892, the Russian biologist Dmitry Ivanovsky (1864–1920) used a Chamberland filter to study what is now known as the tobacco mosaic virus. His experiments showed that crushed leaf extracts from infected tobacco plants remain infectious after filtration. Ivanovsky suggested the infection might be caused by a toxin produced by bacteria, but did not pursue the idea. 

In 1898, the Dutch microbiologist Martinus Beijerinck, a microbiology teacher at the Agricultural School in Wageningen repeated experiments by Adolf Mayer and became convinced that filtrate contained a new form of infectious agent. He observed that the agent multiplied only in cells that were dividing and he called it a contagium vivum fluidum (soluble living germ) and re-introduced the word virus. Beijerinck maintained that viruses were liquid in nature, a theory later discredited by the American biochemist and virologist Wendell Meredith Stanley (1904–1971), who proved that they were in fact, particles. 

Friedrich Loeffler (1852–1915) and Paul Frosch (1860–1928) In 1898 passed the first animal virus through a porous clay filter and discovered the cause of foot-and-mouth disease (FMD). In the aforementioned report, Loeffler and Frosch described their discovery that the causative agent of FMD was neither a known bacterium nor a toxin but an ‘ultravisible, ultrafilterable substance’ which previously had gone unnoticed due to its minute size. In the extensive and very precise experiments undertaken, they were able to transmit the disease from calves suffering from FMD to other susceptible cloven-hoofed animals by transplanting germ-free filtered vesicle lymph. Loeffler and Frosch were able to rule out a toxin as the cause of FMD by serial transmission of filtered vesicle from diseased animals. According to the meticulous calculations which they made, if the cause of FMD was toxin-based, then after several animal-to-animal transmissions the original material would be so diluted that only ‘1 : 2 trillion ’ of the starting substance would remain. ‘A toxic effect of that nature would be unbelievable’ they concluded. Since the disease ran its characteristic course with every round of transmission, the causative agent, therefore, must be some sort of minute agent capable of reproducing itself. Furthermore, Loeffler and Frosch already knew from their initial studies that the infection of animals resulted in a solid immunity. Analogous to Behring’s anti-toxin therapy, they found, in the blood of immune animals, substances which neutralized infectivity and the pathogenic effect of the causative agent, i.e. they found antibodies which protected against infection. Loeffler and Frosch reported that cows and sheep can be artificially immunized. Immunization can be achieved by injecting filtered vesicle fluid which has first been sufficiently pre-heated to destroy infectivity, or by injecting a vesicle lymph immune blood mixture, or passively by the serum of immune calves. Loeffler and Frosch were, therefore, the first to use an inactivated vaccine to fight a virus infection.  In view of their contribution, Loeffler and Frosch can justifiably be regarded as the founders of animal virology.   

Wendell Stanley (1904-1971) In 1935 Wendell Stanley, a young chemist working in the Rockefeller Institute's laboratories in Princeton, NJ, purified a virus in the form of needle-shaped crystals with the chemical properties of a protein. It was perhaps the most startling discovery to date in the Institute's history. How could a virus, with its ability to infect and multiply, also be an inanimate chemical—an inert molecule? Stanley's finding prompted discussion of the question, "what is life?" as well as much criticism. Further research elsewhere, confirmed in Stanley's laboratory, and soon demonstrated that the infectious substance was in fact a combination of protein and the nucleic acid, RNA. The results called attention to the similar reproductive powers of viruses and genes (not yet known to be DNA), and helped lay the groundwork for modern molecular biology. For his achievements, Stanley shared the Nobel Prize in Chemistry in 1946 with his Rockefeller colleague John H. Northrop (1891-1987) and James B. Sumner.

Stanley experimented with tobacco mosaic virus (TMV), which causes spots on the leaves of infected plants. By the time he began his studies in the early 1930s, TMV was well known to laboratory researchers. Others had shown that it could be manipulated chemically without losing its ability to infect. Stanley decided to apply to TMV methods for crystallizing proteins that had been developed by his Rockefeller colleagues John Northrop and Moses Kunitz. Although more than 300 human, animal, and plant viruses were known to science, when Stanley began his research it was not known whether viruses were, in his words, "inorganic, carbohydrate, hydrocarbon, lipid, protein, or organismal in nature."

In addition to opening questions about the basic nature of viruses that set the course for the field of virology, Stanley's own research had a more immediate impact on medicine. During World War II, he turned his attention to developing an inactivated vaccine for viral influenza. He used a Sharples centrifuge—a piece of equipment widely used in the dairy industry at the time—to obtain concentrated and purified quantities of the virus. The centrifuge's large capacity and efficiency made it possible to scale up production of the vaccine, and Stanley's method was used in the development of commercial vaccines. 

Bacteriophage

 

Bacteriophages are the viruses that infect and replicate in bacteria. They were discovered in the early 20th century, by the English bacteriologist Frederick Twort. 

Frederick Twort (1877–1950) In 1915, Frederick Twort discovered the action of bacteriophages on staphylococci bacteria. He noticed that when grown on nutrient agar some colonies of the bacteria became watery or "glassy". He collected some of these watery colonies and passed them through a Chamberland filter to remove the bacteria and discovered that when the filtrate was added to fresh cultures of bacteria, they in turn became watery. He proposed that the agent might be "an amoeba, an ultramicroscopic virus, living protoplasm, or an enzyme with the power of growth". It is interesting that he was actually attempting to grow vaccinia virus in culture, and that it was a contaminating staphylococcus that he noticed was being lysed by his infectious agent.

Félix d'Herelle (1873–1949) was a mainly self-taught French-Canadian microbiologist. In 1917 he discovered that "an invisible antagonist", when added to bacteria on agar, would produce areas of dead bacteria. The antagonist, now known to be a bacteriophage could pass through a Chamberland filter. He accurately diluted a suspension of these viruses and discovered that the highest dilutions (lowest virus concentrations), rather than killing all the bacteria, formed discrete areas of dead organisms. Counting these areas and multiplying by the dilution factor allowed him to calculate the number of viruses in the original suspension. He realised that he had discovered a new form of virus and later coined the term "bacteriophage". Between 1918 and 1921, d'Herelle discovered different types of bacteriophages that could infect several other species of bacteria including Vibrio cholerae. Bacteriophages were heralded as a potential treatment for diseases such as typhoid and cholera, but their promise was forgotten with the development of penicillin. Since the early 1970s, bacteria have continued to develop resistance to antibiotics such as penicillin, and this has led to a renewed interest in the use of bacteriophages to treat serious infections.

Denis Parsons Burkitt (1911–1993) was born in Enniskillen, County Fermanagh, Ireland. He was the first to describe a type of cancer that now bears his name Burkitt's lymphoma. This type of cancer was endemic in equatorial Africa and was the commonest malignancy of children in the early 1960s. In an attempt to find a cause for the cancer, Burkitt sent cells from the tumour to Anthony Epstein (b. 1921) a British virologist, who along with Yvonne Barr and Bert Achong (1928–1996), after many failures, discovered viruses that resembled herpes virus in the fluid that surrounded the cells. The virus was later shown to be a previously unrecognised herpes virus, which is now called Epstein–Barr virus. Surprisingly, Epstein–Barr virus is a very common but relatively mild infection of Europeans. Why it can cause such a devastating illness in Africans is not fully understood, but reduced immunity to virus caused by malaria might be to blame. Epstein–Barr virus is important in the history of viruses for being the first virus shown to cause cancer in humans. 

The first animal viruses

What is often described as the second virus ever discovered was what is now known as Foot and mouth disease virus (FMDV) of farm and other animals, in 1898 by the German scientists Friedrich Loeffler and Paul Frosch. Again, their “sterile” filtered liquid proved infectious in calves, providing the first proof of viruses infecting animals. Indeed, some believe that the true discoverers of viruses were these two scientists, as they concluded the infectious agent was a tiny particle, and was not a liquid agent. The two went further by showing that it was possible to vaccinate cows and sheep against the disease using filtered vesicle extract that had been heated sufficiently to destroy its infectivity: this was possibly the first use of an inactivated virus as a prophylactic vaccine.  

 

The first human virus: yellow fever

The first human virus described was the agent which causes yellow fever: this probably originated in Africa, but was spread along with its mosquito vector Aedes aegyptii to the Americas and neighbouring islands by the slave trade. Indeed, the declaration of independence from France by Haiti in 1804 was made possible in part by the devastating effect of the disease on the French army sent to quell a slave revolt there. The virus was discovered and reported in 1901 by the US Army physician Walter Reed, after pioneering work in Cuba by Carlos Finlay reported in 1881 hypothesising  that mosquitoes transmitted the deadly disease. The agent became the subject of intense study because, in the Spanish-American war in Cuba in the 1890s, about 13 times as many soldiers died of yellow fever as died from wounds. The subsequent eradication of mosquitoes in Panama is probably what allowed the completion of the Panama Canal – stalled because of the death rate among workers from yellow fever and malaria.  

The second half of the 20th century was the golden age of virus discovery and most of the 2,000 recognised species of animal, plant, and bacterial viruses were discovered during these years. 

• In 1963, the hepatitis B virus was discovered by Baruch Blumberg (b. 1925). 

• Reverse transcriptase, the key enzyme that retroviruses use to translate their RNA into DNA, was first described in 1970, independently by Howard Temin and David Baltimore (b. 1938). This was important to the development of antiviral drugs – a key turning-point in the history of viral infections.

• In 1983 Luc Montagnier (b. 1932) and his team at the Pasteur Institute in France, first isolated the retrovirus now called HIV. 

These discoveries have continued in the 21st century as new viral diseases such as SARS and nipah virus have emerged. Despite scientists' achievements over the past one hundred years, viruses continue to pose new threats and challenges.

Among the virus-induced diseases of man: smallpox, yellow fever, dengue fever, poliomyelitis, certain types of encephalitis, chickenpox/shingles, measles, mumps, influenza, virus pneumonia, chikungunya, Human immunodeficiency virus (HIV/AIDS), Human papillomavirus (HPV) and the common cold. Virus diseases of animals include hog cholera, cattle plague, foot-and-mouth disease of cattle, swamp fever of horses, equine encephalitis, rabies, fowl pox, Newcastle disease of chickens, fowl paralysis, and certain benign as well as malignant tumors of rabbits and mice. Plant virus diseases include tobacco mosaic, peach yellows, aster yellows, potato yellow dwarf, alfalfa mosaic, curly top of sugar beets, tomato spotted wilt, tomato bushy stunt, corn mosaic, cucumber mosaic, and sugar cane yellow stripe.