Principles of Virology, Volume 1. Jane Flint

of milkmaids, the reality is more prosaic. As a physician’s apprentice at age 13, Jenner learned about a curious observation of local practitioners who had been variolating farmers with smallpox. No expected skin rash or disease appeared in farmers who had previously suffered a bout with cowpox. This lack of response was typical of individuals who had survived earlier infection with smallpox and were known to be immune to the disease. It was supposed therefore that, like smallpox survivors, these non-responding farmers must somehow be immune to smallpox. Although the phenomenon was first observed and later reported by others, Jenner was the first to appreciate its significance fully and to follow up with direct experiments. From 1794 to 1796, he demonstrated that inoculation with material from cowpox lesions induced only mild symptoms in the recipient but protected against the far more dangerous disease. It is from these experiments that we derive the term vaccination (vacca = “cow” in Latin); Louis Pasteur coined this term in 1881 to honor Jenner’s accomplishments.

Initially, the only way to propagate and maintain the cow-pox-derived vaccine was by serial infection of human subjects. This method was eventually banned, as it was often associated with transmission of other diseases such as syphilis and hepatitis. By 1860, the vaccine had been passaged in cows; later, horses, sheep, and water buffaloes were also used. The origin of the current vaccine virus, vaccinia virus, is now thought to be horsepox virus (Box 1.3).

      The first rabies vaccine was made by Louis Pasteur, although he had no idea at the time that the relevant agent was a virus. In 1885, he inoculated rabbits with material from the brain of a cow suffering from rabies and then used aqueous suspensions of dried spinal cords from these animals to infect other rabbits. After several such passages, the resulting preparations were administered to human subjects, where they produced mild disease but effective immunity against rabies.

      Today, viral vaccine strains selected for reduced virulence are called attenuated, a term derived from the Latin prefix ad, meaning “to,” and tenuis, meaning “weak.” Safer and more efficient methods for the production of larger quantities of these first vaccines awaited the recognition of viruses as distinctive biological entities and parasites of cells in their hosts. Indeed, it took almost 50 years to discover the next antiviral vaccines: a vaccine for yellow fever virus was developed in 1935, and an influenza vaccine was available in 1936. These advances became possible only with radical changes in our knowledge of living organisms and of the causes of disease.

Microorganisms as Pathogenic Agents

      The 19th century was a period of revolution in scientific thought, particularly in ideas about the origins of living things. The publication of Charles Darwin’s The Origin of Species in 1859 crystallized startling (and, to many people, shocking) new ideas about the origin of diversity in plants and animals, until then generally attributed directly to the hand of God. These insights permanently undermined the perception that humans were somehow set apart from all other members of the animal kingdom. From the point of view of the science of virology, the most important changes were in ideas about the causes of disease.

The diversity of macroscopic organisms has been appreciated and cataloged since the dawn of recorded human history. However, a vast new world of organisms too small to be visible to the naked eye was revealed through the microscopes of Antony van Leeuwenhoek (1632–1723). Van Leeuwenhoek’s vivid and exciting descriptions of living microorganisms, the “wee animalcules” present in such ordinary materials as rain or seawater, included examples of protozoa, algae, and bacteria. By the early 19th century, the scientific community had accepted the existence of microorganisms and turned to the question of their origin, a topic of fierce debate. Some believed that microorganisms arose spontaneously, for example, in decomposing matter, where they were especially abundant. Others held the view that all were generated by their reproduction, as are macroscopic organisms. The death knell of the spontaneous-generation hypothesis was sounded with the famous experiments of Pasteur. He demonstrated that boiled (i.e., sterilized) medium remained free of microorganisms as long as it was maintained in special flasks with curved, narrow necks designed to prevent entry of airborne microbes (Fig. 1.6). Pasteur also established that distinct microorganisms were associated with specific processes, such as fermentation, an idea that was crucial in the development of modern explanations for the causes of disease.

      From the earliest times, poisonous air (miasma) was generally invoked to account for epidemics of contagious diseases, and there was little recognition of the differences among causative agents. The association of particular microorganisms, initially bacteria, with specific diseases can be attributed to the ideas of the German physician Robert Koch. He developed and applied a set of criteria for identification of the agent responsible for a specific disease (a pathogen), articulated in an 1890 presentation in Berlin. These criteria, Koch’s postulates, can be summarized as follows.

BOX 1.3

      DISCUSSION

       Origin of vaccinia virus

      Over the years, many hypotheses have been advanced to explain the curious origin of vaccinia virus. However, recent investigations into this mystery by collaborators in the United States, Germany, and Brazil indicate that horsepox, not cowpox, was the likely precursor of vaccine strains of vaccinia virus.

      The proverbial smoking gun was an original wooden and glass container that held capillaries with the smallpox vaccine produced in 1902 by H.K. Mulford in Philadelphia (a company that merged with Sharpe and Dohme in 1929). Sequence analysis of the DNA showed that the core genome of the virus in that vial had the highest degree of similarity (99.7%) to horsepox virus. A review of the historical record shows that during the 19th century, pustular material derived from both cowpox and horsepox lesions was used to immunize against smallpox. The latter technique was called equination. Although the disease is now rare in horses and was never reported in the Americas, it was prevalent in Europe, where most vaccine samples were obtained at the time.

      Most smallpox vaccines used in the United States, Brazil, and many European countries were produced in the United States from calves inoculated with material collected in 1866 from spontaneous cases of cowpox in France. Genetic analysis of existing samples of these early vaccines indicates that they contained a virus more similar to horsepox and vaccinia viruses than to cowpox virus. While naturally occurring vaccinia viruses are found today only in India (in buffalos) and Brazil (in cows), they can infect horses and people, producing pustular lesions similar to those caused by horsepox and cowpox viruses. One hypothesis is that the ancestor of the current vaccine strain was a naturally occurring vaccinia virus present in the widely distributed French preparation. Alternatively, the vaccine strain may have evolved from horsepox virus during animal passage.

      The original wooden (top) and glass (bottom) containers that held capillaries containing the Mulford 1902 smallpox vaccine. Photo kindly provided by Dr. Jose Esparza, Institute of Human Virology, University of Maryland School of Medicine, Baltimore. ©Merck Sharp & Dohme Corp., Merck & Co., Inc.

      It is important to consider that development of the smallpox vaccine took place more than a century before modern concepts of virology were established. One can think of other scenarios to explain why the vaccine strain of vaccinia virus is closely related to horsepox and not cowpox, as originally supposed.

       The milkmaid with lesions that were the source of Jenner’s original inoculum in 1796 was infected with horsepox, not cowpox. Horsepox can be transmitted to cows, and both animals are common on farms.

       Cows from which pustular material was obtained for vaccination were most often infected