Biomedicine and Health: Virology

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Biomedicine and Health: Virology

Introduction

Virology is a twentieth-century science that deals with viruses and viral diseases. Until the 1930s the study of the obscure entities referred to as viruses was inseparable from the science of microbiology, a broader discipline that still encompasses the study of virology, bacteriology, mycology, botany, and zoology. At the beginning of the twentieth century, the term “virus” referred to infectious agents that could not be seen under the microscope, trapped by filters, or grown in laboratory cultures. Today a virus is defined as a minute entity composed of an inner core of nucleic acid contained in a protein envelope. 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.

The ancient Latin word virus has had different meanings during its two millennia of usage. The first and most general meaning was slime, but medical writers used the term in reference to a noxious substance, such as poison or venom, or a mysterious, unknown infectious agent. The Roman medical writer and encyclopedist Aulus Cornelius Celsus (c.25 BC–AD 50) used the term in De re medicina (On medicine), a book that was essentially forgotten until the fifteenth century. Its rediscovery, just in time to be reproduced by Europe's new printing presses, gave Renaissance scholars and physicians access to a source of pure classical Latin.

Although the meaning of “virus” changed considerably from ancient Rome to the early decades of the twentieth century, traces of its original Latin usage can be found in expressions such as “the virus of racism,” where it means something that poisons the mind, rather than a minute particle composed of nucleic acid and protein. After the establishment of germ theory in the late nineteenth century, virus generally referred to unidentifiable entities with infectious properties. In computer jargon it was adopted to describe a noxious bit of code that infects a computer and spreads to other computers.

Historical Background and Scientific Foundations

The infectious agents now known as viruses originally attracted attention because of the diseases they produce in their animal and plant hosts. Long before physicians and scientists had any knowledge about microorganisms of any kind, viral diseases such as smallpox and rabies provided dramatic examples of the possibility of preventing life-threatening diseases through deliberate immunization. Immunity, like virus, is a Latin term with many meanings that has been adapted to biomedical science. Originally, immunitas referred to a special exemption from commonplace obligations, such as taxation or criminal prosecution. In medicine, immunity refers to resistance to infection by disease-causing microbes. Just as a witness can be granted immunity from prosecution when immunized by the court, a person can acquire immunity to disease by means of inoculation.

Smallpox

Smallpox was the first, and so far, the only viral disease to be eradicated by a deliberate worldwide immunization campaign. To understand how smallpox was eradicated in the 1970s, it is necessary to consider both the characteristics of the virus that causes the disease and the evolution of methods used to prevent it. Variola, the smallpox virus, is a member of the family Poxviridae, genus orthopoxvirus, which includes cowpox, camelpox, swinepox, monkeypox, and others. Based on its characteristics and genomic sequencing, the pox viruses probably evolved from a common ancestral virus whose natural host was a rodent. Unlike most viruses, the smallpox virus is fairly stable outside its host. There are several types of smallpox viruses, but the most important distinction occurs between dangerous strains of variola major and weaker variola minor. While mortality rates for smallpox were probably generally about 15–25%, during some epidemics up to 40% of those who contracted the disease died.

Smallpox: Inoculation, Vaccination, and Eradication

For hundreds of years before descriptions of smallpox inoculation appeared in eighteenth-century European scientific journals, people in many parts of Europe, Asia, and Africa attempted to protect their children from deadly smallpox by deliberately exposing them to a person with a mild case. Others employed practitioners who inserted material from smallpox pustules into a cut or scratch on the skin of a healthy individual, a process known as ingrafting, inoculation, or variolation. According to the National Partnership for Immunization, people who are suspicious of modern vaccines have again adopted the practice of exposing their children to “natural” cases of childhood illnesses, such as chickenpox. Since the 1990s, public health officials have repeatedly warned that such practices are much more dangerous than vaccination.

Credit for introducing the English elite to variolation is traditionally ascribed to Lady Mary Wortley Montagu (1689–1762), who observed the practice while in Constantinople, Turkey, where her husband served as the British Ambassador Extraordinary. In 1721, during a smallpox epidemic in London, Lady Mary arranged to have her daughter inoculated. In response to the attacks on the practice launched by physicians and clergymen, she published “A Plain Account of the Inoculating of the Small Pox.” Other descriptions of the Turkish method published in the Royal Society's Philosophical Transactions (1714) attracted the interest of two Americans, the author and minister Cotton Mather (1663–1728) and physician Zabdiel Boylston (1676–1766), who conducted a test of inoculation during a 1721 smallpox outbreak in Boston. Physicians and clergymen denounced these experiments in pamphlets, broadsides, and sermons, but Boylston's statistical evidence demonstrated the relative safety of the process.

Inoculation had important ramifications for medical practitioners, public health officials, and parents. As

the American publisher, writer, diplomat, and scientist Benjamin Franklin (1706–1790) explained, weighing the risks and benefits of inoculation was an awesome responsibility for parents. In 1736, he published a notice in the Pennsylvania Gazette denying rumors that his son Francis had died of inoculated smallpox. Franklin was afraid that false reports about the death of his son would keep other parents from protecting their children. Young Francis had contracted natural smallpox and died of the disease before he could be inoculated. By the end of the eighteenth century, inoculation was a generally accepted medical practice. Moreover, inoculation paved the way for the rapid acceptance of vaccination, a safer practice introduced by English physician Edward Jenner (1749–1823).

In 1798 Jenner published an account of his observations on the relationship between cowpox and smallpox. Milkmaids in Gloucestershire, England, thought that a mild case of cowpox made them immune to smallpox; Jenner's experiments confirmed this folk belief. To distinguish between the old practice of inoculation and his new method, Jenner coined the term vaccination from the Latin vaccines, “from cows.” Critics warned that deliberately transmitting disease from animals to humans was a loathsome, immoral, and dangerous act. His followers, however, called vaccination the greatest discovery in the history of medicine. Within little more than 10 years, vaccination was generally adopted throughout the world.

By the 1960s, for most residents of the wealthiest nations, the odds of suffering ill effects from vaccination became greater than the chance of encountering smallpox. Nevertheless, as long as smallpox existed anywhere in the world, its danger could not be ignored. The worldwide eradication of smallpox offered a humane and economical solution to the vaccination dilemma. Increasing certainty that there was no animal reservoir or human carrier state for smallpox made global eradication a practicable goal. The Smallpox Eradication Program sponsored by the World Health Organization (WHO) began its campaign in the 1960s. In 1977 the last case of naturally acquired smallpox was diagnosed in Somalia. Public health advocates urged WHO to use the lessons of the smallpox campaign to promote global immunization programs for measles, poliomyelitis, and other preventable diseases.

When eradication was achieved, the only known reservoirs of smallpox virus were those remaining in

LOUIS PASTEUR (1822–1895)

As a youth, Louis Pasteur (1822–1895) was a diligent student and a talented artist. His father, a tanner and former soldier in Napoleon's army, hoped that his bright and talented son would distinguish himself as a scholar or artist. Many of the portraits Pasteur made of family and friends are considered quite good, but at 19 years of age he gave up painting and devoted himself strictly to science. Surprisingly, Pasteur earned only mediocre grades in high school chemistry and, in 1838, he had to abandon his first attempt at student life in Paris because of acute homesickness. After a brief recovery, Pasteur went on to study chemistry and physics with distinction.

While still a student, Pasteur became intrigued by new studies of crystal structure, stereoisomerism, and molecular asymmetry. Perhaps the most important lesson Pasteur learned as a student of chemistry and physics was the applicability of the experimental approach to a broad range of questions. This made it possible for him to explore problems in biology and medicine, areas in which he had no specific training.

In 1849 Pasteur was appointed to the University of Strasbourg, where he met and married the daughter of the rector of the academy. Five years later, having achieved considerable recognition for his chemical research on stereochemistry and crystals, he was appointed professor of chemistry and dean of sciences at the University of Lille in northern France. As a university teacher and administrator, Pasteur was expected to offer advice and assistance to local industries and agriculture. Requests for help caused Pasteur to neglect his beloved crystals, but led him to begin his pioneering research on the relationship between fermentation and microorganisms.

In 1857 Pasteur accepted a position at the École Normale in Paris as assistant director for scientific studies. Three years later, the Academy of Sciences awarded its prize for experimental physiology to Pasteur in recognition of the importance of Pasteur's research on fermentation. These studies of what Pasteur called “the diseases of wine and beer” were followed by his research on protective vaccines for the diseases of silkworms, chickens, livestock, and human beings.

Nine specific aspects of Pasteur's research are carved into the marble walls of the chapel at the Institut Pasteur in Paris where he is buried: molecular dissymmetry, fermentations, studies of spontaneous generation, studies of wine, diseases of silkworms, studies of beer, contagious diseases, protective vaccines, and prevention of rabies. The path that led Pasteur from theoretical questions about stereochemistry and the nature of fermentation to medical microbiology involved a progression that he saw as natural and almost inevitable.

Pasteur's lifetime of research, ranging as it did from chemistry to biology and medicine, helped establish the theoretical, methodological, and ideological principles of the new science of microbiology. Despite his many contributions to French science and the adulation of his contemporaries, at the end of his life Pasteur expressed regret for abandoning the theoretical research on crystals that, he thought, might have led to the discovery of a fundamental cosmic asymmetric force.

research laboratories. The danger of maintaining such laboratory stocks was emphasized in 1978 when Janet Parker (1938–1978), a photographer who worked in the Birmingham University Medical School, in Birmingham, England, died of the disease. The virus apparently escaped from a virus research laboratory through the air ducts. WHO planned to destroy all remaining stocks of smallpox virus, but some experts objected. Although the complete genomes of vaccinia and variola were decoded in the 1990s, some scientists thought that samples of the virus might be needed for future research.

With the threat of naturally occurring smallpox eliminated, fears have grown that the virus could be used as an agent of biological warfare. Smallpox has been called an ideal agent for bioterrorism because the virus is stable, easy to grow, easily dispersed as an aerosol, and produces a contagious, potentially lethal disease. Once the possibility of bioterrorism was raised, governments and public health experts were forced to consider how to respond. A smallpox outbreak in the United States could have devastating consequences, because vaccination essentially ended in the 1970s. Nevertheless, the smallpox vaccine is now considered too dangerous to justify its use in the absence of an imminent threat.

Louis Pasteur and the Rabies Vaccine

Louis Pasteur (1822–1895) was not the first scientist to argue that infectious diseases were caused by germs, but his work provided the foundations of virtually every branch of nineteenth-century microbiology. Practical results of Pasteur's research, including the pasteurization process and the development of protective vaccines for infectious diseases, had an obvious impact on human health and welfare. In addition to his genius for selecting research subjects, Pasteur had a genius for publicity and was more than willing to publicly debate his critics.

Among the aphorisms attributed to Pasteur, one of the most quoted is: “Chance favors only the prepared mind.” Chance played a significant role in Pasteur's development of a vaccine for chicken cholera. He isolated a microbe from birds with the disease and, by accident, discovered that weakened cultures of the microbe could be used as protective vaccines. Pasteur realized that he had created a laboratory strain of the chicken cholera microbe that acted like Jenner's cowpox vaccine—the only instance of a “protective vaccine” known at the time. Therefore, Pasteur adopted and expanded the term “vaccine” in honor of Jenner's discovery. As the causative agents of other diseases were discovered, Pasteur and others attempted to create protective vaccines by attenuating (weakening) these pathogens in the laboratory. By extending his work on preventive vaccines to rabies, Pasteur helped establish the foundations of virology.

Although Pasteur hoped that his work on animal vaccines would be extended to human diseases, he thought it would be unethical to experiment on human beings. One way of escaping this ethical dilemma was to choose a deadly disease that attacked both humans and animals. Rabies is invariably fatal, but not all humans bitten by rabid animals contract the disease, and the incubation phase is long and unpredictable.

Pasteur believed that the cause of an infectious disease must be a living entity that multiplies in its victims. He worked to identify and isolate the rabies microbe from preparations that were capable of transmitting the disease, using live animals to culture the virus.

In 1879, Pierre-Victor Galtier (1846–1908), a professor at the Veterinary School of Lyon, demonstrated that rabies could be transmitted from dogs to rabbits, which provided a safer experimental animal. Pasteur and his associates prepared a vaccine by suspending the spinal cords of infected rabbits in a drying chamber to attenuate the rabies virus. After about two weeks the material became essentially harmless. Using these preparations, Pasteur could protect dogs that had been bitten by rabid animals or inoculated with virulent laboratory preparations containing the live virus.

In 1885, nine-year old Joseph Meister was brought to Pasteur after a vicious attack by a rabid dog. Advised by physicians that Meister would inevitably die of rabies, Pasteur began a series of inoculations to save his life. Meister recovered completely, and within a year more than 2,000 people received the Pasteur rabies vaccine. The public glorified Pasteur's achievement as the greatest triumph of biological science, but the crudeness of the vaccine and, in some cases, the long interval between the bite and treatment, led to tragic failures as well as successes.

Successful immunization depends on how soon the inoculations are begun and the individual's reaction to the vaccine. Pasteur's adversaries—conservative physicians, veterinarians, antivivisectionists, and antivaccinators—denounced his methods and accused him of creating a new disease, which they called “laboratory rabies.” Critics also argued that because Pasteur was not a physician, he was not qualified to study or treat human disease. However, thousands of victims of dog or wolf bites preferred the risks of the vaccine to an agonizing death from rabies. The risks and benefits associated with modern vaccines are still subjects of intense debate.

Viral Diseases of Plants, Animals, and Bacteria

Pasteur's studies of rabies showed that the infectious agents of some diseases proved difficult or impossible to identify because they were invisible under the microscope and would not grow in ordinary laboratory media. Various pathogens that could be seen, but not grown in cell-free laboratory cultures, were eventually identified as bacteria and protozoans with unusual growth requirements (e.g., rickettsias, mycoplasmas, brucellas, chlamydias). Microbiologists also discovered that some invisible infectious agents were capable of passing through ceramic filters known to trap even very small bacteria.

In the early twentieth century the term virus, previously used interchangeably with germ and microbe, began to used for filterable, invisible microbes that could pass through filters that trapped bacteria and were invisible to the light microscope. Although filterable and invisible viruses apparently did not to grow in cell-free media, scientists generally did not consider this a fundamental quality because they could not exclude the possibility that exotic microbes might need special media and growth conditions.

Invisible, filterable viruses were thought to cause many important human and animal diseases, but it was the study of a plant virus, the tobacco mosaic disease (TMD) that led to an understanding of their fundamental characteristics. German researcher Adolf Eduard Mayer (1843–1942), Dutch microbiologist Martinus Beijerinck (1851–1931), and Russian biologist Dimitri Ivanovski (1864–1920) are honored as the founders of virology because of their work with TMD.

In 1886 Mayer discovered that he could transmit TMD to healthy plants with filtered extracts of sap from diseased plants. In 1892 Ivanovski also demonstrated that the infectious agent for TMD could pass through the finest filters available. Beijerinck, who had studied with Mayer, suggested that TMD was caused by a contagium vivum fluidum (contagious living fluid) that needed living plant tissues to reproduce. In other words, Beijerinck suggested that the fundamental difference between bacteria and viruses was not a matter of size. Viruses might be infectious agents that could only reproduce within living cells, making them obligate parasites of living organisms that could not be cultured in vitro on any cell-free culture medium.

Tobacco mosaic virus (TMV) was the first to be purified and crystallized, a landmark achievement for which American biochemist Wendell Meredith Stanley (1904–1971) won the 1946 Nobel Prize for chemistry. In the 1930s, Stanley isolated a “crystalline proteinaceous preparation” from infected tobacco plants that retained the infectious power of TMV. His results suggested that viruses could be thought of as living molecules rather than organisms. Using the techniques that became available in the 1940s, including the ultracentrifuge and the electron microscope, scientists found that TMV consisted of a central core of RNA surrounded by a protein coat. These findings brought the study of virology beyond its classical place in medical microbiology and into the domain of chemistry and physics.

Studies of a filterable virus disease of animals were conducted by two German researchers, bacteriologist Friedrich Löffler (1852–1915) and virologist Paul Frosch (1860–1928). In 1897 they reported that foot-and-mouth disease (FMD) could be transmitted to healthy cattle by filtered fluids from the vesicles of infected animals. FMD is generally considered a disease of farm animals, but a few human cases have occurred. All attempts to isolate the causative agent from the lesions of sick animals were unsuccessful. Loeffler and Frosch continued to think of the infectious agent as a very small and unusual microbe rather than a fundamentally novel entity. However, they did suggest that other infectious diseases, such as smallpox, cowpox, and cattle plague, might be caused by similar filterable microbes. Within a few years, filterable viruses were suspected of being the cause of various plant, animal, and human diseases, including certain cancers.

In 1901, American army pathologist Walter Reed (1851–1902) and other members of the U.S. Army Yellow Fever Commission in Cuba demonstrated that yellow fever was caused by a filterable virus transmitted to humans by mosquitoes. Yellow fever became a disease of special interest to the United States as a result of the Spanish-American War and the subsequent occupation of Cuba. In 1927 South African-born American microbiologist Max Theiler (1899–1972) demonstrated that the causative agent was indeed a virus; he won the 1951 Nobel Prize in physiology or medicine for his work and the subsequent development of a safe and effective vaccine. Because yellow fever has a major animal reservoir (jungle primates), unlike smallpox, eradication is not a practical goal.

Bacteriophages: Bacterial Viruses and Molecular Biology

In 1915 English bacteriologist Frederick Twort (1877–1950) discovered that even bacteria can be attacked by filterable viruses. Two years later, French Canadian microbiologist Félix d'Hérelle (1873–1949) discovered the same phenomenon. Convinced that this infectious agent must be an obligate parasite of the dysentery bacillus, d'Hérelle called it a bacteriophage (bacteria-eater). D'Hérelle predicted that bacteriophages would be found for other pathogenic bacteria and that laboratory manipulations could transform naturally occurring phages into specially modified agents that could be used to destroy bacteria that caused human disease, just as Salvarsan, an arsenical drug recently introduced by Nobel laureate Paul Ehrlich (1854–1915), had been used to destroy the bacterial agent that caused syphilis. American novelist Sinclair Lewis (1885–1951), in collaboration with American microbiologist Paul de Kruif (1890–1971), explored this idea in Arrowsmith (1925), a popular and successful novel about an idealistic young American physician hoping to make a significant contribution to biomedical research. Except in the former Soviet Union, research on phage therapy was generally abandoned after the discovery of penicillin.

IN CONTEXT: WHAT IS A VIRUS?

Viruses are small infectious agents that can only multiply within living cells. They are, therefore, defined as “infectious obligate intracellular parasites.” Viruses contain genetic information, but they need to use the biosynthetic machinery of living cells to produce new virus particles. Their origins are obscure, but they may have evolved from microorganisms that lost many of their cellular components and functions or from genes that escaped from an ancestral cell and gained the ability to infect other cells. All viruses consist of a nucleic acid core, which serves as genetic material, surrounded by a protective protein coat. Viruses' genetic material is either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be either single- or double-stranded. In some virus families the nucleic acid-protein complex is surrounded by a lipid membrane.

Despite their small size and relative simplicity, viruses are diverse in size, shape, structure, and their ability to infect host species, which range from bacteria, fungi, and algae to plants, animals, and humans. Some are species specific; others can infect a wide variety of hosts. Viruses are classified according to their structure, nucleic acid, and the presence or absence of a lipid membrane.

When viruses replicate, the whole process from adsorption in the host cell membrane to the release of new viruses may take as little as 30 minutes in the case of some bacteriophages to several days for some animal viruses. Some, such as the herpes simplex virus, are able to enter host cells and establish a latent infection, that is, a condition in which the virus remains dormant. Under appropriate conditions, a latent virus can be reactivated and begin to produce progeny that attack other cells. When this phenomenon occurs in bacteriophage, it is called lysogeny.

By the end of the twentieth century, the growing threat of antibiotic-resistant bacteria had revived interest in phage therapy. An estimated 90,000 Americans died in 2000 of hospital-acquired infections caused by

IN CONTEXT: THE ELECTRON MICROSCOPE

The electron microscope made it possible to see viruses, classify them in terms of their structure, and observe interactions between cells and the viruses that attacked them. Both the ultracentrifuge, which was introduced in the 1920s, and the electron microscope were critical factors in the advance in virology. These instruments facilitated the determination of the size and structure of viruses.

With the light microscope, invented in the 1590s, scientists could see a new world teeming with previously invisible entities. This was so revolutionary that some critics denounced the instrument as an unnatural device that generated false and misleading images. During the seventeenth century, microscopists observed plant and animal cells, molds, protozoa, and bacteria, but viruses remained invisible until the development of the electron microscope.

Microscopists are, of course, concerned with both resolution (the ability to distinguish details) and magnification (the increase in the size of an object). By the end of the nineteenth century, scientists had essentially reached the limits of light microscopy. That is, despite improvements in microscopes and sample preparation, the limiting factor became the wavelength of light. Physicists suggested that using electron beams instead of light could produce microscopes with unprecedented resolution and magnification.

Although the concept seemed reasonable in principle, fundamental problems had to be solved before the electron microscope became a useful tool. Many biologists and physicists assumed that the techniques associated with electron microscopy would destroy the fine structure of biological objects. In addition to technical problems, the development of the electron microscope was obstructed by patent battles involving industrial firms, independent scientists, research laboratories, and universities in Germany, Belgium, and the United States.

During the 1930s, German electrical engineer Ernst Ruska (1906–1988) and colleagues constructed a series of instruments they called “super-microscopes.” Improving on primitive prototypes, Ruska built an instrument in 1933 that provided significantly better magnification and resolution than light microscopes. His brother, Helmut Ruska (1908–1973), studied its applications in medicine and biology. Their work eventually revealed the submicroscopic structures of bacteria, parasites, and the remarkable diversity in size and structure of different viruses. Instead of organizing viruses according to the diseases they caused, Helmut Ruska proposed a classification system based on viral structure, as determined by electron microscopy.

The 1986 Nobel Prize for physics was awarded to Ernst Ruska, German-born physicist Gerd Binnig (1947–) and Swiss physicist Heinrich Rohrer (1933–), who developed the scanning tunneling microscope, an instrument that can provide a topographic map of the surface of viruses, DNA molecules, and other objects.

antibiotic-resistant bacteria. Many researchers are skeptical about phage therapy, but some drug companies are exploring the use of genetic engineering to control potentially useful “therapeutic phages.” Critics warn that phage preparations might contain unknown contaminants and that pathogenic viruses might arise through recombination or mutation, leading to an attack on the patient instead of the bacterial target.

Molecular Biology and Bacteriophage

During the 1930s, virology gradually separated itself from bacteriology and became a scientific discipline in its own right. But it was not until 1953 that Italian-born American biologist Salvador Luria (1912–1991), one of the founders of the “phage group” that established the American school of molecular biology, published General Virology, the first major textbook devoted to the new field, and edited the journal Virology, which debuted in 1955.

Bacteriophages were adopted as “experimental animals” by Luria, German-born American biologist Max Delbrück (1906–1981), and other pioneers of molecular biology. Phage studies led James Watson (1928–) to what was generally accepted as the central dogma of the molecular biology of the gene: “DNA makes RNA makes protein.” Eventually, two American virologists, Howard Temin (1934–1994) and David Baltimore (1938–), proved that information could flow from RNA to DNA by means of an enzyme called reverse transcriptase. This discovery has been critical to understanding a group of viruses called retroviruses, one of which is human immunodeficiency virus (HIV), the virus that causes AIDS (acquired immune deficiency syndrome).

A discovery of great importance to human health, American pathologist Francis Peyton Rous's (1879–1970) report of an association between a virus and cancer, was initially ignored. In 1909 Rous began a series of experiments on a malignant tumor that had appeared on a Plymouth Rock hen. After successfully transplanting the sarcoma, Rous demonstrated that it could also be transferred by cell-free filtrates of tumor tissue, but his viral hypothesis was generally ignored until the 1950s. Rous was awarded a Nobel Prize in 1966 for his pioneering studies of the possible relationship between malignant tumors and viruses. Virologists eventually identified other viruses that appear to cause cancer in humans and other animals, but Rous sarcoma virus, the retrovirus that causes cancer in chickens, was the first tumor virus to be identified. Such viruses are called oncogenic, carcinogenic, tumorigenic, or transforming viruses.

Vaccines and Antivaccine Movements

Despite intense efforts to identify drugs that fight viral diseases, relatively few are available. Because viruses rely on the metabolic apparatus of the host cell, drugs that inhibit virus replication are likely to be harmful to the

IN CONTEXT: HIV/AIDS

The first account of the disease that would eventually be known as AIDS was published in 1981. Studies of the mysterious syndrome were triggered by reports of deadly Pneumocystis cariniipneumonia (PCP) in previously healthy homosexual men in Los Angeles and New York. PCP was generally seen only in severely malnourished children or adults undergoing chemotherapy. By 1982 similar cases were reported throughout the country, often complicated by Kaposi's sarcoma, a form of cancer usually found only in elderly men. Eventually, the collection of symptoms eventually known as AIDS appeared among intravenous drug users, hemophiliacs, the sexual partners of people in these risk groups, and infants born to women with the disease. Within five years of the first reports, more than one million Americans were infected.

AIDS was originally defined in terms of progressively severe symptoms that ultimately led to profound immunodeficiency, opportunistic infections, cancers, and death. The course of the disease was linked to the disappearance of certain kinds of white blood cells (helper T cells) needed to fight off infections. The breakdown of the body's immune system results in vulnerability to many normally harmless microorganisms. Research indicated that the disease was transmitted by sexual intercourse and by contaminated needles.

In 1984 American virologist Robert Gallo (1937–) and French virologist Luc Montagnier (1932–) independently discovered a retrovirus that apparently caused AIDS. After a bitter priority dispute, researchers agreed to call the virus HIV, or human immunodeficiency virus. A blood test to detect HIV carriers was introduced in 1985. The antiviral drug AZT (azidothymidine) was in widespread use by 1987, but it was not effective in advanced cases and caused many adverse reactions. By 2000, complex, expensive multidrug treatments had transformed AIDS from a fatal disease to a chronic, treatable condition, at least in the United States where an estimated 900,000 people were living with HIV/AIDS.

Many bizarre suggestions were offered for the origin of AIDS, but epidemiologists traced the virus to Africa, where deaths from the opportunistic infections that were so unusual in Western nations would have received little or no attention. In the 1990s, some writers suggested that a similar virus (simian immunodeficiency virus, SIV) found in African monkeys might have infected humans who butchered or ate infected primates. Once HIV became established in humans, poor sterile techniques in clinics, hospitals, and mass-vaccinations campaigns could have disseminated the virus. Virologists warn that the expansion of the bush-meat trade in Africa could unleash other obscure animal viruses.

By the time of the Fourteenth International AIDS Conference in 2002, more than 20 million people had died of AIDS. Epidemiologists predicted that unless prevention programs were greatly expanded, AIDS might claim almost 70 million more lives by 2020. In severely affected African countries, more than 30% of adults were HIV-positive. Epidemiologists estimated that of the 40 million HIV-positive people in the world, about 6 million were sick enough to need antiretroviral drugs, but less than 5% got appropriate treatment because of the lack of medical resources. Despite decades of effort, as of early 2008 the virus continued to evade efforts to create a safe, effective vaccine.

patient. Controlling viral diseases, therefore, depends on the availability and widespread use of preventive vaccines.

Debates about the safety and efficacy of vaccines have raged ever since the first experiments on smallpox inoculation and vaccination, long before the establishment of the sciences of virology and immunology. Many arguments about vaccination were more emotional than scientific, such as the assertion that any interference with nature or the will of God was immoral. In the nineteenth to the early twentieth century, leaders of the antivaccination movement denounced smallpox vaccination, especially mandatory vaccination, as an unsanitary procedure that introduced dangerous foreign matter into the body. The movement was broad enough to encompass unorthodox healers, liberal individualists, members of various religious groups, scientists, physicians, and social reformers who argued that vaccination allowed the state, and the rich, to ignore the social roots of disease, such as poverty. Eminent critics of vaccination included the British sociologist and philosopher Herbert Spencer (1820–1903) who disapproved of voluntary vaccination and absolutely detested compulsory measures. Alfred Russel Wallace (1823–1913), English naturalist and codiscover of evolution by natural selection, denounced mandatory vaccination as a crime against liberty, health, and humanity.

In England, where the first compulsory vaccination legislation was passed in 1853, opponents formed anti-vaccination leagues that generated support and publicity by staging popular demonstrations and circulating pamphlets. The movement claimed victory when the right to avoid vaccination was granted in 1907. Critics of mandatory vaccination objected to the enactment of laws that infringed on personal liberty, but German physician Johann Peter Frank (1745–1821), a pioneer of what is now called social medicine, called vaccination medicine's greatest and most important discovery. He predicted that if all nations adopted compulsory vaccination, smallpox would rapidly disappear. In America, future President Thomas Jefferson (1743–1826) vaccinated his entire household, his family and his slaves, in 1800, enthusiastically promoting the practice that had just replaced the more dangerous smallpox

inoculation. Six year later, in a letter to Jenner, Jefferson predicted that future generations would only know about the “loathsome smallpox” through history books, because Jennerian vaccination would have eradicated the disease.

When infectious diseases such as smallpox were highly feared and extremely widespread, the benefits of vaccination were fairly obvious. However, by 2000, many adults had never experienced or observed outbreaks of measles or poliomyelitis and could not comprehend the consequences of rejecting vaccinations. Many people, therefore, underestimate the danger of infectious diseases and overestimate the risks of vaccination. The National Vaccine Information Center, for example, is one of many advocacy groups that question the safety of vaccines. Many Internet sites, newspapers, and magazines publicize stories of autism, seizures, crib deaths, learning disabilities, multiple sclerosis, and Guillain-Barré syndrome allegedly caused by vaccination.

Some opponents of vaccination claim that the global campaign for the eradication of smallpox was responsible for the AIDS epidemic. Public health experts acknowledge that although any medical intervention can cause adverse effects in some people, the side effects associated with vaccination are rare and generally less serious than the effects of the disease. Scientists are often frustrated when reporters fail to explain that adverse effects occurring after vaccination are not necessarily caused by it, a failure that allows readers to draw unwarranted conclusions and increases the suspicion that vaccines are not safe.

According to the CDC's national immunization program, about 1% of all American children are exempt from vaccination. In some states significant numbers of children are not vaccinated against viral diseases such as polio, measles, mumps, rubella, hepatitis B, and chickenpox, because their parents have philosophical or religious exemptions. All 50 states allow medical exemptions for children who are immuno-compromised or allergic to vaccines; almost all states allow religious exemptions; and about 20 allow personal or philosophical exemptions. Public health officials warn that some churches were deliberately established to secure donations from parents seeking certificates that guarantee a religious exemption from mandatory vaccinations against childhood diseases.

In the 1980s, the New York City schools essentially eliminated outbreaks of measles, mumps, and rubella by refusing to admit children without proof of vaccination. If all school systems followed the New York example, such preventable childhood diseases could be eliminated throughout the United States. But in 2006, the worst outbreak of mumps in 20 years spread through at least seven Midwestern states. According to the CDC, about

1,000 people contracted the disease. A vaccine for measles, mumps, and rubella was introduced in the United States in 1967, but, unless children receive two shots, the vaccine seems to be only about 80% effective for mumps. Officials said that there could have been many thousands of cases if the effective two-dose inoculations had not been widely used since the 1970s.

Public health officials are frustrated by religious exemptions that obviously are used to exploit a legal loophole. Many parents have never themselves experienced or observed outbreaks of measles or polio and cannot comprehend the consequences of rejecting vaccinations. Clusters of unvaccinated children pose a threat to the “herd immunity” that is needed to prevent epidemics from developing and spreading to very young infants, the elderly, and those with weakened immune systems who cannot be vaccinated. The public health impact of religious exemptions has been demonstrated many times. The last two American polio outbreaks were in Amish and Mennonite communities in 1979 and in a Christian Science school in Connecticut in 1972. Measles outbreaks in the 1980s affected hundreds of children in Christian Science and Amish communities and killed at least five children. In 1991, 890 cases of rubella, leading to more than a dozen deformed children, hit Amish areas.

Vaccine resistance is not, of course, limited to wealthy industrialized nations where few parents have had any direct experience with diseases like measles, rubella, or polio. In 1988 the WHO began a global vaccination campaign dedicated to eradicating polio, but, despite success in reducing the overall burden of disease, in 2000 polio remained endemic in some areas. By 2004 epidemiologists discovered that the disease was spreading from northern Nigeria to previously polio-free African countries. Nigerian officials stopped polio vaccinations in 2003 because of religious and political opposition. Critics of vaccination claimed that polio vaccine caused sterility in girls and that health organizations sponsored by Western countries were deliberately using tainted vaccine against Muslims.

Viroids and Prions

By the 1970s, when the nature of viruses and other microbes seemed to be fairly well understood, the discovery of radically new infectious agents known as viroids and prions offered an unanticipated challenge to microbiologists. Unlike viruses, viroids appear to be small, single-stranded RNA molecules that have infectious properties, but lack the protein overcoat common to virus particles. In 1971, American plant biologist Theodor O. Diener (1921–) reported that potato spindle tuber disease was caused by a novel infectious agent consisting of naked RNA. Although this claim was originally controversial, by 2001 scientists had discovered about 30 viroid species and hundreds of variants. Despite the absence of a protective protein coat, viroid RNA seems to be remarkably resistant to degradation.

Viroids are primarily associated with plant diseases, but some studies suggest that they may also be involved in tumor formation and other diseases of animals. Although many aspects of viroid replication, transmission, and disease-causing properties are obscure, the discovery of these “naked intruders” has stimulated research into the interaction between foreign RNA molecules and human diseases. Viroids may differ from viruses in many ways, but because they contain nucleic acid, they still seemed to fit into the fundamental framework of molecular biology.

Prions, the most bizarre of all the infectious agents discovered in the twentieth century, raise more fundamental problems for virology, microbiology, and molecular biology. The idea that certain neurological degenerative diseases might be caused by a novel infectious agent was suggested by American physician and medical researcher Carleton Gajdusek's (1923–) studies of kuru, a disease found among the Fore people of New Guinea. Based on epidemiological field studies, Gajdusek came to the conclusion that kuru was transmitted by mourning rituals during which women and children ate the brains of deceased relatives. Using brain tissue from kuru victims, Gajdusek and his associates were able to transmit the disease to chimpanzees. Because symptoms did not appear for about two years, Gajdusek suggested that kuru was caused by a “slow virus.”

In 1972 American neurobiologist Stanley B. Prusiner (1942–) became interested in Creutzfeldt-Jakob disease (CJD) after the death of a patient. CJD seemed to strike sporadically, but some scientists thought that it might be caused by a slow virus like the obscure agents associated with kuru and scrapie, a similar neurological disease previously found only in sheep. Using scrapie as his experimental model, Prusiner found that he could transmit the infectious agent to hamsters. When he isolated the scrapie agent from the brains of diseased hamsters, he was surprised to find that it seemed to consist of a specific protein. Ten years later, Prusiner summarized his work and introduced the term prion, which stood for “proteinaceous infectious particle.” The “protein only” hypothesis was extremely controversial, but skeptics were unable to find any nucleic acid in the scrapie agent. In 1997 Prusiner was awarded the Nobel Prize for discovering prions and establishing a new category of disease-causing agents.

All of the diseases attributed to prions are known as transmissible spongiform encephalopathies (TSEs). Prion diseases of animals include scrapie, transmissible mink encephalopathy, chronic wasting disease of mule deer and elk, feline spongiform encephalopathy, and bovine spongiform encephalopathy (BSE, commonly known as mad cow disease). Human diseases attributed to prions include Creutzfeldt-Jakob disease (CJD) and a new variant (vCJD) that appears to be related to BSE; fatal familial insomnia (FFI); Gerstmann-Sträussler-Scheinker syndrome; and kuru. Unlike bacteria and viruses, prions provoke little or no immune response. They resist alcohol, boiling, hospital detergents, formalin, autoclaving, radiation, exposure to ultraviolet light and other methods that inactivate bacteria and viruses.

Modern Cultural Connections

During the 1960 and 1970s, when the wealthy nations of the world were confident that infectious diseases were under control, the appearance of a series of deadly hemorrhagic fevers in Africa, South America, and the United States provided a clear warning of the dangers of emerging viruses. Marburg, Lassa fever, and Ebola viruses, which originated in Africa, are among the most feared of the newly discovered pathogens. Death rates during small epidemics of these diseases may have reached 80 to 90%, but the source of these outbreaks and the animal reservoirs that harbor the viruses remain obscure.

Ancient Chinese texts suggest the existence of hemorrhagic fevers that might have been caused by hantaviruses, but this family of diseases was apparently unknown to Western doctors until the 1950s. Outbreaks occurred among United Nations troops during the Korean War, and in Japan, Russia, and Europe. Studies of the hantaviruses, Lassa fever virus, Ebola, and other emerging viruses indicate that they tend to mutate frequently and adapt to a wide range of hosts. The appearance of West Nile virus in New York City in 1999 and its subsequent spread to other states demonstrated how easily pathogens could establish themselves in new regions.

West Nile fever, first identified in Uganda in 1937, presented a rare opportunity for scientists to chart the ways in which a virus could establish itself in a new region.

Since 1998, when the first International Conference on Emerging Infectious Diseases met, epidemiologists have emphasized the importance of understanding and reacting to the emergence and reemergence of infectious diseases, especially viral diseases. The catalog of human diseases is likely to grow as new diseases appear and old categories, such as “fevers of unknown origin” (FUOs), are reexamined as a consequence of heightened surveillance and improved diagnostic techniques. Factors that will probably influence the future distribution of emerging diseases include ecological degradation, climate change, the movement of animals from their natural habitats, population growth, migration, war, sexual behaviors, intravenous drug use, international travel and commerce, food processing, livestock handling, organ transplants, and breakdowns in public health measures like sanitation, vaccination, and insect control. Global warming could contribute to changing patterns of disease through the redistribution of host species, vectors, and pathogens. Exotic pets, exotic foods, and bushmeat markets may also contribute to the threat of emerging diseases. For example, monkeypox was found only in Africa until 2003, when more than 70 cases occurred in the United States. The virus was imported within Gambian giant pouched rats shipped from Ghana to American pet stores.

Information about deadly viral diseases, coupled with renewed geopolitical instability and increasing fears of terrorism, have led to calls for creating a “bioterrorism Manhattan Project,” that is, a program as intensive as the effort to produce the atomic bomb during World War II. Viruses are the natural focus for such a project, because no broad-spectrum antivirals analogous to antibiotics exist, though scientists can characterize the genes that code for virulence, synthesize the proteins responsible for virulence, transfer virulence genes from animal viruses to human viruses, and even synthesize whole viruses containing novel combinations of genes.

Research on emerging diseases and diseases now attributed to viroids and prions suggests that other invisible, mysterious, and perhaps menacing creatures might well exist in the submicroscopic world. In addition to developing a research agenda to deal with the hordes of infectious agents already present in nature, virologists and molecular biologists are being called on to find ways to detect and respond to infectious agents that might be used as biological weapons. Because biological weapons, unlike conventional weapons, are fundamentally disease-causing agents that grow and multiply in the victims and spread to others without respecting national borders, the health and safety of all people may well depend on the skills and resources available to virologists and the biomedical community.

Primary Source Connection

In the following article, virologist Jack Woodall recounts memories of belonging to teams that identified and determined the cause of Machupo hemorrhagic fever and also isolating other new viruses from trapping and studying animals in the Amazon rainforest.

Woodall is a graduate of Cambridge University, UK, and received his Ph.D. from London University, UK. During a long and distinguished career, Woodall has served and worked at the East African Virus Research Institute, Entebbe, Uganda; Belem Virus Laboratory in Brazil and the Yale Arbovirus Research Unit in New Haven Connecticut; been head of the Arbovirus Laboratory, New York State Health Department; and served as director of the Centers for Disease Control's (CDC) San Juan Laboratories in Puerto Rico. In 1981 he began work for the World Health Organization (WHO) and was a member of the WHO Gulf Emergency Task Force in support of the UN Special Commission (UNSCOM) in Iraq and leader of the WHO delegation to the Third Review Conference on the Biological Weapons Convention. Until 2007 he served as visiting professor at the Institute of Medical Biochemistry and director of the Nucleus for the Investigation of Emerging Infectious Diseases at the Federal University of Rio de Janeiro, Brazil.

Woodall was a cofounder of ProMED-mail, the online outbreak early warning system of the Program for Monitoring Emerging Diseases (ProMED) of the International Society for Infectious Diseases (ISID), and Web Site Editor and Council member (ex officio), American Society of Tropical Medicine & Hygiene. He is a member of the Biological Weapons Working Group of the Center for Arms Control and Non-Proliferation, Washington, DC, and a board member of the Sabin Vaccine Institute, Washington, DC.

VIRUS HUNTERS

The people of San Joaquim were dying—they were bleeding to death from a disease nobody had ever seen before. They called it “el tifo negro,” the black typhus, later to become known as Bolivian hemorrhagic fever. In this little one-horse garrison town in the lowlands on the Amazonian side of the Bolivian Andes, close to the Brazil border, soldiers and citizens alike were sickening, and no one knew why.

Under the Alliance for Progress established by President John F. Kennedy (1917–1963) between the United States and Latin America, Bolivia asked for help from the United States, and a team was flown in from the National Institute of Health's Middle America Research Unit in the Panama Canal Zone. Heading it was physician Karl Johnson, one of the great virus hunters of the end of the twentieth century. I was greatly privileged to be working with his team, on a traveling fellowship from the Rockefeller Foundation at the time he isolated the causative agent from a human case. It was a new virus, which he named Machupo virus, after a local place name.

Everybody knows that viruses are really nasty pieces of work. They are even smaller than bacteria, and untouched by antibiotics, so for many there is no cure, nor even a preventive vaccine. They are responsible for some of the deadliest diseases on the planet: Ebola, yellow fever, smallpox, AIDS, SARS (Severe Acute Respiratory Syndrome), polio, and influenza, both human and bird, to name only seven. The scientists who tackled them have gone down in history as the “virus hunters,” and some of them paid for their research with their lives.

At the beginning of the twentieth century, the United States Army played a prominent role in the fight against yellow fever. A number of United States military physicians and volunteers died in early tests that proved that the virus was transmitted by mosquitoes. This discovery paved the way for control programs led by General William C. Gorgas, which resulted in the eradication of yellow fever from Havana, Cuba, and of the urban disease from Brazil. It also permitted the completion of the Panama Canal, work on which had been stalled by the huge toll exacted by yellow fever and malaria.

The Rockefeller Foundation's International Health Division set up laboratories in Africa and South America specifically to study yellow fever at its source. Six of their researchers died of the disease, but their work paid off with the isolation of the virus and the development of the 17D yellow fever vaccine, still one of the best vaccines ever invented.

The stories of these pioneer researchers are told in many books, but I want to tell you about two modern virus hunters with whom I had the good fortune to work myself. In San Joaquim, Karl Johnson had set up a breeding colony of hamsters and a separate infected animal room, well screened against mosquitoes and with the individual cages fitted with virus filters in their steel mesh lids. A separate lab had a glove box, inside which the hamsters could be inoculated safely with specimens. The only problem with this was that there was only one glove box, and it had to be sterilized with a disinfectant spray and left for an hour between each litter of hamsters inoculated, which slowed down the work and meant working very late hours. There was also a thatched hut where the zoologist took the rodents he trapped in the town for processing and where the entomologist combed their fur for ectoparasites, such as ticks and mites, in case these were involved in the transmission of the disease (they weren't). There was an autopsy room for humans, but the dead cow that came in at night with a history of bleeding had to be necropsied on a wooden bullock cart in the open air by the light of hurricane lamps. The cow was negative for the virus.

The colonel in charge of the town's garrison invited us to take our meals with him in his quarters. There is a photo of him at the head of the table dining with the team. A week later he was dead from the black typhus. But shortly after that there was euphoria—hamsters inoculated with autopsy material from another victim came down with signs of infection, and a virus was isolated from their brains. Now reagents could be made to test for antibodies in the blood of survivors and wildlife.

The next step was to find out where the disease was coming from and how to stop its transmission. Karl suspected the wild rodents that seemed to have recently overrun the town. He set up a system to trap out all the rats in one half of the place. Lo and behold, after that, no more cases occurred in that area. So the trapping was extended to the whole town, and the epidemic was stopped cold.

What eventually emerged was an extraordinary story. Apparently anti-malaria teams had deluged the town with DDT on a control visit. Cats are highly susceptible to DDT, and they get a fatal dose of it by preening their fur after being caught in the spraying or rubbing against surfaces that have been sprayed. All the cats in the town had died, so the wild rodents from the fields and forest around the town were able to infiltrate the houses in their quest for easy pickings. Some of them were infected with the virus, which they excreted in their urine and droppings inside houses. These dried out in the tropical heat and turned to dust, which, when stirred up by walking through or sweeping the rooms, was inhaled by the inhabitants, giving them the disease. So an intervention to control one fatal disease ended up causing another.

All this was in 1963. Thirteen years later, Karl was working for the United States Public Health Service as head of the Centers for Disease Control's Special Pathogens lab (the euphemism for the lab that handled the most dangerous disease agents known, needing Biosafety Level 4 containment, either in a chain of glove boxes or in negative pressure labs with the researchers wearing space suits). He found himself called out to investigate another hemorrhagic fever epidemic, this time in Yambuku, Zaire (now known as the Democratic Republic of the Congo). The American researchers found that the virus was transmitted by the inadequately sterilized, reused needles and syringes used for giving injections to the patients. The epidemic was being spread in the hospital. Worse, local burial custom demanded that the relatives remove by hand the viscera of the dead person, and of course this was done without any concept of sterile precautions, so that the blood of the deceased infected the relatives. When these practices in the hospital and home were stopped, the epidemic ceased.

Karl's lab showed that the disease agent was a new member of a new family of viruses, the Filoviridae or “thread viruses” because they looked like partially coiled threads (some say more like shepherd's crooks) under the electron microscope. Karl named it Ebola virus, after a nearby river. He couldn't call it Congo virus because that name had already been taken by another, different virus isolated earlier in the same country, which was eventually named as the causative agent of Crimean-Congo hemorrhagic fever.

The labs run by Karl were dynamic places full of eager young researchers bubbling over with ideas about the viruses that cause disease and their epidemiology— where they hide in nature, how they are transmitted, and why they suddenly emerge to cause outbreaks. I would dearly have liked to have joined his lab, but instead I was hired by the Rockefeller Foundation to run their virus lab at the mouth of the Amazon, and so came to know well another modern virus hunter—Bob Shope.

I first met Bob on that same Rockefeller Foundation travel fellowship that took me to Bolivia. His family was away at the time and I was a guest in his home in Belem, Brazil, so we spent many happy hours both in the lab and in his house discussing the riddles of the viruses of the rain forest. His lab had a small mammal recapture program with a grid of traps in the forest at the edge of town, where wild rodents and marsupials were trapped daily, weighed and measured, and obliged to donate a blood sample so that their medical history could be followed. They were exposed to forest mosquitoes that transmitted all sorts of interesting viruses to them, which were then isolated from their blood samples in lab mice. Many of the viruses were new to science. Other mammals such as bats, sloths, and tree porcupines were also caught and studied, and a series of ingenious mosquito traps baited with monkeys or mice were run daily to provide pools of mosquitoes, sorted by species, which also yielded more such viruses. There were even lab workers who volunteered to go out into the forest at night to catch mosquitoes coming to bite them. Some of the human volunteers didn't manage to catch all the mosquitoes before the insects got their bites in, so they came down with jungle fevers. The viruses isolated from their blood provided proof that some of these new viruses could cause disease in people who went into the forest to hunt, collect timber, or clear plantations.

When Bob left Belem for the Yale Arbovirus Research Center (YARU), where he worked as researcher and then director for 30 years, I took over his lab and kept in close touch with him for many years. “Arbovirus” is short for “arthropod-borne virus,” meaning viruses transmitted by fleas, ticks, and mites, as well as mosquitoes. YARU became a World Health Organization Collaborating Center and the world reference center for these and other viruses, because many viruses isolated from wildlife by field labs established around the world by the Rockefeller Foundation, France's Pasteur Institutes, and others turned out not to be transmitted by arthropods—notably Machupo and Ebola. Bob became a living encyclopedia of information on the origins and interrelationships of hundreds of viruses from around the world, including the many viruses from wildlife related to rabies. Some of these have become what we now call emerging diseases. He mentored students and post-docs from around the world, who worked in his lab on Rift Valley fever, Lassa fever, Argentinean, Brazilian, and Venezuelan hemorrhagic fevers, and other dangerous viruses. But he never forgot the lessons from his field experience in Brazil with exotically named viruses such as Caraparu, Oriboca, and Marituba. After his retirement, the YARU lab closed, and he took the world reference collection of arboviruses to the University of Texas Medical Branch at Galveston, where he worked until his death in 2004.

So although it is all the rage now to go into molecular virology and sequencing, I hope that at least some of today's students will be inspired by the examples of Karl and Bob to go out into the field and get their hands dirty trapping wildlife and mosquitoes, finding out what viruses they are carrying and what makes those viruses tick. Because those viruses are the emerging diseases of the future, and we need to know as much as we can about them before they strike.

Jack Woodall

woodall, jack. “virus hunters.” in infectious diseases in context, brenda w. lerner and k. lee lerner, eds. detroit: cengage learning, 2007.

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Lois N. Magner

Scientific Thought: In Context