Monday, 8 July 2019

Viral Pathogenesis

Viral Pathogenesis

Viral Pathogenesis

Introduction to Viral Pathogenesis

While the title of Zinsser’s classic volume Rats, Lice and History may trigger a wry smile, the ideas proposed in this classic volume about pathogens and the diseases they cause remain as relevant today as when they were published in 1934. As Zinsser argued, the global impact of pathogens, including viruses, has shaped human history as much as any war, natural disaster, or invention. This view may seem an exaggeration to today’s student of Virology, who probably perceives most viral infections as annoyances that cause unpleasant side effects and may result in a few missed classes or days of work. But in the context of history, epidemics of smallpox, yellow fever, human immunodeficiency virus, and influenza have resulted in an incalculable loss of life and have changed entire societies. Smallpox alone has killed over 300 million people, more than twice the number of deaths from all the wars in the 20th century. Huge empires fell to a relatively small number of invaders, in part because the conquerors inadvertently introduced viruses that crippled the empires’ defense forces. Although vaccines and antivirals have reduced, and even eliminated, some of these scourges, a recent influenza pandemic, the alarming number of human cases of Ebola virus in Africa, the lack of success in developing a human immunodeficiency virus vaccine, the resurgence of vaccine-preventable infections, and the emergence of “new” human viral pathogens, such as the coronavirus that causes Middle East Respiratory Syndrome, remind us of the challenges we still face. Of equal importance, while populations in resource-rich countries may be generally protected from some former viral foes, infections with vaccine-preventable viruses, including measles, polio, and hepatitis B virus, remain prevalent in countries that lack the money or infrastructure to ensure widespread vaccination.

The ways by which viruses cause diseases in their hosts, the tug-of-war among viruses and the host’s defenses, and the impact that viral epidemics have had on human and animal populations are therefore not just interesting academic pursuits but rather life-and-death issues for all organisms. That said, it is important to bear in mind this critical fact: pathogenesis (the basis of disease) is often an unintended outcome of the parasitic lifestyle of viruses. As is true for humans, selective pressures that control viral evolution act only on the ability to survive and reproduce. From this perspective, one could argue that the most successful viruses are those that cause no apparent disease in their natural host.

In the first article of Volume I, we recounted an abbreviated history of virology and described milestones that established the foundation for our current understanding of viral reproduction. In this article, we return to history, focusing on watershed events that catalyzed the fields of viral epidemiology and pathogenesis. Subsequent articles in this volume will consider the impact of viral infections on individual hosts, tissues, and cells. Our goal is to build on the principles of viral reproduction that were established in Volume I to provide a comprehensive and integrated view of how viruses cause disease in single cells, discrete hosts, and large populations.

A Brief History of Viral Pathogenesis

The Relationships between Microbes
and the Diseases They Cause

Long before any disease-causing microbes were identified, poisonous air (miasma) was generally presumed to cause epidemics of contagious diseases. The association of particular microorganisms, initially bacteria, with specific diseases can be attributed to the ideas of the German physician Robert Koch. With his colleague Friedrich Loeffler, Koch developed four criteria that, if met, would prove a causal relationship between a given microbe and a particular disease. These criteria, Koch’s postulates, were first published in 1884 and are still used today as a standard by which pathogens are identified. The postulates are as follows:
• the microorganism must be associated regularly with the disease and its characteristic lesions but should not be found in healthy individuals;
• the microorganism must be isolated from the diseased host and grown in culture;
• the disease should be reproduced when a pure preparation of the microorganism is introduced into a healthy, susceptible host; and
• the same microorganism must be reisolated from the experimentally infected host.
Guided by these postulates and the methods developed by Pasteur for the sterile culture and isolation of purified preparations of bacteria, researchers identified and classified many pathogenic bacteria (as well as yeasts and fungi) during the latter part of the 19th century. Identifying a cause-and-effect relationship between a microbe and a pathogenic outcome set the stage for transformative therapeutic advances, including the development of antibiotics.

During the last decade of the 19th century, however, it became clear that not all epidemic diseases could be attributed to bacterial or fungal agents. This the breakdown of the paradigm led to the identification of a new class of infectious agents: submicroscopic particles that came to be called viruses (see Volume I, article 1). Koch’s postulates can often be applied to viruses, but not all virus-disease relationships meet these criteria. While compliance with Koch’s principles will establish that a particular virus is the causative agent of a specific disease, failure to comply does not rule out a possible cause-and-effect relationship.

The First Human Viruses Identified
and the Role of Serendipity

The first human virus that was identified as the agent responsible for causing yellow fever. The story of its identification in 1901 is instructive, as it highlights the contributions of creative thinking, collaboration, serendipitous timing, and even heroism in identifying new pathogens.

Yellow fever, widespread in tropical countries since the 15th century, was responsible for devastating epidemics associated with extraordinary rates of mortality (for example, over a quarter of infected individuals died in the New Orleans epidemic of 1853). While the disease can be relatively mild, with transient symptoms that include fever and nausea, more-severe cases result in major organ failure. Destruction of the liver causes yellowing of the skin (jaundice), the symptom from which the disease name is derived. Despite its impact, little was known about how yellow fever was spread, although it was clear that the disease was not transferred directly from person to person. This property prompted speculation that the source of the infection was present in the atmosphere and led to desperate efforts
to “purify” the air, including burning barrels of tar and firing cannons. Others believed that the pathogen was carried on fomites, such as bedding or clothing, although this hypothesis was disproved when volunteers remained healthy after sleeping in the nightwear of yellow fever victims.

The first real advance in establishing the origin, or etiology, of yellow fever, came in 1880, when the Cuban physician Carlos Juan Finlay proposed that a bloodsucking insect, most likely a mosquito, played a part in the transmission of the disease. A commission to study the basis of yellow fever was established in 1899 in Cuba by the U.S. Army under Colonel Walter Reed. This commission was formed in part because of the high incidence of the disease among soldiers who were occupying Cuba. Jesse Lazear, a member of Reed’s commission, confirmed Finlay’s hypothesis when he allowed himself to be bitten by a yellow fever virus-infected mosquito. “I rather think I am on the track of the real germ,” wrote Lazear to his wife, sadly just days before he died of yellow fever himself. The results of the Reed Commission’s study proved conclusively that mosquitoes are the vectors for this disease. In retrospect, a mosquito-borne mode of transmission made sense, as the disease was predominately found in warm and humid regions of the world (e.g., Cuba, New Orleans) where mosquitoes were, and remain, abundant. The members of this courageous team, perhaps the first true epidemiologists, are depicted in a dramatic 1939 painting (Fig. 1.1).

Figure 1.1 Conquerors of yellow fever. This painting by Dean Cornwell (1939) depicts the experimental exposure of James Carroll with infected mosquitoes. Walter Reed, in white, stands at the head of the table, while Jesse Lazear applies the infected mosquitoes to Carroll’s arm. Also depicted in this painting is Carlos Finlay, in a dark suit. Despite the care that Cornwell took to ensure accuracy of his portrayal of the participants and their uniforms, the event documented in this painting never took place; rather, artistic license was used to place all the major players in one depiction of a watershed moment in medical history. Photo courtesy of Wyeth Pharmaceuticals.

The nature of the pathogen was established in 1901, when Reed and James Carroll injected diluted, filtered serum from the blood of a yellow fever patient into three healthy individuals. Two of the volunteers developed yellow fever, causing Reed and Carroll to conclude that a “filterable agent,” which we now know as yellow fever virus, was the cause of the disease. In the same year, Juan Guiteras, a professor of pathology and tropical medicine at the University of Havana, attempted to produce immunity by exposing volunteers to mosquitoes that were allowed to take a blood meal from an individual who showed signs of yellow fever. Of 19 volunteers, 8 contracted the disease and 3 died. One of the deceased was Clara Louise Maass, a U.S. Army nurse. Maass’ story is of interest, as she had volunteered to be inoculated by infected mosquitoes sometime before, developed only mild symptoms, and survived. Her agreement to be infected a second time was to test if her earlier exposure provided protection from a subsequent challenge. This was a prescient idea, because, at that time, virtually nothing was known about immune memory.

Maass’ death prompted a public outcry and helped to end yellow fever experiments in human volunteers. Yellow fever had been endemic in Havana for 150 years, but the conclusions of Reed and his colleagues about the nature of the pathogen and the vector that transmitted it led to the rapid implementation of effective mosquito control measures that dramatically reduced the incidence of disease within a year. To this day, mosquito control remains an important method for preventing yellow fever, as well as other viral diseases transmitted by arthropod vectors.

Other human viruses were identified during the early decades of the 20th century (Fig. 1.2).

Figure 1.2 Pace of discovery of new infectious agents. Koch’s introduction of efficient bacteriology
techniques spawned an explosion of new discoveries of bacterial agents in the early 1880s. Similarly, the discovery of filterable agents launched the field of virology in the early 1900s. Despite an early surge of virus discovery, only 19 distinct human viruses had been reported by 1935. Adapted from K. L. Burdon, Medical Microbiology (MacMillan Co., New York, NY, 1939, with permission.)

However, the pace of discovery was slow, in great part because of the dangers and difficulties associated with experimental manipulation of human viruses so vividly illustrated by the experience with yellow fever virus. Consequently, agents of some important human diseases were not identified for many years and only then with some good luck. A classic example is the identification of the virus reason-
sable for influenza, a name derived in the mid-1700s from the Italian language because of the belief that the disease resulted from the “influence” of contaminated air and adverse astrological signs. Worldwide epidemics ( pandemics ) of influenza had been documented in humans for well over 100 years.

Such pandemics were typically associated with mortality among the very young and the very old, but the 1918-1919 pandemic following the end of World War I was especially devastating. It is estimated that one-fi ft h of the world’s population was infected, resulting in more than 50 million deaths, far more than were killed in the preceding war. Unlike in previous epidemics that affected the elderly and the very young, healthy young adults were often victims (Fig. 1.3).

Figure 1.3 1918 flu consequences. (A) The 1918-1919 influenza pandemic infected a staggering number of people, resulting in the hasty establishment of cavernous quarantines in college gymnasia and large halls, filled with rows and rows of infected patients. Photo courtesy of the Naval History and Heritage Command.
(B) Of particular concern, this epidemic had a high death rate among young, otherwise healthy, individuals compared to those of previous flu seasons. Adapted from R. Ahmed et al., Nat. Immunol. 8:1188–1193, 2007, with permission.

Despite many efforts, a human influenza virus was not isolated until 1933, when Wilson Smith, Christopher Andrewes, and Patrick Laidlaw serendipitously found that the virus could be propagated in an unusual host. Laidlaw and his colleagues at Mill Hill in England were using ferrets in studies of canine distemper virus, a paramyxovirus unrelated to influenza. Despite efforts to keep these ferrets isolated from both the environment and other pathogens (for example, all ferrets were housed separately, and all laboratory personnel had to disinfect themselves before and after entering a room), it is thought that a lab worker infected with influenza transmitted the virus to a ferret. Th is ferret then developed a disease very similar to influenza in humans. Realizing the implications of their observation, Laid- law, and colleagues then infected naive ferrets with throat wash- ings from sick individuals and isolated the virus now known as influenza A virus. (Note the eff ective use of Koch’s postulates in this study!) Subsequently, influenza A virus was shown to also infect adult mice and chicken embryos. The latter proved to be an especially valuable host system, as vast quantities of
the virus are produced in the allantoic sac. Chicken eggs are still used today to produce influenza vaccines.

New Techniques Led to the Study of
Viruses as Causes of Disease

Technological developments propelled advances in our understanding of how viruses are reproduced (Volume I, Article 1) and also paved the way for early insights into viral pathogenesis, the study of how viruses cause disease. The period from approximately 1950 to 1975 was marked by remarkable creativity and productivity, and many experimental procedures developed then are still in use today. With these techniques in hand, scientists performed pioneering studies that revealed how viruses, including mousepox virus, rabies virus, poliovirus, and lymphocytic choriomeningitis virus, caused illness in susceptible hosts.

Revolutionary developments in molecular biology from the mid-1970s to the end of the 20th-century further accelerated the study of viral pathogenesis. Recombinant DNA technology enabled the cloning, sequencing, and manipulation of host and viral genomes. Among other benefits, these techniques allowed investigators to mutate particular viral genes and to determine how specific viral proteins influence cell pathology. The polymerase chain reaction (PCR) was first among the many new offshoots of recombinant DNA technology that transformed the field of virology. PCR is used to amplify extremely small quantities of viral nucleic acid from infected samples. Once sufficient viral DNA has been obtained and the sequence determined, the virus can be more easily identified and studied. The ability to sequence and manipulate DNA also led to major advances in the related field of immunology and, consequently, had an important impact on the investigation of viral pathogenesis. While many of the early studies in immunology focused on immune cell development, others began to address how immune cells recognized and responded to pathogens. The Nobel Prizes of the 1980s and 1990s highlight the importance of this new technologies; they include awards for the establishment of transgenic animals, gene targeting, immune cell recognition of virus-infected cells, and RNA interference. These discoveries and the ways that they helped to shape our current view of the viral disease will be discussed later articles.

The emergence of molecular biology and cell biology as distinct fields marked a transition from a descriptive era to one that focused on the mechanisms by which particular viral processes were controlled, among other advances. Genomes were isolated, proteins were identified, functions were deduced by application of genetic and biochemical methods, and new animal models of disease were developed. These approaches not only defined basic steps in the viral life cycle and functions of virus-encoded proteins but also ushered in practical applications, including the development of diagnostic tests, antiviral drugs, and vaccines. As the 20th century came to a close, another paradigm shift was occurring in virology, as many scientists realized the power of a more holistic strategy to study virus-host relationships. These scientists embraced the concept of systems biology, the notion that all the molecules or reactions that govern a biological process could be identified and monitored during an infection, allowing discovery of new processes that were missed by the more reductionist, one-gene-at-a-time approaches. These ideas were initially developed using microarray technology, which enabled a global and unbiased snapshot of the quantities of both host and viral mRNAs under defined conditions.  

New tools continue to expand our capabilities, and methods once considered cutting edge are eclipsed by more-powerful, faster, or cheaper alternatives. Parallel developments in information technology and computer analyses (often called “data mining”) have been critical to infer meaningful conclusions from the massive data sets now commonly collected. Computer-aided approaches have enabled scientists to define cellular pathways that are triggered during viral infection, to identify common features among seemingly diverse viruses, and to make structural predictions about small-molecule inhibitors that could prevent infection. While these new tools are exciting and powerful, it is likely that traditional approaches will still be required to validate and advance the hypotheses that are emerging from systems biology. New technological developments should be viewed as adding to, rather than replacing, experimental strategies from the past. While the methods that virologists employ may be ever-changing, the fundamental question asked by early pioneers is still with us: how do viruses cause disease? The remainder of this article focuses on the impact of viral infections in large populations and how outbreaks and epidemics begin.

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