Principles of Virology, Volume 2. S. Jane Flint
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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. These ferrets were secluded from 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). Despite such precautions, it is thought that an infected lab worker transmitted the influenza virus to a ferret. When this ferret developed a disease very similar to influenza in humans, Laidlaw and colleagues realized its implications. These researchers then infected naïve ferrets with throat washings from sick individuals and isolated the virus now known as influenza A virus. (Note the effective 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 most influenza virus vaccines.
New Methods Facilitate the Study of Viruses as Causes of Disease
Technological developments propelled advances in our understanding of how viruses are reproduced (Volume I, Chapter 1) and also paved the way for early insights into viral pathogenesis. 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 and beyond 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 can be 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, studied, and manipulated experimentally. The ability to sequence and manipulate DNA also led to major advances in the related field of immunology and greatly improved our understanding of the infected host’s immune response to viral infection. The Nobel Prizes since the 1980s often acknowledge the importance of new technologies and concepts, and include awards for the establishment of transgenic animals, gene targeting methods, and immune cell recognition of virus-infected cells.
Figure 1.3 Consequences of the 1918 influenza pandemic. (A) The 1918–19 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 (Catalog # NH 2654). (B) Of particular concern, this epidemic had a high death rate among young, otherwise healthy individuals compared to deaths in previous flu seasons, in which deaths occurred mostly among the very young and very old (representative data from Massachusetts shown). Based on data from Dauer CC, Serfling RE. 1961. Am Rev RespirDis 83:15–28.
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 underlying viral reproduction, transmission, and disease. 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 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 large-scale, unbiased screens to study virus-host relationships. These scientists embraced 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 molecules and mechanisms that would be overlooked by more reductionist, gene-specific approaches. Large data sets were acquired, initially using microarray technology, which enabled a global and unbiased snapshot of both host and viral RNAs under defined conditions. Today, next-generation strategies, including high-throughput RNA sequencing (RNA-Seq) and nanopore sequencing, are used to reveal the type and quantity of nucleic acid in a biological sample at a given moment (Box 1.3).
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 draw conclusions from the massive data sets, requiring in-depth expertise in bioinformatics and biostatistics. 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 these more global analyses.
Although the methods that virologists employ may be ever-changing, one fundamental question asked by early pioneers remains with us: how do viruses cause disease? The remainder of this chapter focuses on how outbreaks and epidemics begin, and the impact of viral infections in large populations.
Viral Epidemics in History
In the apocalyptic movies I Am Legend (2007), Contagion (2011), and World War Z (2013), fictional epidemics are depicted following introduction of a virus into a naïve human population. (In some of these films, the virus turned the infected victims into zombies; although viruses cause many diverse outcomes, zombification is not among them.) Some of these doomsday films include a scene in which an epidemiologist ominously describes the devastating consequences of uncontrolled, exponential viral spread through a population. These movies were certainly frightening, but ultimately comforting, as humans, with improbable speed, developed strategies to limit viral spread. But how realistic is this Hollywood vision? One could argue that proof of our triumph over viral pathogens can be found in the eradication of smallpox and the development of vaccines to prevent infection by many viruses that historically resulted in much sickness and loss of life. However, there is a risk in becoming self-congratulatory. Doing so makes us ignorant of how quickly a virus can spread in a susceptible population, as the recent SARS-CoV-2 pandemic has taught us. When epidemics and pandemics occur in real life, there is a pervasive feeling of helplessness, and often interventions are not developed in time to mitigate substantial clinical impact. The stories that follow highlight the financial toll, loss of life, and historical ramifications of viral outbreaks, and underscore a new reality: the increased mobility of human and animal populations on the planet has almost certainly accelerated the emergence of epidemics.
METHODS
Nanopore sequencing
A new approach for determining the sequence of a nucleic acid has been developed, referred to as “nanopore sequencing.” This method relies on the use of biological nanopores, such as the bacterial hemolysin, which forms extremely small holes, or pores, in a membrane. These pores have a