Introduction5 but,despite the realization that these new-found agents caused disease in animals as well as plants,people would not accept the idea that they might have anything to do with human diseases.This resistance was finally dispelled in 1909 by Karl Landsteiner and Erwin Popper,who showed that poliomyelitis was caused by a 'filterable agent'-the first human disease to be recognized as being caused by a virus. Frederick Twort (1915)and Felix d'Herelle (1917)were the first to recognize viruses that infect bacteria,which d'Herelle called bacteriophages('eaters of bac- teria').In the 1930s and subsequent decades,pioneering virologists such as Salvador Luria,Max Delbruck,and many others used these viruses as model systems to investigate many aspects of virology,including virus structure(Chapter 2),genet- ics (Chapter 3),and replication (Chapter 4).These relatively simple agents have since proven to be very important to our understanding of all types of viruses including those of humans which are much more difficult to propagate and study. The further history of virology is the story of the development of experimental tools and systems with which viruses could be examined and which opened up whole new areas of biology,including not only the biology of the viruses them- selves but inevitably also the biology of the host cells on which these agents are entirely dependent. LIVING HOST SYSTEMS In 1881,Louis Pasteur began to study rabies in animals.Over several years,he developed methods of producing attenuated virus preparations by progressively drying the spinal cords of rabbits experimentally infected with rabie which when inoculated into other animals,would protect from challenge with virulent rabies virus.In 1885,he inoculated a child,Joseph Meister,with this,the first arti- ficially produced virus vaccine(as the ancient practice of variolation and Jenner use of cowpox virus for vaccination relied on naturally occurring viruses).Whole plants have been used to study the effects of plant viruses after infection ever since tobacco mosaic virus was first discovered by Iwanowski.Usually such studies involve rubbing preparations containing virus particles into the leaves or stem of the plant. During the Spanish-American War of the late nineteenth century and the sub- sequent building of the Panama Canal,the number of American deaths due to yellow fever was colossal.The disease also appeared to be spreading slowly north ward into the continental United States.In 1990,through experimental transmis- sion to mice,Walter Reed demonstrated that yellow fever was caused by a virus spread by mosquitoes.This discovery eventually enabled Max Theiler in 1937 to propagate thevirus in chick embryos andtopodcetteuated vaccthe 17D strain-which is still in use today.The success of this approach led many other
but, despite the realization that these new-found agents caused disease in animals as well as plants, people would not accept the idea that they might have anything to do with human diseases. This resistance was finally dispelled in 1909 by Karl Landsteiner and Erwin Popper, who showed that poliomyelitis was caused by a ‘filterable agent’—the first human disease to be recognized as being caused by a virus. Frederick Twort (1915) and Felix d’Herelle (1917) were the first to recognize viruses that infect bacteria, which d’Herelle called bacteriophages (‘eaters of bacteria’). In the 1930s and subsequent decades, pioneering virologists such as Salvador Luria, Max Delbruck, and many others used these viruses as model systems to investigate many aspects of virology, including virus structure (Chapter 2), genetics (Chapter 3), and replication (Chapter 4). These relatively simple agents have since proven to be very important to our understanding of all types of viruses, including those of humans which are much more difficult to propagate and study. The further history of virology is the story of the development of experimental tools and systems with which viruses could be examined and which opened up whole new areas of biology, including not only the biology of the viruses themselves but inevitably also the biology of the host cells on which these agents are entirely dependent. LIVING HOST SYSTEMS In 1881, Louis Pasteur began to study rabies in animals. Over several years, he developed methods of producing attenuated virus preparations by progressively drying the spinal cords of rabbits experimentally infected with rabies which, when inoculated into other animals, would protect from challenge with virulent rabies virus. In 1885, he inoculated a child, Joseph Meister, with this, the first arti- ficially produced virus vaccine (as the ancient practice of variolation and Jenner’s use of cowpox virus for vaccination relied on naturally occurring viruses).Whole plants have been used to study the effects of plant viruses after infection ever since tobacco mosaic virus was first discovered by Iwanowski. Usually such studies involve rubbing preparations containing virus particles into the leaves or stem of the plant. During the Spanish–American War of the late nineteenth century and the subsequent building of the Panama Canal, the number of American deaths due to yellow fever was colossal. The disease also appeared to be spreading slowly northward into the continental United States. In 1990, through experimental transmission to mice, Walter Reed demonstrated that yellow fever was caused by a virus spread by mosquitoes. This discovery eventually enabled Max Theiler in 1937 to propagate the virus in chick embryos and to produce an attenuated vaccine—the 17D strain—which is still in use today.The success of this approach led many other Introduction 5
6Principles of Molecular Virology investigators from the 1930s to the 1950s to develop animal systems to identify and propagate pathogenic viruses. Eukaryotic cells can be grown in vitro(tissue culture)and viruses can be prop- agated in these cultures,but these techniques are expensive and technically quite demanding.Some viruses will replicate in the living tissues of developing embry- onated hens eggs,such as influenza virus.Egg-adapted strains of influenza virus replicate well in eggand very high virus titres can be obtained.Embryonated hens eggs were first used to propagate viruses in the early decades of the twenti- eth century.This method has proved to be highly effective for the isolation and culture of many viruses,particularly strains of influenza virus and various poxviruses (e.g.,vaccinia virus).Counting the 'pocks'on the chorioallantoic membrane of eggs To produce viruses that cannot be effectively studied in vitro (e.g.,hepatitis B virus) To study the pathogenesis of virus infections (e.g.coxsackieviruses) To test vaccine safety (e.g.,oral poliovirus vaccine) Nevertheless,they are increasingly being discarded for the following reasons: Breeding and maintenance of animals infected with pathogenic viruses is Whole animals are complex systems in which it is sometimes difficult to discern events. Results obtained are not always reproducible due to host variation. Unnecessary or wasteful use of experimental animals is morally repugnant. They are rapidly being overtaken by'modern science'-cell culture and molecu- lar biology. The use of whole plants as host organisms does not give rise to the same moral objections as the use of living animals and continues to play an important part in the study of plant viruses,although such systems are sometimes slow to deliver results and expensive to maintain In recent years,an entirely new technology has been employed to study the effects of viruses on host organisms.This involves the creation of transgenic animals and plants by inserting all or part of the virus genome into the DNA of the experimental organism,resulting in expression of virus mRNA and proteins in somatic cells (and sometimes in the cells of the germ line).Thus,the pathogenic effects of virus proteins,individually and in various combinations,can be studied in living hosts.'SCID-hu'mice have been constructed from immunodeficient lines of animals transplanted with human tissue.These mice form an intriguing model
investigators from the 1930s to the 1950s to develop animal systems to identify and propagate pathogenic viruses. Eukaryotic cells can be grown in vitro (tissue culture) and viruses can be propagated in these cultures, but these techniques are expensive and technically quite demanding. Some viruses will replicate in the living tissues of developing embryonated hens eggs, such as influenza virus. Egg-adapted strains of influenza virus replicate well in eggs and very high virus titres can be obtained. Embryonated hens eggs were first used to propagate viruses in the early decades of the twentieth century. This method has proved to be highly effective for the isolation and culture of many viruses, particularly strains of influenza virus and various poxviruses (e.g., vaccinia virus). Counting the ‘pocks’ on the chorioallantoic membrane of eggs produced by the replication of vaccinia virus was the first quantitative assay for any virus. Animal host systems still have their uses in virology: ■ To produce viruses that cannot be effectively studied in vitro (e.g., hepatitis B virus) ■ To study the pathogenesis of virus infections (e.g., coxsackieviruses) ■ To test vaccine safety (e.g., oral poliovirus vaccine) Nevertheless, they are increasingly being discarded for the following reasons: ■ Breeding and maintenance of animals infected with pathogenic viruses is expensive. ■ Whole animals are complex systems in which it is sometimes difficult to discern events. ■ Results obtained are not always reproducible due to host variation. ■ Unnecessary or wasteful use of experimental animals is morally repugnant. ■ They are rapidly being overtaken by ‘modern science’—cell culture and molecular biology. The use of whole plants as host organisms does not give rise to the same moral objections as the use of living animals and continues to play an important part in the study of plant viruses, although such systems are sometimes slow to deliver results and expensive to maintain. In recent years, an entirely new technology has been employed to study the effects of viruses on host organisms. This involves the creation of transgenic animals and plants by inserting all or part of the virus genome into the DNA of the experimental organism, resulting in expression of virus mRNA and proteins in somatic cells (and sometimes in the cells of the germ line).Thus, the pathogenic effects of virus proteins, individually and in various combinations, can be studied in living hosts. ‘SCID-hu’ mice have been constructed from immunodeficient lines of animals transplanted with human tissue. These mice form an intriguing model 6 Principles of Molecular Virology
Introduction7 to study the pathogenesis of human immunodeficiency virus(HIV)as there is no real alternative to study the properties of this important virus in vivo.While these techniques often raise the same moral objections as 'old-fashioned'experimental infection of animals by viruses,they are immensely powerful new tools for the study of virus pathogenicity.A growing number of plant and animal viruses gene have been analysed in this way,but the results have not always been as expected, and in many cases it has proved difficult to equate the observations obtained with those gathered from experimental infections.Nevertheless,this method will undoubtedly become much more widely used as more of the technical difficulties associated with the construction of transgenic organisms are solved. CELL CULTURE METHODS Cell culture began early in the twentieth century with whole-organ cultures,then progressed to methods involving individual cells,either primary cell cultures (somatic cells from an experimental animal or taken from a human patient which can be maintained for a short period in culture)or immortalized cell lines,which given appropriate conditions,continue to grow in culture indefinitely. In1949John Enders and his colleagues were able to propagate poliovirus in primary human cell cultures.This achievement ushered in what many regard as the 'Golden Age of Virology'and led to the identification and isolation during the 1950s and 1960s of many viruses and their association with human disease example,many enteroviruses and respiratory viruses,such as adenoviruses.Wide- spread virus isolation led to the realization that subclinical virus infections were very common;for example,even in epidemics of the most virulent strains of poliovirus there are approximately 100 subclinical infections for each paralytic case of poliomyelitis. Renato Dulbecco in 1952 was the first to quantify accurately animal viruses using a plaque assay.In this technique,dilutions of the virus are used to infect a cultured cell monolayer,which is then covered with soft agar to restrict diffusion of the virus,resulting in localized cell killing and the appearance of plaques after the monolayer is stained (Figure 1.1).Counting the number of plaques directly determines the number of infectious virus particles applied to the plate.The same technique can also be used biologically to clone a virus (i.e.,isolate a pure form from a mixture of types).This technique had been in use for some time toquan- tify the number of infectious virus particles in bacteriophage suspensions applied to confluent 'lawns'of bacterial cells on agar plates,but its application to viruses of eukaryotes enabled rapid advances in the study of virus replication to be made Plaque assays largely replaced earlier endpoint dilution techniques,such as the tissue culture infectious dose (TCIDso)assay,which are statistical means of measuring virus populations in culture;however,endpoint techniques may still be used in
to study the pathogenesis of human immunodeficiency virus (HIV) as there is no real alternative to study the properties of this important virus in vivo. While these techniques often raise the same moral objections as ‘old-fashioned’ experimental infection of animals by viruses, they are immensely powerful new tools for the study of virus pathogenicity. A growing number of plant and animal viruses genes have been analysed in this way, but the results have not always been as expected, and in many cases it has proved difficult to equate the observations obtained with those gathered from experimental infections. Nevertheless, this method will undoubtedly become much more widely used as more of the technical difficulties associated with the construction of transgenic organisms are solved. CELL CULTURE METHODS Cell culture began early in the twentieth century with whole-organ cultures, then progressed to methods involving individual cells, either primary cell cultures (somatic cells from an experimental animal or taken from a human patient which can be maintained for a short period in culture) or immortalized cell lines, which, given appropriate conditions, continue to grow in culture indefinitely. In 1949, John Enders and his colleagues were able to propagate poliovirus in primary human cell cultures.This achievement ushered in what many regard as the ‘Golden Age of Virology’ and led to the identification and isolation during the 1950s and 1960s of many viruses and their association with human diseases—for example, many enteroviruses and respiratory viruses, such as adenoviruses. Widespread virus isolation led to the realization that subclinical virus infections were very common; for example, even in epidemics of the most virulent strains of poliovirus there are approximately 100 subclinical infections for each paralytic case of poliomyelitis. Renato Dulbecco in 1952 was the first to quantify accurately animal viruses using a plaque assay. In this technique, dilutions of the virus are used to infect a cultured cell monolayer, which is then covered with soft agar to restrict diffusion of the virus, resulting in localized cell killing and the appearance of plaques after the monolayer is stained (Figure 1.1). Counting the number of plaques directly determines the number of infectious virus particles applied to the plate. The same technique can also be used biologically to clone a virus (i.e., isolate a pure form from a mixture of types). This technique had been in use for some time to quantify the number of infectious virus particles in bacteriophage suspensions applied to confluent ‘lawns’ of bacterial cells on agar plates, but its application to viruses of eukaryotes enabled rapid advances in the study of virus replication to be made. Plaque assays largely replaced earlier endpoint dilution techniques, such as the tissue culture infectious dose (TCID50) assay, which are statistical means of measuring virus populations in culture; however, endpoint techniques may still be used in Introduction 7
Principles of Molecular Virology Plateilutions onto susceptible cells. heimatnwhce of virus particle s in loca 0 o Figure 1.1 Plaque assays are performed by applying a suitable dilution of a virus preparation to a confluent or semiconfluent adherent monolayer of susceptible cells After allowing time for virus attachment to and infection of the cells,liquid medium is replaced by a semisolid culture medium containing a polymer such as agarose or carboxymethyl cellulose,which restricts diffusion of virus particles from infected cells.Only direct cell-to-cell spread can occur,resulting in localized destruction of the monolayer.After a suitable period,the medium is usually removed and the cells stained to make the holes in the monolayer (plaques)more easily visible.Each plaque therefore results from infection by a single plaque-forming unit (p.f.u.)
8 Principles of Molecular Virology Make serial dilutions of virus Plate dilutions onto susceptible cells. After virus attachment, overlay cells with semi-solid medium which restricts diffusion of virus particles. Restricted cell-to-cell spread of virus results in localized destruction of cell monolayer visible as “plaques” Figure 1.1 Plaque assays are performed by applying a suitable dilution of a virus preparation to a confluent or semiconfluent adherent monolayer of susceptible cells. After allowing time for virus attachment to and infection of the cells, liquid medium is replaced by a semisolid culture medium containing a polymer such as agarose or carboxymethyl cellulose, which restricts diffusion of virus particles from infected cells. Only direct cell-to-cell spread can occur, resulting in localized destruction of the monolayer. After a suitable period, the medium is usually removed and the cells stained to make the holes in the monolayer (plaques) more easily visible. Each plaque therefore results from infection by a single plaque-forming unit (p.f.u.)
Introduction certain circumstances-for example,for viruses that do not replicate in culture or are not cytopathic and do not produce plaques,(e.g,human immunodeficiency virus). SEROLOGICAL/IMMUNOLOGICAL METHODS As the discipline of virology was emerging.the techniques of immunology were also developing,and,as with molecular biology more recently,the two disciplines have always been very closely linked.Understanding mechanisms of immunity to virus infections has,of course,been very important.Recently,the role that th immune system itself plays in pathogenesis has become known (see Chapter 7). Immunology as a discipline in its own right has contributed many of the classical techniques to virology (Figure 1.2). George Hirst,in 1941,observed haemagglutination of red blood cells by influenza virus (see Chapter 4).This proved to be an important tool in the study of not only influenza but also several other groups of viruses-for example,rubella virus.In addition to measuring the titre (i.e.,relative amount)of virus present in any preparation,this technique can also be used to determine the antigenic type of the virus.Haemagglutination will not occur in the presence of antibodies that bind to and block the virus haemagglutinin.If an antiserum is titrated against a given number of haemagglutinating units,the haemagglutination inhibition titre and specificity of the antiserum can be determined.Also,if antisera of known speci- ficity are used to inhibit haemagglutination,the antigenic type of an unknown virus can be determined.In the 1960s and subsequent years,many improved detection methods for viruses were developed,such as: Complement fixation tests ■Radioimmunoassays Immunofluorescence (direct detection of virus antigens in infected cells or tissue) Enzyme-linked immunosorbent assays (ELISAs) Radioimmune precipitation ■Western blot assays These techniques are sensitive,quick,and quantitative. In 1975,George Kohler and Cesar Milstein isolated the first monoclonal anti- bodies from clones of cells selected in vitro to produce an antibody of a single speci- ficity directed against a particular antigenic target.This enabled virologists to look not only at the whole virus,but at specific regions-epitopes-of individual virus antigens(Figure 1.3).This ability has greatly increased our understanding of the function of individual virus proteins.Monoclonal antibodies are also finding
certain circumstances—for example, for viruses that do not replicate in culture or are not cytopathic and do not produce plaques, (e.g., human immunodeficiency virus). SEROLOGICAL/IMMUNOLOGICAL METHODS As the discipline of virology was emerging, the techniques of immunology were also developing, and, as with molecular biology more recently, the two disciplines have always been very closely linked. Understanding mechanisms of immunity to virus infections has, of course, been very important. Recently, the role that the immune system itself plays in pathogenesis has become known (see Chapter 7). Immunology as a discipline in its own right has contributed many of the classical techniques to virology (Figure 1.2). George Hirst, in 1941, observed haemagglutination of red blood cells by influenza virus (see Chapter 4). This proved to be an important tool in the study of not only influenza but also several other groups of viruses—for example, rubella virus. In addition to measuring the titre (i.e., relative amount) of virus present in any preparation, this technique can also be used to determine the antigenic type of the virus. Haemagglutination will not occur in the presence of antibodies that bind to and block the virus haemagglutinin. If an antiserum is titrated against a given number of haemagglutinating units, the haemagglutination inhibition titre and specificity of the antiserum can be determined.Also, if antisera of known speci- ficity are used to inhibit haemagglutination, the antigenic type of an unknown virus can be determined. In the 1960s and subsequent years, many improved detection methods for viruses were developed, such as: ■ Complement fixation tests ■ Radioimmunoassays ■ Immunofluorescence (direct detection of virus antigens in infected cells or tissue) ■ Enzyme-linked immunosorbent assays (ELISAs) ■ Radioimmune precipitation ■ Western blot assays These techniques are sensitive, quick, and quantitative. In 1975, George Kohler and Cesar Milstein isolated the first monoclonal antibodies from clones of cells selected in vitro to produce an antibody of a single speci- ficity directed against a particular antigenic target. This enabled virologists to look not only at the whole virus, but at specific regions—epitopes—of individual virus antigens (Figure 1.3). This ability has greatly increased our understanding of the function of individual virus proteins. Monoclonal antibodies are also finding Introduction 9