CH 06 Viruses and Prions PDF Biology

6 Viruses and Prions cause severe liver damage. Likewise, vaccines for influenza protect millions each year, and newer vaccines that protect against HPV, chickenpox (varicella-zoster), and rotaviruses (a common cause of viral diarrhea in infants) are now also widely used. However, the fight against...

6 Viruses and Prions cause severe liver damage. Likewise, vaccines for influenza protect millions each year, and newer vaccines that protect against HPV, chickenpox (varicella-zoster), and rotaviruses (a common cause of viral diarrhea in infants) are now also widely used. However, the fight against some types of viruses remains difficult. Due to constant viral evolution, influenza vaccines have to be reformulated and re-administered annually. And if predictions about which strains ought to be included in the flu vaccine prove inaccurate, then vaccinated patients may still catch the disease. Other viruses such as HIV remain without an effective vaccine at all, despite decades of work toward this goal. Most recent emerging diseases are viral: Zika and Ebola are examples in which incidence quickly exploded after formerly isolated outbreaks. Changes in climate allow mosquitoes to thrive in an expanded geographic area, which makes it easier for mosquito-borne The Case of the viruses to spread. Furthermore, wideNCLEX Cancerous Kiss spread and rapid travel means that HESI How can an infection lead isolated viral outbreaks may become TEAS to cancer years later? worldwide pandemics at an alarming speed. The ability of healthcare Scan this code or visit the Mastering Microbiology Study workers to quickly identify and, when Area to watch the case and find necessary, isolate those suffering out how viruses and prions can from particularly deadly viral diseases explain this medical mystery. remains crucial for limiting the scope and impact of these outbreaks. What Will We Explore? Unlike other microbes we study, viruses and prions are nonliving pathogens. Most viruses are smaller than bacteria, outnumber cellular life by more than ten to one, appear in all ecosystems, and can infect every type of life. In humans, viruses cause everything from minor ailments such as the common cold to the deadly and (so far) untreatable disease rabies. Other viruses, such as the human papilloma viruses (HPV), can cause cancer. *In this chapter we investigate the foundations of virology, the study of viruses. We also examine how viruses and prions cause disease and discuss some antiviral drugs. Why Is It Important? For millennia, viruses have been a major source of human disease and suffering. Eradication of the smallpox virus through vaccination was one of the greatest public health victories ever. Following decades of research and development, we finally have drugs that eliminate certain hepatitis C viruses that FPO CLINICAL CASE *Since the early 1990s the association between HPV and cervical cancer has been extensively investigated. There has been a consistent conclusion across the primary literature that there is a causal relationship between certain strains of HPV and cancer. Bosch, F.X., Lorincz, A., Munoz, N., Meijer, C.J., Shah, K.V. (2002).The causal relation between human papillomavirus and cervical cancer. Journal of Clinical Pathology, 55, 244–65. M06_NORM8290_01_SE_C06.indd 164 17/07/17 4:57 PM GENERAL VIRUS CHARACTERISTICS Viruses are nonliving pathogens. After reading this section, you should be able to: In the late 1800s it was discovered that sap from an infected tobacco plant remained infectious to healthy tobacco plants even after it was passed through a specialized filter designed to screen out prokaryotic and eukaryotic cells. The infectious agent, invisible when viewed through a light microscope, was given the name virus. They were described as filterable infectious agents, reflecting their tiny size compared with cells. Today over 5,000 mammal-infecting viral species have been described. Of these, at least 220 infect humans.1 It is estimated that at least 320,000 mammalian viruses remain uncharacterized. Because about 70 percent of viruses that infect humans tend to be harbored in other animals, this number represents a significant pool for the emergence of new viral diseases in humans.2 Therefore, an introduction to the study of viruses, or virology, is essential for any healthcare provider. Extremely small size is one important characteristic of viruses, but not the most distinguishing one. Certain bacteria, such as Mycoplasma, Rickettsia, and Chlamydia species, are also very small intracellular pathogens that enter and infect cells (as opposed to infecting the extracellular spaces in a host, as many larger pathogens do). However, unlike prokaryotes and eukaryotes, viruses are not cells—hence their descriptor, acellular. Most biologists currently consider cells to be the smallest unit of life. Based on this definition, acellular infectious agents are considered nonliving. One reason viruses are considered nonliving is that unlike bacteria and eukaryotic microbes, viruses are incapable of synthesizing their own components, such as nucleic acids or proteins, without the help of the host cells they infect. As such, they are called obligate intracellular pathogens—disease-causing microbes that must invade living cells and hijack their biochemical and cellular tools to replicate. While a number of bacteria are also obligate intracellular pathogens, the bacteria have their own biochemical processes to extract energy from nutrients; in contrast, viruses lack these metabolic processes. TABLE 6.1 compares viruses with prokaryotic and eukaryotic cells. 1 Explain why viruses are classified as nonliving microbes. 2 Compare viruses to prokaryotic and eukaryotic cells. 3 Describe features and functions of viral structures, including capsids, envelopes, and spikes. 4 Describe the genomic variations seen among viruses. 5 Summarize the various ways that DNA and RNA viruses use their genome to make mRNA for protein production. 6 Describe the key contributors to and consequences of viral genome evolution, and state why RNA viruses evolve faster than DNA viruses. 7 Explain antigenic shift and antigenic drift, and state how they impact influenza virus evolution and outbreaks. TABLE 6.1 Comparing Viruses, Prokaryotes, and Eukaryotes Characteristic Viruses Prokaryotes Eukaryotes Cells? No Yes Yes Considered alive? No Yes Yes Relative size Generally smaller than prokaryotes; most require electron microscopy to see Most bigger than viruses and smaller than eukaryotes; usually seen with light microscopy Usually bigger than prokaryotes and viruses; often visible with light microscopy Filterable? Yes No (except for some very small filterable bacteria) No Structure Protein capsid coating and nucleic acid Cells without nuclei or other membranebound organelles Cells with nuclei and membrane-bound organelles Replication Host cell energy and machinery are hijacked to replicate the virus Binary fission (asexual) Mitosis (asexual) Meiosis (sexual) Exhibit metabolism? No Yes Yes Genome composition DNA or RNA DNA DNA 1 Woolhouse, M., Scott, F., Hudson, Z., Howey, R., & Chase-Topping, M. (2012). Human viruses: Discovery and emergence. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 367(1604), 2864–2871. 2 Anthony, S. J., Epstein, J. H., Murray, K. A., et al. (2013). A strategy to estimate unknown viral diversity in mammals. mBio, 4(5), e00598-13 (online only). General Virus Characteristics M06_NORM8290_01_SE_C06.indd 165 165 17/07/17 4:57 PM Viruses exhibit diverse structural and genomic features. Helical capsid Viruses can infect every branch in the tree of life. However, in this chapter we’ll focus on bacteriophages (or phages), which are viruses that only infect bacteria, and animal viruses, which include viruses that infect humans. A single, infectious virus particle is called a virion. All virions have an exterior protective protein capsid, packed with genetic material (DNA or RNA). Some animal viruses also have a lipid envelope around the capsid, which we will discuss later. The structural and genomic features of a virus dictate what sort of host the virus can infect, as well as the progression of the infection. Capsid Capsomeres Viral genome Viral Capsids Icosahedral capsid Viral genome Capsid Capsomeres Complex structure Viral genome Capsid Capsomeres Sheath Baseplate The protein shell that packages and protects the genome and also accounts for the bulk of a virion’s mass is called a capsid (see FIG. 6.1). A capsid’s shape is based on how the individual three-dimensional subunits, called capsomeres, are arranged. The capsids of most animal viruses have either helical or icosahedral symmetry. Helical capsids look like a hollow tube. Icosahedral capsids look like three-dimensional polygons, but may appear fairly spherical––just as a soccer ball is spherical, yet made of multiple hexagon and pentagon shapes. Viruses with less conventional capsids are lumped into the catchall complex structure category. Some animal viruses, such as the Poxviridae family that includes the smallpox virus, have ovoid or brick-shaped capsids and are therefore described as having a complex structure. Bacteriophages also exhibit a complex structure. Although bacteriophages often have capsids with icosahedral symmetry, their capsids are associated with additional complex structures that enable them to act like hypodermic syringes that inject the viral genome into target bacterial cells. The capsomeres that build capsids may be made from a single type of protein or a collection of different proteins. Unlike most cellular proteins, viral capsids often exhibit self-assembly, meaning the amino acid sequence determines how the final proteins fold and come together to make the larger capsid structure. Other more complex viruses use the host cell machinery to put the capsids together properly. Because capsid assembly is such a central part of virion formation, it represents a potential target for antiviral drugs. Viral Envelopes have capsids with helical or icosahedral symmetry. Bacteriophages and certain animal viruses have complex structures. Some animal viruses have a lipid-based envelope that surrounds the capsid. Virions that lack an envelope are said to be naked (or nonenveloped). Enveloped viruses develop by budding from the host, taking a portion of the cell membrane with them as a coating when they go. In contrast, naked viruses lyse (burst) out of host cells and do not coat themselves in an envelope. Animal viruses may be either enveloped or naked. Because all bacteriophages lyse host cells, these complex-shaped viruses are therefore always naked. Critical Thinking How can the basic structure of a virus be compared to the nucleus of a eukaryotic cell? Viral Spikes (Peplomers) Tail fiber Pin FIGURE 6.1 Capsid structures Most animal viruses CHEM NOTE Protein self-assembly As described in Chapter 2, most proteins aren’t physiologically active until they are properly folded into a higher order structure. To achieve proper folding, most proteins enlist the help of specialized cellular tools called chaperones. Proteins that self-assemble, like some capsid proteins, achieve their final form without the help of chaperones. 166 Many viruses have spikes (or peplomers) that may protrude from the viral capsid or, if present, from the viral envelope. These glycoprotein extensions help viruses attach and gain entry to host cells. Because they only bind to specific factors on a given host cell, spikes have an important role in determining what species and tissues the virus can infect, similar to how a lock and key must match. The host immune system may also recognize spikes and mount an immune response to them. Unfortunately, if the host develops an immune response to the spikes and the spikes then change—something certain viruses do rapidly and routinely in their genetic evolution—then the new virus may escape CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 166 17/07/17 4:57 PM immune detection. FIG. 6.2 shows viral envelope varieties and spikes. Influenza viruses frequently mutate and experience small changes in spike proteins. This is such an important issue for influenza A that its spikes hemagglutinin (HA) and neuraminidase (NA) are a key part of the virus subtype naming convention. For example, swine flu’s scientific name is H1N1, while bird flu is called H5N1. Similarly, the Bacteriophage influenza A glycoprotein structures themselves are referred to as HA and NA spikes. (FIG. 6.3). There are at least 18 known variations of hemagglutinin spikes and 11 characterized variations of neuraminidase glycoproteins in influenza A. (Later in this chapter we revisit how changes in influenza spikes impact influenza outbreaks, and Chapter 16 also explores influenza pathology and outbreaks.) Naked icosahedral Spikes Viral genome TEM Capsid Adenovirus Enveloped icosahedral Spikes Envelope TEM Herpes virus Enveloped helical Viral Genomes Spikes Viral genes encode capsomere proteins, various enzymes needed for viral replication, and structural factors. Because viruses do not have to build organelles and they do not have metabolic processes to mediate, they do not require many genes to exist. Most viruses have fewer than 300 genes. The smallest have less than 2,000 base pairs in their whole genome—much smaller than the genomes typically found in eukaryotic or prokaryotic cells. By comparison, humans have about 25,000 genes and about 3.2 billion base pairs in our genome. Envelope TEM Viral genome Helical capsid Ebola virus Hemagglutinin (HA) spike Neuraminidase (NA) spike TEM Envelope FIGURE 6.2 Viral envelope and spikes Animal viruses can be enveloped or naked. Many viruses have spikes (peplomers) that extend from the capsid or envelope. Helical capsid surrounds genome FIGURE 6.3 Influenza’s glycoprotein (HA and NA) spikes Influenza’s hemagglutinin (HA) and neuraminidase (NA) spikes affect how these viruses interact with host cells and how the host’s immune system recognizes these viruses. The colored transmission electron micrograph shows influenza’s HA and NA spikes (red) covering the surface envelope (yellow). General Virus Characteristics M06_NORM8290_01_SE_C06.indd 167 167 17/07/17 4:57 PM DNA or RNA GENOME Genome arrangements: Circular Linear DNA viral genomes Circular or linear Often double stranded May also be single stranded Segmented RNA viral genomes Linear or segmented Often single stranded May also be double stranded FIGURE 6.4 Viral genomes Although cells have larger and more complex genomes than viruses, cells can only have double-stranded DNA genomes. In contrast, viruses can have either an RNA- or DNA-based genome (but usually not both),3 and the nucleic acid present can be either single or double stranded. Furthermore, the viral genome can exist in diverse arrangements. It may be spread over multiple segmented sections, or it could be a single circular or linear molecule. FIG. 6.4 shows possible nucleic acid variations for viruses. While the composition of viral genomes varies greatly, the ultimate goal of all viruses is the same: to get a host cell to make viral proteins, so more virions can be built. As you learned in Chapter 5, DNA is transcribed to make messenger RNA (mRNA), which in turn is translated by ribosomes to build proteins. Consequently, no matter what the viral genome format may be, a virus must ultimately use its genome to direct the production of mRNAs that the host cell then translates into proteins (see FIG. 6.5). If the infecting virus is a double-stranded DNA virus (dsDNA), then transcription to make mRNA is fairly straightforward. The viral DNA is transcribed using host cell RNA polymerases, and the resulting mRNA is then translated into protein. If the virus is a single-stranded DNA virus (ssDNA), then it is converted to a double-stranded form before transcription is performed. RNA viruses have different modes of ultimately making mRNA that the host cell will translate. Three of these methods relate to single-stranded RNA viruses, while the last one relates to double-stranded RNA viruses: The RNA genome functions as an mRNA: A single-stranded RNA genome may be in a form that can be directly translated by host cell ribosomes in the cytoplasm. These are single-stranded, positive, or sense-stranded RNA (ssRNA+) viruses. They include the causative agents of polio, rubella, West Nile encephalitis, and dengue fever. The RNA genome is complementary to mRNA: This group of RNA viruses encompasses a wide variety of pathogens, including the causative agents of influenza, measles, Ebola, and rabies. These single-stranded, antisense, or negative-stranded RNA (ssRNA–) viruses have an RNA genome that is complementary to mRNA. Consequently, their RNA genome must be transcribed into a readable mRNA format before translation. This is accomplished by virally encoded enzymes called RNA-dependent RNA polymerases (RdRPs). Unlike host cell RNA polymerases that build new RNA from a DNA template, RdRPs build new RNA from an existing RNA template. A 3D model of an enveloped icosahedral virus. The RNA genome makes DNA, which is then transcribed to make mRNA: Using the virally encoded enzyme called reverse transcriptase, retroviruses use their single-stranded RNA genome to direct formation of DNA. (See Chapter 5 for more on reverse transcription.) The DNA is usually inserted into the host genome and then transcribed in the nucleus, and the resulting mRNA is translated. HIV (the virus that causes AIDS) as well as other human T-cell leukemia viruses (HTLVs) carry out reverse transcription and operate through a DNA intermediate. Retroviruses are covered in more detail later in this chapter. The double-stranded RNA genome is transcribed to make mRNA: If the virus has a double-stranded RNA (dsRNA) genome, then the RNA has to be unwound, so that RNA polymerases can transcribe it into an mRNA format. The process resembles that of a double-stranded DNA virus, but instead of using host cell RNA polymerases, virally encoded RNA-dependent RNA polymerases are required. Rotaviruses, which cause severe diarrhea, are an example of dsRNA viruses. 3 In 2012 a virus with a hybrid RNA/DNA genome was isolated from a boiling springs lake in Lassen Volcanic National Park. To date, no medically important viruses have been found to have a hybrid genome and the discovered DNA/RNA hybrid virus seems to be a rare exception. 168 CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 168 17/07/17 4:57 PM Translation Transcription Cellular genome Double-stranded DNA (dsDNA) mRNA DNA viral genome Cellular protein Transcription mRNA dsDNA viruses Transcription Complementary DNA is built ssDNA viruses mRNA RNA viral genome ssRNA+: mRNA-like genome immediately ready for translation Translation Transcription (by viral RNA-dependent RNA polymerases) ssRNA-: genome is complementary to mRNA Viral protein mRNA Reverse transcription Retroviruses: mRNA-like genome is converted to a DNA form Transcription dsDNA integrates into a host genome mRNA Transcription (by viral RNA-dependent RNA polymerases) dsRNA viruses: genome is double-stranded RNA (dsRNA) mRNA FIGURE 6.5 Making mRNA from viral genomes All viruses must be able to make mRNA that can be translated by host cell ribosomes to make proteins. DNA viruses tend to closely resemble cells in mRNA production. RNA viruses have four general pathways they may use to get to mRNA. Viral genomes change over time. Because of their relatively quick replication time and the large quantity of virions released within a host, viruses exhibit a faster rate of genomic change than do living infectious agents. Random mutations occur in all types of genomes, but RNA genomes in particular mutate more and evolve faster than their DNA counterparts. This is mainly because RNA polymerases, which copy RNA, do not have the proofreading capabilities of DNA polymerases. As a result, DNA viruses may mutate once per thousand rounds of genome copying, while RNA viruses may mutate as frequently as once every round of genome copying. General Virus Characteristics M06_NORM8290_01_SE_C06.indd 169 169 17/07/17 4:57 PM Viral strain A Viral genome Different strains coinfect the same host cell Virus with new genetic combination Viral strain B FIGURE 6.6 Viral genome reassortment When different viral strains coinfect a host cell, their genomes mix and can generate new viral combinations. Critical Thinking Genetic reassortment is typically between related viral strains. Why do you think this is the case? Reassortment events also contribute to viral evolution. Reassortment may occur when two different viral strains coinfect a single host cell; their genomes can mix and generate new viral strains (FIG. 6.6). As reviewed in Chapter 5, mutations can have neutral, beneficial, or deleterious effects depending on where they occur in the genome. Genetic changes that limit infectivity are unfavorable for viruses because this limits their ability to invade host cells to replicate themselves. Such mutants are called attenuated strains. While these strains don’t cause disease in a host with a normal immune system, they still stimulate an immune response. This makes them ideal candidates for vaccine development. A variety of vaccines, such as the nasal mist influenza vaccines and the oral polio vaccine, use attenuated viral strains. Although a number of mutations may be detrimental to a virus, statistically speaking, an advantageous mutation will eventually occur. Beneficial mutations may allow the virus to escape host immune system detection, broaden host range, expand tropism (the type of cells or tissues the virus infects), or make the virus more infectious so that it is more easily spread from one host to the next. The effects of spontaneous mutations and recombination in viral evolution are classically modeled in influenza viruses. Antigenic Drift and Antigenic Shift Even relatively minor virus mutations may greatly impact public health, as they allow viruses to thwart the “memory” mechanism of our immune response. This is a problem commonly seen with influenza. Following infection (or vaccination), the host immune system recognizes the HA and NA spikes Antigenic drift Influenza virus in one flu season Influenza virus in next flu season Viral RNA genome Viral HA and NA spikes Spontaneous mutation leads to a minor change in HA or NA spikes Immunity Immunity Host immunity after infection or vaccination No immunity to altered spike; host susceptible to infection Antigenic shift Human influenza New highly virulent human strain Reassortment may occur when different strains coinfect a cell (a pig lung cell in this case) Immunity FIGURE 6.7 Antigenic shift and antigenic drift Critical Thinking Why is antigenic shift more likely to lead to expanded host range than antigenic drift? 170 Avian influenza Possible pandemic CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 170 17/07/17 4:57 PM on influenza’s surface (spikes are what the viruses use to attach to host cells before entry). Thus, the spikes also serve as antigens against which the immune system creates specific antibodies. The immune system recognizes a particular antigen profile during the infection, and makes antibodies that will neutralize that pathogen if it reenters the body again later. (See Chapter 12 for more details on the adaptive immune response.) However, since influenza’s RNA genome mutates frequently, minor changes to the HA and NA spikes also routinely occur. These minor changes, referred to as antigenic drift, allow the virus to evade a quick antibody response by making the new strains different enough to go unrecognized by the immune system— even if the host had a prior interaction with a related strain (FIG. 6.7). Antigenic drift is the reason we all need a new, different flu shot every year. Furthermore, antigenic drift means there is always a portion of the population susceptible to the latest virus, which in turn leads to seasonal outbreaks. Occasionally, influenza viruses undergo a major genetic reassortment that dramatically changes HA and NA spikes. These broader mutations, termed antigenic shift, often lead to viral strains with new features, such as increased infectivity or expanded host range. Antigenic shift is what occurred when avian and swine influenza strains “jumped species,” becoming able to infect humans. Not only is a vaccine unlikely to exist at the outset of this sort of strain emergence, but the drastic change in the virus usually means that people have no residual immune protection from prior influenza infections or vaccinations. These factors set the stage for a worldwide outbreak or pandemic. Because of the limited preexisting immunity in the population, pandemics may have a dramatically increased mortality rate compared with epidemics caused by the earlier, progenitor virus strains. (For more on antigenic drift, antigenic shift, and influenza, see Chapter 16.) BUILD YOUR FOUNDATION 1. 2. 3. 4. 5. 6. 7. 8. What is the primary reason that viruses are not considered alive? How do viruses differ from prokaryotes and eukaryotes? Describe the features and functions of viral capsids, spikes, and envelopes. Why are bacteriophages always naked? What are the possible genomic variations for viruses? How do DNA viruses use their genomes to make mRNA? What are four ways that RNA viruses may make mRNA? What are the leading contributors to viral genome evolution and what are their potential effects on viral features? 9. Why do RNA viruses evolve faster than DNA viruses? 10. Define antigenic shift and antigenic drift and describe how they influence influenza evolution and outbreaks. QUICK QUIZ Build your foundation by answering the Quick Quiz: scan this code or visit the Mastering Microbiology Study Area to quiz yourself. CLASSIFYING AND NAMING VIRUSES Diverse features are used to classify and name viruses. The International Committee on Taxonomy of Viruses (ICTV) is a group that works to develop criteria and refine the naming conventions for viruses to limit confusion. Before this committee took over viral taxonomy, naming was haphazard and often misleading. For example, while hepatitis A, B, and C viruses are not related to each other, they all have a common name due to the fact that they all infect and damage the liver. If this was the only system of classification, it could confuse diagnosis and treatments. After reading this section, you should be able to: 8 Describe the main criteria used to classify viruses. 9 Give examples of viral families that are medically important in humans. 10 Define what host range and tropism mean. 11 Explain the conventions for naming viruses. Classifying and Naming Viruses M06_NORM8290_01_SE_C06.indd 171 171 17/07/17 4:57 PM Medically important Capsid shape DNA virus families Icosahedral Naked or enveloped Naked Genome features ssDNA Families (with examples of members) dsDNA circular Parvoviridae Human parovirus B19 (fifth disease) Enveloped dsDNA linear Adenoviridae Papillomaviridae Human papilloma Adenoviruses viruses (warts; some (certain colds) strains cause cervical cancer)* dsDNA circular Hepadnaviridae Hepatitis B virus Medically important Capsid shape Naked Complex None that infect humans Enveloped dsDNA linear dsDNA linear Herpesviridae Herpes simplex viruses (oral and genital herpes) Varicella-zoster virus (chickenpox) Poxviridae Smallpox virus RNA virus families Icosahedral Naked or enveloped Helical Helical Enveloped Enveloped Genome features dsRNA segmented ssRNA+ nonsegmented ssRNA+ nonsegmented ssRNA+ nonsegmented Families (with examples of members) Reoviridae Rotavirus (diarrhea) Calciviridae Hepatitis E virus Norovirus (gastroenteritis) Picornaviridae Poliovirus Hepatitis A virus Rhinoviruses (colds) No reverse transcriptase Coronaviridae SARS virus (severe acute respiratory syndrome) Common cold viruses Flaviviridae Hepatitis C virus West Nile virus Dengue fever virus Togaviridae Rubella virus ssRNA– Nonsegmented Segmented Paramyxoviridae Measles virus Mumps virus Filoviridae Ebola virus Rhabdoviridae Rabies virus Bunyaviridae Hanta virus (hemorrhagic fever) Orthomyxoviridae Influenza viruses Arenaviridae Lassa fever virus Have reverse transcriptase Retroviridae HIV (AIDS) Human T-lymphotropic virus (leukemia) FIGURE 6.8 Medically important DNA and RNA virus families Viruses are now grouped by the following properties: 1. Type of nucleic acid present (DNA or RNA) 2. Capsid symmetry (helical, icosahedral, or complex) 3. Presence or absence of an envelope 4. Genome architecture (ssDNA, ssRNA, etc.) FIG. 6.8 shows the medically important families of viruses that infect humans and how they are grouped into families using the aforementioned criteria. Additional features such as virus size, host range, tropism (the type of cells *Crosbie, E. J., Einstein, M. H., Franceschi, S., & Kitchener, H. C. (2013). Human papillomavirus and cervical cancer. The Lancet, 382(9895), 889–899. 172 CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 172 17/07/17 4:57 PM TRAINING TOMORROW’S HEALTH TEAM Zika: Evolution and Spread Zika virus, a member of the Flaviviridae family, was first discovered in a monkey in the Zika forest of Uganda in 1947. Only about 60 cases of Zika virus were identified in humans before 2013, mostly in Africa. However, in 2013–2014, a French Polynesian outbreak spread throughout the Pacific, leading to 396 confirmed cases and 29,000 suspected cases. By 2015, over one million Zika infections occurred worldwide, with many in Brazil and throughout the Americas. Brazil incidence dropped dramatically by 2017, thanks to aggressive mosquito abatement programs. Despite this, the virus continues to slowly spread to new areas. A variety of mosquito species carry and transmit Zika virus, and it has a wide range of animal hosts, including humans, monkeys, cows, ducks, and bats. Research indicates that Zika mutated rapidly through genetic recombination. The original African virus was known to cause fairly mild disease characterized by joint pain, fever, rash, and conjunctivitis. More virulent recent versions of the virus seem to originate from an Asian strain. The Zika that’s been spreading worldwide can transmit sexually and exhibits neurotropism, meaning it spreads to nervous system tissue, where it can cause microcephaly and brain abnormalities in a developing fetus. In some people the virus is also thought to trigger Guillain Barre Syndrome, an immune disorder that causes damage to nerves. Zika infection during pregnancy can lead to microcephaly (an abnormally small head) of the fetus. Q UE STIO N 6. 1 Zika virus detection has been challenging due to its similarity in both symptoms and virion features to other Flaviviridae members. Based on its family, what other viruses is it related to, and what can you conclude about its genome and other viral structures? or tissues the virus infects), and disease features are often reflected in virus names because they help to refine the grouping of viruses into species. Host Range and Tropism Viruses infect every type of life form on Earth; however, a given virus will exhibit a specific host range or collection of species that it can infect. Some viruses infect more than one species, while others infect only one species. For example, the measles virus is only able to infect humans. Most of the viruses that infect humans are able to infect some other animal. Indeed, most human viruses evolved in other species and then through genetic changes they gained the capacity to infect us. For example, avian influenza (bird flu or H5N1), which primarily infected certain waterfowl species, especially geese and ducks, underwent genetic reassortment that expanded its host range to now include humans. The same holds true for swine flu, which initially infected pigs and can now infect humans. Viruses also exhibit specificity regarding what tissues or cells they will infect in their given host. This tropism is due to virus surface factors only being able to bind to specific surface molecules on certain host cells. Some viruses, like Ebola, can infect a wide range of host cells or tissues and are said to have a broad tropism. Other viruses have a very narrow tropism, and may specifically target only one type of cell or tissue in the given host. For example, the hepatitis viruses target the liver, so they are described as hepatotropic (hepato means liver). Human T-lymphotropic viruses get their name as a result of their partiality for infecting human T cells, which are specialized white blood cells of the immune system— this virus name reveals the virus’s narrow tropism and host range. Virus Sizes Viruses exhibit a wide range of sizes. Rhinoviruses (which typically cause colds) and polioviruses (the cause of polio) may have a diameter as small as 30 nanometers (nm). By comparison, a molecule of hemoglobin, the oxygen carrier in human blood, is around 5 nm. Some viruses, like the Ebola viruses and the pandoraviruses, are relatively large and can have lengths nearing 1,000 nm. Pithovirus, which was discovered in 2014 in ice cores from Siberia, is one of the largest viruses discovered to date; its dimensions of 1,500 nm in length by Classifying and Naming Viruses M06_NORM8290_01_SE_C06.indd 173 173 17/07/17 4:57 PM Bacteriophage T4, 225 nm Poliovirus, 30 nm Rhinovirus, 30 nm HIV, 120 nm E. coli bacterium, 2,000 nm long Pithovirus, 1,500 nm long Pandoravirus, 1,000 nm long Ebola virus, 970 nm long Human red blood cell, 8,000 nm diameter FIGURE 6.9 Virus sizes vary greatly Influenza viruses infect red blood cells. 500 nm in diameter make it close to the size of E. coli (FIG. 6.9). Contrast these larger viruses to the recently discovered new group of marine bacteria that were only approximately 500 nm long, and it’s easy to appreciate that size alone is insufficient for classifying viruses. Viruses are named using standardized rules. Unlike organisms, viruses are not assigned to domains, kingdoms, phyla, or classes––the order level is the highest taxon for viruses. Under the order level we find the following rankings: family (and occasionally subfamily), genus, and species. Within the seven recognized viral orders there are 29 families. As of 2015, at least 82 other families of viruses have not been assigned to an order. Viral taxa and naming conventions are summarized in TABLE 6.2. Classification rankings below the species level are not overseen by the ICTV. BUILD YOUR FOUNDATION Build your foundation by answering the Quick Quiz: scan this code or visit the Mastering Microbiology Study Area to quiz yourself. QUICK QUIZ 11. 12. 13. 14. What criteria are used to classify viruses? Name the virus families that are medically important in humans. What is meant by host range and tropism? What are the naming conventions for viruses? TABLE 6.2 Naming Conventions for Viral Taxonomy Taxon Examples Notes Order Herpesvirales Italicized with first letter capitalized; always ends in “virales” Family Herpesviridae Italicized with first letter capitalized; always end in “viridae” Subfamily Alphaherpesvirinae Italicized with first letter capitalized; always end in “virinae” Genus Simplexvirus Italicized with first letter capitalized; always ends in “virus” Species Human herpesvirus-1, also known as Herpes simplex virus-1 Italicized and first name as well as proper nouns are capitalized; should not be abbreviated Common name human herpes virus-1 (HHV-1), also known as herpes simplex virus-1 (HSV-1) Often the same as the species name except not italicized and only proper nouns are capitalized; may be abbreviated after an initial use of the full name 174 CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 174 17/07/17 4:57 PM INTRODUCTION TO VIRAL REPLICATION PATHWAYS Viruses hijack host cell machinery to multiply. After reading this section, you should be able to: Once inside, a virus commandeers the host cell’s energy, enzymes, organelles, and molecular building blocks such as amino acids to build new virions. In this section we explore the general pathways that bacteriophages use to replicate in bacterial cells and then we’ll explore generalized replication pathways that animal viruses use. 12 Explain the features of bacteriophage lytic and lysogenic replication. 13 Define the term phage conversion and discuss why it is medically important. 14 Describe the generalized steps for animal virus replication. 15 Compare and contrast how enveloped and naked animal viruses differ in their replication pathways. 16 Define the term persistent infection and explain how one may develop through chronic or latent infection mechanisms. 17 Explain what makes a virus oncogenic and name examples of oncogenic viruses and the cancers they may cause. Generalized Bacteriophage Replication Although bacteriophages don’t infect human cells, they’re still medically important because they serve as a means for bacteria to evolve. Bacteriophages facilitate specialized and generalized transduction, which allow bacteria to develop new genetic combinations despite their inability to sexually reproduce. (See Chapter 5.) Also, bacteriophages impact bacterial population levels––an important consideration, in light of the role that microbiome bacteria play in human physiology. Lytic replication Bacteriophages are diverse and use different mechanisms for host cell infection and subsequent viral replication. Some, like the T-even bacteriophages, infect the host bacterial cell and immediately build new virions. These phages use a lytic replication pathway. This pathway kills the host cell as newly made bacteriophages are released. The lytic replication pathway involves five key steps, which are described below and depicted in FIG. 6.10. 1 Attachment Phage binds to bacterial cell. 1. Attachment (adsorption4): Since viruses can’t move on their own, the bacteriophage and host cell usually come together from random contact. Bacteriophage tail fibers help the virus adhere to the specific protein on the bacterial cell wall surface. The specificity of these interactions means that certain bacteriophages only infect certain bacteria. 2 Penetration Phage injects its genome into the host; empty capsids remain outside the cell. 3 Replication Protein synthesis makes phage parts and genome is replicated; host cell DNA is broken down by bacteriophage DNAases. 4 Assembly Genome packed into capsid and phage structures assembled. 5 Release Bacterial cell lyses and new phages are released. 2. Penetration (entry): Like a hypodermic needle, the bacteriophage injects genetic material into the cell, through the host’s cell wall and plasma membrane. The empty capsid remains outside the cell. 3. Replication (synthesis): Once the viral genome is inside, the bacteriophage commandeers host cell factors to transcribe and translate viral genes. Among the early viral proteins that get made are DNAases (DNA-degrading enzymes) that break up the host cell’s DNA. The viral genome also encodes proteins to build new phage particles and enzymes that will copy the viral genome. 4. Assembly (maturation): Once all the parts of the bacteriophage are replicated, viral factors pack the viral genome into the capsid and the remaining phage parts are assembled. Hundreds to thousands of new bacteriophages may be generated in this step. Bacteriophage 5. Release: Bacteriophages encode an enzyme called lysozyme, which breaks down host cell walls and causes bacterial cell lysis (bursting) once the newly assembled phages are mature. The released bacteriophages can then infect other cells. Lysogenic replication Bacteriophages such as lambda phages (l phages) are called temperate phages. These bacteriophages can use either a lytic or a lysogenic pathway. The first two steps of the lysogenic cycle, attachment and 4 Language note: It can be easy to confuse absorption with adsorption; one letter changes the word’s meaning. Absorption is the process of soaking up/absorbing something while adsorption is the process of sticking to something/adhering to something. Bacterial chromosome Phage genome + + + FIGURE 6.10 Bacteriophage lytic replication Introduction to Viral Replication Pathways M06_NORM8290_01_SE_C06.indd 175 = 175 17/07/17 4:57 PM Lytic replication 1 Temperate phage Bacterial chromosome Attachment Lysogenic replication Integration Phage DNA integrates into host genome, forming a prophage. Prophage Phage genome 2 3 Penetration Cell division Host and phage genomes are copied before cell division; despite a single infection event, multiple cells now carry the phage genome. Lytic cycle entered Phage may enter lytic cycle if the host is stressed. Replication FIGURE 6.11 Temperate bacteriophage lytic and lysogenic replication 4 5 Assembly + + + = Critical Thinking What evolutionary advantages does the lysogenic pathway confer on temperate phages? penetration, are the same as in the lytic pathway. However, following penetration, the phage genome is incorporated into the host cell genome, forming a prophage. As the infected bacterial cell divides, it copies its own genome as well as the prophage’s genome. Therefore, a single infection event ultimately results in many cells carrying the bacteriophage’s genome. If a host cell carrying a prophage is stressed, the prophage may excise itself from the host genome and enter the lytic replication pathway. This “abandon ship” approach allows the bacteriophage to replicate and find a new host before its current host cell dies. The lytic and lysogenic paths that temperate phages can exploit are illustrated in FIG. 6.11. Prophages are medically important due to their ability to confer new pathogenic properties to bacterial cells, a situation called phage conversion. Phage conversion can provide new pathogenicity factors to a bacterium, such as the capacity to make certain toxins. The bacterium that causes diphtheria, Corynebacterium diphtheria, gained its ability to make the potent diphtheria toxin from a prophage. Similarly, the botulinum toxin made by the bacterium that causes botulism, Clostridium botulinum, is encoded by a prophage. Release Generalized Animal Virus Replication Bring the art to life! Watch the Concept Coach animation and master lytic and lysogenic replication. 176 CONCEPT COACH Animal viruses and bacteriophages share many features in their replication pathways. The generalized animal virus replication pathway has six main steps: attachment, penetration, uncoating, replication, assembly, and release. Except for the uncoating step, the names of these steps are exactly the same as for bacteriophages––however, the precise details differ. FIG. 6.12 summarizes the process. CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 176 17/07/17 4:57 PM 1 Attachment Viral surface proteins interact with host plasma membrane proteins. 2 Penetration Occurs by fusion or endocytosis (fusion shown). Virus Plasma membrane protein 3 ANIMAL CELL + Capsid proteins Uncoating Genome released from capsid. 4 + Genome Spike proteins 5 6 Replication Genome is replicated and viral proteins are made. Assembly New virions are assembled. Release Virions are released by budding or lysis (budding shown). FIGURE 6.12 Overview of animal virus replication Animal viruses use six general steps in their replication. The finer specifics of these steps vary from one viral family to the next. Viral envelope 1. Attachment: Naked viruses attach to host cell membranes through capsid proteins. Other viruses (both naked and enveloped) use spikes (FIG. 6.13). As described earlier, specificity of this binding is why viruses exhibit host range and tissue tropism. This makes attachment proteins a target for drug therapy to limit or prevent infection. One drug called maraviroc (Sezentry) works by blocking HIV’s attachment to host cell proteins. Spike off viral envelope (alternatively could be a spike off the capsid) 2. Penetration (Entry): Enveloped animal viruses enter the host cell through endocytosis or membrane fusion (see Chapter 4 for more on endocytosis). Naked viruses mainly enter by endocytosis. In membrane fusion, the cell’s plasma membrane and the viral envelope blend together, releasing the viral capsid into the cytoplasm. In endocytosis, viral binding to host cell surface receptors triggers uptake of the virus into vesicles (FIG. 6.14). Host plasma membrane 3. Uncoating: Unlike bacteriophage capsids, animal virus capsids enter the host cells. The capsid is then entirely or partially broken down, releasing the viral genome. This uncoating varies by process and location. A virus that entered by endocytosis usually has its capsid digested away by enzymes in the endocytic vesicle. Poliovirus is an exception: It enters via endocytosis, but spits out its genome through a pore that forms in the capsid and the surrounding endocytic vesicle.5 Viruses entering by fusion 5 Brandenburg, B., Lee, L. Y., Lakadamyali, M., Rust, M. J., Zhuang, X., et al. (2007). Imaging poliovirus entry in live cells. PLoS Biology, 5(7), e183. doi: 10.1371/journal.pbio.0050183 (online only). Receptor Capsomere Receptor Host plasma membrane FIGURE 6.13 Viral attachment to host cells Viral envelope or capsid proteins attach to host cell proteins to facilitate attachment. Critical Thinking Some antiviral drugs block viral attachment. Blocking viral proteins is typically preferred to blocking host proteins. Why do you think this is the case? Introduction to Viral Replication Pathways M06_NORM8290_01_SE_C06.indd 177 177 17/07/17 4:57 PM Fusion: Only enveloped animal viruses Endocytosis: Enveloped and naked animal viruses Enzymes 1 Virus binds to specific receptors on the host cell plasma membrane. 2 Viral envelope and host cell membrane fuse. 3 Uncoating: Host cell cytoplasmic enzymes break down viral capsid and the viral genome is released. 1 Virus binds to specific receptors on the host cell plasma membrane. 2 Virus is engulfed into endocytic vesicle. 3 Uncoating: Enzymes in the endocytic vesicle break down the viral capsid and the viral genome is released. FIGURE 6.14 Animal virus penetration and uncoating Critical Thinking Why are naked viruses unable to enter using fusion? HOST CELL Virus (not yet enveloped) Plasma membrane may have their capsid degraded by enzymes in the host cell cytoplasm (Figure 6.14). Many DNA viruses don’t undergo uncoating until their capsid is safely delivered to the host nucleus. 4. Replication (Synthesis): As stated earlier, DNA viruses import their genome into the host cell nucleus to be transcribed and replicated. The resulting mRNA is then shipped from nucleus to cytoplasm, where it is translated to make viral proteins. Most RNA viruses direct genome replication and protein synthesis from the cytoplasm, never entering the nucleus. Notable exceptions include the orthomyxoviruses (which cause influenza) and retroviruses such as HIV, which are RNA viruses but have a replication process that takes place in the nucleus. 5. Assembly: Sometimes new capsids are partially built and packed with the viral genome before finishing and sealing them. However, usually the capsid assembles around the genome. Enveloped viruses often require viral proteins to be embedded in the host cell plasma membrane before virion release. Budding virus Enveloped virus TEM 6. Release: Newly assembled enveloped viruses are released by budding off the host cell. As they do so, they usually take a portion of the cell’s plasma membrane, enriched with viral surface proteins, with them (FIG. 6.15). Naked viruses rupture the host cell during release, usually killing the cell. Some animal viruses have unique replication mechanisms that cause persistent infections. HIV budding off a white blood cell FIGURE 6.15 Release of enveloped animal viruses Enveloped viruses are released by budding. Viral envelope proteins must be embedded in the host cell plasma membrane before budding occurs. Critical Thinking Although viruses that bud from the host cells do not lead to cell lysis, they are no less dangerous to the host. Explain this statement. 178 The steps of animal virus replication just described apply to a productive infection––one where a virus infects a host cell and immediately starts making new virions. Viruses that employ this replication strategy cause acute infections, which run their course and are cleared by the host immune system. The common cold and influenza are examples of acute viral infections. Other viruses have different replication strategies that allow them to avoid immune system clearance. They cause persistent infections. Persistent viruses tend to remain in the host for long periods––from many weeks to a lifetime. Most persistent infections can be described as chronic or latent (FIG. 6.16). CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 178 17/07/17 4:57 PM Chronic Persistent Infections Acute Nonpersistent Infection Chronic infections are characterized by continuous release of virions over time and a slow progression of disease. In some cases, a period of quiet infection for months or years precedes a period of active viral replication. In the quiet period, host cells produce a small number of virions. Disease is not evident, but the patient can still pass the virus to others. Human immunodeficiency virus (HIV), which impacts the immune system by killing T-cells, follows this slow progressing chronic model. Those infected in the quiet stage are said to be “HIV positive.” Without treatment, eventually the quiet stage ends. Virus count increases and healthy T-cell levels drop, causing loss of immune function. The end stage of HIV infection is called acquired immune deficiency syndrome, or AIDS. (For more on HIV/AIDS, see Chapter 21.) Some viruses that cause persistent infections will integrate their genome into the host cell, forming a provirus that resembles the prophage made by temperate bacteriophages that we discussed earlier. The distinct difference is that proviruses don’t excise themselves from the genome to employ the “abandon ship” approach seen in lysogenic bacteriophages. Retroviruses like HIV are notorious for forming a provirus in infected cells and then remaining silent for years before releasing sufficient virions to destroy the host’s immune system (FIG. 6.17). Similarly, the papillomaviruses, which are perhaps best known for causing cervical cancer, form proviruses and generate persistent infections. Still other viruses, like those that cause hepatitis B and C, cause persistent infections, but they do not form proviruses. 1 VIRAL REPLICATION TIME (DAYS) Latent Persistent Infection VIRAL REPLICATION TIME (DAYS, MONTHS, OR YEARS) Chronic Persistent Infection VIRAL REPLICATION TIME (DAYS, MONTHS, OR YEARS) Attachment and penetration HIV attaches to host cell and enters by fusion. 2 Uncoating 3 Viral RNA genome Reverse transcriptase Reverse transcription Upon entry, HIV genome is reverse transcribed by viral reverse transcriptase to make DNA. FIGURE 6.16 Acute, latent, and chronic infections (generalized graphs) In an acute (nonpersistent) infection, viral replication peaks, followed by immune clearance of the virus. Latent and chronic infections evade immune clearance and persist in the host. Latent infections exhibit bursts of viral replication with intermittent (silent) periods. Chronic infections exhibit steady viral production that may increase over time. Critical Thinking It is impossible to make a single graph that would accurately model the precise pattern of viral replication for all latent persistent infections. Explain why. DNA HOST CELL 4 Viral integration DNA version of the HIV genome integrates into the host genome to make a provirus. 5 Replication Provirus is transcribed to make viral genome and translated to make viral proteins. Provirus Host genome Viral RNA genome NUCLEUS 6 7 Assembly Release HIV provirus directs the production of new virions, which bud from the host cell. FIGURE 6.17 HIV retroviral provirus formation Introduction to Viral Replication Pathways M06_NORM8290_01_SE_C06.indd 179 179 17/07/17 4:57 PM TRAINING TOMORROW’S HEALTH TEAM Keeping HIV Quiet In the 1980s, when HIV first emerged worldwide, patients usually only lived for about 12 years following infection. Today, HIV-positive patients may have an almost normal life expectancy thanks to daily “cocktails” of antiviral medications. Some HIV drugs slow viral replication by targeting the viral genome. Others directly attack viral replication proteins. One class of drug stops HIV from inserting itself into the genome of T cells. Ultimately, all of these drugs aim to extend the “quiet,” symptom-free stage of the disease, so that an infection doesn’t progress to AIDS. Other anti-HIV drugs interfere with the virus’s ability to infect new cells. One prevents the maturation of essential viral proteins to limit the infectivity of newly made virions. Another prevents viral attachment by blocking the receptors on human immune system cells. The final drug class prevents HIV entry by blocking viral fusion with host plasma membranes. For healthcare providers, one of the most important advances in HIV treatment is postexposure prophylaxis (PEP), or preventing the infection from starting. This is administered to people after an incident like an accidental needle stick. PEP must start within 72 hours of the initial exposure. It consists of three different drugs that are taken for at least a month. TEM of HIV Q UE STIO N 6. 2 Based on the previous sections, what step in viral replication might a new class of anti-HIV drugs target? How might they work? Latent Persistent Infections FPO CLINICAL CASE The Case of the Cancerous Kiss NCLEX HESI TEAS Practice applying what you know clinically: scan this code or visit the Mastering Microbiology Study Area to watch Part 2 and practice for future exams. Latent infections are distinguished by flare-ups with intermittent periods of dormancy (latency). During the flare-up, virions are shed and the infected person experiences symptoms. When the flare-up concludes, the virus retreats into a period of inactivity, during which virion levels fall drastically and may be difficult to detect. Flare-ups can be triggered by any stress to the host, including an infection with another pathogen, fever, sunburn, hormone level changes, and immune suppression. Members of the Herpesviridae family are also notorious for causing latent infections by going dormant in certain host cells. However, these viruses do not integrate themselves into the host genome; instead they exist episomally—that is, their genome remains outside the host’s genome. Human herpes virus-1 (HHV1, also known as HSV-1) is known to lie dormant in host nerve cells and then reemerge to generate cold sores (FIG. 6.18). HHV-2, which causes genital herpes, follows a similar pattern of latency and active replication. Another Herpesviridae member, the varicella-zoster virus (HHV-3) that causes chickenpox, persists episomally in host nerve cells and reemerges to cause shingles. Intermittent flare-ups and retreats make it hard for the immune system to eliminate the virus. They also provide episodic bursts of virions that allow for transmittal to a new host. The oral antiviral drug valacyclovir (trade names Valtrex and Zelitrex) is a drug that limits HHV-1, HHV-2, and varicella-zoster (HHV-3) lytic cycle progression by interfering with viral genome replication. Although the drug doesn’t eliminate these viruses from the host and therefore is not a cure for cold sores, genital herpes, shingles, or chickenpox, it does reduce the extent to which they enter active replication to cause symptoms. (See Chapter 17 for more on varicella-zoster virus and Chapters 17 and 20 for more on herpes viruses.) Persistent Infections That Can Lead to Cancer FIGURE 6.18 HSV-1 cold sores HSV-1 causes cold sores when it transitions from a latent to an active replication state. Most people have been infected with this virus by young adulthood, but most people will not have repeated outbreaks of cold sores. 180 Viruses that cause persistent infections are often associated with cancer. Those that can cause cancer are called oncogenic viruses (or oncoviruses). They include a number of DNA viruses as well as the RNA viruses. Some of them integrate into the host genome, while others are maintained episomally––that is, separate from the host chromosome and maintained either in the nucleus or cytoplasm. The six human viruses that are associated with cancers are reviewed CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 180 17/07/17 4:57 PM TABLE 6.3 The Six Main Human Viruses Associated with Cancers Integrates with Host Genome Cancer-Causing Mechanism Virus Viral Genome Human papilloma viruses (HPVs) DNA; Papillomaviridae family Yes Cervical, oropharyngeal, anal, and rare vaginal and penile cancers Human herpes virus-8 DNA; Herpesviridae family No Kaposi sarcoma Epstein-Barr virus (also causes mononucleosis) DNA; Herpesviridae family No Associated with a number of malignancies to include B- and T-cell lymphomas and Hodgkin’s disease Human T-lymphotropic viruses (HTLVs) RNA; Retroviridae family Yes Adult T-cell leukemia Hepatitis B virus DNA; Hepadnaviridae family No RNA; Flaviviridae family No Hepatitis C viruses Cancer Link Viral genes cause uncontrolled cell division Liver cancer (hepatocellular carcinoma) Hypothesized that chronic inflammation from the virus triggers host cell DNA damage and mutations, leading to cancer in TABLE 6.3. Oncogenic viruses are thought to cause between 10 to 15 percent of cancers. In general terms, oncogenic viruses cause cancer by stimulating uncontrolled host cell division, and/or decreasing host cell responsiveness to death signals. Cells that ignore death signals are said to be “immortalized.” Unlike normal cells that have a limited life span and can be stimulated to undergo apoptosis (cell suicide), immortalized cells may undergo repeated division and survive indefinitely. It should be noted that infection with an oncogenic virus increases risk, but does not guarantee cancer will develop. Some of the most well-known oncogenic viruses are the human papilloma viruses (HPVs). There are over 200 different types of HPVs, at least 40 of which spread through sexual contact. The vast majority cause benign warts. About a dozen HPV types have been linked to cancer, with HPV-16 and HPV-18 causing the majority of cases. Even the HPV viruses most associated with cancer are usually cleared by the immune system within one or two years without further problems. Nonetheless, about 90 percent of all cervical cancer cases are linked to the virus, according to the Centers for Disease Control and Prevention (CDC). Due to this, the CDC recommends all children 11–12 years old receive the Gardasil vaccine that protects against the HPV types that are most frequently associated with cancer. (For more on HPV and cervical cancer, see Chapter 20.) Other oncogenic viruses include the human T-lymphotropic viruses (HTLV). HTLV-1 and HTLV-2 are clinically important. These retroviruses form a provirus and can quietly persist in host cells for more than a decade before emerging to cause leukemia or lymphoma. Worldwide, approximately 20 million people are infected with these viruses.6 The primary modes of transmission are through breast milk, sexual contact, and blood contact. BUILD YOUR FOUNDATION 15. 16. 17. 18. Compare and contrast lytic and lysogenic bacteriophage replication. How does phage conversion impact bacterial pathogens? What are the steps in animal virus replication and what occurs in each step? How does the presence or absence of an envelope impact viral entry and release? 19. Compare and contrast latent and chronic infections. 20. Give three examples of oncogenic viruses and the cancers they cause. 21. In general, how do oncogenic viruses generate cancer? QUICK QUIZ Build your foundation by answering the Quick Quiz: scan this code or visit the Mastering Microbiology Study Area to quiz yourself. 6 Proietti, F. A., Carneiro-Proietti, A. B., Catalan-Soares, B. C., & Murphy, E. L. (2005). Global epidemiology of HTLV-I infection and associated diseases. Oncogene, 24(39), 6058–6068. Introduction to Viral Replication Pathways M06_NORM8290_01_SE_C06.indd 181 181 17/07/17 4:58 PM CLINICAL ASPECTS OF VIRUSES AND PRIONS After reading this section, you should be able to: 18 Discuss the various laboratory methods for growing bacteriophages and animal viruses. 19 Explain what the plaque assay is and why it is useful. 20 Describe several methods for detecting viral proteins and genetic material and state their advantages and limitations. 21 Explain the different drug approaches to managing viral infections and name several antiviral drugs. 22 Describe what prions are, name the diseases they cause in humans, and explain how they can be transmitted. 1 The initial virus stock is sequentially diluted. 0.1 ml 0.1 ml Concentrated virus stock 2 A portion of the diluted samples is mixed with bacteria and melted agar and poured into a petri plate. 3 Clear zones, or plaques, form where host cells are killed by viruses. 4 Following incubation, plaques are counted and the initial viral titer is calculated and presented as PFU/ml. Dilute virus stock Plaque (clear zone) Viruses can be grown in the laboratory. In order to develop vaccines and drugs to combat viruses, researchers must be able to grow viruses in a laboratory setting. This requires a host cell for the virus. Here we review some of the basic techniques for cultivating viruses. Growing Bacteriophages Bacteriophages can be grown in the laboratory with relative ease. While specific techniques vary, generally all that’s needed is a combination of the desired bacteriophage, an appropriate bacterial host cell, and the right medium to support the growth of the bacteria. The bacteria can be grown and infected with bacteriophages in a liquid broth culture or using solid agar in a petri plate. The advantage of the solid agar approach is that it allows for a technique called the plaque assay. When lytic bacteriophages, like the Teven phages that we reviewed earlier, lyse out of the host cell at the end of their replication pathways, they kill the host cell. This killing leaves a clear zone called a plaque on the growing plate of bacteria (FIG. 6.19). A higher initial virus level will lead to more plaques following incubation. In theory, each plaque represents a single bacteriophage in the initial sample. Thus, the quantity of bacteriophages in an initial volume of sample can be noted in plaque-forming units (PFUs). The plaque assay is also adaptable to animal viruses and is a useful way of determining how much of a given animal virus is present. In addition to determining viral titer, or quantity of virus present in a given volume of sample, the plaque assay can be used to purify specific viruses. Some limitations of the plaque assay are that the virus must cause cell lysis and also the cells used as the viral host have to grow in a plate format. Growing Animal Viruses Animal viruses are more difficult to cultivate than bacteriophages. Most animal viruses are grown using tissue culture techniques. A variety of commercially available human and animal cell lines can be used to support viral replication. For example, HeLa cells, which are cancer cells that were derived from a patient named Henrietta Lacks, are among the most common commercially available human cells used in research today. Unfortunately, commercially available cell lines don’t always support the growth of a given virus. This is because cells that are well adapted to being cultured outside of the animal they originally came from may lose the ability to make specific surface factors the virus requires for attachment. In this event, primary cell lines derived directly from the preferred host can often be used. For example, white blood cells from donated blood can serve as a primary cell line, as can samples from tissue biopsies. A lot of specialized equipment, technical experience, and reagents go into supporting tissue culture, making this an expensive and time-consuming endeavor. Live animal hosts such as mice, rats, guinea pigs, or other animals may also be required to support the study of certain viruses. Animal models are essential to clinical trials for antiviral drug and vaccine development, and are unlikely to ever be fully replaced by tissue culture methods. FIGURE 6.19 Plaque assay Only diluted samples are plated because high concentrations would kill all cells. Critical Thinking Would the plaque assay be useful for calculating lambda phage titers? Explain your reasoning. 182 CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 182 17/07/17 4:58 PM Embryonated eggs (fertilized eggs) are also useful for growing certain viruses (FIG. 6.20). Active virions are injected into the egg and then the egg is incubated to allow for viral replication. The site of injection in the egg determines what cells in the embryonated egg become infected. The resulting virions are purified from the eggs and used for a number of applications. For example, they can be used in certain vaccines. Because it is possible that miniscule amounts of egg protein will remain in the purified viral preparation, people allergic to eggs can’t be vaccinated with this type of vaccine. This is why patients are asked about allergies to eggs when getting certain vaccines. (See Chapter 14 for more on vaccines and their development.) Amniotic cavity injection Influenza virus Mumps virus Allantoic cavity injection Influenza virus Mumps virus Adenovirus Yolk sac injection HHV Diagnostic tests determine the presence of certain viruses. The ability to detect the presence of a virus is central to studying them, but it’s also important for determining appropriate patient care. Accurate diagnostics are necessary to ensure safe, virus-free transplant tissues, pharmaceutical products, and clinical samples (tissue, blood, saliva, etc.). With such diversity in what needs testing, it stands to reason there are many methods available for identifying agents as well. Because viruses do not have their own biochemical processes and aren’t viewable via standard light microscopy, most detection techniques use molecular methods to identify viral genetic material, viral proteins, or antibodies that a patient may have against viral proteins. When a clinical sample (sputum, cerebrospinal fluid, blood, etc.) is sent to the lab, it is usually filtered or centrifuged (rapidly spun in a specialized machine) so that only particles as small as viruses are left in the test fluid. Then the fluid is subjected to diagnostic tests or used to inoculate cell lines. The most clinically useful detection tests are specific, sensitive, and have short turnaround times. Specificity means that the diagnostic test reliably detects only the virus(es) of interest without producing false positive results. Sensitivity means the test detects very low levels of the target to limit false negative results. Finally, it goes without saying that a highly specific and sensitive test that returns results after the patient is permanently impaired or dead is not the best clinical tool. The shortest turnaround time possible for receiving results is important for any diagnostic test. Holding a light up to the egg allows for specific location of the areas for injection. FIGURE 6.20 Using embryonated eggs to grow viruses Critical Thinking Not all viruses can be grown in chicken eggs. In general terms, give a reason why this is the case. TRAINING TOMORROW’S HEALTH TEAM Hold the Eggs: Egg-Free Influenza Vaccines For the past 70 years, the majority of the 500 million flu vaccine doses made each year come from the egg-based production system. But although egg-based systems for growing viruses have a large yield, they are very slow and don’t allow for rapid increases in production if the need arises. Additionally, people with an egg allergy can’t receive this type of vaccine. Select manufacturers are now using eggfree vaccine development methods, and the first egg-free influenza vaccines are now available. Production of the newest vaccine, Flublok®, grows influenza proteins (not whole viruses) in insect cells. Because the vaccine is based on influenza proteins instead of whole virions, developers can readily engineer the vaccine against new strains and quickly increase vaccine production in the event of a pandemic. Q UE STIO N 6. 3 Based on what you have read in the preceding sections, what criteria do you think vaccine manufacturers use to decide how to grow a particular virus? Clinical Aspects of Viruses and Prions M06_NORM8290_01_SE_C06.indd 183 183 17/07/17 4:58 PM Detecting Viral Proteins Detecting virus Antibodies Bead Virus + Beads coated with antibodies = Virions bearing antigen Agglutination reaction Detecting patient antibodies to a virus Viral protein Bead Antibodies + Beads coated with viral protein antigen Positive result (agglutination) = Antibodies from patient serum Agglutination reaction Negative result (no agglutination) FIGURE 6.21 Latex agglutination test to detect antigens or antibodies Latex beads linked to antibodies can be used to detect the presence of viruses in samples. Modifying the beads so that they are linked to viral proteins (viral antigens) is useful for determining if a patient has had an infection with the virus in question. Critical Thinking Which version of the latex agglutination test would be used to determine if a patient who is no longer exhibiting symptoms had been exposed to the varicellazoster virus? Some of the most common virus detection methods involve searching for viral proteins in a sample. These techniques tend to rely on purified antibodies–– specialized proteins that are made by the immune system to combat infections. Antibodies are a specific and sensitive way to detect proteins. They recognize and bind to even miniscule amounts of a target protein. The proteins that antibodies target and bind are called antigens. Here we’ll review some of the most common antigen–antibody-based methods, also called immune assays, for detecting viruses. There are commercially available kits using these methods for most common viruses. These types of tests are also adapted for detecting other pathogens and are explored further in Chapter 14. Agglutination tests Purified antibodies can be linked to tiny latex beads that are no larger than a fine grain of sand. These antibody-coated beads can then be mixed with a sample. If the sample contains the viral antigen being sought, then the antibodies will bind to the antigen and the beads will clump, or agglutinate. The top of FIG. 6.21 shows how this agglutination occurs. This version of the latex agglutination test is useful for determining if a sample contains a specific virus. It can be used to screen bodily fluids such as urine, cerebrospinal fluid, or blood for specific viruses. However, this form of the test is useless if virions are not actively being produced in a patient. Fortunately, a minor modification of the agglutination test makes it possible to determine if a prior exposure to a virus occurred––even if the virions in question aren’t actively present in the patient. Our immune system “remembers” antigens and can make the same antibodies against them later, even if the virus has long since been cleared from the host or is latent. So testing for an antibody response to a specific antigen serves as evidence of a prior infection. The latex agglutination test is one commonly performed test that can do this. To perform this test, latex beads are coated with viral proteins. If the patient has antibodies to the viral proteins coating the beads, then the beads will agglutinate when exposed to the patient’s serum (the part of blood that contains antibodies). The bottom of Figure 6.21 shows how this agglutination occurs. Enzyme-linked immunosorbent assays (ELISAs) Another clinical mainstay in pathogen detection is the ELISA assay. Like agglutination assays, ELISA methods exploit the specificity and sensitivity of antibody–antigen interactions and they can be adapted to detect either antigens or antibodies in a sample. Instead of clumping beads, ELISAs adhere the antigen or antibody to a surface, and usually change color if there is binding. (ELISA methods are further discussed in Chapter 14.) Limitations of ELISA and agglutination assays Clinical labs use a wide variety of agglutination and ELISA methods to detect diverse viruses. Most viruses that cause human disease can be detected using some version of agglutination or ELISA method. As useful as these tests are, they have some important limitations. In both cases the sample being tested must be a liquid and the antigens being detected must be fairly well characterized to ensure detection specificity. Another major consideration is that if a virus undergoes an antigenic shift, or even antigenic drift in just the right antigen, it may no longer be detectable using existing ELISA or agglutination methods. Furthermore, tests that detect patient antibodies are ineffective during early infection, since it takes at least a couple of weeks following infection for detectable antibodies to develop, a time period called the seroconversion window. For HIV, the seroconversion window can be several weeks to months. This makes methods that detect patient antibodies against HIV an unreliable tool to assess infection status. For this reason, it is helpful to use a combination of detection methods, which brings us to our next topic: detecting viral genetic material. 184 CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 184 17/07/17 4:58 PM Detecting Viral Genetic Material Before genetic techniques were developed, viruses were mainly characterized using serology studies that analyze patient antibodies, electron microscopy to view viral structures, and animal and cell culture infection methods. Today, genetic sequencing broadens our ability to identify new viruses and detect infections before seroconversion. These desirable features, along with decreasing costs of reagents and equipment, make detecting viral nucleic acids a growing trend in diagnostics. Nucleic acid detection techniques are more sensitive and sometimes more rapid than the previously described antigen–antibody-based tests. Also, they can detect new viruses and early-stage infections that antibody–antigen tests are likely to miss. To perform the test, DNA and RNA are extracted from a clinical sample such as sputum, blood, cerebrospinal fluid, or tissue. Then very specific segments of viral nucleic acid, usually those coding for a unique viral gene, can be detected by using fluorescent-labeled probes, by sequencing the nucleic acids, or by carrying out a process called PCR (polymerase chain reaction) that can amplify specific parts of a genetic sequence. (Nucleic acid detection methods are discussed in more detail in Chapter 14.) FPO CLINICAL CASE NCLEX The Case of the Cancerous Kiss HESI TEAS Practice applying what you know clinically: scan this code or visit the Mastering Microbiology Study Area to watch Part 3 and practice for future exams. Antiviral drugs treat infections, but don’t typically cure them. Most viral diseases are self-limiting and do not require treatment. But others, like HIV or certain influenza strains, cause serious disease and warrant therapy. Any step in the viral replication pathway is a potential drug target. As such, basic research into how a given virus gains entry into a host cell and then replicates is essential to developing antiviral drugs. Studying how our own immune systems successfully end certain viral infections also gives us a model for developing antiviral treatments. More and more antivirals are coming on the market to treat a myriad of conditions, from herpes to hepatitis. But it is important to acknowledge that in most cases (excepting the new hepatitis C treatments) antiviral drugs only limit infections rather than cure them. Because viruses are not cells, they cannot be eliminated through typical drug actions that affect bacterial, fungal, or protist pathogens. There are special difficulties that come into play when designing antiviral drugs. Because viral pathogens exist inside host cells much of the time, drugs cannot always reach them. And to be an effective drug rather than just a poison, antivirals should be selectively toxic, stopping the pathogen but leaving host cells unharmed. Ideally, a drug targets processes that are unique to the pathogen—but that goal is difficult to achieve when viruses use the cell’s own machinery and metabolism. Lastly, being so simple in their structure, viruses have fewer chemically distinct targets than living pathogens like bacteria, protists, or fungi. The difficulty in developing antiviral drugs is the main reason we have so few effective antiviral agents and why prevention of serious viral diseases through vaccination is so incredibly important. Vaccines train the immune system to recognize viruses and are effective means to limit infection. (Vaccines are covered in more detail in Chapter 14.) Drugs that Block Viral Attachment, Penetration, and Uncoating A number of antiviral drugs prevent viral entry into cells by blocking attachment or penetration (FIG. 6.22). For example, a laboratory-prepared mixture of injectable antibodies to the rabies virus (known as HRIG, human rabies immunoglobulin) prevents the virus from binding to and entering host cells. This therapy is a key postexposure prophylaxis, a prevention treatment applied after an exposure to limit infection. It is administered if a rabid animal bites a person who has not been vaccinated against rabies. While rabies is vaccine preventable, the vaccine is not routinely used in humans unless they are at an increased risk for exposure, Attachment blocked by: HRIG (Rabies) Maraviroc (HIV) + Release blocked by: Oseltamivir (influenza) Zanamivir (influenza) Penetration blocked by: Interferon-alfa (HBV, HCV) Enfuvirtide (HIV) Docosanol (HHV-1) Palivizumab (RSV) + Uncoating blocked by: Amantadine (influenza) Rimantadine (influenza) Vapendavir (rhinoviruses) Replication and assembly blocked by: NRTIs/reverse transcriptase inhibitors like AZT (HIV) Ribavirin (RSV, HCV, and certain hemorrhagic fevers) Protease inhibitors (HIV) FIGURE 6.22 Mechanisms of action of select antiviral drugs Every aspect of viral replication is a potential target. Ideally, antiviral drugs should not induce significant damage to host cells. Note the acronyms for hepatitis B virus (HBV), hepatitis C virus (HCV), human herpes virus (HHV), and respiratory syncytial virus (RSV). Critical Thinking Most influenza A strains are now resistant to amantadine. What do you think contributed to this evolutionary change in the virus? Clinical Aspects of Viruses and Prions M06_NORM8290_01_SE_C06.indd 185 185 05/10/17 11:42 AM TRAINING TOMORROW’S HEALTH TEAM The Virome When we hear the word “microbiome,” we tend to think about bacteria. But viruses are part of the microbiome, too. Our “virome” includes bacteriophages that infect the bacteria that live within us, and viruses that infect our own cells. Bacteriophages are best known for conferring virulence genes to bacteria. For example, a phage gives Corynebacterium diptheriae the ability to produce the toxin that makes human diphtheria infections so deadly. But phages may indirectly affect a human host. Some studies suggest they kill off bacteria that rapidly overtake a niche in the human body, serving as a buffer against changes in our bacterial microbiomes. Also, phages bind to the epithelial layers of human gastrointestinal tracts, potentially preventing pathogenic bacterial adhesion. Some preliminary studies also show that when combined with antibiotics, phages may help control the emergence of antibioticresistant bacteria. Furthermore, viral genes make up as much as 1.5 percent of the human genome. Some of these viral elements stimulate the immune system while others increase our risk for certain cancers. Q U EST I ON 6.4 Given what you know about the evolution of antibiotic resistance, why would it be helpful to administer bacteriophages alongside an antibiotic? O N O¯ HO P O¯ O O P N O¯ O O P O NH NH2 N O O Active form of acyclovir that when added to DNA will halt replication O O HO P OH O O O P N O OH P OH O O N NH N NH2 OH Guanine nucleotide that is normally added to DNA FIGURE 6.23 Active form of acyclovir Inside infected cells, viral enzymes activate acyclovir to the represented form. The activated drug is very similar to guanine nucleotides that are the “G” of the G-C pairs in DNA. 186 such as veterinarians or people that research the rabies virus. Even if the rabies vaccine was administered immediately upon a suspected exposure to a rabid animal, it takes weeks before the patient makes sufficient antibodies to protect from infection. Having a viral attachment blocker is an important part of the medical toolkit against rabies, since there have only been a handful of people who have ever survived the full-blown infection. (See Chapter 18 for more on rabies.) Blocking viral penetration is a key strategy in postexposure prohylaxis against HIV. The injected peptide drug enfuvirtide is administered to people who suspect they have been exposed to HIV, such as a healthcare worker who gets an accidental needle stick while caring for an AIDS patient. The drug binds to proteins essential for HIV to enter new cells. It works by blocking fusion of the viral envelope with host cell membranes. Docosanol is not a postexposure prophylactic, but, like enfuvirtide, it blocks viral entry into host cells. As a topically applied agent that blocks viral fusion, its main clinical use is to treat cold sores caused by HHV-1. Lastly, palivizumab is an injectable antibody preparation that blocks fusion of the respiratory syncytial virus (RSV)––a virus that causes potentially dangerous respiratory distress in premature babies. If viral entry can’t be targeted, then targeting other steps that precede viral replication may be useful. One drug that is in late-stage clinical trials to prevent the common cold is vapendavir. This orally administered drug includes a small compound that binds to capsid proteins of rhinoviruses to inhibit viral uncoating. Thus, even if the virus enters host cells, the viral genome is not accessible for replication. Other inhibitors of viral uncoating include amantadine and its chemically related cousin rimantadine; both of these block influenza A uncoating. Unfortunately, as of 2009, most influenza A isolates are now resistant to these compounds. Drugs that Target Viral Replication, Assembly, and Release The most common drugs that block replication are nucleoside analogs. There are at least a dozen drugs in this class, but all of them tend to work by blocking successful nucleic acid production to limit viral replication. In certain virus-infected cells, nucleoside analogs are activated into compounds that mimic normal nucleotides (adenine, guanine, cytosine, thymine, and uracil). However, these analogs are chemically different from natural nucleotides and represent a chemical dead end for nucleic acid replication. If a DNA or RNA polymerase uses an analog in place of a natural nucleotide, nucleic acid replication will be interrupted. One of the best known nucleoside analogs is the compound acyclovir, the active ingredient in brand name antiviral drugs such as Zovirax and Valtrex (FIG. 6.23). This compound blocks DNA replication in cells infected with HHV-1, HHV-2, or varicellazoster virus (HHV-3). Blocking DNA replication sounds like a dangerous prospect, and it definitely would be if this drug did not specifically target cells infected with these viruses. Only cells infected with these HHV viruses can chemically modify the drug to activate it. The activated drug mimics guanosine nucleotides that DNA polymerases use to make DNA. If this nucleotide analog is incorporated, it prematurely ends DNA replication. The activated form of the drug preferentially interacts with viral DNA polymerases over host DNA polymerases, but that preference is not absolute, so the drug can also lead to collateral host cell damage or even cell death. While acyclovir targets DNA-based viruses, another nucleoside analog, ribavirin, targets RNA polymerases and is used to combat certain RNA viruses. It is mostly effective against human respiratory syncytial virus and hepatitis C virus; it may also have some efficacy in early infections with Lassa virus, hanta virus, and some others that cause certain hemorrhagic fevers. Unfortunately, it is not effective against hemorrhagic fevers like Ebola. Nucleoside reverse transcriptase inhibitors (NRTIs), target reverse transcriptase enzymes. Recall that reverse transcriptase is an enzyme that retroviruses require to convert their RNA genome into a DNA format, so that the virus can integrate into the host genome and perpetuate viral replication. HIV treatment usually CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 186 05/10/17 11:43 AM includes administration of the NRTI drug called azidothymidine (also known as AZT, or by brand names like Retrovir and Zidovudine). Originally, AZT was developed as a potential anticancer drug, but it failed in that capacity, only to be resurrected later as a successful drug for fighting HIV. Because AZT has about a 100-fold preference for binding to viral reverse transcriptase as opposed to human polymerases, at therapeutic doses it has minimal effects on noninfected host cells. Other enzymes besides reverse transcriptase can also be targeted to limit HIV replication. For example, HIV requires a number of enzymes called proteases (enzymes that cut proteins) to make mature infectious virions. Over 25 protease inhibitors have been developed to target HIV proteases to limit viral assembly. Lastly, viral replication can be blocked by antisense antivirals. These agents are short sequences of nucleotides that are complementary to the RNA transcribed by specific viruses. After viral mRNA is made, antisense antivirals can bind to it, preventing the host ribosome from translating the viral mRNA. Additionally, the targeted RNA is destroyed by cellular enzymes. So far the only antisense drug approved to treat viral associated pathologies is Vitravene. In 1998 this drug was FDA approved for the local treatment of cytomegalovirus (CMV)induced retinitis. Because this drug has largely been replaced by safer and more effective therapies, it is no longer manufactured. Naturally occurring substances called interferons are released by cells in response to viral infections. Interferons signal the presence of a virus, causing neighboring, uninfected cells to make defensive changes that limit viral entry and/or replication. These molecules can be produced in the lab and administered to help limit the progression of certain viral infections. Interferon therapies have been used to treat hepatitis B and C infections in combination with other drugs. Unfortunately, they tend to generate flu-like symptoms as side effects, making them a fairly unpleasant treatment option. (Chapter 11 reviews the role of interferons in immune responses.) Lastly, if viral entry or replication can’t be blocked, there is always the last step––virus release. Drugs like oseltamivir (Tamiflu) and zanamivir (Relenza) prevent influenza A and influenza B virions from budding off the host cell surface. CHEM NOTE Nucleotides are the basic building blocks of the nucleic acids DNA and RNA. They include a fivecarbon sugar, a nitrogenous base, and phosphate groups. In contrast, a nucleoside is basically a nucleotide that is missing the phosphate groups. Most antiviral drugs are administered as a nucleoside form and then virally encoded enzymes that are only found in infected cells convert the drug to the active nucleotide form. Nucleoside analogs only mimic natural nucleosides, so they may not contain all the same parts as a naturally occurring nucleoside. Prions are infectious proteins. Even though viruses are not cells and therefore are nonliving by current biology definitions, some virologists debate over whether or not viruses should be considered alive, since they act like a life form in some ways (albeit indirectly, via a host). But another type of infectious agent, prions, are clearly not living and (like viruses) are acellular. Prions are infectious proteins. They prove that causing disease doesn’t require much biochemical sophistication. Unlike viruses, they do not have any genetic material and they do not replicate. Prions cause a class of diseases called transmissible spongiform encephalopathies (TSEs), which destroy brain tissue. These extremely rare diseases have a worldwide incidence of about one case per million people. Diagnosis is only confirmable during autopsy by the characteristic spongy holes in the brain tissue of those infected. Some patients with a TSE develop dementia at an older age, whereas others demonstrate psychological and behavioral symptoms at a median age of 27. The normal prion protein is found throughout the nervous system, but it is especially prevalent in the brain. In their normal form, prions have a neuroprotective role, but when misfolded, these proteins cause serious damage to the nervous system. The current theory is that the misfolded, infectious version of the prion protein makes contact with the normal version, causing changes to the normal protein’s shape. This leads to a chain reaction of protein misfolding that spreads throughout the brain’s prion proteins. As misshapen prion proteins clump together, they cause brain tissue degeneration through an unknown mechanism. As the brain tissue deteriorates, spongy holes are left in the brain, hence the name “spongiform” encephalopathy (FIG. 6.24). (For a more detailed discussion of TSEs, see Chapter 18.) FIGURE 6.24 Prions Prions infect nervous tissue and cause sponge-like holes. Clinical Aspects of Viruses and Prions M06_NORM8290_01_SE_C06.indd 187 187 17/07/17 4:58 PM BENCH to BEDSIDE Safeguarding Healthcare Workers Who Treat Ebola Patients An article in the journal The Lancet evaluated the effect of postexposure prophylaxis for healthcare workers after exposure to Ebola. Study participants were given a combination of favipiravir, a viral RNA-polymerase inhibitor; TKM-Ebola, which binds and inactivates the viral mRNA; and ZMapp, immunotherapy with specific combinations of three monoclonal anti-Ebola virus antibodies. None of the treated patients developed Ebola. Despite the availability of potentially useful postexposure prophylaxis treatments, the goal is to prevent an exposure in the first place. Despite the fact that Ebola has landed on U.S. shores, most healthcare workers still have only limited training on how to manage patients infected with this deadly biolevel 4 agent. The need for enhanced training on this front became all too clear when two American nurses, Nina Pham and Amber Vinson, contracted Ebola while caring for patients. The CDC requires that people treating Ebola patients wear coveralls with a hood and integrated face shield as well as a respirator. Extensive training and diligent observation to ensure proper doffing and donning of gear as well as equipment maintenance is central to prevention. Sources: Jacobs, M., Aarons, E., Bhagani, S., Buchanan, R., Cropley, I., Hopkins, S., et al. (2015). Postexposure prophylaxis against Ebola virus disease with experimental antiviral agents: A case-series of healthcare workers. The Lancet Infectious Diseases, 15(11), 1300–1304. doi:10.1016/s1473-3099(15)00228-5. Ebola healthcare worker Types of Spongiform Encephalopathies There are many forms of spongiform encephalopathies; some are inherited, while others are acquired. Gerstmann-Straussler-Schienker syndrome and a condition called fatal familial insomnia are inherited when a parent passes on a gene that encodes a mutated form of the prion protein to his or her children. CreuzfeldtJakob disease (CJD) is probably the best known prion-based disease. It has four forms: variant, sporadic, inherited, and iatrogenic. Variant CJD is caused by a prion that has a different protein sequence than other CJDs. It was given the infamous moniker “mad cow disease” after a study linked human infections to consumption of meat contaminated with the form of the prion that primarily affects cattle. Sporadic CJD is linked to a spontaneous mutation in the normal cellular prion protein and affects 200–400 people in the United States every year. Inherited CJD is uncommon, accounting for only 15 percent of human prion disease cases. Iatrogenic CJD, which is accidental transmission of CJD to a patient as a result of a medical intervention, is much rarer and has been detected in only 400 people worldwide. Most iatrogenic CJD cases can be traced to either contaminated surgical instruments or tissue transplants derived from infected cadavers. Dura mater grafts used for head injury and brain surgery patients and corneal transplants have been associated with iatrogenic CJD. In the past, human growth hormone treatments were also a risk factor for iatrogenic CJD. This is because the hormone was formerly derived from cadaver pituitary tissue. As of 1985, human growth hormone is no longer derived from cadavers and is instead produced in genetically engineered cells in a lab, so people who get human growth hormone therapies are no longer at risk for iatrogenic CJD. BUILD YOUR FOUNDATION Build your foundation by answering the Quick Quiz: scan this code or visit the Mastering Microbiology Study Area to quiz yourself. 188 QUICK QUIZ 22. How are bacteriophages and animal viruses grown in the laboratory? 23. What is the plaque assay and what information does it reveal? 24. What are the different main methods for detecting if a patient has been exposed to a virus or if a virus is present in a sample? What are the limitations of the methods you named? 25. What are three different antiviral drugs and their modes of action? 26. What are prions and what diseases do they cause? 27. How is iatrogenic Creuzfeldt-Jakob disease (CJD) contracted versus variant CJD? CHAPTER 6 Viruses and Prions M06_NORM8290_01_SE_C06.indd 188 17/07/17 4:58 PM VISUAL SUMMARY | Viruses and Prions Viral Genomes Viral Structure Viruses can have double- or single-stranded DNA or RNA. Naked Helical capsid Icosahedral capsid Complex structure DNA viral genomes Circular or linear Often double stranded May also be single stranded Enveloped Helical capsid RNA viral genomes Linear or segmented Often single stranded May also be double stranded Viruses can have special enzymes. Icosahedral capsid Reverse transcriptase Viral RNA Viral Replication Bacteriophage + + Viral DNA Antiviral Drugs Animal virus Examples of drugs that block steps in animal virus replication: Attachment Attachment Maraviroc (HIV) Penetration Penetration Docosanol (HHV-1) Uncoating (in animal viruses) Uncoating Amantadine (influenza) Replication Vapendavir (rhinoviruses) Assembly Assembly AZT and protease inhibitors (HIV) Release Release Replication + Reverse transcription = + + Some viruses integrate their genome into the host cell genome. Temperate phage HIV can form a provirus Oseltamivir (influenza) Prions Prions are infectious proteins that cause transmissible spongiform encephalopathies. Provirus Prophage Human T cell Light micrograph of prion-induced sponge-like holes in brain tissue. 189 M06_NORM8290_01_SE_C06.indd 189 05/10/17 11:43 AM CHAPTER 6 OVERVIEW General Virus Characteristics Viruses are infectious, acellular particles that must invade living cells to replicate. The viral genome is encased in a capsid that’s made of repeating protein units called capsomeres. Capsids can be helical, complex, or icosahedral. Outside the capsid some viruses have a lipid-based envelope. Those without an envelope are called naked. Many viruses have spikes (peplomers) that help them infect a host cell. Viruses can have DNA or RNA genomes. RNA viruses have different ways of making viral mRNA: 0 Single-stranded RNA genomes that are positive/sense-stranded RNA (ssRNA+) can be used directly by the host’s ribosomes to make protein. 0 Single-stranded RNA genomes that are antisense/negative-stranded RNA (ssRNA–) use RNA-dependent RNA polymerases to make mRNA. 0 Retroviruses convert their RNA genome into DNA and integrate into the host genome. 0 Double-stranded RNA genomes use virally encoded RNAdependent RNA polymerases after unwinding the strands in order to make mRNA. Rapid replication promotes viral evolution. Because they use errorprone polymerases to copy their genome, RNA viruses tend to mutate more frequently than DNA viruses. Antigenic drift occurs due to small mutations in antigen-coding genes; it is responsible for seasonal influenza variations. Antigenic shift occurs when two different strains infect the same cell and undergo reassortment to make a new virus strain; this is often responsible for pandemic strains. Classifying and Naming Viruses Viruses are assigned to orders, families (and occasionally subfamilies), genus, and species. They are mainly classified by the type of nucleic acid present (DNA or RNA); capsid symmetry; the presence or absence of an envelope; and genome architecture (ssDNA, ssRNA, etc.). Size, host range, tissue/cell tropism, and disease features help to refine viral groupings into species. Viruses exhibit characteristic host ranges and tissue tropisms. Introduction to Viral Replication Pathways Viruses use a host cell’s energy, enzymes, organelles, and molecular building blocks to build new virions. Bacteriophages are viruses that infect bacteria. They perform either lytic or lysogenic replication pathways. 0 Lytic pathways kill the host cell. They involve attachment, penetration, replication, assembly, and release from the host. 0 Lysogenic replication involves insertion of the phage genome into the host’s chromosome to form a prophage. Through phage conversion, prophages confer new properties onto a host 190 bacterium. At any point the prophage can excise itself from the host genome to continue into the lytic pathway. Animal virus replication has six general steps: (1) Attachment (often aided by spikes or other capsid proteins); (2) penetration (naked viruses mainly enter by endocytosis, while enveloped viruses use membrane fusion or endocytosis); (3) uncoating; (4) replication; (5) assembly; and (6) release (enveloped viruses bud out of host cells, while naked viruses lyse the cell). Acute viral infections have a rapid increase in virions and then resolve. Persistent infections last longer. Some are caused by viruses that integrate into the host genome, forming a provirus. Some persistent infections are caused by oncoviruses, viruses that can cause cancer. Latent persistent infections involve viruses that lie dormant for long periods of time between outbreaks with large numbers of free virions. Chronic persistent infections have a slow buildup of virions over time, eventually leading to a more rapid increase in viral replication. Clinical Aspects of Viruses and Prions The number of infectious virions, or viral titer, can be determined using a plaque assay. Animal viruses may be grown in animals, cell culture, and fertilized chicken eggs. Detection tests are evaluated based on specificity, sensitivity, and turnaround time. Specificity means that the diagnostic test reliably detects only the virus(es) of interest without producing false positive results. Sensitivity means the test detects very low levels of the target to limit false negative results. The two major classes of tests for detecting viruses are an