Chapter 6
Acellular Pathogens
Figure 6.1 The year 2014 saw the first large-scale outbreak of Ebola virus (electron micrograph, left) in human populations in West Africa (right). Such epidemics are now widely reported and documented, but viral epidemics are sure to have plagued human populations since the origin of our species. (credit left: modification of work by Thomas
W. Geisbert)
Chapter Outline
Introduction
Public health measures in the developed world have dramatically reduced mortality from viral epidemics. But when epidemics do occur, they can spread quickly with global air travel. In 2009, an outbreak of H1N1 influenza spread across various continents. In early 2014, cases of Ebola in Guinea led to a massive epidemic in western Africa. This included the case of an infected man who traveled to the United States, sparking fears the epidemic might spread beyond Africa.
Until the late 1930s and the advent of the electron microscope, no one had seen a virus. Yet treatments for preventing or curing viral infections were used and developed long before that. Historical records suggest that by the 17th century, and perhaps earlier, inoculation (also known as variolation) was being used to prevent the viral disease smallpox in various parts of the world. By the late 18th century, Englishman Edward Jenner was inoculating patients with cowpox to prevent smallpox, a technique he coined vaccination.[1]
Today, the structure and genetics of viruses are well defined, yet new discoveries continue to reveal their complexities. In this chapter, we will learn about the structure, classification, and cultivation of viruses, and how they impact their hosts. In addition, we will learn about other infective particles such as viroids and prions.
S. Riedel “Edward Jenner and the History of Smallpox and Vaccination.” Baylor University Medical Center Proceedings 18, no. 1 (January 2005): 21–25.
Viruses
Learning Objectives
Describe the general characteristics of viruses as pathogens
Describe viral genomes
Describe the general characteristics of viral life cycles
Differentiate among bacteriophages, plant viruses, and animal viruses
Describe the characteristics used to identify viruses as obligate intracellular parasites
Despite their small size, which prevented them from being seen with light microscopes, the discovery of a filterable component smaller than a bacterium that causes tobacco mosaic disease (TMD) dates back to 1892.[2] At that time, Dmitri Ivanovski, a Russian botanist, discovered the source of TMD by using a porcelain filtering device first invented by Charles Chamberland and Louis Pasteur in Paris in 1884. Porcelain Chamberland filters have a pore size of 0.1 µm, which is small enough to remove all bacteria ≥0.2 µm from any liquids passed through the device. An extract obtained from TMD-infected tobacco plants was made to determine the cause of the disease. Initially, the source of the disease was thought to be bacterial. It was surprising to everyone when Ivanovski, using a Chamberland filter, found that the cause of TMD was not removed after passing the extract through the porcelain filter. So if a bacterium was not the cause of TMD, what could be causing the disease? Ivanovski concluded the cause of TMD must be an extremely small bacterium or bacterial spore. Other scientists, including Martinus Beijerinck, continued investigating the cause of TMD. It was Beijerinck, in 1899, who eventually concluded the causative agent was not a bacterium but, instead, possibly a chemical, like a biological poison we would describe today as a toxin. As a result, the word virus, Latin for poison, was used to describe the cause of TMD a few years after Ivanovski’s initial discovery. Even though he was not able to see the virus that caused TMD, and did not realize the cause was not a bacterium, Ivanovski is credited as the original discoverer of viruses and a founder of the field of virology.
Today, we can see viruses using electron microscopes (Figure 6.2) and we know much more about them. Viruses are distinct biological entities; however, their evolutionary origin is still a matter of speculation. In terms of taxonomy, they are not included in the tree of life because they are acellular (not consisting of cells). In order to survive and reproduce, viruses must infect a cellular host, making them obligate intracellular parasites. The genome of a virus
H. Lecoq. “[Discovery of the First Virus, the Tobacco Mosaic Virus: 1892 or 1898?].” Comptes Rendus de l’Academie des Sciences – Serie III – Sciences de la Vie 324, no. 10 (2001): 929–933.
enters a host cell and directs the production of the viral components, proteins and nucleic acids, needed to form new virus particles called virions. New virions are made in the host cell by assembly of viral components. The new virions transport the viral genome to another host cell to carry out another round of infection. Table 6.1 summarizes the properties of viruses.
|
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Infectious, acellular pathogens |
Obligate intracellular parasites with host and cell-type specificity |
DNA or RNA genome (never both) |
Genome is surrounded by a protein capsid and, in some cases, a phospholipid membrane studded with viral glycoproteins |
Lack genes for many products needed for successful reproduction, requiring exploitation of host-cell genomes to reproduce |
Table 6.1
Figure 6.2 (a) Tobacco mosaic virus (TMV) viewed with transmission electron microscope. (b) Plants infected with tobacco mosaic disease (TMD), caused by TMV. (credit a: modification of work by USDA Agricultural Research Service—scale-bar data from Matt Russell; credit b: modification of work by USDA Forest Service, Department of Plant Pathology Archive North Carolina State University)
Why was the first virus investigated mistaken for a toxin?
Hosts and Viral Transmission
Viruses can infect every type of host cell, including those of plants, animals, fungi, protists, bacteria, and archaea. Most viruses will only be able to infect the cells of one or a few species of organism. This is called the host range. However, having a wide host range is not common and viruses will typically only infect specific hosts and only specific cell types within those hosts. The viruses that infect bacteria are called bacteriophages, or simply phages. The word phage comes from the Greek word for devour. Other viruses are just identified by their host group, such as animal or plant viruses. Once a cell is infected, the effects of the virus can vary depending on the type of virus.
Viruses may cause abnormal growth of the cell or cell death, alter the cell’s genome, or cause little noticeable effect in the cell.
Viruses can be transmitted through direct contact, indirect contact with fomites, or through a vector: an animal that transmits a pathogen from one host to another. Arthropods such as mosquitoes, ticks, and flies, are typical vectors for viral diseases, and they may act as mechanical vectors or biological vectors. Mechanical transmission occurs when the arthropod carries a viral pathogen on the outside of its body and transmits it to a new host by physical contact. Biological transmission occurs when the arthropod carries the viral pathogen inside its body and transmits it to the new host through biting.
In humans, a wide variety of viruses are capable of causing various infections and diseases. Some of the deadliest emerging pathogens in humans are viruses, yet we have few treatments or drugs to deal with viral infections, making them difficult to eradicate.
Viruses that can be transmitted from an animal host to a human host can cause zoonoses. For example, the avian influenza virus originates in birds, but can cause disease in humans. Reverse zoonoses are caused by infection of an animal by a virus that originated in a human.
Micro Connections
Fighting Bacteria with Viruses
The emergence of superbugs, or multidrug resistant bacteria, has become a major challenge for pharmaceutical companies and a serious health-care problem. According to a 2013 report by the US Centers for Disease Control and Prevention (CDC), more than 2 million people are infected with drug-resistant bacteria in the US annually, resulting in at least 23,000 deaths.[3] The continued use and overuse of antibiotics will likely lead to the evolution of even more drug-resistant strains.
One potential solution is the use of phage therapy, a procedure that uses bacteria-killing viruses (bacteriophages) to treat bacterial infections. Phage therapy is not a new idea. The discovery of bacteriophages dates back to the early 20th century, and phage therapy was first used in Europe in 1915 by the English bacteriologist Frederick Twort.[4] However, the subsequent discovery of penicillin and other antibiotics led to the near abandonment of this form of therapy, except in the former Soviet Union and a few countries in Eastern Europe. Interest in phage therapy outside of the countries of the former Soviet Union is only recently re-emerging because of the rise in antibiotic-resistant bacteria.[5]
Phage therapy has some advantages over antibiotics in that phages kill only one specific bacterium, whereas antibiotics kill not only the pathogen but also beneficial bacteria of the normal microbiota. Development of new antibiotics is also expensive for drug companies and for patients, especially for those who live in countries with high poverty rates.
Phages have also been used to prevent food spoilage. In 2006, the US Food and Drug Administration approved the use of a solution containing six bacteriophages that can be sprayed on lunch meats such as bologna, ham, and turkey to kill Listeria monocytogenes, a bacterium responsible for listeriosis, a form of food poisoning. Some consumers have concerns about the use of phages on foods, however, especially given the rising popularity of organic products. Foods that have been treated with phages must declare “bacteriophage preparation” in the list of ingredients or include a label declaring that the meat has been “treated with antimicrobial solution to reduce microorganisms.”[6]
US Department of Health and Human Services, Centers for Disease Control and Prevention. “Antibiotic Resistance Threats in the United States, 2013.” http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf (accessed September 22, 2015).
M. Clokie et al. “Phages in Nature.” Bacteriophage 1, no. 1 (2011): 31–45.
A. Sulakvelidze et al. “Bacteriophage Therapy.” Antimicrobial Agents and Chemotherapy 45, no. 3 (2001): 649–659.
US Food and Drug Administration. “FDA Approval of Listeria-specific Bacteriophage Preparation on Ready-to-Eat (RTE) Meat and Poultry Products.” http://www.fda.gov/food/ingredientspackaginglabeling/ucm083572.htm (accessed September 22, 2015).
Why do humans not have to be concerned about the presence of bacteriophages in their food?
What are three ways that viruses can be transmitted between hosts?
Classification and Taxonomy of Viruses
Although viruses are not classified in the three domains of life, their numbers are great enough to require classification. Since 1971, the International Union of Microbiological Societies Virology Division has given the task of developing, refining, and maintaining a universal virus taxonomy to the International Committee on Taxonomy of Viruses (ICTV). Since viruses can mutate so quickly, it can be difficult to classify them into a genus and a species epithet using the binomial nomenclature system. Thus, the ICTV’s viral nomenclature system classifies viruses into families and genera based on viral genetics, chemistry, morphology, and mechanism of multiplication. To date, the ICTV has classified known viruses in seven orders, 96 families, and 350 genera. Viral family names end in -viridae (e.g, Parvoviridae) and genus names end in −virus (e.g., Parvovirus). The names of viral orders, families, and genera are all italicized. When referring to a viral species, we often use a genus and species epithet such as Pandoravirus dulcis or Pandoravirus salinus.
The Baltimore classification system is an alternative to ICTV nomenclature. The Baltimore system classifies viruses according to their genomes (DNA or RNA, single versus double stranded, and mode of replication). This system thus creates seven groups of viruses that have common genetics and biology.
Aside from formal systems of nomenclature, viruses are often informally grouped into categories based on chemistry, morphology, or other characteristics they share in common. Categories may include naked or enveloped structure, single-stranded (ss) or double-stranded (ds) DNA or ss or ds RNA genomes, segmented or nonsegmented genomes, and positive-strand (+) or negative-strand (−) RNA. For example, herpes viruses can be classified as a dsDNA enveloped virus; human immunodeficiency virus (HIV) is a +ssRNA enveloped virus, and tobacco mosaic virus is a +ssRNA virus. Other characteristics such as host specificity, tissue specificity, capsid shape, and special genes or enzymes may also be used to describe groups of similar viruses. Table 6.2 lists some of the most common viruses that are human pathogens by genome type.
Common Pathogenic Viruses
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dsDNA, enveloped | Poxviridae |
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Poxviridae |
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| |
Herpesviridae |
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| |
dsDNA, naked | Adenoviridae |
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Papillomaviridae |
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| |
Reoviridae |
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| |
ssDNA, naked | Parvoviridae |
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Parvoviridae |
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| |
dsRNA, naked | Reoviridae |
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+ssRNA, naked | Picornaviridae |
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Picornaviridae |
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| |
Picornaviridae |
|
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Table 6.2
Common Pathogenic Viruses
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+ssRNA, enveloped | Togaviridae |
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Togaviridae |
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| |
Retroviridae |
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| |
−ssRNA, enveloped | Filoviridae |
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Orthomyxoviridae |
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| |
Rhabdoviridae |
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|
Table 6.2
What are the types of virus genomes?
Isolation, Culture, and Identification of Viruses
Learning Objectives
Discuss why viruses were originally described as filterable agents
Describe the cultivation of viruses and specimen collection and handling
Compare in vivo and in vitro techniques used to cultivate viruses
At the beginning of this chapter, we described how porcelain Chamberland filters with pores small enough to allow viruses to pass through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to isolate and identify viruses.
Isolation of Viruses
Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate (see Figure 6.16).
Figure 6.16 Membrane filters can be used to remove cells or viruses from a solution. (a) This scanning electron micrograph shows rod-shaped bacterial cells captured on the surface of a membrane filter. Note differences in the comparative size of the membrane pores and bacteria. Viruses will pass through this filter. (b) The size of the pores in the filter determines what is captured on the surface of the filter (animal [red] and bacteria [blue]) and removed from liquid passing through. Note the viruses (green) pass through the finer filter. (credit a: modification of work by U.S. Department of Energy)
What size filter pore is needed to collect a virus?
Cultivation of Viruses
Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (see Figure 6.17). The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected (see Figure 6.17). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.
Figure 6.17 (a) Flasks like this may be used to culture human or animal cells for viral culturing. (b) These plates contain bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells have been lysed. Viral titers increase on the plates to the left. (credit a: modification of work by National Institutes of Health; credit b: modification of work by American Society for Microbiology)
Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.
The embryo or host animal serves as an incubator for viral replication (see Figure 6.18). Location within the embryo or host animal is important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.
Figure 6.18 (a) The cells within chicken eggs are used to culture different types of viruses. (b) Viruses can be replicated in various locations within the egg, including the chorioallantoic membrane, the amniotic cavity, and the yolk sac. (credit a: modification of work by “Chung Hoang”/YouTube)
For in vitro studies, various types of cells can be used to support the growth of viruses. A primary cell culture is freshly prepared from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an enzymatic method using trypsin or collagenase to break up tissue and release single cells into suspension. Because of anchorage-dependence requirements, primary cell cultures require a liquid culture medium in a Petri dish or tissue-culture flask so cells have a solid surface such as glass or plastic for attachment and growth. Primary cultures usually have a limited life span. When cells in a primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact with other cells. When this cell-to-cell-contact occurs, mitosis is
triggered to stop. This is called contact inhibition and it prevents the density of the cells from becoming too high. To prevent contact inhibition, cells from the primary cell culture must be transferred to another vessel with fresh growth medium. This is called a secondary cell culture. Periodically, cell density must be reduced by pouring off some cells and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous cell lines, usually derived from transformed cells or tumors, are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in suspension) and may have lost their contact inhibition. As a result, continuous cell lines can grow in piles or lumps resembling small tumor growths (see Figure 6.19).
Figure 6.19 Cells for culture are prepared by separating them from their tissue matrix. (a) Primary cell cultures grow attached to the surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense and begin touching each other. At this point, growth can only be sustained by making a secondary culture. (b) Continuous cell cultures are not affected by contact inhibition. They continue to grow regardless of cell density. (credit “micrographs”: modification of work by Centers for Disease Control and Prevention)
An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first continuous tissue- culture cell line and were used to establish tissue culture as an important technology for research in cell biology, virology, and medicine. Prior to the discovery of HeLa cells, scientists were not able to establish tissue cultures with any reliability or stability. More than six decades later, this cell line is still alive and being used for medical research. See Eye on Ethics: The Immortal Cell Line of Henrietta Lacks to read more about this important cell line and the controversial means by which it was obtained.
What property of cells makes periodic dilutions of primary cell cultures necessary?
Eye on Ethics
The Immortal Cell Line of Henrietta Lacks
In January 1951, Henrietta Lacks, a 30-year-old African American woman from Baltimore, was diagnosed with cervical cancer at John Hopkins Hospital. We now know her cancer was caused by the human papillomavirus (HPV). Cytopathic effects of the virus altered the characteristics of her cells in a process called transformation, which gives the cells the ability to divide continuously. This ability, of course, resulted in a cancerous tumor that eventually killed Mrs. Lacks in October at age 31. Before her death, samples of her cancerous cells were taken without her knowledge or permission. The samples eventually ended up in the possession of Dr. George Gey, a biomedical researcher at Johns Hopkins University. Gey was able to grow some of the cells from Lacks’s sample, creating what is known today as the immortal HeLa cell line. These cells have the ability to live and grow indefinitely and, even today, are still widely used in many areas of research.
According to Lacks’s husband, neither Henrietta nor the family gave the hospital permission to collect her tissue specimen. Indeed, the family was not aware until 20 years after Lacks’s death that her cells were still alive and actively being used for commercial and research purposes. Yet HeLa cells have been pivotal in numerous research discoveries related to polio, cancer, and AIDS, among other diseases. The cells have also been commercialized, although they have never themselves been patented. Despite this, Henrietta Lacks’s estate has never benefited from the use of the cells, although, in 2013, the Lacks family was given control over the publication of the genetic sequence of her cells.
This case raises several bioethical issues surrounding patients’ informed consent and the right to know. At the time Lacks’s tissues were taken, there were no laws or guidelines about informed consent. Does that mean she was treated fairly at the time? Certainly by today’s standards, the answer would be no. Harvesting tissue or organs from a dying patient without consent is not only considered unethical but illegal, regardless of whether such an act could save other patients’ lives. Is it ethical, then, for scientists to continue to use Lacks’s tissues for research, even though they were obtained illegally by today’s standards?
Ethical or not, Lacks’s cells are widely used today for so many applications that it is impossible to list them all. Is this a case in which the ends justify the means? Would Lacks be pleased to know about her contribution to science and the millions of people who have benefited? Would she want her family to be compensated for the commercial products that have been developed using her cells? Or would she feel violated and exploited by the researchers who took part of her body without her consent? Because she was never asked, we will never know.
Figure 6.20 A multiphoton fluorescence image of HeLa cells in culture. Various fluorescent stains have been used to show the DNA (cyan), microtubules (green), and Golgi apparatus (orange). (credit: modification of work by National Institutes of Health)
Detection of a Virus
Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo, or tissue-culture cell, a sample can be prepared from the infected host, embryo, or cell line for further analysis under a brightfield, electron, or fluorescent microscope. Cytopathic effects (CPEs) are distinct observable cell abnormalities due to viral infection. CPEs can include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis (see Figure 6.21).
Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas. The type or severity of the CPE depends on the type of virus involved. Figure 6.21 lists CPEs for specific viruses.
Figure 6.21 (credit “micrographs”: modification of work by American Society for Microbiology)
Hemagglutination Assay
A serological assay is used to detect the presence of certain types of viruses in patient serum. Serum is the straw- colored liquid fraction of blood plasma from which clotting factors have been removed. Serum can be used in a direct assay called a hemagglutination assay to detect specific types of viruses in the patient’s sample. Hemagglutination is the agglutination (clumping) together of erythrocytes (red blood cells). Many viruses produce surface proteins or spikes called hemagglutinins that can bind to receptors on the membranes of erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the microscope, but this method does not always differentiate between infectious and noninfectious viral particles, since both can agglutinate erythrocytes.
To identify a specific pathogenic virus using hemagglutination, we must use an indirect approach. Proteins called antibodies, generated by the patient’s immune system to fight a specific virus, can be used to bind to components such as hemagglutinins that are uniquely associated with specific types of viruses. The binding of the antibodies with the hemagglutinins found on the virus subsequently prevent erythrocytes from directly interacting with the virus. So when erythrocytes are added to the antibody-coated viruses, there is no appearance of agglutination; agglutination has been inhibited. We call these types of indirect assays for virus-specific antibodies hemagglutination inhibition (HAI) assays. HAI can be used to detect the presence of antibodies specific to many types of viruses that may be causing or have caused an infection in a patient even months or years after infection (see Figure 6.22). This assay is described in greater detail in Agglutination Assays.
Figure 6.22 This chart shows the possible outcomes of a hemagglutination test. Row A: Erythrocytes do not bind together and will sink to the bottom of the well plate; this becomes visible as a red dot in the center of the well. Row B: Many viruses have hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination forms a lattice structure that results in red color throughout the well. Row C: Virus-specific antibody, the viruses, and the erythrocytes are added to the well plate. The virus-specific antibodies inhibit agglutination, as can be seen as a red dot in the bottom of the well. (credit: modification of work by Centers for Disease Control and Prevention)
What is the outcome of a positive HIA test?
Nucleic Acid Amplification Test
Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient samples. Polymerase chain reaction (PCR) is an NAAT used to detect the presence of viral DNA in a patient’s tissue or body fluid sample. PCR is a technique that amplifies (i.e., synthesizes many copies) of a viral DNA segment of interest. Using PCR, short nucleotide sequences called primers bind to specific sequences of viral DNA, enabling identification of the virus.
Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that the enzyme reverse transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acid in patient specimens.
Enzyme Immunoassay
Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules called antigens. The detecting antibody attaches to the target antigen with a high degree of specificity in what might be a complex mixture of biomolecules. Also included in this type of assay is a colorless enzyme attached to the detecting antibody. The enzyme acts as a tag on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end product. EIAs often rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (see Figure 6.23). EIAs for viral antigens are often used as preliminary screening tests. If the results are positive, further confirmation will require tests with even greater sensitivity, such as a western blot or an NAAT. EIAs are discussed in more detail in EIAs and ELISAs.
Figure 6.23 Similar to rapid, over-the-counter pregnancy tests, EIAs for viral antigens require a few drops of diluted patient serum or plasma applied to a membrane filter. The membrane filter has been previously modified and embedded with antibody to viral antigen and internal controls. Antibody conjugate is added to the filter, with the targeted antibody attached to the antigen (in the case of a positive test). Excess conjugate is washed off the filter.
Substrate is added to activate the enzyme-mediated reaction to reveal the color change of a positive test. (credit: modification of work by “Cavitri”/Wikimedia Commons)
What typically indicates a positive EIA test?
Viroids, Virusoids, and Prions
Learning Objectives
Describe viroids and their unique characteristics
Describe virusoids and their unique characteristics
Describe prions and their unique characteristics
Research attempts to discover the causative agents of previously uninvestigated diseases have led to the discovery of nonliving disease agents quite different from viruses. These include particles consisting only of RNA or only of protein that, nonetheless, are able to self-propagate at the expense of a host—a key similarity to viruses that allows them to cause disease conditions. To date, these discoveries include viroids, virusoids, and the proteinaceous prions.
Viroids
In 1971, Theodor Diener, a pathologist working at the Agriculture Research Service, discovered an acellular particle that he named a viroid, meaning “virus-like.” Viroids consist only of a short strand of circular RNA capable of self- replication. The first viroid discovered was found to cause potato tuber spindle disease, which causes slower sprouting and various deformities in potato plants (see Figure 6.24). Like viruses, potato spindle tuber viroids (PSTVs) take control of the host machinery to replicate their RNA genome. Unlike viruses, viroids do not have a protein coat to protect their genetic information.
Figure 6.24 These potatoes have been infected by the potato spindle tuber viroid (PSTV), which is typically spread when infected knives are used to cut healthy potatoes, which are then planted. (credit: Pamela Roberts, University of Florida Institute of Food and Agricultural Sciences, USDA ARS)
Viroids can result in devastating losses of commercially important agricultural food crops grown in fields and orchards. Since the discovery of PSTV, other viroids have been discovered that cause diseases in plants. Tomato planta macho viroid (TPMVd) infects tomato plants, which causes loss of chlorophyll, disfigured and brittle leaves, and very small tomatoes, resulting in loss of productivity in this field crop. Avocado sunblotch viroid (ASBVd) results in lower yields and poorer-quality fruit. ASBVd is the smallest viroid discovered thus far that infects plants. Peach latent mosaic viroid (PLMVd) can cause necrosis of flower buds and branches, and wounding of ripened fruit, which leads to fungal and bacterial growth in the fruit. PLMVd can also cause similar pathological changes in plums, nectarines, apricots, and cherries, resulting in decreased productivity in these orchards, as well. Viroids, in general, can be dispersed mechanically during crop maintenance or harvesting, vegetative reproduction, and possibly via seeds and insects, resulting in a severe drop in food availability and devastating economic consequences.
What is the genome of a viroid made of?
Virusoids
A second type of pathogenic RNA that can infect commercially important agricultural crops are the virusoids, which are subviral particles best described as non–self-replicating ssRNAs. RNA replication of virusoids is similar to that of viroids but, unlike viroids, virusoids require that the cell also be infected with a specific “helper” virus. There are currently only five described types of virusoids and their associated helper viruses. The helper viruses are all from the family of Sobemoviruses. An example of a helper virus is the subterranean clover mottle virus, which has an associated virusoid packaged inside the viral capsid. Once the helper virus enters the host cell, the virusoids are released and can be found free in plant cell cytoplasm, where they possess ribozyme activity. The helper virus undergoes typical viral replication independent of the activity of the virusoid. The virusoid genomes are small, only 220 to 388 nucleotides long. A virusoid genome does not code for any proteins, but instead serves only to replicate virusoid RNA.
Virusoids belong to a larger group of infectious agents called satellite RNAs, which are similar pathogenic RNAs found in animals. Unlike the plant virusoids, satellite RNAs may encode for proteins; however, like plant virusoids, satellite RNAs must coinfect with a helper virus to replicate. One satellite RNA that infects humans and that has been described by some scientists as a virusoid is the hepatitis delta virus (HDV), which, by some reports, is also called hepatitis delta virusoid. Much larger than a plant virusoid, HDV has a circular, ssRNA genome of 1,700 nucleotides and can direct the biosynthesis of HDV-associated proteins. The HDV helper virus is the hepatitis B virus (HBV).
Coinfection with HBV and HDV results in more severe pathological changes in the liver during infection, which is how HDV was first discovered.
What is the main difference between a viroid and a virusoid?
Prions
At one time, scientists believed that any infectious particle must contain DNA or RNA. Then, in 1982, Stanley Prusiner, a medical doctor studying scrapie (a fatal, degenerative disease in sheep) discovered that the disease was caused by proteinaceous infectious particles, or prions. Because proteins are acellular and do not contain DNA or RNA, Prusiner’s findings were originally met with resistance and skepticism; however, his research was eventually validated, and he received the Nobel Prize in Physiology or Medicine in 1997.
A prion is a misfolded rogue form of a normal protein (PrPc) found in the cell. This rogue prion protein (PrPsc), which may be caused by a genetic mutation or occur spontaneously, can be infectious, stimulating other endogenous normal proteins to become misfolded, forming plaques (see Figure 6.25). Today, prions are known to cause various forms of transmissible spongiform encephalopathy (TSE) in human and animals. TSE is a rare degenerative disorder that affects the brain and nervous system. The accumulation of rogue proteins causes the brain tissue to become sponge- like, killing brain cells and forming holes in the tissue, leading to brain damage, loss of motor coordination, and dementia (see Figure 6.26). Infected individuals are mentally impaired and become unable to move or speak. There is no cure, and the disease progresses rapidly, eventually leading to death within a few months or years.
Figure 6.25 Endogenous normal prion protein (PrPc) is converted into the disease-causing form (PrPsc) when it encounters this variant form of the protein. PrPsc may arise spontaneously in brain tissue, especially if a mutant form of the protein is present, or it may originate from misfolded prions consumed in food that eventually find their way into brain tissue. (credit b: modification of work by USDA)
Figure 6.26 Creutzfeldt-Jakob disease (CJD) is a fatal disease that causes degeneration of neural tissue. (a) These brain scans compare a normal brain to one with CJD. (b) Compared to a normal brain, the brain tissue of a CJD patient is full of sponge-like lesions, which result from abnormal formations of prion protein. (credit a (right): modification of work by Dr. Laughlin Dawes; credit b (top): modification of work by Suzanne Wakim; credit b (bottom): modification of work by Centers for Disease Control and Prevention)
TSEs in humans include kuru, fatal familial insomnia, Gerstmann-Straussler-Scheinker disease, and Creutzfeldt- Jakob disease (see Figure 6.26). TSEs in animals include mad cow disease, scrapie (in sheep and goats), and chronic wasting disease (in elk and deer). TSEs can be transmitted between animals and from animals to humans by eating contaminated meat or animal feed. Transmission between humans can occur through heredity (as is often the case with GSS and CJD) or by contact with contaminated tissue, as might occur during a blood transfusion or organ transplant. There is no evidence for transmission via casual contact with an infected person. Table 6.3 lists TSEs that affect humans and their modes of transmission.
Transmissible Spongiform Encephalopathies (TSEs) in Humans
|
|
---|---|
Sporadic CJD (sCJD) | Not known; possibly by alteration of normal prior protein (PrP) to rogue form due to somatic mutation |
Variant CJD (vCJD) | Eating contaminated cattle products and by secondary bloodborne transmission |
Familial CJD (fCJD) | Mutation in germline PrP gene |
Iatrogenic CJD (iCJD) | Contaminated neurosurgical instruments, corneal graft, gonadotrophic hormone, and, secondarily, by blood transfusion |
Kuru | Eating infected meat through ritualistic cannibalism |
Gerstmann-Straussler- Scheinker disease (GSS) | Mutation in germline PrP gene |
Table 6.3
National Institute of Neurological Disorders and Stroke. “Creutzfeldt-Jakob Disease Fact Sheet.” http://www.ninds.nih.gov/disorders/ cjd/detail_cjd.htm (accessed December 31, 2015).
Transmissible Spongiform Encephalopathies (TSEs) in Humans
|
|
---|---|
Fatal familial insomnia (FFI) |
|
Table 6.3
Prions are extremely difficult to destroy because they are resistant to heat, chemicals, and radiation. Even standard sterilization procedures do not ensure the destruction of these particles. Currently, there is no treatment or cure for TSE disease, and contaminated meats or infected animals must be handled according to federal guidelines to prevent transmission.
Does a prion have a genome?
Summary
Viruses are generally ultramicroscopic, typically from 20 nm to 900 nm in length. Some large viruses have been found.
Virions are acellular and consist of a nucleic acid, DNA or RNA, but not both, surrounded by a protein capsid. There may also be a phospholipid membrane surrounding the capsid.
Viruses are obligate intracellular parasites.
Viruses are known to infect various types of cells found in plants, animals, fungi, protists, bacteria, and archaea. Viruses typically have limited host ranges and infect specific cell types.
Viruses may have helical, polyhedral, or complex shapes.
Classification of viruses is based on morphology, type of nucleic acid, host range, cell specificity, and enzymes carried within the virion.
Like other diseases, viral diseases are classified using ICD codes.
Many viruses target specific hosts or tissues. Some may have more than one host.
Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating, biosynthesis, maturation, and release.
Bacteriophages have a lytic or lysogenic cycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads to integration of phage into the host genome.
Bacteriophages inject DNA into the host cell, whereas animal viruses enter by endocytosis or membrane fusion.
Animal viruses can undergo latency, similar to lysogeny for a bacteriophage.
The majority of plant viruses are positive-strand ssRNA and can undergo latency, chronic, or lytic infection, as observed for animal viruses.
The growth curve of bacteriophage populations is a one-step multiplication curve and not a sigmoidal curve, as compared to the bacterial growth curve.
Bacteriophages transfer genetic information between hosts using either generalized or specialized transduction.
Isolation, Culture, and Identification of Viruses
Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture).
Viruses can be isolated from samples by filtration.
Viral filtrate is a rich source of released virions.
Bacteriophages are detected by presence of clear plaques on bacterial lawn.
Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay).
Viroids, Virusoids, and Prions
Other acellular agents such as viroids, virusoids, and prions also cause diseases. Viroids consist of small, naked ssRNAs that cause diseases in plants. Virusoids are ssRNAs that require other helper viruses to establish an infection. Prions are proteinaceous infectious particles that cause transmissible spongiform encephalopathies.
Prions are extremely resistant to chemicals, heat, and radiation.
There are no treatments for prion infection.
Review Questions
Multiple Choice
The component(s) of a virus that is/are extended from the envelope for attachment is/are the:
capsomeres
spikes
nucleic acid
viral whiskers
Which of the following does a virus lack? Select all that apply.
ribosomes
metabolic processes
nucleic acid
glycoprotein
The envelope of a virus is derived from the host’s
nucleic acids
membrane structures
cytoplasm
genome
In naming viruses, the family name ends with
and genus name ends with .
−virus; −viridae
−viridae; −virus
−virion; virus
−virus; virion
What is another name for a nonenveloped virus?
enveloped virus
provirus
naked virus
latent virus
Which of the following leads to the destruction of the host cells?
lysogenic cycle
lytic cycle
prophage
temperate phage
A virus obtains its envelope during which of the following phases?
attachment
penetration
assembly
release
Which of the following components is brought into a cell by HIV?
a DNA-dependent DNA polymerase
RNA polymerase
ribosome
reverse transcriptase
A positive-strand RNA virus:
must first be converted to a mRNA before it can be translated.
can be used directly to translate viral proteins.
will be degraded by host enzymes.
is not recognized by host ribosomes.
What is the name for the transfer of genetic information from one bacterium to another bacterium by a phage?
transduction
penetration
excision
translation
Which of the followings cannot be used to culture viruses?
tissue culture
liquid medium only
embryo
animal host
Which of the following tests can be used to detect the presence of a specific virus?
EIA
RT-PCR
PCR
all of the above
Which of the following is NOT a cytopathic effect?
transformation
cell fusion
mononucleated cell
inclusion bodies
Which of these infectious agents do not have nucleic acid?
viroids
viruses
bacteria
prions
Which of the following is true of prions?
They can be inactivated by boiling at 100 °C.
They contain a capsid.
They are a rogue form of protein, PrP.
They can be reliably inactivated by an autoclave.
True/False
True or False: Scientists have identified viruses that are able to infect fungal cells.
Fill in the Blank
A virus that infects a bacterium is called a/an .
A/an virus possesses characteristics of both a polyhedral and helical virus.
A virus containing only nucleic acid and a capsid is called a/an virus or
virus.
The on the bacteriophage allow for binding to the bacterial cell.
An enzyme from HIV that can make a copy of DNA from RNA is called .
For lytic viruses, is a phase during a viral growth curve when the virus is not detected.
Viruses can be diagnosed and observed using a(n) microscope.
Cell abnormalities resulting from a viral infection are called .
Both viroids and virusoids have a(n) genome, but virusoids require a(n) to reproduce.
Short Answer
Discuss the geometric differences among helical, polyhedral, and complex viruses.
What was the meaning of the word “virus” in the 1880s and why was it used to describe the cause of tobacco mosaic disease?
Briefly explain the difference between the mechanism of entry of a T-even bacteriophage and an animal virus.
Discuss the difference between generalized and specialized transduction.
Differentiate between lytic and lysogenic cycles.
Briefly explain the various methods of culturing viruses.
Describe the disease symptoms observed in animals infected with prions.
Critical Thinking
Name each labeled part of the illustrated bacteriophage.
In terms of evolution, which do you think arises first? The virus or the host? Explain your answer.
Do you think it is possible to create a virus in the lab? Imagine that you are a mad scientist. Describe how you would go about creating a new virus.
Label the five stages of a bacteriophage infection in the figure:
Bacteriophages have lytic and lysogenic cycles. Discuss the advantages and disadvantages for the phage.
How does reverse transcriptase aid a retrovirus in establishing a chronic infection?
Discuss some methods by which plant viruses are transmitted from a diseased plant to a healthy one.
Label the components indicated by arrows.
Figure 6.27 (credit: modification of work by American Society for Microbiology)
What are some characteristics of the viruses that are similar to a computer virus?
Does a prion replicate? Explain.