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Nearly all productively infected cells die (see further for exceptions), presumably because the viral genetic program is dominant and precludes the continuation of normal cell functions required for survival. In many cases, direct viral interference with normal cellular metabolic processes leads to cell death. For example, picornaviruses shut off host protein synthesis soon after infection, and many DNA human viruses interfere with normal cell-cycle controls. In many cases, the end result of such insults is a triggering of a cellular stress response called programmed cell death or apoptosis. Some viruses are known to code for proteins that block or delay apoptosis, probably to stave off cell death until the virus replication cycle has been completed. Ultimately, the cell lysis that accompanies cell death is responsible for the release of naked capsid viruses into the environment.
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Naked capsid viruses lacking specific lysis mechanisms are released with cell death
Some viruses block or delay apoptosis to allow completion of the virus replication cycle
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Most enveloped human viruses acquire their membrane by budding either through the plasma membrane or, in the case of herpesviruses, through the nuclear membrane; however, in some other viruses such as coronaviruses and poxviruses, budding occurs through cytoplasmic membranes. Thus, for these viruses, release from the cell is coupled to the final stage of virion assembly. The herpesviruses ultimately escape from the cell when the membrane of the exocytic vesicle fuses with the plasma membrane. The poxviruses appear to program the formation of membrane structures and acquire membrane from Golgi apparatus that is lost upon the release of extracellular enveloped virions.
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Most enveloped viruses acquire an envelope during release by budding
Poxviruses program the formation of envelope membrane structure and acquire membrane from the Golgi apparatus
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The membrane changes that accompany budding appear to be just the reverse of the entry process described before for those viruses that enter by direct fusion (compare Figure 6–9 and Figure 6–15). The region of the cellular membrane where budding is to occur acquires a cluster of viral glycoprotein spikes. These proteins are synthesized by the pathway that normally delivers cellular membrane proteins to the surface of the cell by way of the Golgi apparatus. At the site of the glycoprotein cluster, the inside of the membrane becomes coated with a virion structural protein called the matrix or M protein. The accumulation of the matrix protein at the proper location is probably facilitated by the presence of a binding site for the matrix protein on the cytoplasmic side of the transmembrane glycoprotein spike. The matrix protein attracts the completed nucleocapsid that triggers the envelopment process leading to the release of the completed particle to the outside (Figure 6–15).
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The membrane site for budding first acquires virus-specified spikes and then matrix protein that attracts nucleoprotein complex (nucleocapsids)
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For viruses that bud, it is important to note that the plasma membrane of the infected cell contains virus-specific glycoproteins that represent foreign (viral) antigens. This means that infected cells become targets for the immune system. In fact, cytotoxic T lymphocytes that recognize these antigens can be a significant factor in combating a virus infection.
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The process of initial viral budding usually does not lead directly to cell death because the plasma membrane can be repaired after budding. It is likely that cell death for most enveloped viruses, as for naked capsid viruses, is related to the loss of normal cellular functions required for survival or as a result of apoptosis. Unlike most retroviruses that do not kill the host cell, HIV-1 is cytopathic. Although the mechanism of HIV-1 cell killing is not entirely understood, factors such as the accumulation of viral DNA in the cytoplasm, the toxic effects of certain viral proteins, alterations in plasma membrane permeability, apoptosis, and cell–cell fusion are believed to contribute to the cytopathic potential of the virus.
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The initial budding process rarely causes cell death; however, too many daughter viruses released may result in loss of cell membrane permeability
Most retroviruses (except HIV) reproduce without cell death
HIV causes cytopathic effects (cell death)
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For retroviruses (except HIV-1 and other lentiviruses) and the filamentous bacteriophages, virus reproduction and cell survival are compatible. Retroviruses convert their RNA genome into double-stranded DNA, which integrates into a host cell chromosome and is transcribed just like any other cellular gene (see Chapter 18). Thus, the impact on cellular metabolism is minimal. Moreover, these retroviruses bud through the plasma membrane without any permanent damage to the cell (except HIV). How the cell escapes permanent damage in this case is unknown. As with the retroviruses, the infected cell continues to produce virus indefinitely.
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QUANTITATION OF VIRUSES
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Hemagglutination Assay
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For some human viruses such as influenza viruses, red blood cells from one or more human species contain receptors for the virion attachment proteins. Because the receptors and attachment proteins are present in multiple copies on the cells and virions, respectively, an excess of virus particles coats the cells and causes them to aggregate. This aggregation phenomenon was first discovered with influenza virus and is called hemagglutination. The virion attachment protein on the influenza virion is appropriately called the hemagglutinin. Furthermore, the presence of the hemagglutinin in the plasma membrane of the infected cell means that the cells as well as the virions bind the red blood cells. This reaction, called hemadsorption, is a useful indicator of infection by certain viruses.
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Virion and infected cell–attachment proteins also bind red blood cells
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Hemagglutination can be used to estimate the titer of virus particles in a virus-containing sample. Serially diluted samples of the virus preparation are mixed with a constant amount of red blood cells, and the mixture is allowed to settle in a test tube. Agglutinated red blood cells settle to the bottom to form a thin, dispersed layer. If there is insufficient virus to agglutinate the red blood cells, they will settle to the bottom of the tube and form a tight pellet. The difference is easily scored visually, and the endpoint of the agglutination is used as a relative measure of the virus concentration in the sample. Furthermore, hemagglutination can be inhibited by virus attachment protein-specific antibodies known as hemagglutination inhibition (HI), which can be used to determine titer of the antibody.
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The plaque assay is a method for determining the titer of infectious virions in a virus preparation or lysate. The sample is diluted serially, and an aliquot of each dilution is added to a vast excess of susceptible host cells. For a human virus, the host cells are usually attached to the bottom of a plastic Petri dish; for bacterial cells, adsorption is typically carried out in a cell suspension. In both cases, the cells are then immersed in a semisolid medium such as agar, which prevents the released virions from spreading throughout the entire cell population. Thus, the virus released from the initial and subsequent rounds of infection can invade only the cells in the immediate vicinity of the initial infected cell on the plate. The end result is an easily visible clearing of dead cells at each of the sites on the plate where one of the original infected cells was located. The clearing is called a plaque (Figure 6–16). Visualization in the case of human cells usually requires staining the cells. By counting the number of plaques and correcting for the dilution factor, the virus titer in the original sample can be calculated. The titer is usually expressed as the number of plaque-forming units per milliliter (pfu/mL).
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Plaque assay: Dilutions of virus are added to excess cells immobilized in agar
Replicated virus infects only neighboring cells, producing countable plaques
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Viral antigen can be quantified by using antigen–antibody specificity, as measured by enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), and immunofluoresence assay (IFA). Similar to other assays, in immunologic assays the antigen–antibody specificity and conditions should be worked out. For most viruses, commercial antibodies are available and can be used to detect or quantify the antigen of viruses in culture and body fluids, tissue biopsies, serum, plasma, and cerebrospinal fluid (CSF). The most common example is the detection and sometimes quantification of RSV by IFA in which RSV antigens are be measured in nasopharyngeal and throat washing, sputum, or bronchoalveolar lavage. In addition, viral antigens can be detected and quantified in blood (plasma or serum), which can then provide information on the amount of virus present in the blood. For example, HIV can be quantified by the levels of p24 (capsid) antigen in the culture fluid or blood. On the other hand, these immunoassays can also be used to detect antibodies produced during infection and are very powerful tools to diagnose infection. In this scenario, commercial antigens-coated plates are available that can be used to detect antibodies in patients’ samples for diagnosis of infection and monitoring the effectiveness of vaccines.
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Using antigen–antibody specificity, viral antigens can be quantified by ELISA
EIA or ELISA can be used to detect antibodies produced during infection
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Viral genomes, both RNA and DNA, can be quantified to determine the amount of virus (viral load) in blood (serum or plasma) or any given samples. The RNA genomes of the viruses are first reversely transcribed to cDNA by reverse transcriptase enzyme and then amplified by polymerase chain reaction (PCR). However, viral DNA genomes can be directly amplified by PCR to quantify the viral genomes. On the basis of the number of copies of the viral genomes, the amount of virus in any sample can be determined. This is the most sensitive and specific method to detect and quantify viral genomes. PCR is routinely used to determine viral load in HIV, hepatitis C virus, and other viral and microbial infections.
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DNA and RNA genomes of viruses can be quantified by PCR
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Viruses generally use two mechanisms—mutation and recombination—by which viral genomes change during infection and there are virologic, immunologic, and clinical consequences of some of these changes. Typically, the majority of the virus particles derived from a cell infected with a human virus are noninfectious in other cells as determined by a plaque assay. Although some of this discrepancy may be attributable to inefficiencies in the assay procedures, it is clear that many defective particles are being produced. In part, this production of defective particles arises because the mutation rates for human viruses are unusually high and many infections occur at high multiplicities, where defective genomes are complemented by nondefective viruses and therefore propagated.
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Majority of the human virus particles from an infected cell are defective
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Many DNA viruses use the host DNA synthesis machinery for replicating their genomes. Therefore, they benefit from the built-in proofreading and other error-correcting mechanisms used by the host cell. However, the large human viruses (adenoviruses, herpesviruses, and poxviruses) code for their own DNA polymerases, and these enzymes are not as effective at proofreading as the cellular polymerases. The resulting higher error rates in DNA replication endow the viruses with the potential for a high rate of mutation, but they are also partially responsible for the high frequency of defective viral particles.
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The replication of RNA viruses is characterized by even higher error rates of mutation because viral RNA polymerases do not possess any proofreading capabilities. The result is that error rates for RNA viruses commonly approach one mistake for every 2500 to 10 000 nucleotides polymerized. Such a high misincorporation rate means that, even for the smallest RNA viruses, virtually every round of replication introduces one or more nucleotide changes somewhere in the genome. If it is assumed that errors are introduced at random, most of the members of a clone (eg, in a plaque) are genetically different from all other members of the clone. The resulting mixture of different genome sequences for a particular RNA virus has been referred to as quasispecies to emphasize that the level of genetic variation is much greater than what normally exists in a species.
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Because of the redundancy in the genetic code, some mutations are silent and are not reflected in changes at the protein level, but many occur in essential genes and contribute to the large number of defective particles found for RNA human viruses. The concept of genetic stability takes on a new meaning in view of these considerations, and the RNA virus population as a whole maintains some degree of homogeneity only because of the high degree of fitness exhibited by a subset of the possible genome sequences. Thus, strong selective forces continually operate on a population to eliminate most mutants that fail to compete with the few very successful members of the population. However, any time the environment changes (eg, with the appearance of neutralizing antibodies), a new subset of the population is selected and maintained as long as the selective forces remain constant.
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High error rates for RNA viruses produce genetically heterogeneous populations
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The high mutation rates found for RNA viruses endow them with a genetic plasticity that leads readily to the occurrence of genetic variants and permits rapid adaptation to new environmental conditions. The large number of serotypes of rhinoviruses causing the common cold, for instance, likely reflects the potential to vary by mutation. Although rapid genetic change occurs for most if not all viruses, no medically important RNA virus has exhibited this phenomenon as conspicuously as influenza virus. Point mutations accumulate in the influenza genes coding for the two envelope proteins (hemagglutinin and neuraminidase), resulting in changes in the antigenic structure of the virions. These changes lead to new variants not recognized by the immune system of previously infected individuals. This phenomenon is called antigenic drift (see Chapter 9). Figure 6–17 shows the effect of mutations resulting in antigenic drift. Apparently, the domains of the two envelope proteins that are most important for immune recognition are not essential for virus entry and, as a result, can tolerate amino acid changes leading to antigenic variation. This feature may distinguish influenza from other human RNA viruses that possess the same high mutation rates, but do not exhibit such high rates of antigenic drift. Antigenic drift in epidemic influenza viruses from year to year requires continual updating of the strains used to produce annual influenza vaccines.
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High mutation rates permit adaptation to changed conditions
Mutations are responsible for antigenic drift in influenza viruses that allow escape from preexisiting immunity
Antigenic drift in influenza viruses requires updating or change of influenza strains for annual influenza vaccines
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The retroviruses likewise show high rates of variation because of error-prone reverse transcriptase enzyme that converts retroviral RNA into double-stranded DNA. For example, error rate for HIV-1 reverse transcriptase is approximately four to five errors per reverse transcription of the genome. After the viral DNA has integrated into the chromosome of the host cell, the retroviral DNA is transcribed by the host RNA polymerase II, which is also capable of generating errors. Accordingly, HIV-1 exhibits a high rate of mutation, and this property gives HIV-1 the ability to evolve rapidly in response to changing conditions in the infected host. Genetic variation has resulted in several clades or subtypes of HIV-1 worldwide.
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High rates of mutation in retroviruses are due to error-prone reverse transcriptase
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Retroviruses that exhibit high rates of antigenic variation such as HIV-1 pose particularly difficult problems for the development of effective vaccines. Attempts are being made to identify conserved and, therefore, presumably essential domains of the envelope proteins for these viruses, which might be useful in developing a genetically engineered vaccine.
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HIV-1 antigenic variation makes vaccine development difficult
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Von Magnus Phenomenon and Defective Interfering Particles
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In early studies with influenza virus, it was noted that serial passage of virus stocks at high multiplicities of infection led to a steady decline of infectious titer with each passage. At the same time, the titer of noninfectious particles increased. As discussed later, the noninfectious genomes interfere with the replication of the infectious virus and so are called defective interfering (DI) particles. Later, these observations were extended to include virtually all RNA and DNA human viruses. The phenomenon is now named after von Magnus, who described the initial observations with the influenza virus.
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Defective interfering particles accumulate at high multiplicities of infection
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A combination of two separate events leads to von Magnus phenomenon. First, deletion mutations occur at a significant frequency for all viruses. For DNA viruses, the mechanisms are not well understood, but deletions presumably occur as a result of mistakes in replication or by nonhomologous recombination. The basis for the occurrence of deletions in RNA viruses is better understood. All RNA polymerases (replicases) have a tendency to dissociate from the template RNA, but remain bound to the end of the growing RNA chain. By reassociating with the same or a different template at a different location, the replicase “finishes” replication, but, in the process, creates a shorter or longer RNA molecule. A subset of these variants possesses the proper signals for initiating RNA synthesis and continues replicating. Because the deletion variants in the population require less time to complete a replication cycle, they eventually predominate and constitute the DI particles.
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Deletions result from mistakes in replication, recombination, or the dissociation–reassociation of replicases (polymerases)
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Second, as their name implies, the DI particles interfere with the replication of nondefective or normal (wild-type) particles. Interference occurs because the DI particles successfully compete with the nondefective genomes for a limited supply of replication enzymes. The virions released at the end of the infection are therefore enriched for the DI particles. With each successive infection, the DI particles can predominate over the normal particles as long as the multiplicity of infection is high enough that every cell is infected with at least one normal infectious particle. If this condition is satisfied, then the normal virus particle can complement any defects in the DI particles and provide all of the viral proteins required for the infection. This process is called complementation. Eventually, however, as serial passage is continued, the multiplicity of infectious particles drops below one, and the majority of the cells are infected only with DI particles. When this happens, the proportion of DI particles in the progeny virus decreases.
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Defective interfering particles compete with infectious particles for replication enzymes
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Besides mutation, genetic recombination between related viruses is a major source of genomic variation. Bacterial cells as well as the nuclei of human cells contain the enzymes necessary for homologous recombination of DNA. Thus, it is not surprising that recombinants arise from mixed infections involving two different strains of the same type of DNA virus. The larger bacteriophages such as λ and T4 code for their own recombination enzymes, a fact that attests to the importance of recombination in the life cycles and possibly the evolution of these viruses. The fact that recombination has also been observed for cytoplasmic poxviruses suggests that they too code for their own recombination enzymes.
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Homologous recombination is common in DNA viruses
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As far as is known, cells do not possess the machinery to recombine RNA molecules. However, recombination among at least some RNA viruses has been observed by two different mechanisms. The first, which is unique to the viruses with segmented genomes (orthomyxoviruses and reoviruses), involves reassortment of segments during a mixed infection involving two different viral strains. Recombinant progeny viruses that differ from either parent can be accounted for by the formation of new combinations of the genomic segments that are free to mix with each other at some time during the infection. Reassortment of this type occurring during infections of the same cell by human and certain animal influenza viruses is believed to account for the occasional drastic change in the antigenicity of the human influenza A virus. These dramatic changes, called antigenic shifts (Figure 6–18), produce strains to which much of the human population lacks immunity and, thus, can have enormous epidemiologic and clinical consequences (see Chapter 9).
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Recombination for viruses with segmented RNA genomes involves reassortment of segments
Segment reassortment in mixed infections probably accounts for antigenic shifts in influenza virus
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The second mechanism of RNA virus recombination is exemplified by the genetic recombination between different forms of poliovirus. Because the poliovirus RNA genome is not segmented, reassortment cannot be invoked as the basis for the observed recombinants. In this case, it appears that recombination occurs during replication by a “copy choice” type of mechanism. During RNA synthesis, the replicase (polymerase) dissociates from one template and resumes copying a second template at the exact place where it left off on the first. The end result is a progeny RNA genome containing information from two different input RNA molecules. Strand switching during replication, therefore, generates a recombinant virus. Although this is not frequently observed, it is likely that most of the RNA human viruses are capable of this type of recombination.
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Poliovirus replicase (polymerase) switches templates to generate recombinants
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A “copy choice” mechanism has also been invoked to explain a high rate of recombination observed with retroviruses. Early after infection, the reverse transcriptase within the virion synthesizes a DNA copy of the RNA genome by a process called reverse transcription. In the course of reverse transcription, the enzyme is required to “jump” between two sites on the RNA genome (see Chapter 18). This propensity to switch templates apparently explains how the enzyme generates recombinant viruses. Because reverse transcription takes place in subviral particles, free mixing of RNA templates brought into the cell in different virus particles is not permitted. However, retroviruses are diploid, because each particle carries two copies of the genome. This arrangement appears to be a situation readymade for template switching during DNA synthesis, and most likely accounts for retroviral recombination.
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The diploid nature of retroviruses permits template switching and recombination during DNA synthesis
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Occasionally, animal retroviruses package a cellular mRNA into the virion rather than a second RNA genome. This arrangement can lead to copy choice recombination between the viral genome and a cellular mRNA. The end result is, sometimes, the incorporation of a cellular gene into the viral genome. This mechanism is believed to account for the production of highly oncogenic retroviruses containing modified cellular genes (see later).
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Occasional incorporation of host mRNA into retroviral particles may produce oncogenic variants
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Mixing of two closely related viruses, A and B, may result in generation of a hybrid virus with genome of virus A and outer surface protein of B. Upon infection of the new appropriate cells, the surface protein determines the tropism (means binds to the receptor on the host cells). However, the progeny viruses produced from this hybrid virus will be of type A virus because the genome in this hybrid virus belonged to type A virus.
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Temperate viruses, which can establish both productive and nonproductive responses, can infect a cell and enter a latent state that is characterized by little or no virus production. The viral DNA genome is replicated and segregated along with the cellular DNA when the cell divides. There exist two possible states for the latent viral genome. It can exist extrachromosomally (herpesviruses) like a bacterial plasmid, or it can become integrated into the chromosome (retroviruses) like the bacterial F factor in the formation of a high-frequency recombination (HFR) strain (see Chapter 21). Because the latent genome is usually capable of reactivation and entry into the lytic cycle, it is called a provirus or, in the case of bacteriophages, a prophage. In many cases, viral latency goes undetected; however, limited expression of proviral genes can occasionally endow the cell with a new set of properties. For example, the latent herpes simplex virus infection is characterized by the presence of viral DNA (extrachromosomal) in the nerve ganglion without any production of infectious virus particles as well as no symptoms (clinical latency) in infected individuals. However, this latent state can be reactivated characterized by the presence of infectious virus particles and symptoms. For instance, lysogeny (latent state) can lead to the production of virulence-determining toxins in some bacteria (lysogenic conversion— details in Chapter 21) and latency by a human virus may produce oncogenic transformation.
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The latent state involves infection of a cell with little or no virus production
Latent virus may be silent, change cell phenotype, or be induced to enter the lytic cycle
Latent genomes can exist extrachromosomally or can be integrated
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The significance of lysogeny and lysogenic conversion is described in Chapter 21. Diphtheria, scarlet fever, and botulism all are caused by toxins produced by bacteria that have been “converted” by a temperate bacteriophage. In each case, the gene that codes for the toxin protein resides in the phage DNA and is expressed together with the repressor gene in the lysogenic state.
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Lysogenic conversion results from expression of a prophage gene that alters cell phenotype
Several bacterial exotoxins are encoded in temperate bacteriophages