Viruses are obligate intracellular parasites. They consist of a DNA or RNA genome surrounded by protein. They may also have an outer-membrane lipoprotein envelope. Viruses can replicate only within cells because their nucleic acid does not encode many enzymes necessary for the metabolism of proteins, carbohydrates, or lipids or for the generation of high-energy phosphates. Typically, viral nucleic acids encode messenger RNA (mRNA) and proteins necessary for replicating, packaging, and releasing progeny virus from infected cells.
Viruses differ from virusoids, viroids, and prions. Virusoids are nucleic acids that depend on cells and helper viruses for packaging their nucleic acids into virus-like particles. Viroids are naked, cyclical, mostly double-strand small RNAs that appear to be restricted to plants, spread from cell to cell, and are replicated by cellular RNA polymerase II. Prions (Chap. 453e) are abnormal proteins that propagate and cause disease by altering the structure of a normal cell protein. Prions cause neurodegenerative diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straüssler disease, kuru, and human or bovine spongiform encephalopathy (“mad cow disease”).
Viral genomes may consist of single- or double-strand DNA, single- or double-strand RNA, single-strand or segmented antisense RNA, or double-strand segmented RNA. Viral nucleic acids may encode only a few genes or more than 100. Sense-strand viral RNA genomes can be translated directly into protein, whereas antisense RNAs must be copied into translatable RNA. Sense and antisense genomes are also referred to as positive-strand and negative-strand genomes, respectively. Viral nucleic acid is usually associated with virus-encoded nucleoprotein(s) in the virus core. Viral nucleic acids and nucleoproteins are almost always enclosed in a protein capsid. Because of the limited genetic complexity of viruses, their capsids are usually composed of multimers of identical capsomeres made up of one or a few proteins. Capsids have icosahedral or helical symmetry. Icosahedral capsid structures approximate spheres and have two-, three-, or fivefold axes of symmetry, whereas helical capsid structures have only a twofold axis of symmetry. The nucleic acid, nucleoprotein(s), and protein capsid together are called a nucleocapsid.
Many viruses are composed of a nucleic acid core and a capsid. For these viruses, the outer capsid surface mediates contact with uninfected cells’ plasma membranes. Other viruses are more complex and have an outer phospholipid, cholesterol, glycoprotein, and glycolipid envelope that is derived from virus-modified infected cell membranes. Cell nuclear, endoplasmic reticulum, Golgi, or plasma membranes that become parts of the viral envelope have usually been modified during infection by the insertion of virus-encoded glycoproteins, which mediate contact of enveloped virus with uninfected cell surfaces. Matrix or tegument proteins may fill the space between the nucleocapsid and the outer envelope of the virus.
Enveloped viruses are usually sensitive to lipid solvents or detergents that can dissolve the envelope, whereas viruses with protein nucleocapsid exteriors may be somewhat detergent resistant. A schematic diagram of large and complex herpesviruses is shown in Fig. 214e-1. Structures of prototypical pathogenic human viruses are described in Table 214e-1. The relative sizes and structures of typical pathogenic human viruses are shown in Fig. 214e-2.
Schematic diagram of an enveloped herpesvirus with an icosahedral nucleocapsid. The approximate respective dimensions of the nucleocapsid and the enveloped particles are 110 and 180 nm. The capsid is composed of 162 capsomeres: 150 with sixfold and 12 with fivefold axes of symmetry.
TABLE 214e-1Virus Families Pathogenic for Humans ||Download (.pdf) TABLE 214e-1Virus Families Pathogenic for Humans
|Family ||Representative Viruses ||Type of RNA/DNA ||Lipid Envelope |
|RNA Viruses |
|Picornaviridae ||Poliovirus ||(+) RNA ||No |
| ||Coxsackievirus || || |
| ||Echovirus || || |
| ||Enterovirus || || |
| ||Rhinovirus || || |
| ||Hepatitis A virus || || |
|Caliciviridae ||Norovirus ||(+) RNA ||No |
| ||Hepatitis E virus || || |
|Togaviridae ||Rubella virus ||(+) RNA ||Yes |
| ||Eastern equine encephalitis virus || || |
| ||Western equine encephalitis virus || || |
|Flaviviridae ||Yellow fever virus ||(+) RNA ||Yes |
| ||Dengue virus || || |
| ||St. Louis encephalitis virus || || |
| ||West Nile virus || || |
| ||Hepatitis C virus || || |
| ||Hepatitis G virus || || |
|Coronaviridae ||Coronavirusesa ||(+) RNA ||Yes |
|Rhabdoviridae ||Rabies virus ||(−) RNA ||Yes |
| ||Vesicular stomatitis virus || || |
|Filoviridae ||Marburg virus ||(−) RNA ||Yes |
| ||Ebola virus || || |
|Paramyxoviridae ||Parainfluenza virus ||(−) RNA ||Yes |
| ||Respiratory syncytial virus || || |
| ||Newcastle disease virus || || |
| ||Mumps virus || || |
| ||Rubeola (measles) virus || || |
|Orthomyxoviridae ||Influenza A, B, and C viruses ||(−) RNA, 8 segments ||Yes |
|Bunyaviridae ||Hantavirus ||(−) RNA, 3 circular segments ||Yes |
| ||California encephalitis virus || || |
| ||Sandfly fever virus || || |
|Arenaviridae ||Lymphocytic choriomeningitis virus ||(−) RNA, 2 circular segments ||Yes |
| ||Lassa fever virus || || |
| ||South American hemorrhagic fever virus || || |
|Reoviridae ||Rotavirus ||ds RNA, 10–12 segments ||No |
| ||Reovirus || || |
| ||Colorado tick fever virus || || |
|Retroviridae ||Human T lymphotropic virus types 1 and 2 ||(+) RNA, 2 identical segments ||Yes |
| ||Human immunodeficiency virus types 1 and 2 || || |
|DNA Viruses |
|Hepadnaviridae ||Hepatitis B virus ||ds DNA with ss portions ||Yes |
|Parvoviridae ||Parvovirus B19 ||ss DNA ||No |
|Human papillomaviruses ||ds DNA ||No |
|JC virus || || |
|BK virus || || |
| ||Merkel cell polyoma virus || || |
|Adenoviridae ||Human adenoviruses ||ds DNA ||No |
|Herpesviridae ||Herpes simplex virus types 1 and 2b ||ds DNA ||Yes |
| ||Varicella-zoster virusc || || |
| ||Epstein-Barr virusd || || |
| ||Cytomegaloviruse || || |
| ||Human herpesvirus 6 || || |
| ||Human herpesvirus 7 || || |
| ||Kaposi’s sarcoma–associated herpesvirusf || || |
|Poxviridae ||Variola (smallpox) virus ||ds DNA ||Yes |
| ||Orf virus || || |
| ||Molluscum contagiosum virus || || |
Schematic diagrams of the major virus families including species that infect humans. The viruses are grouped by genome type and are drawn approximately to scale. Prototype viruses of each family that cause human disease are listed in Table 214e-1.
TAXONOMY OF PATHOGENIC HUMAN VIRUSES
As is apparent from Table 214e-1 and Fig. 214e-2, the classification of viruses into orders and families is based on nucleic acid composition, nucleocapsid size and symmetry, and presence or absence of an envelope. Viruses of a single family have similar structures and may be morphologically indistinguishable in electron micrographs. Subclassification into genera depends on similarity in epidemiology, biologic effects, and nucleic acid sequence.
Most viruses that infect humans have a common name related to their pathologic effects or the circumstances of their discovery. In addition, formal species names—consisting of the name of the host followed by the family or genus of the virus and a number—have been assigned by the International Committee on Taxonomy of Viruses. This dual terminology can cause confusion when viruses are referred to by either name—e.g., varicella-zoster virus (VZV) or human herpesvirus 3 (HHV-3).
STAGES OF VIRAL INFECTION OF CELLS IN CULTURE
Viral Interactions with Cell Surfaces and Cell Entry
To deliver its nucleic acid payload to the cell cytoplasm or nucleoplasm, a virus must overcome barriers posed by the cell’s plasma and cytoplasmic membranes. Infection is frequently initiated by weak electrostatic or hydrophobic interactions with the cell surface. Subsequent stronger, more specific attachment to a cell plasma membrane protein, carbohydrate, glycolipid, heparan sulfate proteoglycan, or sialic acid enables stable binding to a specific cell surface “receptor” that mediates fusion with the cell plasma membrane (see Table 145e-1). Receptor binding is often augmented by a viral surface protein interaction with more than one cell surface protein or co-receptor. Receptors and co-receptors are important determinants of the species and cell type that a virus can infect. For example, the HIV envelope glycoprotein binds to the T cell surface protein CD4 and then engages a chemokine receptor that is the definitive co-receptor for the virus and mediates entry into the cell cytoplasm. The Epstein-Barr virus (EBV) glycoprotein gp350 binds to the B lymphocyte complement receptor CD21 and then uses a major histocompatibility complex (MHC) class II molecule as a co-receptor and an integrin for definitive entry.
Viruses have evolved a wide range of strategies to enter cells. Influenza virus has an outer-membrane hemagglutinin glycoprotein that binds to sialic acid on respiratory tract cell plasma membranes. The hemagglutinin mediates adsorption to cell membranes, receptor aggregation, and endocytosis. As the endosome pH decreases in the cell cytoplasm, the influenza hemagglutinin conformation changes, enabling hydrophobic helices, which are initially at the base of the hemagglutinin, to extend, interacting and fusing with the endosome membrane and thereby releasing the viral genome into the cell cytoplasm. The influenza virus M2 membrane channel protein has a key role in lowering endosome pH and permitting virus and cell membrane fusion.
Nonenveloped viruses (e.g., human papillomaviruses [HPVs]) and some enveloped viruses have evolved to partially fuse with cell plasma membrane receptors and be internalized into endosomes. The low pH in an endosome can then trigger virus membrane or capsid fusion with the endocytic membrane, releasing viral DNA into the cytoplasm to initiate infection.
Hydrophobic interactions required for fusion can be susceptible to chemical inhibition or blockade. The HIV envelope glycoprotein gp120 is associated with gp41 on the viral surface. HIV gp120 binding to CD4 and then to specific chemokine receptors results in conformational changes that allow gp41 to initiate cell membrane fusion. The anti-HIV drug enfuvirtide is a small peptide derived from the gp41 structure. Enfuvirtide binds to gp41 and prevents conformational changes required for fusion. In contrast, maraviroc prevents virus entry by binding to the CCR5 receptor, thereby blocking gp120 binding to CCR5 and preventing gp120 fusion with CCR5.
Viral Gene Expression and Replication
After uncoating and release of viral nucleoprotein into the cytoplasm, the viral genome is transported to sites of expression and replication. To produce infectious progeny, viruses must produce proteins necessary for replicating their nucleic acids as well as structural proteins necessary for coating their nucleic acids and for assembling nucleic acids and proteins into progeny virus. Different viruses use different strategies and gene repertoires to accomplish these goals. Most DNA viruses, except for poxviruses, replicate their nucleic acid and assemble into nucleocapsids in the cell nucleus. RNA viruses, except for influenza viruses, transcribe and replicate their RNA and assemble in the cytoplasm before envelopment at the cell plasma membrane. The replication strategies of DNA and RNA viruses and of positive- and negative-strand RNA viruses are presented and discussed separately below. Medically important viruses of each group are used for illustrative purposes.
POSITIVE-STRAND RNA VIRUSES
RNA viruses of medical importance include positive-strand picornaviruses, flaviviruses, togaviruses, caliciviruses, and coronaviruses. Genome RNA from positive-strand RNA viruses is released into the cytoplasm without associated enzymes. Cell ribosomes recognize and associate with the viral genome’s internal ribosome entry sequence and translate a virus-encoded polyprotein. Proteases within the polyprotein cleave out the viral RNA polymerase and other viral proteins necessary for replication. Antigenomic RNA is next transcribed from the genome RNA template. Positive-strand genomes and mRNAs are then transcribed from the antigenome RNA by the viral RNA polymerase and are translated into capsid proteins. Genomic RNA is encapsidated in the cytoplasm and released as the infected cell undergoes lysis.
NEGATIVE-STRAND RNA VIRUSES
Medically important negative-strand RNA viruses include rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and bunyaviruses. The genomes of negative-strand viruses are frequently segmented. Negative-strand RNA viral genomes are released into the cytoplasm with an associated RNA polymerase and one or more polymerase accessory proteins. The viral RNA polymerase transcribes mRNAs as well as full-length antigenome RNA, which is the template for genome RNA replication. Viral mRNAs encode the viral RNA polymerase and accessory factors as well as viral structural proteins. Except for influenza virus, which transcribes its mRNAs and antigenome RNAs in the cell nucleus, negative-strand RNA viruses replicate entirely in the cytoplasm. All negative-strand RNA viruses, including influenza viruses, assemble in the cytoplasm.
DOUBLE-STRAND SEGMENTED RNA VIRUSES
Double-strand RNA viruses are taxonomically grouped in the family Reoviridae. The medically important viruses in this group are rotaviruses and Colorado tick fever virus. Reovirus genomes have 10–12 RNA segments. Reovirus particles contain an RNA polymerase complex. These viruses replicate and assemble in the cell cytoplasm.
Medically important DNA viruses include parvoviruses, which have small single-strand DNA genomes and cause transient arthritis, and polyomaviruses, including the smaller polyomaviruses such as JC virus, which causes progressive multifocal leukoencephalopathy in immunocompromised patients; BK virus; and Merkel cell polyomavirus. The larger HPVs cause warts as well as cervical, penile, and oral carcinomas. The next larger DNA viruses are adenoviruses, which mostly cause transient respiratory tract and ocular inflammatory disease. The herpesviruses include eight viruses that cause a wide range of inflammatory and malignant diseases in humans. EBV is an important cause of lymphomas and Hodgkin’s disease in both immunocompromised and immunocompetent people and of nasopharyngeal carcinoma in southern Chinese and northern African populations. Cytomegalovirus (CMV) is an important cause of transplacental infections and neonatal neurologic impairment. Poxviruses, the largest DNA viruses and the largest viruses that infect humans (barely visible by light microscopy), cause smallpox, monkeypox, and molluscum contagiosum. Aside from those of poxviruses, other DNA virus genomes enter the cell nucleus and are transcribed by cellular RNA polymerase II.
After receptor binding and fusion with plasma membranes or endocytic vesicle membranes, herpesvirus nucleocapsids are released into the cytoplasm with tegument proteins and are transported along microtubules to a nuclear pore. Capsids then release DNA into the nucleus.
DNA virus transcription and mRNA processing depend on both viral and cellular proteins. For herpes simplex virus (HSV), a viral tegument protein enters the nucleus and activates immediate-early genes, the first genes expressed after infection. Transcription of immediate-early genes requires the viral tegument protein and cell transcription factors. HSV becomes nonreplicating, or latent, in neurons because essential cell transcription factors for expression of viral immediate-early genes are docked in the cytoplasm in neurons. Heat shock or other cell stresses can cause these cell factors to enter the nucleus, activate viral gene expression, and initiate replication. This information explains HSV-1 latency in neurons and activation of replicative infection.
For adenoviruses and herpesviruses, transcription of immediate-early genes results in expression of early proteins necessary for viral DNA replication. Viral DNA synthesis is required to turn on late-gene expression and production of viral structural components. The HPVs, polyomaviruses, and parvoviruses are not dependent on transactivators encoded from the viral genome for early-gene transcription. Instead, their early genes have upstream enhancing elements that bind cell transcription factors. The early genes encode proteins that are necessary for viral DNA synthesis and late-gene transcription. DNA virus late genes encode structural proteins necessary for viral assembly and for viral egress from the infected cell. Late-gene transcription is continuously dependent on DNA replication. Therefore, inhibitors of DNA replication also stop late-gene transcription.
Each DNA virus family uses unique mechanisms for replicating its DNA. Adenovirus and herpesvirus DNAs are linear in the virion. Adenovirus DNA remains linear in infected cells and replicates as a linear genome, using an initiator protein–DNA complex. In contrast, herpesvirus DNA circularizes in the infected cell, and genomes replicate into linear concatemers through a “rolling-circle” mechanism. Full-length DNA genomes are cleaved and packaged into virus. Herpesviruses encode a DNA polymerase and at least six other viral proteins necessary for viral DNA replication. Acyclovir and ganciclovir prevent viral DNA synthesis when they are phosphorylated and incorporated into DNA by the viral polymerase. Herpesviruses also encode enzymes that increase the deoxynucleotide triphosphate pools. HPV and polyomavirus DNAs are circular both within the virus and in infected cells. These genomes are reproduced by cellular DNA replication enzymes and remain circular through replication and packaging. HPV and polyomavirus early proteins are necessary for DNA replication in both latent and viral replicative phases. Early viral proteins stimulate cells to remain in cycle, facilitating viral DNA replication.
Parvoviruses have negative single-strand DNA genomes and are the smallest DNA viruses. Their genomes are half the size of HPV genomes and include only two genes. The replication of autonomous parvoviruses, such as B19, depends on cellular DNA replication and requires the virus-encoded Rep protein. Other parvoviruses, such as adeno-associated virus (AAV), are not autonomous and require helper viruses of the adenovirus or herpesvirus family for their replication. AAV is being used as a potentially safe human gene therapy vector because its replication protein causes integration at a single chromosome site. The small genome size limits the range of proteins that can be expressed from AAV vectors.
As stated above, poxviruses are the largest DNA viruses. They are unique among DNA viruses in replicating and assembling in the cytoplasm. To accomplish cytoplasmic replication, poxviruses encode transcription factors, an RNA polymerase II orthologue, enzymes for RNA capping, enzymes for RNA polyadenylation, and enzymes for viral DNA synthesis. Poxvirus DNA also has a unique structure. The double-strand linear DNA is covalently linked at the ends, making a covalently closed double-strand circular genome. Replication of the circular genomes is initiated by nicking in inverted repeats at the ends of the linear DNA. During DNA replication, the genome is cleaved within the terminal inverted repeats, and the inverted repeats self-prime complementary-strand synthesis by the virus-encoded DNA polymerase. Like herpesviruses, poxviruses encode several enzymes that increase deoxynucleotide triphosphate precursor levels and thus facilitate viral DNA synthesis.
VIRUSES THAT USE BOTH RNA AND DNA GENOMES IN THEIR LIFE CYCLE
Retroviruses, including HIV, are RNA viruses that use a DNA intermediate to replicate their genomes. In contrast, hepatitis B virus (HBV) is a DNA virus that uses an RNA intermediate to replicate its genome. Thus these viruses are not purely RNA or DNA viruses. Retroviruses are RNA viruses with two identical sense-strand genomes and associated reverse transcriptase and integrase enzymes. Retroviruses differ from all other viruses in that they reverse-transcribe themselves into partially duplicated double-strand DNA copies and then routinely integrate into the host genome as part of their persistence and replication strategies. Inhibitors of reverse transcriptase (e.g., zidovudine) or integrase (e.g., raltegravir) are now commonly used as antiviral treatments for HIV infection. Integration of remnants and even complete copies of simple retrovirus DNAs into the human genome raises the possibility of replication-competent simple human retroviruses. However, endogenous human retrovirus replication has not been documented or associated with any disease. Integrated, replication-competent retroviral DNAs are also present in many animal species, such as pigs. These porcine retroviruses are a potential cause for concern in xenotransplantation because retrovirus replication could cause disease in humans.
Cellular RNA polymerase II and transcription factors regulate transcription from the integrated provirus DNA genome. Some retroviruses also encode regulators of transcription and RNA processing, such as Tax and Rex in human T lymphotropic virus (HTLV) types 1 and 2. HIV-1 and HIV-2 have orthologous Tat and Rev genes as well as the additional accessory proteins Vpr, Vpu, and Vif, which are important for efficient infection and immune escape. Full-length proviral transcripts are made from a promoter in the viral terminal repeat and serve as both genome RNAs that are packaged in the nucleocapsids and differentially spliced mRNAs that encode for the virus Gag protein, polymerase/integrase protein, and envelope glycoprotein. The Gag protein includes a protease that cleaves it into several components, including a viral matrix protein that coats the viral RNA. Viral RNA polymerase/integrase, matrix protein, and cellular tRNAs are key components in the viral nucleocapsid.
Protease inhibitors have been developed as effective agents against infections caused by HIV (e.g., saquinavir) or hepatitis C virus (HCV) (e.g., telaprevir).
HBV replication is unique in several respects. The HBV genome is a partially double-strand DNA genome that is repaired in infected cells to a fully double-strand circular DNA by the virion polymerase. Viral mRNAs are transcribed from the closed circular viral episome by the cellular RNA polymerase II and are translated to yield HBV proteins, including core protein, surface antigen, and polymerase. In addition, a full-genome-length mRNA is packaged into viral core particles in the cytoplasm of infected cells as an intermediate for viral DNA replication. This RNA associates with the viral polymerase, which also has reverse transcriptase activity and converts the full-length encapsidated RNA genome into partially double-strand DNA. Thus, nucleos(t)ide analogs that inhibit reverse transcription (e.g., tenofovir) are commonly used to treat HBV infection. HBV is believed to mature by budding through the cell’s plasma membrane, which has been modified by the insertion of viral surface antigen protein.
Viral Assembly and Egress
For most viruses, nucleic acid and structural protein synthesis is accompanied by the assembly of protein and nucleic acid complexes. The assembly and egress of mature infectious virus mark the end of the eclipse phase of infection, during which infectious virus cannot be recovered from the infected cell. Nucleic acids from RNA viruses and poxviruses assemble into nucleocapsids in the cytoplasm. For all DNA viruses except poxviruses, viral DNA assembles into nucleocapsids in the nucleus. In general, the capsid proteins of viruses with icosahedral nucleocapsids can self-assemble into densely packed and highly ordered capsid structures. Herpesviruses require an assemblin protein as a scaffold for capsid assembly. Viral nucleic acid then spools into the assembled capsid. For herpesviruses, a full unit of the viral DNA genome is packaged into the capsid, and a capsid-associated nuclease cleaves the viral DNA at both ends. In the case of viruses with helical nucleocapsids, the protein component appears to assemble around the nucleic acid, which contributes to capsid organization.
Viruses must egress from the infected cell and not bind back to their receptor(s) on the outer surface of the plasma membrane. Viruses can acquire envelopes from cytoplasmic membranes or by budding through the cell’s plasma membrane. Excess viral membrane glycoproteins are synthesized to saturate cell receptors and facilitate separation of the virus from the infected cell. Some viruses encode membrane proteins with enzymatic activity for receptor destruction. Influenza virus, for example, encodes a glycoprotein with neuraminidase activity. Neuraminidase destroys sialic acid on the infected cell’s plasma membrane so that newly released virus does not get stuck to the dying cell. Oseltamivir and zanamivir are neuraminidase inhibitors that are used to treat or provide prophylaxis for influenza virus infection. Herpesvirus nucleocapsids acquire an initial envelope by assembling in the nucleus and then budding through the nuclear membrane into the endoplasmic reticular space. The initially enveloped herpesvirus is then de-enveloped and released from the cell either by exocytosis or by re-envelopment at the plasma membrane. Nonenveloped viruses depend on the death and dissolution of the infected cell for their release.
FIDELITY OF VIRAL REPLICATION
Hundreds or thousands of progeny may be produced from a single virus-infected cell. Many particles partially assemble and never mature into virions. Many mature-appearing virions are imperfect and have only incomplete or nonfunctional genomes. Despite the inefficiency of assembly, a typical virus-infected cell releases 10–1000 infectious progeny. Some of these progeny may contain genomes that differ from those of the virus that infected the cell. Smaller, “defective” viral genomes have been noted with the replication of many RNA and DNA viruses. Virions with defective genomes can be produced in large numbers through packaging of incompletely synthesized nucleic acid. Adenovirus packaging is notoriously inefficient, and a high ratio of particle to infectious virus may limit the amount of recombinant adenovirus that can be administered for gene therapy since the immunogenicity of defective particles may contribute to adverse effects.
Changes in viral genomes can lead to mutant viruses of medical significance. In general, viral nucleic acid replication is more error-prone than cellular nucleic acid replication. RNA polymerases and reverse transcriptases are significantly more error-prone than DNA polymerases. Mutations can also be introduced into the HIV genome by APOBEC3G, a cellular protein that is packaged in the virion. APOBEC3G deaminates cytidine in the virion RNA to uridine. When reverse transcriptase subsequently uses the altered virion RNA as a template in the infected cell, a guanosine-to-adenosine mutation is introduced into the proviral DNA. Mutations resulting in less efficient viral growth, or fitness, may be detrimental to the virus. HIV-encoded Vif blocks APOBEC3G activity in the virion, inhibiting the debilitating effects of hypermutation on genetic integrity. Nevertheless, mutations resulting in evasion of the host immune response or resistance to antiviral drugs are preferentially selected in patients, with the consequent perpetuation of infection. Viral genomes can also be altered by recombination or reassortment between two related viruses in a single infected cell. Although this occurrence is unusual under most circumstances of natural infection, the genome changes can be substantial and can significantly alter virulence or epidemiology. Reassortment of the avian or mammalian influenza A hemagglutinin gene into a human influenza background can result in the emergence of new epidemic or pandemic influenza A strains.
VIRAL GENES NOT REQUIRED FOR VIRAL REPLICATION
Viruses frequently have genes encoding proteins that are not directly involved in replication or packaging of the viral nucleic acid, in virion assembly, or in regulation of the transcription of viral genes involved in those processes. Most of these proteins fall into five classes: (1) proteins that directly or indirectly alter cell growth; (2) proteins that inhibit cellular RNA or protein synthesis so that viral mRNA can be efficiently transcribed or translated; (3) proteins that promote cell survival or inhibit apoptosis so that progeny virus can mature and escape from the infected cell; (4) proteins that inhibit the host interferon response; and (5) proteins that downregulate host inflammatory or immune responses so that viral infection can proceed in an infected person to the extent consistent with the survival of the virus and its efficient transmission to a new host. More complex viruses of the poxvirus or herpesvirus family encode many proteins that serve these functions. Some of these viral proteins have motifs similar to those of cellular proteins, while others are quite novel. Virology has increasingly focused on these more sophisticated strategies evolved by viruses to permit the establishment of long-term infection in humans and other animals. These strategies often provide unique insights into the control of cell growth, cell survival, macromolecular synthesis, proteolytic processing, immune or inflammatory suppression, immune resistance, cytokine mimicry, or cytokine blockade.
MicroRNAs (miRNAs) are small noncoding RNAs that can regulate gene expression at the posttranscriptional level by targeting—and usually silencing—mRNAs. miRNAs were initially discovered in plants and plant viruses, where they alter expression of cell defensins. Herpesviruses are especially rich in miRNAs; for example, at least 23 miRNAs have been identified in EBV and 11 in CMV. Adenovirus and polyomavirus miRNAs have also been described. Increasing data indicate that animal viruses encode miRNAs to alter the growth and survival of host cells and the innate and acquired immune responses.
The concept of host range was originally based on the cell types in which a virus replicates in tissue culture. For the most part, the host range is limited by specific cell-surface proteins required for viral adsorption or penetration—i.e., to the cell types that express receptors or co-receptors for a specific virus. Another common basis for host-range limitation is the degree of transcriptional activity from viral promoters in different cell types. Most DNA viruses depend not only on cellular RNA polymerase II and the basal components of the cellular transcription complex but also on activated components and transcriptional accessory factors, both of which differ among differentiated tissues, among cells at various phases of the cell cycle, and between resting and cycling cells.
The importance of host range factors is illustrated by the effects of specific host determinants that limit the replication of influenza virus with avian or porcine hemagglutinins in humans. These viral proteins have adapted to bind avian or porcine sialic acids, and spread of avian or porcine influenza viruses in human populations is limited by their ability to infect human cells.
VIRAL CYTOPATHIC EFFECTS AND INHIBITORS OF APOPTOSIS
The replication of almost all viruses has adverse effects on the infected cell, inhibiting cellular synthesis of DNA, RNA, or proteins through efficient competition for key substrates and enzymatic processes. These general inhibitory effects enable viruses to nonspecifically limit components of innate host resistance, such as interferon (IFN) production. Viruses can specifically inhibit host protein synthesis by attacking a component of the translational initiation complex—frequently, a component that is not required for efficient translation of viral RNAs. Poliovirus protease 2A, for example, cleaves a cellular component of the complex that ordinarily facilitates translation of cellular mRNAs by interacting with their cap structure. Poliovirus RNA is efficiently translated without a cap because it has an internal ribosome entry sequence. Influenza virus inhibits the processing of mRNA by snatching cap structures from nascent cellular RNAs and using them as primers in the synthesis of viral mRNA. HSV has a virion tegument protein that inhibits cellular mRNA translation.
Apoptosis is the expected consequence of virus-induced inhibition of cellular macromolecular synthesis and viral nucleic acid replication. Although the induction of apoptosis may be important for the release of some viruses (particularly nonenveloped viruses), many viruses have acquired genes or parts of genes that enable them to forestall infected-cell death. This delay increases the yield from viral replication. Adenoviruses and herpesviruses encode analogues of the cellular Bc12 protein, which block mitochondrial enhancement of proapoptotic stimuli. Poxviruses and some herpesviruses also encode caspase inhibitors. Many viruses, including HPVs and adenoviruses, encode proteins that inhibit p53 or its downstream proapoptotic effects.
The capsid and envelope of a virus protect the genome and enable efficient transmission of the virus from cell to cell and to new prospective hosts. Most common viral infections are spread by direct contact, by ingestion of contaminated water or food, or by inhalation of aerosolized particles. In all these situations, infection begins on an epithelial or mucosal surface and spreads along the mucosa and into deeper tissues. Infection may spread to cells that can enter blood vessels, lymphatics, or neural circuits. HBV, HCV, HTLV, and HIV are dependent on transmission by parenteral inoculation. Some viruses are transmitted only between humans. The dependence of smallpox virus and poliovirus infections on interhuman transmission makes it feasible to eliminate these viruses from human circulation by mass vaccination. Herpesviruses also survive by interhuman transmission but may be more difficult to eliminate because they establish persistent latent infection in humans and continuously reactivate to infect new and naïve generations.
Animals are also important reservoirs and vectors for transmission of viruses causing human disease. Insect vectors can mediate parenteral transfer of viruses that reach high titers in animal or human hosts. Arboviruses are parenterally transmitted from mammalian species to humans by mosquito vectors. Herpes B, monkeypox, rabies, and viral hemorrhagic fevers are other examples of zoonotic infections caused by direct contact with animals, animal tissues, or arthropod vectors.
Initial viral infections usually last for several days or weeks. During this period, the concentration of virus at sites of infection rises and then falls, usually to unmeasurable levels. The rise and fall of viral replication at a given site depend on local innate immune responses and the access of systemic antibody and cell immune effectors to the virus. Typically, primary infections with enteroviruses, mumps virus, measles virus, rubella virus, rotavirus, influenza virus, AAV, adenovirus, HSV, and VZV are cleared from almost all sites within 3–4 weeks. Some viruses are especially proficient in altering or evading innate and acquired immune responses. Primary infection with AAV, EBV, or CMV can last for several months. Characteristically, primary infections due to HBV, HCV, hepatitis D virus (HDV), HIV, HPV, and molluscum contagiosum virus (MCV) extend beyond several weeks. For some of these viruses (e.g., HPV, HBV, HCV, HDV, and MCV), the manifestations of primary infection are almost indistinguishable from the persistent phase.
Disease manifestations usually arise as a consequence of viral replication, infected-cell injury or death, and local inflammatory and innate immune responses. Disease severity may not necessarily correlate with the level of viral replication alone. For example, the clinical manifestations of intense primary infection with poliovirus, enterovirus, rabies virus, measles virus, mumps virus, or HSV at mucosal surfaces may be inapparent or relatively mild, whereas limited replication in neural cells can have dramatic consequences. Similarly, rubella virus or CMV infections in utero or neonatal HSV infections may have much more devastating effects than infections in adults.
Primary infections are cleared by nonspecific innate and specific adaptive immune responses. Thereafter, an immunocompetent host is usually immune to the disease manifestations of reinfection by the same virus. Immunity frequently does not prevent transient surface colonization on reexposure, persistent colonization, or even limited deeper infection.
PERSISTENT AND LATENT INFECTIONS
Relatively few viruses cause persistent or latent infections. HBV, HCV, rabies virus, measles virus, HIV, HTLV, HPV, HHVs, and MCV are notable exceptions. The mechanisms for persistent infection vary. HCV RNA polymerase and HIV reverse transcriptase are error-prone and generate variant genomes. Genome variation can be sufficient to permit evasion of host immune responses, thereby allowing persistent infection. HIV is also directly immunosuppressive, depleting CD4+ T lymphocytes and compromising CD8+ cytotoxic T cell immune responsiveness. Moreover, HIV encodes the Nef protein, which downmodulates MHC class I expression, rendering HIV-infected cells partially resistant to immune CD8+ T cell lysis.
DNA viruses have low mutation rates. Their persistence in human populations usually depends on their ability to establish latent infection in some cells, to reactivate from latency, and then to replicate at epithelial surfaces. Latency is defined as a state of infection in which virus is not replicating, viral genes associated with lytic infection are not expressed, and infectious virus is not made. The complete viral genome is present and may be replicated by cellular DNA polymerase in conjunction with replication of the cell’s genome. HPVs establish latent infection in basal epithelial cells. The latently infected basal cell replicates, along with the HPV episome, by using cellular DNA polymerase. Some of the progeny cells provide new latently infected basal cells, whereas others go on to squamous differentiation. Infected cells that differentiate to squamous cells become permissive for lytic viral infection. Herpesviruses establish latent infection in nonreplicating neural cells (HSV and VZV) or in replicating cells of hematopoietic lineages (EBV, CMV, HHV-6, HHV-7, and Kaposi’s sarcoma–associated herpesvirus [KSHV, also known as HHV-8]). In their latent stage, HPV and herpesvirus genomes are largely hidden from the normal immune response. Reactivated HPV and herpesvirus infections escape immediate and effective immune responses in highly immune hosts by inhibiting host innate immune and inflammatory responses. In addition, HPV, HSV, and VZV are somewhat protected because they replicate in the middle and upper layers of the squamous epithelium—sites not routinely visited by cells that mediate or amplify immune and inflammatory responses. HSV and CMV are also known to encode proteins that downregulate MHC class I expression and antigenic peptide presentation, enabling infected cells to escape recognition by and cytotoxic effects of CD8+ T lymphocytes.
Like other poxviruses, MCV cannot establish latent infection. This virus causes persistent infection in hypertrophic skin lesions that last for months or years. MCV encodes a chemokine homologue that probably blocks inflammatory responses, an MHC class I analogue that blocks cytotoxic T lymphocyte attack, and inhibitors of cell death that prolong infected-cell viability.
PERSISTENT VIRAL INFECTIONS AND CANCER
Persistent viral infection is estimated to be the root cause of as many as 20% of human malignancies. Cancer is an accidental and highly unusual or long-term effect of oncogenic human viral infection. With most “oncogenic viruses,” infection is a critical and ultimately determinative early step in carcinogenesis. Latent HPV infection can block cell death and cause cervical cells to proliferate. A virus-infected cell with an integrated HPV genome overexpressing E6 and E7 undergoes subsequent cellular genetic changes that enhance autonomous malignant cell growth.
Most hepatocellular carcinoma is believed to be caused by chronic inflammatory, immune, and regenerative responses to HBV or HCV infection. Epidemiologic data firmly link HBV and HCV infections to hepatocellular carcinoma. These infections elicit repetitive cycles of virus-induced liver injury followed by tissue repair and regeneration. Over decades, chronic viral infection, repetitive tissue regeneration, and acquired chromosomal changes can result in proliferative nodules. Further chromosomal mutations can lead to the degeneration of cells in a proliferating nodule into hepatocellular carcinoma. In rare instances, HBV DNA integrates into cellular DNA, promoting overexpression of a cell gene that can also contribute to oncogenesis.
Most cervical carcinoma is caused by persistent infection with “high-risk” HPV type 16 or 18. In contrast to HBV and HCV infections, which stimulate cell growth as a consequence of virus-induced cell death, HPV type 16 or 18 proteins E6 and E7 destroy p53 and pRB, respectively. Elimination of these key tumor-suppressive cell proteins increases cell growth, cell survival, and cell genome instability. However, like HBV and HCV infections, HPV infection alone is not sufficient for carcinogenesis. Cervical carcinoma is inevitably associated with persistent HPV infection and integration of the HPV genome into chromosomal DNA. Integrations that result in overexpression of E6 and E7 from HPV type 16 or 18 cause more profound changes in cell growth and survival and permit subsequent chromosomal changes that result in cervical carcinoma.
EBV is the most unusual oncogenic virus in that normal B cell infection results in latency with expression of viral proteins that can cause endless B lymphocyte growth. In almost all humans, strong CD4+ and CD8+ T cell immune responses to the antigenic EBV latent-infection nuclear proteins prevent uncontrolled B cell lymphoproliferation. However, when humans are severely immunosuppressed by transplantation-associated medication, HIV infection, or genetic immune deficiencies, EBV-induced B cell malignancies can emerge.
EBV infection also has a role in the long-term development of B lymphocyte and epithelial cell malignancies. Persistent EBV infection with expression of an EBV latency-associated integral membrane protein (LMP1) in latently infected epithelial cells appears to be a critical early step in the evolution of anaplastic nasopharyngeal carcinoma, a common malignancy in populations in southern China and northern Africa. Genomic instability and chromosomal abnormalities also contribute to the development of EBV-associated nasopharyngeal carcinoma. EBV is an important cause of Hodgkin’s lymphoma. High-level expression of LMP1 or LMP2 in Reed-Sternberg cells is a hallmark in up to 50% of Hodgkin’s lymphoma cases. LMP1-induced nuclear factor-κB (NF-κB) activity may prolong the survival of defective B cells that are normally eliminated by apoptosis, thereby allowing other genetic changes leading to the development of malignant Reed-Sternberg cells.
The HTLV-1 Tax and Rex proteins are critical to the initiation of cutaneous adult T cell lymphoma/leukemias that occur long after primary HTLV-1 infection. Tax-induced NF-κB activation may contribute to cytokine production, infected-cell survival, and eventual outgrowth of malignant cells.
Molecular data confirm the presence of KSHV DNA in all Kaposi’s tumors, including those associated with HIV infection, transplantation, and familial transmission. KSHV infection is also etiologically implicated in pleural-effusion lymphomas and multicentric Castleman’s disease, which are more common among HIV-infected than among HIV-uninfected people. KSHV also has a virus-encoded cyclin, an IFN regulatory factor, and a latency-associated nuclear antigen that are implicated in increased-cell proliferation and survival.
Evidence supporting a causal role for viral infection in all of these malignancies includes (1) epidemiologic data, (2) the presence of viral DNA in all tumor cells, (3) the ability of the viruses to transform human cells in culture, (4) the results of in vitro cell culture–based assays that reveal transforming effects of specific viral genes on cell growth or survival, (5) pathologic data indicating the expression of transforming viral genes in premalignant or malignant cells in vivo, (6) the demonstration in animal models that these viral genes can cause malignant cell growth, and (7) the ability of virus-specific vaccines to reduce the incidence of virus-associated malignancy.
Virus-related malignancies provide an opportunity to expand our understanding of the biologic mechanisms important in the development of cancer. They also offer unique opportunities to develop diagnostics, vaccines, or therapeutics that could prevent or specifically treat cancers associated with viral infection. Widespread immunization against hepatitis B has resulted in a decreased prevalence of HBV-associated hepatitis and will probably prevent most HBV-related liver cancers. Current HPV vaccines can reduce rates of colonization with high-risk HPV strains and thereby decrease the risk of cervical cancer. The successful use of in vitro–expanded EBV-specific T cell populations to treat or prevent EBV-associated posttransplantation lymphoproliferative disease demonstrates the potential of immunoprevention or immunotherapy against virus-associated cancers.
RESISTANCE TO VIRAL INFECTIONS
Resistance to viral infections is initially provided by factors that are not virus-specific. Physical protection is afforded by the cornified layers of the skin and by mucous secretions that continuously sweep over mucosal surfaces. Once the first cell is infected, IFNs are induced and confer resistance to RNA virus replication. Viral infection may also trigger the release of other cytokines from infected cells. These cytokines may be chemotactic to inflammatory and immune cells. Viral protein epitopes expressed on the cell surface in the context of MHC class I and II proteins can stimulate the expansion of T cell populations with receptors that can recognize virus-encoded peptides presented on the cell surface by MHC class I proteins. Cytokines and antigens released by virus-induced cell death further attract inflammatory cells, dendritic cells, granulocytes, natural killer (NK) cells, and B lymphocytes to sites of infection and to draining lymph nodes. IFNs and NK cells are particularly important in containing viral infection for the first several days. Granulocytes and macrophages are also important in the phagocytosis and degradation of viruses, especially after an initial antibody response.
By 7–10 days after infection, virus-specific antibody responses, virus-specific human leukocyte antigen (HLA) class II–restricted CD4+ helper T lymphocyte responses, and virus-specific HLA class I–restricted CD8+ cytotoxic T lymphocyte responses develop. These responses, whose magnitude typically increases over the second and third weeks of infection, are important for rapid recovery. Also between the second and third weeks, the antibody type usually changes from IgM to IgG; IgG or IgA antibody can then be detected at infected mucosal surfaces. Antibody may directly neutralize virus by binding to its surface and preventing cell attachment or penetration. Complement can significantly enhance antibody-mediated virus neutralization. Antibody and complement can also lyse virus-infected cells that express viral membrane proteins on the cell surface. Cells infected with a replicating enveloped virus usually express the virus-envelope glycoproteins on the cell plasma membrane. Specific antibodies can bind to the glycoproteins, fix complement, and lyse the infected cell.
Antibody and CD4+/CD8+ T lymphocyte responses to viral infection can remain at high levels for several months after primary infection but usually wane over time. Low-level persistence of antibody-producing B lymphocytes and CD4+ or CD8+ T lymphocyte responses as memory cells can provide a rapid response to a second infection or an early barrier to reinfection with the same virus. Redevelopment of T cell immunity may take longer than secondary antibody responses, particularly when many years have elapsed between primary infection and reexposure. However, persistent infections or frequent reactivations from latency can result in sustained high-level T cell responses. EBV and CMV typically induce high-level CD4+ and CD8+ T cell responses that are maintained for decades after primary infection.
Some viruses have genes that alter innate and acquired host defenses. Adenoviruses encode small RNAs that inhibit IFN-induced, protein kinase R (PKR)–mediated shutoff of infected-cell protein synthesis. Adenovirus E1A can also directly inhibit IFN-mediated changes in cell gene transcription. Moreover, adenovirus E3 proteins prevent tumor necrosis factor (TNF)–induced cytolysis and block HLA class I synthesis by the infected cell. HSV ICP47 and CMV US11 also block class I antigen presentation. EBV encodes an interleukin (IL) 10 homologue that inhibits NK and T cell responses. Vaccinia virus encodes a soluble receptor for IFN-α and binding proteins for IFN-γ, IL-1, IL-18, and TNF, which inhibit host innate and adaptive immune responses. Vaccinia virus also encodes a caspase inhibitor that inhibits the ability of CD8+ cytotoxic T cells to kill virus-infected cells. Some poxviruses and herpesviruses encode chemokine-binding proteins that inhibit cell inflammatory responses. The adoption of these strategies by viruses highlights the importance of the corresponding host resistance factors in containing viral infection and the importance of redundancy in host resistance.
The host inflammatory and immune responses to viral infection do not come without a price. These responses contribute to the symptoms, signs, and other pathophysiologic manifestations of viral infection. Inflammation at sites of viral infection can subvert an effective immune response and induce tissue death and dysfunction. Moreover, immune responses to viral infection could, in principle, result in immune attack upon cross-reactive epitopes on normal cells, with consequent autoimmunity.
All human cells can synthesize IFN-α or IFN-β in response to viral infection. These IFN responses are usually induced by the presence of double-strand viral RNA, which can be made by both RNA and DNA viruses and sensed by double-strand RNA binding proteins (e.g., PKR and RIG-I) in the cell cytoplasm. IFN-γ is not closely related to IFN-α or IFN-β and is produced mainly by NK cells and by immune T lymphocytes responding to IL-12. IFN-α and -β bind to the IFN-α receptor, whereas IFN-γ binds to a different but related receptor. Both receptors signal through receptor-associated JAK kinases and other cytoplasmic proteins, including “STAT” proteins, which are tyrosine-phosphorylated by JAK kinases, translocate to the nucleus, and activate promoters for specific cell genes. Three types of antiviral effects are induced by IFN at the transcriptional level. The first effect is attributable to the induction of 2′-5′ oligo(A) synthetases, which require double-strand RNA for their activation. Activated synthetase polymerizes oligo(A) and thereby activates RNAse L, which in turn degrades single-strand RNA. A second effect results from the induction of PKR, a serine and threonine kinase that is also activated by double-strand RNA. PKR phosphorylates and negatively regulates the translational initiation factor eIF2α, shutting down protein synthesis in the infected cell. A third effect is initiated through the induction of Mx proteins, a family of GTPases that is particularly important in inhibiting the replication of influenza virus and vesicular stomatitis virus. These IFN effects are mostly directed against the infected cell, causing virus and cell dysfunction and thereby limiting viral replication.
A wide variety of methods are used to diagnose viral infection. Serology and virus isolation in tissue culture remain important standards. Acute- and convalescent-phase sera with rising titers of antibody to virus-specific antigens and a shift from IgM to IgG antibodies are generally accepted as diagnostic of acute viral infection. Serologic diagnosis is based on a more than fourfold rise in IgG antibody concentration when acute- and convalescent-phase serum samples are analyzed at the same time.
Immunofluorescence, hemadsorption, and hemagglutination assays for antiviral antibodies are labor-intensive and have been replaced by enzyme-linked immunosorbent assays (ELISAs), which generally use the specific viral proteins most frequently targeted by the antibody response. The proteins are purified from virus-infected cells or produced by recombinant DNA technology and are attached to a solid phase, where they can be incubated with serum, washed to eliminate nonspecific antibodies, and allowed to react with an enzyme-linked reagent to detect human IgG or IgM antibody specifically adhering to the viral antigen. The amount of antibody can then be quantitated by the intensity of a color reaction mediated by the linked enzyme. ELISAs can be sensitive and automated. Western blots can simultaneously confirm the presence of antibody to multiple specific viral proteins. The proteins are separated by size and transferred to an inert membrane, where they are incubated with serum antibodies. Western blots have an internal specificity control because the level of reactivity for viral proteins can be compared with that for cellular proteins in the same sample. Western blots require individual evaluation and are inherently difficult to quantitate or automate.
Isolation of virus in tissue culture depends on infection and replication in susceptible cells. Growth of virus in cell cultures can frequently be identified by effects on cell morphology under light microscopy. For example, HSV produces a typical cytopathic effect in rabbit kidney cells within 3 days. Other viral cytopathic effects may not be as diagnostically distinctive. Identification usually requires confirmation by staining with virus-specific monoclonal antibodies. The efficiency and speed of virus identification can be enhanced by combining short-term culture with immune detection. In assays with “shell vials” of tissue culture cells growing on a coverslip, viral infection can be detected by staining with a monoclonal antibody to a specific viral protein expressed early in viral replication. Thus, virus-infected cells can be detected within hours or days of inoculation, whereas several rounds of infection would be required to produce visible cytopathic effects.
Isolation of virus in tissue culture also depends on the collection of specimens from appropriate sites and the rapid transport of these specimens in appropriate medium to the virology laboratory (Chap. 150e). Rapid transport maintains viral viability and limits bacterial and fungal overgrowth. Enveloped viruses are generally more sensitive to freezing and thawing than nonenveloped viruses. The most appropriate site for culture depends on the pathogenesis of the virus in question. Nasopharyngeal, tracheal, or endobronchial aspirates are most appropriate for the identification of respiratory viruses. Sputum cultures generally are less appropriate because bacterial contamination and viscosity threaten tissue-culture cell viability. Aspirates of vesicular fluid are useful for isolation of HSV and VZV. Nasopharyngeal aspirates and stool specimens may be useful when the patient has fever and a rash and an enteroviral infection is suspected. Adenoviruses can be cultured from the urine of patients with hemorrhagic cystitis. CMV can frequently be isolated from cultures of urine or buffy coat. Biopsy material can be effectively cultured when viruses infect major organs, as in HSV encephalitis or adenovirus pneumonia.
The isolation of a virus does not necessarily establish disease causality. Viruses can persistently or intermittently colonize normal human mucosal surfaces. Saliva can be positive for herpesviruses, and normal urine samples can be positive for CMV. Isolations from blood, cerebrospinal fluid (CSF), or tissue are more often diagnostic of significant viral infection.
Another method aimed at increasing the speed of viral diagnosis is direct testing for antigen or cytopathic effects. Virus-infected cells from the patient may be detected by staining with virus-specific monoclonal antibodies. For example, epithelial cells obtained by nasopharyngeal aspiration can be stained with a variety of specific monoclonal antibodies to identify the specific infecting respiratory virus. Antigen and serologic assays can be multiplexed to detect multiple analytes simultaneously by coupling of reagents to color-coded beads for each analyte and detection by flow cytometry.
Nucleic acid amplification techniques bring speed, sensitivity, and specificity to diagnostic virology. The ability to directly amplify minute amounts of viral nucleic acids in specimens means that detection no longer depends on viable virus and its replication. For example, amplification and detection of HSV nucleic acids in the CSF of patients with HSV encephalitis is a more sensitive detection method than culture of virus from CSF. The extreme sensitivity of these tests can be a problem, because subclinical infection or contamination can lead to false-positive results. Detection of viral nucleic acids does not necessarily indicate virus-induced disease.
Measurement of the amount of viral RNA or DNA in peripheral blood is an important means for determining whether a patient is at increased risk for virus-induced disease and for evaluating clinical responses to antiviral chemotherapy. Nucleic acid technologies for RNA quantification are routinely used in AIDS patients to evaluate responses to antiviral agents and to detect viral resistance or noncompliance with therapy. Virus-load measurements are also useful for evaluating the treatment of patients with HBV and HCV infections. Nucleic acid testing or direct staining with CMV-specific monoclonal antibodies to quantitate virus-infected cells in the peripheral blood (CMV antigenemia) is useful for identifying immunosuppressed patients who may be at risk for CMV-induced disease.
DRUG TREATMENT FOR VIRAL INFECTIONS
Multiple steps in the life cycles of viruses can be effectively targeted by antiviral drugs (Chaps. 215e and 216). Nucleoside and nonnucleoside reverse transcriptase inhibitors prevent HIV provirus synthesis, whereas protease inhibitors block maturation of the HIV and HCV polyprotein after infection of the cell. Enfuvirtide is a small peptide derived from HIV gp41 that acts before cell infection by preventing a conformational change required for initial fusion of the virus with the cell membrane. Raltegravir is an integrase inhibitor that is approved for use with other anti-HIV drugs. Amantadine and rimantadine inhibit the influenza M2 protein, preventing release of viral RNA early during infection, whereas zanamivir and oseltamivir inhibit the influenza neuraminidase, which is necessary for the efficient release of mature virions from infected cells.
Viral genomes can evolve resistance to drugs by mutation and selection, by recombination with a drug-resistant virus, or (in the case of influenza virus and other segmented RNA viral genomes) by reassortment. The emergence of drug-resistant strains can limit the efficacy of antiviral therapy. As in antibacterial therapy, excessive and inappropriate use of antiviral therapy can select for the emergence of drug-resistant strains. HIV genotyping is a rapid method for identifying drug-resistant viruses. Resistance to reverse transcriptase or protease inhibitors has been associated with specific mutations in the reverse transcriptase or protease genes. Identification of these mutations by polymerase chain reaction amplification and nucleic acid sequencing can be clinically useful for determining which antiviral agents may still be effective. Drug resistance also can arise in herpesviruses but is a less common clinical problem.
IMMUNIZATION FOR THE PREVENTION OF VIRAL INFECTIONS
Viral vaccines are among the outstanding accomplishments of medical science. Smallpox has been eradicated except as a potential weapon of biological warfare or bioterrorism (Chap. 261e). Poliovirus eradication may soon follow. Measles can be contained or eliminated. Excess mortality due to influenza virus epidemics can be prevented, and the threat of influenza pandemics can be decreased by contemporary killed or live attenuated influenza vaccines. Mumps, rubella, and chickenpox are well controlled by childhood vaccination in the developed world. Reimmunization of mature adults can be used to control herpes zoster. New rotavirus vaccines can have a major impact on this leading cause of gastroenteritis and prominent cause of childhood death worldwide. Widespread HBV vaccination has dramatically lowered the frequency of acute and chronic hepatitis and is expected to lead to a dramatic decrease in the incidence of hepatocellular carcinoma. The HPV vaccine was the first vaccine specifically licensed to prevent virus-induced cancer. Use of purified proteins, genetically engineered live-virus vaccines, and recombinant DNA–based strategies will make it possible to immunize against severe infections with other viruses. The development of effective HIV and HCV vaccines is complicated by the high mutation rate of viral RNA polymerase and reverse transcriptase, the population-based and individual divergence of HIV or HCV genomes, and repeated high-level exposure in some populations. Concerns about the use of smallpox and other viruses as weapons necessitate maintenance of immunity to agents that are not encountered naturally.
VIRUSES AS NOVEL THERAPEUTIC TOOLS OR AGENTS
Viruses are being used experimentally to deliver biotherapeutic agents or novel vaccines. Foreign genes can be inserted into viral nucleic acids, and the recombinant virus vectors can be used to infect the patient or the patient’s cells ex vivo. Retrovirus integration into the cell genome has been used to functionally replace the abnormal gene in T cells of patients with severe combined immunodeficiency, thereby restoring immune function. Recombinant adenovirus, AAV, and retroviruses are being explored for use in diseases due to single-gene defects, such as cystic fibrosis and hemophilia. AAV carrying a lipoprotein lipase gene is now being used in Europe to treat a rare lipid-processing disease and is the first gene therapy approved for clinical use. Recombinant poxviruses, adenoviruses, and influenza viruses are also being used experimentally as vaccine vectors. Viral vectors are being tested experimentally for the expression of cytokines that can enhance immunity against tumor cells or for the expression of proteins that can increase the sensitivity of tumor cells to chemotherapy. HSV deficient for replication in resting cells is being used to selectively kill proliferating glioblastoma cells after injections into CNS tumors. For improved safety, nonreplicating viruses are frequently used in clinical trials. Potential adverse events associated with virus-mediated gene transfer include the induction of inflammatory and antiviral immune responses. Instances of retrovirus-induced human malignances have raised concerns about the safety of retroviral gene therapy vectors.