Section 11: Viral Diseases: General Considerations

DEFINING A VIRUS

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. 430) 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”).

VIRUS STRUCTURE

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. 185-1. Structures of prototypical pathogenic human viruses are described in Table 185-1. The relative sizes and structures of typical pathogenic human viruses are shown in Fig. 185-2.

TAXONOMY OF PATHOGENIC HUMAN VIRUSES

As is apparent from Table 185-1 and Fig. 185-2, 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).

VIRAL INFECTION IN VITRO

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 116-1). 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.

DNA VIRUSES

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.

HOST RANGE

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.

VIRAL INFECTION IN VIVO

TRANSMISSION

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.

PRIMARY INFECTION

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 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.

INTERFERONS

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.

DIAGNOSTIC VIROLOGY

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. S13). 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. 186 and 197). 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. 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 malignancies have raised concerns about the safety of retroviral gene therapy vectors.

FURTHER READING

INTRODUCTION

The field of antiviral therapy—both the number of antiviral drugs and our understanding of their optimal use—historically has lagged behind that of antibacterial treatment, but significant progress has been made in recent years on new drugs for several viral infections. The development of antiviral drugs poses several challenges. Viruses replicate intracellularly and often use host cell enzymes, macromolecules, and organelles for synthesis of viral particles. Therefore, useful antiviral compounds must discriminate between host and viral functions with a high degree of specificity; agents without such selectivity are likely to be too toxic for clinical use.

Significant progress has also been made in the development of laboratory assays to assist clinicians in the appropriate use of antiviral drugs. Phenotypic and genotypic assays for resistance to antiviral drugs are becoming more widely available, and correlations of laboratory results with clinical outcomes are being better defined. Of particular note has been the development of highly sensitive and specific methods that measure the concentration of virus in blood (viral load) and permit direct assessment of the antiviral effect of a given drug regimen in that host site. Viral load measurements have been useful in recognizing the risk of disease progression in patients with viral infections and in identifying patients for whom antiviral chemotherapy might be of greatest benefit. As with any in vitro laboratory test, results are highly dependent on and likely vary with the laboratory techniques used.

Information regarding the pharmacodynamics of antiviral drugs, and particularly the relationship of concentration effects to efficacy, has been slow to develop but is also expanding. However, assays to measure concentrations of antiviral drugs, especially of their active moieties within cells, are still primarily research procedures not widely available to clinicians. Thus, there are limited guidelines for adjusting dosages of antiviral agents to maximize antiviral activity and minimize toxicity. Consequently, clinical use of antiviral drugs must be accompanied by particular vigilance for unanticipated adverse effects.

Like that of other infections, the course of viral infections is profoundly affected by interplay between the pathogen and a complex set of host defenses. The presence or absence of preexisting immunity, the ability to mount humoral and/or cell-mediated immune responses, and the stimulation of innate immunity are important determinants of the outcome of viral infections. The state of the host’s defenses needs to be considered when antiviral agents are used or evaluated.

As with any therapy, the optimal use of antiviral compounds requires a specific and timely diagnosis. For some viral infections, such as herpes zoster, the clinical manifestations are so characteristic that a diagnosis can be made on clinical grounds alone. For other viral infections, such as influenza A, epidemiologic information (e.g., the documentation of a community-wide influenza outbreak) can be used to make a presumptive diagnosis with a high degree of accuracy. However, for most of the remaining viral infections, including herpes simplex encephalitis, cytomegalovirus (CMV) infections other than retinitis, and enterovirus infections, diagnosis on clinical grounds alone cannot be accomplished with certainty. For such infections, rapid viral diagnostic techniques are of great importance. Considerable progress has been made in recent years in the development of such tests, which are now widely available for a number of viral infections.

Despite these complexities, the efficacy of a number of antiviral compounds has been clearly established in rigorously conducted and controlled studies. As summarized in Table 186-1, this chapter reviews the antiviral drugs that are currently approved or are likely to be considered for approval in the near future for use against viral infections other than those caused by HIV. Antiretroviral drugs are reviewed in Chap. 197.

ANTIVIRAL DRUGS ACTIVE AGAINST RESPIRATORY INFECTIONS

(See Also Chaps. 194 and 195)

ZANAMIVIR, OSELTAMIVIR, PERAMIVIR, AND LANINAMIVIR

Zanamivir and oseltamivir are inhibitors of the influenza virus neuraminidase enzyme, which is essential for release of virus from infected cells and for its subsequent spread throughout the respiratory tract of the infected host. The enzyme cleaves terminal sialic acid residues and thus destroys the cellular receptors to which the viral hemagglutinin attaches. Zanamivir and oseltamivir are sialic acid transition-state analogues and are highly active and specific inhibitors of the neuraminidases of both influenza A and B viruses. The antineuraminidase activity of the two drugs is similar, although zanamivir has somewhat greater in vitro activity against influenza B virus. Zanamivir may also be active against certain strains of influenza A virus that are resistant to oseltamivir. Both zanamivir and oseltamivir act through competitive and reversible inhibition of the active site of influenza A and B viral neuraminidases and have relatively little effect on mammalian cell enzymes.

Oseltamivir phosphate is an ethyl ester prodrug that is converted to oseltamivir carboxylate by esterases in the liver. Orally administered oseltamivir has a bioavailability of >60% and a plasma half-life of 7–9 h. The drug is excreted unmetabolized, primarily by the kidneys. Zanamivir has low oral bioavailability and is administered orally via a hand-held inhaler. By this route, ~15% of the dose is deposited in the lower respiratory tract, and low plasma levels of the drug are detected. The toxicities most frequently encountered with orally administered oseltamivir are nausea, gastrointestinal discomfort, and (less commonly) vomiting. Gastrointestinal discomfort is usually transient and is less likely if the drug is administered with food. Neuropsychiatric events (delirium, self-injury) have been reported in children who have been taking oseltamivir, primarily in Japan. Zanamivir is orally inhaled and is generally well tolerated, although exacerbations of asthma may occur. An IV formulation of zanamivir is under development and is available from GlaxoSmithKline as part of clinical trials.

Inhaled zanamivir and orally administered oseltamivir have been effective in the treatment of naturally occurring, uncomplicated influenza A or B in otherwise healthy adults. In placebo-controlled studies, illness has been shortened by 1.0–1.5 days of therapy with either of these drugs when treatment is administered within 2 days of onset of symptoms. Pooled analyses of clinical studies of oseltamivir suggest that treatment may reduce the likelihood of hospitalizations and of certain respiratory tract complications associated with influenza, and observational studies suggest that oseltamivir may reduce mortality rates associated with influenza A outbreaks (Chap. 195). Once-daily inhaled zanamivir or once-daily orally administered oseltamivir can provide prophylaxis against laboratory-documented influenza A– and influenza B–associated illness.

Resistance to the neuraminidase inhibitors may develop by changes in the viral neuraminidase enzyme, by changes in the hemagglutinin that make it more resistant to the actions of the neuraminidase, or by both mechanisms. Isolates that are resistant to oseltamivir—most commonly through the H275Y mutation, which leads to a change from histidine to tyrosine at that residue in the neuraminidase—remain sensitive to zanamivir. Certain mutations impart resistance to both oseltamivir and zanamivir (e.g., I223R, which leads to a change from isoleucine to arginine). Because the mechanisms of action of the neuraminidase inhibitors differ from those of the adamantanes (see below), zanamivir and oseltamivir are active against strains of influenza A virus that are resistant to amantadine and rimantadine.

Appropriate use of antiviral agents against influenza viruses depends on a knowledge of the resistance patterns of circulating viruses. As of this writing, currently circulating influenza A/H1N1 and H3N2 viruses (2013–2014) were sensitive to zanamivir and oseltamivir, with a few exceptions for oseltamivir. Up-to-date information on patterns of resistance to antiviral drugs is available from the Centers for Disease Control and Prevention (CDC) at www.cdc.gov/flu.

Zanamivir and oseltamivir have been approved by the U.S. Food and Drug Administration (FDA) for treatment of influenza in adults and in children (those ≥7 years old for zanamivir and those ≥1 year old for oseltamivir) who have been symptomatic for ≤2 days. Oseltamivir is approved for prophylaxis of influenza in individuals ≥1 year of age and zanamivir for those ≥5 years of age (Table 186-1). Guidelines for the use of oseltamivir in children <1 year of age can be accessed through the CDC website (www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm).

Peramivir (BCX-1812) is a neuraminidase inhibitor that can be administered intravenously. It has been approved in Japan, China, and South Korea but not in the United States, where it has been available in clinical trials through BioCryst Pharmaceuticals and previously as part of an emergency use authorization in response to the influenza A(H1N1)pdm09 virus pandemic in 2009–2010. Oseltamivir-resistant viruses generally exhibit reduced sensitivity to peramivir.

Laninamivir octanoate is a neuraminidase that has been approved in Japan for the treatment and prevention of influenza A and B. It is the prodrug of laninamivir, which is administered by oral inhalation and has a prolonged half-life of ~3 days. In limited studies, it has been investigated as single-dose therapy for influenza; its effects were similar to those obtained with multiple doses of zanamivir or oseltamivir.

AMANTADINE AND RIMANTADINE

Amantadine and the closely related compound rimantadine are primary symmetric amines that have antiviral activity limited to influenza A viruses. Amantadine and rimantadine have a long history of efficacy in the prophylaxis and treatment of influenza A infections in humans. However, high frequencies of resistance to these drugs were noted among influenza A/H3N2 viruses in the 2005–2006 influenza season and continued to be seen in 2013–2014. The pandemic A/H1N1 viruses that circulated in 2009–2010 were also resistant to amantadine and rimantadine, and circulating influenza A/H1N1 viruses in the 2013–2014 season were largely resistant. Therefore, these agents are no longer recommended unless the sensitivity of the particular isolate of influenza A virus is known, in which case their use may be considered. Amantadine and rimantadine act through inhibition of the ion channel function of the influenza A M2 matrix protein, on which uncoating of the virus depends. A substitution of a single amino acid at critical sites in the M2 protein can result in a virus that is resistant to amantadine and rimantadine.

Amantadine and rimantadine have been shown to be effective in the prophylaxis of influenza A in large-scale studies of young adults and in less extensive studies of children and elderly persons. In such studies, efficacy rates of 55–80% in the prevention of influenza-like illness were noted, and even higher rates were reported when virus-specific attack rates were calculated. Amantadine and rimantadine have also been found to be effective in the treatment of influenza A infection in studies involving predominantly young adults and, to a lesser extent, children. Administration of these compounds within 24–72 h after the onset of illness has resulted in a reduction of the duration of signs and symptoms by ~50% compared with that in placebo recipients. The effect on signs and symptoms of illness is superior to that of commonly used antipyretic–analgesic agents. Only anecdotal reports are available concerning the efficacy of amantadine or rimantadine in the prevention or treatment of complications of influenza (e.g., pneumonia).

Amantadine and rimantadine are available only in oral formulations and are ordinarily administered to adults once or twice daily, with a dosage of 100–200 mg/d. Despite their structural similarities, the two compounds have different pharmacokinetics. Amantadine is not metabolized and is excreted almost entirely by the kidneys, with a half-life of 12–17 h and peak plasma concentrations of 0.4 μg/mL. In contrast, rimantadine is extensively metabolized to hydroxylated derivatives and has a half-life of 30 h. Only 30–40% of an orally administered dose of rimantadine is recovered in the urine. The peak plasma levels of rimantadine are approximately half those of amantadine, but rimantadine is concentrated in respiratory secretions to a greater extent than amantadine. For prophylaxis, the compounds must be administered daily for the period at risk (i.e., duration of the exposure). For therapy, amantadine or rimantadine is generally administered for 5–7 days.

Although these compounds are generally well tolerated, 5–10% of amantadine recipients experience mild central nervous system side effects consisting primarily of dizziness, anxiety, insomnia, and difficulty in concentrating. These effects are rapidly reversible upon cessation of the drug’s administration. At a dose of 200 mg/d, rimantadine is better tolerated than amantadine; in a large-scale study of young adults, adverse effects were no more frequent among rimantadine recipients than among placebo recipients. Seizures and worsening of congestive heart failure have also been reported in patients treated with amantadine, although a causal relationship has not been established. The dosage of amantadine should be reduced to 100 mg/d in patients with renal insufficiency—i.e., a creatinine clearance rate (Cr_Cl) of <50 mL/min—and in the elderly. A rimantadine dose of 100 mg/d should be used for patients with a Cr_Cl of <10 mL/min and for the elderly.

RIBAVIRIN

Ribavirin is a synthetic nucleoside analogue that inhibits a wide range of RNA and DNA viruses. The mechanism of action of ribavirin is not completely defined and may be different for different groups of viruses. Ribavirin-5′-monophosphate blocks the conversion of inosine-5′-monophosphate to xanthosine-5′-monophosphate and interferes with the synthesis of guanine nucleotides as well as with that of both RNA and DNA. Ribavirin-5′-monophosphate also inhibits capping of virus-specific messenger RNA in certain viral systems.

Ribavirin administered as a small-particle aerosol to young children hospitalized with respiratory syncytial virus (RSV) infection has been clinically beneficial and has improved oxygenation in some studies (7 of 11). Although ribavirin has been approved for treatment of infants hospitalized with RSV infection, the American Academy of Pediatrics has recommended that it be considered on an individual basis rather than used routinely in that setting. Aerosolized ribavirin has also been administered to older children and adults (including immunosuppressed patients) with severe RSV and parainfluenza virus infections and to older children and adults with influenza A or B infection, but the benefit of this treatment, if any, is unclear. In RSV infections in immunosuppressed patients, ribavirin has been given in combination with anti-RSV immunoglobulins.

Orally administered ribavirin has not been effective in the treatment of influenza A virus infections. IV or oral ribavirin has reduced mortality rates among patients with Lassa fever; it is thought to be more effective in this regard when given within the first 6 days of illness. IV ribavirin has been reported to be of clinical benefit in the treatment of hemorrhagic fever with renal syndrome caused by Hantaan virus and as therapy for Argentinean hemorrhagic fever. Oral ribavirin has also been recommended for the treatment and prophylaxis of Congo-Crimean hemorrhagic fever. Use of IV ribavirin in patients with hantavirus pulmonary syndrome in the United States has not been associated with clear-cut benefits.

Oral administration of ribavirin reduces serum aminotransferase levels in patients with chronic hepatitis C virus (HCV) infection; because it appears not to reduce serum HCV RNA levels, the mechanism of this effect is unclear. The drug provides added benefit when given by mouth in doses of 800–1200 mg/d in combination with interferon (IFN) α2b or α2a (see below), and the triple combination of ribavirin, IFN, and sofosbuvir or simeprevir has been approved for the treatment of patients with chronic HCV infection (see below). Recent data suggest that oral ribavirin may be beneficial in resolution of chronic hepatitis E infection (largely genotype 3) associated with organ transplantation. Larger oral doses of ribavirin (800–1000 mg/d) have been associated with reversible hematopoietic toxicity. This effect has not been observed with aerosolized ribavirin, apparently because little drug is absorbed systemically. Aerosolized administration of ribavirin is generally well tolerated but occasionally is associated with bronchospasm, rash, or conjunctival irritation. It should be administered under close supervision—particularly in the setting of mechanical ventilation, where precipitation of the drug is possible. Health care workers exposed to the drug have experienced minor toxicity, including eye and respiratory tract irritation. Because ribavirin is mutagenic, teratogenic, and embryotoxic, its use is generally contraindicated in pregnancy. Its administration as an aerosol poses a risk to pregnant health care workers. Because clearance of ribavirin is primarily renal, dose reduction is required in the setting of significant renal dysfunction.

AGENTS OF INVESTIGATIVE INTEREST

Favipiravir (T-705) is a viral RNA–dependent RNA polymerase inhibitor active against influenza viruses, including neuraminidase inhibitor–resistant strains. It is approved in Japan for the treatment of influenza. Given favipiravir’s in vitro activity against a broad range of viruses, including arenaviruses, phleboviruses (e.g., Rift Valley Fever virus), hantaviruses, flaviviruses, and filoviruses (e.g., Ebola virus), its use has been considered in the context of outbreaks of disease caused by these viruses even though their clinical activity remains uncertain. DAS181 is an investigational antiviral agent with activity against influenza A and B and parainfluenza viruses. A sialidase fusion protein, DAS181 cleaves the terminal sialic acid residues on human respiratory cells, reducing the binding of influenza and parainfluenza viruses. It is interesting to note that this agent targets host cellular rather than microbial protein. DAS181 is administered by oral inhalation and is being evaluated in the treatment of parainfluenza type 3 infections in recipients of lung and stem cell transplants. Three investigational agents with activity against RSV are being studied: (1) GS-5806, a small molecule that blocks fusion of the viral envelope with the host cell membrane, thus inhibiting viral entry, and has shown promising activity in human challenge studies; (2) ALS-008176, a prodrug of a cytidine nucleoside analogue that inhibits RSV replication by means of chain termination; and (3) ALN-RSV01, which works via RNA interference and is directed against the conserved region encoding the nucleocapsid (N) protein.

ANTIVIRAL DRUGS ACTIVE AGAINST HERPESVIRUS INFECTIONS

ACYCLOVIR AND VALACYCLOVIR

Acyclovir is a highly potent and selective inhibitor of the replication of certain herpesviruses, including herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). This drug is relatively ineffective in the treatment of human CMV infections; however, some studies have indicated effectiveness (at higher doses) in the prevention of CMV-associated disease in immunosuppressed patients. Valacyclovir, the L-valyl ester of acyclovir, is converted almost entirely to acyclovir by intestinal and hepatic hydrolysis after oral administration. Valacyclovir offers pharmacokinetic advantages over orally administered acyclovir: it exhibits significantly greater oral bioavailability, results in higher blood levels, and can be given less frequently than acyclovir (two or three rather than five times daily).

The high degree of selectivity of acyclovir is related to its mechanism of action, which requires that the compound first be phosphorylated to acyclovir monophosphate. This phosphorylation occurs efficiently in herpesvirus-infected cells by means of a virus-coded thymidine kinase. In uninfected mammalian cells, little phosphorylation of acyclovir occurs, and the drug is therefore concentrated in herpesvirus-infected cells. Acyclovir monophosphate is subsequently converted by host cell kinases to a triphosphate that is a potent inhibitor of virus-induced DNA polymerase but has relatively little effect on host cell DNA polymerase. Acyclovir triphosphate can also be incorporated into viral DNA, with early chain termination.

Acyclovir is available in IV, oral, and topical forms, while valacyclovir is available in an oral formulation. IV acyclovir is effective in the treatment of mucocutaneous HSV infections in immunocompromised hosts, in whom it reduces time to healing, duration of pain, and virus shedding. When administered prophylactically during periods of intense immunosuppression (e.g., related to chemotherapy for leukemia or transplantation) and before the development of lesions, IV acyclovir reduces the frequency of HSV-associated disease. After prophylaxis is discontinued, HSV lesions recur. IV acyclovir is also effective in the treatment of HSV encephalitis.

Because VZV is generally less sensitive to acyclovir than is HSV, higher doses of acyclovir must be used to treat VZV infections. In immunocompromised patients with herpes zoster, IV acyclovir reduces the frequency of cutaneous dissemination and visceral complications and—in one comparative trial—was more effective than vidarabine. Acyclovir, administered at oral doses of 800 mg five times a day, had a modest beneficial effect on localized herpes zoster lesions in both immunocompromised and immunocompetent patients. Combination of acyclovir with a tapering regimen of prednisone appeared to be more effective than acyclovir alone in terms of quality-of-life outcomes in immunocompetent patients aged >50 years with herpes zoster. A comparative study of acyclovir (800 mg PO five times daily) and valacyclovir (1 g PO three times daily) in immunocompetent patients with herpes zoster indicated that the latter drug may be more effective in eliciting the resolution of zoster-associated pain. Orally administered acyclovir (600 mg five times a day) reduced complications of herpes zoster ophthalmicus in a placebo-controlled trial.

In chickenpox, a modest overall clinical benefit is attained when oral acyclovir therapy is begun within 24 h of the onset of rash in otherwise healthy children (20 mg/kg, up to a maximum of 800 mg, four times a day) or adults (800 mg five times a day). IV acyclovir has also been reported to be effective in the treatment of immunocompromised children with chickenpox.

A common use of acyclovir is in the treatment of genital HSV infections. IV or oral acyclovir or oral valacyclovir has shortened the duration of symptoms, reduced virus shedding, and accelerated healing when used for the treatment of primary genital HSV infections. Oral acyclovir and valacyclovir have also had a modest effect in treatment of recurrent genital HSV infections. However, the failure of treatment of either primary or recurrent disease to reduce the frequency of subsequent recurrences has indicated that acyclovir is ineffective in eliminating latent infection. Documented chronic oral administration of acyclovir for up to 6 years or of valacyclovir for up to 1 year has reduced the frequency of recurrences markedly during therapy; once the drug is discontinued, lesions recur. In one study, suppressive therapy with valacyclovir (500 mg once daily for 8 months) reduced transmission of HSV-2 genital infections among discordant couples by 50%. A modest effect on herpes labialis (i.e., a reduction of disease duration by 1 day) was seen when valacyclovir was administered upon detection of the first symptom of a lesion at a dose of 2 g every 12 h for 1 day. In AIDS patients, chronic or intermittent administration of acyclovir has been associated with the development of HSV and VZV strains resistant to the action of the drug and with clinical failures. The most common mechanism of resistance is a deficiency of the virus-induced thymidine kinase. Patients with HSV or VZV infections resistant to acyclovir have frequently responded to foscarnet.

With the availability of the oral and IV forms, there are few indications for topical acyclovir, although treatment with this formulation has been modestly beneficial in primary genital HSV infections and in mucocutaneous HSV infections in immunocompromised hosts.

Overall, acyclovir is remarkably well tolerated and is generally free of toxicity. The most frequently encountered form of toxicity is renal dysfunction because of drug crystallization (which is pH dependent), particularly after rapid IV administration or with inadequate hydration. Central nervous system changes, including lethargy and tremors, are occasionally reported, primarily in immunosuppressed patients. However, whether these changes are related to acyclovir, to concurrent administration of other therapy, or to underlying infection remains unclear. Acyclovir is excreted primarily unmetabolized by the kidneys via both glomerular filtration and tubular secretion. Approximately 15% of a dose of acyclovir is metabolized to 9-Carboxymethoxymethylguanine or other minor metabolites. Reduction in dosage is indicated in patients with a Cr_Cl of <50 mL/min. The half-life of acyclovir is ~3 h in normal adults, and the peak plasma concentration after a 1-h infusion of a dose of 5 mg/kg is 9.8 μg/mL. Approximately 22% of an orally administered acyclovir dose is absorbed, and peak plasma concentrations of 0.3–0.9 μg/mL are attained after administration of a 200-mg dose. Acyclovir penetrates relatively well into the cerebrospinal fluid (CSF), with concentrations approaching half of those found in plasma.

Acyclovir causes chromosomal breakage at high doses, but its administration to pregnant women has not been associated with fetal abnormalities. Nonetheless, the potential risks and benefits of acyclovir should be carefully assessed before the drug is used in pregnancy.

Valacyclovir exhibits three to five times greater bioavailability than acyclovir. The concentration–time curve for valacyclovir, given as 1 g PO three times daily, is similar to that for acyclovir, given as 5 mg/kg IV every 8 h. The safety profiles of valacyclovir and acyclovir are similar, although thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome has been reported in immunocompromised patients who have received high doses of valacyclovir (8 g/d). Valacyclovir is approved for the treatment of herpes zoster, of initial and recurrent episodes of genital HSV infection, and of herpes labialis in immunocompetent adults as well as for suppressive treatment of genital herpes. Although it has not been extensively studied in other clinical settings involving HSV or VZV infections, many consultants use valacyclovir rather than oral acyclovir in settings where only the latter has been approved because of valacyclovir’s superior pharmacokinetics and more convenient dosing schedule.

CIDOFOVIR

Cidofovir is a phosphonate nucleotide analogue of cytosine. Its major use is in CMV infections, but it is active against a broad range of herpesviruses, including HSV, human herpesvirus (HHV) types 6A and 6B, HHV-8, and certain other DNA viruses such as polyomaviruses, papillomaviruses, adenoviruses, and poxviruses, including variola (smallpox) and vaccinia. Cidofovir does not require initial phosphorylation by virus-induced kinases; the drug is phosphorylated by host cell enzymes to cidofovir diphosphate, which is a competitive inhibitor of viral DNA polymerases and, to a lesser extent, of host cell DNA polymerases. Incorporation of cidofovir diphosphate slows or terminates nascent DNA chain elongation. Cidofovir is active against HSV isolates that are resistant to acyclovir because of absent or altered thymidine kinase and against CMV isolates that are resistant to ganciclovir because of UL97 phosphotransferase mutations. CMV isolates resistant to ganciclovir on the basis of UL54 mutations are usually resistant to cidofovir as well. Cidofovir is usually active against foscarnet-resistant CMV, although cross-resistance to foscarnet has been described, typically in the UL54 polymerase.

Cidofovir has poor oral availability and is administered intravenously. It is excreted primarily by the kidney and has a plasma half-life of 2.6 h. Cidofovir diphosphate’s intracellular half-life of >48 h is the basis for the recommended dosing regimen of 5 mg/kg once a week for the initial 2 weeks and then 5 mg/kg every other week. The major toxic effect of cidofovir is proximal renal tubular injury, as manifested by elevated serum creatinine levels and proteinuria. The risk of nephrotoxicity can be reduced by vigorous saline hydration and by concomitant oral administration of probenecid. Neutropenia, rashes, and gastrointestinal tolerance may also occur.

IV cidofovir has been approved for the treatment of CMV retinitis in AIDS patients who are intolerant of ganciclovir or foscarnet or in whom those drugs have failed. In a controlled study, a maintenance dosage of 5 mg/kg per week administered to AIDS patients reduced the progression of CMV retinitis from that seen at 3 mg/kg. Intravitreal cidofovir has been used to treat CMV retinitis but has been associated with significant toxicity. IV cidofovir has been reported anecdotally to be effective for treatment of acyclovir-resistant mucocutaneous HSV infections. Likewise, topically administered cidofovir is reportedly beneficial against mucocutaneous HSV infections in HIV-infected patients. Anecdotal use of IV cidofovir has been described in disseminated adenoviral infections in immunosuppressed patients and in genitourinary infections with BK virus in renal transplant recipients; however, its efficacy, if any, in these circumstances is not established.

CMX-001 (brincidofovir) is an ester prodrug of cidofovir that can be administered orally and may be less nephrotoxic than IV cidofovir. It has not shown efficacy in the prevention of CMV infection in stem cell transplant recipients but is being studied for treatment of BK nephropathy and adenovirus infections.

FOMIVIRSEN

Fomivirsen is the first antisense oligonucleotide approved by the FDA for therapy in humans. This phosphorothioate oligonucleotide, 21 nucleotides in length, inhibits CMV replication through interaction with CMV messenger RNA. Fomivirsen is complementary to messenger transcripts of the major immediate early region 2 of CMV, which codes for proteins regulating viral gene expression. In addition to its antisense mechanism of action, fomivirsen may exert activity against CMV through inhibition of viral adsorption to cells as well as direct inhibition of viral replication. Because of its different mechanism of action, fomivirsen is active against CMV isolates that are resistant to nucleoside or nucleotide analogues, such as ganciclovir, foscarnet, or cidofovir.

Fomivirsen has been approved for intravitreal administration in the treatment of CMV retinitis in AIDS patients who have failed to respond to other treatments or cannot tolerate them. Injection of two doses of 330 mg 2 weeks apart, followed by maintenance doses of 330 mg monthly, significantly reduces the rate of progression of CMV retinitis. The major toxicity is ocular inflammation, including vitritis and iritis, which usually responds to topically administered glucocorticoids.

GANCICLOVIR AND VALGANCICLOVIR

An analogue of acyclovir, ganciclovir is active against HSV and VZV and is markedly more active than acyclovir against CMV. Ganciclovir triphosphate inhibits CMV DNA polymerase and can be incorporated into CMV DNA, whose elongation it eventually terminates. In HSV- and VZV-infected cells, ganciclovir is phosphorylated by virus-encoded thymidine kinases; in CMV-infected cells, it is phosphorylated by a viral kinase encoded by the UL97 gene. Ganciclovir triphosphate is present in tenfold higher concentrations in CMV-infected cells than in uninfected cells. Ganciclovir is approved for the treatment of CMV retinitis in immunosuppressed patients and for the prevention of CMV disease in transplant recipients. It is widely used for the treatment of other CMV-associated syndromes, including pneumonia, esophagogastrointestinal infections, hepatitis, and “wasting” illness.

Ganciclovir is available for IV or oral administration. Because its oral bioavailability is low (5–9%), relatively large doses (1 g three times daily) must be administered by this route. Oral ganciclovir has largely been supplanted by valganciclovir, which is the L-valyl ester of ganciclovir. Valganciclovir is well absorbed orally, with a bioavailability of 60%, and is rapidly hydrolyzed to ganciclovir in the intestine and liver. The area under the curve for a 900-mg dose of valganciclovir is equivalent to that for 5 mg/kg of IV ganciclovir, although peak serum concentrations are ~40% lower for valganciclovir. The serum half-life is 3.5 h after IV administration of ganciclovir and 4.0 h after PO administration of valganciclovir. Ganciclovir is excreted primarily by the kidneys in an unmetabolized form, and its dosage should be reduced in cases of renal failure. Ganciclovir therapy at the most commonly used initial IV dosage—i.e., 5 mg/kg every 12 h for 14–21 days—can be changed to valganciclovir (900 mg PO twice daily) when the patient can tolerate oral therapy. The maintenance dose is 5 mg/kg IV daily or five times per week for ganciclovir and 900 mg by mouth once a day for valganciclovir. Dose adjustment in patients with renal dysfunction is required. Intraocular ganciclovir, given by either intravitreal injection or intraocular implantation, has also been used to treat CMV retinitis.

Ganciclovir is effective as prophylaxis against CMV-associated disease in organ and bone marrow transplant recipients. Oral ganciclovir administered prophylactically to AIDS patients with CD4+ T cell counts of <100/μL has provided protection against the development of CMV retinitis. However, the long-term benefits of this approach to prophylaxis in AIDS patients have not been established, and most experts do not recommend the use of oral ganciclovir for this purpose. As already mentioned, valganciclovir has supplanted oral ganciclovir in settings where oral prophylaxis or therapy is considered.

The administration of ganciclovir has been associated with bone marrow suppression, particularly neutropenia, which significantly limits the drug’s use in many patients. Bone marrow toxicity is potentiated in the setting of renal dysfunction and when other bone marrow suppressants, such as zidovudine or mycophenolate mofetil, are used concomitantly. This toxicity is typically dose and duration sensitive and is reversible with cessation of ganciclovir use.

Resistance has been noted in CMV isolates obtained after therapy with ganciclovir, especially those from patients with AIDS or from patients receiving prolonged ganciclovir therapy after organ transplantation. Such resistance may develop through a mutation in either the viral UL97 gene or the viral DNA polymerase. Ganciclovir-resistant isolates are usually sensitive to foscarnet (see below) or may be sensitive to cidofovir, depending on the mechanism of resistance (see above).

FAMCICLOVIR AND PENCICLOVIR

Famciclovir is the diacetyl 6-deoxyester of the guanosine analogue penciclovir. This agent is well absorbed orally, has a bioavailability of 77%, and is rapidly converted to penciclovir by deacetylation and oxidation in the intestine and liver. Penciclovir’s spectrum of activity and mechanism of action are similar to those of acyclovir. Thus, penciclovir usually is not active against acyclovir-resistant viruses. However, some acyclovir-resistant viruses with altered thymidine kinase or DNA polymerase substrate specificity may be sensitive to penciclovir. This drug is phosphorylated initially by a virus-encoded thymidine kinase and subsequently by cellular kinases to penciclovir triphosphate, which inhibits HSV-1, HSV-2, VZV, and EBV as well as hepatitis B virus (HBV). The serum half-life of penciclovir is 2 h, but the intracellular half-life of penciclovir triphosphate is 7–20 h—markedly longer than that of acyclovir triphosphate. The latter is the basis for the less frequent (twice-daily) dosing schedule for famciclovir than for acyclovir. Penciclovir is eliminated primarily in the urine by both glomerular filtration and tubular secretion. The usually recommended dosage interval should be adjusted for renal insufficiency.

Clinical trials involving immunocompetent adults with herpes zoster showed that famciclovir was superior to placebo in eliciting the resolution of skin lesions and virus shedding and in shortening the duration of postherpetic neuralgia; moreover, administered at 500 mg every 8 h, famciclovir was at least as effective as acyclovir administered at an oral dose of 800 mg five times daily. Famciclovir was also effective in the treatment of herpes zoster in immunosuppressed patients. Clinical trials have demonstrated its effectiveness in the suppression of genital HSV infections for up to 1 year and in the treatment of initial and recurrent episodes of genital herpes. Famciclovir is effective as therapy for mucocutaneous HSV infections in HIV-infected patients. Application of a 1% penciclovir cream reduces the duration of signs and symptoms of herpes labialis in immunocompetent patients (by 0.5–1 day) and has been approved for that purpose by the FDA. Famciclovir is generally well tolerated, with occasional headache, nausea, and diarrhea reported in frequencies similar to those among placebo recipients. The administration of high doses of famciclovir for 2 years was associated with an increased incidence of mammary adenocarcinomas in female rats, but the clinical significance of this effect is unknown.

FOSCARNET

Foscarnet (phosphonoformic acid) is a pyrophosphate-containing compound that potently inhibits herpesviruses, including CMV. This drug inhibits DNA polymerases at the pyrophosphate binding site at concentrations that have relatively little effect on cellular polymerases. Foscarnet does not require phosphorylation to exert its antiviral activity and is therefore active against HSV and VZV isolates that are resistant to acyclovir because of deficiencies in thymidine kinase as well as against most ganciclovir-resistant strains of CMV. Foscarnet also inhibits the reverse transcriptase of HIV and is active against HIV in vivo.

Foscarnet is poorly soluble and must be administered intravenously via an infusion pump in a dilute solution over 1–2 h. The plasma half-life of foscarnet is 3–5 h and increases with decreasing renal function because the drug is eliminated primarily by the kidneys. It has been estimated that 10–28% of a dose may be deposited in bone, where it can persist for months. The most common initial dosage of foscarnet is 60 mg/kg every 8 h for CMV and 40 mg/kg every 8 h for HSV. Once the infection is controlled, a maintenance dose of 90–120 mg/kg once a day has been used by some.

Foscarnet is approved for the treatment of CMV retinitis in patients with AIDS and of acyclovir-resistant mucocutaneous HSV infections. In a comparative clinical trial, the drug appeared to be about as efficacious as ganciclovir against CMV retinitis but was associated with a longer survival period, possibly because of its activity against HIV. Intraocular foscarnet has been used to treat CMV retinitis. In addition, foscarnet has been employed to treat acyclovir-resistant HSV and VZV infections as well as ganciclovir-resistant CMV infections, although resistance to foscarnet has been reported in CMV isolates obtained during therapy. Foscarnet has also been used to treat HHV-6B infections in immunosuppressed patients.

The major form of toxicity associated with foscarnet is renal impairment. Thus renal function should be monitored closely, particularly during the initial phase of therapy. Because foscarnet binds divalent metal ions, hypocalcemia, hypomagnesemia, hypokalemia, and hypo- or hyperphosphatemia can develop. Saline hydration and slow infusion appear to protect the patient against nephrotoxicity and electrolyte disturbances. Although hematologic abnormalities have been documented (most commonly anemia), foscarnet is not generally myelosuppressive and can be administered concomitantly with myelosuppressive medications.

TRIFLURIDINE

Trifluridine is a pyrimidine nucleoside active against HSV-1, HSV-2, and CMV. Trifluridine monophosphate irreversibly inhibits thymidylate synthetase, and trifluridine triphosphate inhibits viral and, to a lesser extent, cellular DNA polymerases. Because of systemic toxicity, trifluridine’s use is limited to topical therapy. Trifluridine is approved for treatment of HSV keratitis, against which trials have shown that it is more effective than topical idoxuridine but similar in efficacy to topical vidarabine. The drug has benefited some patients with HSV keratitis who have failed to respond to idoxuridine or vidarabine. Topical application of trifluridine to sites of acyclovir-resistant HSV mucocutaneous infection has also been beneficial in some cases.

VIDARABINE

Vidarabine is a purine nucleoside analogue with activity against HSV-1, HSV-2, VZV, and EBV. Vidarabine inhibits viral DNA synthesis through its 5′-triphosphorylated metabolite, although its precise molecular mechanisms of action are not completely understood. IV-administered vidarabine has been shown to be effective in the treatment of herpes simplex encephalitis, mucocutaneous HSV infections, herpes zoster in immunocompromised patients, and neonatal HSV infections. Its use has been supplanted by that of IV acyclovir, which is more effective and easier to administer. Production of the IV preparation has been discontinued by the manufacturer, but vidarabine is available as an ophthalmic ointment, which is effective in the treatment of HSV keratitis.

AGENTS OF INVESTIGATIVE INTEREST

Maribavir is a benzimidazole that inhibits CMV and EBV. This drug inhibits the CMV UL97 kinase and does not require intracellular phosphorylation for its antiviral activity. Its mechanism of action involves blocking viral DNA synthesis and virion egress. Maribavir is orally administered and has been associated with taste disturbance and diarrhea. In phase 3 studies, it was not efficacious in the prevention of CMV infection in recipients of hematopoietic stem cell and adult liver transplants. However, when used at somewhat higher doses, it may be efficacious for the treatment of refractory or resistant CMV infections in transplant recipients.

Letermovir is an investigational drug with activity against CMV. It is a dihydroquinazoline that acts through inhibition of the viral terminase enzyme complex. This mechanism of action differs from that of ganciclovir, foscarnet, and cidofovir, which inhibit viral DNA polymerase; therefore, letermovir is active against CMV isolates that are resistant to those drugs. It is orally administered and is reportedly well tolerated. Letermovir demonstrated significant activity in preventing CMV reactivation in a recent phase 3 trial in adults undergoing hematopoietic stem cell transplantation and may be clinically available soon.

Inhibition of the helicase–primase heterotrimeric complex of HSV-1 and HSV-2 represents a novel mechanism of action of amenamevir and pritelivir. These drugs are being assessed for prevention and treatment of HSV genital infection. The efficacy of amenamevir, administered as a single oral dose of 1200 mg for recurrent genital herpes, was comparable to that of valacyclovir given for 3 days. Pritelivir has a longer half-life (up to 80 h) and was studied in a placebo-controlled trial of suppression of genital HSV infections. Compared with placebo, pritelivir—a loading dose followed by either a daily oral dose of 75 mg for 4 weeks or a weekly dose of 400 mg for 4 weeks—reduced HSV shedding and days of genital lesions. Additional clinical studies of the helicase–primase inhibitors of HSV are planned.

ANTIVIRAL DRUGS ACTIVE AGAINST HEPATITIS VIRUSES

LAMIVUDINE

Lamivudine is a pyrimidine nucleoside analogue that is used primarily in combination therapy against HIV infection (Chap. 197). Its activity against HBV is attributable to inhibition of the viral DNA polymerase. This drug has also been approved for the treatment of chronic HBV infection. At doses of 100 mg/d given for 1 year to patients positive for hepatitis B e antigen (HBeAg), lamivudine is well tolerated and results in suppression of HBV DNA levels, normalization of serum aminotransferase levels in 40–75% of patients, and reduction of hepatic inflammation and fibrosis in 50–60% of patients. Loss of HBeAg occurs in 30% of patients. Lamivudine also appears to be useful in the prevention or suppression of HBV infection associated with liver transplantation. Resistance to lamivudine develops in 24% of patients treated for 1 year and is associated with changes in the YMDD motif of HBV DNA polymerase. Because of the frequency of development of resistance, lamivudine has been largely supplanted by less-resistance-prone drugs for the treatment of HBV infection.

ADEFOVIR DIPIVOXIL

Adefovir dipivoxil is the oral prodrug of adefovir, an acyclic nucleotide analogue of adenosine monophosphate that is active against HBV, HIV, HSV, CMV, and poxviruses. It is phosphorylated by cellular kinases to the active triphosphate moiety, which is a competitive inhibitor of HBV DNA polymerase and results in chain termination after incorporation into nascent viral DNA. Adefovir is administered orally and is eliminated primarily by the kidneys, with a plasma half-life of 5–7.5 h. In clinical studies, therapy with adefovir at a dose of 10 mg/d for 48 weeks resulted in normalization of serum alanine aminotransferase (ALT) levels in 48–72% of patients and improved liver histology in 53–64%; it also resulted in a 3.5- to 3.9-log₁₀ reduction in the number of HBV DNA copies per milliliter of plasma. Adefovir was effective in treatment-naïve patients as well as in those infected with lamivudine-resistant HBV. Resistance to adefovir appears to develop less readily than that to lamivudine, but adefovir resistance rates of 15–18% have been reported after 192 weeks of treatment and may reach 30% after 5 years. This agent is generally well tolerated. Significant nephrotoxicity attributable to adefovir is uncommon at the dose used in the treatment of HBV infections (10 mg/d) but is a treatment-limiting adverse effect at the higher doses used in therapy for HIV infections (30–120 mg/d). In any case, renal function should be monitored in patients taking adefovir, even at the lower dose. Adefovir is approved only for treatment of chronic HBV infection.

TENOFOVIR

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir, a nucleotide analogue of adenosine monophosphate with activity against both retroviruses and hepadnaviruses. In both immunocompetent and immunocompromised patients (including those co-infected with HIV and HBV), tenofovir given at a dose of 300 mg/d for 48 weeks reduced HBV replication by 4.6–6 log₁₀, normalized ALT levels in 68–76% of patients, and improved liver histopathology in 72–74% of patients. Resistance develops uncommonly during ≥2 years of therapy, and tenofovir is active against lamivudine-resistant HBV. The safety profile of tenofovir is similar to that of adefovir, but nephrotoxicity has not been encountered at the dose used for HBV therapy. Tenofovir is approved for the treatment of HIV and chronic HBV infections. Tenofovir alafenamide (TAF) has recently been approved for use in the treatment of HIV and chronic HBV infections. TAF is dosed at 25 mg orally per day and has better renal and bone safety than TDF. Lactic acidosis is an important side effect associated with tenofovir use. For a more detailed discussion of tenofovir, see Chap. 197.

ENTECAVIR

Entecavir is a cyclopentyl 2′-deoxyguanosine analogue that inhibits HBV through interaction of entecavir triphosphate with several HBV DNA polymerase functions. At a dose of 0.5 mg/d given for 48 weeks, entecavir reduced HBV DNA copies by 5.0–6.9 log₁₀, normalized serum aminotransferase levels in 68–78% of patients, and improved histopathology in 70–72% of patients. Entecavir inhibits lamivudine-resistant viruses that have M550I or M550V/L526M mutations but only at serum concentrations twenty- or thirtyfold higher than those obtained with the 0.5-mg/d dose. Thus, higher doses of entecavir (1 mg/d) are recommended for the treatment of patients infected with lamivudine-resistant HBV. Development of resistance to entecavir is uncommon in treatment-naïve patients but does occur at unacceptably high rates (43% after 4 years) in patients previously infected with lamivudine-resistant virus. Entecavir-resistant strains appear to be sensitive to adefovir and tenofovir.

Entecavir is highly bioavailable but should be taken on an empty stomach because food interferes with its absorption. The drug is eliminated primarily in unchanged form by the kidneys, and its dosage should be adjusted for patients with Cr_Cl values of <50 mL/min. Overall, entecavir is well tolerated, with a safety profile similar to that of lamivudine. As with other anti-HBV treatments, exacerbation of hepatitis may occur when entecavir therapy is stopped. Entecavir is approved for treatment of chronic hepatitis B, including infection with lamivudine-resistant viruses, in adults. Entecavir has some activity against HIV-1 (median effective concentration, 0.026 to >10 μM) but should not be used as monotherapy in HIV-positive patients because of the potential for development of HIV resistance due to the M184V mutation.

TELBIVUDINE

Telbivudine is a β-L enantiomer of thymidine and is a potent, selective inhibitor of HBV. Its active form is telbivudine triphosphate, which inhibits HBV DNA polymerase and causes chain termination but has little or no activity against human DNA polymerase. Administration of telbivudine at an oral dose of 600 mg/d for 52 weeks to patients with chronic hepatitis B resulted in reduction of HBV DNA by 5.2–6.4 log₁₀ copies/mL along with normalization of ALT levels in 74–77% of recipients and improved histopathology in 65–67% of patients. Telbivudine-resistant HBV is generally cross-resistant with lamivudine-resistant virus but is usually susceptible to adefovir. After 2 years of therapy, resistance to telbivudine was noted in isolates from 22% of HBeAg-positive patients and in those from 9% of HBeAg-negative patients.

Orally administered telbivudine is rapidly absorbed; because it is eliminated primarily by the kidneys, its dosage should be reduced in patients with a Cr_Cl value of <50 mL/min. Telbivudine is generally well tolerated, but increases in serum levels of creatinine kinases as well as fatigue and myalgias have been observed. As with other anti-HBV drugs, hepatitis may be exacerbated in patients who discontinue telbivudine therapy. Telbivudine has been approved for the treatment of adults with chronic hepatitis B who have evidence of viral replication and either persistently elevated serum aminotransferase levels or histopathologically active disease, but it has not been widely used because of the frequency of development of resistance, as noted above.

INTERFERONS

IFNs are cytokines that exhibit a broad spectrum of antiviral activities as well as immunomodulating and antiproliferative properties. IFNs are not available for oral administration but must be given IM, SC, or IV. Early studies with human leukocyte IFN demonstrated an effect in the prophylaxis of experimentally induced rhinovirus infections in humans and in the treatment of VZV infections in immunosuppressed patients. DNA recombinant technology has made available highly purified α, β, γ, and λ IFNs that have been evaluated in a variety of viral infections. Results of such trials have confirmed the effectiveness of intranasally administered IFN in the prophylaxis of rhinovirus infections, although its use has been associated with nasal mucosal irritation. Studies have also demonstrated a beneficial effect of intralesionally or systemically administered IFNs on genital warts. The effect of systemic administration consists primarily of a reduction in the size of the warts, and this mode of therapy may be useful in persons who have numerous warts that cannot easily be treated by individual intralesional injections. However, lesions frequently recur after either intralesional or systemic IFN therapy is discontinued.

IFNs have undergone extensive study in the treatment of chronic HBV infection. The administration of standard IFN-α2b (5 million units daily or 10 million units three times a week for 16–24 weeks) to patients with stable chronic HBV infection resulted in loss of markers of HBV replication, such as HBeAg and HBV DNA, in 33–37% of cases; 8% of patients also became negative for hepatitis B surface antigen. In most patients who lose HBeAg and HBV DNA markers, serum aminotransferases return to normal levels, and both short- and long-term improvements in liver histopathology have been described. Predictors of a favorable response to standard IFN therapy include low pretherapy levels of HBV DNA, high pretherapy serum levels of ALT, a short duration of chronic HBV infection, and active inflammation in liver histopathology. Poor responses are seen in immunosuppressed patients, including those with HIV infection.

In pegylated IFNs, IFN alphas are linked to polyethylene glycol. This linkage results in slower absorption, decreased clearance, and more sustained serum concentrations, thereby permitting a more convenient, once-weekly dosing schedule; in many instances, pegylated IFN has supplanted standard IFN. After 48 weeks of treatment with 180 μg of pegylated IFN-α2a, HBV DNA was reduced by 4.1–4.5 log₁₀ copies/mL, with normalization of serum ALT levels in 39% of patients and improved histology in 38%. Response rates were somewhat higher when lamivudine was administered with pegylated IFN-α2a. Adverse effects of IFN are common and include fever, chills, myalgia, fatigue, neurotoxicity (manifested primarily as somnolence, depression, anxiety, and confusion), and leukopenia. Autoantibodies (e.g., antithyroid antibodies) can also develop. IFN-α2b and pegylated IFN-α2a are approved for the treatment of patients with chronic hepatitis B. Data supporting the therapeutic efficacy of pegylated interferon-α2b in HBV infection have been published; the drug has not been approved for this indication in the United States but has been approved for treatment of chronic HBV infection in other countries.

Several IFN preparations, including IFN-α2a, IFN-α2b, IFN-alfacon-1, and IFN-αm1 (lymphoblastoid), have been studied as therapy for chronic HCV infections. A variety of monotherapy regimens have been studied, of which the most common for standard IFN is IFN-α2b or -α2a at 3 million units three times per week for 12–18 months. The addition of oral ribavirin to IFN-α2b—either as initial therapy or after failure of IFN therapy alone—results in significantly higher rates of sustained virologic and/or serum ALT responses (40–50%) than are obtained with monotherapy. Comparative studies indicate that pegylated IFN-α2b or -α2a therapy is more effective than standard IFN treatment against chronic HCV infection. The combination of SC pegylated IFN and oral ribavirin results in sustained virologic responses (SVRs) in 42–51% of patients with HCV genotype 1 infection and in 76–82% of patients with genotype 2 or 3 infection. Ribavirin appears to have a small antiviral effect in HCV infection but may also be working through an immunomodulatory effect in combination with IFN. Optimal results with ribavirin appear to be associated with weight-based dosing. Prognostic factors for a favorable response include an age of <40 years, a short duration of infection, low levels of HCV RNA, a lesser degree of liver histopathology, and infection with HCV genotypes other than 1. IFN-alfacon, a synthetic “consensus” α interferon, appears to produce response rates similar to those elicited by standard IFN-α2a or -α2b alone. In 2014, the approval of a polymerase inhibitor, sofosbuvir, and a second-generation protease inhibitor, simeprevir, as well as the successful development of other direct-acting antiviral agents (DAAs) active against HCV led to revised recommendations for treatment of hepatitis C with DAA regimens not requiring IFN or ribavirin in most cases. DAA regimens have been developed that are active against all HCV genotypes (see below and Table 186-1).

IFN-α and pegylated IFN-α are active against hepatitis D, but high doses are required (9 million units three times per week for 48 weeks). IFN-α elicited an SVR in 25–30% of patients, whereas pegylated IFN-α had a variable effect, evoking an SVR in 17–43% of patients. However, long-term biochemical and histologic improvements have been seen, even in the absence of sustained inhibition of viral replication.

POLYMERASE INHIBITORS

Sofosbuvir is the prodrug of a uridine nucleoside inhibitor of HCV RNA NS5B polymerase. Its metabolism to the active uridine nucleoside triphosphate results in chain termination. Sofosbuvir is active against all HCV genotypes (1–6) and has a median effective concentration (EC₅₀) of 0.7–2.6 μM against NS5B. Resistance to sofosbuvir is conferred by an S282T substitution in NS5B, but clinically expressed resistance to treatment has only rarely been encountered in patients who receive sofosbuvir.

Sofosbuvir is administered orally and is unaffected by food. After oral administration, plasma concentrations of sofosbuvir and of its active metabolite peak in 0.5–2 h and 2–4 h, respectively. Approximately 61–65% of sofosbuvir is bound in plasma proteins, but very little of the active metabolite is bound. Both sofosbuvir and its active metabolite are cleared renally, with t_1/2 values of 0.4 and 27 h, respectively. Sofosbuvir is relatively free from clinically significant drug interactions, although P-glycoprotein inducers can reduce sofosbuvir concentrations.

Sofosbuvir is generally well tolerated and has not been associated with significant toxicity. The most common side effects in recipients of sofosbuvir have been attributable to concomitant administration of IFN and ribavirin in combination clinical trials (see below).

Sofosbuvir has been studied in a variety of controlled and open-label clinical trials. In late 2013, the results of these trials led to its recommendation—in triple combination with pegylated IFN and ribavirin—as first-line treatment for chronic hepatitis due to HCV genotypes 1, 4, 5, and 6, in which SVR rates among treatment-naïve patients were 89–97%. For HCV genotypes 2 and 3, IFN-free regimens consisting of sofosbuvir and ribavirin have been recommended, with SVR rates among treatment-naïve patients of 93% for genotype 2 and 61% for genotype 3.

PROTEASE INHIBITORS

BOCEPREVIR, TELAPREVIR

This drug class is specifically designed to inhibit the 3/4A (NS3/4A) HCV protease. These agents resemble the HCV polypeptide and, when processed by the viral protease, form a covalent bond with the catalytic NS3 serine residues, block further activity, and prevent proteolytic cleavage of the HCV polyprotein into NS4A, NS4B, NS5A, and NS5B proteins. Boceprevir and telaprevir are linear ketoamide compounds that are active against HCV genotype 1 (1b > 1a) and much less so against genotypes 2 and 3. These first-generation protease inhibitors received approval for combination therapy (with IFN and ribavirin) for genotype 1 infection. Neither boceprevir nor telaprevir is now recommended for the treatment of hepatitis C. These drugs have been supplanted by sofosbuvir and by simeprevir, a second-generation protease inhibitor with improved pharmacokinetic properties, fewer drug–drug interactions, and less overall toxicity (see below).

SIMEPREVIR

Simeprevir is a second-generation NS3/4A protease inhibitor with antiviral activity against HCV genotype 1 (1b > 1a); the EC₅₀ is 9.4 nM in an HCV genotype 1b replicon. The NS3 polymorphism Q80K, which is present in approximately one-third of patients carrying HCV genotype 1b, increases the EC₅₀ by elevenfold and results in clinical resistance to simeprevir. Thus testing for Q80K should be carried out if treatment with simeprevir is being considered. Cross-resistance occurs between simeprevir and the first-generation protease inhibitors boceprevir and telaprevir.

Simeprevir is orally administered as a 150-mg capsule, and its bioavailability is increased by administration with food. The serum concentration peaks 4–6 h after oral administration. The drug’s elimination half-life is 10–13 h in healthy individuals and 41 h in patients with hepatitis C. Simeprevir is nearly entirely bound by plasma proteins and cleared by biliary excretion. Because there is no renal excretion, dose adjustments are not required in the presence of renal dysfunction. Simeprevir is metabolized by hepatic CYP3A and therefore should not be administered to patients with decompensated liver function.

Because of its metabolism by cytochrome P450 3A (CYP3A), simeprevir interacts with drugs that induce or inhibit CYP3A, and these interactions may concomitantly increase or reduce plasma concentrations of simeprevir. Administration of simeprevir may also increase plasma concentrations of drugs that are substrates for hepatic organic anion-transporting polypeptide 1B1 or 1B3 or for P glycoprotein transporters.

Toxicity observed during clinical trials with simeprevir included photosensitivity (usually mild or moderate) in 28% of recipients and reversible hyperbilirubinemia (both conjugated and unconjugated), which was generally mild to moderate. Most of the other adverse effects seen in clinical trials with simeprevir were attributable to concomitant administration of IFN and ribavirin.

Simeprevir has been recommended as a component of alternative treatment—in combination with pegylated IFN and ribavirin—of chronic infection with HCV genotypes 1 and 4. Daily simeprevir, daily ribavirin, and weekly pegylated IFN for 12 weeks followed by another 12 weeks of pegylated IFN and ribavirin resulted in an SVR of 80% in the absence of the Q80K variant. In general, simeprevir-based triple therapy appeared to be 10% less likely to yield an SVR than sofosbuvir-based therapy and more likely to cause adverse effects. However, for prior nonresponders or partial responders to pegylated IFN, the IFN-free regimen of simeprevir, sofosbuvir, and ribavirin shows promise.

PARITAPREVIR/RITONAVIR AND GRAZOPREVIR

These drugs are more recently developed NS3/4A protease inhibitors. Paritaprevir is used with ritonavir and ombitasvir (an NS5A inhibitor; see below) as a fixed-dose combination and may be used with dasabuvir and ribavirin. This combination is active against HCV genotypes 1a and 1b. Ritonavir is used to increase the levels of paritaprevir. Ritonavir is a potent CYP3A inhibitor and may impact the metabolism of other medications handled by this pathway. Grazoprevir is used with elbasvir (an NS5A inhibitor; see below) in a fixed-dose combination and is approved by the FDA for treatment of HCV genotypes 1 and 4.

NS5A INHIBITORS

NS5A is a membrane-associated phosphoprotein that is part of the HCV RNA replication complex and is essential for viral replication and assembly. Ledipasvir, velpatasvir, daclatasvir, elbasvir, and ombitasvir are all NS5A inhibitors. Each of these agents has largely been developed and studied with specific partner drugs as noted above (Table 186-1).

Treatment of HCV has been associated with flaring of chronic HBV infection. Monitoring for HBV activation in this context is warranted. In the setting of significant renal dysfunction (Cr_Cl, <30 mL/min), few data are available to guide use of these newer DAAs. However, studies are ongoing to assess elbasvir/grazoprevir in this context, as these agents are eliminated through the feces and are not renally handled. Emergence of HCV resistance-associated substitutions to the DAAs have been documented. The impact on treatment is under active investigation and at this time is relevant mostly to those patients in whom prior treatment has failed.

These newer DAA regimens allow shorter courses of therapy, improved tolerability, and reduced resistance. For updated information, readers should consult http://www.hcvguidelines.org/.

ACKNOWLEDGMENT

The author thanks Raphael Dolin, MD, for his contributions to prior versions of this chapter and for years of mentorship.

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