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DEFINING A VIRUS

Viruses consist of a nucleic acid surrounded by one or more proteins. Some viruses also have an outer-membrane envelope. Viruses are obligate intracellular parasites: they can replicate only within cells since their nucleic acids do not encode the many enzymes necessary for protein, carbohydrate, or lipid metabolism and for the generation of highenergy phosphates. Typically, viral nucleic acids encode proteins necessary for replicating and packaging their nucleic acids within the biochemical milieu of host cells. Viruses differ from viroids, prions, and virusoids. Virusoids are nucleic acids that depend on helper viruses to package their nucleic acids into virus-like particles. Viroids are naked, cyclical, mostly double-stranded, small RNAs.Viroids appear to be restricted to plants, spread from cell to cell, and are replicated by cellular RNA polymerase II. Prions (Chap. 101) are abnormal protein molecules that can spread. These molecules reproduce by changing the structure of their normal cellular protein counterparts. Prions have been implicated in neurodegenerative conditions such as Creutzfeldt-Jakob disease, Gerstmann-Straüssler disease, kuru, and human bovine spongiform encephalopathy (“mad cow disease”).


VIRAL STRUCTURE

Viruses have from a few to several hundred genes.These genes may be in a single-strand or double-strand DNA genome or in a single-strand sense, a single-strand or segmented antisense, or a double-strand segmented RNA genome. Sense-strand RNA genomes can be translated directly into protein. Sense and antisense genomes are also referred to as positive-strand and negative-strand genomes, respectively. The viral nucleic acid is usually associated with one or more virus-encoded nucleoproteins in the core of the viral particle. The viral nucleic acid and nucleoproteins are almost always enclosed in a protein shell called a capsid. Because of the limited genetic complexity of viruses, their capsids are usually composed of multimers of identical capsomeres. Capsomeres are in turn composed of one or a few proteins. Capsids have icosahedral or helical symmetry. Icosahedral structures approximate spheres but have two-, three-, and fivefold axes of symmetry, whereas helical structures have only a twofold axis of symmetry.The entire structural unit of nucleic acid, nucleoprotein(s), and capsid is called a nucleocapsid. Many human viruses are simply composed of a core and a capsid. For these viruses, the outer surface of the capsid mediates contact with uninfected cells.Other viruses are more complex and have an outer lipid-containing envelope derived from virus-modified membranes of the infected cell.


The piece of infected-cell membrane that becomes the viral envelope has usually been modified during infection by the insertion of virus-encoded glycoproteins, which usually mediate contact of enveloped viruses with uninfected cells. Matrix or tegument proteins fill the space between the nucleocapsid and the envelope in many enveloped viruses. In general, enveloped viruses are sensitive to lipid solvents and nonionic detergents that can dissolve the envelope, whereas viruses that consist only of nucleocapsids are somewhat resistant. A schematic diagram for large and complex herpesviruses is shown in Fig. 78-1. Prototypical pathogenic human viruses are listed in Table 78-1. The relative sizes and structures of typical pathogenic human viruses are shown in Fig. 78-2.


TAXONOMY OF PATHOGENIC HUMAN VIRUSES

As is apparent from Table 78-1 and Fig. 78-2, 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 types of genomes and are often morphologically indistinguishable in electron micrographs. Further subclassification into genera depends on similarities in epidemiology and biologic effects and on the degree of colinear nucleic acid sequence homology. Most human viruses have a common name related to their pathologic effects or the circumstances of their  discovery. Formal species names have been assigned by the International Committee on Taxonomy of Viruses. The formal designation consists of the name of the host followed by the family or genus of the virus and a number. This dual terminology has created a confusing situation in which viruses are referred to and referenced by either name—e.g., varicella-zoster virus (VZV) or human herpesvirus (HHV) 3.


VIRAL INFECTION IN VITRO STAGES OF VIRAL INFECTION AT THE CELLULAR LEVEL

Viral Interactions with the Cell Surface and Cell Entry Viral infection is initiated by adsorption of the virus to the cell surface. Adsorption results from the molecular interaction of viral surface proteins with receptors on the cell’s plasma membrane (see Table 2-1). For example, a poliovirus capsid protein binds to a cell plasma-membrane protein of the immunoglobulin superfamily type. A rhinovirus capsid protein binds to intercellular adhesion molecule 1. An echovirus capsid protein binds to an integrin. The influenza A virus envelope hemagglutinin protein binds to sialic acid.The HIV envelope glycoprotein binds to CD4 and then engages one of several chemokine receptors that function as co-receptors for the virus. Herpes simplex virus (HSV) envelope glycoproteins bind to heparan sulfate on cell surfaces and then engage one of several immunoglobulin superfamily or tumor necrosis factor (TNF) receptors. Epstein-Barr virus (EBV) glycoprotein gp350 binds to the B lymphocyte complement receptor CD21 and then engages major histocompatibility complex (MHC) class II molecules as a co-receptor.Adsorption characteristically proceeds almost as well at 4°C as at 37°C. Adsorbed virus can still be neutralized by antibody.


Adsorption frequently initiates changes in virion surface proteins that destabilize the viral surface proteins and prepare the way for the next stage of entry into the cell. After adsorption, viruses penetrate the cell membrane by fusing with it.The fusion reaction results in the virus’s partial decomposition. The virus becomes insensitive to neutralizing antibody as it penetrates, becomes uncoated, and enters the cytoplasm. Penetration and uncoating result in viral nucleocapsid or nucleoprotein entry into the cytoplasm. Penetration and uncoating as well as subsequent steps in viral replication depend on the cell’s energy metabolism and on biochemical changes in the cell’s plasma membrane and cytoskeleton. Therefore, penetration proceeds slowly at temperatures <37°C. Interaction of viral surface proteins with cell receptors can induce receptor aggregation at the site of adsorption. Receptor aggregation can trigger signaling events within the cytoplasm and changes in the plasma membrane. The cell frequently misperceives that the receptor has encountered its “normal ligand.” Aggregated receptor may be internalized with the attached virus in an endocytic process. Viral endocytosis may proceed through clathrin-coated pits. Endocytosis is important in the entry of viruses as diverse as picornaviruses, influenza viruses, HIV, adenoviruses, and herpesviruses.


In many cases, entry of the virus into the cytoplasm depends on acidification of the viral endosome. Influenza virus provides a well-studied example of the effect of low pH on viral penetration. Influenza hemagglutinin mediates adsorption, receptor aggregation, and endocytosis. In low-pH endosomes, changes in the conformation of the hemagglutinin expose amphipathic domains that interact chemically with the cell membrane and initiate fusion of the viral and cellular membranes. (The HIV envelope glycoprotein undergoes similar conformational changes after interaction with CD4 and chemokine receptors.) For influenza virus, the M2 membrane protein also plays a key role in the uncoating of the viral envelope by providing an ion channel in the envelope. The fusion of viral proteins with cell membranes is a crucial step in viral infection, resulting in the mixture of viral envelope lipids and proteins with cell membrane lipids and proteins and (in this case) in the penetration of the influenza nucleocapsid into the cytoplasm.


Viral glycoproteins other than the protein that mediates initial adsorption may be critical in mediating envelope fusion with cell membranes, which involves hydrophobic interactions. The hydrophobic interactions required for fusion can be susceptible to chemical inhibition or blockade. Viral Gene Expression and Replication After uncoating and release of viral nucleoprotein into the cytoplasm, the viral genome is transported to a site for expression and replication. In order to produce infectious progeny, viruses must (1) produce proteins necessary to replicate their nucleic acid, (2) produce structural proteins, and (3) assemble the nucleic acid and proteins into progeny virions. Different viruses use different strategies and gene repertoires to accomplish these goals. DNA viruses, except for poxviruses, replicate their nucleic acid and assemble into nucleocapsid complexes in the cell nucleus. RNA viruses, except for influenza viruses, transcribe and replicate their nucleic acid and assemble entirely in the cytoplasm. Thus the replication strategies of DNA and RNA viruses are presented separately below. Positive-strand and negative-strand RNA viruses are discussed separately. Medically important viruses of each group are used for illustrative purposes.


Positive-Strand RNA Viruses Medically important positive-strand RNA viruses include picornaviruses, flaviviruses, togaviruses, caliciviruses, and coronaviruses. Genomic RNA from positive-strand RNA viruses is released into the cytoplasm without associated enzymes. Cell ribosomes recognize and associate with an internal ribosome entry sequence in the viral genomic RNA and translate a polyprotein that is a fusion of many or all of the viral proteins. The viral RNA polymerase and other viral proteins are cleaved from the polyprotein by protease components of the polyprotein.Antigenomic RNA is then transcribed from the genomic RNA template. Positive-strand genomes and mRNAs are next transcribed from the antigenomic RNA by the viral RNA polymerase. Positive-strand genomic RNA is encapsidated in the cytoplasm. Negative-Strand RNA Viruses Medically important negative-strand RNA viruses include rhabdoviruses, filoviruses, paramyxoviruses, myxoviruses, and bunyaviruses.


Negative-strand RNA virus genomes are released into the cytoplasm with an associated RNA polymerase and one or more accessory proteins. Some of these genomes are segmented. Except for influenza viruses, negative-strand RNA viruses replicate entirely in the cytoplasm.The viral RNA polymerase transcribes messenger RNAs (mRNAs) as well as full-length antigenomic RNA, which is the template for replication of genomic RNA. These mRNAs encode for the viral RNA polymerase and accessory factors as well as for viral structural proteins. Influenza virus is an unusual negativestrand RNA virus that transcribes its mRNAs and antigenomic RNAs in the cell’s nucleus. All negativestrand RNA viruses, including influenza viruses, assemble in the cytoplasm. Double-Strand Segmented RNA Viruses These viruses, which are taxonomically grouped in the reovirus family, have 10–12 RNA segments that make up their genome. The medically important viruses in this group are rotaviruses and Colorado tick fever virus. Reovirus virions include an RNA polymerase complex. Reoviruses replicate and assemble in the cytoplasm.


DNA Viruses Medically important DNA viruses include parvoviruses, papovaviruses [e.g., human papillomaviruses (HPVs) and polyomaviruses], adenoviruses, herpesviruses, and poxviruses. Other than poxviruses, most DNA virus genomes must get to the cell’s nucleus for transcription by cellular RNA polymerase II. For example, after receptor binding and fusion, herpesvirus nucleocapsids are released into the cytoplasm along with tegument proteins. The complex is then transported along microtubules to nuclear pores, and the DNA is released into the nucleus. Transcriptional regulation and mRNA processing for nuclear DNA viruses depend on both viral and cellular proteins. For HSVs, a virus tegument protein activates transcription of viral immediate-early genes, a class of genes expressed immediately after infection. Transcription of immediate-early genes requires the viral tegument protein and preexisting cellular transcription factors. One of the key preexisting cellular factors for HSV-1 immediate- early gene transcription is docked in the cytoplasm in neurons. Nuclear absence of this cell factor important for viral gene transcription may explain why HSV-1 goes into a latent state in neurons and how lytic infection is activated by signaling in a latently infected cell. DNA virus gene transcription is usually regulated and proceeds in an organized cascade. Transcription and expression of adenovirus and herpesvirus immediateearly genes turn on the promoters for early genes, whereas poxvirus virions carry all the factors necessary for early-gene transcription. Smaller DNA viruses are not as dependent on transactivators encoded from the viral genome for early-gene transcription. Most early genes encode proteins that are necessary for viral DNA synthesis and for the turn-on of late-gene transcription. Late genes encode mostly viral structural proteins or viral proteins necessary for the assembly and egress of the virus from the infected cell. Late-gene transcription is continuously dependent on DNA replication.


Therefore, inhibitors of DNA replication also stop lategene transcription. Each DNA virus family uses unique mechanisms for replicating its DNA. Herpesvirus DNAs are linear in the virion but circularize in the infected cell. In lytic virus infection, circular herpesvirus genomes are replicated into linear concatemers through a “rolling-circle” mechanism. Herpesviruses encode a DNA polymerase and at least six other viral proteins necessary for viral DNA replication; these viruses also encode several enzymes that increase the pool of precursor deoxynucleotide triphosphates. Adenovirus genomes are linear in the virion and are replicated into complementary linear copies by a virus-encoded DNA polymerase and an initiator protein complex.The double-strand circular papovavirus genomes are replicated into progeny circular DNA molecules by cellular DNA replication enzymes.Two viral early proteins contribute to viral DNA replication and to the persistence of papovavirus DNA in latently infected cells. Early papovavirus proteins stimulate cells to remain in cycle, thus facilitating viral DNA replication.


Parvoviruses are the smallest DNA viruses: their genomes are half the size of the papovavirus genomes and include only two genes. Parvoviruses have negative singlestrand DNA genomes. 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 has been touted as a potentially safe human gene therapy vector because its Rep protein causes its integration at a single chromosomal site. Poxviruses are the largest DNA viruses and are unique among DNA viruses in replicating and assembling in the cytoplasm. Poxviruses encode transcription factors and an RNA polymerase as well as enzymes for RNA capping and polyadenylation and for DNA synthesis. Poxvirus DNA also has a unique structure. The two strands of the double-strand linear DNA are covalently linked at the ends so that the genome is also a covalently closed single-strand circle.


In addition, there are inverted repeats at the ends of the DNA. During DNA replication, the genome is cleaved within the terminal inverted repeat, 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 with Both RNA and DNA Genomes Retroviruses and hepatitis B virus (HBV) are not purely RNA or DNA viruses. Retroviruses are enveloped 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 replication strategy. Cellular RNA polymerase II and transcription factors regulate transcription from the integrated provirus genome. Some retroviruses also encode for regulators of transcription and RNA processing, such as Tax and Rex in human T-lymphotropic virus (HTLV) types I and II and Tat and Rev in HIV-1 and HIV-2. HIV genomes also encode for 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 genomic RNAs that will be packaged in the nucleocapsids and mRNAs that encode for the viral 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 tRNA are key components of the viral nucleocapsid. The HIV Gag protease has been an important target for inhibition of HIV replication. Remnants and even complete copies of simple retroviral DNA in the human genome indicate that there may be replication-competent simple human retroviruses. However, replication has not been documented or associated with any disease. Integrated retroviral DNAs are also present in other animal species, such as pigs. These porcine retroviruses are a potential cause for concern in xenotransplantation because retroviral replication could cause disease in humans. Since the retroviral DNA is integrated into the porcine genome, special pathogen-free breeding practices cannot cleanse the donor herd of retroviral infection.


HBV is unique because virion DNA expression in infected cells results in the packaging of reverse transcriptase and genomic RNA in the virion.The genomic RNA is then copied into an incomplete double-strand circular DNA genome before the virion matures and is released from the infected cell. On entry of HBV into the cytoplasm of an infected cell, the virion reverse transcriptase/ DNA polymerase completes DNA synthesis, and the covalently closed circular genome resides in the nucleus. Viral mRNAs are transcribed from the closed circular viral episome by cellular RNA polymerase II.A capped and polyadenylated, full-genome-length, terminally redundant transcript is packaged into virus core particles in the cytoplasm of infected cells. This RNA associates with the viral reverse transcriptase.The reverse transcriptase converts the full-length, terminally redundant, core-particle, encapsidated RNA genome into partially double-strand DNA. 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. In many cases, enveloped viruses simply egress and acquire their envelope by budding through the cell’s plasma membrane. Excess viral membrane glycoproteins are synthesized to saturate cell receptors and facilitate virus separation 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, which destroys sialic acid on the infected cell’s plasma membrane. Herpesvirus nucleocapsids acquire their initial envelope by assembling in the nucleus and then budding through the nuclear membrane into the endoplasmic reticular space.The enveloped herpesvirus is then released from the cell either by maturation in cytoplasmic vesicles, which fuse with the plasma membrane and release the virus by exocytosis, or by “de-envelopment” into the cytoplasm and “re-envelopment” at the Golgi or plasma membrane. In most instances, nonenveloped viruses appear to 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 matureappearing 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” virus 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. Mutant viral genomes are also produced and can be 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.


Mutant viruses can be virulent and may preferentially cause disease through evasion of the host immune response or through resistance to antiviral drugs. Persistent hepatitis C virus (HCV) infection is due in part to genome mutation and persistent immune escape.Viral nucleic acids can also mutate 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 changes can be substantial and can significantly alter virulence or epidemiology. Reassortment of an avian or mammalian influenza A hemagglutinin gene into a human influenza background is believed to play a role in the emergence of new epidemic 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 virus 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 cell proteins, whereas 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. HOST RANGE The concept of host range was originally based on the cell types in which a virus replicated 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 identification of determinants that prevent certain animal viruses from infecting humans.The SARS coronavirus and the influenza virus strain from the 1918 pandemic are believed to have originated from animal viruses in which minor genetic mutations resulted in more efficient human infection or enhanced transmission among humans.


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. This inhibitory effect probably stems from the viruses’ need to prevent or limit nonspecific, innate host resistance factors, including interferon (IFN). Most commonly, viruses 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 cell mRNAs by interacting with their cap structure. Poliovirus RNA is efficiently translated without a cap since it has an internal ribosome entry sequence. Influenza virus inhibits the processing of mRNA by snatching cap structures from nascent cell 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 apoptosis.This delay may be advantageous in allowing the completion of viral replication. Adenoviruses and herpesviruses encode analogues of the cellular Bc12 protein, which blocks mitochondrial enhancement of proapoptotic stimuli. Poxviruses and some herpesviruses 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 its genome and permit its efficient transmission from cell to cell and to 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 it or from it to deeper tissues. Infection may then spread through the body via the bloodstream, lymphatics, or neural circuits. Parenteral inoculation can also transmit some viral infections among humans or from animals (including insects) to humans. Some viruses are transmitted only between humans. The dependence of smallpox and poliovirus infections on interhuman transmission makes it feasible to eliminate these viruses from human circulation by mass vaccination. In contrast, herpesviruses survive over time by establishing persistent infection in humans for decades, with eventual reactivation and infection of new and naïve generations.


Animals are important reservoirs and vectors for transmission of viruses causing human disease. Herpes B, monkeypox, and viral hemorrhagic fevers are examples of zoonotic infections caused by direct contact with animals or transmission from animals through other vectors. These infections may not be sustainable among humans alone because of the lack of efficient interhuman transmission. SARS resulted when an animal coronavirus apparently gained access to the human population concomitant with a mutation that enhanced its pathogenicity and spread in humans. Avian influenza viruses have drawn increased public attention because of their potential to undergo genetic changes and contribute to human disease.


PRIMARY INFECTION

The first (primary) episode of viral infection usually lasts from several days to several weeks. During this period, the concentration of virus at sites of infection rises and then falls, usually to unmeasurable levels. The rate at which the intensity of viral infection rises and falls at a given site depends on the accessibility of that organ or tissue to both the virus and systemic immune effectors, the intrinsic ability of the virus to replicate at that site, and endogenous nonspecific and specific resistance.Typically, infections with enterovirus, 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 of these viruses are especially proficient in altering or evading the innate and acquired immune responses; thus primary infection with AAV, EBV, or cytomegalovirus (CMV) can last for several months. Characteristically, primary infections due to HBV, HCV, hepatitis D virus (HDV), HIV, HPV, and molluscum contagiosum virus extend beyond several weeks. For some of these viruses (e.g., HPV, HBV, HCV, HDV, and molluscum contagiosum virus), the primary phase of infection is almost indistinguishable from the persistent phase. Disease manifestations usually arise as a consequence of viral replication and the resultant inflammatory response at a specific site, but do not necessarily correlate with levels of replication at that site.


For example, the clinical manifestations of limited infection with poliovirus, enterovirus, rabies virus, measles virus, mumps virus, or HSV in neural cells are severe relative to the level of viral replication at mucosal surfaces. Similarly, significant morbidity may accompany in utero fetal infection with rubella virus or CMV. 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 deep infection.


PERSISTENT AND LATENT INFECTIONS

Relatively few viruses cause persistent or latent infections. HBV,HCV, rabies virus, measles virus, HIV, HTLV, HPV,HHV, and some poxviruses are notable exceptions. The mechanisms for persistent infection vary widely. HCV RNA polymerase and HIV reverse transcriptase have high mutation rates, and the generation of variant genomes that evade the host immune response facilitates persistent infection. HIV is also directly immunosuppressive, depleting CD4+ T lymphocytes and compromising CD8+ cytotoxic T-cell immune responsiveness. Moreover, HIV encodes a Nef protein that downmodulates MHC class I expression, rendering HIV-infected cells partially resistant to immune CD8+ cytolysis. In contrast, DNA viruses have much lower mutation rates.Their persistence in human populations can be due to their ability to establish latent infection and to reactivate from latency. In this instance, latency is defined as a state of infection in which the 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 the cell genome replication. HPVs establish latent infection in basal epithelial cells, which replicate. Some of the progeny cells provide a stable supply of latently infected basal cells, whereas others go on to squamous differentiation and, in the process, become permissive for lytic viral infection. For herpesviruses, latent infection is established in nonreplicating neural cells (HSV and VZV) or in replicating cells of hematopoietic lineages [EBV and probably 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. It is still not fully understood how partially latent and reactivated HPV and herpesvirus infections escape immediate and effective immune responses in highly immune hosts. HPV, HSV, and VZV may be somewhat protected because they replicate in middle and upper layers of the squamous epithelium—sites not routinely visited by immune and inflammatory cells.


HSV and CMV are also known to encode proteins that downregulate MHC class I expression and antigenic peptide presentation on infected cells, thereby enabling these cells to escape CD8+T-lymphocyte cytotoxicity. Like other poxviruses, molluscum contagiosum virus cannot establish latent infection but rather causes persistent infection in hypertrophic lesions that last for months or years. This virus encodes a chemokine homologue that probably blocks inflammatory responses and an MHC class I analogue that may block cytotoxic T-lymphocyte attack.


PERSISTENT VIRAL INFECTIONS AND CANCER

Persistent viral infection is estimated to be the root cause of as many as 20% of human malignancies. For the most part, cancer is an accidental and highly unusual or longterm effect of infection with oncogenic human viruses. In these malignancies, viral infection is a critical and ultimately determinative early step, forcing infected cells to enter the cell cycle and enhancing their survival. An unusual virus-infected cell undergoes the subsequent genetic changes that permit the enhanced autonomous growth and survival characteristic of a malignant cell.  Most hepatocellular carcinoma is now 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 virusinduced liver injury followed by tissue repair and regeneration. Over decades, chronic virus infection, repetitive tissue regeneration, and acquired chromosomal changes can result in enhanced cell proliferation and survival and eventually in hepatocellular carcinoma.


In rare instances, HBV DNA integrates into cellular DNA—an event that probably contributes to the development of some tumors. Almost all cervical carcinoma is caused by persistent infection with “high-risk” genital HPV strains.Whereas HBV and HCV infections stimulate cell growth indirectly in response to virus-induced injury, proteins E6 and E7 of HPV type 16 or 18 can directly affect cell growth by causing the loss of p53 and RB, two cell proteins with tumor-suppressive function.These viral proteins can also increase genomic 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 can cause profound changes in cell growth and survival, and subsequent chromosomal changes accumulating over ensuing cycles of cell growth can lead to malignant conversion and cervical carcinoma. EBV infection and expression of the latent-infection viral proteins can immortalize B lymphocyte growth in tissue culture. In most humans, the immune response to the strongly antigenic EBV latent-infection proteins prevents uncontrolled B-cell lymphoproliferation. However, when humans are immunosuppressed by posttransplantation medications, HIV infection, or genetic immunodeficiencies, EBV-induced B-cell malignancies can emerge. EBV infection also plays a role in the long-term development of certain B lymphocyte and epithelial cell malignancies. Persistent EBV infection and expression of the EBV oncogene LMP1 in latently infected epithelial cells appear to be critical early steps in the evolution of anaplastic nasopharyngeal carcinoma, a common malignancy in Chinese and North African populations. As in other virus-associated malignancies, genomic instability and chromosomal abnormalities contribute to the development of EBV-associated nasopharyngeal carcinomas.


High-level LMP1 expression in Reed-Sternberg cells is also a hallmark of many cases of Hodgkin’s disease. LMP1- induced nuclear factor êB (NF-êB) activity may rescue and prolong the survival of defective B cells that are normally eliminated by apoptosis, thereby allowing the acquisition of other genetic changes leading to malignant Reed-Sternberg cells. The HTLV-I Tax and Rex proteins appear to be critical to the initiation of cutaneous adult T-cell lymphoma/ leukemias that may occur long after primary HTLV-I infection.Tax-induced NF-êB 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 HIVuninfected people. Several KSHV proteins that can be expressed in latently infected cells, such as v-cyclin, vinterferon regulatory factor (v-IRF), and latency-associated nuclear antigen (LANA), are implicated in increased cell proliferation and survival.


Evidence supporting a causal role of viral infection in 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 assays for transforming effects of specific viral genes on cell growth or survival, and (5) pathologic data indicating the expression of transforming viral genes in premalignant or malignant cells in vivo.Virusrelated malignancies provide an opportunity to expand our understanding of the biologic mechanisms important in the development of cancer; they also offer unique opportunities for the development of vaccines and therapeutics that could prevent or specifically treat cancers associated with virus infection.Widespread immunization against hepatitis B has resulted in a decreased prevalence of HBV-associated hepatitis and will likely prevent most HBV-related liver cancers. Studies of an HPV vaccine have shown reduced rates of colonization with high-risk HPV strains and a decreased 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 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 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 stimulate the expansion of T-cell populations with T-cell receptors that can recognize the virus-encoded peptides. Cytokines, inflammatory agents, and antigens released by virus-induced cell death further attract inflammatory cells, dendritic cells, granulocytes, natural killer (NK) cells, and B lymphocytes to the sites of initial 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 HLA class II–restricted CD4+ helper T-lymphocyte responses, and virus-specific HLA class I–restricted CD8+ cytotoxic T-lymphocyte responses have developed.


These responses, whose magnitude typically increases over the second and third weeks of infection, are important in 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 its adsorption or penetration. Complement usually enhances antibody-mediated virus neutralization. Antibody and complement can also lyse virus-infected cells that express viral proteins on their surface. A cell infected with a replicating enveloped virus usually expresses 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 tend to persist for several months after primary infection. Antibody-producing lymphocytes persist in small numbers as memory cells and begin to proliferate rapidly in response to a second infection, providing an early barrier to reinfection with the same virus. Immunologic memory for T-cell responses appears to be shorter-lived. 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 highlevel T-cell responses. For example, EBV and CMV typically induce high-level CD4+ and CD8+T-cell responses that are sustained for decades after primary infection. Some viruses have genes that alter innate and acquired host defenses.


Adenoviruses encode small RNAs that inhibit IFN-induced, PKR-mediated shutoff of infectedcell protein synthesis. Furthermore, adenovirus E1A can directly inhibit IFN-mediated changes in cell gene transcription. Moreover, adenovirus E3 proteins prevent TNFinduced cytolysis and block HLA class I antigen synthesis by the infected cell. HSV ICP47 and CMV US11 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-á and binding proteins for IFN-ã, 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 cells to kill virus-infected cells. Some poxviruses and herpesviruses also encode chemokine-binding proteins that inhibit cellular 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 response to viral infection does not come without a price. This response contributes 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. Although such effects have been demonstrated in experimental models, their role in the autoimmune manifestations of primary or recurrent human viral infections is uncertain.


INTERFERONS

All human cells can synthesize IFN-á or -â 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 in the cell cytoplasm, such as PKR and RIG-I. IFN-ã is not highly related to IFN-á or -â and is produced mainly by NK cells and by immune T lymphocytes responding to IL-12. IFN-á and -â bind to the IFN-á receptor, whereas IFN-ã binds to a different but related receptor. Both receptors signal through receptor-associated JAK kinases and other cytoplasmic proteins, including “STAT” proteins. STAT proteins 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 singlestrand RNA.


The second effect takes place through 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 eIF2-á, 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 (VSV). These IFN effects are mostly directed against the infected cell, causing both viral and cellular dysfunction and thereby limiting viral replication.


DIAGNOSTIC VIROLOGY

A wide variety of methods are now used to diagnose viral infection. Serology and viral 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 convalescentphase serum samples are analyzed at the same time. Immunofluorescence, hemadsorption, and hemagglutination assays for antiviral antibodies are labor-intensive and are being replaced by enzyme-linked immunosorbent assays (ELISAs). ELISAs generally use specific viral proteins that are 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 confirm the presence of antibody to multiple specific viral proteins simultaneously. 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, since 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. Virus isolation in tissue culture depends on infection of susceptible cells and amplification by replication in infected cells.Virus growth in cell cultures can frequently be identified by its 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 useful. 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 of the culture 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; several rounds of infection would be required to produce a visible cytopathic effect. Virus isolation in tissue culture also depends on the collection of specimens from the appropriate site and the rapid transport of these specimens in the appropriate medium to the virology laboratory. Rapid transport maintains viral viability and limits bacterial and fungal overgrowth. Enveloped viruses are generally much 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. Virus isolation 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; e.g., epithelial cells obtained by nasopharyngeal aspiration can be stained with a variety of monoclonal antibodies to respiratory viruses. Nucleic acid amplification techniques bring speed, sensitivity, and specificity to diagnostic virology.The ability to directly amplify minute amounts of viral nucleic acids present 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, since 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 of determining which patients are at increased risk for virus-induced disease and of 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 virus resistance or noncompliance with therapy. Viral-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 viral life cycle can be effectively targeted by antiviral drugs. Nucleoside and nonnucleoside reverse transcriptase inhibitors prevent synthesis of the HIV provirus, whereas protease inhibitors block maturation of the HIV polyprotein after infection of the cell. Enfuvirtide is a small peptide derived from HIV gp41 that acts before infection by preventing a conformational change required for virus fusion. Integrase inhibitors are now in clinical testing. 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. Virus genomes can evolve resistance to drugs by mutation and selection, by recombination with a drugresistant virus, or (in the case of influenza virus and other multicomponent RNA virus genomes) by reassortment. The emergence of drug-resistant strains can limit therapeutic efficacy. As with 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 in herpesviruses is a more unusual problem.


IMMUNIZATION FOR THE PREVENTION OF VIRAL INFECTIONS

Viral vaccines are among the outstanding accomplishments of medical science. Smallpox has been eradicated except as a potential weapon of biological warfare or bioterrorism (Chap. 6). 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 are entering the market.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. 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 vaccines against HIV and HCV is complicated by the high mutation rate of RNA polymerase and reverse transcriptase, the evolutionary and individual divergence of HIV and 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 naturally encountered.


VIRUSES AS NOVEL THERAPEUTIC TOOLS OR AGENTS

Viruses are being experimentally developed for the delivery of biotherapeutics 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. Retroviruses integrate into the cell genome and have 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. Recombinant poxviruses and adenoviruses are also being used experimentally as vaccine vectors.Viral vectors are being tested experimentally for expressing cytokines that can enhance immunity against tumor cells or for expressing proteins that can increase the sensitivity of tumor cells to chemotherapy. Live HSV is now being used experimentally to kill glioblastoma cells after injections into tumors. For improved safety, nonreplicating viruses are frequently employed in clinical trial settings. Potential adverse events associated with virus-mediated gene transfer include the induction of inflammatory and antiviral immune responses. Integration is useful for permanent gene therapy, but integrations can induce disease by enhancing or interrupting the expression of important cellular genes.  ​