Human Enterovirus Infections

Salient organizing principles for understanding the epidemiology of enterovirus infection and disease include evolutionary and adaptational strategies, the interaction of the virus with its human host and the environment, and characteristic modes of transmission. The epidemiology of a particular agent, or of a disease associated with a particular agent, reflects not only the properties of the agent, host, and environment but also the mode of transmission. The spectrum of enteroviral infection and disease can be better understood and categorized according to epidemiologic features that include modes of transmission as well as evolutionary-adaptational aspects of the relationships between the viral agents, their human hosts, and the environment. The chapter provides a discussion on molecular epidemiology that focuses on three of the most useful techniques: partial genome sequencing, analysis of RNA genome relationships by screening oligonucleotide mapping of the entire genome, and analysis of viral epitope distribution by use of monoclonal antibodies. In recent years, molecular techniques have been increasingly used to study the epidemiology of other polioviruses. The technique of oligonucleotide mapping produces characteristic patterns for given strains of viruses that have been referred to as "fingerprints." Fingerprint studies of wild and vaccine polioviruses have revealed many fascinating aspects of poliovirus molecular epidemiology. Molecular epidemiology promises new insights into the origin, evolution, and prevention of human enteroviruses.

The genetic analysis of enteroviruses was revolutionized by the advent of genetic engineering techniques and more specifically by two discoveries. First, the genetic map of the poliovirus genome was determined through sequence analysis of viral RNA and virus-encoded proteins. Second, cDNA copies of picornavirus genomes were found to yield infectious virus when they were introduced into mammalian cells either as DNA or, more efficiently, as RNA transcripts. This development has permitted facile generation and analysis of mutant viruses at the molecular level. Genetic studies based on these two developments have contributed substantially to our understanding of the biology and pathogenic properties of enteroviruses. Genetic dissection of the enteroviral 5' nontranslated region (NTR) and polyprotein has been facilitated by the recent development of a strategy that involves insertion of an internal ribosomal entry site (IRES) element into the enteroviral genome. The evolutionary role of recombination in the generation of enterovirus genomes is discussed in detail in this chapter. Generation of enterovirus mutants is the first critical step in their genetic analysis. Mutants with single defined genetic alterations are preferred starting materials for genetic experiments, and they can now be generated readily by manipulation of infectious cDNA clones, and genotypes can be confirmed by recloning and sequencing. Enteroviral mutants have been generated by a variety of strategies. The chapter talks about genetic complementation, reversion, recombination, and genetics of pathogens.

As knowledge of the early events in infection with enteroviruses is limited, this chapter focuses on studies with poliovirus but examines the emerging data on several other enteroviruses. The early events in virus infection, from virus binding to uncoating of the viral genome, are well characterized for a number of enveloped viruses. The poliovirus-receptor interaction is a particularly good model for studying virus entry because of the experimental manipulations that are possible given the known structure of the virus and our ability to mutagenize both the virus and its cellular receptor. Despite our increasing genetic and structural understanding of early events in poliovirus infection, many problems, such as the location of the uncoating event, remain unsolved. Perhaps imminent studies on the entry of echoviruses (ECVs) and coxsackieviruses, stimulated by the identification of their receptors, will provide clues. The roles of receptors in host range and pathogenesis have been extensively studied for poliovirus, but many questions, such as the basis of tissue tropism, remain. A study of the cell functions of virus receptors may provide information on their role in virus replication. It has been suggested that virus binding to cell receptors may lead to activation of cell events that lead to disease, and there is evidence that receptors may regulate virus-induced cytopathic effects. Studies of the interactions of cell receptors with their natural cell ligands may therefore provide clues about cell processes that are activated upon virus binding and govern the outcome of virus infections.

This chapter summarizes what is known about how a single viral RNA molecule can be selectively amplified into thousands of RNA progeny in infected cells. It specifically provides the roles of viral proteins and RNA sequences in RNA replication, and describes the kinetics and products of RNA replication in infected cells. Next, it explains the sites and compositions of viral replication complexes (RCs) in infected cells. Then, the chapter discusses the models that have been proposed to explain how viral positive and negative RNA species are made by the viral RNA-dependent RNA polymerase. Finally, it describes the coupling between translation and replication processes in infected cells. Poliovirus is used as the prototype of an enterovirus because most of the research has been performed with poliovirus infected cell. To accomplish the unique task of RNA-dependent RNA polymerization in infected cells, enteroviruses encode several proteins required for viral RNA synthesis. Open questions about the mechanism of viral synthesis include the nature o f the RNA primers for positive- and negative-strand RNA synthesis, the source of specificity for the viral template RNA, and the relationship between translation and RNA synthesis, which may occur simultaneously in the infected host cell cytoplasm. Some of these questions may be studied with the recently discovered cellfree system.

Poliovirus represents the prototypic enterovirus, and thus, much of the information available on the mechanism of protein synthesis of enteroviruses has been derived from its study. The other members of the genus Enterovirus, such as coxsackieviruses A and B and enteric cytopathic human orphan (ECHO) virus, presumably display similar modes of translation initiation. Enteroviruses, like all members of the Picornaviridae family, have a positive-sense (i.e., message-sense), single-stranded RNA genome that is translated in the cellular cytoplasm immediately after the virions have been uncoated. Enteroviral RNAs resemble cellular mRNAs to such an extent that the viral mRNAs are translated efficiently by the host cell translation machinery, although the mechanism for initiation of protein synthesis appears to be distinct from that employed for most eukaryotic mRNAs. In poliovirus-infected cells, eIF-4F is targeted for proteolysis, thereby causing a shutoff of host cell translation, but the processed form of eIF-4F is still capable of stimulating poliovirus protein synthesis.

Poliovirus, coxsackievirus, and echoviruses are cytopathic in most cell types, and the cytopathic effect (CPE) of enteroviral infection has been frequently described. This chapter discusses alterations in chromatin structure and transcription, rearrangement of cytoskeleton, accumulation of membranous vesicles, inhibition of protein secretion, and eventual lysis of cells infected with cytopathic enteroviruses. Wherever possible, the known or suspected viral proteins involved in these processes are identified, and the evidence for their involvement is presented. The chapter first discusses nuclear effects of enteroviral infection. While many changes in the cell biology of infected cells may result directly from the inhibition of cellular translation, it is also true that several preexisting proteins in the infected cell are specifically degraded during infection by poliovirus. The cell cytoskeleton is composed of three distinct but interconnected filament systems: actin microfilaments, microtubules, and intermediate filaments. A variety of findings support the hypothesis that the synthesis of viral RNA is membrane associated. First, subcellular fractions of poliovirus-infected cells that contain virus-induced membranes are able to synthesize viral RNA. Second, a complex of proteins containing the RNA-dependent RNA polymerase was shown to be membrane associated: when such complexes were isolated from poliovirus-infected cells under conditions that disrupted the membranes, many proteins, including the RNA polymerase, associated spontaneously with liposomes in vitro. Third, membranous complexes isolated from poliovirus-infected cells contain all of the viral proteins thought to be involved in RNA replication.

This chapter describes the structures of enterovirus capsid proteins and virions and the process by which virions are assembled and the genome is encapsidated. Enterovirus capsid proteins are translated in the order VP4-VP2-VP3-VP1 as part of a large polyprotein and are separated by proteolytic cleavage. The four poliovirus capsid proteins and their precursors have been mapped onto the genome by alignment of its nucleotide sequence with partial amino acid sequences derived from the amino and carboxy termini of these polypeptides. The RNA genomes of poliovirus and other enteroviruses are translated into single large polyproteins that are subsequently proteolytically processed to yield diverse structural and nonstructural proteins. Proteolytic cleavage of enterovirus capsid proteins from the P1 precursor, assembly, and eventual maturation of the virion are regulated, sequential processes that appear to be intimately connected. Two models have been proposed for virion formation. The first involves encapsidation into procapsids and is supported by the reported association of procapsids with the viral replication complex and the conversion of procapsids into virions in pulse-chase experiments. The second model involves the association of pentamers or pentamer assemblies around virion RNA.

Poliomyelitis is an acute, febrile illness characterized by aseptic meningitis and weakness or paralysis of one or more extremities. This chapter reviews the historical background, pathophysiology, and clinical manifestations of poliomyelitis. It also reviews current issues regarding both inactivated and live, attenuated poliovirus vaccines. The clinical, epidemiologic, and scientific foundations for the control of poliomyelitis were laid in the first half of the 20th century, and eradication has since been achieved through routine immunization programs that use two very effective vaccines, each of which possesses unique advantages and disadvantages.

This chapter summarizes information about the effects of enteroviral infections during gestation and the neonatal period. Pathology of the human fetus infected in utero with enteroviruses has been described in several reports. The majority of enterovirus (poliovirus and nonpoliovirus)-infected newborns are presumed to be infected intrapartum or postnatally via exposure to maternal blood, vaginal secretions, or oropharyngeal secretions or feces of mothers or other infectious contacts. Maternal poliovirus infection during gestation is associated with an increased risk of fetal loss, stillbirth, intrauterine growth retardation, and prematurity, particularly when maternal infection occurs early in pregnancy. Fetal loss was greatest with maternal infection in the first trimester, occurring in almost half of clinically affected pregnancies in one series, and with severe maternal disease, although fetal loss with mild, nonparalytic maternal illness was also observed. Researchers have reported several cases of abortion between the third and fifth months of pregnancy and stillbirths during the ninth month associated with enteroviral infection; echovirus 27, echovirus 33, coxsackievirus B2, and coxsackievirus B6 were implicated by placental and/or fetal cultures (brain, liver, heart, kidney, adrenal glands, and/or spleen). Viral culture is the standard technique for diagnosis of neonatal enteroviral infections. The highest-yield specimens in the newborn are rectum or stool and cerebrospinal fluid. Symptomatic newborns most often require hospitalization both for diagnostic evaluation and for empiric treatment of possible bacterial and/or herpes simplex virus infection, because the symptoms of enteroviral infection are nonspecific.

This chapter focuses on the epidemiology, clinical features, and laboratory aspects of nonpolio enteroviral infection in infants, primarily those less than 3 months of age. Despite many common clinical features, enteroviruses vary considerably in their organ tropism. The clinical manifestations of enteroviral infection are strikingly similar, despite the serotype-dependent differences in pathogenesis. On the basis of these clinical features, one cannot predict the presence or absence of enteroviral meningitis. Furthermore, there is considerable overlap in the signs and symptoms caused by the various viruses that are associated with febrile illness in young infants. The distinctive as well as overlapping features of disease caused by enteroviruses, respiratory syncytial virus, and influenza viruses in the young infant are illustrated. Despite the increasing emphasis on rapid diagnostic techniques for direct detection of viral antigens and nucleic acid, virus isolation in cell culture remains the only reliable way of demonstrating most enteroviruses in clinical specimens. Virus culture of specimens from multiple sites, perhaps with the addition of PCR techniques, can provide a sensitive, rapid, often specific diagnosis of enteroviral disease in symptomatic young infants.

Respiratory infections are the most frequent and universal illnesses suffered by children and adults. Evidence that enteroviruses cause respiratory disease has been obtained from three main sources: (i) surveys of patients during outbreaks of respiratory disease, with or without parallel study of healthy subjects; (ii) longitudinal studies of populations during both health and illness; and (iii) experimental inoculation of susceptible hosts with the viruses. This chapter first discusses epidemiology of respiratory disease. Respiratory diseases associated with enteroviruses are usually mild and self-limited. Therefore, opportunities to study the pathogenesis and pathology of these diseases have been limited. Then, the chapter describes clinical presentations of common cold, otitis media, pharyngitis, pharyngotonsillitis, tonsillitis, herpangina, croup, and bronchitis. Due to the mild and self-limited nature of respiratory disease caused by enteroviruses, specific treatment is not generally indicated. Conventional viral culture remains the mainstay for laboratory diagnosis of enteroviral infections of the respiratory tract. Supportive and symptomatic treatment depends on the spectrum and severity of clinical manifestations involved.

This chapter reviews the two most common central nervous system (CNS) enterovirus (EV) infections, meningitis and encephalitis. Many patients exhibit less discrete signs and symptoms and are described as having ''meningoencephalitis.'' The EVs are the most common cause of aseptic meningitis in the United States as well as an important cause of encephalitis. Autoimmune diseases, malignancies, and reactions to certain drugs are occasionally manifested as aseptic meningitis as well. Children are the primary victims of CNS EV infections. Attack rates for aseptic meningitis among athletes have been higher than those among other students during EV outbreaks. A male-to-female incidence ratio for EV infections of 1.3 to 1.5:1 has been reported. EVs are acquired by fecal-oral contamination and, less commonly, by respiratory droplet. The benign nature of EV meningitis has made human pathologic data for this disease sparse. Disseminated intravascular coagulation and other findings of sepsis result in patients with illness indistinguishable from that due to overwhelming bacterial infection. One of these drugs, disoxaril, protects mice from developing meningoencephalitis due to echoviruses and cured mice of chronic EV meningitis in an experimental model. The most common CNS manifestations of the nonpolio EVs, meningitis and encephalitis, result in significant morbidity for tens of thousands of people in the United States each year.

This chapter presents epidemiologic data on the incidence of myocarditis and dilated cardiomyopathy (DCM) and their associations with enteroviruses. The pathogenesis of enterovirus-induced heart disease is comprehensively reviewed, with particular attention given to experimental studies elucidating the viral and immunologic mechanisms of the disease process. The chapter gives an overview of pathology, and talks about viral replication in the heart, host defense mechanisms, cell-mediated destruction of myofibers, virus participation in chronic heart disease, autoimmunity, viral infection and cardiac dysfunction, and factors affecting disease severity. Next, it reviews the detection of enteroviral infection in patients with myocarditis and DCM. It also describes the clinical features of myocarditis and DCM, and discusses evidence that these diseases are two ends of the same disease spectrum. Finally, the chapter reviews conventional treatments for patients with myocarditis and DCM. In addition, the role of immunosuppression and future avenues of therapy are also examined.

Insulin-dependent diabetes mellitus (IDDM) is the most common severe chronic childhood illness, affecting an estimated 123,000 children in the United States. Picornaviruses have long been examined for their role as the primary etiologic agent in the pathogenesis of IDDM. Encephalomyocarditis virus (EMCV), a murine picornavirus, produces acute lytic infection of pancreatic β cells and diabetes whose severity correlates with the degree of β-cell lysis. An alternative innocent bystander theory proposes that local inflammation in the peri-insular tissue may induce molecules on the surface of β-cells that enteroviruses could use as cellular receptors. Well-established models of persistent viral infection leading to IDDM include reovirus type 1-induced diabetes and lymphocytic choriomeningitis virus (LCMV)-induced diabetes in mice as well as congenital rubella syndrome (CRS) in humans. Much of what is known concerning tropism of enteroviruses to the human pancreas has been learned from autopsy studies of newborns and infants who died of fulminant enteroviral infection. A large prospective study of young children monitored for concurrent development of enteroviral infections and β-cell autoimmunity is needed to test the hypothesis that symptomatic and/or serologically detectable enteroviral infection is associated with development of IDDM or β-cell autoimmunity. At present, it is not known, whether a single pathogenic mechanism is responsible for diabetes induced by various picornaviruses or whether diabetes can result from a spectrum of picornavirus-induced lesions, e.g., acute β-cell lysis, chronic autoimmunity, or insulin secretion defect. The rate of spontaneous mutation for enteroviruses is as high as 10-4 .

Coxsackieviruses, echoviruses, and polioviruses have been implicated in the pathogenesis of human neuromuscular diseases because o f their association with certain acute and chronic acquired myopathies and paralytic motor neuron syndromes. The advent of new molecular techniques has added to both the capability of establishing and the controversy surrounding the role of enteroviruses in various acquired inflammatory muscle diseases and motor neuron syndromes such as polymyositis (PM), dermatomyositis (DM), chronic fatigue syndrome (CFS), postpolio syndrome (PPS), and amyotrophic lateral sclerosis (ALS), each of which is discussed in this chapter. The author has studied muscle biopsy specimens from 39 patients (16 with PM, 12 with DM, and 11 with inclusion body myositis) who fulfilled the strict diagnostic clinicopathologic criteria of active inflammatory myopathy. Two recent studies using in situ hybridization, PCR, and sequence analysis of the amplified product have shown the presence of an enterovirus that differs from the typical strains in the 5' non coding region, suggesting a virus defective or mutated in that region. Biologic plausibility coupled with preliminary laboratory data has focused much attention on the enteroviruses as causative agents in numerous acute and chronic human muscle diseases.

In the United States alone, the enteroviruses (EVs) are estimated to cause 5 to 10 million symptomatic infections annually. Laboratory diagnosis of the EVs has exploited many of the distinctive characteristics. The predictable and well-studied growth properties of the EVs in in vitro culture systems or animals have made viral culture the mainstay of diagnosis for the past four decades. Recent insights into the surface features of these agents have revitalized efforts to design immunoassays and serologic tests for both initial detection and subsequent serotyping. Recognition of genetic homology among all of the EVs spawned the development of sensitive and specific nucleic acid detections systems, particularly the PCR, promises to revolutionize EV diagnosis. Other EV detection techniques include electron microscopy, immunoassays, and nucleic acid hybridization. The chapter ends with a discussion on serotype identification, diagnosis, and evaluation and interpretation of results.

The discovery and development of antiviral agents for the treatment of picornavirus infections have been the focus of extensive research for more than 50 years. This chapter discusses the pharmaceutical advantages of newer molecules with respect to drug development. In reviewing the historical progression toward potent picornavirus antiviral agents, two classes of inhibitors (interferon and capsid-binding molecules) stand out as having demonstrated efficacy in clinical trials of picornavirus infections. This chapter reviews the activities of these agents and their corresponding clinical data. A discussion of problems in the further development of antipicornavirus agents is also presented in this chapter. Experimental approaches to modulation of enteroviral infections have included research on chemotherapy, monoclonal antibodies, vaccines, interferons, and capsid-binding agents. Several factors appear to correlate with the abilities of a compound to bind to the drugbinding pocket and to manifest antiviral activity. Examples of the potential influence of these parameters on compound binding and antiviral activity are discussed in this chapter. The chapter talks about mechanism of viral inhibition by capsid-binding molecules, preclinical biology of capsidbinding molecules, and clinical studies with capsidbinding molecules. The majority of molecules studied have limited solubility in aqueous environments. Significant progress has been made in the discovery and development of chemotherapeutic agents for picornaviruses.