The Picornaviruses

Picornavirus proteins and their precursors take their names from sequential locations in the polyprotein open reading frame (ORF). Polyprotein processing occurs through a three tiered cascade of primary, secondary, and maturation cleavages. The first, or primary, cleavage is almost always cotranslational, as ribosomes traverse the middle (P2) region of the genome. For hepatoviruses, the first (2A/2B junction) and most subsequent cleavages within the polyprotein require downstream synthesis of viral protease 3Cpro, the central enzyme in the overall cleavage cascade. The third (final) tier of polyprotein cleavage, maturation of the 1AB peptide (VP4/VP2), is normally observed in vivo only during the final stages of virion morphogenesis and is believed to occur concomitantly with RNA assembly into large capsid structures. Unlike negative-strand or double-strand RNA viruses, which require nucleoproteins or preattached polymerases for infectivity, picornavirus genomes are infectious as naked RNA. The most recently described additions to the picornavirus RNA structural library are cis-acting replication elements (CREs). Proteolytic cleavage of the P1 precursor generates the proteins VP0 (precursor of VP4 and VP2), VP1, and VP3. The leader proteins encoded by various picornaviruses can differ considerably in length and function, even for viruses within the same genus. Viruses with the smallest 2A apparently maintain only the minimum segment required for this activity and for subsequent upstream cleavage of P1/2A by 3Cpro. The essential genome organization, especially with regard to the L-4-3-4 layout of the polyprotein, remains canonical and is a diagnostic identifier for any virus in this family.

The work of taxonomy is carried out by groups of specialist virologists, each group being responsible for monitoring developments in a particular field of virology, usually a virus family (e.g., the Picornaviridae), and for proposing new taxonomy. The ultimate aim of taxonomy is to construct a hierarchical classification (species < genus < family < order) that reproduces phylogeny. Members of most picornavirus species can be grown in cell culture, but fastidious strains and even species also exist. The effects of virus replication on cellular morphology and physiology vary from cytolysis (common) or strong inhibition of cellular macromolecular synthesis to an apparently minimal influence. Human rhinovirus infections appear to be restricted to respiratory mucosa and result in local disease symptoms, while human enteroviruses, irrespective of the species, show a range of disease severity from asymptomatic infections to life-threatening systemic infections of the newborn or to poliomyelitis or other neurological diseases with potentially persisting sequelae. Equine rhinitis A virus (ERAV) causes upper respiratory tract infections in horses but may also infect other species, including humans (causing an influenza-like illness). In humans, the Hepatitis A virus is transmitted feco-orally and especially through contaminated water and seafood, causing outbreaks of acute hepatitis. Combining the cardio- and aphthoviruses has also been suggested and would presumably include the erbo-, seneca-, tescho-, and cosaviruses. These changes would only be necessary if phylogenies were compromised by the taxonomy.

Picornavirus genomes have a unique structure, and they developed mechanisms of gene expression different from those of their prokaryotic counterparts. Genetic analyses have played a crucial role in deciphering the genome function of picornaviruses. One of the hallmarks in RNA virus research was the discovery of genetic recombination, an accomplishment received with great skepticism for several years. Cell-free synthesis, which duplicates essential steps of viral proliferation in the living cell, opened new strategies for studying individual steps of picornavirus replication in the absence of cell membrane barriers. In poliovirus (PV), the cloverleaf (CL) is followed by a short spacer of 24 nt that does not appear to engage in any base-pairing with adjacent nucleotides, as deduced from a detailed study with coxsackievirus B3 (CVB3). In cardioviruses and aphthoviruses, the genome segment preceding the internal ribosome entry site (IRES) differs completely from that of the enteroviruses. There is no CL; instead, a succession of elaborate RNA structures is followed by a long stretch of poly(C) that in encephalomyocarditis virus (EMCV) can exceed 600 nt (and plays a role in mouse pathogenesis). The most interesting trait of the poly proteins is that they contain the information for cis-cleavages, either as self-processing oligopeptide sequences (in aphtho- and cardioviruses) and/or as proteinases that are able to clip the polypeptide chain at their own N termini. Genetic complementation has been firmly established in picornavirus replication.

Picornaviruses were the first animal viruses whose structure was determined in atomic detail and, as of October 2009, the Protein Data Bank (PDB) registered 53 structure depositions for picornaviruses. These data have contributed significantly to the understanding of picornavirus evolution, assembly, host-cell interaction, host adaptation, and antigenic variation and are providing the basis for novel therapeutic strategies. Subsequently classified as a picornavirus, the general morphology of FMDV could not be visualized until the advent of the electron microscope, when negative-stained images to a resolution of 4 to 5 nm revealed rather smooth round particles of ~30 nm diameter. The current classification of picornaviruses is based on genome and protein sequence properties which are derived from the interplay of the error-prone replication mechanism of the virus with the process of natural selection. Differences in physical properties, such as buoyant density in cesium chloride and pH stability, underpinned the early classification of picornaviruses. Virus capsids recognize susceptible cells by attachment to specific receptors on the host cell membrane, thereby determining the host range and tropism of infection. The majority of antibodies are weak neutralizers that appear to operate by using the two arms of the antibody to cross-link different virus particles, causing aggregation.

Clathrin is critical for internalization of many viruses, yet no one would consider it a receptor; similarly, although encephalomyocarditis virus binds to glycophorin A on nonpermissive red blood cells, glycophorin is not expressed on most permissive cell lines and is unlikely to be the real receptor. The author suggests that a putative receptor molecule should fulfill two general criteria. First, the molecule must interact with virus at the cell surface. Second, interaction with the molecule must promote infection. The typical picornavirus capsid is an icosahedral structure constructed of 12 pentamers, with each pentamer composed of five copies of each of the four viral structural proteins, VP1 to -4. The first and simplest function of a receptor is to permit virus attachment and to concentrate the virus at the cell surface so that subsequent events in infection can occur. Unlike most enteroviruses, which have evolved to resist gastric acidity as they move through the enteric tract, foot-and-mouth disease virus (FMDV) and some rhinoviruses are destabilized by acid. Virus receptors may also transmit intracellular signals that are important for infection. The same surface loop that displays the RGD motif is also a major site recognized by neutralizing antibodies. Coxsackievirus and adenovirus receptor (CAR) functions in cell-cell adhesion, mediating both homotypic and heterotypic interactions. The murine CAR homolog is a functional coxsackie B viruses (CVB) receptor and is most likely responsible for the susceptibility of mice to CVB infection.

This chapter begins with a discussion of some of the obstacles that have hampered progress in studying cell entry pathways for picornaviruses and other non-enveloped viruses. The chapter reviews what is known about the early steps leading to internalization of the viruses into intracellular vesicles, focusing on examples (for key members of the family) that point out the diversity in the cell entry pathways used as well as the common themes. It finishes with an exploration (admittedly poliovirus centered) of what we know about the machinery that facilitates translocation of the genome across the membrane once the virus has been internalized. The virion- to-135S (or A particle) transition has not been observed in the aphthoviruses (foot-and-mouth disease viruses [FMDVs] and equine rhinitis virus) or cardioviruses (encephalomyocarditis virus, mengovirus). Although clathrin-mediated endocytosis may serve as the predominant entry pathway for some picornaviruses (including FMDV, minor group rhinoviruses, and probably major group rhinoviruses), it is clear that other picornaviruses use a variety of other endocytic pathways. In a recent study it was shown that the coxsackievirus B3 first binds to DAF (CD55) on the apical surfaces of the cells. This study clearly demonstrates the importance of virus-induced signaling in cell entry. Advances in detectors and optics that should become available in the future will improve the resolution achievable in both cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) approaches, making it possible to approach near-atomic resolution from cryo-EM studies of intermediates in vitro and unprecedented resolution for structures determined in situ.

This chapter reviews the viral and host players in the viral RNA replication process and the forms of picornavirus RNA utilized, as well as the RNA/protein complexes that facilitate viral RNA synthesis. Cleavage of poly(rC)-binding protein 2 (PCBP2) by 3CD contributes to a switch from translation to RNA replication for poliovirus, as full-length PCBP2 functions in viral translation but the truncated PCBP2 cleavage product can only function in RNA replication. The cis- acting replication element (CRE) is an RNA structure required for picornavirus RNA replication and was first discovered in the HRV14 genome by McKnight and Lemon. The picornavirus polymerase will homodimerize, oligomerize, and interact with the viral proteins 3AB, VPg, and 3CD. Although research efforts designed to elucidate the mechanisms of RNA replication utilized by picornaviruses have been comprehensive during the past several decades, there is still much to understand about the protein players and viral RNA sequences involved in replication. It is still unknown how 3Dpol can bind to sequences as disparate as the 3’ NCR/poly(A) tract of picornavirus genomic RNAs and those found at the 3’ ends of negative-strand RNA intermediates. Importantly, additional inhibitors of picornavirus replication that target specific players involved in RNA replication complex assembly, initiation, and chain elongation need to be developed as potential therapeutics against this important class of human and animal viruses.

In this chapter the authors attempt to dissect the mechanisms that underlie picornavirus RNA replication by first addressing how the viral template RNA may be specifically recognized. They then discuss the coordination of viral translation with negative-strand RNA synthesis. This discussion is followed by a description of the organization of proteins in the RNA replication complex on two-dimensional membrane surfaces, with an emphasis on viral proteins that rearrange cytoplasmic membrane structures and tether RNA replication complexes to the rearranged membranous vesicles as well as viral polymerase proteins and the proteins with which they interact. Then, the chapter describes how the cascade of proteolytic processing contributes to the formation and maturation of picornavirus RNA replication complexes. Finally, steps in the synthesis and utilization of the protein-nucleotidyl primer for initiation of picornavirus RNA synthesis are presented as a lead-in to a discussion of RNA chain elongation and the topology of the RNA in the viral RNA replication complex. Given the unique viral protein-protein and protein-RNA interfaces highlighted in the processes, picornavirus RNA replication remains an attractive target for the development of small-molecule inhibitors that disrupt this crucial part of the viral replication cycle.

This chapter addresses the main features concerning picornavirus gene expression. Picornavirus genomes are tightly packed; the RNA encodes a single poly protein whose translation is governed by the internal ribosome entry site (IRES) element using a cap-independent mechanism that hijacks the translation machinery. Picornavirus IRES activity depends on the coordination of RNA structure and RNA-protein interactions. RNA probing of the entire element revealed long-distance interactions within the 5' untranslated region (UTR) of coxsackievirus B3 (CVB3), thereby providing information on overall IRES structure. Despite the fact that many IRES trans-acting factors (ITAFs) are promiscuous RNA-binding proteins, IRESs exhibit distinct requirements in terms of functional RNA-protein associations. Ribonucleoprotein complexes assembled on IRESs share various components with the spliceosome, as in the case of SRp20, polypyrimidine tract-binding protein (PTB), or hnRNP A1. Most of the knowledge on factors required for IRES activity comes from in vitro assays. The study of IRES-ribonucleoprotein complexes in living cells has been addressed using reagents that are permeable to the cell membrane and recognize RNA molecules in a structure-dependent manner. Picornaviral genome RNAs encode their proteins in a single, long open reading frame (ORF), translated into a single poly protein. The presence of 3C and 3C-like proteinase domains in a wide range of positive-stranded RNA virus poly proteins argues strongly that this proteolytic domain was acquired at an early stage in the evolution of these viruses.

This chapter focuses on new advances in understanding viral inhibition of host gene expression at four levels: transcription, nucleocytoplasmic trafficking, translation initiation, and manipulation of mRNA granules that store or process mRNA. Blockage of host gene expression serves multiple functions of liberating ribonucleotides, charged amino- acyl tRNAs, and ribosomal machinery for viral use and also restricting expression of innate immune response polypeptides that could counter viral replication. Further, blockage of host gene expression can hamper premature cell apoptosis and promote cell lysis after viral assembly. Recently, viral interference with RNA metabolism has been shown to extend to spliceosome assembly. Interestingly, in contrast to enteroviruses, cardioviruses appear to inhibit mRNA export, and this difference may be due to the different mechanisms utilized by these viruses to inhibit nuclear transport. In addition to the cleavages of eIF4G and PABP, which have major functional consequences, picornavirus infection leads to the proteolytic processing of other accessory translation factors that likely contribute to host cell translation shutoff. Mechanistically, various cellular stresses, such as oxidative stress, heat shock, or nutrient deprivation, induce SG formation by driving phosphorylation of eIF2α, which causes generalized translational arrest, and accumulation of mRNPs with stalled 40S ribosome subunits in stress granules (SGs). In the future, a more complete understanding of the mechanisms by which these fascinating viruses manipulate host gene expression and linkage to specific pathologies could lead to the rational design of novel antiviral drugs and therapies to combat these viruses and limit or interrupt disease progression.

The extensive cellular membrane remodeling exerts major effects on cellular metabolic and physiologic functions, which may be important for viral suppression of antiviral responses within the infected host. This chapter describes the current status of our understanding of the induction and formation of membranous viral RNA replication sites for the different picornaviruses. Picornaviruses replicate their genomes in close association with cellular membranes. Fractionation of cytoplasmic extracts from infected cells demonstrated that viral RNA replication activity was associated with these new structures. Subsequently, improved imaging technology greatly increased our knowledge of the morphological changes occurring in cells infected with poliovirus as well as other members of the Picornaviridae family. Picornavirus 3A proteins show a variable size, ranging from 73 to 153 aa. They all contain at least one hydrophobic domain, which most likely serves to anchor the protein in the vesicles at which viral RNA replication takes place. Parechovirus and foot-and-mouth disease virus (FMDV) proteins interact with early secretory pathway membranes, but none of them interacts with GBF1 or interferes with membrane traffic or perturbs the secretory pathway organelle structure. We are now only beginning to understand the details of the three-dimensional structures that ultimately comprise the viral RNA replication complexes and the interactions of viral and cellular factors that lead to the dramatic remodeling of cellular membranes to form these structures. The Picornaviridae are a diverse group of viruses that infect a wide range of hosts; they are responsible for a broad spectrum of disease patterns.

Viral pathogenesis is not alien to the evolutionary history of a virus. Picornaviruses, simply by the fact of sharing a phylogenetic position, need not be associated with similar diseases, reflecting that the nature of the interactions with their host organisms may depend in a subtle manner on minimal genetic change of the virus. Picornaviruses have served to establish core concepts in the understanding of viruses as mutated collectivities and in establishing the relevance of quasispecies for viral pathogenesis. The adaptive potential of RNA viruses is also manifested in the response to selective agents administered to inhibit their replication. High mutation rates result in the almost-systematic selection of viral mutants resistant to antiviral inhibitors, either because resistant mutants are present in mutant spectra or because they are rapidly generated during viral replication. The participation of interfering genomes in virus extinction constitutes the basis of the lethal defection model of virus extinction by enhanced mutagenesis. The initial experiments to test the validity for RNA viruses of the error threshold concept consisted of documenting an adverse effect on viral infectivity as a result of increasing the mutation rate of poliovirus (PV) and vesicular stomatitis virus by chemical mutagens and base and nucleoside analogues added during viral RNA replication. Genetic modifications upon extensive passage of FMDV in BHK- 21 cells included genomes with internal in-frame deletions that were infectious by complementation in the absence of standard, wildtype genomes.

That RNA viruses have extreme mutation frequencies that permit them to rapidly adapt and evolve to changing environments is a well-established fact. The first description of viral polymerase fidelity variants and their effects on mutation frequency was made in 1974, with mutator and antimutator strains of DNA bacteriophage T4. Since RNA viruses are notorious for generating resistance to virtually every antiviral compound, it was not surprising that the isolation of ribavirin-resistant poliovirus would soon follow the demonstration of this compound as an RNA mutagen. The reasoning was that a population that was passaged several times in tissue culture would have had the opportunity to expand into a diverse quasispecies. Lower-fidelity viruses, which would expectedly be more sensitive to RNA mutagens, would likely be the first variants to be removed from the population in the screens used above to identify higher-fidelity polymerases. The identification and characterization of G64S polymerase have already unlocked a wealth of knowledge on how viral RdRps dictate copying fidelity and mutation rate. Importantly, the data obtained using these first RdRp fidelity variants revealed that the polymerase error rate does indeed play a key role in the observed mutation frequencies of RNA viruses. The recent isolation and characterization of higher- and lower-fidelity RdRps of picornaviruses suggest that viral RNA polymerase fidelity is more flexible than once thought and that nature has indeed selected for a less-than-perfect fidelity to benefit adaptation.

In viruses, recombination may allow foreign genes to be acquired or may create a composite genome through recombination between different virus variants. The ability to identify a recombinant virus and the positions where recombination occurred is only as certain as the identification of the component parental viral genomes from which it was generated. Recombination detection thus shares many elements and is ultimately dependent on evolutionary reconstructions and, most importantly, on methods for the delineation of separate phylogenetic groups. The structure of the 5’ untranslated region (5’ UTR) of picornaviruses provides a further example of modular exchange through recombination during the evolution of separate genera within the picornavirus family. Members of the same picornavirus genus show conserved gene order and content, and over the much shorter evolutionary time scale in which species and serotypes developed, gene exchange is best documented as homologous recombination events. One of the problems with conceptualizing the process of recombination of enteroviruses and other picornaviruses revolves around the fundamentally different sequence relationships between serotypes in structural gene and nonstructural (NS) region sequences. From the evidence we have from the current range of picornaviruses infecting humans and other mammals, recombination has been a pervasive influence on both the early and contemporary evolution of these viruses. The wide range of molecular tools developed in picornavirus research, reverse genetics, and methods for in vitro and in vivo culture provides unprecedented future opportunities to explore the causes and consequences of recombination in RNA viruses.

The possibility of recombination should obviously depend on the fate and status of the viral RNA in the infected cell. Two fundamentally different but not mutually exclusive mechanisms of RNA recombination were proposed at an early step of investigation of this phenomenon. The preciseness of recombination may also be interpreted in favor of the replicative model. Perhaps the most formidable problem of replicative RNA recombination is finding the proper anchoring site on the secondary template. The crossovers during RNA recombination can be distributed over the whole viral genome, indicating that no specific sequences or structures are required. Numerous examples of natural interserotype picornavirus recombinants have been described in this chapter. The chapter focuses on an aspect especially important from the evolutionary perspective, namely, the possible role of RNA recombination in viral speciation. Remarkably, IRESs of some picornaviruses exhibit marked structural similarities to respective cis-elements of viruses belonging to other families of RNA viruses, e.g., Flaviviridae. In addition to picornaviruses, it is very common in nidoviruses, in particular, coronaviruses and toroviruses. It should be noted that mechanisms of RNA recombination other than the aforementioned classical template switch and apparently host protein-dependent nonreplicative mode have been proposed to occur in some systems. Another promising approach consists of using small interfering RNA to identify host genes involved in RNA recombination, a technique not yet attempted for picornaviruses.

This chapter briefly reviews our current understanding of the origin and evolutionary dynamic of the picornavirus proteome. There is a large body of literature on protein evolution recorded during picornavirus outbreaks and on picornavirus passaging in cells and animals in the absence or presence of a selective factor, e.g., a drug. Before discussing picornavirus proteins, it is useful to recall that they were originally named without regard to evolutionary considerations, which is a common framework in contemporary studies. Two processes, mutation and homologous recombination, have been shown to be involved in generating these changes in the most conserved proteins. Special cases of nonhomologous recombination are gene duplication and loss in progeny of a single parent. In the case of gene duplication, a genetic locus is repeatedly copied, while gene loss is a result of skipping a genetic locus from copying; both are considered to be aberrations of template-mediated replication in picornaviruses. The origin of the N-terminal amphipathic helix of 2C is another case open to different evolutionary interpretations. Gene loss along with repeated introduction of a protein variety may be invoked for explaining phylogenetic discontinuity of the presence of the protein variety in picornaviruses.

Codon usage bias has also been described for DNA and RNA viruses. Among the latter, poliovirus (PV) has selected through a codon bias similar to that of its human host species (“optimized”), while the codon bias of hepatitis A virus (HAV) is very different from that of its host (“deoptimized”). Picornavirus internal ribosome entry site (IRES) types have probably evolved by gradual addition of domains and elements that improved their function in ribosome recruitment or otherwise conferred regulation to the process of viral protein synthesis in a specific cell environment. The highly inefficient IRES combined with the lack of a mechanism to induce cellular shutoff leads in HAV to an unfair competition for the cellular translational machinery. An intriguing connection exists between codon bias, codon pair bias, and dinucleotide bias in mammalian genomes. Viral genomes, especially of RNA viruses and retroviruses, are short enough to make them amenable to whole-genome synthesis with currently available technology. Such freedom of design can provide tremendous power to reengineer DNA- and RNA-coding sequences at will to study the impact on viral fitness of large-scale changes in codon bias, codon pair bias, dinucleotide biases, GC content, RNA secondary structures, and other sequence signatures, with the aim to develop a new platform for vaccine design and genetic engineering. The codon usage selected through evolution by PV, HAV and its contribution to their in vivo fitness are still not completely elucidated, but it is certainly remarkable how they follow clearly different strategies.

When viruses enter the host, they are first detected by the innate immune system, which comprises cytokines, sentinel cells (dendritic cells [DCs] and macrophages), complement, and natural killer (NK) cells. A second family of pattern recognition receptors comprises the cytoplasmic sensors of viral nucleic acids, MDA-5 (melanoma differentiation associated gene-5), RIG-I (retinoic acid-inducible gene I), and LGP2. Interferon (IFN) production after infection of cultured cells with paramyxoviruses, VSV, influenza virus, and the flavivirus Japanese encephalitis virus is impaired in fibroblasts from rig-I -1- mice. RIG- I- like receptors (RLRs) play central roles in viral recognition and induction of antiviral innate responses in cDCs, macrophages, and fibroblasts. Infection of mda-5 -1- mice revealed that this protein is critical for detection of infection with picornaviruses, but not flaviviruses, influenza viruses, or paramyxoviruses. Mice lacking the nitric oxide synthase gene have reduced macrophage activation and β-cell apoptosis and consequently reduced virus-induced diabetes. Understanding viral countermeasures not only improves one's understanding of innate sensing pathways but may also suggest avenues for therapeutic intervention. Infection of cells with several picornaviruses leads to cleavage or degradation of MDA-5 and RIG-I. We survive only because we possess powerful intrinsic, innate, and adaptive immune defense systems. The study of how picornaviruses interact with the innate immune system is in its infancy. Understanding innate signaling pathways and how viruses counteract them should be a productive area of research for many years.

The main protection against virus-induced disease is the immune system. This chapter provides a brief overview of the adaptive immune response and discusses its relevance to picornavirus infection. The immune response can be classified in several ways, but it may be most logical to do so by antigen specificity. Thus, immune responses may be termed non-antigen-specific or antigen-specific responses. A small subset of T cells expresses a different T-cell receptor (TCR), with one γ and one δ chain; the function and target antigens of these γ δ T cells are less well defined but studies, described later in this chapter, suggest that these cells may regulate picornavirus pathogenesis. Memory B cells probably do not secrete antibody; instead, they maintain cell surface expression of their immunoglobulin receptors so that they can recognize their specific antigen. The benefits of antiviral antibodies in almost all viral infections (and antiviral vaccines) are abundantly clear. High levels of neutralizing antibody can protect animals from infection and disease, and in certain instances (e.g., rabies virus, hepatitis B virus, and Junin virus infections) postexposure antibody therapy is often recommended and is efficacious. The chapter summarizes the events that follow virus infection in naïve and immune hosts.

Once considered rare, persistent virus infections in animals and humans are no longer thought uncommon. Such persistent infections may culminate in pathological changes or, after a prolonged incubation period, overt disease. Persistent virus infections in livestock, such as foot-and-mouth disease virus (FMDV) infection, also can have substantial economic consequences. Well-characterized models are needed to improve the understanding of the mechanisms underlying persistent viral infections. Several members of the Picornaviridae family produce persistent infections in their natural hosts, with FMDV infection in cloven-hoofed cattle and Theiler's murine encephalomyelitis virus (TMEV) infection in mice as the best-characterized examples. CD155-negative mouse LM cells transfected with mutated CD155 cDNA from cured IMR-32 cells also showed partial resistance to poliovirus (PV)-induced cytolysis and apoptosis compared to cells transfected with nonmutated CD155 cDNA. Noncytolytic FMDV mutants were isolated after repeated transfers and established persistent infections in BHK-21 cells without cell crisis. Enteroviruses, primarily coxsackievirus B (CVB), have been associated with the development of dilated cardiomyopathies, for which about 30% of patients have a history of acute mycocarditis. Once considered rare, persistent virus infections in animals and humans are no longer uncommon. Persistent picornavirus infections in cells in culture have provided important models for elucidating the mechanisms underlying virus persistence.

Poliovirus (PV) is the causative agent of poliomyelitis, an acute human disease of the central nervous system (CNS). This chapter provides a review of recent advances in our understanding of PV pathogenicity. The host range of most PV strains is restricted to primates, with humans as the natural host. The PV receptor (PVR) was identified by taking advantage of the species-specific nature of infection. Mouse cells are not susceptible to PV infection but permit PV replication when PV RNA is transfected, circumventing infection through the cell surface. The infected mice exhibit clinical signs and pathological lesions that resemble human poliomyelitis after intracerebral, intraperitoneal, intravenous, intramuscular, or intranasal inoculation of PV. In addition to monkeys, PVR-Tg21 mice are recognized by the World Health Organization as an animal model of poliomyelitis. Provocation poliomyelitis was experimentally reproduced in transgenic (tg) mice, with results that suggested that skeletal muscle injury stimulates retrograde axonal transport of PV and thereby facilitates viral invasion of the CNS, with resultant spinal cord damage. The quasispecies of PV plays an important role in PV pathogenesis. PV, as well as other RNA viruses, has a high error rate in RNA replication, and therefore each viral genome in the population differs from others by one or more mutations.

Two groups of coxsackieviruses, A and B, were subsequently defined according to their pathogenicity in suckling mice: the group A coxsackieviruses caused generalized myositis, while the coxsackieviruses group B (CVB) resulted in multiorgan infections. This chapter focuses on CVB, because this group includes major human pathogens and because extensive pathogenesis studies have been carried out on a number of viruses in this group. The chapter reviews salient aspects of CVB pathogenesis with an emphasis on recent findings related to CVB4 and CVB3. Recent reviews have examined the cell's responses to CVB infection in depth. Given the distance that an RNA virus genome can randomly wander in sequence space in a short length of time, comparing contemporary strains in such studies may be necessary when mapping genetic determinants of virulence. A mechanism by which enteroviruses can persist at low levels has been identified by recent work in which enteroviral RNA with genomic terminal deletions was detected in naturally infected human heart as well as in an experimental murine model of chronic myocarditis. Evidence from a murine model of myocarditis as well as a human case of myocarditis shows that the form in which CVB persist in the heart is through selection of a defective form of the virus with 5'- terminal deletions.

Human rhinovirus (HRV) has long been known to infect the upper respiratory tract, i.e., nasal passages, paranasal sinuses, and pharynx. However, it has recently become clear that HRV infection also causes exacerbations of chronic respiratory diseases affecting the lower respiratory tract, i.e., the larynx, trachea, bronchi, bronchioles, and alveoli. Recent data hint at the possibility that HRV infections not only cause exacerbations of asthma but also influence the severity of the disease, and they may even be involved in its initial pathogenesis. Eosinophils and eosinophil cationic protein have been detected in the airways following experimental HRV infection, and asthma patients show more eosinophils than controls. In vitro studies have noted attachment of HRV to peripheral blood monocytes and airway macrophages, with subsequent secretion of numerous proinflammatory cytokines, chemokines, and IFNs. Cultured HeLa cells were infected with lung homogenates from HRV1B-treated mice. Together, these results strongly suggest that HRV1B causes viral RNA replication with a productive infection in wild- type mice. HRV has been detected in secretions and epithelial cells of the segmental bronchial epithelium and is associated with exacerbations of chronic respiratory diseases affecting the lower airways, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF). New animal models demonstrate that HRV infection and allergen exposure have synergistic effects on bronchial inflammation and hyperreactivity. The large number of serotypes of HRV indicates that a novel strategy may be necessary for generating an effective vaccine.

The most common clinical manifestation of hepatitis A virus (HAV) infection is acute hepatitis, which typically presents with rapid onset of nausea, loss of appetite, fever, abdominal pain, dark urine, and jaundice. Importantly, HAV causes only acute hepatitis, and long-term persistent infection has never been well documented or clearly associated with chronic liver disease. The internal ribosome entry site (IRES) structure is unique, but overall resembles the aphthovirus IRES more than the enterovirus IRES. Importantly, the HAV IRES is distinguished from other picornaviral IRES elements by its apparent requirement for all eukaryotic translation initiation factors, including intact eIF- 4G, to direct internal entry of the 40S ribosome on the viral RNA. In vivo, the virus is thought to replicate primarily within hepatocytes. The infecting HAV particle presumably arrives at the basolateral membrane surface of the hepatocyte, within the space of Disse, via the portal circulation from the intestines and diffusion through the hepatic sinusoids. Viremia is present throughout much of the incubation period and into the acute phase of illness but is reduced in magnitude (as is the fecal shedding of virus) with the onset of hepatitis and the appearance of antibodies to HAV.

Foot-and-mouth disease (FMD) is a highly infectious viral disease of domestic cloven-hoofed animals, including cattle, swine, goats, and sheep, as well as some species of wild animals. The viral agent, FMD virus (FMDV), is the type species of the Aphthovirus genus of the Picornaviridae family. The major consequence of FMD is a high degree of morbidity, including fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats, but there is generally low mortality in infected animals except when the disease affects the young. Additional research with naturally susceptible animals is necessary to understand the mechanism of immunosuppression FMDV has developed and to identify the viral protein(s) that participates in this process. In addition to the high genetic variation, other mechanisms involving interaction of specific viral proteins or interactions of 5’ and 3’ untranslated regions (UTRs) with cellular targets have been shown to contribute to its virulence. In vitro studies have shown that after FMDV infection of BHK cells several proteins, including poly(A)-binding protein, polypyrimidine tract-binding protein (PTBP), and two subunits of the translation factor eIF3 (eIF3a and -b) undergo proteolytic cleavage. It is hoped that new information obtained from comprehensive viral pathogenesis studies will enable researchers to develop more effective disease control strategies, including induction of rapid protective responses to inhibit or at least limit the spread of this disease.

The Cardiovirus genus is divided into two species: the Encephalomyocarditis species, which includes encephalomyocarditis virus (EMCV) and mengovirus, and the Theilovirus species, which includes Theiler’s murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), and Saffold virus (SAFV). This chapter discusses members of the Cardiovirus genus and distinctive characteristics of these viruses. It focuses on the pathogenesis of TMEV-induced disease because of the extensive investigations of this topic as well as the remarkable ability of this virus to establish lifelong infection of the central nervous system (CNS) and to produce an immune-mediated demyelinating disease. There are many features of cardioviruses, and especially TMEV, that make them attractive for pathogenesis studies, including the following: the unusual and rather unique characteristics of TMEV strains and TMEV-induced diseases (neurovirulent versus persistent strains, acute versus chronic disease, virus-induced versus immune-mediated pathology); the ability of some strains to persist in the host in the absence of latency or genome integration; the substantial knowledge concerning cardioviruses along with powerful tools for their study; the experimental model system of TMEV (and EMCV) infection in the mouse, which is the virus’ natural host. The recent identification of SAFV infection as a frequent one in humans has focused attention on whether this virus causes disease in humans. Furthermore, the identification of the human SAFV indicates that efforts investigating the involvement of cardioviruses as a cause of Vilyuisk encephalomyelitis should be renewed.

Vaccination is one of the most cost-effective methods to control and prevent infectious diseases and has had a major impact on human and animal health over the last century. Commercial vaccines have been developed for just three picornavirus-associated diseases. Two are human afflictions, poliomyelitis and hepatitis A, and the third is foot-and-mouth disease (FMD), a major disease of domestic livestock. The antigenic material for use in vaccines is subject to rigorous regulatory controls to minimize unwanted side effects. All current picornavirus vaccines are produced in tissue culture under tightly controlled conditions. The presence of multiple serotypes is obviously a complicating issue for the development and deployment of vaccines. In the early development of FMD vaccines, formaldehyde was used to chemically inactivate the virus. However, the inactivation kinetics in the relatively crude virus suspensions used for vaccine production are not linear, and a small persistent fraction of infectious virus may be detected even after prolonged inactivation times, as was also seen in some early polio vaccines. The culture of hepatitis A virus in vitro proved difficult, and much of the early work that led to the eventual development of a vaccine relied on human volunteer studies or work in New World primates or chimpanzees. Quantitative improvements would be expected to reduce the frequency at which booster immunizations are required and, qualitatively, broadening of the immune response to include cell-mediated responses and secretory immunity is desirable.

Poliomyelitis is a neurological disease characterized by paralysis of the extremities and occasionally other muscles, leading to transient or permanent impairment, and rarely, in the most severe bulbar cases, death. Interest in poliomyelitis and poliovirus increased dramatically at the beginning of the 20th century because a relatively rare sporadic illness changed to an epidemic disease with a clear trend to global spread. Due to the very low paralysis-to-infection ratio, some limited circulation of wild poliovirus (WPV) without overt clinical manifestations cannot be fully excluded as the explanation for orphan isolates. Since the early days of oral poliovirus vaccine (OPV), it has been known that the use of this vaccine can cause vaccine associated paralytic poliomyelitis (VAPP) among vaccine recipients and their immediate contacts. Another category of vaccine-derived poliovirus (VDPV), called immunodeficiency-associated VDPVs (iVDPVs), have been isolated from immunodeficient patients chronically infected with poliovirus. The most serious challenge, however, is to greatly strengthen routine immunization within the Expanded Program on Immunization (EPI), which is clearly inadequate in many low-income countries. The successful reintegration of polio eradication activities into the EPI, by also helping to reinvigorate the EPI, can be one of the enduring legacies of the Global Polio Eradication Initiative (GPEI) and serve as a model for other global infectious disease control and eradication initiatives.

Although picornaviruses have an enormous impact on human health, the effective control of poliovirus (PV) and hepatitis A virus (HAV) infections with specific vaccines has largely restricted the search for antiviral drugs to those active in combating the common cold, caused by human rhinoviruses (HRVs). Today, there is growing interest in the development of antipicornavirus drugs for several reasons. Inhibitors of picornavirus replication that target different (structural as well as nonstructural) viral proteins have been reported during the last decades. This chapter presents an overview of the most relevant antipicornavirus compounds, according to their viral target. One of the earliest discovered classes of picornavirus inhibitors belongs to a series of compounds that were later designated WIN compounds, referring to Sterling-Winthrop, where they were developed. Several structural analogs of DPBR0Z-194 with enhanced antiviral activity were synthesized, including DBPR 103 and compound 28b, the latter being a highly selective inhibitor of enterovirus 71. A systematic evaluation of several reported HRV capsid-binding compounds active against all serotypes of HRV was performed by Andries. This study revealed the existence of two groups of HRVs, designated antiviral groups A and B. The activity of the gliotoxin compound was shown to be dependent on the presence of a disulfide bridge that is formed when the compound exists in its native oxidized state. For coxsackievirus, it was demonstrated that the in vitro efficacy of RNAi is highly dependent on two critical factors, namely, target selection and emergence of viral escape mutants.