Introduction to the Human Enteroviruses and Parechoviruses

Physical Characteristics

Enterovirus virions are nonenveloped, icosahedral capsids of approximately 30 nm composed of 60 structural subunits; the subunits are formed from four polypeptides, with an aggregate molecular weight of 80 to 140 kDa, that surround the single-stranded RNA genome. In contrast to other picornaviruses (e.g., rhinoviruses), the human enteroviruses are stable over a wide range of pH (3–10), permitting them to retain infectivity during transit through the gastrointestinal tract. Lacking a lipid envelope, enteroviruses are resistant to ether, chloroform, and alcohol. However, they are readily inactivated by ionizing radiation, formaldehyde, or phenol. Molar magnesium chloride reduces the thermolability of enteroviruses across a wide range of temperatures; this feature allows live-attenuated oral poliomyelitis (OPV) vaccines to maintain potency when refrigeration is suboptimal or unavailable.

The capsid encloses a linear RNA genome of approximately 7.5 kb that is divided into four regions: a 5′ nontranslated region of approximately 740 nucleotides, a continuous-coding region of approximately 6625 nucleotides, a 3′ nontranslated region of approximately 70 nucleotides, and a 3′ poly(A) tail of 70 to 100 nucleotides in length. The 5′ terminus is covalently linked to a small virus-coded protein (VPg), which is required for the initiation of RNA synthesis. The 5′ nontranslated region contains genomic elements and sequences essential for viral RNA replication, translation, and, for some enteroviruses, virulence. The most conserved region of the genome among the human enteroviruses is the 5′ nontranslated region. The regions coding for the structural proteins show the greatest intraserotypic sequence variability, in particular, within the coding regions for epitopes that bind neutralizing antibody. The 3′ nontranslated region plays a role in viral RNA replication. Removal of the poly(A) 3′ terminus renders the RNA noninfectious.

Molecular Biology

The RNA genomes of naturally occurring polioviruses, attenuated polioviruses, and many nonpolio enteroviruses have been fully sequenced, and the replication of polioviruses in primates and transgenic mice has been studied in extensive detail. Full-length genomic clones of the polioviruses and several enteroviruses have provided the tools for the dissection of many of the molecular events of infection. The molecular structure and intracellular replicative events appear to be similar for all four enteroviruses species A through D.

Host cell susceptibility to enteroviral infection is defined in large part by the presence of specific membrane receptor proteins that serve as cellular receptors or coreceptors that bind enteroviruses generally along taxonomic lines ( Table 170.3 ). The three poliovirus serotypes share a common receptor (poliovirus receceptor [PVR] or CD155), a member of the immunoglobulin superfamily that is coded on human chromosome 19. The receptor and coreceptor proteins have been identified for many of the human enteroviruses. Both decay-accelerating factor (DAF, or CD55), a complement regulatory protein, and intercellular adhesion molecule 1 (ICAM-1) play a role in CV-A21 cell entry. The CV-B serotypes also interact with two different cell membrane proteins, the 49-kDa coxsackievirus-adenovirus receptor (CAR) and DAF. The presence of CAR permits binding and cell entry by all six CV-B serotypes, whereas antibodies to DAF block binding and infection by serotypes 1, 3, and 5. DAF also appears to be a major echovirus receptor, binding multiple echovirus serotypes, whereas serotypes E-1 and E-8 bind to the α ₂ -subunit of very late antigen 2 (VLA2) integrin molecule. P-selectin glycoprotein ligand 1 (PSGL-1) and the scavenger receptor class B member 2 (SCARB2) are both used as receptors for EV-A71. The receptor for EV-D68 has been identified as ICAM-5/telencephalin.

The processes of penetration, uncoating, and release of the nucleic acid into the cytoplasm occur within minutes at 37°C. Binding of poliovirus with its cellular receptor leads to conformational changes in the viral capsid that allow for extrusion of VP4 and the formation of a channel through which the RNA genome enters the cytoplasm of the cell. RNA synthesis begins within 30 minutes, leading to an exponential increase of minus-strand complementary and plus-strand progeny RNA until 2.5 hours after infection, when there is a switch to a linear accumulation of mainly progeny RNA. The full-length RNA functions as a monocistronic messenger whose translational product, a polyprotein of 250-kDa molecular weight, is encoded by a single open reading frame involving about 90% of the entire genome. By convention, the enteroviral polypeptide is organized into three large regions, organized 5′ to 3′ as P1, P2, and P3. The nascent polypeptide is cleaved as it emerges from the host ribosome by viral proteins acting initially in cis and later in trans to yield the proteins, and protein intermediates are essential to the viral life cycle. Cleavage of the P2 and P3 regions results in eight nonstructural proteins and several protein intermediates, whose known functions include polymerase activity, proteolytic cleavage of the translational products, inhibition of host cell protein synthesis, and cellular remodeling.

Following cleavage of P1 from the nascent polypeptide, it undergoes further cleavages to form the viral capsid, which proceeds by aggregation of five copies each of VP1, VP3, and VP0 (the precursor of VP2 and VP4) into subunits and assembly of 12 of these pentamers into the complete dodecahedral capsid shell. Encapsidation of the viral RNA is associated with a final cleavage of the VP0 protein to VP2 and VP4. The latter is an internal protein closely associated with the RNA. The complete virion contains 60 copies of each of the four structural proteins.

Host protein and RNA synthesis are severely compromised by 3 hours after infection. After about 6 to 7 hours, virions are visible by electron microscopy within the cytoplasm, and they are subsequently released by lysis of the cell, resulting in a yield of 10 ⁴ to 10 ⁵ virions per cell. The number of infectious virions is 10- to 1000-fold lower.

Pathogenesis

The pathogenesis of poliovirus infection has been extensively investigated in primates experimentally infected with neurovirulent strains and in humans infected with vaccine strains, and it is widely assumed that the early pathophysiologic events of nonpolio enterovirus infections are similar. Studies of coxsackievirus infection in mice have produced much information about the influence of various host and environmental factors on the ability of the virus to replicate in the heart, brain, and other organs and about the mechanism of vertical transmission of enteroviruses from infected pregnant animals to their offspring.

Enteroviruses infect humans via direct or indirect contact with virus shed from the gastrointestinal tract or upper respiratory tract. Whereas ingested virus implants and replicates in the pharynx and the distal small bowel, volunteer studies have shown that attenuated polioviruses replicate most efficiently in the distal small intestine. The precise site of viral entry has long been the subject of conjecture. Studies have demonstrated that microfold cells (M cells) expressing the PVR serve to transport polioviruses across the intestinal mucosa. Enteroviral replication in ileal lymphoid tissue is detectable 1 to 3 days after the ingestion of virus. The quantity of virus recoverable from the tonsils is much less than that in Peyer patches, where it may reach 10 ⁷ to 10 ⁸ tissue culture median infective doses (TCID ₅₀ ) per 1 g. In healthy individuals, the duration of viral excretion is typically less than 3 to 4 weeks from the pharynx and 5 to 6 weeks in the feces. Longer periods are reported in individuals with B-cell immunodeficiencies and, occasionally, in healthy children.

After multiplication in submucosal lymphatic tissues, enteroviruses pass to regional lymph nodes and give rise to a transient minor viremia. This leads to infection and viral replication in reticuloendothelial tissue including liver, spleen, and bone marrow. The most common result is a subclinical infection, in which viral replication is contained by host defense mechanisms. In a minority of infected persons, however, further replication of virus occurs in these reticuloendothelial sites, leading to a sustained major viremia that coincides with the onset of the minor illness of poliomyelitis and probably of the nonspecific febrile illnesses associated with other enterovirus infections. Prodromal viremia has been demonstrated with wild strains of poliovirus and E-9 but is uncommon with Sabin OPV strains except for type 2.

The major viremia results in dissemination to target organs such as the central nervous system, heart, and skin, where tissue necrosis and inflammation occur in proportion to the level of viral replication. Histopathologic lesions are usually not seen in the gastrointestinal tract, even though the small bowel is the site of initial viral replication. The severity of infection in experimental animals can be enhanced by induced exercise, cold exposure, malnutrition, pregnancy, and immune suppression with corticosteroids or radiation.

Viral Mutation During Natural Infection

Enteroviruses undergo a high rate of mutation during replication in the human gastrointestinal tract, and transcription errors occur with a frequency of 1 per 10 ⁴ bases, approximately one error per genome. As a result, single-site mutations are commonly observed in the 5′ noncoding region of attenuated polioviruses within days after feeding to young infants, and such changes are associated with longer excretion and increased neurovirulence. Serial isolates of the same enterovirus serotype excreted over many years by patients with B-cell immunodeficiency syndromes represent a heterogenetic “swarm” with continuous genetic variation, which can be characterized by oligonucleotide fingerprinting and RNA sequencing and permits estimation of the duration of infection. In addition, RNA sequencing has led to the discovery of virulent circulating vaccine-derived polioviruses (cVDPVs) that have caused outbreaks of paralytic disease among underimmunized populations in a number of countries previously free of poliomyelitis.

Dual infection with different enteroviral and parechovirus strains may produce recombinant progeny virus if the parent strains are in the same species. Intertypia can be demonstrated in 1 in 10 ⁴ to 1 in 10 ⁵ infectious virions in vitro and in the feces of infants given trivalent OPV. Most cVDPVs isolated to date are OPV viruses that have recombined with the nonpolio serotypes in the enterovirus C species.

Immunity and the Immune Response

Immunity to enteroviral infections is serotype specific. Antibody-mediated immune mechanisms operate in the alimentary tract to prevent mucosal infection and in the blood to prevent dissemination to target organs. Neutralizing antibodies target epitopes primarily located on VP1 and less so on VP2 and VP3. VP1 possesses the largest number of epitopes as a result of being the dominantly exposed viral capsid protein. As early as 1 to 3 days after enteroviral challenge, immunoglobulin M (IgM) humoral antibodies are produced; they predominate in serum during the first month and disappear within 2 to 3 months. IgG antibody, which is typically detected 7 to 10 days after infection, is mostly of the IgG ₁ and IgG ₃ subtypes. Neutralizing IgG antibodies in serum persist for life after natural infection with enteroviruses.

Small concentrations of humoral type-specific neutralizing antibodies prevented poliovirus viremia and paralysis in experimentally infected primates, and passive immunity to paralytic disease in humans can be achieved by the administration of immune serum globulin before exposure to neurovirulent polioviruses. However, passively administered immune globulin does not modify the outcome of established central nervous system poliovirus disease ; at this late stage of infection, patients have detectable serum antibody. There is no proven role for immune globulin treatment of other systemic enterovirus infections.

IgA antibody appears in nasal and alimentary secretions 2 to 4 weeks after the administration of live-attenuated OPV and persists for at least 15 years. However, mucosal immunity is relative: On reexposure to infectious virus, high titers of secretory IgA antibodies prevent or substantially reduce poliovirus shedding, whereas lower titers are associated with more extensive oropharyngeal replication of virus and longer viral shedding. The elaboration of virus-specific IgA antibodies by the small intestine depends on local immunocompetent tissues, as was demonstrated in experiments in infants with double-barrel colostomies who were fed live-attenuated poliovirus through the colostomy and generated secretory IgA antibodies only in the distal loop of the colostomy, not in the pharynx or the proximal loop. Antibodies are present in the colostrum and milk of immune women who are nursing and may interfere with the replication of OPV given to breastfed neonates. Maternal antibodies passively acquired transplacentally or via milk prevent or modify enteroviral infections of early infancy. Humoral antibodies have an important role in recovery from enteroviral infection, as evidenced by the development of persistent infections in individuals with significant B-cell immunodeficiency.

There is both clinical and laboratory evidence that humoral antibody alone is not sufficient to limit enteroviral replication in vivo. It has been known that macrophage function is critical for viral clearance and that macrophage ablation enhances the severity of CV-B infections. Host innate immunity appears to play a crucial role in limiting the severity of enteroviral infections. Findings in transgenic mice expressing PVR have shown that the host interferon (IFN) response inhibits poliovirus replication in extraneural tissues and limits the spread of poliovirus in the host. This activity is likely mediated via interaction with Toll-like receptor 3 (TLR3) and the Toll/interleukin-1 receptor domain-containing adaptor-inducing IFN-β pathway. The TLR3 pathway appears to have a central role in the immune response to human enteroviral infections as well. TLR3 variants with reduced responsiveness to double-stranded RNA have been associated with enteroviral myocarditis or dilated cardiomyopathy in humans (see Chapter 84 ). A report examining the role of TLR3 gene polymorphism (TLR3c.1377C/T) in EV-A71 disease severity among Chinese children showed that the TT allele was found more frequently in children with severe rather than mild infection. Children possessing TT allele demonstrated decreased plasma IFN-γ levels and decreased IFN-γ/interleukin-4 ratio compared with children with mild disease.

Inhibition of T-lymphocyte function has little effect on virus replication in vivo, and persons with abnormal cell-mediated immunity are not predisposed to serious or prolonged enterovirus infections unless they have accompanying B-cell dysfunction. Although T lymphocytes do not contribute to the inhibition of enteroviral replication, there is evidence that certain immunopathologic events after enterovirus infection are mediated by T-cell activity. In the murine myocarditis model, expression of proinflammatory cytokines and acute inflammatory infiltration follow peak viral replication, and induction of natural killer cell activity and T-lymphocyte immune responses contributes to necrosis of infected cardiac myocytes (see Chapter 84 ). An inflammatory response may persist long after viral replication has ceased, and ongoing cardiac damage may be mediated by virus-induced antibodies against cardiac antigens or by cytotoxic T-lymphocyte–mediated myocyte lysis.