**3.1. Classification of human enteroviruses**

The Picornaviridae family consists of 9 genera: Erbovirus, Kobuvirus, Teschovirus, Aphtovi‐ rus, Cardiovirus Hepatovirus, Parechovirus, Enterovirus. Human pathogens are in the four last-mentioned genera. Human enteroviruses were previously classified on the basis of sero‐ logic criteria into 64 serotypes distributed as: poliovirus (PV), coxsackievirus A (CV-A), cox‐ sackievirus B (CV-B), echovirus and other enteroviruses. The International Committee on Taxonomy of Viruses (ICTV) proposed a classification based on their phylogenetic relations encompassing 4 species, HEV-A, B, C, D, which include various serotypes (table 2). Recent‐ ly, the former human rhinovirus species have been moved to the Enterovirus genus.

roll" topology. In the case of enteroviruses and rhinoviruses, VP1 contains a cavity, or pock‐ et, accessible from a depression on the outer surface of the virus capsid. VP4 is a shorter pro‐ tein around 70 residues (7 kDaltons) lies across the inner face of the capsid with its Nterminus close to the icosahedral fivefold axis and its C-terminal close to the threefold axis [105]. The N-terminal residue of VP4 in all picornaviruses is covalently linked to the inner

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

http://dx.doi.org/10.5772/52087

35

**Figure 1.** Organisation of the enterovirus genome, polyprotein processing cascade and architecture of enterovirus capsid The genome of enteroviruses contains one single open reading frame flanked by a 5′-and 3' untranslated re‐ gions (UTR). A small viral protein, VPg, is covalently linked to the 5′ UTR. The 3'UTR encoded poly(A) tail. The transla‐ tion of the genome results in a polyprotein which is cleaved into four structural proteins (dark gray) and seven nonstructural proteins (light gray and yellow). The sites of cleavage by viral proteinases are indicated by arrows. The four structural proteins adopte an icosahedral symmetry with VP1, VP2 and VP3 located at the outer surface of the capsid

All picornaviruses have a similar genome organization consisting of a molecule of approxi‐ mately 7,500 to 8,000 nucleotides (figure 1). The RNA genome is organized with one single large open reading frame preceded by a long 5′-untranslated region (5' UTR) [97]. It contains a 3' poly(A) tail with a variable length from 65 to 100 nt. The virion RNA has a virus-encoded peptide, VPg, covalently linked to the 5′ end of the viral genome. Translation of the RNA ge‐ nome yields a polyprotein of approximately 2,200 amino acids. An early cotranslational cleav‐ age of the polyprotein by the viral 2A protease (2Apro) releases a precursor protein P1 from the N terminus of the polyprotein. The P1 protein contains all the capsid protein sequences. Sub‐ sequent cleavage of P1 by the viral 3CD protease (3CDpro) produces the capsid proteins VP1 and VP3 and the immature capsid protein VP0. Finally, the immature protein VP0 is cleaved to VP4 and VP2. There is no known protease requirement for this cleavage. From the P2 region, protein 2A may have an unidentified function in viral RNA synthesis. Protein 2B and its pre‐

and VP4 at the inner surface. The single strand genomic RNA is located inside the capsid.

**3.3. Viral proteins of enterovirus: synthesis and functions**

surface of the capsid defining a channel through the inner and outer surfaces.


**Table 2.** Classification of human enteroviruses, adapted from www.picornaviridae.com

#### **3.2. Structure of enterovirus particles**

Picornaviridae particles are small (30 nm), icosahedral, non-enveloped viruses with a singlestrand positive RNA genome (approxymatly 7 000- 8 500 nucleotides) (figure 1). The crystal structure of diverses representatives of the family have been solved [69, 2]. The fundamental capsid architecture is the same in all members of the family. The capsid is composed of 60 copies of each four structural proteins VP1 to VP4 in icosahedral symetry and protects the single strand genomic RNA and associated viral proteins [138]. In each case, VP1, VP2 and VP3 made of 240 to 290 residues (32.4-39.1 kDaltons) constitute the outer surface of the cap‐ sid. They are taking the form of eight-stranded antiparallel ß sheet structures with a "jelly roll" topology. In the case of enteroviruses and rhinoviruses, VP1 contains a cavity, or pock‐ et, accessible from a depression on the outer surface of the virus capsid. VP4 is a shorter pro‐ tein around 70 residues (7 kDaltons) lies across the inner face of the capsid with its Nterminus close to the icosahedral fivefold axis and its C-terminal close to the threefold axis [105]. The N-terminal residue of VP4 in all picornaviruses is covalently linked to the inner surface of the capsid defining a channel through the inner and outer surfaces.

**Figure 1.** Organisation of the enterovirus genome, polyprotein processing cascade and architecture of enterovirus capsid The genome of enteroviruses contains one single open reading frame flanked by a 5′-and 3' untranslated re‐ gions (UTR). A small viral protein, VPg, is covalently linked to the 5′ UTR. The 3'UTR encoded poly(A) tail. The transla‐ tion of the genome results in a polyprotein which is cleaved into four structural proteins (dark gray) and seven nonstructural proteins (light gray and yellow). The sites of cleavage by viral proteinases are indicated by arrows. The four structural proteins adopte an icosahedral symmetry with VP1, VP2 and VP3 located at the outer surface of the capsid and VP4 at the inner surface. The single strand genomic RNA is located inside the capsid.

#### **3.3. Viral proteins of enterovirus: synthesis and functions**

**3. Presentation of enteroviruses**

34 Type 1 Diabetes

**Species (number of serotypes)**

Human rhinoviruses A (75) Human rhinoviruses B (25) Human rhinoviruses C (48)

**3.2. Structure of enterovirus particles**

**3.1. Classification of human enteroviruses**

The Picornaviridae family consists of 9 genera: Erbovirus, Kobuvirus, Teschovirus, Aphtovi‐ rus, Cardiovirus Hepatovirus, Parechovirus, Enterovirus. Human pathogens are in the four last-mentioned genera. Human enteroviruses were previously classified on the basis of sero‐ logic criteria into 64 serotypes distributed as: poliovirus (PV), coxsackievirus A (CV-A), cox‐ sackievirus B (CV-B), echovirus and other enteroviruses. The International Committee on Taxonomy of Viruses (ICTV) proposed a classification based on their phylogenetic relations encompassing 4 species, HEV-A, B, C, D, which include various serotypes (table 2). Recent‐

ly, the former human rhinovirus species have been moved to the Enterovirus genus.

Human enterovirus 71

Human enterovirus 69

Human coxsackievirus B1-6

Unclassified enteroviruses (over 50)

Picornaviridae particles are small (30 nm), icosahedral, non-enveloped viruses with a singlestrand positive RNA genome (approxymatly 7 000- 8 500 nucleotides) (figure 1). The crystal structure of diverses representatives of the family have been solved [69, 2]. The fundamental capsid architecture is the same in all members of the family. The capsid is composed of 60 copies of each four structural proteins VP1 to VP4 in icosahedral symetry and protects the single strand genomic RNA and associated viral proteins [138]. In each case, VP1, VP2 and VP3 made of 240 to 290 residues (32.4-39.1 kDaltons) constitute the outer surface of the cap‐ sid. They are taking the form of eight-stranded antiparallel ß sheet structures with a "jelly

Human echovirus 1–7, 9, 11–21, 24–27, 29–33

Human coxsackieviruses A1, A11, A13, A15, A17–22, A24

Human enteroviruses A (12) Human coxsackievirus A2-8, A10, A12, A14, A16

**Table 2.** Classification of human enteroviruses, adapted from www.picornaviridae.com

Human enteroviruses B (36) Human coxsackievirus A9

Human enteroviruses C (11) Human polioviruses 1-3

Human enteroviruses D (2) Human enterovirus 68, 70

**Representatives**

All picornaviruses have a similar genome organization consisting of a molecule of approxi‐ mately 7,500 to 8,000 nucleotides (figure 1). The RNA genome is organized with one single large open reading frame preceded by a long 5′-untranslated region (5' UTR) [97]. It contains a 3' poly(A) tail with a variable length from 65 to 100 nt. The virion RNA has a virus-encoded peptide, VPg, covalently linked to the 5′ end of the viral genome. Translation of the RNA ge‐ nome yields a polyprotein of approximately 2,200 amino acids. An early cotranslational cleav‐ age of the polyprotein by the viral 2A protease (2Apro) releases a precursor protein P1 from the N terminus of the polyprotein. The P1 protein contains all the capsid protein sequences. Sub‐ sequent cleavage of P1 by the viral 3CD protease (3CDpro) produces the capsid proteins VP1 and VP3 and the immature capsid protein VP0. Finally, the immature protein VP0 is cleaved to VP4 and VP2. There is no known protease requirement for this cleavage. From the P2 region, protein 2A may have an unidentified function in viral RNA synthesis. Protein 2B and its pre‐ cursor 2BC have been suggested to be responsible for membranous alteration in infected cells. From the P3 region, two precursors are synthesized: 3AB and 3BC. The precursor 3AB is a mul‐ tifunctional protein principally involved in the membrane association of replication complex. Protein 3A is a membrane binding protein that plays a role in inhibiting cellular protein secre‐ tion. Protein 3B (VPg) is a small peptide containing 21 to 23 amino acids, which is covalently linked with the 5'UTR. The precursor 3CD exhibits protease activity and is capable of process‐ ing the P1 precursor region. Protein 3C is the protease responsible for the majority of polypro‐ tein cleavages. Protein 3D has the RNA-dependent-RNA polymerase activity and is one of the major components of the viral RNA replication complex.

ceptor with virus capsid induce conformationnal modifications of capsid proteins and cellular receptor that initiate the process of viral entry and genome delivery to the cyto‐ plasm. In brief, receptor binding triggers capsid rearrangements that result in the external‐ ization of VP4 and the N-terminus of VP1. At the same time, released VP4 also interacts with the membrane. VP1 and/or VP4 form a membrane pore through which the genom‐

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

http://dx.doi.org/10.5772/52087

37

The enterovirus genome is a positive stranded RNA that can be used as messenger RNA and immediately translated by the host cell to produce specific viral proteins. The viral ge‐ nomic RNA is then transcribed into a complementary negative RNA, which is used as a template to synthezise new strands of genomic positive RNA. Enterovirus infection induces vesicles in infected cell that are localized in the perinuclear region of the cell and are thought to be the sites of RNA replication. These vesicles clusters where viral RNA can be detected have been refered to as replication complexs (RCs). Viral RNA replication occurs at the sur‐ face of vesicles. RCs derive from cellular membranes participating to endoplasmic reticu‐ lum-to-golgi traffick, hijacked by viral proteins [19]. The viral protein 3A plays a role in the formation of the RCs. Viral proteins synthesis and genomic RNA replication are catalyzed by RNA-dependent RNA polymerase 3D and several other viral proteins, 2B, 2C and 3AB, also participate in RNA replication [4]. It has been suggested that genome replication and encapsidation are coupled [111]. To date, the VPg protein has been implicated as a determi‐ nant of encapsidation.The encapsidation of the RNA is associated to the processing of the immature protein VP0 to yield VP4 and VP2. There is no known protease requirement for this cleavage, and it is thought to be autocatalytic, depending only on the capsid proteins themselves and perhaps the viral RNA. The cleavage of VP0 to form the virion is associated with a significant increase in the stability of the particle [68]. It is commonly accepted that enteroviruses exits the cell by lysis of the host cell. However, newly synthesized virus can be detected long before lysis. In addition, enteroviruses are able to establish persitent infection without killing the cell [32]. Both observations argue in favor of alternative exit pathways probably with activation of the apototic program in enterovirus-infected cells. Enteroviruses have a large distribution in the world. Fecal-oral route via the ingestion of contaminated wa‐ ter or food is the major way of enterovirus transmission. Enterovirus infections are generally asymptomatic, but some of them, especially the one due to coxsackievirus B (CV-B), have been associated with acute manifestations (around 20 diseases such as non-specific febrile disease, cutaneous symptoms, meningitis, encephalitis, pericarditis etc.). In addition, their role in chronic diseases, like chronic myocarditis, dilated cardiomyopathy, and T1D is

**4. Relationship between enteroviruses and autoimmune type 1 diabetes:**

Enterovirus infections are among the main environmental risk factors for autoimmune T1D and they have been diagnosed more frequently in T1D patients than in healthy subjects. In this section we report the different studies conducted to investigate the relationship between

ic RNA is transported into the cytoplasm [159.].

strongly suspected [81].

**clinical studies**

#### **3.4. Enterovirus lifecycle**

The first stage of picornavirus infection of susceptible cells is mediated by the interactions of viral capsid with specific receptor on the cell membrane (figure 2). Receptors used by differ‐ ent picornaviruses include members of the immunoglobulin-like family, the low density lip‐ oprotein receptor (LDLR) family (used by minor group of rhinovirus), the complement control family (used by certain rhinovirus), the integrin family of cell adhesion molecules (receptors for aphtovirus family) and the T cell immunoglobulin domain mucin-like domain receptors (TIM-1), receptor for hepatitis A virus, [159].

**Figure 2.** Enterovirus lifecycle.

The group of immunoglobulin-like molecules includes several well characterized recep‐ tors for viruses of the enterovirus genus. For example, intercellular adhesion molecule-1 (ICAM-1) is the receptor for major group human rhinoviruses (HRVs), the Coxsackie and adenovirus receptor (CAR), a component of the tight junction between cells in intact ep‐ ithelium, is the common receptor for Adenoviruses and Coxsakieviruses. These mole‐ cules are all type 1 membrane glycoproteins encompasing, for CAR, two extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain, and a cytoplasmic do‐ main. The first Ig-like domain is responsible for virus binding. The interactions of the re‐ ceptor with virus capsid induce conformationnal modifications of capsid proteins and cellular receptor that initiate the process of viral entry and genome delivery to the cyto‐ plasm. In brief, receptor binding triggers capsid rearrangements that result in the external‐ ization of VP4 and the N-terminus of VP1. At the same time, released VP4 also interacts with the membrane. VP1 and/or VP4 form a membrane pore through which the genom‐ ic RNA is transported into the cytoplasm [159.].

cursor 2BC have been suggested to be responsible for membranous alteration in infected cells. From the P3 region, two precursors are synthesized: 3AB and 3BC. The precursor 3AB is a mul‐ tifunctional protein principally involved in the membrane association of replication complex. Protein 3A is a membrane binding protein that plays a role in inhibiting cellular protein secre‐ tion. Protein 3B (VPg) is a small peptide containing 21 to 23 amino acids, which is covalently linked with the 5'UTR. The precursor 3CD exhibits protease activity and is capable of process‐ ing the P1 precursor region. Protein 3C is the protease responsible for the majority of polypro‐ tein cleavages. Protein 3D has the RNA-dependent-RNA polymerase activity and is one of the

The first stage of picornavirus infection of susceptible cells is mediated by the interactions of viral capsid with specific receptor on the cell membrane (figure 2). Receptors used by differ‐ ent picornaviruses include members of the immunoglobulin-like family, the low density lip‐ oprotein receptor (LDLR) family (used by minor group of rhinovirus), the complement control family (used by certain rhinovirus), the integrin family of cell adhesion molecules (receptors for aphtovirus family) and the T cell immunoglobulin domain mucin-like domain

The group of immunoglobulin-like molecules includes several well characterized recep‐ tors for viruses of the enterovirus genus. For example, intercellular adhesion molecule-1 (ICAM-1) is the receptor for major group human rhinoviruses (HRVs), the Coxsackie and adenovirus receptor (CAR), a component of the tight junction between cells in intact ep‐ ithelium, is the common receptor for Adenoviruses and Coxsakieviruses. These mole‐ cules are all type 1 membrane glycoproteins encompasing, for CAR, two extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain, and a cytoplasmic do‐ main. The first Ig-like domain is responsible for virus binding. The interactions of the re‐

major components of the viral RNA replication complex.

receptors (TIM-1), receptor for hepatitis A virus, [159].

**3.4. Enterovirus lifecycle**

36 Type 1 Diabetes

**Figure 2.** Enterovirus lifecycle.

The enterovirus genome is a positive stranded RNA that can be used as messenger RNA and immediately translated by the host cell to produce specific viral proteins. The viral ge‐ nomic RNA is then transcribed into a complementary negative RNA, which is used as a template to synthezise new strands of genomic positive RNA. Enterovirus infection induces vesicles in infected cell that are localized in the perinuclear region of the cell and are thought to be the sites of RNA replication. These vesicles clusters where viral RNA can be detected have been refered to as replication complexs (RCs). Viral RNA replication occurs at the sur‐ face of vesicles. RCs derive from cellular membranes participating to endoplasmic reticu‐ lum-to-golgi traffick, hijacked by viral proteins [19]. The viral protein 3A plays a role in the formation of the RCs. Viral proteins synthesis and genomic RNA replication are catalyzed by RNA-dependent RNA polymerase 3D and several other viral proteins, 2B, 2C and 3AB, also participate in RNA replication [4]. It has been suggested that genome replication and encapsidation are coupled [111]. To date, the VPg protein has been implicated as a determi‐ nant of encapsidation.The encapsidation of the RNA is associated to the processing of the immature protein VP0 to yield VP4 and VP2. There is no known protease requirement for this cleavage, and it is thought to be autocatalytic, depending only on the capsid proteins themselves and perhaps the viral RNA. The cleavage of VP0 to form the virion is associated with a significant increase in the stability of the particle [68]. It is commonly accepted that enteroviruses exits the cell by lysis of the host cell. However, newly synthesized virus can be detected long before lysis. In addition, enteroviruses are able to establish persitent infection without killing the cell [32]. Both observations argue in favor of alternative exit pathways probably with activation of the apototic program in enterovirus-infected cells. Enteroviruses have a large distribution in the world. Fecal-oral route via the ingestion of contaminated wa‐ ter or food is the major way of enterovirus transmission. Enterovirus infections are generally asymptomatic, but some of them, especially the one due to coxsackievirus B (CV-B), have been associated with acute manifestations (around 20 diseases such as non-specific febrile disease, cutaneous symptoms, meningitis, encephalitis, pericarditis etc.). In addition, their role in chronic diseases, like chronic myocarditis, dilated cardiomyopathy, and T1D is strongly suspected [81].
