The 3-dimensional play of human parechovirus infection ; Cell , virus and antibody Westerhuis

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The HPeV genome is ~7300 bases in length, and encodes a single polyprotein flanked by 5' and 3' untranslated regions (UTRs) (Figure 1). The covalently linked protein VPg (also designated 3B) is attached to the long 5'UTR of around 700 nucleotides preceding an open reading frame (ORF) of 2200 codons, followed by the 3'UTR of around 80 nucleotides and the poly(A) tail. Translation of the ORF, initiated by a cap-independent mechanism, is driven by an internal ribosome entry site (IRES), which results in a large polyprotein (2,3). Picornavirus polyproteins are cleaved by virus-encoded proteases to give precursors and the final structural (capsid) and non-structural (NS) proteins. HPeVs together with kobuviruses have a different organisation of their structural proteins compared to other picornaviruses. The structural protein VP0, which is a precursor for VP4 and VP2 in most picornaviruses, is not cleaved; this VP0 maturation is thought to be critical for capsid stability. By that, HPeVs as well as Kobuvirus have only 3 structural proteins (VP0, VP3 and VP1) (4) ( Figure  1). As in the other picornaviruses, the capsid is composed of 60 copies of each of the capsid proteins.
HPeVs non-structural 2A lacks the proteolytic activity seen in other picornaviruses, indicating incapability of the cleavage of VP1 and 2A proteins (5,6). In the case of HPeVs, it seems that only one protease, 3Cpro, is involved in processing. Although it has been shown that HPeV1 2A binds to viral 3'UTR RNA, suggesting that 2A exerts an important function during HPeV1 replication, the exact function remains unknown (7). The 2C protein has been shown to have ATP hydrolysis and AMP kinase activities, which may contribute to functions in the viral replication cycle. The 2B protein is an integral membrane protein that is mainly co-localized at the Golgi complex, mediating calcium release and increasing plasma permeability (8). Thereby co-localization with the ER is also observed. The 3A protein has also been observed to interfere with ER-to-Golgi transport. The function of the 3Dpol protein is well known as RNA dependent RNA polymerase.
Intra-species genome recombination is a well-known phenomenon among HEVs (9,10). Analysis of full-length HPeV1-6 genomes has shown that the genomes between the different types are highly mosaic, showing type-specific clustering on the VP1, where this typespecific segregation is lost in the non-structural (NS) region 3Dpol. In case of HPeV3 this is different, where clustering is shown in both the VP1 and 3Dpol region of the different HPeV3 strains. Between HPeV1, -4, -5 and -6 strains recombination was frequently observed within the NS region, but only one HPeV3 sequence showed a loss of type-specific segregation (11,12), which might suggest a different cell tropism.

Epidemiology of HPeVs
HPeVs are widespread pathogens, infecting mainly young children worldwide (13)(14)(15). The first HPeVs were discovered over 50 years ago during a diarrhoea outbreak in the USA (16), and were described as echovirus 22 and 23 in the Enterovirus genus, based on their HEV like cytophatic effect (CPE) in cell culture and non-pathogenicity in both mice and monkeys. However, based on evident differences in genome organization and biological characteristics they were reclassified into their own genus and renamed as HPeV1 and 2 in 1999 (17)(18)(19). The US National Enterovirus Surveillance System at the Centers for Disease Control reported for the period 1970-2005 that HPeV1 and 2 accounted for a total of 1.8% of enterovirus detections (20) In addition, data extracted from monthly reports from all virological laboratories in Finland (between 1971-1992) showed a HPeV1 prevalence of 8% (21). From the US National Enterovirus Surveillance it was shown that between 1983 and 2005 73% of the HPeV1 and 68% of the HPeV2 infections occurred in children less than 1 year old (20). More extensive studies on HPeV1 revealed similar results; a Finnish followup study showed that at the age of 12 months 20% of the children experienced their first infection, and at the age of 36 months about 98% of the children had at least one HPeV infection (22). In a Norwegian longitudinal study it was observed that at 12 months of age 43% of the infants had experienced at least one HPeV infection, and at age 24 months 86% had encountered the virus (23).
Not until 2004, a third genotype was described, isolated from a stool specimen from a 1-year old child with transient paralysis (24). Since then, an additional 13 types have been described; a fourth type was identified in the Netherlands (25). Further phylogenetic analysis of previously isolated HPeV showed a fifth cluster within the genus. In 2007 a sixth type was isolated from a child with Reye's syndrome (26), and in 2008 several new types were characterized in Pakistan (HPeV7) (27), Brazil (HPeV8) (28), Thailand (Oberste et al. , unpublished) and the Netherlands (HPeV14) (29). Prevalence studies including new PCRbased detection methods and including the newer types HPeV3-6 showed high prevalence of HPeVs, and HPeV3 being the second most prevalent type. In a 3-year study period in the Netherlands a high prevalence of 16% was found by direct PCR screening of feces from hospitalized children under the age of 5 years (29). In feces from healthy children in Finland a prevalence of 6,4% was found (30). In both countries the circulation of HPeV1 is yearly prominent. In the Netherlands and confirmed by a study in the United Kingdom a clear periodicity is found for HPeV3 with seasonal peaks in 2000, 2002, 2004, 2006 and 2008 (29, 31-33), occurring mostly late summer.
Most studies show prevalence of HPeVs based on cell culture and PCR, but seroprevalence data remain rare. The available seroprevalence data do show that HPeVs are very common. For HPeV1 two Finnish studies showed seroprevalences of >97% in adults, and of the 21 neonates tested 95% had aHPeV1 antibodies (Abs), which are most likely maternal (22,34). A Japanese study showed HPeV3 seroprevalence among women of childbearing age to be 68% (24). A second Japanese study showed antiHPeV3 Abs present in a group of 22 adults after infection with HPeV3 (86%), however, only low neutralizing titers were found up to 71 days after onset of illness (35). Seroprevalence data for the other HPeV types are not available. Determining seroprevalence can give a good insight on virus circulation and the existing immunity within different populations, which is important for understanding epidemiology and outbreaks.

Clinical aspects and diagnosis of HPeV infections
The transmission route of HPeVs remains to be elucidated, but as for the closely related HEVs, transmission seems to occur via the fecal-oral and respiratory routes. HPeV are associated with a wide range of clinical symptoms from mild disease such as gastro-enteritis and respiratory tract infections to a variety of severe disease like myocarditis, sepsis-like illness, paralysis, encephalitis and meningitis. For HPeV1 infection diarrhoea is the most common symptom followed by respiratory symptoms (17,34,36). In an evaluation of the clinical picture of children with an HPeV4, 5 or 6 infection the main symptoms were gastrointestinal and respiratory symptoms as well (37). Occasionally HPeV1, 4, 5 and 6 have been associated with severe disease (38)(39)(40). A recent study showed two cases of neonatal sepsis suspected to be caused by HPeV4, which was not shown before for this genotype (41). Although most severe disease cases associated with HPeV infection are caused by the HPeV3 genotype (24,31,(42)(43)(44)(45)(46). A study on the clinical relevance of HPeV1 and HPeV3 detection in stool showed that a HPeV3 positive stool sample is commonly associated with clinical disease, whereas in case of HPeV1 there was only clinical relevance in case of underlying disease (36). Several reports showed that HPeV3 is the predominant type detected in cerebrospinal fluid (CSF), showing the importance of HPeV3 in CNS infections (31,32,(47)(48)(49)(50). In a big screening of CSF samples in the United Kingdom HPeV3 was even identified as the most common picornavirus type (32). A recent study showed that seizures and CNS symptoms were more common among the HPeV infected children compared to those infected with HEV, both virus positive in CSF (51). Additionally, HPeV3-infected children are generally below 6 months of age (median age 1.3 months), which is significantly younger than HPeV1-infected children who are mostly around 6 months (median age 6.6 months) (29,33,52). Although HPeV3 infections have been mostly detected in young infants in Europe and the US, in 2008 a symptomatic HPeV3 outbreak was reported in Japan with 22 cases among adults, suffering from myalgia, muscular weakness, sore throat and orchiodynia (35,53).  (26). For HPeV3 it was shown that initial culturing of 3 clinical specimen showed induction of CPE on the LLCMK2 cell line after 14-18 days, albeit after passaging the virus to Vero cells, CPE appeared after 4-5 days (43). Benschop et al., showed that the HT29 cell line is an efficient cell line to propagate most HPeVs from clinical samples except for HPeV3, which could only be isolated on A549 and Vero cells (54). Nowadays for the detection of picornaviruses in clinical specimens reverse transcriptase PCR (RT-PCR) is shown to be faster and more sensitive than cell culture (54). PCRs targeting the 5'UTR are commonly used to diagnose HPeV infection. Since the 5'UTR is highly conserved all HPeV types can be detected, and this method is highly sensitive (42,56). Typing can subsequently be performed on the clinical sample by sequencing of the VP1 region (56,57).
HPeV infections are now recognized as clinically relevant and highly sensitive and fast methods are implemented for detection. However, antiviral treatment is still not available. Successful vaccines have been developed against poliovirus, hepatitis A virus, the veterinary virus foot and mouth disease virus (FMDV), and recently against EV71 (58,59). The development of antiviral therapy against HEVs and HPEV has not yet been successful. Therefore treatment options are limited and therapy is mainly supportive. One option for treatment is the use of intravenous immunoglobulins (IVIg); one blinded randomized controlled study showed that only neonates receiving IVIg with high specific Ab titers (>1:800) were able to clear the HEV (55). Despite these occasional positive results, treatment with IVIg is non-specific and efficacy has not been proven (60). A promising drug in HEV treatment was the capsid inhibitor pleconaril, showing clinical activity in some patient groups, and a favourable safety profile (reviewed in [60][61][62]. Concerns about possible drug interactions and resistance resulted in rejection by the US FDA, and production was abandoned. HPeV1 and HPeV3 were resistant against this capsid inhibitor (64). Next to capsid inhibitors another promising option for antiviral therapy are compounds targeting the viral factors, like protease or polymerase inhibitors. A drug with wide anti-picornaviral activity would be preferable, and given the high clinical relevance of HPeVs they should be included into the test panels for antiviral compound development.

The role of the RGD motif in infection
Sequence analyses of the HPeV genome revealed that the C-terminus of VP1 contains the arginine-glycine-aspartic acid (RGD) motif. This motif is conserved in HPeV1, 2, 4, 5 and 6, while HPeV3 and the newer identified HPeV7-16 lack this motif (1). The RGD motif has been shown to bind to the αvβ3, αvβ6 integrins as their receptors (65)(66)(67)(68), resulting in virus entry into the host cell by a clathrin-dependent endocytic pathway (67). For HPeV1 it has been shown that viral infection could be blocked by RGD containing peptides or by removal of the RGD motif from the HPeV1 genome (4,65). The lack of this motif in HPeV3 and HPeV7-16 implies different receptor usage and mechanism of entry, possibly related to a different tropism, but these have not yet been determined. The RGD motif is also found in the enteroviruses CAV9 and E9 as well in FMDV (Apthovirus genus), and is shown to play a role in attachment and entry (69)(70)(71). For CAV9 it has been shown that removal of the RGD motif is not fully inhibiting viral infection (72), showing an efficient RGD independent entry process of CAV9 (73). The RGD containing strain E9 strain was shown to be pathogenic for newborn mice, while the RGD lacking strains were not (71,(74)(75)(76). In case of CAV9, mutants without an RGD motif were found to be less pathogenic (77,78). Both studies showed that pathogenicity of these strains was different in older mice. In case of E9 a paralytic response shown in newborn mice remained absent in older mice (74). Therefore it seems that for CAV9 and E9 RGD loss is associated with loss of pathogenicity, in contrast to what is found for HPeV3 being the most pathogenetic HPeV up to now. In HEVs major neutralizing antigenic sites reside on the VP1 capsid protein and to a lesser extent on VP2 and VP3 (79)(80)(81). For the RGD containing HEVs CAV9 and FMDV, the interaction of the antibody with the RGD motif is important for virus neutralization (80,82). This antigenic site has also been shown to be important for HPeV1, by blocking of infection with RGD containing peptides (4,83). Full neutralization was obtained with rabbit antiserum against VP1, but antiserum against VP0 could inhibit infection up to 80% as well (84). Peptide scanning of the viral capsid proteins with immune serum from an HPeV1 immunized rabbit, revealed several immunogenic sites. The rabbit immune serum clearly showed immunogenic reactivity with a site in the N-terminus of VP0 (aa82-99), and two regions in the VP3 capsid protein, one located at the N-terminus of VP3 (aa15-35) and the other at the C-terminal region (aa183-195) (83). Antisera raised against the RGD containing peptide was able to neutralize 51% of HPeV1 infection, and the antisera raised against the VP0 peptides neutralized 43% of HPeV1 infection (83), indicating that HPeV1 contains several antigenic sites. These VP0 and VP3 antigenic sites for HPeV1 have never further been characterized, nor are the antigenic sites of the other HPeV genotypes.

Humoral immunity and antibody protection
The immune response against picornaviruses is mainly humoral, and is supposed to be type specific. The humoral immune response involves antibodies (Abs) produced by B cells, that prevent further spread of the infection by 1) coating the virus and preventing it from entering the cells, 2) by coating the surface to enhance phagocytosis, or 3) by complement activation. The role of the humoral immune system in defence against picornavirus infections is illustrated by the vulnerability of newborns and patients with humoral immune deficiencies for severe HEV infections, while T cell deficient patients do not seem to experience problems with these infections (64). During pregnancy IgGs are transported over the placenta, resulting in high Ab levels protecting the child until the newborn can produce its own Abs. This Ab protection will last up to a several months. Failure in Ab protection due to the absence of specific Abs may lead to severe disease and even to death. In neonates with severe HEV infection, the maternal Ab titers against the specific HEV serotype were shown to be absent or low (85,86).
Patients with a deficient humoral immune response, such as X-linked agammaglobulinemia, are at great risk of chronic enteroviral infections (87). In immunodeficient patients prolonged poliovirus replication was shown (88). In patients with agammaglobulinemia, coxsackievirus (CV) infection could spread to and stay persistent in the central nervous system (89)(90)(91). For CV it has been shown that the humoral immune response plays a prominent role in limiting virus spread and in viral clearance (92); CBV3 infection in B cell deficient mice results in chronic infections with high viral titers (93). The role of the humoral immune response seems to be clear, but the role of the T cell response in CV infections is not clear, showing discordant data with different strains and in mice (92,(94)(95)(96). As antiviral drugs against HEV are currently lacking, IVIg is often used as a replacement therapy in patients with a primary humoral immune deficiency, preventing chronic enteroviral infections (87,97,98). IVIg administration remains an unspecific treatment against HEV infections, and it cannot protect against lowly circulating or newly introduced HEV or HPeV strains against which there is no immunity in the population. A more targeted option for antiviral treatment of HEV and HPeVs would therefore be the use of specific monoclonal Abs. New approaches for generating human monoclonal Abs have been successful against influenza virus (99) and respiratory syncytial virus (100). For HEVs neutralizing Abs are supposed to be type specific, which makes cross-neutralization probably rare among over a 100 serotypes, and the development of monoclonal Abs against so many types is unattractive. In contrast, the HPeV group is small, depicting high similarity among the different genotypes, whereas several antigenic epitopes are described and cross neutralization is observed (83,84). This would make HPeVs an ideal target for development of monoclonal Ab (mAb) treatment.
To achieve this, localization of antibody binding sites is important for obtaining information concerning the mechanism of neutralization and receptor binding. For HRV2, HRV14 and FMDV virus Ab neutralization is extensively studied using cryo-electron microscopy (cryo-EM) (101)(102)(103)(104)(105)(106)(107). For Ab neutralization of FMDV the VP1 C-terminal loop is important, where direct interaction is shown between the RGD motif and several complementary regions of the Ab molecule (82). Structural 3D reconstructions of HPeV1 by Cryo-EM revealed a typical pseudo T=3 organization seen in picornaviruses, and confirmed the binding of αvβ3, αvβ6 integrins to the RGD loop of HPeV1 VP1 (68). Ab neutralization of HPeV1 has never been studied using these techniques. For HPeV3 the 3D virus structure has not yet been revealed. Structural studies are important to gain more insight in the differences between HPeV1 and HPeV3, leading to better understanding of receptor usage and Ab binding, and thus of the pathogenic difference between type 3 and other HPeVs.

Thesis outline
HPeV3 has been shown to be the 'odd one out' compared to the other genotypes; HPeV3 is more often associated with severe disease, infects significantly younger infants, seems to be more difficult to culture, and does not recombine with other HPeV types. The aim of this thesis is to study whether these differences could be explained by differences in viral tropism and Ab protection between the subsequent HPeV types.
The first part of the thesis focuses on differences in viral tropism among HPeV types. In Chapter 2, we describe the growth characteristics of the different genotypes on different cell lines. To more extensively look into the differences between HPeV3 an HPeV1 related to the clinical outcome of infection, we describe the specific cell tropism and neutralization of human parechovirus types 1 and 3 in Chapter 3. To study the importance of the human airway as a primary replication site of HPeVs and differences in virus tropism, we determined replication kinetics of different HPeVs types in a human respiratory primary cell culture system (HAE) (Chapter 4).
The second part of the manuscript focuses on neutralization of HPeVs. Chapter 5 describes the seroprevalence of neutralizing (protective) Abs among different groups in Finland and the Netherlands. The (cross-) neutralization of HPeVs by different HPeV1 and HPeV3 polyclonal and monoclonal Abs is extensively described in Chapter 6 and 8. Using cryo-EM, we determined the different neutralizing epitopes for HPeV1 (Chapter 7) and we revealed a high-resolution structure of HPeV3 (Chapter 9).