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Clinical and molecular insights into human parechovirus infection

Benschop, K.S.M.

Publication date2009Document VersionFinal published version

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Citation for published version (APA):Benschop, K. S. M. (2009). Clinical and molecular insights into human parechovirus infection.

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Clinical and Molecular Insights into Human

Parechovirus Infection

Clinical and Molecular Insights into Human Parechovirus Infection

© Kimberley Benschop, 2009. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior permission of the author. Published papers and figures were reprinted with permission from the publishers. The printing of this thesis was financially supported by the University of Amsterdam.

Cover: Veronique A. Veldhuis playing with her blocks Printed by: Proefschriftenmaken.nl ISBN: 978-90-8-891-112-5

Clinical and Molecular Insights into Human

Parechovirus Infection

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie,

in het openbaar te verdedigen in de agnietenkapel op donderdag 8 oktober 2009, te 14:00 uur

door

Kimberley Samantha Meriaha Benschop

geboren te Paramaribo, Suriname

Promotiecommissie Promotores: Prof. dr. M.D. de Jong Prof. dr. C.M.J.E. Vandenbroucke-Grauls Co-promotor: Dr. K.C. Wolthers Overige Leden Prof. dr. J. Schuitemaker Prof. dr. T.W. Kuijpers Dr. C.M. van der Hoek Prof. dr. J.M.D. Galama Prof. dr. M.P.G. Koopmans Prof. dr. P. Simmonds Faculteit der Geneeskunde

The research described in this thesis was performed at the Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Table of contents

Chapter 1 General introduction 7

Part I Clinical relevance

Chapter 2 Human parechovirus infections in Dutch children 33

and the association between serotype and disease

severity.

Clin.Infect.Dis. 2006. 42:204-210.

Chapter 3 Rapid detection of Human Parechoviruses in clinical 49

samples by real time PCR.

J. Clin. Virol. 2007. 41 (2): 69-74.

Chapter 4 Human parechoviruses as an important viral cause of 65

sepsislike illness and meningitis in young children.

Clin Infect Dis. 2008. 47(3):358-63.

Chapter 5 High prevalence of Human Parechovirus genotypes in 79

the amsterdam region and the identification of specific

HPeV variants by direct genotyping of stool samples.

J. Clin Microbiol. 2008. 46 (12) 3965-70.

Chapter 6 Clinical characteristics of human parechoviruses 97

infections 4-6 in young children.

Ped.Infect.Dis.J. Accepted for publication.

Chapter 7 Detection of Enterovirus and Human Parechovirus 109

genotypes from clinical stool samples: PCR and

direct molecular typing, culture characteristics and

serotyping.

Manuscript in preparation.

Part II. Phylogeny and evolution

Chapter 8 Fourth Human Parechovirus Serotype. 131

Emerg.Infect.Dis. 2006.12:1572-1575.

Chapter 9 Widespread recombination within human parechoviruses; 141

analysis of temporal dynamics and constraints.

J.Gen.Virol. 2008. 89: 1030-35

Chapter 10 Comprehensive full length sequence analyses of human 155

parechoviruses; diversity and recombination.

Submitted for publication.

Summary and discussion

Chapter 11 Summary 177

Chapter 12 Discussion 183

Appendices

Samenvatting voor niet ingewijden 197

List of publications 205

Curriculum Vitae 209

Dankwoord/Acknowledgements 213

General introduction

Adapted from Emerging Infections 8, 2008

Chapter 4: New Human Parechoviruses: six and counting

Kimberley S.M. Benschop1, Glyn Stanway

2,

and Katja C. Wolthers1

1 Lab. of Clinical Virology, Dept. of Medical Microbiology, Academic Medical

Center, Amsterdam. 2 Dept. of Biological Sciences, University of Essex, Colchester, UK.

9


Human parechoviruses (HPeVs) have been recognized since 1992 as a

separate group in the family Picornaviridae on the basis of distinct

molecular and biological properties. They have predominantly been

associated with mild gastrointestinal and respiratory symptoms.

Although more severe symptoms have been associated with HPeV1

infections, the relevance of this small group was not significantly

recognized. It was not until the identification of HPeV3 and its

association with severe disease, that HPeV infections were considered

relevant pathogens in young children. However, epidemiological and

clinical data remained limited.

PICORNAVIRUSES

Picornaviruses are small non-enveloped viruses containing a single-stranded

RNA with positive polarity. The Picornaviridae family is one of the largest

RNA virus families and contains an array of pathogens that infect both

humans and animals. At present the family is divided into 8 genera, but

current proposals made by the International Committee on Taxonomy of

Viruses (ICTV) would increase this to 11: Enterovirus, Parechovirus,

Hepatovirus, Kobuvirus, Aphthovirus, Erbovirus, Teschovirus, Cardiovirus,

Tremovirus, Sapelovirus and Senecavirus (Fig. 1) of which the Enterovirus,

Hepatovirus, Kobuvirus and Parechovirus genera include several important

human pathogens.

The Enterovirus genus contains over 100 types, those identified earliest

being subdivided into polioviruses (PV; 3 types), Coxsackie A virus (CAV; 23

types) and Coxsackie B virus (CBV; 6 types), and echoviruses (28 types),

based on their ability to replicate in human or primate cells, their infectivity

and pathogenicity in different animal species and their antigenic differences

(82). New types were later identified and numerically classified as

enterovirus 68-102 (58,71-73). Advances in molecular techniques and the

accumulation of sequence data allowed for a more precise classification

based on molecular rather than phenotypic characteristics and four distinct

human enterovirus (HEV) clusters were identified (30,75) later forming the

basis for defining the species HEV-A, HEV-B, HEV-C (containing PV) and

HEV-D (97). Enteroviruses isolated from animals were classified in four

additional clusters. Similar studies showed rhinoviruses to be close relatives

of the enteroviruses, and rhinoviruses were reclassified by the ICTV as 3

new species within the Enterovirus genus; Human Rhinovirus A to C.

Chapter 1

10

Figure 1. Unrooted phylogenetic tree, showing the relationship between human parechoviruses

and other Picornaviridae genera. The proposed genera Sapelovirus, Senecavirus and

Tremovirus are given in italics. The Rhinovirus genus, proposed to be merged with Enterovirus,

is encircled in a hatched line. The tree was constructed based on amino acid differences, using

the neighbour joining method. The following nucleotide sequences were obtained from

GenBank according to recent proposals by the ICTV. Parechovirus: (HPeV1 (S45208); HPeV1

BNI-788St (EF051629); HPeV2 (AJ005695); HPeV3 A308-99 (AB084913) and Can82853-01

(AJ889918); HPeV4 K251176-02 (DQ315670) and T75-4077 (AM235750); HPeV5 CT86-6760

(AF055846) and T92-15 (AM235749); HPeV6 NII561-2000 (AB252582) and BNI-67/03

(EU024629); Ljungan virus 174F (AF327921), 87-012 (AF327920) and 145SL (AF327922);

Enterovirus: PV1 (V01149); PV2 (M12197); PV3 (K01392); CAV10 (AY421767); CAV16

(U05876); EV71 (U22521); CBV3 (M16572); CBV6 (AF114384); echovirus 30 (AF311938);

echovirus 11 (AJ577589); CAV9 (D00627); CAV20 (AF465514); CAV24 (D90457); EV68

(AY426531); EV70 (D00820); Simian EV (SEV, NC003988); Rhinovirus 1B (HRV-1B, D00239);

HRV 14 (K02121); Aphthovirus: Foot and mouth disease virus-A (FMDV-A, NC011450);

FMDV-O (AY686687); FMDV-SAT1 (NC011451); equine rhinitis A virus (ERAV, DQ272577);

Cardiovirus: Encephalomyocarditis virus (EMCV, X87335); Theilovirus (TMEV, NC001366);

Hepatovirus: Hepatitis A virus (HAV, AJ299464); Teschovirus: porcine teschovirus (PTV,

NC003985); Erbovirus: equine rhinitis B virus (ERBV, AF3612153); Kobuvirus: Aichivirus

(AIV, AB010145); Bovine kobuvirus (BKV, AB084788); Sapelovirus: Porcine EV (new

proposed name: Avian sapelovirus, PEV/ASV, AF406813) Senecavirus: Seneca Valley virus

(SVV, DQ641257); Tremovirus: avain encephalomyelitis-like virus (AEV, AY275539).


11

Enteroviruses and rhinoviruses are common human pathogens (21,54) and

are responsible for a wide variety of diseases and clinical manifestations.

Rhinoviruses are predominantly associated with the common cold, whereas

enteroviruses have been associated with meningitis, myocarditis, and

poliomyelitis. Poliomyelitis, caused by PV, is expected to be eradicated over

the next few years due to efficient vaccination programs introduced by the

World Health Organization (WHO) in 1988. However, no vaccines are

available for other enteroviruses, and these still constitute a significant

clinical problem. Although enteroviruses are transmitted via the fecal-oral

route, gastrointestinal and respiratory symptoms are reported less frequently

than the more severe symptoms (21).

HPeVs were previously classified as members of the Enterovirus genus.

Together with Ljungan virus isolated from rodents, HPeVs form a separate

genus, Parechovirus, within the family Picornaviridae (97). Ljungan virus was

identified in 1999, during a search for an infectious agent that could be

linked to myocarditis in humans. The virus was isolated from bank voles

(Clethrionomys glareolus) and was most closely related to HPeVs (63).

Molecular techniques are now frequently used to identify and type different

human picornaviruses from clinical samples. Typing of enteroviruses and

parechoviruses is of great importance to elucidate the clinical and

epidemiological characteristics of these viruses. With respect to the WHO

poliovirus eradication campaign, it is essential to differentiate between PV

and non-PV enteroviruses to ensure that wild type PVs or revertant PV

vaccine strains responsible for vaccine derived poliomyelitis are not

circulating in populations where PV has been successfully eradicated.

Moreover, the use of molecular techniques allows the identification of new

types or variants, in contrast to traditional typing methods such as

serotyping, where the antisera used are only directed against known types.

To maintain consistency with the traditional typing of known HEVs, as well

as HPeVs, molecular methods have been directed against the capsid region,

in particular, the VP1 region (31,67,68,99).

The identification of new HEV and HPeV types has dramatically increased

since the turn of the century. Molecular data are rapidly being generated and

submitted to data banks, allowing for more precise classifications and

reclassification of different viruses within new genera. This will increase our

understanding of the virus diversity in relation to pathogenesis and evolution.

Chapter 1

12

GENOME

HPeVs have a single-stranded, positive-sense RNA genome of 7300

nucleotides which has a typical picornavirus organization (Fig. 2) (95,97). A

5’untranslated region (UTR) of around 700 nucleotides precedes an open

reading frame of 2200 codons. This is followed by a 3’UTR (80 nucleotides)

and a poly(A) tail. As in other picornaviruses, the open reading frame

encodes structural proteins at its 5’end and nonstructural proteins

downstream. Picornavirus polyproteins are cleaved by virus-encoded

proteases to give precursors and the final proteins. In the case of HPeV, it

seems likely that only one protease, 3Cpro, is involved in processing. In

addition to 3Cpro, the functions of the picornavirus 3Dpol protein (the RNA-

dependent RNA polymerase) and 3B protein (VPg, a protein primer of RNA

replication) are well-documented. 2C is relatively well-conserved in

picornaviruses and appears to function in RNA replication and possibly

capsid assembly (49), but its modes of action are not fully understood. In

HPeV, 2C appears to have ATP hydrolysis and AMP kinase activities (86),

which may be involved in RNA replication. The proteins 2B and 3A are both

Figure 2. The genome organisation of a typical picornavirus, together with differences in the polyprotein between different genera. These are mainly limited to the L protein and 2A protein. Different shading of these proteins indicates distinct structural classes in the genera. The genera shown are those which will exist if current taxonomic proposals are accepted by ICTV.


13

small proteins containing hydrophobic regions. They appear to interact with

membranes and mediate cellular changes necessary for virus replication and

release (45).

In addition to the proteins present in all picornaviruses, there are two loci

which are highly diverse between different picornaviruses. These are the L

and 2A regions (Fig. 2). The L region encodes a leader protein and occurs in

only around half of picornavirus genera. Parechoviruses lack an L protein.

Four different types of the 2A protein have been identified (Fig. 2). That in

the Enterovirus (and probably Sapelovirus) genus, is a chymotrypsin-like

protease involved in cleaving the polyprotein at its own N-terminus and also

in host-cell protein synthesis shutoff (82). Although diverse in sequence, the

2A proteins of the Parechovirus, Kobuvirus and Tremovirus genera are

related and share conserved motifs with a group of cellular proteins involved

in the control of cell growth (26). The significance of this observation has not

been established, but one function reported for the HPeV protein is RNA

binding (85).

Parechoviruses, together with kobuviruses, have another major difference

from other picornaviruses in that the structural protein VP0, usually a

precursor of VP4 and VP2, is not cleaved, and so there are only three

structural proteins rather than the four typically seen. As this VP0 maturation

cleavage is thought to be critical for capsid stability and the acquisition of

infectivity, this raises questions about these parameters in parechoviruses.

Possibly other structural changes are involved in parechovirus maturation,

but these remain obscure. Another distinct feature of kobuviruses and

parechoviruses is the absence of VP0/VP4 myristoylation, a modification

which is seen in most other picornaviruses (94).

HISTORIC OVERVIEW OF ECHOVIRUSES 22 AND 23

HPeVs were first isolated in 1956 by Wigand and Sabin (102) from children

with diarrhea and were classified in the genus Enterovirus as echovirus 22

and echovirus 23. In this first report it was noted that these viruses were also

identified in two patients with aseptic meningitis and three patients with

febrile respiratory disease. When first isolated, they exhibited distinct growth

characteristics from other enteroviruses, such as difficulty in adapting to

monkey kidney cells. However, the cytopathogenic effect (CPE) seen in

monkey kidney cells was generally similar to that seen with enteroviruses.

Early reports identified echovirus 22 by cell culture or increase in neutralizing

antibodies in children with gastrointestinal symptoms or respiratory infections

(6,8,59). These associations were confirmed by WHO data from 1967 to

Chapter 1

14

1974 showing that in patients with echovirus 22 infections, gastrointestinal

symptoms and respiratory infections occurred at about the same frequencies

(29% and 26%, respectively) while for HEVs these frequencies were much

lower (9% and 15%, respectively). Central nervous system (CNS) symptoms

were also reported but occurred less often, in 12% of the echovirus 22

infections, compared to 46% in other echovirus infections (21,39,96). Severe

conditions associated with echovirus 22 infections, such as encephalitis,

paralysis, and, myocarditis have been described occasionally (18,44,55,84).

One report suggested an association with hemolytic uremic syndrome on the

basis of 10 patients (74), and a publication from 1997 describing an outbreak

of echovirus 22 infection in 19 neonatal intensive care unit patients

suggested that for gastrointestinal disease with features of necrotizing

entrocolitis, echovirus 22 infection should be considered (9).

A large Swedish study from 1993 retrospectively identified during a 25-year

observation period (1966 to 1990) 109 patients with echovirus 22 infection.

Clinical data were collected from 57 patients. Again, diarrhea was found

most frequently, followed by respiratory infections. In 9% of the patients,

encephalitis was clinically suspected, and one case of myocarditis was found

(15). During the same study period, only five patients were identified with

echovirus 23 infection, showing mild signs of gastroenteritis and/or

respiratory infection (16). Until then, only one report had described echovirus

23 spread in a neonatal unit (5). Ehrnst et al. were the first to describe the

specific epidemiological features of echovirus 22 and 23 infections (15,16).

From these studies it was concluded that infections with echovirus 22

behaved differently from other HEV infections. Indeed, sequence analysis of

the full-length genomes showed echovirus 22 and 23 to be distinct from

other members within the Enterovirus genus (20,29), and further studies

emphasized their characteristic molecular and biological properties

(39,95,96). They were also genetically distinct from other picornavirus

genera and consequently were renamed in 2000 human parechovirus 1 and

2 and classified as members of the human parechovirus species within a

new genus Parechovirus (43).

THE EXPANDING HUMAN PARECHOVIRUS SPECIES

The establishment of a new genus within the picornavirus family was the

focus of two key reviews describing the biology and clinical relevance of the

new group of HPeVs, which contained two members named HPeV1 and -2

(39,96). From data available at that time, it was concluded that HPeV1

occurred frequently, predominantly in children with mild respiratory and


15

gastrointestinal symptoms. Occasionally, HPeV1 infection could give rise to

severe symptoms such as myocarditis, encephalitis, pneumonia, meningitis,

and flaccid paralysis. HPeV2 infections, however, appeared to be rare

(39,96).

At the same time, molecular techniques were rapidly becoming state-of-the-

art methodologies in many laboratories. The complete sequence of HPeV2

was published in 1998 by two independent groups (20,65). However, the

genome of HPeV2 type CT86-6760 (65) appeared to be different from that of

HPeV2 type Williamson, previously known as echovirus 23 (20).

In 2004, a third HPeV type was isolated in Japan by cell culture from a stool

specimen of a 1-year-old child with transient paralysis. This new strain,

designated A308/99, could not be neutralized with known antisera against

human picornaviruses (including antisera against HPeV1 and HPeV2).

Shortly after the identification of HPeV3, a fourth HPeV type was identified in

both The Netherlands (chapter 8) and the United Kingdom (3). In addition,

phylogenetic analysis of all known HPeV types, including the second HPeV

type 2 (CT86-6760) showed the CT86-6760 strain to be genetically distinct

from the prototype HPeV2 strain, forming a fifth HPeV cluster along with 4

additional strains isolated in California between 1973 and 1992 (89). In

2007, a sixth HPeV type was identified following isolation from a child with

Reye’s syndrome (101), and in 2008, 8 new HPeV types were genotypically

characterized in Pakistan (HPeV7) (50), Brazil (HPeV8) (14), Thailand

(Oberste et al, unpublished) and The Netherlands (HPeV14) (Chapter 5),

thereby bringing the number of HPeV types to fourteen (Table 1).

Table 1 Prototype HPeV strains.

Type Strain Origin References

HPeV1 Harris/echovirus 22 Ohio, USA (29,102)

HPeV2 Williamson/echovirus 23 Ohio, USA (20,102)

HPeV3 A308/99 Aichi, Japan (31)

HPeV4 K251176-02 Amsterdam, The Netherlands (chapter 8)

HPeV5 CT86-6760 Conneticut, USA (65)

T92-15 California, USA (3)

HPeV6 NII561-2000 Niigata, Japan (101)

HPeV7 Pak5045 Pakistan (50)

HPeV8 Br/217/2006 Salvador de Bahia, Brazil (13)

HPeV9 BAN2004-10902 Bangkok, Thailand unpublished





HPeV14 451564 Amsterdam, The Netherlands (chapter 5)

Chapter 1

16

EPIDEMIOLOGY OF HPeV INFECTIONS

HPeV1 is a widespread pathogen that occurs globally, mainly infecting

young children (38,39,96). Data reported to the U.S. National Enterovirus

Surveillance System at the Centers for Disease Control and Prevention

during 1983 to 2003 showed that HPeV1 accounted for 0.8% of the detected

non-PV HEV and HPeVs in neonates and for 2.3% in the older age group.

HPeV2 was not reported (42). In comparison, echovirus 30, one of the most

common HEV serotypes, was detected in 6 to 12% of the HEVs isolated

from neonates and older age groups. In a French surveillance over 2000 to

2004, HPeV1 was found in 0.6% of 2.757 patients reported with HEV or

HPeVs (4). In contrast, a surveillance of HEV and HPeV during 1971 to 1992

in Finland reported that HPeV1 was one of the six most common HEVs and

HPeVs and was isolated in 8% of the patients (24).

The idea that HPeV infections are frequent and widespread is illustrated by

the high seroprevalences found in different parts of the world

(1,38,59,88,96). In neonates, 95% had antibodies against HPeV1, evidently

of maternal origin. At around 6 months of age, about 50% had antibodies,

increasing rapidly to >90% in children >1 year of age (59,96). Among adults,

seroprevalence for HPeV1 was 97%, while antibodies against echovirus 30

were found in 30% of the Finnish adults (38). In 219 pregnant mothers from

children followed in a Finnish cohort study on type 1 diabetes, HPeV1

seroprevalence was 99% (98). In this prospective birth cohort study, HPeV1

antibody prevalence was 20% at 12 months and 72% at 24 months, which

was lower than in the previous cross-sectional studies. At 36 months, almost

all children (98%) in this study had antibodies against HPeV1. Therefore,

from all studies it can be concluded that most individuals become infected

with HPeV1 before adolescence. The seroprevelance for HPeV3 was found

to be much lower, where HPeV3 seroprevalence had increased to 87%,

above the age of 40 (31). Unfortunately, seroprevalence data on the newer

HPeV types are lacking.

LABORATORY DIAGNOSIS OF HPeV INFECTION

The classical method for diagnosis of infection with HEVs or HPeVs has

been virus isolation in cell culture from different clinical samples such as

stool, throat swabs, cerebral spinal fluid (CSF), and blood. The standard cell

culture for isolation of HEVs and HPeVs involves at least three cell lines,

usually including monkey kidney cells and human fibroblasts. When a CPE is

observed, the isolated virus can be identified by neutralization with a panel


17

of specific antibodies (including antisera against HPeV1 and -2). CPE

produced by HPeV is not that different from that produced by HEV and

HPeVs may therefore easily be identified as HEVs when specific serotyping

is not routinely performed.

For detection of picornaviruses in stool samples and throat swabs,

conventional cell culture is still widely used. However, reverse transcriptase

PCR (RT-PCR) to detect HEV in CSF has shown to be faster and more

sensitive than cell culture (80,81,100). Therefore, PCR is the preferred

method for detection of viruses in CSF (17,77). RT-PCRs that detect HEV

target the 5’UTR, which is highly conserved and therefore suitable to detect

all HEV serotypes (7,68). Since the nucleotide sequences of the HPeVs are

quite divergent from the HEVs, pan-EV RT-PCR fails to detect HPeVs

(7,27,28,66). HPeV infections of the CNS will therefore be missed if only an

HEV-specific RT-PCR is performed. Several conventional end point RT-PCR

assays have been developed for detection of HPeV1 and -2 (48,66,77,90).

Nowadays, in most diagnostic laboratories real-time PCR has become state-

of-the-art. This testing method combines amplification by PCR with

fluorescent probe detection of amplified product in a closed tube format,

therefore eliminating the need of post-PCR analysis and decreasing

contamination risk.

Detection of HEV and HPeV by real-time PCR is faster and less laborious

than conventional cell culture or endpoint PCRs. For HEV it has been shown

extensively that PCR is more sensitive than cell culture (17,81), not only for

CSF but also for other clinical samples (2,91,100). For HPeVs, comparative

studies have not been published yet. In a 10-month-old boy with

encephalomyelitis, HPeV1 could be detected in CSF by PCR but not by cell

culture (47), indicating that PCR is more sensitive.

RECEPTOR USAGE AND REPLICATION

The first HPeV sequence revealed the presence of an arginine-glycine-

aspartic acid (RGD) motif close to the C-terminus of VP1 (Fig. 3). As this

motif is found in a number of cellular and viral proteins which recognise

integrin molecules, it suggests a role for the motif in the initial interaction with

these cell surface receptors. All subsequent evidence confirms this initial

supposition (11,40,94). The RGD motif, although in a relatively variable

context, is itself absolutely conserved in HPeV1, -2, -4, -5, and -6 (Fig. 3).

This motif has also been identified in two enteroviruses, CAV9 and echovirus

9 and also in FMDV, a member of the Aphthovirus genus (12,19,104). There

is some conservation of flanking residues of the motif seen in these

Chapter 1

18

picornaviruses and in HPeV, but while mutation and deletion studies show

this region to be nonessential for their replication (23, 25, 83,105) deletion

of the motif is lethal to HPeV1 (17).

Several papers have indicated that HPeV1 is recognised by integrins

including αvβ1 and αvβ3, which recognize the RGD motif, and this

interaction is followed by internalisation via endosomes (40,76,77,94). The

motif was found to be essential for HPeV1 replication through mutation and

deletion studies (17). Interestingly, the motif was found to be absent in

HPeV3 to 14 (Fig. 3) and they are thought to enter the cell via an RGD-

independent pathway.

Following release of the virus RNA into the cell cytoplasm, it is translated to

give the polyprotein containing all the virus proteins. Picornaviruses use a

cap-independent mechanism for initiation of translation, driven by an internal

ribosome entry site (IRES) in the 5’UTR (Fig. 4) (60). Potentially this allows

the majority of host cellular mRNA translation to be shut off, as this proceeds

by a cap-dependent mechanism, although there is little evidence that HPeVs

bring about shutoff (94). Following translation and processing of the

polyprotein, the RNA genome is replicated through interactions within

specific domains within the 5’UTR (60,61) and 3’UTR. The role of the latter

region in picornaviruses has not been fully elucidated and has not been

studied in HPeVs.

Figure 3. Alignment of the region flanking the RGD sequence (shaded grey) in the

picornaviruses which have this motif. One representative sequence is shown for each

virus serotype. Sites showing characteristic patterns of conservation and located

downstream of the RGD motif are shown in bold. The integrins reported to be

recognised by at least one virus in each species are also indicated: HPeV (40), CAV9

(78,103), echovirus 9 (62) and FMDV (32-36).


19

Another region of critical importance in RNA replication is the cis-acting

replication element (CRE), the site of uridylation of VPg to give the primer

required for RNA synthesis which is predicted to be in the VP0 gene in

HPeVs (3).

EVOLUTION

HPeVs exhibit several unique molecular features but also attain features

commonly found in other picornaviruses, making these viruses interesting in

terms of evolutionary studies on the group itself and on their place in the

picornavirus family.

RNA viruses are known to evolve rapidly within a population due to the

genetic flexibility of the genome. Mutations, recombination, and segment

reassortment all contribute to the genetic variation and evolution of RNA

viruses and can result in a changed spread and pathogenicity within a

population (41,57). The different genomic regions of picornaviruses each

have different functions, which are reflected by their evolution. The protein

capsid is under constant immune pressure, and in order to evade immunity

the virus has to constantly change its appearance. Due to the gradual

accumulation of mutations, the capsid region is known to be the most

diverse region within the genome (92,93). The nonstructural region is driven

by functional pressure, due to the functional requirements of the proteins

Figure 4. Schematic diagram of the HPeV 5’UTR showing the key IRES domains

(continuous oval) and RNA replication determinants (dotted oval).

Chapter 1

20

encoded within this region. While phylogenetic analysis of the capsid region

can distinguish the HEV types according to their classification, phylogenetic

analysis of the nonstructural region of HEV shows inconsistent clustering of

types. This was attributed to recombination between nonstructural regions of

different types, rather than convergent evolution of the nonstructural region

(92). Lukashev et al. recently proposed that HEV species can exist as a pool

of a finite array of capsid genes and an infinite number of nonstructural

genes which can freely evolve and recombine independently from another

(52,53).

Recombination has been extensively studied in PVs (10,46,56,79), in

particular, in vaccine-derived PV (13,22,37) and non-PV enteroviruses

(51,53,64,69,70,87,92,93) and can have a profound impact on clinical

outcome. As the HPeV group is a very small group containing only 14

members, studies on the evolution of the HPeV genome have been limited.

With the identification of new HPeV types, recombination was suggested to

play a role in the evolution of HPeVs (3,50,92,106, and chapter 8).

Moreover, Simmonds et al. showed HPeVs to exhibit similar characteristics

as other frequently recombining viruses (93).

THESIS OUTLINE

This aim of this thesis is to study the clinical and molecular characteristics of

the HPeV group.

Part 1 points out the clinical relevance of HPeV infection in infants and the

need for more specific diagnostics of both HPeV and enteroviruses. In

chapter 2 the clinical symptoms associated with HPeV1 and HPeV3

infections are studied and it becomes apparent that HPeV infections are

relevant pathogens in young children. A specific HPeV real time PCR to

rapidly detect HPeV infection is described in chapter 3. This assay is used

to retrospectively screen for HPeV in CSF and stool samples over a 3 year

period (2004-2006) to establish the prevalence of HPeV infection among

children (chapters 4 and 5). To determine the prevalence of the different

HPeV types circulating, a method is developed to directly genotype HPeV

from clinical material (chapter 5 and 7). In chapter 7 the detection of HPeV

and HEV by PCR and genotyping is further evaluated in relation to culture

characteristics and serotyping.

The clinical manifestations of the newer HPeV types, HPeV4 to -6 are

explained in chapter 6.


21

Part 2 describes the identification of new HPeV types and the likelihood of

recombination among HPeV strains. Within this thesis, 2 new types are

described (HPeV4 (chapter 8) and HPeV14 (chapter 5)). To study

recombination among HPeV, two distant regions (VP1, used for typing, and

3Dpol, the polymerase nonstructural protein) are analysed to determine

whether type-specific segregation observed within the capsid, is lost or

carries over within the 3Dpol gene. The occurrence of recombination among

HPeV is compared to that of HEV (chapter 9). These studies are extended

with the generation and analysis of additional full length sequences of the

predominantly circulating strains HPeV1 and -3 (chapter 10) to study

diversity, dynamics and recombination breakpoints within the HPeV group.

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A. C. Palmenberg, and T. Skern. 2005. Picornaviridae, p. 757-778.

In C.M.Fauquet, M.A.Mayo, J.Maniloff, U.Desselberger, and L.A.Ball

(ed.), Virus Taxonomy. Classification and Nomenclature of Viruses,

Eight Report of the ICTV. Elsevier/Academic Press, London.

98. Tauriainen, S., M. Martiskainen, S. Oikarinen, M. Lonnrot, H.

Viskari, J. Ilonen, O. Simell, M. Knip, and H. Hyoty. 2007. Human

parechovirus 1 infections in young children--no association with type

1 diabetes. J. Med. Virol. 79:457-462.

99. Thoelen, I., E. Moes, P. Lemey, S. Mostmans, E. Wollants, A. M.

Lindberg, A. M. Vandamme, and M van Ranst. 2004. Analysis of

the serotype and genotype correlation of VP1 and the 5' noncoding

region in an epidemiological survey of the human enterovirus B

species. J. Clin. Microbiol. 42:963-971.

100. Van Doornum, G. J., M. Schutten, J. Voermans, G. J.

Guldemeester, and H. G. Niesters. 2007. Development and

implementation of real-time nucleic acid amplification for the

detection of enterovirus infections in comparison to rapid culture of

various clinical specimens. J. Med. Virol. 79:1868-1876.

101. Watanabe, K., M. Oie, M. Higuchi, M. Nishikawa, and M. Fujii.

2007. Isolation and characterization of novel human parechovirus

from clinical samples. Emerg. Infect. Dis. 13:889-895.

102. Wigand, R. and A. B. Sabin. 1961. Properties of ECHO types 22,

23 and 24 viruses. Arch. Gesamte Virusforsch. 11:224-247.

103. Williams, C. H., T. Kajander, T. Hyypia, T. Jackson, D. Sheppard,

and G. Stanway. 2004. Integrin alpha v beta 6 is an RGD-

dependent receptor for coxsackievirus A9. J. Virol. 78:6967-6973.

104. Zimmermann, H., H. J. Eggers, and B. Nelsen-Salz. 1996.

Molecular cloning and sequence determination of the complete

genome of the virulent echovirus 9 strain barty. Virus Genes.

12:149-154.

105. Zimmermann, H., H. J. Eggers, and B. Nelsen-Salz. 1997. Cell

attachment and mouse virulence of echovirus 9 correlate with an

RGD motif in the capsid protein VP1. Virology. 233:149-156.

106. Zoll J., J. M. Galama, and F. J. van Kuppeveld. Identification of

potential recombination breakpoints in human parechoviruses. 2009.

J. Virol 83(7):3379-83.

P A R T 1

Clinical relevance

Human Parechovirus Infections in

Dutch Children and the Association

between Serotype and Disease Severity

K. S. M. Benschop1, J. Schinkel1, R. P. Minnaar1, D. Pajkrt3,

L. Spanjerberg5, H. C. Kraakman4, B. Berkhout2,

H. L. Zaaijer1, M. G. H. M. Beld1, and K. C. Wolthers1,2

Clinical Infectious Diseases 2006; 42:204-210


Center, Amsterdam. 2 Lab. of Human Retrovirology, Dept. of Medical Microbiology, Academic

Medical Center, Amsterdam. 3 Dept. of Pediatrics, Academic Medical Center, Amsterdam.

4 Dept. of Pediatrics, Onze Lieve Vrouwe Gasthuis, Amsterdam.

5 Dept. of Pediatrics, Ziekenhuis Amstelland, Amstelveen.

35

Human Parechovirus Infections in Dutch Children and the

Association between Serotype and Disease Severity

Background. Human parechoviruses (HPeVs) are members of the

family Picornaviridae and are classified into 3 known serotypes: HPeV1,

HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections

are most commonly associated with mild respiratory or gastrointestinal

symptoms and occasionally with severe disease conditions, such as

flaccid paralysis and encephalitis. HPeV3 infection has been

associated with transient paralysis and neonatal infection and has until

now only been reported in Japan and Canada.

Methods. Culture isolates considered to be enterovirus on the basis of

cell culture but that were found to be enterovirus negative by

5’untranslated region reverse-transcriptase polymerase chain reaction

(5’UTR RT-PCR) during the period December 2000 through January

2005 were selected. Isolates were tested by HPeV 5’UTR RT-PCR and

were genotyped by sequencing the VP1 region. Phylogenetic analysis

was performed, and the association with clinical symptoms was

established.

Results. Thirty-seven (12%) of the 303 isolates that tested positive for

enterovirus by cell culture were in fact HPeV. The majority of the HPeV-

positive isolates (n=27) could be identified as HPeV1. The remaining 10

isolates, which were grown from samples obtained in 2001, 2002, and

2004, could be typed as the recently identified HPeV3. HPeV was

exclusively detected in children aged <3 years. Children infected with

HPeV3 were significantly younger than children infected with HPeV1,

and sepsis-like illness and central nervous system involvement were

more frequently reported in children infected with HPeV3.

Conclusions. We report HPeV infections in young children during the

period of 2000–2005 and show an association between HPeV3 infection

and sepsis-like illness and central nervous system involvement in

neonates.

INTRODUCTION

Picornaviruses form a group of small, nonenveloped, positive single-

stranded RNA viruses that are grouped into 9 genera, including

enteroviruses and parechoviruses. The human parechoviruses (HPeVs)

were previously known as enteroviruses echo22 and echo23, which were

first isolated in 1956 [1, 2]. When molecular techniques were designed for

Chapter 2

36

the detection of enterovirus, echo22 and echo23 could not be detected, and

sequence analysis revealed that they were genetically distinct from all other

picornaviruses. Therefore, they were renamed “HPeV1” and “HPeV2,”

belonging to a new genus of parechoviruses within the Picornaviridae [3].

The HPeV genome is similar to that of other picornaviruses, except for the

fact that the capsid protein VP0 is not cleaved for maturation into VP2 and

VP4 [1, 2].

HPeV1 is considered to be a widely spread pathogen that mainly affects

young children [1]. HPeV1 infections are most commonly associated with

mild gastrointestinal or respiratory symptoms. Severe disease conditions,

such as flaccid paralysis [4] and encephalitis [5] have also been reported,

but they appear to occur less frequently in cases of HPeV1 infection than in

cases of enterovirus infection [2]. HPeV2 infections rarely occur and are

mostly associated with gastrointestinal symptoms [6].

Recently, a new HPeV serotype (HPeV3) was isolated from a 1-year-old

patient with transient paralysis in Japan [7]. HPeV3 has also been found in 3

patients aged <1 month who had neonatal sepsis in Canada [8].

HPeV can grow on tertiary monkey kidney cells, human embryonic lung cells,

and African green monkey kidney (Vero) cells, and it has the same

cytopathogenic effect as enterovirus. With the development of molecular

techniques, many diagnostic laboratories replaced cell culture with RT-PCR

that is based on the highly conserved 5’untranslated region (5’UTR) to detect

enterovirus in CSF specimens. However, PCR assays based on the 5’UTR

region of enterovirus will not detect HPeV. RT-PCR assays for HPeV are

available [9], but they are not routinely used to detect HPeV in clinical

samples. Therefore, infections with HPeV are either missed or may be

misdiagnosed as enterovirus infection.

Here, we studied whether enterovirus infections that were diagnosed by cell

culture in our laboratory during the period of 2000–2005 could, in fact, have

been HPeV infections. We also investigated whether the newly described

HPeV3 serotype could be found in our clinical isolates by genotyping of the

VP1 region. Classification of enteroviruses by VP1 genotyping closely

relates to the classification by serotyping [10–14]. This has also been

described for another picornavirus, the foot-and-mouth disease virus [15].

For HPeV, the variability within the VP1 region allows one to distinguish

between the 3 serotypes of HPeV [7, 8]. Because it has been suggested that

HPeV3 infection can lead to severe morbidity in young children, we studied

the association between the different HPeV serotypes and various clinical

symptoms.

Human parechovirus infection in children

37

METHODS

Clinical samples

Fecal samples and throat swab specimens that are sent to our laboratory for

enterovirus diagnosis are routinely cultured on tertiary monkey kidney cells,

Vero cells, and human embryonic lung cells. Viral culture is performed by

cocultivation of patient material with tertiary monkey kidney cells, Vero cells,

and human embryonic lung cells in tubes containing 1 mL of minimum

essential medium Hanks 8% fecal cow serum. These viral cultures are

examined twice weekly for the appearance of enterovirus-specific

cytopathogenic effect. Preliminary identification of isolates is performed

according to either the unstained cytopathogenic effect or

immunofluorescence with a specific monoclonal antibody (DAKO-Enterovirus

monoclonal antibody 5-D8/1; DAKO) [16]. Viral cultures are incubated for a

maximum of 21 days before they are considered negative. All culture

isolates that are determined by cell culture to be enterovirus are routinely

sent to a reference laboratory (National Institute for Public Health and the

Environment, Bilthoven, The Netherlands) for poliovirus surveillance.

From December 2000 through January 2005, a total of 284 enterovirus-

positive culture isolates were sent to the National Institute for Public Health

and the Environment. Thirty-five culture isolates that then tested enterovirus

negative by the 5’UTR RT-PCR at the National Institute for Public Health and

the Environment were selected for this study. Additionally, 19 samples that

were found to be enterovirus positive in cell culture but were not yet sent to

the National Institute for Public Health and the Environment were also

included.

RNA extraction

RNA was extracted from culture isolates using the method described by

Boom et al. [17]. Twenty-five µL of a 100-fold dilution in PBS of the culture

isolates was added to 900 µL of lysis buffer and 20 µL of size-fractioned

silica coarse particles. The extraction mixture was spiked with copies of

armored enterovirus internal control RNA [16] when testing for the presence

of HPeV or enterovirus by the 5’UTR RT-PCR, and it was not spiked when

genotyping by sequencing of the VP1 region had to be performed. As

positive controls, we included culture isolates that have previously been

typed by the National Institute for Public Health and the Environment as

echo22 (HPeV1) and echo23 (HPeV2). The negative control consisted of

calf thymus DNA (20 ng/µL; Sigma).

Chapter 2

38

5’UTR RT-PCR and detection

Forty µL of extracted RNA was used for reverse transcription. The final

reverse-transcription mixture (50 µL) contained 1x reverse-transcriptase

buffer CMB1 (10 mmol/L TRIS-HCl [pH, 8.3], 50 mmol/L KCl, 0.1% Triton-

X100; Sigma), 5.0 mmol/L MgCl2 (Sigma); 1.5 µg of random hexamers

(Roche Diagnostics), 120 µmol/L (each) dNTPs (Applied Biosystems), 280

ng/µL α-caseine (lot number 17H9551; Sigma), 4 U of RNAsin (Promega),

and 20 U of Superscript II (Invitrogen Life Technologies). The mixture was

incubated for 30 min at 42oC, and 25 µL of the generated cDNA was used as

input in the PCR. The PCR was performed in a 50-µL volume containing 1x

PCR II buffer (Applied Biosystems); 200 µmol/L of each dATP, dCTP, and

dGTP (Applied Biosystems); 400 µmol/L dUTP (Applied Biosystems); 0.1

µg/µL bovine serum albumin (Roche Diagnostics); 400 ng/µL α-caseine; 0.5

U of AmpErase (Uracil-N-glycolase; Applied Biosystems); and 2.5 U of

Amplitaq Gold (Applied Biosystems). The final MgCl2 concentration was 2.5

mmol/L [16].

For the 5’UTR enterovirus PCR, 200 ng of Entero-1 primer (5’-

CCCTGAATGCGGCTAAT-3’; nt 452–468) and 200 ng of Bio-entero-2

primer (5’-ATTGTCACCATAAGCAGCC-3’; 5’biotinylated; nt 597–579) [16]

were used. For the 5’UTR HPeV PCR, 200 ng of the K29 primer and 200 ng

of the K30 primer [18] were used. Both 5’UTR PCRs were performed in an

Applied Biosystems 9600 thermocycler, with the method described by Beld

et al. [16].

The enterovirus 5’UTR amplicons were analyzed by hybridization and

electrochemiluminescence measurement [16]. The HPeV 5’UTR amplicons

were analyzed by gel electrophoresis.

VP1 one-step RT-PCR

To detect all 3 known HPeV serotypes, new primers were designed just

outside the VP1 region, amplifying the complete VP1 region (760 bp).

Primers were designed using the following reference strains: HPeV1 strain

Harris (S45208), HPeV2 strain Williamson (AJ005695), and HPeV3 isolate

A308-99 (AB084913) (table 1). The PCR was performed in a 50-µL volume

containing 0.5 µmol/L primer VP1-parEchoF1 and 0.5 µmol/L primer VP1-

parEchoR1 (table 1), 1x RT-PCR mix (67 mmol/L Tris [pH, 8.8], 17 mmol/L

(NH4)2SO4, 6 µmol/L EDTA, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol;

(Sigma), 200 µmol/L (each) dNTPs, 400 ng/µL α-caseine, 10 U of RNAsin, 3

U of AMV-RT (Roche), and 2.5 U of Taq DNA polymerase (Applied

Biosystems). The RT-PCR was performed in an Applied Biosystems 9600

thermocycler by the method described by Oberste et al. [19].


39

Table 1. VP1 human parechovirus primers designed for genotyping of human parechoviruses,

2000-2005.

Primer Sequence 5’-3’ Positiona

VP1-parEchoF1 CCAAAATTCRTGGGG TTC 2332-2349

VP1-parEchoR1 AAACCYCTRTCTAAATAWGC 3090-3071

NOTE. Degeneracy: standard International Union of Biochemistry nucleotide ambiguity codes. a The reference strain was human parechovirus serotype 1 Harris (Genbank accession number

S45208).

HPeV genotyping and phylogenetic analysis

The HPeV VP1 amplicons were gel purified and sequenced. The sequencing

PCR was performed in a 20-mL volume containing 10 ng of either forward

primer VP1-parEchoF1 or reverse primer VP1-parEchoR1 (table 1), 5 ng of

(purified) amplicon, 1 mL of Big Dye Terminator ready reaction mix (Applied

Biosystems), 7 mL of dilution buffer (400 mmol/L Tris HCl [pH, 8.0] and 5

mmol/L MgCl2). The sequencing PCR is performed in an Applied Biosystems

9600 thermocycler, as follows: 1 min at 96oC, followed by 25 cycles that

each consisted of 10 s at 96oC, 5 s at 50

oC, and 4 min at 60

oC. The

sequences were analyzed on an ABI 3730/3100 DNA analyzer (Applied

Biosystems). Sequences were aligned using Clustal-W included in the

Vector NTI suite 7 software package (InforMax). Sequences were edited

using Genedoc software, version 2.6.02 [20]. Phylogenetic analyses were

performed by the neighbor-joining method [21], as implemented in the

Molecular Evolutionary Genetics Analysis software package, version 2.1 [22].

Jukes and Cantor distances [23] were estimated for the nucleotide

sequences, and P-distances were used for amino acid sequences. One

thousand bootstrap replicates were analyzed. The use of other methods for

distance estimation did not influence the tree topology. As reference

strains/isolates, we used HPeV1 strain Harris (S45208) and HPeV1 isolates

A1086-99 (AB112485), A942-99 (AB112486), and A10987-00 (AB112487);

and HPeV2 strain Williamson (AJ005695) and HPeV3 isolates A308-99

(AB084913), A317-99 (AB112482), A354-99 (AB112483), A628-99

(AB112484), and Can82853-01 (AJ889918). The previously typed echo22

(HPeV1) and echo23 (HPeV2) isolates from a National Institute for Public

Health and the Environment panel were also included as a control that VP1

genotyping correlates with serotyping. HPeV genotype was assigned on the

basis of phylogenetic clustering analysis. The nucleotide sequences of the

VP1 region from the HPeV1 and HPeV3 isolates are deposited in GenBank

under the accession numbers DQ172416-DQ172441 (HPeV1), and

DQ172442-DQ172451 (HPeV3).

Chapter 2

40

Clinical data and statistical analysis

Clinical data were collected from medical records and letters of discharge

from the 37 patients infected with HPeV. The patient’s age at time of virus

isolation, sex, duration of hospitalization, underlying illnesses, and the ward

of admission were documented. The patients were scored for the presence

or absence of the following clinical symptoms: fever (temperature, >38oC),

sepsis-like illness (signs of circulatory or respiratory dysfunction), respiratory

symptoms (rhinorrhea, cough, upper respiratory tract infections, or lower

respiratory tract infections), gastrointestinal symptoms (diarrhea and/or

vomiting alone or in combination with abdominal distension), and

neurological symptoms (clinically suspected meningitis, lethargy,

convulsions, or paralysis).

Statistical analysis was performed to compare the 2 groups of patients. To

compare age distribution, the Mann-Whitney U test was used. Clinical

symptoms were compared using Fisher’s exact test.

RESULTS

Molecular identification of HPeV

From December 2000 to January 2005, a total of 303 clinical samples were

determined to be positive for enterovirus by cell culture in our laboratory, and

284 of these culture isolates were sent to the reference laboratory for

poliovirus surveillance. Of those, the reference center reported that 35

samples were found to be negative for enterovirus by 5’UTR RT-PCR. To

identify whether these culture isolates could be HPeV, these 35 isolates and

the additional 19 isolates that had not yet been sent to the reference

laboratory (total number of isolates, 54) were tested by our 5’UTR RT-PCRs,

which are specific for HPeV and enterovirus. All 37 isolates that had positive

HPeV RT-PCR results had negative results of the enterovirus RT-PCR. The

other 17 culture isolates tested positive for enterovirus and negative for

HPeV by RT-PCR. Thus, all 54 culture isolates tested by RT-PCR could be

identified as either HPeV or enterovirus, and no double infections were

found in this subset. Remarkably, 5 isolates that had negative 5’UTR RT-

PCR results at the reference center were found to be positive for enterovirus

by our 5’UTR RT-PCR, indicating that the RT-PCR for enterovirus described

by Beld et al. [16] is more sensitive. In conclusion, 37 (12%) of our 303

clinical isolates that were determined to be enterovirus on the basis of cell

culture results were in fact HPeV.


41

Molecular typing of HPeV

Because genotyping based on VP1 sequencing has been shown to correlate

with serotyping for both enterovirus and foot-and-mouth disease virus [10-

15], we designed degenerate primers (table 1) to sequence the entire VP1

region, to distinguish between HPeV1, HPeV2, and HPeV3. All 37 HPeV

culture isolates were amplified and sequenced. Culture isolates from a panel

typed by the reference laboratory as echo22 (HPeV1) and echo23 (HPeV2)

were also amplified and sequenced with these primers. The reference

strains for HPeV1 (strain Harris), HPeV2 (strain Williamson), and HPeV3

(A308-99) and published isolates for HPeV1 and HPeV3 were included in

the analysis. Figure 1 shows the phylogenetic tree based on amino acid

EC

HO

23

HP

EV

2A

WIL

LIA

MS

ON

HP

EV

1A

1086-9

9

350757

350642

452538

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350918

15259

8152478

HPEV1A10987-00HPEV1A942-99252485

452176

152824

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252521

550163

45

034

33

50

01

4

25

222

8 25

258

11

53

231

251949

350841

1514

42

451294

452473

452252152212054330152329

HPEV1HARRISECHO22

HPEV

3A31

7-99

HPEV3A354-99

HPEV3A628-99

HPEV3A308-99

152037

HPEV3CAN82853-01251360451371

450936

451517

252277

251

181

251407

251393

250956

0.02

HPeV1

HPeV3

EC

HO

23

HP

EV

2A

WIL

LIA

MS

ON

HP

EV

1A

1086-9

9

350757

350642

452538

450976

350918

15259

8

152478

HPEV1A10987-00HPEV1A942-99252485

452176

152824

452712

252521

550163

45

034

33

50

01

4

25

222

8 25

258

11

53

231

251949

350841

1514

42

451294

452473

452252152212054330152329

HPEV1HARRISECHO22

HPEV

3A31

7-99

HPEV3A354-99

HPEV3A628-99

HPEV3A308-99

152037

HPEV3CAN82853-01251360451371

450936

451517

252277

251

181

251407

251393

250956

0.02

HPeV1

HPeV3

EC

HO

23

HP

EV

2A

WIL

LIA

MS

ON

HP

EV

1A

1086-9

9

350757

350642

452538

450976

350918

15259

8

152478

HPEV1A10987-00HPEV1A942-99252485

452176

152824

452712

252521

550163

45

034

33

50

01

4

25

222

8 25

258

11

53

231

251949

350841

1514

42

451294

452473

452252152212054330152329

HPEV1HARRISECHO22

HPEV

3A31

7-99

HPEV3A354-99

HPEV3A628-99

HPEV3A308-99

152037

HPEV3CAN82853-01251360451371

450936

451517

252277

251

181

251407

251393

250956

0.020.02

HPeV1

HPeV3

Figure 1. Unrooted phylogenetic tree showing the relationship between clinical isolates

from January 2000-January 2005 and HPeV1 strain Harris (S45208) and isolates

A1086-99 (AB112485), A942-99 (AB112486) and A10987-00 (AB112487); HPeV2

strain Williamson (AJ005695) and HPeV3 isolates A308-99 (AB084913), A317-99

(AB112482), A354-99 (AB112483), A628-99 (AB112484) and Can82853-01

(AJ889918) based on amino acid differences in the capsid protein VP1. The tree was

constructed by using the neighbour–joining method. Numbers represent the frequency

of occurrence of nodes in 1000 bootstrap replicas.

Chapter 2

42

sequences. The tree based on nucleotide sequences revealed the same

topology (data not shown). The previously typed echo22 and echo23

clustered with their respective reference HPeV genotype. The majority of the

isolates (n=27) could be identified as HPeV1. In addition, 10 isolates could

be typed as the recently identified HPeV3. The Dutch HPeV1 isolates (n=27)

were only 91% homologous to the HPeV1 strain Harris. However, they

clustered closely with the recently genotyped and serotyped Japanese

HPeV1 sequences (98% homology) [7]. The Dutch HPeV3 isolates (n=10)

were 97% homologous to the HPeV3 isolate A308-99 [7]. The Dutch HPeV3

isolates (n=10) clustered closely together (99% homology) and were more

closely related to the Canadian isolate (CAN82853-01; 99% homology) than

to the Japanese isolates (figure 1). Homology between the HPeV1 cluster

and the HPeV3 cluster was 73%. Clustering of HPeV1 and HPeV3 isolates

could not be related to year of isolation, hospital or ward of admission, or

clinical features.

HPeV3 was already present in a patient in July 2001, whereas 6 isolates

were cultured from patients in 2002, and the remaining 3 were cultured in

2004 (data not shown). For HPeV1, most isolates were cultured in the

autumn-winter season, with the peak occurring in October, whereas none

were cultured in June through July. For HPeV3, the opposite was observed

(figure 2).

Figure 2. Seasonal distribution of culturing HPeV1 (white bars) and HPeV3 (hatched

bars) isolates from patients over the period 2000-2005.

0

1

2

3

4

5

6

7

jan

feb

mar

april

may

june

july

august

sept

oct

nov

dec

Month

No

. o

f is

ola

tes

HPeV1

HPeV3


43

Table 2. Clinical symptoms in human parechovirus (HPeV) infection in Dutch children, 2000-

2005.

No. of patients with symptoms/ no. of

patients with available data (%)

characteristics HPeV

serotype 1

(n= 27)

HPeV

serotype 3

(n= 10)

P

Fever 16/25 (64) 9/10 (90) .2

Sepsis-like-illness 2/26 (8) 7/10 (70) <.001a

Respiratory tract symptoms 13/27 (48) 3/10 (30) .5

Gastrointestinal tract symptoms 21/26 (81) 7/10 (70) .7

Central Nervous System symptoms 3/26 (12) 5/10 (50) .02a

NOTE. P values were determined by Fisher’s exact test. a statistically significant.

Characterization of clinical symptoms

We identified 27 patients infected with HPeV1 and 10 infected with HPeV3.

All patients were children aged <3 years. HPeV3 was exclusively present in

young infants (median age, 1.3 months), whereas the median age of the

children infected with HPeV1 was significantly higher (6.6 months; P=.0039).

Remarkably, almost all HPeV3 infections were observed in boys (ratio of

male to female children for HPeV1 and HPeV3 infections, 1.5:1 and 9:1,

respectively).

In total, 23 children were admitted to the academic hospital, and 10 were

admitted to a general hospital in the region. Most children were admitted to a

general children’s ward or a children’s surgery ward, but 1 child was treated

on an intensive care unit because of convulsions.

HPeV1 infections are generally associated with mild gastrointestinal or

respiratory disease; however, HPeV3 has been found in children with more-

severe clinical symptoms, such as paralysis [7] and neonatal sepsis [8].

Therefore, we compared the 2 groups of children for the presence or

absence of fever, sepsis-like illness, respiratory tract symptoms,

gastrointestinal tract symptoms, and neurological (CNS) symptoms (table 2).

The majority of children in both groups were found to have fever for at least

1 day. In both groups, gastrointestinal tract symptoms were more frequent

than respiratory tract symptoms. For the children infected with HPeV1,

clinical symptoms were mainly gastrointestinal tract and/or respiratory tract

symptoms, whereas sepsis-like illness and CNS symptoms were reported in

only a minority of patients. However, in significantly more children infected

with HPeV3, sepsis-like illness and CNS symptoms were reported. All

children with sepsis-like illness were treated with antibiotics, which was

usually stopped after 48–72 h because no bacterial agent could be cultured

Chapter 2

44

and because the children improved clinically. There was no mortality among

the children with sepsis-like illness or CNS symptoms. None of the children

with CNS symptoms developed paralysis.

DISCUSSION

The newly identified HPeV3 was first isolated in 2004 and has since then

only been reported in 4 children from Japan [7] and 3 children from Canada

[8]. Until now, HPeV3 has not been reported in Europe. Seroepidemiological

studies by Ito et al. [7] showed that HPeV3 existed long before its first

isolation in 2004, because 87% of Japanese adults aged >40 years have

antibodies against HPeV3. Here, we describe 10 children from The

Netherlands with HPeV3 infection during the period of 2000–2005.

Typing of our clinical isolates was done by sequencing the entire VP1 region.

The majority of the samples (n=27) clustered in a subgroup of HPeV1

isolates that was more closely related to the HPeV1 isolates identified in

Japan in 2004 [7] than to the reference strain Harris, which was sequenced

in 1992 [3] and isolated in 1956. The remaining 10 isolates clustered with the

Japanese and Canadian HPeV3 isolates. The amino acid similarity of the

VP1 region between the HPeV1 cluster and the HPeV3 cluster was 73%,

which is in accordance with that found by Ito et al. [7]. The high amino acid

homology of the Dutch HPeV3 isolates to the Canadian HPeV3 isolate

suggests that HPeV3 circulating in The Netherlands is more closely related

to HPeV3 circulating in Canada than in Japan. However, more data on

geographical spread of HPeV3 are needed. When clustering of HPeV1 or

HPeV3 strains was observed, it could not be related to the year of isolation,

admittance to the same hospital (ward), or specific clinical features.

The previously described HPeV3 infections in children suggested a more

severe clinical spectrum than occurs with HPeV1 infection, including

transient paralysis [7] and neonatal sepsis [8]. This is the first report that

directly compares the clinical symptoms in a group of children infected with

HPeV3 with the symptoms in children infected with HPeV1. Although the

groups are small, our analysis shows that the children infected with HPeV3

were significantly younger and that sepsis-like illness and CNS symptoms

were observed more often in HPeV3-infected children than in HPeV1-

infected children. It remains unclear why HPeV3 would infect younger

children. This could be related to the presence of maternal antibodies for

HPeV1 but not for HPeV3, although the reason for this is also unclear. In

addition, it cannot be excluded that the severity of symptoms is related to the

young age at which the infection tends to occur.


45

The seasonal distribution of our HPeV1 isolates is in agreement with data

from a Swedish study, which showed that HPeV1 occurs in late summer to

autumn and in winter to early spring [24]. The Dutch HPeV3 isolates were

isolated in spring and the early summer. Originally, HPeV3 was found in a

child admitted to the hospital in Japan in August. Two Canadian HPeV3

isolates were isolated from children admitted in September and from 1 child

admitted in December. Our study indicates a different pattern of seasonal

circulation for HPeV1 and HPeV3; however, more data are needed to

confirm this trend.

We showed that 12% of our clinical isolates that were determined to be

enterovirus by cell culture were, in fact, HPeV. All HPeV isolates were

recovered from young children (mean age, 5.7 months). We confirm that

HPeV mainly infects children before the age of 3 years, with the majority of

children infected before the first year of age [9], whereas enterovirus

infections occur during a much broader age range (mean age for the 17

children with enterovirus infection, 20 months; data not shown). Symptoms

vary from mild gastrointestinal tract and respiratory tract symptoms to

sepsis-like illness and meningitis. This indicates that HPeV is a relevant

pathogen in young children. However, molecular techniques for diagnosis of

enterovirus infection do not include HPeV. Thus, when only PCR for

enterovirus is performed, HPeV infections will not be detected; therefore, we

suggest the implementation of an RT-PCR for the detection of HPeV in

clinical specimens obtained from children aged <3 years.

Acknowledgments

We would like to acknowledge Hetty van Eijk, for providing assistance with

cultures and for critically reading the manuscript, and Alex van Breda and

Alwin van der Ham, for providing technical support. We would also like to

acknowledge Georgios Pollakis for his assistance in the statistical analysis.

REFERENCES

1. Joki-Korpela P, Hyypia T. Parechoviruses, a novel group of human

picornaviruses. Ann Med 2001; 33:466-71.

2. Stanway G, Hyypia T. Parechoviruses. J Virol 1999; 73:5249-54.

3. Hyypia T, Horsnell C, Maaronen M, et al. Distinct picornavirus group

identified by sequence analysis. Proc Natl Acad Sci U S A 1992;

89:8847-51.

Chapter 2

46

4. Figueroa JP, Ashley D, King D, Hull B. An outbreak of acute flaccid

paralysis in Jamaica associated with echovirus type 22. J Med Virol

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5. Koskiniemi M, Paetau R, Linnavuori K. Severe encephalitis

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6. Ehrnst A, Eriksson M. Echovirus type 23 observed as a nosocomial

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7. Ito M, Yamashita T, Tsuzuki H, Takeda N, Sakae K. Isolation and

identification of a novel human parechovirus. J Gen Virol 2004; 85:391-

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8. Boivin G. Human parechovirus 3 and neonatal infections. Emerg Infect

Dis 2005; 11:103-5.

9. Stanway G, Joki-Korpela P, Hyypia T. Human parechoviruses -

biology and clinical significance. Rev Med Virol 2000; 10:57-69.

10. Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. Molecular

evolution of the human enteroviruses: correlation of serotype with VP1

sequence and application to picornavirus classification. J Virol 1999;

73:1941-8.

11. Thoelen I, Moes E, Lemey P, et al. Analysis of the serotype and

genotype correlation of VP1 and the 5' noncoding region in an

epidemiological survey of the human enterovirus B species. J Clin

Microbiol 2004; 42:963-71.

12. Oberste MS, Maher K, Kilpatrick DR, Flemister MR, Brown BA,

Pallansch MA. Typing of human enteroviruses by partial sequencing

of VP1. J Clin Microbiol 1999; 37:1288-93.

13. Norder H, Bjerregaard L, Magnius LO. Homotypic echoviruses share

aminoterminal VP1 sequence homology applicable for typing. J Med

Virol 2001; 63:35-44.

14. Muir P, Kammerer U, Korn K, et al. Molecular typing of enteroviruses:

current status and future requirements. The European Union

Concerted Action on Virus Meningitis and Encephalitis. Clin Microbiol

Rev 1998; 11:202-27.

15. Vangrysperre W, De Clercq K. Rapid and sensitive polymerase chain

reaction based detection and typing of foot-and-mouth disease virus in

clinical samples and cell culture isolates, combined with a

simultaneous differentiation with other genomically and/or

symptomatically related viruses. Arch Virol 1996; 141:331-44.


47

16. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen

PM, van der Noordaa J. Rapid and simple method for purification of

nucleic acids. J Clin Microbiol 1990; 28:495-503.

17. Beld M, Minnaar R, Weel J, et al. Highly sensitive assay for detection

of enterovirus in clinical specimens by reverse transcription-PCR with

an armored RNA internal control. J Clin Microbiol 2004; 42:3059-64.

18. Oberste MS, Maher K, Pallansch MA. Specific detection of

echoviruses 22 and 23 in cell culture supernatants by RT-PCR. J Med

Virol 1999; 58:178-81.

19. Oberste MS, Nix WA, Maher K, Pallansch MA. Improved molecular

identification of enteroviruses by RT-PCR and amplicon sequencing. J

Clin Virol 2003; 26:375-7.

20. Nicholas KB, Nicholas HB Jr, Deerfield DW II. GeneDoc: Analysis

and Visualization of Genetic Variation. Embnew News 1997; 4:14.

21. Saitou N, Nei M. The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4:406-25.

22. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular

evolutionary genetics analysis software. Bioinformatics 2001;

172:1244-45.

23. Jukes, T. H. and C. R. Cantor. Evolution of protein molecules. In:

Munro HN, ed. Mammalian Protein Metabolism. New York: Academic

Press, 1969: 21-132.

24. Ehrnst A, Eriksson M. Epidemiological features of type 22 echovirus

infection. Scand J Infect Dis. 1993; 25:275-81.

Rapid Detection of Human

Parechoviruses in Clinical Samples by

Real-time PCR

Kimberley Benschop, Richard Molenkamp,

Alwin van der Ham, Katja Wolthers, and Marcel Beld

Journal of Clinical Virology 2008; 41:69-70

Lab. of Clinical Virology, Dept. of Medical Microbiology, Academic Medical

Center, Amsterdam.

51

Rapid Detection of Human Parechoviruses in Clinical

Samples by Real-time PCR

Background: Human parechoviruses (HPeVs) have been associated

with severe conditions such as neonatal sepsis and meningitis in

young children. Rapid identification of an infectious agent in such

serious conditions in these patients is essential for adequate decision

making regarding treatment and hospital stay.

Objectives: We have developed an HPeV specific real-time PCR assay

based on the conserved 5’untranslated region.

Study design: To determine the detection limit of the assay, serial

dilutions of HPeV in vitro RNA were tested in a background of HPeV

and EV RNA-negative cerebrospinal fluid (CSF). The specificity was

tested by analyzing culture isolates of HPeV 1–6, enterovirus (EV)

types, human rhinoviruses (HRVs) and hepatitis A virus (HAV). To

establish diagnostic relevance, 522 CSF samples from children <5

years were tested.

Results: The detection limit of the assay was 75 copies of HPeV cDNA

per reaction. The assay was highly specific for HPeV, detecting all

HPeV types. We identified HPeV infections in CSF of 20 children (3.8%),

all with severe conditions such as sepsis and meningitis.

Conclusions: These results suggest that HPeV screening of paediatric

clinical samples should be included in viral diagnostics in suspected

cases of neonatal sepsis and meningitis.

INTRODUCTION

Human parechoviruses (HPeVs) are members of the Parechovirus genus,

which forms a separate genus within the family Picornaviridae. In addition to

the previously known enteroviruses (EV) echovirus22 (HPeV1) and

echovirus23 (HPeV2), HPeV types 3, 4, 5 and 6 have been identified in

Japan, The Netherlands and the United States (Al-Sunaidi et al., 2006;

Benschop et al., 2006b; Ito et al., 2004; Watanabe et al., 2007). HPeV

infections have commonly been associated with mild gastrointestinal or

respiratory symptoms in young children. However, severe conditions such as

flaccid paralysis (Figueroa et al., 1989), bronchiolitis (Abed and Boivin, 2006)

and neonatal sepsis (Boivin et al., 2005) have also been reported. In 2006,

we showed infections with HPeV3 to be associated with more severe illness,

at a younger age compared to infections with HPeV1 (Benschop et al.,

2006a).

Chapter 3

52

For detection of EV in cerebral spinal fluid (CSF), cell culture is replaced by

RT-PCR based on the conserved 5’UTR region. However, such specific EV

RT-PCR assays will not detect HPeV and thus infections with HPeV will be

missed. A rapid HPeV or EV diagnosis is needed to avoid inappropriate

treatment of suspected bacterial meningitis or herpes encephalitis and to

decrease duration of hospital stay (Foray et al., 1999; Nigrovic and Chiang,

2000; Rotbart, 1995). For rapid detection of all known HPeV types, we have

developed and validated an HPeV specific real-time assay based on the

5’UTR.

METHODS

Viral strains

EV strains, Coxsackie virus A (CVA) 2, 5, 7, 10, 16 and EV 71 (EV-A);

Coxsackie virus B (CVB) 1, 3, 5, 6 and echovirus 1, 6, 13, 16, 20, 25, 30, 33

(EV-B); CVA 1, 11, 13, 15, 18, 22, 24 (EV-C); EV 68 and 70 (EV-D) and

HPeV1 and 2 were provided by the National Institute of Public Health and

the Environment (RIVM). Culture isolates containing either HPeV1, HPeV3

and the recently identified fourth serotype K251176-02 were obtained from

clinical specimens (Benschop et al., 2006a,b). HPeV4 and HPeV5 strains

including the prototype strains HPeV4 T75-4077 and HPeV5 T92-15 (Al-

Sunaidi et al., 2006; Schnurr et al., 1996) were provided by the Department

of Biological Sciences at the University of Essex. HPeV6 was isolated from

background population in Finland by Professor Heikki Hyoty group

(Department of virology, University of Tampere) and was provided by the

Department of Biological Sciences. HRV strains 1A, 1B, 3, 8, 11, 13, 14, and

15 were provided by the Department of Virology of the Utrecht Medical

Center. Hepatitis A virus (HAV) was provided by the Municipal Health

Service, Amsterdam.

Primers and probes

Reverse transcriptase (RT-)PCR was performed as described before (Beld

et al., 2004). The primer pair used for amplification of HPeV is located in the

5’UTR and consists of forward primer ParechoF31 (5’-

CTGGGGCCAAAAGCCA-3’; 441–457) and reverse primer K30 (Oberste et

al., 1999). Nucleotide numbering is relative to HPeV1 Harris. The primer pair

used for amplification of a heterologous internal control (IC) was essentially

as described by Beld et al. (2004) with minor modifications: entero-1-TM (5’-

ggCCCTGAATGCGGCTAAT-3’) and entero-2-TM (5’-

gggATTGTCACCATAAGCAGCC-3’). For detection of HPeV and IC, we

HPeV real time PCR

53

designed two differentially labelled probes containing either a FAM or VIC®-

label at the 5’-end and a nonfluorescent quencher (NFQ) and a minor groove

binding site (MGB) at the 3’-end as follows: (6’FAM)-HPeV-WT-MGB (5’-

AAACACTAGTTGTA(A/T)GGCCC-3’) and (VIC®)-IC-MGB (5’-

CTTGAGACGTGCGTGGTAACC-3’) (Beld et al., 2004). All primers were

obtained from Biolegio. The probes were obtained from Applied Biosystems.

Construction of the HPeV in vitro RNA positive control

A plasmid containing sequences of HPeV1 Harris strain (nucleotide 441–584)

was generated by cloning the RT-PCR amplicon generated by primers

ParechoF31 and K30 into PCRII-TOPO vector (Promega) as described by

the manufacturer. In vitro transcription was performed on BamHI-linearized

plasmid samples using the T7 MEGAscript® High Yield Transcription Kit

(Ambion). Synthesized RNA was purified as described by Boom et al. (1999)

and quantified by measuring the optical density at 260 nm. The in vitro RNA

was stored in TE-buffer, containing 20 ng/µL CT-DNA (Sigma) at −80oC.

HPeV specific duplex and single-target real-time

RT-PCR HPeV RNA was purified from clinical samples and culture isolates

as described by Boom et al. (1990). Forty microliters of extracted RNA

(corresponding to 2/5 of the extracted RNA) was used for RT using random

hexamers (Beld et al., 2004).To allow detection of both HPeV and IC cDNA

(duplex assay), the PCR was performed in a 25 µL volume containing 900

nM of each primer (ParechoF31 and K30; entero-1-TM and entero-2-TM);

200 nM of HPeV-WT-MGB probe and 200 nM of the IC-MGB probe; 400

ng/µL of bovine alpha-casein (Lot no. 17H9551, Sigma); 1x TaqMan®

universal PCR mastermix (Applied Biosystems); and 5 µL of the cDNA

reaction (corresponding to 1/25 of the extracted RNA). The real-time PCR

was performed in an Applied Biosystems 7000 sequence analyzer, as

follows: 2 min at 50oC and 10 min at 95

oC, followed by 45 cycles each

consisting of 15 s at 95oC, and 1 min at 60

oC.

Individual detection of HPeV RNA was done in a single target format, using

the initial cDNA. The primers and probe for detection of the IC were

excluded from the PCR mixture and the mixture was processed as described

above.

Clinical specimens

All CSF samples stored in our laboratory from patients under the age of 5

years were obtained from the period 2000–2005. All samples tested

Chapter 3

54

negative in the EV specific RT-PCR and were internally controlled with the

IC (Beld et al., 2004).

RESULTS

The minimal input of IC within the HPeV real-time assay was determined

with serial dilutions of armored IC RNA control, spiked into lysis buffer

containing HPeV/EV-negative CSF prior to extraction by the method of

Boom et al. (1990). A detection limit corresponding to 10 copies of IC

cDNA/PCR was found with a 100% hit-rate and a mean Ct value of 37.29

(Table 1, Fig. 1). However, to allow reliable detection and to minimize the

number of false negative results, a minimal input corresponding to 50 copies

IC cDNA/PCR (mean Ct value: 35.18) was chosen as standard input for

every extraction.

Table 1. Sensitivity of the internal control.

No. of copies in PCRa/

200 µL CSF in extraction

No. of positives (%); Mean Ct; Standard Deviation.

1000 6/6 (100%); 30.08; 0.36

500 6/6 (100%); 31.20; 0.14

250 6/6 (100%); 32.59; 0.55

100 6/6 (100%); 33.66; 0.49

50 6/6 (100%); 35.18; 0.45

25 6/6 (100%); 36.13; 0.61

10 6/6 (100%); 37.29; 1.17

0 0/6 (0%)

a Number of copies cDNA/PCR has been calculated as 1/25 of the extracted RNA, assuming

100% efficiency in RT and PCR.

Figure 1. Lower limit of

detection of the IC.

Slope: 3.82.

Regression coefficient:

0.951. The minimal input

of the IC with a Ct value

below 36.00 was 50

copies of IC cDNA in

PCR.

50

Ct

10 100 1.000

copies HPeV copies cDNA in PCR

28,00

30,00

32,00

34,00

36,00

38,00

40,00

25 250 500

HPeV real time PCR

55

The dynamic range and linearity of the duplex assay was analyzed by testing

serial 10-fold dilutions of HPeV WT in vitro RNA in HPeV and EV-negative

CSF background. A dynamic range from 106 to 10

3 copies HPeV cDNA/PCR

with a regression coefficient of 0.907 (Table 2, Fig. 2a) was found. However,

linearity was only observed from 106 to 10

4 copies HPeV cDNA/PCR with a

regression coefficient of 0.965 (Fig. 2a). The IC was detected with a 100%

hit-rate in each of the dilutions of HPeV in vitro RNA.

To determine the lower limit of detection of the assay, we analyzed serial

twofold dilutions of HPeV in vitro RNA in HPeV and EV-negative CSF

background. We could detect 625 copies HPeV cDNA/PCR with a 100% hit-

rate (Table 3). Subsequent twofold dilutions were all found negative. The IC

was detected with a 100% hit-rate in each of the dilutions of HPeV in vitro

RNA. To fine tune the detection limit, a further analysis between 625 and

310 copies HPeV cDNA/PCR was done. A detection of 500 and 400 copies

HPeV cDNA/PCR was found with a hit-rate of 76% (mean Ct: 42.82, S.D.:

0.87) and 50% (mean Ct: 42.33, S.D.: 1.85), respectively, following a

Poisson distribution (Diaco, 1995). This showed a detection limit of 400

copies HPeV cDNA/PCR with a 50% hit-rate.

Table 2. Dynamic range/Linearity and inter assay variation of the HPeV duplex and single target

real-time assay.

No. of positives (%); Mean Ct; Standard Deviation. No. of copies in PCRa/

200 µL CSF in extraction Duplex format 50 copies

armored IC RNA

Single target assay,

no detection of IC

106 12/12 (100%); 25.96; 0.50 12/12 (100%); 25.73; 0.55

105 12/12 (100%); 29.27; 0.42 12/12 (100%); 28.81; 0.29

104 12/12 (100%); 32.90; 0.69 12/12 (100%); 32.37; 0.26

103 10/12 (83%); 39.90; 2.58 12/12 (100%); 36.07; 0.76

102 0/12 (0%) 6/12 (50%); 39.90; 0.91

10 0/12 (0%) 0/12 (0%)

1 0/12 (0%) 0/12 (0%)

0 0/12 (0%) 0/12 (0%)

a Number of copies cDNA/PCR has been calculated as 1/25 of the extracted RNA, assuming


Chapter 3

56

20,0

22,5

25,0

27,5

30,0

32,5

35,0

37,5

40,0

42,5

45,0

6 5 4 3 2

Log copies HPeV copies cDNA in PCR

b.

Ct

Figure 2. Dynamic range and linearity of the HPeV duplex assay (a) and the single

target assay (b). (a). Duplex format: Slope: 3.88; regression coefficient (106-10

3): 0.907.

Linearity was found between 106-10

4, slope: 3.47 and regression coefficient: 0.965. (b).

Single target assay: Slope: 3.42; regression coefficient (106-10

2): 0.984. Linearity was

found between 106-10

2.

20,0

22,5

25,0

27,5

30,0

32,5

35,0

37,5

40,0

42,5

45,0

6 5 4 3 2

Log copies HPeV copies cDNA in PCR

Ct

a.

HPeV real time PCR

57

Table 3. Lower limit of detection of HPeV RNA of the HPeV duplex and single target real-time

assay.

No. of positives (%); Mean Ct; Standard Deviation. No. of copies in PCRa/

200 µL CSF in extraction Duplex assay,

50 copies armored IC RNA

Single target assay,

no detection of IC

10.000 6/6 (100%); 33.61; 0.62 6/6 (100%); 32.79; 0.59

5000 6/6 (100%); 34.46; 0.53 6/6 (100%); 33.90; 0.40

2500 6/6 (100%); 35.84; 0.43 6/6 (100%); 34.84; 0.67

1250 6/6 (100%); 37.82; 1.04 6/6 (100%); 35.88; 0.90

625 6/6 (100%); 39.79; 1.62 6/6 (100%); 36.80; 0.94

310 0/6 (0%) 5/6 (83%); 38.06; 0.99

155 0/6 (0%) 5/6 (83%); 39.85; 0.98

75 0/6 (00%) 3/6 (50%); 41.16; 2.56

38 0/6 (0%) 0/6 (0%)

0 0/6 (0%) 0/6 (0%) a Number of copies cDNA/PCR has been calculated as 1/25 of the extracted RNA, assuming


Chapter 3

58

The loss in linearity and high detection limit in our duplex PCR described

above might be the results of a preferential amplification and detection of the

IC. The same cDNA dilution series were tested in a single-target assay, in

which the primers and probe of the IC were omitted from the PCR and only

HPeV specific primers and probe were used. In this assay, the dynamic

range improved and was linear from 106 to 10

2 copies HPeV cDNA/PCR with

a regression coefficient of 0.984 (Table 2, Fig. 2b). In comparison to the

duplex assay, the detection limit was found to be much lower: 75 copies

HPeV cDNA were detected in 50% of the cases (Table 3).

Both the duplex and single-target assay showed stable Ct values for each

serial dilutions of HPeV in vitro RNA when analyzed in triplicate divided over

4 days in 4 separate runs (Table 4).

To determine whether the assay was specific, all known HPeV strains (1–6),

and other available picornaviruses (EVs, RHVs and HAV) were tested by our

HPeV real-time RT-PCR in both the duplex and single target assay. The

extraction of every single isolate was done alternately with negative

extraction controls. All 6 HPeV types were found positive, whereas the

samples containing other picornaviruses and all negative controls were

negative (Table 5). The negative results were truly negative, as the IC was

detected in all samples containing EVs, RHVs and HAV.

Table 5. Specificity and sensitivity of HPeV real-time RT-PCR.

Virus Results Mean Ct (Ct-range)

HPeV1 Harris strain Positive 29.19

HPeV1 (550163, 252485, 252228, 253231, 152478, 152329)

Positive 28.58 (25.44 - 31.20)

HPeV2 Williamson strain Positive 30.34

HPeV3 (451517, 251407, 251360, 252277, 251181)

Positive 26.36 (24.25 - 28.75)

HPeV4 (K251176-02, T75-4077, T82-0203, T75-4080) Positive 23.61 (21.41 – 25.56)

HPeV5 (T92-15, T82-1115, T82-659, T82-2192, T82-0169) Positive 20.66 (17.87 – 24.47)

HPeV6 (69960)* Positive 28.62

EV Group A (CVA2,5,7,16) Negative Undet.

EV Group B (CVB1,3,5,6, Echo1,6,13,16,20,25,30,33) Negative Undet.

EV Group C (CVA1,11,13,15,18,22,24) Negative Undet.

EV Group D (EV 68,70) Negative Undet.

Rhino virus (1A,1B,3,8,11,13,14,15) Negative Undet.

HAV Negative Undet.

Negative control Negative Undet.

Positive control (10.000 copies HPeV cDNA/PCR) Positive 33.19 (32.35-34.27)

Internal control (50 copies HPeV cDNA/PCR) Positive 34.14 (31.32-36.50)

All isolates, with the exception of HPeV6 (*) where obtained from cell culture. HPeV6 was

obtained from a stool sample. The negative control consisted of TE with 20 mg/mL CT-DNA.

HPeV real time PCR

59

S.D

.

betw

een

runs

0.1

8

0.3

8

0.5

0

0.1

8

0.1

8

Undet

Undet

Undet

Mean C

t

Run 4

25.4

1

29.8

4

31.8

1

35.8

4

40.2

3

Undet

Undet

Undet

Mean C

t

Run 3

25.7

9

29.4

4

32.8

7

35.8

1

40.4

9

Undet

Undet

Undet

Mean C

t

Run 2

25.5

9

29.0

9

32.2

7

35.5

1

Undet

Undet

Undet

Undet

Sin

gle

targ

et

assay, no d

ete

ction o

f IC

Mean C

t

Run 1

25.4

2

28.5

2

32.3

2

36.0

4

Undet

Undet

Undet

Undet

S.D

.

betw

een

runs

0.2

5

0.2

6

0.2

9

1.0

7

Undet

Undet

Undet

Undet

Mean C

t

Run 4

26.0

4

29.1

6

32.9

6

36.5

4

Undet

Undet

Undet

Undet

Mean C

t

Run 3

26.3

5

29.7

4

33.3

8

37.7

6

Undet

Undet

Undet

Undet

Mean C

t

Run 2

27.7

5

29.4

2

32.7

6

38.9

2

Undet

Undet

Undet

Undet

Duple

x a

ssay, 50 c

opie

s a

rmore

d IC

RN

A

Mean C

t

Run 1

25.9

3

29.2

2

33.3

1

38.6

2

Undet

Undet

Undet

Undet

Table

4. In

tra a

ssay v

ariation o

f th

e H

PeV

duple

x a

nd s

ingle

targ

et

real-tim

e a

ssay.

No.

of copie

s in P

CR

a/

200 µ

L C

SF

in e

xtr

action

10

6

10

5

10

4

10

3

10

2

10

1

0

a N

um

ber

of copie

s c

DN

A/P

CR

has b

een c

alc

ula

ted a

s 1

/25 o

f th

e e

xtr

acte

d R

NA

, assum

ing 1

00%

effic

iency in R

T a

nd P

CR

.

Chapter 3

60

To establish clinical relevance, 522 CSF samples obtained in 2000–2005

were tested. The initial cDNA samples that had already been monitored with

an IC within the EV specific RT-PCR (Beld et al., 2004), were tested with the

sensitive single-target assay. We found 20 (3.8%) samples to be positive for

HPeV with Ct values ranging from 16.18 to 38.80. All children positive for

HPeV presented with suspected sepsis like illness and or meningitis.

DISCUSSION

Diagnosis of viral meningitis and sepsis-like illness routinely relies on the

detection of a specific viral pathogen within CSF samples. The recent

association of HPeV with severe disease within young children has

warranted the need for a specific diagnostic assay. Therefore, we developed

a rapid HPeV specific real-time PCR. A specific primer pair and a single

degenerate probe were used to detect all 6 known HPeV types. Evaluation

of the real-time assay as a duplex assay (detection of both the wild type

target and the internal control) showed a loss in linearity within the dynamic

range and a low sensitivity. The loss in linearity and low sensitivity was most

likely due to competition of the IC, since the detection of HPeV improved

considerably when primers and probe for IC amplification and detection were

excluded. Now, linearity was observed over a broader dynamic range and a

sensitivity of 75 copies of HPeV cDNA/PCR could be observed. This was

comparable to the dynamic range and sensitivity found within the EV specific

duplex real-time assay, where detection of the same IC was included (data

not shown). The differential amplification effect of the IC within the EV and

HPeV duplex assays might be due to difference in PCR efficiency of IC to

the EV or HPeV amplification. The PCR efficiency for amplification of the IC

might thus be lower to the EV target, but higher to the HPeV target. Attempts,

such as other primers/probe combination or other real-time formats (LC480),

to upgrade the PCR efficiency of the HPeV target in relation in the IC failed.

However, for diagnostic purposes a clinical sample is first screened for an

EV infection with the EV specific duplex assay, and in addition the same

cDNA sample used in the first PCR can be screened for an HPeV infection in

the HPeV single-target assay. The sample is thus internally controlled

without loss of sensitivity in either PCR.

Although, several conventional end-point RT-PCR assays have been

developed for detection of HPeV1 and 2 (Corless et al., 2002; Legay et al.,

2002; Oberste et al., 1999), in most diagnostic laboratories, real-time PCR

has become state of the art. Corless et al. (2002) developed a real-time

assay for the detection of HPeV1 and 2. However, the probe used shows

HPeV real time PCR

61

several mismatches with the new HPeV types 3–6 and might not detect

these HPeV types. In comparison, Corless et al. (2002) found only 1.5% of

CSF samples positive, while we found a percentage of approximately 4% in

CSF samples. This might be due to selection of the patient groups tested, or

to the inability of the previous assay to detect newer HPeV types.

The high percentage of CSF positive samples we found shows that HPeV is

an important pathogen in infants that justifies the introduction of a rapid and

specific real-time PCR for HPeV that can be done in combination with EV

PCR for the diagnosis of viral meningitis or neonatal sepsis.

Acknowledgements

We thank Prof. G. Stanway from the Department of Biological Sciences at

the University of Essex (Colchester, UK) for providing HPeV4, 5 and 6

strains and Prof. H. Hyoty from the Department of Virology at the University

of Tampere (Tampere, Finland) for permission to use their HPeV6 strain.

Furthermore, we would like to thank the National Institute of Public Health

and the Environment (RIVM), the Department of Virology of the Utrecht

Medical Center (Utrecht, The Netherlands) and the Municipal Health Service,

Amsterdam for providing the prototype HPeV1 and 2, EVs, HRVs and HAV

strains. We would also like to thank Rene Minnaar for outstanding technical

assistance.

REFERENCES

Abed Y, Boivin G. Human parechovirus types 1, 2 and 3 infections in

Canada. Emerg Infect Dis 2006;12:969–75.

Al-Sunaidi M, Williams CH, Hughes PJ, Schnurr DP, Stanway G.

Analysis of a new human parechovirus allows the definition of

Parechovirus types and the identification of RNA structural domains. J

Virol 2006;81(2):1013–21.

Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-Van Dillen PM,

van der Noordaa J. Rapid and simple method for purification of nucleic

acids. J Clin Microbiol 1990;28:495–503.

Boom R, Sol C, Weel J, Gerrits Y, de Boer M, Wertheim-Van Dillen PM.

A highly sensitive assay for detection and quantitation of human

cytomegalovirus DNA in serum and plasma by PCR and

electrochemiluminescence. J Clin Microbiol 1999;37:1489–97.

Beld M, Minnaar R, Weel J, Sol C, Damen M, van der Avoort H, et al.

Highly sensitive assay for detection of enterovirus in clinical specimens by

Chapter 3

62

reverse transcription-PCR with an armored RNA internal control. J Clin

Microbiol 2004;42:3059–64.

Benschop KS, Schinkel J, Minnaar RP, Pajkrt D, Spanjerberg L,

Kraakman HC, et al. Human parechovirus infections in Dutch children

and the association between serotype and disease severity. Clin Infect

Dis 2006a;42:204–10.

Benschop KSM, Schinkel J, Luken ME, van den Broek PJM, Beersma

MFC, Menelik N, et al. Fourth human parechovirus serotype. Emerg

Infect Dis 2006b;12:1572–5.

Boivin G, Abed Y, Boucher FD. Human parechovirus 3 and neonatal

infections. Emerg Infect Dis 2005;11:103–5.

Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ,

Kaczmarski EB, et al. Development and evaluation of a ‘real-time’ RT-

PCR for the detection of enterovirus and parechovirus RNA in CSF and

throat swab samples. J Med Virol 2002;67:555–62.

Diaco R. Practical considerations for the design of quantitative PCR assays.

In: Innis MA, Gelfand DH, Sninky JJ, editors. PCR strategies. NewYork,

NY: Academic Press, Inc.; 1995. p. 84–108.

Figueroa JP, Ashley D, King D, Hull B. An outbreak of acute flaccid


1989;29:315–9.

Foray S, Pailloud F, Thouvenot D, Floret D, Aymard M, Lina B.

Evaluation of combining upper respiratory tract swab samples with

cerebrospinal fluid examination for the diagnosis of enteroviral meningitis

in children. J Med Virol 1999;57:193–7.

Ito M, Yamashita T, Tsuzuki H, Takeda N, Sakae K. Isolation and

identification of a novel human parechovirus. J Gen Virol 2004;85:391–8.

Legay V, Chomel JJ, Lina B. Specific RT-PCR procedure for the detection

of human parechovirus type 1 genome in clinical samples. J Virol

Methods 2002;102:157–60.

Nigrovic LE, Chiang VW. Cost analysis of enteroviral polymerase chain

reaction in infants with fever and cerebrospinal fluid pleocytosis. Arch

Pediatr Adolesc Med 2000;154:817–21.

Oberste MS, Maher K, Pallansch MA. Specific detection of echoviruses 22

and 23 in cell culture supernatants by RT-PCR. J Med Virol 1999;58:178–

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Rotbart HA. Enteroviral infections of the central nervous system. Clin Infect

Dis 1995;20:971–81.

Schnurr D, Dondero M, Holland D, Connor J. Characterization of

echovirus 22 variants. Arch Virol 1996;141:1749–58.

HPeV real time PCR

63

Watanabe K, Oie M, Higuchi M, Nishikawa M, Fujii M. Isolation and

characterization of novel human parechovirus from clinical samples.

Emerg Infect Dis 2007;13:889–95.

Human Parechoviruses as an Important

Viral Cause of Sepsislike Illness and

Meningitis in Young Children

K. C. Wolthers1, K. S. M. Benschop1, J. Schinkel1,

R. Molenkamp1, R. M. Bergevoet1, I. J. B. Spijkerman 3,

H. C. Kraakman4, and D. Pajkrt2

Clinical Infectious Diseases 2008; 47:356-363


Center, Amsterdam. 2 Dept. of Pediatric Infectious Diseases, Emma Children’s Hospital,

Academic Medical Center, Amsterdam. 3 Dept. of Medical Microbiology, Onze Lieve Vrouwe Gasthuis, Amsterdam.

4 Dept. of Pediatrics, Onze Lieve Vrouwe Gasthuis, Amsterdam.

67

Human Parechoviruses as an Important Viral Cause of

Sepsislike Illness and Meningitis in Young Children

Background. Enteroviruses (EVs) belong to the family Picornaviridae

and are a well-known cause of neonatal sepsis and viral meningitis.

Human parechoviruses (HPeVs) type 1 and 2, previously named

echovirus 22 and 23, have been associated with mild gastrointestinal

or respiratory symptoms in young children. Six HPeV genotypes are

currently known, of which HPeV3 is associated with neonatal sepsis

and meningitis.

Methods. Cerebrospinal fluid samples from children aged <5 years

previously tested by EV-specific polymerase chain reaction (PCR)

during 2004–2006 were selected (n=761). Samples from 716 of those

children were available for retrospective testing by HPeV-specific real-

time PCR. The prevalence of EV and HPeV in these samples was

compared. Data on clinical presentation of children infected with HPeV

were retrospectively documented.

Results. HPeV was found in cerebrospinal fluid samples from 33 (4.6%)

of the children. Yearly prevalence of HPeV in cerebrospinal fluid varied

remarkably: 8.2% in 2004, 0.4% in 2005, and 5.7% in 2006. EV was

detected in 14% (108 of 761 samples), with no variation in yearly

prevalence. Children with HPeV in cerebrospinal fluid presented with

clinical symptoms of sepsislike illness and meningitis, which led to

hospitalization and antibiotic treatment.

Conclusion. EV-specific PCRs do not detect HPeVs. The addition of an

HPeV-specific PCR has led to a 31% increase in detection of a viral

cause of neonatal sepsis or central nervous system symptoms in

children aged <5 years. HPeV can be considered to be the second

cause of viral sepsis and meningitis in young children, and rapid

identification of HPeV by PCR could contribute to shorter duration of

both antibiotic use and hospital stay.

INTRODUCTION

Enteroviruses (EVs) belong to the family Picornaviridae and are a well-

known cause of sepsis and meningitis in young children [1, 2]. Two former

EV serotypes, known as echovirus 22 and echovirus 23, have been

reclassified in the newly assigned genus Parechovirus of the Picornaviridae

as human parechovirus (HPeV) types 1 and 2 [3]. New HPeV types 3–6

were recently identified in Japan, The Netherlands, and the United States

Chapter 4

68

[4–7]. HPeV1 is considered to be a widely spread pathogen that affects

mainly young children [8, 9]. HPeV1 infections are most commonly

associated with mild gastrointestinal or respiratory symptoms. HPeV2

infections rarely occur and are associated mostly with gastrointestinal

symptoms [10]. HPeV3 has been associated with more-severe disease, such

as neonatal sepsis and meningitis [11, 12]. Symptoms of CNS involvement,

as with encephalitis and paralysis, have been reported for HPeV1 as well [13,

14] but have been reported less frequently than have infections with EV [15]

or HPeV3 [12, 16]. To date, hardly any clinical data are available on the

more recently discovered HPeV types 4, 5, and 6.

PCR based on the highly conserved 5’untranslated region has been to

shown to be a rapid and sensitive method for diagnosing EV as a cause of

meningitis and sepsis [2, 17, 18]. However, molecular assays for diagnosing

EV will not detect HPeV, because of the lack of sequence conservation

between HPeV and EV at the 5’end of the genome [19, 20]. Therefore,

infections in which HPeV causes severe disease like meningitis or sepsis

may be underdiagnosed, because viral culture of CSF is insensitive and

culture of stool samples or throat swabs is often not performed. Therefore,

the relative contribution of HPeV, compared with EV, as a causative agent of

viral meningitis or sepsislike illness in children is unknown.We developed a

real-time TaqMan PCR assay directed at the 5’untranslated region, to detect

HPeV directly from clinical samples [21]. Here, we retrospectively studied the

prevalence of HPeV in CSF samples from children obtained during 2004–

2006, and we studied the clinical symptoms associated with HPeV detection

in CSF.

METHODS

Clinical specimens

Since 2004, CSF samples that had been referred to the Laboratory of

Clinical Virology for viral diagnostics were routinely stored at -80oC. CSF

samples from children <5 years of age previously tested for EV by RT-PCR

were selected (840 samples obtained from 761 children). Available

complementary DNA (cDNA) samples were retrospectively tested for HPeV

(793 samples obtained from 716 children).

RNA extraction

CSF samples (200 µL) were extracted, as described by Boom et al. [22],

with use of 20 µL of sizefractionated silica particles in combination with 900

µL of lysis buffer L6. Samples were coextracted with 6250 copies armored

Human parechovirus infection in CSF

69

internal control RNA, corresponding to 500 copies internal control cDNA in

PCR [23]. RNA was eluted in 100 µL of Tris-EDTA buffer.

Detection of EV by 5’untranslated region RT-PCR

Forty microliters of extracted RNA was used for reverse transcription, as

described elsewhere [23], with use of random hexamers (Roche

Diagnostics). Twenty-five microliters of the cDNA sample was used for the

EV-specific end-point PCR, as described by Beld et al. [23]. The remaining

25 µL of cDNA sample was stored at -80oC.

Detection of HPeV by real-time PCR

Five microliters of the initial cDNA sample was used for the previously

described HPeV-specific single-target real-time PCR [21]. The HPeV PCR

was performed in a 25-µL volume containing 900 nM of each primer

(ParechoF31 and K30) [20], 200 nM of the HPeV-WT MGB probe (Applied

Biosystems), 400 ng/µL of bovine alpha-caseine (lot number 17H9551;

Sigma), 1x TaqMan universal PCR mastermix (Applied Biosystems), and 5

µL of the RT reaction. The PCR was performed in an Applied Biosystems

7000 sequence analyzer, as follows: 2 min at 50oC and 10 min at 95

oC,

followed by 45 cycles, each consisting of 15 s at 95oC and 1 min at 60

oC.

Clinical data

Data on clinical presentation of children infected with HPeV were

retrospectively collected using a questionnaire. The patient’s age at time of

virus isolation, sex, hospital or ward of admission, and duration of hospital

stay were documented. Letters of discharge and medical records were used

to document data on presence and duration of fever (temperature, >38oC),

irritability (as judged by the examining physician), sepsislike illness (fever or

hypothermia with signs of circulatory and/or respiratory dysfunction defined

by tachycardia or bradycardia, low blood pressure, and decreased

saturation), neurological symptoms (clinically suspected meningitis,

encephalitis, seizures, or paralysis), cell count determined in sample

obtained by lumbar puncture (cell count, <10 cells/ mm3), CSF protein level

(<0.35 g/L), glucose level (2.8–4.4 mmol/L), presence of other microbial

pathogens in CSF, and abnormalities revealed on diagnostic imaging of the

brain. In addition, symptoms of respiratory infections (rhinorrhea, cough,

tachypneu, apneu, wheezing, and/or abnormalities on radiograph of the

thorax), and symptoms of gastrointestinal infections (diarrhea and/or

vomiting, alone or in combination with abdominal distension), rashes, use of

antibiotics, and diagnosis at discharge were recorded. If the presence or

Chapter 4

70

absence of a specific symptom was not clearly mentioned in the medical

record or letter of discharge, the symptom information was labeled as

“missing.”

Statistical analysis

Analysis was performed using SPSS for Windows, version 12.1 (SPSS).

Statistical analysis was performed on the first sample available per child.

The Mann-Whitney U test was used to compare age distribution between

categorical variables. Categorical variables were analyzed by Chi2 test. A P

value <.05 was considered to be significant.

RESULTS

HPeV and EV detection in CSF samples

During 2004–2006, 761 children <5 years of age were tested, by PCR, for

EV in their CSF, and 108 children had positive results (table 1). At the time

of our study, samples from 716 children were still available for HPeV testing;

33 children had test results positive for HPeV. Double infections were not

observed. HPeV infection and EV infection were found in very young

children, as presented in table 1. Ninety-seven percent of the children

infected with HPeV were <24 months of age, and 46% were neonates (age,

<28 days). For children infected with EV, 95% were <24 months of age, and

50% were neonates. There was no statistical difference in age between

children positive for HPeV or children positive for EV. Of note, most samples

(92.5%) that had been sent to the laboratory were obtained from children <2

years of age.

The majority of the children infected with HPeV or EV were boys (70% and

62%, respectively) (table 1). When the fact that 61% of the children tested

were boys was taken into account, we could not find a higher risk for boys

becoming positive for either HPeV or EV (OR, 0.9; 95% CI, 0.6–1.3).

Table 1. Characteristics of children tested for HPeV and/or EV.

Characteristic Total group

(n=761)

HPeV pos

(n=33a)

EV pos

(n=108)

Age, median months (IQR) 0,9 (0,3-5,0) 1,2 (0,6-2,6) 0,9 (0,4-1,8)

Sex, no. (%)

Female (%)

294 (39)

10 (30)

41 (38)

Male (%) 467 (61) 23 (70) 67 (62)

IQR: 25-75% interquartile range. a33 out of 716 children tested for HPeV.


71

Table 2. HPeV and EV prevalence in CSF per year.

Virus(es) Proportion (%) of virus positive-patients positive in:

2004 2005 2006 Total

HPeV 16/196 (8,2) 1/239 (0,4) 16/281 (5,7) 33/716 (4,6)

EV 31/216 (14,4) 37/262 (14,1) 40/283 (14,1) 108/761 (14,2)

The yearly prevalence of EV in CSF was 14% during 2004–2006 (table 2). In

contrast, the yearly prevalence of HPeV in CSF varied considerably. HPeV

was detected in 8.2% and 5.7% of patients during 2004 and 2006,

respectively, but was detected in only 0.4% of patients during 2005. Figure 1

illustrates the monthly prevalence of HPeV and EV in CSF during 2004–

2006. Yearly and seasonal distribution varied between EV and HPeV. EV

infections of the CNS could be found throughout the year but were most

prevalent in summer and fall, with the highest peak occurring in October

2004 (50% of patients tested positive). HPeV infections of the CNS were

observed in spring, summer, and fall, with the highest peak in May 2004

(21% of patients tested positive), whereas no HPeV infections were detected

in December or January of 2004, 2005, or 2006. During 2004–2006, HPeV

was detected in 4.6% of the CSF samples, compared with detection of EV in

14.2% of samples (table 2). The percentage of infected children who had

HPeV infection was 23.4% (33 of 141 children with either EV or HPeV

infection) (table 2). By use of the HPeV-specific PCR, the percentage of

0

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HPEV

Figure 1. Monthly prevalence of HPeV and EV in CSF. The y-axis shows the monthly

prevalence as percentage of positive patients per month for HPeV (hatched) and EV

(black) in the total group of patients tested.

Chapter 4

72

children positive for infection increased by 31% (i.e., 33 HPeV-positive

children in addition to the 108 EV-positive children reported at the time of

their illnesses).

Clinical characteristics of children with HPeV in CSF

Clinical data about the children who tested positive for HPeV in CSF were

available for 29 children (88%; 10 girls and 19 boys). The majority of the

children were born at term (23 children [79%]) and were healthy before

admittance to the hospital (25 children [86%]). Twenty (69%) of the 29

children were admitted to a general hospital, and 9 (41%) were admitted to

the academic hospital (2 of whom were admitted to an intensive care unit).

The mean duration of hospital stay was 7 days, and 82% of the children

were given antibiotics for a mean of 5.7 days (table 3). Fever was present in

97% of the children, and irritability was described by the examining

pediatrician in 86% of the children. Fifteen (54%) of the children showed

signs of sepsislike illness, and another 6 children (21%) received a diagnosis

of sepsislike illness from the examining pediatrician (“suspected SLI”) but did

Table 3. Clinical characteristics of 29 patients with human parechovirus in CSF.

Variable Finding

Age, months

Mean

Median (range)

3.7

1.2 (0.2-58)

Hospital stay, days

Mean

Median (range)

7.2

5.0 (1.0-39)

Antibiotic treatment 23/28 (82)

Duration of antibiotic treatment, days

Mean

Median (range)

5.7

7.0 (3.0-10)

Fever 28/29 (97)

Irritability 24/28 (86)

Sepsislike illness 15/28 (54)

Suspected sepsislike illness 6/28 (21)

Meningitis 3/26 (12)

Seizures 2/28 (7)

Encephalitis 1/26 (4)

Paralysis 1/27 (4)

CSF

Cell count, mean no. of cells/mm3 4.6 (3.1-22)

Normal glucose level 19/25 (76)

Elevated protein level 13/21 (62)

Rash 5/29 (17)

Gastrointestinal tract symptoms 11/28 (39)

Respiratory tract symptoms 10/28 (36)


73

not meet our definition of sepsislike illness. The maximum cell count in CSF

was 22 cells/mm3, and overall normal glucose values but elevated protein

levels were found in the CSF. Meningitis was diagnosed in 3 children (12%),

and encephalitis with seizures was diagnosed in 1 child (4%). In 1 child with

severe meningitis, cerebral infarction determined by CT of the brain could

not be explained by other causes. In 2 children, underlying disease was the

most likely cause for the scored symptoms in the questionnaires. Paralysis

was noted for 1 child, but that child received a diagnosis of neurological

trauma after an accident. One child with preexisting developmental brain

damage developed seizures. In those children, abnormalities revealed on

diagnostic imaging were found as expected, in accordance with the

underlying disease. Other clinical symptoms that were recorded were

symptoms of gastrointestinal infections (39%), respiratory infections (36%),

and rash (17%). Bacterial sepsis and meningitis were excluded on the basis

of negative blood and CSF culture results. In 1 of 2 CSF cultures performed

for one child, Staphylococcus epidermidis was found; in another child,

culture of skin samples were positive for Staphylococcus aureus. No other

pathogens were found in our study group.

DISCUSSION

Here, we show, in a retrospective analysis, that HPeV could be detected by

real-time PCR in 4.6% of CSF samples obtained from children <5 years of

age who were referred to our laboratory. Compared with EV infection, HPeV

infection of the CNS was detected less frequently in our study, which is in

agreement with previous observations [15, 24]. Children with HPeV in CSF

presented mainly with clinical symptoms of sepsis, accompanied by signs of

respiratory or gastrointestinal infection. CNS symptoms were reported to a

lesser extent, and CSF cell counts were not significantly increased. The

clinical presentation of children with HPeV in CSF described here closely

resembled the clinical symptoms described for EV infection in children

reported elsewhere [1, 25].

The mean duration of hospital stay for children with HPeV in CSF was 1

week, and antibiotic therapy was given to 82% of the children for at least 3

days. We demonstrated that the addition of HPeV-specific PCR increased

the detection of infection in children by 31% in our laboratory. It has been

shown that rapid diagnosis of EV infection by PCR can reduce hospital stay

and duration of antibiotic use [26–28]; therefore, the addition of an HPeV-

specific PCR could further reduce duration of antibiotic use and duration of

hospital stay in children with sepsislike illness or CNS symptoms. However,

Chapter 4

74

additional studies are needed to determine the effect of rapid viral diagnosis

on reduction of hospital stay and reduced use of antibiotics.

Only a few reports have been published on the epidemiology of HPeVs, and

these are all based on HPeV detection by cell culture. HPeV infections are

considered to be widely prevalent, mainly affecting children [7–10, 16, 29].

For HPeV1, it has been shown that a minority of patients show signs of CNS

involvement [9, 15]. Recent studies of HPeV3 all show an association with

more-severe disease than HPeV1 infection - that is, neonatal sepsis,

meningitis, and paralysis [4, 7, 12, 16]. In our study, HPeV was not isolated

by cell culture; it was detected directly from CSF samples by real-time PCR

through use of primers and probes validated for all 6 known genotypes [21].

Previously, 1 other study detected HPeV directly from CSF samples by real-

time PCR, reporting that 1% of the samples were positive for HPeV [24]. In

that study, CSF samples that were negative for meningococcal PCR or were

negative in cell culture but that were suspected to be positive for viral

meningitis were selected without age restriction, decreasing the likelihood of

finding HPeV. In addition, Corless et al. [24] might have missed HPeVs,

because the probe of their real-time PCR, which was described in 2002,

shows mismatches with the newer HPeV types 3–6.

We showed that the yearly prevalence of HPeV in CSF varied remarkably, in

contrast to EV, for which the yearly prevalence in CSF was stable during

2004–2006. The difference in yearly prevalence of HPeV could be

attributable to absence of HPeV circulation during 2005. However, we

cannot exclude the possibility that our HPeV-specific PCR was not able to

detect an unidentified HPeV that could have been circulating in 2005. Both

possibilities seem unlikely, because HPeV could be detected in feces

samples from 2005 [30]. However, different HPeV genotypes might circulate

in different years or seasons, as has been suggested elsewhere [7, 10, 12,

29]. Thus, it could be anticipated that HPeV types able to infect the CNS -

presumably HPeV3 - circulate only in specific years. Whether the HPeVs

found in the CSF will be predominantly HPeV3 needs to be further

elucidated. Genotyping of HPeV can be done by sequencing of the VP1

gene [6, 12], but this has been performed only on HPeV isolates obtained

from cell culture. The combination of small volumes of CSF obtained from

young children and insensitivity of the technique limited the possibility of

genotyping in our study.

There are other potential limitations to our study. First, the samples selection

is not random but biased, from a pediatric population referred for virological

testing. To circumvent this bias, we compared HPeV prevalence with that of

EV, the most important viral cause of neonatal sepsis and meningitis.


75

Another potential bias the inability to test 45 of our samples for HPeV (29

EV-positive and 16 EV-negative samples). Exclusion of these samples from

the analysis would thus underestimate the EV prevalence. If the 29 EV-

positive samples were considered to be HPeV negative, the HPeV

prevalence in the total study group would vary between 4.3% (if all 16 EV-

negative samples were HPeV negative) and 6.4% (if all 16 EV-negative

samples were HPeV positive). Therefore, the HPeV prevalence of 4.6%

could be a slight over- or underestimation of the real prevalence in our study

group. Despite these limitations, we conclude that HPeV is another important

cause of viral sepsis and meningitis in young children that has frequently

been undetected. PCR, together with a sensitive molecular typing method,

will elucidate further the epidemiology of HPeV relative to clinical symptoms

and genotypes. HPeV-specific PCR should be included in viral diagnostic

testing for CSF samples and needs to be further evaluated for use in other

clinical samples, such as blood, throat swabs and feces.

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Rapid detection of human parechoviruses in clinical samples by

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24. Corless CE, Guiver M, Borrow R, et al. Development and evaluation

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Nurnberg: Eurovirology, 2007:91.

High Prevalence of Human

Parechovirus (HPeV) Genotypes in the

Amsterdam Region and Identification

of Specific HPeV Variants by Direct

Genotyping of Stool Samples

K. Benschop, X. Thomas, C. Serpenti, R. Molenkamp, and

K. Wolthers

Journal of Clinical Microbiology 2008; 46 (12):3965-3970


Center, Amsterdam.

81

High Prevalence of Human Parechovirus (HPeV) Genotypes

in the Amsterdam Region and Identification of Specific

HPeV Variants by Direct Genotyping of Stool Samples

Human parechoviruses (HPeV) are widespread pathogens belonging to

the Picornavirus family. Six genotypes, which have predominantly

been isolated from children, are known. Data on prevalence of HPeV

genotypes are generally based on cell culture, which may

underestimate the prevalence of certain HPeV strains that are difficult

to grow. We studied 1,824 stool samples from 1,379 children (<5 years

old) sent to the Academic Medical Center, Amsterdam, The

Netherlands, between 2004 and 2006. Samples were screened using

specific human enterovirus (HEV) and HPeV real-time PCRs based on

the 5’untranslated region. A high percentage of HPeV infections

(16.3%), comparable to the percentage of HEV infections (18.4%), were

found by PCR in stool samples. HPeV-positive stool samples were

directly genotyped based on the VP1 region for the first time to avoid a

culture bias. HPeV1 was found to be the most prevalent type. The

majority of the HPeV1 strains clustered separately from the prototype

strain, Harris, which has not been reported to circulate lately. However,

we could identify three strains as HPeV1 Harris. HPeV3 was identified

as the second most predominant type during 2004 and 2006 but was

not found in 2005. HPeV4 to -6 were found in smaller numbers. One

strain could not be associated with a known HPeV type (VP1 gene

nucleotide similarity: 71%), possibly indicating a new genotype. Also,

we report the first identification of three HPeV5 strains and one HPeV1

strain with a different motif at the C-terminal end of VP1, where the

arginine-glycine-aspartic acid (RGD) motif is normally located.

INTRODUCTION

Human parechovirus 1 (HPeV1) and HPeV2 were first identified in the mid-

1960s as human enteroviruses (HEVs) and were classified as echovirus 22

and 23 (30). Sequence analyses showed that these viruses were genetically

distinct from the entire Enterovirus genus, and they were reclassified as the

new genus Parechovirus in the family Picornaviridae (24). There are now six

known HPeV genotypes (2, 8, 16, 29), which have been isolated mainly from

young children. Infections with HPeVs have been shown to be widespread

(17, 18, 25), but different HPeV genotypes were found to differ

epidemiologically and clinically from each other (1, 6, 29).

Chapter 5

82

Epidemiological data on HPeV prevalence are predominantly based on cell

culture isolation of different HPeV strains. As HPeVs, in particular HPeV3,

are found to be difficult to culture in standard diagnostic cell lines and their

isolation is largely determined by the cell lines used (1, 29, 30), the exact

HPeV prevalence might be underestimated.

Recent studies show that HPeV1 is still the most frequently isolated (1, 3, 6,

13, 29). However, genotyping showed the recently circulating HPeV1 strains

to be phylogenetically distinct from the prototype strain, designated Harris,

first isolated in 1956 (1, 6, 12, 29). Both the Harris strain and the recently

isolated HPeV1 strains can be serotyped using antisera directed against the

original Harris strain (1). Since the older studies depended on serotyping

methods, while the more recent studies are based on genotyping, it is

difficult to establish the time frame in which the prototype strain ceased

circulation. The benefits of molecular typing instead of classical serotyping

methods to type different HPeVs have already been proven by the

reclassification of some HEVs as HPeVs (17, 26). In addition, genotyping

has led to the reclassification of the second HPeV2 serotype, represented by

the CT86-6760 strain (20), as the HPeV5 genotype (2), following the

identification of HPeV3 and -4 (2, 8, 16).

Infections with HPeV3 have been associated with more severe symptoms

than other HPeV infections, and HPeV3 has been reported to be either the

second most predominant strain (1, 6, 16) or equal to HPeV1 in prevalence

(13, 29). As HPeV3 was found to display different cell tropisms in different

cell culture assays (1, 29) and as these studies were based on cell culture

isolation of HPeVs, the use of different cell lines within different studies might

account for these discrepancies.

Direct screening and typing of clinical samples would exclude such cell

culture bias and therefore would provide a better estimation of the

prevalences of different HPeV types. In addition, by screening over 700

cerebrospinal fluid (CSF) samples by real-time PCRs, HPeVs were identified

in almost 5% of the children under the age of 5 years, in comparison to 14%

for HEVs (5, 31). Since higher prevalences were found in 2004 and 2006

than in 2005, a 2-year cycle was suggested for HPeVs with neuroinvasive

characteristics. Unfortunately, due to low HPeV viral load these CSF

samples could not be genotyped. In order to gain insight into the circulation

of different HPeV types within the Amsterdam region in those years, we

retrospectively screened and directly typed stool samples from children

under the age of 5 years from 2004 to 2006 by realtime PCR.

HPeV prevalence by direct genotyping

83

METHODS

Clinical samples

Stool samples from children under the age of 5 years obtained between

2004 and 2006 from the Amsterdam region were collected. Stool samples

were suspended in 2% broth (Oxoid, Drongen, Belgium) and stored at -

20°C. In total, 1,824 stool samples from 1,379 children were available for

testing (808 boys and 571 girls).

RNA extraction

Broth-suspended stool samples (50 µl) were incubated for 10 min in lysis

buffer L6 (10) and centrifuged for 2 min to remove any stool debris. The

supernatant was further extracted using 20 µl of size-fractionated silica

particles in combination with 900 µl of lysis buffer L6 as previously described

by Boom et al. (9, 10). When testing for the presence of an HPeV and/or

HEV infection, samples were coextracted with 6,250 copies of armored RNA

of an internal control (IC), corresponding to 500 copies of IC cDNA in PCR

(4). The IC was omitted from extraction when genotyping by sequencing of

the VP1 region had to be performed (6). During the study period we changed

from manual extraction to automatic extraction using the total nucleic acid

isolation kit with the MagnaPure LC instrument (Roche Diagnostics, Almere,

The Netherlands). Retesting of samples showed 100% recovery compared

to the manual extraction. RNA was eluted in 50 µl of Tris-EDTA buffer in

both extraction methods.

Detection of HPeV and HEV by real-time reverse transcription-PCR (RT-

PCR)

Forty microliters of extracted RNA was used for reverse transcription using

random hexamers (Applied Biosystems, Niewerkerk a/d IJssel, The

Netherlands) as previously described (4). Five microliters of cDNA was used

for both the HEV-specific duplex assay and the HPeV-specific single-target

assay (5). The HEV-specific duplex assay was performed in a 25 µl volume

containing 900 nM of each primer (entero-1-TM and entero-2-TM [5]), 200

nM of the EV-WT-MGB probe and IC-MGB probe (5), 400 ng/µl of bovine α-

casein (lot number 17H9551; Sigma, Zwijndrecht, The Netherlands), and 1x

TaqMan universal PCR master mix (Applied Biosystems). The HPeV-specific

single-target assay was performed in a 25-µl volume containing 900 nM of

each primer (ParechoF31 [5] and K30 [20]), 200 nM of the HPeV-WT-MGB

probe (5), 400 ng/µl of bovine α-casein (Sigma), and 1x TaqMan universal

PCR master mix (Applied Biosystems). The primers and probes were

Chapter 5

84

obtained from Biolegio (Nijmegen, The Netherlands) and Applied

Biosystems, respectively. The real-time PCRs were performed in an Applied

Biosystems 7000 sequence analyzer as follows: 2 min at 50°C and 10 min at

95°C, followed by 45 cycles, each consisting of 15 s at 95°C and 1 min at

60°C.

Genotyping by VP1 sequencing

The previously described one-step VP1 RT-PCR (6) was adapted into a two-

step assay. As the previously described primer set might not detect HPeV5

(13), a second primer set was designed to include degeneracy to all known

HPeV types (VP1-parEchoF12 [5’-CCA RAA YTC ITG GGG YTC-3’] and

VP1-parEchoR12 [5’-AAI CCY CTR TCY ARR TAW GC-3’]). Two hundred

sixty-one HPeV-positive samples from 216 children could be retrieved for

typing, of which 168 samples from 130 children could be successfully

genotyped using both primers sets. Forty microliters of newly extracted RNA

(omission of IC) was used for reverse transcription as previously described

using random hexamers (4). Twenty-five microliters of cDNA was used for

the VP1 PCR. The PCR was performed in a 50-µl volume containing 1x PCR

II buffer (Applied Biosystems); 200 µmol/liter each of dATP, dCTP, and

dGTP (Applied Biosystems); 400 µmol/liter dUTP (Applied Biosystems); 0.1

µg/µl bovine serum albumin (Roche Diagnostics); 400 ng/µl α-casein; 1 µM

VP1-parEchoF1/VP1-parEchoF12 and 1 µM VP1-parEchoR1/VP1-

parEchoR12 (6); and 2.5 U of Amplitaq Gold (Applied Biosystems). The final

MgCl2 concentration was 2.5 mmol/liter. The HPeV VP1 amplicons were gel

purified and sequenced as previously described (6).

Phylogenetic and statistical analysis

The sequences were analyzed on an ABI 3730/3100 DNA analyzer (Applied

Biosystems). Sequences were aligned using Clustal-W (28), included in the

VectorNTI suite 10 software package (Invitrogen) and edited using

Simmonics v1.62 (http://www2.warwick.ac.uk/fac/sci/bio/research/devans/

bioinformatics/simmonics, 23). Phylogenetic analyses were performed by the

neighbor-joining method (22), as implemented in the Molecular Evolutionary

Genetics Analysis software package, version 3.1 (19). P-distances were

estimated for amino acid sequences. One thousand bootstrap replicates

were analyzed. The HPeV genotype was assigned on the basis of

phylogenetic clustering. HPeVs from nine children were previously typed

from culture isolates obtained between 2000 and 2005 (6). The HPeV type

was confirmed by genotyping the original stool samples.


85

Statistical analysis was performed using SPSS 12.1 for Windows based on

the number of children. Children with more than one sample were identified

as follow-up subjects if samples were less than 11 weeks apart (11). In

addition, infections of children from whom multiple samples containing

identical sequences were obtained were characterized as single HPeV

infections. To compare age distributions, we used the Wilcoxon-Mann-

Whitney test. To compare HPeV and HEV prevalences over the years, we

used the chi-square distribution with 95% confidence intervals (CI).

Nucleotide sequence accession numbers

The nucleotide sequences of the VP1 gene region are deposited in

GenBank under accession numbers FJ373059 to FJ373179.

RESULTS

HPeV in stool samples from 2004 to 2006

Stool samples (n = 1,824) from 1,379 children under the age of 5 years were

obtained from 2004 to 2006 and screened retrospectively using HPeV- and

HEV-specific real-time PCRs. HPeV infections were detected in 270 samples

obtained from 225 (16.3%) children, and HEV infections were detected in

265 samples from 253 (18.4%) children (Table 1). Forty-one children

showed a double infection with HPeV and HEV (Table 1).

The yearly prevalences for both HPeV and HEV varied during the 3 years

studied. HPeV was detected in 17.5% (80/456), 13.2% (59/447), and 18.2%

(86/476) of the children in 2004, 2005, and 2006, respectively (Table 1).

HEV was detected in 20% (91/456), 14.2% (65/447), and 20.5% (97/476) of

the children, respectively, in these 3 years (Table 1). The lower prevalence

observed in 2005 was not significant for HPeV (P=0.09) and was just

significant for HEV (P=0.04).

Table 1. Human Parechovirus (HPeV) and Human Enterovirus (HEV) infections detected in

stool samples.

Number (%) of virus-positive patients in:

Virus(es) 2004

(n=456)

2005

(n=447)

2006

(n=476)

Total

(n=1379)

HPeV 80 (17.5) 59 (13.2) 86 (18.2) 225 (16.3)

HEV 91 (20) 65 (14.5) 97 (20.6) 253 (18.4)

HPeV and HEV 12 (2.6) 8 (1.8) 21 (4.4) 41 (2.9)

Chapter 5

86

Table 2. Number of neonates (< 28 days) found positive for Human Parechovirus (HPeV) and

Human Enterovirus (HEV).

Virus(es) No. of virus-positive neonates/no. tested (%) in:

2004 2005 2006 total

HPeV 9/80 (11.3) 2/59 (3.4) 7/86 (8.1) 18/225 (8.0)

HEV 16/91 (17.6) 8/65 (12.3) 12/97 (12.4) 36/253 (14.2)

The majority of the children infected with HPeV (84%, 189/225) were <2

years old, and 8% (18/225) were neonates (<28 days old). Of the children

infected with HEV, 77% (196/253) were <2 years old and 14% (36/253) were

neonates. As shown in Table 2, the number of neonates infected with HPeV

was much lower in 2005 than in 2004 and 2006.

We found more boys infected with either HPeV or HEV than girls. However,

taking into account that the ratio of boys to girls tested (1.4:1) was similar to

the ratios of HPeV- and HEV-positive boys to girls (1.4:1 and 1.3:1,

respectively), the relative risk (RR) for infection of boys with either virus was

not higher than that for girls (RRHEV = 1.1, 95% CI, 0.9 to 1.4; RRHPeV = 1.0,

95% CI, 0.8 to 1.3).

Prevalence of HPeV types

In total, 168 samples from 130 children were successfully genotyped directly

from stool samples as either HPeV1, -3, -4, -5, or -6 (Fig. 1). HPeV1 was the

predominant type identified in all 3 years; 64.6% (84/130) of the children

were found to be infected with strains of this type (Fig. 2). During 2004 and

2006, HPeV3 was identified as the second most predominant type (22.3%,

29/130). However, HPeV3 was not identified in 2005, nor was HPeV4 (Fig.

2). In total, HPeV4 was found in 9 children (7%). HPeV5 and -6 were

identified at least once a year in the 3 years studied.

HPeVs could be isolated during the entire year. However, differences in

seasonal distribution between the genotypes could be observed. HPeV1, -4,

-5, and -6 were mostly identified during autumn and winter. However, HPeV3

was mainly found in summer. Interestingly, all HPeV types were less

prevalent in spring.

In comparison to HPeV1 infections, more HPeV3 infections were found at

the younger ages. For HPeV3, 66% (19/29) of the infected children were

younger than 6 months, of which 24% (n = 7) were less than 28 days old. In

contrast, for HPeV1, only 40% (34/84) of the infected children were younger

than 6 months, of which 2.3% (n = 2) were neonates. Of the 261 samples

available for typing, we were unable to type 93 samples from 86 children.

Fifty samples from 49 children were found to have a low viral load (cycle


87

Figure 1. Rooted phylogenetic tree based on amino acid differences in the capsid protein VP1

(236 aa). The tree was constructed by using the neighbor–joining method. Numbers represent

the frequency of occurrence of nodes in 1000 bootstrap replicas. The Dutch strains sequenced

within this study contain a 6 digit numbering, and have been color-coded by year of isolation

(2004: red, 2005: green, and 2006: blue). The different HPeV genotype clusters have been

color-coded within the tree: HPeV1 (purple), HPeV2 (orange), HPeV3 (light brown), HPeV4

(light blue), HPeV5 (pink), HPeV6 (light green). As an outgroup, Ljungan Virus 145 SL

(AF327922) was used. Reference strains and isolates were obtained from GenBank: HPeV1

Harris (S45208), BNI788st (EF051629), 450343 (DQ172430), 550163 (DQ172425), 450976

(DQ172417), 451294 (DQ172440), 452176 (DQ172431), 4522252 (DQ172435), A317/99,

A354/99, A628/99, A1086/99, A10987/00 (AB112482-AB112487), A301/01 (AB300943)

650648

651654

652780

651128

651625

652920

674450

550505

451729

651898

452786

550182

550193

HPeV1 452176

450111

HPeV1 A151/05

HPeV1 A150/05

HPeV1 BNI-R21/03

452344

HPeV1 A486/00

HPeV1 A295/02

HPeV1 A136/02

HPeV1 A241/05

HPeV1 A191/05

550435

450734

650606

650750

HPeV1 A62/06

HPeV1 A65/05

HPeV1 A669/99

HPeV1 A942/99

676323

HPeV1 A573/00

HPeV1 A477/00

HPeV1 A248/04

HPeV1 A249/04

451858

HPeV1 450976

550332

HPeV1 A244/04

452530

550652

653096

HPeV1 BNI-R90/30

HPeV1 A1086/99

HPeV1

A10987

/99

452643

677008

652536

652643

652445

450296

550320

552314

650163HPeV1 45129

4

552154HPeV1 452252HPeV1 BNI788st

650164650258650989652568652499676406451821550252451302HPeV1 BNI-R32/03

452208HPeV1 BNI-R09/03

HPeV1 BNI-R04/03

HPeV1 A336/02

HPeV1 A351/04

677033

HPeV1 A329/04

HPeV1 A301/01

HPeV1 A708/99

HPeV1 A233/04HPeV1 A258/04

450308

HPeV1 A258/05

HPeV1 A234/05

HPeV1 A291/05

451125

650941HPeV1 BN

I-R15/03

551612

652467

452666

552444

451669

451660

550981

550290

450829

650081

552762

HPeV1 A584/00

650854

651108

650151

552001

652146

451894

HPeV1 A222/05

452476

HPeV1 550163

676271

HPeV1 A242/05

HPeV1 A177/03

450653

HPeV1 A177/01

HPeV1 A657/99

HPeV1 BNI-R30/03

651934

675458

551907

HPeV1 450343

450342

450559

450823

HPeV1 A322/04

HPeV1 A527/99

652281

HPeV1 HARRIS

452568

550328

HPeV6 NII561-2000

451701

650045

HPeV6 BNI67-03

HPeV6 2005/823

550389

HPeV6 A231/01

HPeV2 WILLIAMSON

552106

HPeV5 T82-659

HPeV5 T92-15

HPeV5 2000/1108

HPeV5 T83-2051

HPeV5 CT86-6760

HPeV5 T82-0619

676618

652444

452373

HPeV4 T75-4077

HPeV4 T82-203

653046

HPeV4 T73-838

652598

652872

652580

HPeV4 A374/06

452323

675149450369

HPeV4 K251176-02

45252445

2674451564

651689451

513675391

HPeV3

A1027/

99

HPeV3

A354/9

9451653

HPeV3 A3

17/99

HPeV3 A31

9/99HPeV3 A492

/99HPeV3 A628/9

9HPeV3 A531/99

HPeV3 A606/99

HPeV3 A683/99HPeV3 A153/04

HPeV3 A680/99HPeV3 A308/99HPeV3 A390/01

HPeV3 A141/02

HPeV3 A415/01

HPeV3 A256/06

HPeV3 A265/02

450900

451987

HPeV3 A188/05

HPeV3 A287/06

HPeV3 A246/06

HPeV3 A281/06

HPeV3 A264/06

HPeV3 A257/06

451297

HPeV3 A259/06

HPeV3 A320/06

HPeV3 A285/06

HPeV3 A255/06

HPeV3 A417/06

451377

652880

HPeV3 CAN82853-01

451692

HPeV3 451371

HPeV3 A225/06

652545

652761

676401

676053

674271

652649

677146

451550

451677

451393

451512

451935

451610

451425

451678

452000

HPeV3 450936

HPeV3 451517

Ljungan145SL

0.05

98

99

95

74

66

79

85

99

99

97

99

650648

651654

652780

651128

651625

652920

674450

550505

451729

651898

452786

550182

550193

HPeV1 452176

450111

HPeV1 A151/05

HPeV1 A150/05

HPeV1 BNI-R21/03

452344

HPeV1 A486/00

HPeV1 A295/02

HPeV1 A136/02

HPeV1 A241/05

HPeV1 A191/05

550435

450734

650606

650750

HPeV1 A62/06

HPeV1 A65/05

HPeV1 A669/99

HPeV1 A942/99

676323

HPeV1 A573/00

HPeV1 A477/00

HPeV1 A248/04

HPeV1 A249/04

451858

HPeV1 450976

550332

HPeV1 A244/04

452530

550652

653096

HPeV1 BNI-R90/30

HPeV1 A1086/99

HPeV1

A10987

/99

452643

677008

652536

652643

652445

450296

550320

552314

650163HPeV1 45129

4

552154HPeV1 452252HPeV1 BNI788st

650164650258650989652568652499676406451821550252451302HPeV1 BNI-R32/03

452208HPeV1 BNI-R09/03

HPeV1 BNI-R04/03

HPeV1 A336/02

HPeV1 A351/04

677033

HPeV1 A329/04

HPeV1 A301/01

HPeV1 A708/99

HPeV1 A233/04HPeV1 A258/04

450308

HPeV1 A258/05

HPeV1 A234/05

HPeV1 A291/05

451125

650941HPeV1 BN

I-R15/03

551612

652467

452666

552444

451669

451660

550981

550290

450829

650081

552762

HPeV1 A584/00

650854

651108

650151

552001

652146

451894

HPeV1 A222/05

452476

HPeV1 550163

676271

HPeV1 A242/05

HPeV1 A177/03

450653

HPeV1 A177/01

HPeV1 A657/99

HPeV1 BNI-R30/03

651934

675458

551907

HPeV1 450343

450342

450559

450823

HPeV1 A322/04

HPeV1 A527/99

652281

HPeV1 HARRIS

452568

550328

HPeV6 NII561-2000

451701

650045

HPeV6 BNI67-03

HPeV6 2005/823

550389

HPeV6 A231/01

HPeV2 WILLIAMSON

552106

HPeV5 T82-659

HPeV5 T92-15

HPeV5 2000/1108

HPeV5 T83-2051

HPeV5 CT86-6760

HPeV5 T82-0619

676618

652444

452373

HPeV4 T75-4077

HPeV4 T82-203

653046

HPeV4 T73-838

652598

652872

652580

HPeV4 A374/06

452323

675149450369

HPeV4 K251176-02

45252445

2674451564

651689451

513675391

HPeV3

A1027/

99

HPeV3

A354/9

9451653

HPeV3 A3

17/99

HPeV3 A31

9/99HPeV3 A492

/99HPeV3 A628/9

9HPeV3 A531/99

HPeV3 A606/99

HPeV3 A683/99HPeV3 A153/04

HPeV3 A680/99HPeV3 A308/99HPeV3 A390/01

HPeV3 A141/02

HPeV3 A415/01

HPeV3 A256/06

HPeV3 A265/02

450900

451987

HPeV3 A188/05

HPeV3 A287/06

HPeV3 A246/06

HPeV3 A281/06

HPeV3 A264/06

HPeV3 A257/06

451297

HPeV3 A259/06

HPeV3 A320/06

HPeV3 A285/06

HPeV3 A255/06

HPeV3 A417/06

451377

652880

HPeV3 CAN82853-01

451692

HPeV3 451371

HPeV3 A225/06

652545

652761

676401

676053

674271

652649

677146

451550

451677

451393

451512

451935

451610

451425

451678

452000

HPeV3 450936

HPeV3 451517

Ljungan145SL

0.05

98

99

95

74

66

79

85

99

99

97

99

Chapter 5

88

A177/01, A584/00, A573/00, A486/00, A477/00 (AB300937-AB300941), A708/99

(AB300935), A669/99 (AB300932), A657/99 (AB300931), A527/99 (AB300928),

A233/04, A244/04, A248/04, A249/04, A258/04, A322/04, A329/04, A351/04, A62/05,

A65/05, A150/05, A151/05 (AB300954-AB300965), A295/02, A336/02, A177/03

(AB300949-AB300951), A136/02 (AB300946), A347/06 (AB300985), A191/05, A222/05,

A229/05, A234/05, A241/05, A242/05, A258/05 (AB300966-AB300972), BNI-R90/03,

BNI-R04/03, BNI-R09/03, BNI-R15/03, BNI-R21/03, BNI-R30/03, BNI-R32/03

(EU024630-EU024636); HPeV2 Williamson (AF055846); HPeV3 A308/99 (AB084913),

CAN82853-01 (AJ889918), 451371 (DQ172449), 451517 (DQ172447), 450936

(DQ172446), A390/01 (AB300944), A415/01 (AB300945), A1027/99 (AB300936),

A683/99 (AB300934), A680/99 (AB300933), A606/99 (AB300930), A531/99

(AB300929), A492/99 (AB300927), A319/99 (AB300926), A153/04 (AB300952),

A141/02, (AB300947) A265/02 (AB300948), A471/06 (AB300986), A188/05, A225/06,

A246/06, A255/06, A257/06, A259/06, A264/06, A265/06, A281/06, A285/06, A287/06,

A320/06 (AB300973-AB300984); HPeV4 K251176-02 (DQ315670), T75-4077

(AM235750), T82-203 (AM234727), T73-838 (AM234725); HPeV5 CT86-6760

(AJ005695), T92-15 (AM235749), T820169 (AM234728), T82-659 (AM234726), T83-

2051 (AM234724); HPeV6 NII561-2000 (AB252582), BNI67-03 (EU024629), 2005/823

(EU077518), A231/01 (AB300942).

Figure 2. The prevalence, of the known Human Parechovirus (HPeV) types in 2004

(HPeV1, n=30; HPeV3, n=19; HPeV4, n=4; HPeV5, n=1 and HPeV6, n=1), 2005

(HPeV1, n=19; HPeV5, n=1 and HPeV6, n=1) and 2006 (HPeV1, n=35; HPeV3, n=10;

HPeV4, n=5; HPeV5, n=2; HPeV6, n=1) identified in the Amsterdam region. The

different types have been numerically numbered according to the HPeV genotype.


89

threshold [CT] value by real-time PCR > 38) and could therefore not be

typed. Forty-three samples from 37 children were found to have a high

enough viral load for genotyping based on the CT value by real-time PCR.

As stool samples can be very incongruent, these samples were tested

multiple times. However, after several tests, signs of degradation could be

observed, as the CT values increased considerably after freeze-thawing

steps (mean increase of CT value, 9; range, 7 to 11).

Phylogenetic characterization of HPeV types

Based on specific clustering of the isolates with known HPeV types obtained

from GenBank, we could identify HPeV genotypes 1, 3, 4, 5, and 6 (Fig. 1).

HPeV2 was not found. HPeV1 comprised the largest cluster. The majority of

the HPeV1 strains (n = 81) were found to cluster closely together with strains

identified in Japan (strains having five-digit numbers prefixed with “A”) (Fig.

1) and Germany (strains having three-digit numbers prefixed with “BNI-R”)

(Fig. 1) and formed a separate cluster from the prototype strain, identified in

1956 (Harris). The amino acid similarity between the recently isolated

HPeV1 strains, including those identified in Germany and Japan, and the

prototype strain, Harris, was only 89.2%. Three strains were found to cluster

closely with the Harris strain (amino acid similarity, 93.7%). We found no

specific geographical or temporal separation between the different HPeV1

strains (Fig. 1). HPeV3 comprised the second-largest cluster, containing 29

Dutch stains. As seen within the HPeV1 cluster, two separate lineages,

which previously had not been found, could be seen within the HPeV3

cluster based on the VP1 gene. The majority of the Dutch strains were found

to form a tight cluster with the Japanese strains (amino acid similarity,

96.2%). However, one strain (651689; 2006) clustered outside the larger

HPeV3 group and had 96.2% amino acid similarity to this group. In contrast

to findings for HPeV1, we could identify geographical or temporal separation

between the different HPeV3 strains (Fig. 1); however, due to the close

similarities, more diverse sequences are needed for confirmation.

Based on cluster analyses, strains were further identified as either HPeV4, -

5, or -6. The isolate from one positive HPeV stool obtained in 2004 could not

be assigned to a specific HPeV cluster. Strain 451564 had the best

nucleotide identity, 71.2% (79.8% amino acid identity), to the prototype

strain, HPeV3 A308-99, identified in 2004. The second-best match was

found to be less than 70% (66% identity to HPeV1 Harris). Based on

previous proposed criteria to assign HPeV types (21), further

characterization is needed. Strain 451564 was also found to lack the

Chapter 5

90

arginine-glycine-aspartic acid (RGD) motif (Fig. 3), as previously seen for

other HPeV3 genotypes.

Interestingly, the RGD motif was not found in three HPeV5 strains (452373

[2004], 652444 [2006], and 676618 [2006]) and one HPeV1 strain (652281

[2006]), which was most closely related to Harris. Instead of the deletion

seen in HPeV3 and the unidentified strain 451564, all four strains contained

a specific sequence at the C-terminal end of VP1, preceded by the well-

conserved domain proline-alanine-proline-lysine (PAPK) and followed by the

conserved sequences predicted to be part of the cleavage site seen in all

known HPeV genotypes, (D/N)-(E/Q)ˆ(S)-(P/L) (Fig. 3).

DISCUSSION

In the 3-year study period, we found a high percentage (16%) of HPeV-

positive children by direct PCR screening from stool samples. Previous

studies on the prevalence of HPeV were primarily based on culture isolates,

which can bias the data due to the inability of virus variants to replicate in

certain cell lines (1, 29). Only one other study also directly screened from

stool samples (3). In that study a much lower prevalence of HPeV was found

(1.3%). However, that study involved both children and adults, while our

study was performed on stool samples obtained exclusively from children.

When the prevalence of HPeV is calculated solely on the basis of samples

from children less than 2 years old, the study by Baumgarte et al. (3) and the

Figure 3. Alignment of the VP1 region flanking the RGD motif. The strains containing the

RGD motif are shown in black. The strains lacking the RGD motif are shown in blue. The

three HPeV5 strains and one HPeV1 strain, containing the different motif at the C

terminal end of the VP1 gene are shown in red. The arrowhead marks the cleavage site

of the VP1-2A junction.

VP1 2A

HPeV1 HARRIS P A P K - - - V T S S R A L R G D M A N L T N Q S P

652281 (1) P A P K - - - V T N T S R A I S N N P – F E D E S P

HPeV2 WILLIAMSON P A P K - - - P A T – R K Y R G D L A T W S D Q S P

HPeV3 A308/99 P A P K - - - P T G S R A - - - - - T A L S D E S P

HPeV4 K251176-02 P A P K - - - P A T S R A L R G D M A N F S D Q S L

HPeV5 CT86-6760 P A P K - - - E K T S R A L R G D L A N F I D Q S P

452373 (5) P A P K - - - E K S S R S I Q G N P - - F E D E S P

652444 (5) P A P K - - - E K S S R S I T S N P - - F E D E S P

676618 (5) P A P K - - - Q K S S R S I T S N P - - F E D E S P

HPeV6 NII561-2000 P A P K N T P R S Q S R A L R G D M A N L T N Q S P

451564 (x) P A P K - - - P E N T K R - - - - - I A L H D E S P


91

study presented here show similar prevalences (11.6% and 13.4%,

respectively).

In concordance with seroepidemiological data on HPeV1 (18, 25, 27) and

HPeV3 (16), HPeV1 was the most common genotype found. The HPeV1

strains could be grouped in two lineages, of which one lineage comprises

the prototype HPeV1 strain, designated Harris (30). HPeV1 Harris has not

been identified in recent years (1, 2, 6, 29). The fact that we identified three

strains as Harris shows that the “old” strain is either still circulating or

recirculating, albeit at low frequency.

The study by Baumgarte et al. (3) found a third HPeV1 lineage intermediate

between the recently circulating HPeV1 strains and the old Harris strain and

suggested that this strain formed a transition group between the recent and

old HPeV1 lineages (3). On the basis of the VP1 regions that we analyzed,

we could observe several of these “transition” strains. Whether the recently

circulating strains have gradually evolved from the old strain or are the result

of several recombination events (7, 12) needs to be investigated further.

In contrast to the yearly circulation of HPeV1, HPeV3 was not observed in

2005. Remarkably, in that same year the HPeV prevalence in CSF was low

(0.4%) (31). During the same study period, HPeV was found in almost 5% of

CSF samples, and it was suggested that HPeV was the second most

frequent viral cause of sepsis-like illness and meningitis after HEV, which

was found in 14% of the children. However, screening of stool samples

showed HPeV infections to be as prevalent as HEV infections, which is

related to the fact that both viruses are transmitted through the fecal-oral

route. In addition, double infections were found; these were not found within

CSF. Although data on type-specific prevalence in CSF are lacking, the low

prevalence in CSF and the absence of HPeV3 seen in stool samples in 2005

strongly suggest that HPeV3 might be the predominant genotype infecting

the central nervous system. In addition, the number of HPeV-infected

neonates was also lowest in 2005 (3%), again underlining the association

between HPeV3 and infection at a younger age (6, 7, 29).

Although seroprevalence data are lacking for HPeV4 to -6, we speculate that

these recent types circulate to a lesser extent than HPeV1 and -3, both in

the Amsterdam region and globally.

To directly perform genotyping on stool samples, we optimized our VP1 RT-

PCR (6) and a second primer pair was designed to be able to amplify all

known HPeV genotypes. However, we were not able to type all available

samples due to low virus titers or degradation. Thus, the prevalences of

certain HPeV genotypes (indicated in Fig. 2 legend) might be slightly

underestimated. We cannot exclude the possibility that these samples

Chapter 5

92

contained unidentified HPeV genotypes which our assay might not be able

to pick up. However, a majority of these samples (21/26) could still be typed

using a nested approach based on the VP3/VP1 region as belonging to one

of the known HPeV genotypes 1, 4, and 5 (15). Therefore, we speculate that

both the incongruity of stool samples and the sensitivity of our assay caused

the limitations of our assay. Despite these limitations, the use of this direct

genotyping method comprising the entire VP1 gene has resulted in the

identification of four interesting strains lacking the RGD motif. The RGD motif

has been identified in all known HPeV genotypes, with the exception of

HPeV3. This is the first report describing two HPeV genotypes whose VP1

regions have consistently been found to contain the RGD motif in different

isolates and yet to also contain a different specific consensus sequence at

the C-terminal end. The identification of two different variants of the same

genotype is not uncommon within the family Picornaviridae. The echovirus 9

strain designated Barty (14) was shown to contain different consensus

sequences, including the RGD motif, in comparison to the echovirus 9 strain

designated Hill (14).

It has been proposed that the RGD motif is a key factor in defining cell

tropism of the different HPeV genotypes (7, 16). Its absence in HPeV3

suggests an RGD-independent entry pathway.

The insertion of the specific sequence found in the four strains identified

could indicate a second RGD-independent pathway. Preliminary culture data

already showed these strains to be difficult to culture in standard diagnostic

cell lines such as African green monkey kidney (Vero), human colon

carcinoma (HT-29), and human lung carcinoma (A549) (1, 29; our

unpublished data) cells. This was also observed for the unidentified HPeV

strain 451564, which also lacked the RGD motif. In order to identify what

specific cell entry pathways the different HPeV types use and what effect

these different pathways have on their clinical outcome, more research is

needed. In summary, this is the first study where HPeVs were directly typed

from stool samples without being isolated first by cell culture. This resulted in

the identification of an unidentified HPeV genotype and “RGD-absent”

HPeV5 and HPeV1 strains. In addition direct screening from stool samples

showed HPeV1 to be the most prevalent type, followed by HPeV3. As

HPeVs are transmitted via the fecal-oral route, analysis of stool samples

provided an unbiased analysis of different HPeV types.


93

Acknowledgements

We thank Hetty van Eijck for providing culture data on the new HPeV

variants. This work was supported by the Department of Medical

Microbiology, Academic Medical Center, Amsterdam, The Netherlands.

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Clinical Characteristics of Human

Parechoviruses 4-6 Infections in Young

Children

Dasja Pajkrt1, Kimberley S.M. Benschop2,Brenda

Westerhuis2, Richard Molenkamp2, Louise Spanjerberg3,

Katja C. Wolthers2

Pediatric Infectious Disease Journal, In press

1 Dept. of Pediatric Infectious Diseases, Emma Children’s Hospital,

Academic Medical Center, Amsterdam. 2 Lab. of Clinical Virology, Dept. of Medical Microbiology, Academic Medical

Center, Amsterdam. 3 Dept. of Pediatrics, Amstelland Hospital, Amstelveen.

99

Clinical Characteristics of Human Parechoviruses 4-6

Infections in Young Children

Background. Human parechoviruses (HPeVs) and enteroviruses (EVs)

belong to the family Picornaviridae. EVs are known to elicit a wide

range of disease such as meningitis, encephalitis, and sepsis. HPeV1

and 2 have been associated with mild gastrointestinal or respiratory

symptoms in young children. HPeV3 is associated with more severe

neonatal infection. Little is known about the epidemiology and

pathology of HPeV4-6 in children.

Methods. We evaluated the clinical symptoms of the children with an

HPeV 4, 5 or 6 infection. The patients with positive HPeV 4-6 in stool

samples were selected and available plasma or cerebrospinal fluid

samples from these patients were tested for HPeV. Data on clinical

symptoms, diagnosis, presence and duration of fever, medical history,

mean age, use of antibiotics of the children infected with HPeV4-6 were

retrospectively documented.

Results. HPeV 4-6 were found in 31 of the 277 HPeV positive children

(11%). Co-infection with EV was seen in 8 patients. Fever was seen in

13 (42%) patients. Of the HPeV 4-6 positive patients, 20 of the 31

children (64%) presented with gastrointestinal complaints and 18 of 31

(58%) patients had respiratory symptoms. The mean age was 11

months, 58% of the patients had an underlying disorder such as

bronchomalacia or a cardiac disorder.

Conclusions: Symptomatic HPeV4-6 infections are seen in relative

young children and are associated with respiratory and/or

gastrointestinal symptoms. HPeV type 4 was detected more frequently

than HPeV types 5 and 6.

INTRODUCTION

Human Parechoviruses (HPeV) and Enteroviruses (EVs) belong to the family

Picornaviridae. EVs are well established causes of a variety of diseases

such as sepsis, encephalitis and meningitis in children [1], but studies on the

clinical pathology of HPeV infections are scarce. HPeV type 1 and 2 were

previously known as echovirus 22 and echovirus 23 [2], and are associated

with respiratory tract and gastrointestinal infections, and with otitis media in

young children [3-6]. Encephalitis [7,8] and paralysis [9] have also been

associated with a HPeV1 infection. HPeV types 3, 4, 5 and 6 have only

recently been discovered [10-13] and little is known of the clinical pathology

Chapter 6

100

associated with infection by these viruses. HPeV3 has been associated with

more severe disease such as neonatal sepsis and meningitis [10, 14,15].

Most recently, encephalitis with white matter injury caused by HPeV was

described in ten neonates [16]. HPeV genotyping directly from CSF showed

that all HPeVs that could be typed (80%) were HPeV3. HPeV4 was first

identified in an infant with fever and feeding problems [11]. HPeV6 was

isolated from a child with Reye’s syndrome and subsequently 10 children

with gastroenteritis, respiratory symptoms and a rash were found to be

infected with HPeV6 as was also a child with flaccid paralysis [13].

In clinical practice diagnosing EV in a patient clinically suspected of sepsis or

meningitis is accomplished by RT-RT-PCR using the conserved 5’

Untranslated Region (UTR) [2,17,18]. Because of distinct sequences at the

5’ end in the EV and HPeV genes [19,20], HPeV will not be detected with

these molecular assays for EV. We have recently developed a real time

Taqman RT-PCR assay directed at the 5’UTR to detect HPeV directly from

clinical samples [18]. For genotyping, the VP1 region is sequenced directly

from stool samples [21].

This method was used to study retrospectively the prevalence of HPeV 4-6

in fecal samples from children younger than 5 years of age obtained from

2004 until 2007 and clinical characteristics of the children infected with HPeV

4-6 were documented. Additionally, from patients with positive HPeV 4-6

feces samples, available samples from plasma, cerebrospinal fluid (CSF)

and nasopharyngeal aspirates (NPA) were tested for HPeV presence.

METHODS

Detection and genotyping of HPeV by real-time RT-PCR

From 2004 until 2007, feces samples that had been referred to the

Laboratory of Clinical Virology for viral diagnostics were routinely stored at -

80°C. Samples (n=2,372), obtained between 2004 and 2007 from children

<5 years (n=1,809) were respectively screened using an HPeV specific real

time RT-PCR [5].

Positive HPeV samples were genotyped by sequencing the complete VP1

region as previously described [21].

From patients that had positive HPeV 4-6 detection in the feces, additional

HPeV testing of available materials from ethylenediaminetetraacetic acid

(EDTA) plasma (n=5), NPA (n=5) and CSF (n=4) was performed as

described before [18,22].

Human parechoviruses 4-6

101

Furthermore all available materials from patients with positive HPEV4-6

feces samples were tested for EV presence according to previously

published RT-PCR method [18].

Clinical data

The clinical characteristics of children with positive HPeV4-6 in the fecal

samples were obtained using a questionnaire. The following items were

retrospectively registered using the medical records or letters of discharge:

age at time of virus isolation, sex, medical history (including prematurity) and

length of hospital stay. The presence and duration of fever (temperature

>38°C), sepsis-like illness (fever or hypothermia with signs of circulatory

and/or respiratory dysfunction defined by tachycardia or bradycardia, low

blood pressure and decreased saturation), neurologic symptoms (meningitis

with elevated CSF cell count (> 10 cells/mm3), with or without raised CSF

protein level (>0.35 g/L) or decreased CSF glucose level (< 2.8 mmol/L),

irritability, encephalitis, seizures, or paralysis were documented. In addition,

respiratory symptoms (rhinorrhoea, cough, tachypnea, inter- or subcostal

retractions, wheezing, inspiratory stridor, and abnormalities on chest

radiograph), otitis and gastrointestinal symptoms (diarrhea, nausea and/or

vomiting), rashes, use of antibiotics and diagnosis at discharge were

recorded. If a specific symptom was not clearly mentioned in the medical

record or letter of discharge, the symptom was labeled as ‘missing’.

RESULTS

HPeV samples

Between 2004 and 2007, all fecal samples of children younger than 5 years

were tested for HPeV by RT-PCR (n=2,372), and 277 children tested

positive. Of all the HPeV positive samples, 31 patients were diagnosed with

HPeV4-6 (11%), 20 patients were positive for HPeV genotype 4, six patients

with HPeV5 and five with HPeV6 (Table 1). In only two patients HPeV was

detected in plasma and NPA. Of the 31 patients, 8 children had positive EV

feces samples at the same time (five HPeV4 patients, two HPeV5 and one

HPeV6 patient). Only one patient, that is described in more detail below, had

a positive EV infection in plasma. There was no relation between the

detection of EV and the occurrence of fever, gastro-intestinal or respiratory

symptoms. There were 22 boys and 9 girls. The mean age of the children

was 11 months (median 10 months) There were two neonates of two weeks

old. More than half of the patients (58%) had an underlying illness and 22%

of the patients was born prematurely. In 26% (n=8) of the patients another

Chapter 6

102

illness was present besides the HPeV infection. The mean duration of

hospital stay was 8 days (median, 4 days) and 55% of the children were

given antibiotics for an average of 5 days. All but one patients were

admitted: 9 to the general hospital, and 21 children to the academic hospital,

of whom 5 to the intensive care unit.

Clinical characteristics of children with HPeV4-6 in feces

Clinical data of the children tested positive for HPeV4-6 in feces were

available from all children (Table 2). Fever was present in 13 (42%) children

with a mean duration of 3 days. There was only one patient with signs of a

sepsis-like illness. In 2 children meningitis (with 1552 and 91 cells/3 µl in

CSF respectively and protein levels of 2.28 and 0.80 g/l respectively) was

diagnosed. The first of the two patients had a medical history of

hydrocephalus with a ventriculo-peritoneal shunt. The CSF of this child was

tested on herpes simplex virus, EV, HPeV by RT-PCR and a bacterial

culture of the CSF was performed; no viral or bacterial pathogen was

detected. In the other child S. pneumoniae was cultured from the CSF.

Table 1. Baseline characteristics of 31 patients with HPeV4-6 in fecal samples.

Variable Finding

Sex males/females 22/9

Age, months

Mean

Median (range)

11

10 (3-64)

Underlying condition 18 (58)

Prematurity 7 (22)

Other acute illness at time of HPeV infection 8 (26)

Hospital stay, days

Mean

Median (range)

8

4 (0-40)

Admission to general hospital 9 (29)

Admission to academic hospital 21 (68)

Admission on IC unit with artificial ventilation 5 (16)

Use of antibiotics 17 (55)

Duration of antibiotic treatment, days

Mean

Median (range)

5

7 (3-14)

HPeV 4 20 (65)

HPeV 5 6 (19)

HPeV 6 5 (16)

EV 8 (25)

Note: Data are amount followed by (%) of patients with symptom, unless stated otherwise. IC

(Intensive Care), HPeV (human parechovirus), EV (enterovirus).


103

Table 2. Clinical data of 31 patients with HPeV4-6 in fecal samples.

Variable Finding

Fever 13 (42)

Duration of fever, days

Mean

Median (range)

3

2,5 (1-7)

Sepsislike illness 1 (3)

Meningitis 2 (7)

GI tract symptoms 20 (64)

Diarrhea 18 (58)

Duration of diarrhea, days

Mean

Median (range)

4

2,5 (1-21)

Respiratory tract symptoms 18 (58)

Coughing 7 (23)

Rhinitis 13 (42)

Sub-or intercostal retractions 5 (16)

Wheezing 1 (3)

Pneumonia 6 (19)

Otitis 6 (19)

Rash 3 (10)

Note: Data are amount followed by (%) of patients with symptom, unless stated otherwise.

In 20 children gastrointestinal symptoms, 18 with diarrhea were present, with

a mean duration of 4 days. In 58 percent of the patients respiratory

symptoms such as rhinorrhea, pneumonia, retractions or wheezing were

present. In 6 patients otitis media was diagnosed. A rash was noted in 3

patients.

In table 3 the detection of HPeV4 and EV from different samples is depicted

together with the clinical symptoms of one patient. The patient was a

prematurely born boy with a omphalocele and bronchopulmonary dysplasia

that was hospitalized all his life and that presented with respiratory and

gastrointestinal symptoms at the age of 3 months and 11 days. On day 0

HPeV4 was first detected in the NPA and was associated with a pneumonia

followed by recovery from feces on day 3. An increase in respiratory

symptoms coincided with the presence of HPeV4 in plasma, a pneumonia

was simultaneously diagnosed at this time point. Subsequently the patient

was infected with EV as was demonstrated by the detection of EV in plasma

and feces. Simultaneously with a decrease in clinical symptoms, EV and

HPeV4 were no longer detected. Both HPeV4 and EV could be detected

from feces samples up to 40 days after the initial detection.

Chapter 6

104

pla

sm

a

Nasophary

ngeal

aspira

te

Feces

Respira

tory

sym

pto

ms

GI s

ym

pto

ms

Day

EV

-

HP

eV

+

0

EV

-

HP

eV

+

pneum

onia

3

EV

-

HP

eV

-

6

EV

-

HP

eV

+

EV

-

HP

eV

+

pneum

onia

23

EV

+

HP

eV

+

27

EV

+

HP

eV

+

29

EV

+

HP

eV

-

EV

+

HP

eV

+

43

EV

+

HP

eV

-

69

On d

ay =

0 H

PeV

4 c

ould

be d

ete

cte

d fro

m N

PA

for th

e firs

t time (p

atie

nt w

as 3

month

s a

nd 1

1 d

ays o

ld). A

t time p

oin

ts w

here

there

are

no re

sults

depic

ted, th

ere

were

no s

am

ple

s a

vaila

ble

for te

stin

g. G

I= g

astro

inte

stin

al, U

RT

I= u

pper re

spira

tory

tract in

fectio

n

EV

-

HP

eV

-

EV

-

HP

eV

-

Recurre

nt U

RT

I

Recurre

nt g

astro

ente

ritis

93

Table

3. R

ela

tion o

f clin

ical s

ym

pto

ms a

nd d

ete

ctio

n o

f HP

eV

4 in

one p

atie

nt.


105

DISCUSSION

In this study we describe for the first time the characteristics and clinical

symptoms of young children with an HPeV4-6 infection. The majority of the

children were boys, a finding that is seen before in studies on HPeV [22].

More than half of the patients had an underlying illness, which suggests that

HPeV4-6 could cause more severe illness in these children in comparison to

previously healthy children.

The mean duration of hospital stay for children with HPeV4-6 in feces was 8

days and antibiotic treatment was given to more than half of the cases. It has

been demonstrated that reduction in hospital stay and antibiotic treatment

can be achieved by rapid diagnostic testing for EV [23], but data involving

rapid diagnostic tests for HPEV for hospital stay reduction and antibiotic use

are lacking.

In 25% of the patients with positive HPeV4-6 in feces, EV could be detected

in plasma, feces or NPA. As the questionnaires did not contain a severity

scale for the clinical signs, we do not know whether a dual infection with

HPeV and EV elicited more severe symptoms in comparison with only an EV

or HPeV infection.

The majority of the children with a HPeV4-6 infections had gastrointestinal or

respiratory symptoms. Interestingly, the 6 cases of otitis media were

associated with all the three different types studied on this study (3 patients

with HPeV4, 2 patients with HPeV5 and 1 patient with HPeV6). Otitis media

has also been associated in children infected with HPeV1 [6]. Additional

studies are needed to determine whether HPeV4-6 are causally associated

with otitis media in childhood. Sepsis or sepsis-like illness was diagnosed in

only one of the children and although two patients had clinical signs of a

meningitis and increased CSF cell count and CSF protein value, HPEV could

not be detected in the CSF of these children. It is well known that EV can

cause sepsis-like illness and aseptic meningitis in young children [1,24] and

recently we demonstrated that patients with HPeV in the CSF presented with

sepsis-like illness and meningitis [22]. HPeV3 infections are associated with

more severe disease such as neonatal sepsis and meningitis, even when

isolated only form stool samples [14,15]. Patients with a HPeV1 and HPeV2

infection present mainly with mild gastrointestinal or respiratory complaints

[25,26] and our data suggest that HPeV4-6 can be associated with similar

symptoms. A limitation of our study is that the sample collection is biased as

they derive from a pediatric population referred for virologic diagnostic

testing. We did not include a control group, so it is unknown whether

asymptomatic shedding of HPeV4-6 in feces occurred in our study. In a

Chapter 6

106

recent study, HPeV6 was found in 9% of non-hospitalized children without

causing any illness whereas HPeV4 and HPeV5 were not detected in this

study [27].

There are no reports on the possible duration of excretion of HPeV in fecal

samples. One study showed the duration of enteroviruses to last up to 11

weeks after infection [28]. Here we report one case of a young infant

infected with HPeV4 followed by EV. Both viruses could be detected in

various samples for as long as 40 days. In this one case detection of HPeV4

and EV was associated with clinical symptoms at all testing times, but a

clear contribution of HPeV4 or EV cannot be given.

In summary, we conclude that HPeV4-6 are associated with gastrointestinal

and respiratory symptoms in young infants. In our study, HPeV4-6 infections

are not associated with sepsis-like illness or aseptic meningitis.

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2. King AMQ, Brown F, Christian P et al. Picornaviridae. 2000;657-673.


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4. Stanway G, Joki-Korpela P, Hyypia T. Human parechoviruses--

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6. Tauriainen S, Oikarinen S, Taimen K, et al. Relationship between

Human Parechovirus 1 Infection and Otitis Media in Young Children. J

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associated with disseminated echovirus 22 infection. Scand J Infect

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8. Legay V, Chomel JJ, Fernandez E, et al. Encephalomyelitis due to

human parechovirus type 1. J Clin Virol. 2002; 25:193-195.


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1989;29:315-319.


identification of a novel human parechovirus. J Gen Virol. 2004;85:391-

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107

11. Benschop KSM, Schinkel J, Luken ME et al. Fourth Human

Parechovirus Serotype. Emerg Infect Dis. 2006;12:1572-1575.

12. Al-Sunaidi M, Williams CH, Hughes PJ, et al. Analysis of a new

human parechovirus allows the definition of parechovirus types and the

identification of RNA structural domains. J Virol. 2007;81:1013-1021.

13. Watanabe K, Oie M, Higuchi M, Nishikawa M, Fujii M. Isolation and


Emerg Infect Dis. 2007;13:889-895.

14. Boivin G, Abed Y, Boucher FD. Human parechovirus 3 and neonatal

infections. Emerg Infect Dis. 2005;11:103-105.

15. Benschop KS, Schinkel J, Minnaar RP et al. Human parechovirus

infections in Dutch children and the association between serotype and

disease severity. Clin Infect Dis. 2006;42:204-210.

16. Verboon-Maciolek MA, Groenendaal F, Hahn CD, et al. Human

Parechovirus causes encephalitis with white matter injury in neonates.

Ann Neurol. 2008;64:226-273.

17. Romero JR. Reverse-transcription polymerase chain reaction

detection of the enteroviruses. Arch Pathol Lab Med. 1999;123:1161-

1169.

18. Benschop K, Molenkamp R, van der Ham A, et al. Rapid detection

of Human Parechoviruses in clinical samples by real-time PCR. J Clin

Virol. 2008;41:69-74.

19. Hyypia T, Auvinen P, Maaronen M. Polymerase chain reaction

detection for human picornaviruses. J Gen Virol. 1989;70:3261-3268.


echoviruses 22 and 23 in cell culture supernatants by PCR. J Med

Virol. 1999;58:178-181.

21. Benschop K, Thomas X, Serpenti C, et al. High prevalence of human

parechovirus (HPeV) genotypes in the Amsterdam region and

indentification of specific HPeV variants by direct genotyping of stool

samples. J Clin Microbiol. 2008;46:3965-3970

22. Wolthers KC, Benschop KSM, Schinkel J, et al. Human

Parechoviruses as an important viral cause of sepsis like illness and

meningitis in young children. Clin Inf Dis. 2008;47:358-363

23. Robinson CC, Willis M, Meagher A et al. Impact of rapid polymerase

chain reaction results on management of pediatric patients with

enteroviral meningitis. Pediatr Infect Dis J. 2002;21:283-286.

24. Rotbart HA, McCracken GH, Jr, Whitley RJ, et al. Clinical

significance of enteroviruses in serious summer febrile illnesses of

children. Pediatr Infect Dis J. 1999;18:869-874.

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25. Joki-Korpela P, HyypiaT. Diagnosis and epidemiology of echovirus 22

infections. Clin Infect Dis.1998;27:129-136.

26. Birenbaum E, Handsher R, Kuint J, Dagan R. Echovirus 22 outbreak

associated with gastro-intestinal disease in a neonatal intensive care

unit. Am J Perinatol. 1997;14:469-473.

27. Tapia G, Cinek O, Witso E, et al. Longitudinal observation of

parechovirus in stool samples from Norwegian infants. J Med Virol.

2008;80:1835-42.

28. Chung P W, Huang YC, Chang LY, et al. Duration of enterovirus

shedding in stool. J Microbiol Immunol Infect. 2001;34:167–170.

Detection of Enterovirus and Human

Parechovirus Genotypes from Clinical

Stool Samples: PCR and Direct

Molecular Typing, Culture

Characteristics and Serotyping

K. S. M. Benschop, R. P. Minnaar, G. Koen,

H. W. M. van Eijk, K. Dijkman, B. Westerhuis,

R. Molenkamp, K. C. Wolthers

Manuscript in preparation


Center, Amsterdam.

111

Detection of Enterovirus and Human Parechovirus

Genotypes from Clinical Stool Samples: PCR and Direct

Molecular Typing, Culture Characteristics and Serotyping

Introduction: Human Enteroviruses (HEV) and Human Parechovirues

(HPeV) are common human pathogens associated with a wide array of

clinical manifestations ranging from mild respiratory or gastrointestinal

symptoms to severe central nervous system (CNS)-associated

symptoms such as meningitis. Molecular methods are increasingly

being used to detect these viruses in diagnostics. As the gold

standard, cell culture is also still widely used, however, the use of

different cell panels to isolates these viruses may give varying results

between laboratories due to difference in culture characteristics

between types.

Methods and results: By use of real time PCR we found 248/1465 (17%)

of stool samples collected between 2007 and 2008 positive for

HEV/HPeV. By cell culture, we isolated 107 (42%) of the PCR positive

samples using 6 different cell lines; tertiary monkey kidney cells (tMK),

Vero cells, human embryonic lung (Hel), human colon carcinoma cells

(HT29), Rhabdomyosarcoma cells (RD), and human lung fibroblasts

cells (A549). RD and HT29 cells efficiently propagated HEVs in addition

to tMK and Hel cells, whereas HPeV cultured well on HT29 and tMK

cells, but could not be cultured on Hel. HPeV3 could only be cultured

on A549 and Vero cells. The ability to efficiently isolate virus from PCR

positive samples correlated with the virus load, measured by Ct values.

By molecular genotyping we characterized 120 samples (49%).

Serotyping was limited by the ability to isolate the virus by cell culture

and only 46 PCR positive samples were successfully typed by

neutralization assay, while 13 samples were typed by serotyping alone.

Conclusion: Molecular typing in combination with genotyping is of

value to determine the prevalence of HEV and HPeV genotypes

independent of culture characteristics that may limit detection and

serotyping. However, cell culture can still be valuable for virus

isolation. In addition, validation of molecular typing techniques on

clinical samples is needed for introduction in a diagnostic setting.

Chapter 7

112

INTRODUCTION

Human enteroviruses (HEVs) and parechoviruses (HPeVs) belong to

Picornaviridae family of small, non-enveloped RNA viruses. HEVs were

originally subdivided into poliovirus (PV, 3 serotypes), coxsackievirus A

(CAV, 23 serotypes), coxsackievirus B (CBV, 6 serotypes) and echovirus (28

serotypes), and newer HEVs were numerically classified as enterovirus (EV)

68-102 (24,30-32). Based on phylogenetic analysis, HEVs are now classified

into 4 species HEV-A, HEV-B, HEV-C (containing PV) and HEV-D (18,22).

HPeVs were originally classified within the Enterovirus genus as echovirus

22 and 23, but were recently assigned a separate genus of Parechovirus,

which also includes Ljungan virus (26). Currently 14 HPeV genotypes are

known.

HEVs and HPeVs are associated with a wide array of clinical manifestations

ranging from mild respiratory or gastrointestinal symptoms, hand-foot and

mouth disease, myocarditis, neonatal sepsis, and infections of the central

nervous system (CNS) such as meningitis, acute flaccid paralysis and

encephalitis (3,5,39). HEVs and HPeVs have a seasonal distribution that can

vary between the genera as well as between the types (9,21,35,42). In

addition, different types can co-circulate in different variety depending on the

year and place. Recent outbreaks of severe disease caused by Coxsackie B

viruses (CBV) in the US and Europe and enterovirus 71 in Asia emphasize

the importance of this group of small RNA viruses (2,13).

HEVs and HPeVs are widespread and are commonly found in routine

diagnostics (5). The classical method for diagnosis of infection with HEVs or

HPeVs has been virus isolation in cell culture from different clinical samples

such as stool, throat swabs, cerebral spinal fluid (CSF), and blood. The

standard cell culture for isolation of HEV/HPeVs involves at least three cell

lines, usually including monkey kidney cells and human fibroblasts

(34,41,43). When a cytopathogenic effect (CPE) is observed, the isolated

virus can be identified by neutralization with a panel of specific antibodies

(including antisera against HPeV1 and 2) (20). CPE induced by HPeV is not

that different from that induced by HEV, and HPeVs may therefore easily be

identified as HEVs when specific serotyping is not routinely performed (9).

PCR for HEV diagnosis target the 5’UTR, which is highly conserved and

therefore suitable to detect all HEV serotypes (4,19,43). Since the nucleotide

sequences of the HPeVs are quite divergent from the HEVs, pan-EV RT-

PCR fails to detect HPeVs (9,17). HPeV infections will therefore be missed if

only an HEV specific PCR is performed.

Human parechovirus and enterovirus detection

113

The advantage of conventional cell culture techniques lies in the ability to

identify the different serotypes by neutralization, which is a commonly found

technique in the diagnostic laboratories for surveillance purposes. However,

some HEV and HPeV types are difficult to grow in cell culture or cannot be

cultured at all, and antibody pools for HEV and HPeV typing only include

HPeV type 1 and 2. Therefore, specific HEV and HPeV types will be missed

or underdiagnosed when only cell culture is performed.

Molecular typing of the different HEV/HPeV types is done by sequencing

part of the capsid region, VP1. It has been shown that typing by sequencing

part of the VP1 region is in good agreement with serotyping by

neutralization, since type-specific antibodies are also directed against the

VP1 region (29). Typing methods may also involve sequencing of the capsid

genes, VP4 and/ or VP2. However, sequencing of these regions was found

to be less discriminatory to define the different types (12,33).

Many laboratories are now rapidly introducing PCR as the method of first

choice for detection of viral pathogens in CSF (14,45), but this is also a rapid

and sensitive method to detect HEV and HPeVs in other clinical samples as

stool or throat swabs (4,8,28,43). However, the clinical relevance of more

sensitive detection of HEV or HPeV from stool samples by PCR has still to

be determined. In addition, many laboratories are not familiar with molecular

typing techniques and performance of these techniques needs to be

validated as well. Furthermore, conventional cell culture can still have its

advantages as in isolating the virus for typing or other purposes. Although

much is known already for culturing HEV on different sets of cell lines, much

less is known about the culture characteristics of HPeVs. Therefore, we

performed an analysis in which we compared our cell culture system with

real-time PCR for detecting HEV and HPeV in stool samples from

hospitalized patients, describe the efficacy of different cell lines for culturing

different HEV and HPeV types, and compare the yield of genotyping directly

from stool with serotyping by neutralisation of culture isolates.

METHODS

Clinical samples

Stool samples obtained between 2007 and 2008 from patients of different

age groups sent to the Academic Medical Center were collected. Stool

samples were suspended in 2% broth (Oxoid, Drongen, Belgium) and stored

at -80°C.

Chapter 7

114

Viral Culture and serotyping

Viral culture was performed by co-cultivation of patient material with tertiary

monkey kidney cells (tMK), Vero cells, human embryonic lung cells (Hel),

human colon carcinoma cells (HT29), Rhabdomyosarcoma cells (RD), and

human lung fibroblasts cells (A549). Monolayers of the cell lines were

cultured in 24-wells plates. The broth suspended stool was filtrated and a

volume of 150 µl was inoculated and centrifuged at 3500g for 15 min at room

temperature. The cultures were incubated at 37oC, 5% CO2. The viral

cultures were examined twice a week for the appearance of CPE and

incubated for a maximum of 21 days before they were considered negative.

Cell culture isolates were characterized by neutralisation assay with

enterovirus specific antisera pools A-G and H-R (20).

RNA extraction and detection

All stool samples were extracted by automatic extraction using the total

nucleic acid isolation kit with the MagnaPure LC instrument (Roche

Diagnostics). Broth-suspended stool samples (50 µl) were incubated for 10

min. in lysis buffer provided by the MagnaPure extraction kit and centrifuged

for 2 min. to remove any stool debris. RNA was eluted in 50 µl elution buffer

and reverse transcribed as previously described (6,7). Five µl of cDNA was

used for both the HEV-specific duplex assay and the HPeV-specific single-

target assay (6,7). When testing for the presence of an HPeV and/or HEV

infection, samples were co-extracted with 6,250 copies of armored RNA of

an internal control (IC), corresponding to 500 copies of IC cDNA in PCR

(6,7). The IC was omitted from extraction when genotyping by sequencing of

the VP1 region had to be performed (6).

Direct genotyping

For genotyping of HPeV positive samples, the complete VP1 gene was

amplified and sequenced using the newly developed primer set VP1-

parEchoF12 and VP1-parEchoR12 (8). For genotyping of the HEV positive

samples, the seminested approach by Nix et al. (27) was adopted. The first

round involved the amplification of a 992 bp fragment encompassing the

VP3 and VP1 genes followed by a second round, resulting in a 350 to 400

bp fragment of the VP1 gene. The amplicons were purified out of gel and

sequenced using the Big Dye Terminator reaction kit on an ABI 3730/ 3100

DNA analyzer (Applied Biosystems). The amplicons were compared with the

VP1 sequences of HEV and HPeV reference strains and phylogenetically

characterized based on cluster analyses (8).


115

Statistical analysis

Statistical analysis was performed using the wilcoxon-mann-whitney test

(95% confidence interval) implemented in SPSS 12.1 for Windows.

RESULTS

Patients positive for HEV and/or HPeV by PCR

Over 2007 and 2008, 1465 samples from 1177 patients could be analyzed

by HPeV PCR and 1458 samples from 1171 patients could be analyzed by

HEV PCR. We found 131 samples from 116 patients positive for HPeV and

134 samples from 120 patients positive for HEV. From these patients, 15

had a double infection (in 17 samples) with HPeV and HEV (Table 1). In

total, 19.3% of the patients were found positive for HEV/HPeV by PCR.

We found similar percentages males and females to be infected in both

groups. Patients positive for HEV/HPeV were generally younger than 5 years

old (109/120 (90,8%) and 105/116 (90,5%) respectively), of which 10,8%

(13/120) and 12% (14/116) respectively were neonates. The median age of

the patients positive for HEV was 9,34 months (0,20 months-71 years) and

was higher than that found for HPeV (median age 7,37 months (0,07

months-68 years), but this difference was not statistically significant. We

found only 8 adults (>19 years) positive for either HPeV (n=4) or HEV (n=4).

Among the children under the age of 5 years, the HPeV prevalences in 2007

and 2008 were found to be 12,2% and 13,1% respectively. In the same age

group, the HEV prevalence was 17,2% in 2007 and 8.6% in 2008, which

differed significantly (p<0.05) over the 2 years.

In both years, an HEV peak was observed in the summer (Fig. 1). HPeV

infections predominantly peaked in the winter of late 2007 and early 2008. A

smaller peak was observed in 2008 in the summer.

Table 1. HEV and HPEV infections in stool between 2007 and 2008.

No. of virus-positive patients/no.tested (%) in:

Virus(es) 2007 2008 Total

HEV 81/609 (13.3) 39/562 (6.0) 120/1171 (10.2)

HPeV 60/614 (9.8) 56/563 (9.9) 116/1177 (9.9)

HEV and HPeV 9/609 (1.5) 6/562 (1.1) 15/1171 (1.3)

Chapter 7

116

HEV/HPeV detected by cell culture

From January 2007 till May 2008, clinical samples (n=997) were primarily

cultured on tMK and Hel cells as a first line of diagnostics. In total, 4.2%

(n=42) had been found positive. During this time, 185 samples had been

found positive for HEV/HPeV by PCR (91 HEV and 82 HPeV, 12 double

positive). Of these samples, 31 HEV positive and 9 HPeV positive samples

had been cultured. Two positive cultures were identified as double positive

by PCR.

To optimize virus isolation, additional cell culture was performed. HT29,

Vero, A549 and RD cells were used as additional cell lines and all 248

HEV/HPeV PCR positive samples from 2007 and 2008 were (re)cultured

(117 HEV, 11 4 HPeV and 17 double positive). In total, 107 (43.1%) were

positive in cell culture on the entire cell panel; 56 (47.9%) PCR positive HEV

samples, and 41 (36%) HPeV PCR positive samples could be cultured. Of

the 17 PCR double positive infections, 10 could be cultured.

Thus, more than 50% of the PCR positive samples remained negative in

culture. The ability of HEV and HPeV PCR positive samples to propagate in

cell culture correlated significantly with the viral load expressed as Ct value

obtained by real-time PCR (p<0.05) (Fig. 2). However, 43 samples with a Ct

value <32 cycli still remained negative in culture (19 HEV and 24 HPeV,

including the double positive samples).

Figure 1. Number of children positive for human parechovirus (HPeV, white bars) and/or

enterovirus (HEV, black bars) in stool per month.

0

2

4

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117

EV and HPeV genotyping

By amplification and sequencing part of the VP1 capsid region directly from

the stool sample, 49/117 (41.9%) of the HEV PCR positive samples could be

typed (Table 2). Based on the same technique, 62/114 (4.4%) HPeV PCR

positive samples could be typed (Table 2). Nine samples identified as double

positive could be typed by either HPeV or HEV VP1 PCR (Table 2).

Amplification of VP1 is less sensitive than detection based on amplification

of the ‘well’-conserved 5’UTR, and none of the samples with Ct values > 37

cycli in the 5’UTR PCR could be typed (n=31; 16 HEV and 15 HPeV). From

the samples with Ct values between 32 and 37 cycli (n=84), only 16 (19%)

could be typed; 7 HEV and 9 HPeV. When Ct values were <32 cycli in the

5’UTR (n=148), the majority of the samples (n=112, 75.7%) could be typed;

54 HEV and 58 HPeV. Despite the low Ct value, which indicates a good viral

load for genotyping, 36 samples (26 HEV and 10 HPeV positives) with a Ct

value <32 could still not be genotyped.

Figure 2. Cell culture results of human enterovirus (HEV) and parechovirus (HPeV)

PCR positive samples. Culture positive samples are given in grey diamonds. Culture

negative samples are given in black diamonds. The results are plotted against the Ct

value obtained by real time PCR. The mean Ct value of samples that remained culture

negative was 33.56 and 35.37 for HEV and HPeV respectively. The mean Ct value of

samples that could be cultured was 28.21 and 25.08 for HEV and HPeV respectively.

The mean values were found to be statistically significant (P<0.05).

17

22

27

32

37

42

-

HEV HPeV

+ - +Cell culture Cell culture

PC

R: C

t V

alu

e

17

22

27

32

37

42******

17

22

27

32

37

42

-

HEV HPeV

+ - +Cell culture Cell culture

PC

R: C

t V

alu

e

17

22

27

32

37

42******

Chapter 7

118

Table 2. Genotyping of HEV and HPeV positive stool samples between 2007 and 2008.

positive for types 2007 2008 Total

HPeV HPeV1 19 18 37

HPeV3 0 4 4

HPeV4 15 4 19

HPeV5 1 0 1

HPeV6 1 0 1

Total 36 26 62

EV CBV1 0 1 1

CBV2 1 0 1

CBV3 0 5 5

CBV4 0 1 1

CAV1 1 1 2

CAV2 4 0 4

CAV4 1 1 2

CAV5 1 0 1

CAV6 1 0 1

CAV9 3 0 3

CAV16 0 1 1

echovirus 3 0 3 3

echovirus 6 2 0 2

echovirus 9 4 0 4

echovirus 11 4 0 4

echovirus 25 2 1 3

echovirus 30 5 2 7

echovirus 33 1 0 1

enterovirus 71 3 0 3

Total 33 16 49

HEV and HPeV HPeV3 0 1 1

HPeV4 1 0 1

HPeV4 and CAV6 1 0 1

HPeV6 and CAV5 1 0 1

CBV4 0 1 1

CAV2 1 0 1

echovirus 6 0 1 1

echovirus 30 0 1 1

HPeV3 and echovirus 13 0 1 1

Total 4 5 9


119

HPeV1 was found the most frequently (n=37), followed by echoviruses

(n=27), HPeV4 (n=21), CAV (n=17), CBV (n=9), HPeV3 (n=6), enterovirus

71 (n=3), HPeV6 (n=2) and HPeV5 (n=1) (Table 2).

The HEV types identified in 2007 and 2008 belonged to three of the 4 HEV

species; HEV-A: CAV2, -4, -5, -6, and -16, HEV-B: CBV1-4, CAV9, and

echovirus 3, 6, 9, 11, 25, 30, and 33, and HEV-C, CAV1. In the HEV group,

echovirus 30 was most commonly found. Within 2007, we could observe

HPeV1, -4, -5 and -6 infections, but did not observe any HPeV3 infections.

Within 2008 we observed 5 types, HPeV1, -3, -4, -5 and -6.

Of the 43 samples that were unable to grow in cell culture, despite a Ct value

of <32 cycli, 28 could be genotyped. In total, we found 9 HPeV types; HPeV1

(n=7), HPeV 4 (n=1) and HPeV6 (n=1); and 19 enteroviruses; 7 echoviruses

(types 9 (n=2), 11 (n=1) 25 (n=2) 30 (n=1), and 33 (n=1)) and 12 CAVs

(types 1 (n=1), 2 (n=5), 4 (n=1), 5 (n=1), 5 (n=1), and 6 (n=1)). Therefore,

the inability to grow in our cell culture system despite high copy numbers for

HEV was partly type-specific due to difficult-to-culture CAVs, and partly

random: the echoviruses should have been able to propagate in cell culture.

Culture characteristics and serotyping of HEV/HPeV

RD and HT29 cells proved to be good cell lines to propagate HEVs in

addition to tMK and Hel cells, whereas HPeV cultured well on HT29 and

tMK, but could not be cultured on Hel (Table 3).

Table 3. Cell culture yield of HEV and HPeV PCR positive samples.

Cell culture PCR positive Total

positive HPeV HEV HPeV and

HEV

neg or not

done

Not done 478 478

Negative 67 59 7 796 929

HT29 31 15 6 3* 55

RD 3 21 5 4* 33

Vero 7 0 0 3* 10

tMK 13 11 4 5* 33

Hel 0 8 2 5* 15

A549 6 0 0 0 6

tMK and/or Hel 0 29 0 0 29

Total 127 143 24 1294 1588

Note: numbers are based on the results of a sample on different cell lines.

*) HEV/HPeV negative, adenovirus positive.

Chapter 7

120

CBV and echoviruses could be cultured on the HT29, RD, tMK and Hel

cerlls. Enterovirus 71 was only found to grow on tMK and/or Hel and the

CAV strains were exclusively cultured on RD cells (Table 4). Use of the

HT29 cell line led to an increase in the detection of HPeV of almost 20%.

Predominantly HPeV1 and -4 cultured well on this cell line. HPeV3 could

exclusively be cultured on Vero and A549. In comparison, HPeV1 and -4

could be cultured on HT29, RD, Vero, tMK, and A549 cells. HPeV5 and 6

were identified only once of which HPeV6 could be cultured on HT29 cells.

The HPeV5 strain was not cultured. However, these strains were identified

only once to clearly determine on what cell lines they can be cultured well.

From the 107 samples cultured (56 HEV, 41 HPeV PCR, and 10 double

positive), 85 samples were analyzed by neutralization assay. Echoviruses

were found in 20 cultures, followed by HPeV1 (n=18), CAV (n=10), CBV

(n=9) and enterovirus 71 (n=1). Eleven samples showed no growth of the

virus strain in the assay, despite previous isolation by culture. Sixteen

samples could not be neutralized by the RIVM pool (table 5), and 5 of these

were HPeV types (HPeV3 and -4) that are not included in the antisera pool,

while six of these samples could be genotyped as enterovirus strains

included in the pool (echovirus 3 and 13, enterovirus 71, CBV3, and

CAV16). Serotyping provided 13 additional types that could not be typed by

direct genotyping (Table 5). Sixteen samples could not be typed by either

method. In the 45 stool samples that were both serotyped and genotyped,

results were 100% identical (Table 5).

Table 4. Cell line specific culture per virus type.

Virus(es) Cell lines Total

HT29 RD Vero tMK Hel A549

HPeV 29 2 7 14 0 4 56

CBV 7 5 0 8 4 0 24

CAV 0 10 0 0 0 0 10

Echovirus 8 6 0 21 21 0 56

Enterovirus 71 0 0 0 3 3 0 6

Total 44 23 7 46 28 4 152


121

Table 5. Genotyping compared to serotyping.

genotyping

HPeV

(n=67)

CBV

(n=9)

CAV

(n=17)

Echovirus

(n=28)

HEV 71

(n=3) Total

n (%) n (%) n (%) n (%) n (%) n

Serotyped 16 (24) 7 (78) 7 (35) 15 (54) 1 (33) 46

Culture negative 28 (42) 1 (11) 6 (35) 8 (29) 0 (0) 43

No neutralisation 5 (7)1

1 (11) 1 (6) 2 (7)1

2 (67) 111

No gowth in assay 7 (10) 0 (0) 0 (0) 1 (4) 0 (0) 8

Not done 11 (16)2

0 (0) 3 (18)2

2 (7) 0 (0) 162

Serotyping alone (n) 2 2 4 5 0 13

Total typed (n) 69 11 21 33 3 137 n) Number of double positive samples for which and HEV and HPeV types were successfully

identified.

DISCUSSION

Here, we describe the value of direct stool screening by PCR and

genotyping to detect HEV and HPeV genotypes. A higher percentage of

HPeV and HEV positive samples were identified by PCR in comparison to

culture, and this was correlated to the Ct value in PCR. Thus, detection by

PCR is more sensitive than cell culture as has been described before

(19,36,43). For detection of HEV in CSF, this has been proven an advantage

(25,38,40). However, since HEV and HPeV are shedded for weeks after

infection from the gastrointestinal tract, the clinical advantage of detecting

low amounts of HEV/HPeV needs to be determined.

One advantage of PCR detection from stool is the ability to detect types that

are difficult to culture, which is especially the case for CAV and certain HPeV

types. Therefore, PCR and genotyping will give a more complete picture of

the circulating strains and will be of particular use in years with high CAV or

HPeV prevalence (9,41). The overall percentage of HEV PCR positive stool

samples in our study was comparable to that found by another Dutch group

(11.6% positive stools) (43), but was significantly lower that that found in Iran

(22,8%) (37) and Germany (11). Although in the latter study the percentage

PCR positive stools varied considerably over the years (14%-27%).

However, when comparing HEV prevalence data, it should be considered

that the percentage of HEV infections can vary between years, and

countries. It is therefore essential to relate the regional data to the data

obtained from the general population over different years. Data from the

national surveillance system from 2007 and 2008 showed a marked

decrease of HEV infection reported in 2008, in comparison to the previous

years, which is in agreement with our findings in 2007 and 2008 and our

Chapter 7

122

previous data over 2004-2006 (8). In contrast to the HEV data, the

percentage HPeV PCR positive samples showed no marked differences

between the years and was comparable to our previous study (8).

The variability over the years observed for HEV and HPeV, has been

correlated to the number of circulating types that may vary between years

and also seasons (8,10,16,21). By direct genotyping we identified commonly

circulating HEV and HPeV types (21,41,43), with echovirus 30 and HPeV1

identified as the predominant strains..

In both years, an HEV peak was observed in the summer; but this was not

related to a specific HEV type. HPeV infections predominantly peaked in the

winter of late 2007 and early 2008, correlating with a majority of the HPeV1

types. A smaller peak was observed in 2008 in the summer that may be

related to the finding of HPeV3 within that year, in contrast to 2007 when no

HPeV3 could be found, confirming the biannual cycle of HPeV3 (8,15,16,42).

Even though PCR is now the common method for detection of HEV and

HPeV in clinical samples (19,25,36,38,40,41,43), cell culture is still beneficial

for surveillance and environmental purposes and also in relation to cost

effectiveness and laboratory experience.

For the isolation of HEV, the use of at least three cell line is recommended

since different types grow on different cell lines. However, there is no

standard cell panel known that is generally used by all laboratories. tMK and

Hel cells are routinely used, but may be limited to detect certain HEV and

also HPeV types. By use of these cell lines the recovery of HEV and HPeV

samples found positive by stool was only 4%, also observed by Van

Doornum et al. (43). We evaluated and used 6 different cell lines known to

propagate either virus, with the emphasis on the ability to detect HPeV

(1,41,44). Inclusion of these cell lines led to an increase of 37% more HEV

and HPeV positive samples by cell culture. The study by Terletskaia-Ladwig

et al. (41), achieved a 71% detection rate by culture in comparison to PCR,

using only RD and Caco-2 cells. The RD cell line is one of the few cell lines

that can propagate most HEV types. In our study, the RD cell line was the

only one to efficiently support the growth of CAVs, whereas CBV and

echoviruses could be cultured on the HT29, RD, tMK and Hel cells.

However, a combination of cell lines could lead to the identification of other

types that will not propagate on RD cells, such as enterovirus 71.

Moreover, in our experience the RD cell line performed poorly with respect to

HPeV isolation. In contrast, the HT29 cell lines proved ideal to culture

HPeV1 and HPeV4. Although 5/6 cell lines supported HPeV growth, HPeV3

could exclusively be cultured on Vero and A549. HPeV5 and -6 were only


123

identified once and the culture characteristics of these viruses need to be

further evaluated. The study by Watanabe et al. (44) did not show HPeV6 to

exclusively grow on specific cell lines.

The major advantage of PCR in combination with genotyping is, that it is

more rapid than cell culture combined with serotyping, which will take at

least 2-3 weeks. For HEV and HPeV detection, PCRs are increasingly used.

This reflects the need for good and easy-to-perform molecular typing

techniques. In our study, we used VP1 sequencing as the method of choice

for genotyping directly from clinical samples. However, only 49% of the

samples could be typed, mainly due to much lower sensitivity than our

5’UTR PCR. In addition, this method could not type HEV and HPeVs directly

from CSF (45, and unpublished results). More sensitive typing methods have

been developed. By use of a (semi-)nested approach comprising the VP3-

VP1 region, 97.7% to 100% of HEV an HPeV types could be typed (15,23).

Thus, compared to our data obtained with VP1 sequencing, semi-nested

typing of the VP3-VP1 region is more sensitive and may be preferred.

Although serotyping by neutralization assay is generally reliable, it is labour-

intensive and time-consuming. Furthermore, it may fail to detect new types

or variants of known types as the antisera used are only directed against

known types and have been manufactured in the sixties (20). In contrast to

direct genotyping, serotyping depends on culture isolates. The advantage of

direct genotyping lies within the ability to identify difficult-to-culture types

such as CAV and HPeV3. Remarkably, we could type 13 additional samples

by serotyping alone. This could be a reflection of an increase in viral load by

culture isolation. The ability to culture samples with a low viral load, given by

their Ct value, which may have resulted in the lack of genotyping results, is

not absolute.

In an era, where molecular technology, increasingly becomes the method of

choice for detection HEV an HPeV, and cell culture is not performed,

genotyping methods that can be performed directly from clinical samples are

essential. These methods will be of direct use during outbreaks of a specific

type when decision making is depended on these results. In addition, direct

genotyping has led to the identification of new types and variants (8, and

Oberste et al. unpublished). On the other hand, cell culture provides us with

the tools to isolate new viruses or variants that may not be picked up by

specific PCRs. As different HEV and HPeV types have different culture

characteristics, a wide range of different cell lines may improve the yield of

detecting HEV and HPeV.

Chapter 7

124

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36. Shoja, Z. O., H. Tabatabai, M. Sarijloo, S. Shahmahmoodi, T. M.

Azad, and R. Nategh. 2007. Detection of enteroviruses by reverse-

transcriptase polymerase chain reaction in cell culture negative stool

specimens of patients with acute flaccid paralysis. J.Virol.Methods

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37. Shoja, Z. O., H. Tabatabie, S. Shahmahmoudi, and R. Nategh. 2007.

Comparison of cell culture with RT-PCR for enterovirus detection in

stool specimens from patients with acute flaccid paralysis. J.Clin.Lab

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38. Siafakas, N., P. Markoulatos, and S. Levidiotou-Stefanou. 2004.

Molecular identification of enteroviruses responsible for an outbreak of

Chapter 7

128

septic meningitis; implications in clinical practice and epidemiology.

Mol.Cell Probes 18:389-398.


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69.

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41. Terletskaia-Ladwig, E., S. Meier, R. Hahn, M. Leinmuller, F.

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43. van Doornum, G. J., M. Schutten, J. Voermans, G. J.

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45. Wolthers, K. C., K. S. Benschop, J. Schinkel, R. Molenkamp, R. M.

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P A R T 2

Phylogeny and evolution

Fourth Human Parechovirus Serotype

Kimberley S.M. Benschop1, Janke Schinkel1,

Manon E. Luken3, Peter J.M. van den Broek3,

Matthias F.C. Beersma4, Negassi Menelik5, Hetty W.M. van

Eijk1, Hans L. Zaaijer1, Christina M.J.E. VandenBroucke-

Grauls2, Marcel G.H.M. Beld1, and Katja C. Wolthers1

Emerging Infectious Diseases 2006; 12:1572-1575

1 Lab. of Clinical Virology,

2Dept. of Medical Microbiology, Academic Medical

Center, Amsterdam. 3 Primagen, Amsterdam.

4 Dept. of Medical Microbiology, Leiden University Medical Center, Leiden.

5 Department of Pediatrics, Boven IJ Ziekenhuis, Amsterdam.

133

Fourth Human Parechovirus Serotype

We identified a novel human parechovirus (HPeV) type (K251176-02)

from a neonate with fever. Analysis of the complete genome showed

K251176-02 to be a new HPeV genotype. Since K251176-02 could not

be neutralized with antibodies against known HPeV serotypes 1–3, it

should be classified as a fourth HPeV serotype.

INTRODUCTION

Infections with human parechoviruses (HPeVs) are commonly associated

with mild gastrointestinal and respiratory symptoms in young children (1–3),

but more severe conditions, such as flaccid paralysis (4) and encephalitis (5),

have also been described. Recently, a new serotype (HPeV3) has been

isolated, which has been associated with transient paralysis (6) and neonatal

sepsis (7).

HPeV1 and HPeV2 were previously known as the enteroviruses echovirus

22 and 23 but were reclassified into a new genus within the family

Picornaviridae after phylogenetic analysis showed that parechoviruses were

distinct from other picornaviruses (1–3,8–11). HPeVs have predominantly

been isolated from young children, and increasing evidence shows that

HPeV can cause serious illness in these patients.

We recently showed that infection with HPeV3 is associated with younger

age and more severe disease than is infection with HPeV1 (12). During the

screening of patient samples, we identified 1 aberrant HPeV type.

Phylogenetic analysis of the full-length sequence and viral neutralization

assays showed that the isolate designated K251176-02 is a new HPeV

genotype and serotype.

THE STUDY

Viral culture of the stool of a 6-day-old patient with a 2-day history of high

fever and poor feeding and no history of gastrointestinal or respiratory

symptoms showed enterovirus cytopathic effects. However, PCR targeted at

the 5′ untranslated region (UTR) of enterovirus (13) was negative, whereas a

5′ UTR PCR specific for HPeV (12) was positive.

Results of sequencing the VP1 region (12) suggested that K251176-02 was

a novel HPeV genotype. Therefore, the full-length sequence was determined.

Combinations of consensus primers were used to generate partially

overlapping amplicons that covered the complete genome.

Chapter 8

134

Fig

ure

1. U

nro

ote

d p

hylo

genetic

trees s

how

ing th

e re

latio

nship

betw

een K

251176-0

2 (D

Q315670) a

nd

the p

roto

type s

train

s H

PeV

1 H

arris

(S45208); H

PeV

2

Willia

mson (A

J005695); H

PeV

2 C

T86-6

760 (A

F055846); H

PeV

3 A

308-9

9 (A

B084913), C

an82853-0

1 (A

J889918) b

ased o

n n

ucle

otid

e J

ukes a

nd C

anto

r

substitu

tion m

odel

(16) fo

r the c

apsid

re

gio

n (a

) and th

e non-s

tructu

ral

regio

n (b

). T

he tre

e w

as c

onstru

cte

d by th

e neig

hbour–

join

ing m

eth

od (1

7)

as

imple

mente

d in

the M

EG

A 3

.1 s

oftw

are

package (1

8). G

aps in

troduced fo

r optim

al a

lignm

ent w

ere

not c

onsid

ere

d in

form

ativ

e a

nd h

ence e

xclu

ded fro

m th

e

analy

ses b

y c

om

ple

te d

ele

tion. N

um

bers

repre

sent th

e fre

quency o

f occurre

nce o

f nodes in

1000 b

oots

trap re

plic

as. T

he

us

e o

f oth

er e

vo

lutio

n m

od

els

did

not in

fluence th

e tre

e to

polo

gy.

HP

eV

1 H

arris

HPeV2 Williamson

HPeV2 CT86-6760K251

176-0

2

HPeV3 A308-99

HPeV3 C

an82853-01

0.0

5

790

100

0

100

0

HP

eV

3 A

308-9

9

HPeV3 Can82853-01

K251176-02

HP

eV

2 C

T86-6

760

HPeV1 Harris

HPeV2 Williamson

0.0

2

100

0

100

0

920

AB

HP

eV

1 H

arris

HPeV2 Williamson

HPeV2 CT86-6760K251

176-0

2

HPeV3 A308-99

HPeV3 C

an82853-01

0.0

5

790

100

0

100

0

HP

eV

1 H

arris

HPeV2 Williamson

HPeV2 CT86-6760K251

176-0

2

HPeV3 A308-99

HPeV3 C

an82853-01

0.0

5 HP

eV

1 H

arris

HPeV2 Williamson

HPeV2 CT86-6760K251

176-0

2

HPeV3 A308-99

HPeV3 C

an82853-01

HP

eV

1 H

arris

HPeV2 Williamson

HPeV2 CT86-6760K251

176-0

2

HPeV3 A308-99

HPeV3 C

an82853-01

0.0

50.0

5

790

100

0

100

0

HP

eV

3 A

308-9

9

HPeV3 Can82853-01

K251176-02

HP

eV

2 C

T86-6

760

HPeV1 Harris

HPeV2 Williamson

0.0

20.0

2

100

0

100

0

920

AB

Fourth human parechovirus serotype

135

Amplicons were sequenced according to a primer walking strategy. The 5′

UTR was amplified by using the 5′ RACE System (Invitrogen, Carlsbad, CA,

USA). Because a primer composed of the first 22 nucleotides (nt) of

published consensus parechovirus sequences was used to amplify the 5′

UTR proximal end, these 22 nt could not be determined with absolute

certainty (8). The 3′ UTR end was amplified with a tagged oligo-dT primer.

The complete genome of K251176-02 was 7,348 nt long, containing a 5′

UTR of 708 nt, a large single open reading frame (ORF) of 6,549 nt, and a 3′

UTR of 91 nt followed by a poly(A) tract. The full-length sequence of

K251176-02 has been deposited in GenBank under accession no.

DQ315670.

We found a best-match nucleotide identity (14) of 72.2% in the VP1 gene

with HPeV2 CT86-6760, which suggests that K251176-02 is most closely

related to HPeV2 CT86-6760. Indeed, phylogenetic analysis of the capsid

nucleotide sequence based on Jukes and Cantor distances showed

K251176-02 to cluster with HPeV2 CT86-6760 (Figure 1A). However, the

genetic distance was considerable (0.327) and comparable to the genetic

distance between HPeV1 Harris and HPeV2 Williamson (0.332).

Phylogenetic analysis of the nonstructural region showed that K251176-02

clustered with the HPeV3 prototypes A308-99 and Can82853-01 (Figure 1B).

To identify recombination events between the different HPeV prototypes, a

SimPlot analysis was performed on the known full-length nucleotide HPeV

genomes against K251176-02. The SimPlot analysis (Figure 2) showed the

differential similarity of K251176-02 with HPeV2 CT86-6760 in the highly

variable P1 region and with HPeV3 in the more conserved P2–P3 region.

This finding may be the result of a recombination event.

The secondary structure of the 5′ UTR of K251176-02, determined by the

Mfold program of Zuker and Turner (http://mfold2.wustl.edu), was predicted

to be highly structured and was characterized by a stable hairpin at the

proximal end that was also found in known HPeV prototypes (8,11, data not

shown). The predicted secondary structure of the 3′ UTR of K251176-02

contained the same 1-stem loop organization as the HPeV prototypes and

was similar to the secondary structure of HPeV1 Harris and HPeV2

Williamson and CT86-6760 (15).

A comparison of the complete ORF of K251176-02 with the HPeV prototypes

showed an amino acid identity of 86.9% to 90.1% (Table 1). This amount is

in the same range of amino acid identity as observed between known HPeV

protoypes. For the VP1 gene, the greatest amino acid identity was observed

with HPeV2 CT86-6760 (80.4%). In the nonstructural region, identity was

greater to HPeV3, with 98.1% identity in the polymerase gene (3Dpol).

Chapter 8

136

Comparison of the deduced amino acid sequence in the capsid region of

K251176-02 with the HPeV prototypes showed that the sequences that are

predicted to be part of the β-barrel structure (6,10,11) are well conserved in

K251176-02. Like HPeV1 and HPeV2, K251176-02 also contained an RGD

motif at the C-terminal end of the VP1 gene, which was absent in HPeV3

(6,7,15). K251176-02 also contained the common motifs X2GXGK(S/T) and

DDLXQ (2C gene), which are predicted to have a helicase function. The

active-site cysteine of the protease 3C is in the context of GXCG, and the

active site of polymerase 3Dpol contains the conserved sequence YGDD.

The wellconserved motifs within the 3Dpol gene (KDELR, PSG, and FLKR)

were also found in K251176-02 (6,9,11).

Figure 2. Similarity plot of HPeV1 Harris (S45208); HPeV2 Williamson (AJ005695);

HPeV2 CT86-6760 (AF055846); HPeV3 A308-99 (AB084913) and Can82853-01

(AJ889918) against K251176-02. Each curve is a comparison between the K251176-02

genome and a HPeV prototype. Each point represents the percent identity within a

sliding window of 600bp wide, centered on the position plotted, with a step size of 20

bp. Positions containing gaps were excluded from the comparison by gap stripping and

Jukes and Cantor (16) correction was applied. Similarity plots of the full length sequences

of the HPeV prototypes were generated using the SimPlot v2.5 software (19).

Window: 600 bp, Step: 20 bp, GapStrip: On, J-C Correction: On

Position (bp)

7.0006.5006.0005.5005.0004.5004.0003.5003.0002.5002.0001.5001.000500

Sim

ilarity

(%

)

100

98

96

94

92

90

88

86

84

82

80

78

76

74

72

70

68

66

64

62

60

58

56

54

52

50

Position (bp)

7.0006.5006.0005.5005.0004.5004.0003.5003.0002.5002.0001.5001.000500

Sim

ilarity

(%

)

100

95

90

85

80

75

70

65

60

55

50

VP0 VP3 VP1 2A 2B 2C 3A 3C 3D

HPeV 1 HarrisHPeV 2 CT86-6760HPeV 2 WilliamsonHPeV 3 A308-99HPeV 3 Can82853

Window: 600 bp, Step: 20 bp, GapStrip: On, J-C Correction: On

Position (bp)

7.0006.5006.0005.5005.0004.5004.0003.5003.0002.5002.0001.5001.000500

Sim

ilarity

(%

)

100

98

96

94

92

90

88

86

84

82

80

78

76

74

72

70

68

66

64

62

60

58

56

54

52

50

Position (bp)

7.0006.5006.0005.5005.0004.5004.0003.5003.0002.5002.0001.5001.000500

Sim

ilarity

(%

)

100

95

90

85

80

75

70

65

60

55

50

VP0 VP3 VP1 2A 2B 2C 3A 3C 3D

HPeV 1 HarrisHPeV 2 CT86-6760HPeV 2 WilliamsonHPeV 3 A308-99HPeV 3 Can82853


137

Table 1. Amino acid identity matrix of all known HPeV full length sequences.

HPeV1 (H) HPeV2 (W) HPeV2 (CT) HPeV3 (A308) HPeV3 (Can)

ORF

K251176-02 88,7 86,9 90,1 89,1 89,3

HPeV1 (H) - 88,6 88,1 87,2 87,3

HPeV2 (W) - - 85,8 85,1 85,0

HPeV2 (CT) - - - 86,7 86,9

HPeV3 (A308) - - - - 98,2

HPeV3 (Can) - - - - -

P1

K251176-02 78,3 78,6 81,3 74,7 75,1

HPeV1 (H) - 81,6 76,4 74,9 75,3

HPeV2 (W) - - 74,2 73,9 73,9

HPeV2 (CT) - - - 73,0 73,5

HPeV3 (A308) - - - - 97,4

HPeV3 (Can) - - - - -

P2-P3

K251176-02 94,3 91,5 94,9 97,0 97,1

HPeV1 (H) - 92,4 94,5 93,8 93,9

HPeV2 (W) - - 92,2 91,2 91,1

HPeV2 (CT) - - - 94,2 94,2

HPeV3 (A308) - - - - 98,6

HPeV3 (Can) - - - -

Amino acid identities for the open reading frame (ORF), the capsid region (P1) and the non-

structural region (P2-P3) are based on p-distance models between K251176-02 (DQ315670)

and the HPeV prototypes, HPeV1 (H) (Harris, S45208); HPeV2 (W) (Williamson, AJ005695);

HPeV2 (CT) (CT86-6760, AF055846); HPeV3 (A308) (A308-99, AB084913) and HPeV3 (Can)

(Can82853-01, AJ889918). The full length sequence of K251176-02 was aligned with the HPeV

prototypes using Clustal-W included in the Vector NTI Advance 10 software package

(Invitrogen). The alignment was edited using GeneDoc software (version 2.6.02) (21). The

matrix was constructed using MEGA 3.1 software package (18). The 5’UTR and 3’UTR are

excluded form the analysis as the regions are non-coding.

Table 2. Neutralization assay using LLcMk2 cells.

Virus Antiserum

α-HPeV1

(Harris)

α-HPeV2

(Williamson)

α-HPeV3

(A308-99)

Viral Controls

HPeV1 Harris - ++++ ++++ ++++

HPeV2 Williamson ++++ - ++++ ++++

K251181-02 (HPeV3) ++++ ++++ - ++++

K251176-02 ++++ ++++ ++++ ++++

Culture isolates of K251176-02, HPeV1 (echovirus 22) and HPeV2 (echovirus 23) from a

reference panel (National Institute for Public Health and the Environment, Bilthoven, the

Netherlands) and K251181-02 that was previously genotyped as HPeV3 (13) were incubated

with antisera (20 U/ml in EMEM) directed against HPeV1 Harris, HPeV2 Williamson and HPeV3

A308-99. The antisera to HPeV1 and HPeV2 were raised in horses. The antiserum to HPeV3

was raised in guinea pigs (Courtesy of Dr. Hiroyuki Shimizu, Japan). The neutralization is done

on a 96-microtitre plate containing a monolayer of LLcMk2 cells that have been incubated for 3

days. The assay is determined after the viral controls (no antisera used) of the 4 culture isolates

showed a CPE of more than 50% (++++).

Chapter 8

138

In summary, K251176-02 represents a new genotype in the genus

Parechovirus. To confirm that K251176-02 is also a new serotype, a

neutralization assay was performed. Table 2 shows that K251176-02 could

not be neutralized by antisera directed against HPeV1 Harris, HPeV2

Williamson, and HPeV3 A308-99, which confirms that K251176-02 is a new

genotype that can be classified as a fourth HPeV serotype.

CONCLUSIONS

HPeVs are classified in the genus Parechovirus in the family Picornaviridae.

The recently identified HPeV3 has been associated with severe illness in

young children in several studies (6,7,12). This association has increased

the awareness of HPeVs as relevant pathogens in young children.

We identified a new HPeV genotype in a stool specimen from a neonate with

high fever. Since classification criteria based on genotyping have not been

defined for HPeVs, we used the criteria proposed by Oberste et al. (14) for

the classification of new enteroviral genotypes. According to these criteria, a

new genotype is defined when a best-match nucleotide identity of <70% is

found in the VP1 gene. A 70%–75% best-match nucleotide identity indicates

further characterization is needed. Therefore, neutralization assays were

conducted; these assays showed that K251176-02 did not neutralize with

antisera directed against the 3 known HPeV serotypes. This finding indicates

that K251176-02 is a new genotype that can be classified as a fourth HPeV

serotype.

The patient from whom K251176-02 was isolated had high fever but no

signs of neonatal sepsis, as has been found in infections with HPeV3

(6,7,12). Previous data suggest differences in severity of disease between

the different HPeV serotypes (12); however, more data are needed to

elucidate epidemiologic and pathogenic features of the different HPeV

serotypes, including K251176-02. HPeV2 CT86–6760 was genotypically as

distinct from HPeV2 Williamson as from other HPeV types (Table 1). The

existence of 2 genotypically divergent HPeV serotypes 2 is surprising and

needs to be elucidated further. This finding, however, argues in favor of a

universal typing method that is based on molecular characteristics

(genotyping) instead of serotyping, provided classification criteria are well

defined.


139

Acknowledgements

We thank Georgios Pollakis for his assistance in the phylogenetic analysis

and for technical support in sequencing the fulllength genome, Rene

Minnaar for further technical support, and Hiroyuki Shimizu for antisera used

in the neutralization assay.

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enteroviruses. Virus Res. 1998;56:217–23.

9. Hyypia T, Horsnell C, Maaronen M, Khan M, Kalkkinen N, Auvinen

P, et al. A distinct picornavirus group identified by sequence analysis.

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10. Stanway G, Kalkkinen N, Roivainen M, Ghazi F, Khan M, Smyth M,

et al. Molecular and biological characteristics of echovirus 22, a

representative of a new picornavirus group. J Virol. 1994;68:8232–8.

11. Ghazi F, Hughes PJ, Hyypia T, Stanway G. Molecular analysis of

human parechovirus type 2 (formerly echovirus 23). J Gen

Virol.1998;79:2641–50.

12. Benschop KSM, Schinkel J, Minnaar RP, Pajkrt D, Spanjerberg L,

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Dis. 2006;42:204–10.

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Clin Microbiol. 2004;42:3059–64.

14. Oberste MS, Michele SM, Maher K, Schnurr D, Cisterna D, Junttila

N, et al. Molecular identification and characterization of two proposed

new enterovirus serotypes, EV74 and EV75. J Gen Virol.

2004;85:3205–12.

15. Abed Y, Boivin G. Molecular characterization of a Canadian human

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Widespread Recombination within

Human Parechoviruses: Analysis of

Temporal Dynamics and Constraints

K. S. M. Benschop1, C. H. Williams2, K. C. Wolthers1,

G. Stanway2 and P. Simmonds3

Journal of General Virology 2008; 89:1030-1035

1, Lab.of Clinical Virology, Dept. of Medical Microbiology, Academic Medical

Center, Amsterdam. 2 Dept.of Biological Sciences, University of Essex, Colchester, UK.

3 Virus Evolution Group, Centre for Infectious Diseases, University of

Edinburgh, Edinburgh, UK.

143

Widespread Recombination within Human Parechoviruses:

Analysis of Temporal Dynamics and Constraints

Human parechoviruses (HPeVs), members of the family Picornaviridae,

are classified into six types. To investigate the dynamics and likelihood

of recombination among HPeVs, we compared phylogenies of two

distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3–5)

and prototype sequences (types 1–6). Evidence for frequent

recombination between HPeV1, 4, 5 and 6 was found. The likelihood of

recombination was correlated with the degree of VP1 divergence and

differences in isolation dates, both indicative of evolutionary times of

divergence. These temporal dynamics were found to be most similar to

those of human enterovirus species B variants. In contrast, HPeV3

remained phylogenetically distinct from other types throughout the

genome. As HPeV3 is equally divergent in nucleotide sequence from

the other HPeV types, its genetic isolation may reflect different biology

and changed cellular tropisms, arising from the deletion of the RGD

motif, and likely use of a non-integrin receptor.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are

EU170302–EU170304 and AM933167– AM933171 (VP1) and EU170312–EU170340 and

AM933159–AM933166 (3Dpol). A supplementary table showing HPeV sequence data is

available with the online version of this paper.

Human parechoviruses (HPeVs) belong to a large family of positive-sense,

single-stranded RNA viruses, the Picornaviridae (Stanway & Hyypiä, 1999).

The parechovirus genome is approximately 7400 nt in length and encodes a

large polyprotein within a single open reading frame flanked by 5’ and 3’

untranslated regions (UTRs). The polyprotein is cleaved post-translationally

into three structural proteins (VP0, VP3 and VP1) and seven non-structural

proteins (2A–2C and 3A–3D).

There are now six HPeV types classified based on their genetic sequences.

HPeV1 and 2 were originally assigned to the genus Enterovirus as echovirus

serotypes 22 and 23, respectively. However, these two viruses were shown

to be genetically distinct from the entire genus Enterovirus and also from

other genera within the family Picornaviridae, prompting their current

classification as members of the genus Parechovirus along with Ljungan

viruses isolated from rodents (Stanway et al., 2005). A further HPeV variant,

originally described as ‘type 2’ (CT86-6760; Oberste et al., 1998), was

shown to be different from the prototype HPeV2 sequence (Ghazi et al.,

1998) and was reclassified as type 5 (Al-Sunaidi et al., 2007). Novel HPeV

Chapter 9

144

types have subsequently been classified as HPeV3 (Ito et al., 2004), HPeV4

(Al-Sunaidi et al., 2007; Benschop et al., 2006a) and HPeV6 (Watanabe et

al., 2007). HPeVs have predominantly been isolated from young infants and

are commonly associated with mild gastrointestinal and respiratory

symptoms, but more severe conditions, such as paralysis (Figueroa et al.,

1989; Ito et al., 2004), neonatal sepsis (Benschop et al., 2006a; Boivin et al.,

2005) and bronchiolitis (Abed & Boivin, 2006), have also been reported.

Although HPeVs are considered to be a widespread pathogen, they remain

largely undiagnosed due to the lack of HPeV-specific screening tools, such

as RT-PCR. Therefore, little is known about the spread and pathogenicity of

these viruses.

Previous analysis of novel HPeV types has indicated that recombination

might play a role in the evolution of HPeV (Al-Sunaidi et al., 2007; Benschop

et al., 2006b). The occurrence of recombination may have a profound impact

on the spread and pathogenicity of RNA viruses in a population (Kendal,

1987; Minor, 1992; Robertson et al., 1995). Recombination has been

documented extensively in human enteroviruses and was found to play a

major role in the evolution of these viruses (Lindberg et al., 2003; Lukashev,

2005; Oberste et al., 2004a, b; Santti et al., 1999; Simmonds & Welch, 2006).

To study the likelihood and dynamics of recombination within parechoviruses,

we analysed several HPeV sequences obtained from two distant regions

within the genome (VP1 and 3Dpol). In total, 37 HPeV isolates were

obtained from The Netherlands (n=29), California (n=6) and Finland (n=2). A

full listing of the HPeV sequences used is available as Supplementary Table

S1 in JGV Online. Twenty-nine samples were sequenced and typed

previously based on the VP1 region and submitted to GenBank under the

accession numbers DQ172416, DQ172418, DQ172420–DQ172421,

DQ172424–DQ172428, DQ172430, DQ172432–DQ172433, DQ172435–

DQ172446, DQ172448, DQ172451 (Dutch isolates containing six-digit

numbers, prefixed with NL; Benschop et al., 2006a), AM234724, AM234726

and AM234728 (Californian isolates; Al-Sunaidi et al., 2007; Schnurr et al.,

1996). The remaining samples were typed within the VP1 region as

described previously (Al-Sunaidi et al., 2007; Benschop et al., 2006b). To

determine partial 3Dpol sequences of each HPeV isolate, RNA was

extracted by using a QIAamp Viral RNA mini kit according to the

manufacturer’s instructions (Qiagen). The extracted RNA was reverse-

transcribed and amplified by using a SuperScript III One-Step RT-PCR kit

(Invitrogen) according to the manufacturer’s instructions. The following

primers were designed within the conserved region within the 3Dpol

sequence by aligning all known HPeV types: OL1502 (5’-

Widespread recombination within human parechoviruses

145

GTGTACAGGATGATCATGATGGA-3’, nt 6419–6441) and OL1501 (5’-

CTTAGTCAAACACCATGGGCA-3’, nt 7253–7233), where nucleotide

numbering is relative to that of the HPeV1 Harris strain (GenBank accession

no. S45208). Amplicons were isolated by agarose-gel electrophoresis and

purified by using a QIAquick gel extraction kit according to the

manufacturer’s instructions (Qiagen), using a spincolumn protocol. DNA was

eluted in 50 µl water. Big Dye Terminator sequencing reactions were

performed by GeneService, Cambridge, UK (http://www.geneservice.co.uk).

The HPeV VP1 and 3Dpol sequences were aligned by using CLUSTAL W

(Thompson et al., 1994) and edited manually by using the SIMMONICS

sequence editor (version 1.6; http://www2.warwick.ac.uk/fac/sci/bio

/research/devans/bioinformatics/simmonics/). Neighbour-joining phylogenetic

trees based on the nucleotide sequence were constructed separately for the

VP1 and 3Dpol regions of the HPeV genome by using the MEGA 3.1

software package (Kumar et al., 2004) with Jukes–Cantor (J-C)-corrected

distances (Jukes & Cantor, 1969) (Fig. 1). Sequence data were bootstrap-

resampled 1000 times to determine robustness of the observed grouping;

branches supported by >70% of replicate trees are indicated. All available

full-length sequences for HPeV were obtained from GenBank and were

included in the analysis: HPeV1 strains Harris (S45208) and BNI-788St

(EF051629); HPeV2 strain Williamson (AJ005695); HPeV3 strains A308-99

(AB084913) and Can82853-01 (AJ889918); HPeV4 strains K251176-02

(DQ315670) and T75-4077 (AM235750); HPeV5 strains CT86-6760

(AF055846) and T92-15 (AM235749); HPeV6 strainsNII561-2000

(AB252582) and BNI-67/03 (EU024629). All HPeV sequences were

monophyletic and formed phylogenetic groups that corresponded to their

type designation in the VP1 region (609 nt) (Fig. 1). However, within the

3Dpol region (706 nt), a breakdown of the type-specific segregation was

observed; sequences in this region of HPeV1, 4, 5 and 6 frequently failed to

group according to type. The majority of recently isolated HPeV1 strains

showed 3Dpol sequences distinct from that of the prototype HPeV1 strain

Harris, first isolated in 1956. As an initial indication of the time-related nature

of the recombination events in HPeV, isolates that clustered closely together

in the VP1 region (pairwise-compared J-C distances of <0.0601) almost

invariably remained together in the 3Dpol region. When a greater VP1

divergence between HPeV1 isolates was observed, a loss of segregation

was consistently observed in the 3Dpol region. These findings are consistent

with the previously observed incongruent phylogenetic relationships within

Chapter 9

146

VP1HPEV1 BNI788St, 2003

HPEV1 NL650258, 2006

HPEV1 NL350014, 2003

HPEV1 NL152212, 2001

HPEV1 NL151442, 2001

HPEV1 FIN70094, 2000

HPEV1 NL251949, 2002

HPEV1 NL153231, 2001

HPEV1 NL650941, 2006

HPEV1 FIN69960, 2000

HPEV1 NL252581, 2000

HPEV1 NL152478, 2001

HPEV1 NL152598, 2001

HPEV1 NL450343, 2004

HPEV1 NL550163, 2005

HPEV1 NL452252, 2004

HPEV1 NL651108, 2006

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL152824, 2001

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL054330, 2000

HPEV1 HARRIS, 1956

HPEV2 WILLIAMSON, 1956

HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV4 T73-510, 1973

HPEV4 K251176-02, 2002

HPEV5 T83-456, 1983

HPEV5 T92-15, 1992

HPEV5 CT86-6760, 1986

HPEV5 T82-0169, 1982

HPEV5 T82-659, 1982

HPEV5 T83-2051, 1983

HPEV6 BNI67-03, 2003

HPEV6 NII561-2000, 2000

HPEV3 A308-99, 1999

HPEV3 NL152037, 2001

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL251360, 2002

HPEV3 NL252277, 2002

HPEV3 NL251181, 2002

HPEV3 NL250956, 2002

HPEV3 NL251393, 2002

HPEV3 NL251407, 2002

99

100

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100

71

70

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100

100

82

97

99

99

91

92

85

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100

98

80

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86

87

80

0.05

HPEV1 BNI788St, 2003

HPEV1 NL650258, 2006

HPEV1 NL350014, 2003

HPEV1 NL152212, 2001

HPEV1 NL151442, 2001

HPEV1 FIN70094, 2000

HPEV1 NL251949, 2002

HPEV1 NL153231, 2001

HPEV1 NL650941, 2006

HPEV1 FIN69960, 2000

HPEV1 NL252581, 2000

HPEV1 NL152478, 2001

HPEV1 NL152598, 2001

HPEV1 NL450343, 2004

HPEV1 NL550163, 2005

HPEV1 NL452252, 2004

HPEV1 NL651108, 2006

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL152824, 2001

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL054330, 2000

HPEV1 HARRIS, 1956


HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV4 T73-510, 1973

HPEV4 K251176-02, 2002

HPEV5 T83-456, 1983

HPEV5 T92-15, 1992

HPEV5 CT86-6760, 1986

HPEV5 T82-0169, 1982

HPEV5 T82-659, 1982

HPEV5 T83-2051, 1983

HPEV6 BNI67-03, 2003

HPEV6 NII561-2000, 2000

HPEV3 A308-99, 1999

HPEV3 NL152037, 2001

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL251360, 2002

HPEV3 NL252277, 2002

HPEV3 NL251181, 2002

HPEV3 NL250956, 2002

HPEV3 NL251393, 2002

HPEV3 NL251407, 2002

99

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99

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86

87

80

0.05

VP1HPEV1 BNI788St, 2003

HPEV1 NL650258, 2006

HPEV1 NL350014, 2003

HPEV1 NL152212, 2001

HPEV1 NL151442, 2001

HPEV1 FIN70094, 2000

HPEV1 NL251949, 2002

HPEV1 NL153231, 2001

HPEV1 NL650941, 2006

HPEV1 FIN69960, 2000

HPEV1 NL252581, 2000

HPEV1 NL152478, 2001

HPEV1 NL152598, 2001

HPEV1 NL450343, 2004

HPEV1 NL550163, 2005

HPEV1 NL452252, 2004

HPEV1 NL651108, 2006

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL152824, 2001

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL054330, 2000

HPEV1 HARRIS, 1956


HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV4 T73-510, 1973

HPEV4 K251176-02, 2002

HPEV5 T83-456, 1983

HPEV5 T92-15, 1992

HPEV5 CT86-6760, 1986

HPEV5 T82-0169, 1982

HPEV5 T82-659, 1982

HPEV5 T83-2051, 1983

HPEV6 BNI67-03, 2003

HPEV6 NII561-2000, 2000

HPEV3 A308-99, 1999

HPEV3 NL152037, 2001

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL251360, 2002

HPEV3 NL252277, 2002

HPEV3 NL251181, 2002

HPEV3 NL250956, 2002

HPEV3 NL251393, 2002

HPEV3 NL251407, 2002

99

100

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100

71

70

100

100

100

82

97

99

99

91

92

85

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100

100

98

80

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86

87

80

0.05

HPEV1 BNI788St, 2003

HPEV1 NL650258, 2006

HPEV1 NL350014, 2003

HPEV1 NL152212, 2001

HPEV1 NL151442, 2001

HPEV1 FIN70094, 2000

HPEV1 NL251949, 2002

HPEV1 NL153231, 2001

HPEV1 NL650941, 2006

HPEV1 FIN69960, 2000

HPEV1 NL252581, 2000

HPEV1 NL152478, 2001

HPEV1 NL152598, 2001

HPEV1 NL450343, 2004

HPEV1 NL550163, 2005

HPEV1 NL452252, 2004

HPEV1 NL651108, 2006

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL152824, 2001

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL054330, 2000

HPEV1 HARRIS, 1956


HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV4 T73-510, 1973

HPEV4 K251176-02, 2002

HPEV5 T83-456, 1983

HPEV5 T92-15, 1992

HPEV5 CT86-6760, 1986

HPEV5 T82-0169, 1982

HPEV5 T82-659, 1982

HPEV5 T83-2051, 1983

HPEV6 BNI67-03, 2003

HPEV6 NII561-2000, 2000

HPEV3 A308-99, 1999

HPEV3 NL152037, 2001

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL251360, 2002

HPEV3 NL252277, 2002

HPEV3 NL251181, 2002

HPEV3 NL250956, 2002

HPEV3 NL251393, 2002

HPEV3 NL251407, 2002

99

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99

100

100

71

70

100

100

100

82

97

99

99

91

92

85

77

100

100

98

80

100

86

87

80

0.05

Figure 1. Unrooted phylogenetic trees of VP1 (609 bp) and 3Dpol (706 bp) regions of HPeVs based

on the neighbour-joining method with J-C corrected distances. HPeV types are colour coded. Loss

of type specific segregation in 3Dpol was observed for HPeV1 (red), HPeV4 (blue), HPeV5 (green)

and HPeV6 (brown), while HPeV3 (purple) remained monophyletic in both regions.


147

3DpolHPEV3 NL251360, 2002

HPEV3 NL251393, 2002

HPEV3 NL250956, 2002

HPEV3 NL251181, 2002

HPEV3 NL251407, 2002

HPEV3 NL252277, 2002

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL152037, 2001

HPEV3 A308-99, 1999

HPEV1 NL252581, 2002

HPEV1 NL152598, 2001

HPEV4 K251176-02, 2002

HPEV1 NL651108, 2006

HPEV1 NL452252, 2004

HPEV1 NL152824, 2001

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL152212, 2001

HPEV1 NL054330, 2000

HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV1 NL251949, 2002

HPEV1 FIN70094, 2000

HPEV1 FIN69960, 2000

HPEV1 NL151442, 2001

HPEV1 NL153231, 2003

HPEV1 NL350014, 2003

HPEV1 BNI-788St, 2003

HPEV1 NL650258, 2006

HPEV1 NL152478, 2001

HPEV1 NL550163, 2005


HPEV4 T73-510, 1973

HPEV5 T82-0169, 1982

HPEV1 NL650941, 2006

HPEV6 NII561-2000, 2000

HPEV5 CT86-6760, 1986

HPEV1 NL450343, 2004

HPEV1 HARRIS, 1956

HPEV6 BNI67-03, 2003

HPEV5 T83-456, 1983

HPEV5 T83-2051, 1983

HPEV5 T82-659, 1982

HPEV5 T92-15, 1992

100

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98

99

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74

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93

77

0.02

HPEV3 NL251360, 2002

HPEV3 NL251393, 2002

HPEV3 NL250956, 2002

HPEV3 NL251181, 2002

HPEV3 NL251407, 2002

HPEV3 NL252277, 2002

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL152037, 2001

HPEV3 A308-99, 1999

HPEV1 NL252581, 2002

HPEV1 NL152598, 2001

HPEV4 K251176-02, 2002

HPEV1 NL651108, 2006

HPEV1 NL452252, 2004

HPEV1 NL152824, 2001

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL152212, 2001

HPEV1 NL054330, 2000

HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV1 NL251949, 2002

HPEV1 FIN70094, 2000

HPEV1 FIN69960, 2000

HPEV1 NL151442, 2001

HPEV1 NL153231, 2003

HPEV1 NL350014, 2003


HPEV1 NL650258, 2006

HPEV1 NL152478, 2001

HPEV1 NL550163, 2005


HPEV4 T73-510, 1973

HPEV5 T82-0169, 1982

HPEV1 NL650941, 2006

HPEV6 NII561-2000, 2000

HPEV5 CT86-6760, 1986

HPEV1 NL450343, 2004

HPEV1 HARRIS, 1956

HPEV6 BNI67-03, 2003

HPEV5 T83-456, 1983

HPEV5 T83-2051, 1983

HPEV5 T82-659, 1982

HPEV5 T92-15, 1992

100

80

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94

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89

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100

98

99

72

74

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99

97

76

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93

77

0.02

3DpolHPEV3 NL251360, 2002

HPEV3 NL251393, 2002

HPEV3 NL250956, 2002

HPEV3 NL251181, 2002

HPEV3 NL251407, 2002

HPEV3 NL252277, 2002

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL152037, 2001

HPEV3 A308-99, 1999

HPEV1 NL252581, 2002

HPEV1 NL152598, 2001

HPEV4 K251176-02, 2002

HPEV1 NL651108, 2006

HPEV1 NL452252, 2004

HPEV1 NL152824, 2001

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL152212, 2001

HPEV1 NL054330, 2000

HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV1 NL251949, 2002

HPEV1 FIN70094, 2000

HPEV1 FIN69960, 2000

HPEV1 NL151442, 2001

HPEV1 NL153231, 2003

HPEV1 NL350014, 2003


HPEV1 NL650258, 2006

HPEV1 NL152478, 2001

HPEV1 NL550163, 2005


HPEV4 T73-510, 1973

HPEV5 T82-0169, 1982

HPEV1 NL650941, 2006

HPEV6 NII561-2000, 2000

HPEV5 CT86-6760, 1986

HPEV1 NL450343, 2004

HPEV1 HARRIS, 1956

HPEV6 BNI67-03, 2003

HPEV5 T83-456, 1983

HPEV5 T83-2051, 1983

HPEV5 T82-659, 1982

HPEV5 T92-15, 1992

100

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98

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93

77

0.02

HPEV3 NL251360, 2002

HPEV3 NL251393, 2002

HPEV3 NL250956, 2002

HPEV3 NL251181, 2002

HPEV3 NL251407, 2002

HPEV3 NL252277, 2002

HPEV3 NL450936, 2004

HPEV3 CAN82853-01, 2001

HPEV3 NL152037, 2001

HPEV3 A308-99, 1999

HPEV1 NL252581, 2002

HPEV1 NL152598, 2001

HPEV4 K251176-02, 2002

HPEV1 NL651108, 2006

HPEV1 NL452252, 2004

HPEV1 NL152824, 2001

HPEV1 NL452538, 2004

HPEV1 NL452712, 2004

HPEV1 NL252228, 2002

HPEV1 NL350757, 2003

HPEV1 NL350918, 2003

HPEV1 NL152212, 2001

HPEV1 NL054330, 2000

HPEV4 T75-4077, 1975

HPEV4 T75-4080, 1975

HPEV1 NL251949, 2002

HPEV1 FIN70094, 2000

HPEV1 FIN69960, 2000

HPEV1 NL151442, 2001

HPEV1 NL153231, 2003

HPEV1 NL350014, 2003


HPEV1 NL650258, 2006

HPEV1 NL152478, 2001

HPEV1 NL550163, 2005


HPEV4 T73-510, 1973

HPEV5 T82-0169, 1982

HPEV1 NL650941, 2006

HPEV6 NII561-2000, 2000

HPEV5 CT86-6760, 1986

HPEV1 NL450343, 2004

HPEV1 HARRIS, 1956

HPEV6 BNI67-03, 2003

HPEV5 T83-456, 1983

HPEV5 T83-2051, 1983

HPEV5 T82-659, 1982

HPEV5 T92-15, 1992

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Chapter 9

148

the non-structural region of HPeV4 and 5 (Al-Sunaidi et al., 2007; Benschop

et al., 2006b). To test formally whether the loss of segregation between variants within

each of the parechovirus types was related to their degree of evolutionary

and epidemiological separation (as indicated by their divergence in VP1),

members of the same HPeV type were classified further by their bootstrap

supported phylogenetic groupings within the 3Dpol region. Subsequent

pairwise comparison of sequences recorded their VP1 sequence divergence

(evolutionary separation) and whether the two variants remained clustered in

3Dpol. The likelihood of recombination (i.e. separate grouping of two types in

the 3Dpol region) increased steadily with VP1 sequence divergence (Fig. 2a),

indicative of time-related recombination comparable to that observed for

human enterovirus species A and B sequences (HEV-A, -B; Simmonds &

Welch, 2006). Although different HPeV isolates were obtained from different

geographical locations and over different collection periods, measurement of

VP1 divergence provided an independent measure of their temporal

separation from each other, and thus provided a robust comparator with

recombination frequency. Furthermore, this independent measure of

divergence time allowed us to compare the dynamics of recombination

directly with those of HEV-A and -B.

Comparison of the dynamics of recombination of HPeV was carried out by

parallel investigation of VP1 sequence divergence and recombination

frequency in datasets of HEV-A and -B sequences (Simmonds & Welch,

2006) containing newly published, full-length sequences from 2006–2007. A

full listing of the HEV sequences used is available as Supplementary Table

S1 in JGV Online. It was not possible to perform a parallel analysis of

recombination frequency in HEV-C or HEV-D, because of a lack of published

complete-genome sequences of epidemiologically independent isolates

(although there are several available HEV-C sequences, many are from

vaccine-derived poliovirus strains). The dynamics of recombination for HPeV

variants were remarkably similar to those of the larger HEV-B sequence

dataset (Fig. 2a, black and white bars). This similarity extended to a second

comparison of recombination frequency and differences in isolation dates

(Fig. 2b), an alternative measure of temporal separation of isolates, but one

that does not take geographical separation into account.

Consistent with previous findings, the time course (measured by both VP1

divergence and isolation-time differences) of recombination of HEV-A

variants was substantially slower than that of HEV-B. Separate analysis of

specific HPeV and HEV types showed a similar pattern of increase in

recombination when types were analysed within a species (data not shown).


149

Despite the detection of frequent recombination events in parechoviruses,

recombination was never observed among HPeV3 sequences (Fig. 1).

Although the majority of the HPeV3 isolates were isolated within the same

year (see Supplementary Table S1, available in JGV Online) and clustered

tightly together, a measurable proportion of recombination was detected

among HPeV1, 4, 5 and 6 types that were similarly divergent in VP1 (approx.

one-fifth of pairwise comparisons where VP1 divergence <0.025 showed

recombination). In addition, 50% of non-HPeV3 variants isolated in the same

year were recombinant, compared with a frequency of zero for HPeV3 (Figs

1 and 2). These observations suggest possible biological constraints that

limit HPeV3 recombination events. In HEVs and other picornavirus genera,

the high degree of sequence divergence between species in the non-

structural region previously appeared to be the main factor limiting inter-

species recombination (Simmonds, 2006). However, analysis of all available

ful-llength sequences (n=11; Fig. 3) showed that HPeV3 was similarly

divergent from other parechovirus types (green line) as the latter were from

0

20

40

60

80

100

0-0,025 0,025-0,075 0,075-0,2 >0.2

0

20

40

60

80

100

0 1-3 4-10 >10

VP1 Distance Range

Difference in Isolation Year

Pro

port

ion o

f com

parisons r

ecom

bin

ant (%

)P

roport

ion o

f com

parisons r

ecom

bin

ant (%

)

A)

B)

HPeV

HEV-A

HEV-B

HPeV

HEV-A

HEV-B

Figure 2. (A) VP1 divergence and (B)

isolation time differences and

frequency of recombination. J-C

distances in the VP1 region (A) and

differences in isolation date (B) were

calculated for each pairwise

comparison of study samples or

complete genome sequences of the

same HPeV type, and categorized

according to whether they grouped

together in the 3Dpol region. The

proportion of pairwise comparisons

showing different 3Dpol groupings

indicative of recombination (y-axis)

was plotted for different ranges of

VP1 pairwise distances (A) and

isolation times (B). The same

analysis was performed for available

VP1/3Dpol sequences of HEV

species A and B.

Chapter 9

150

each other (red line). The only exception was the slightly greater sequence

divergence between HPeV3 and other types at the C terminus of VP1. This

very local region of greater divergence corresponds to the part of the HPeV3

VP1 sequence where the RGD integrin-binding sequence is absent. As the

RGD motif in other HPeV types was found to be critical for replication

0

0.1

0.2

0.3

0.4

0.5

0.6

0 1000 2000 3000 4000 5000 6000 7000

Se

gre

ga

tio

n

VP0 VP3 VP1 2A 2B 2C 3A 3C 3D5’UTR 3’UTR3B

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Am

ino

acid

se

qu

en

ce

va

riab

ility

0 1000 2000 3000 4000 5000 6000 7000

Genome position

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

dN

/dS

Intr

a-t

yp

e

Inte

r-ty

pe

Intra

-typ

e

Inte

r-typ

eTyp

e 3

v. Oth

ers

0.40

0.45

A)

B)

Figure 3. (A) Segregation score (blue line) plotted against amino acid variability

between types (red line) and within types (pink). For the segregation scan, phylogenetic

trees were drawn from consecutive sequence fragments across the genome, using a

window size of 300 bases, incrementing 24 bases between trees. Segregation scores

for each tree (left hand y-axis) were plotted according to their position in the genome

(x-axis). Segregation scores (blue) are represented by 0, perfect segregation by HPeV

types, and where 1 represents absence of type specific phylogenetic grouping. Amino

acid sequence variability between (red) and within (pink) HPeV types was plotted on

the right hand y-axis (window size 100 codons, incrementing by 8 codons). A separate

analysis of sequence divergence between HPeV3 and other HPeV types is shown in

dark green. (B) dN/dS ratios between (dark green) and within (light green) HPeV types

were calculated using window size 100 codons, incrementing by 8 codons between

values. A genome diagram drawn to scale has been included to assist localisation of

changes in recombination frequency and amino acid sequence divergence.


151

(Boonyakiat et al., 2001), its absence in HPeV3 suggests the use of a

different (non-integrin) receptor for entry. Different receptor use can result in

a change in cellular tropism and might account for different clinical outcomes

observed in HPeV infections, such as more severe disease and central

nervous system involvement (Benschop et al., 2006a; Boivin et al., 2005; Ito

et al., 2004). Infection of different cell types in vivo might additionally reduce

the opportunity for recombination between HPeV3 and other HPeV types to

occur, and therefore potentially account for the failure to detect such

recombinants in our genetic survey.

Scans of sequence variability across parechovirus genomes revealed much

greater amino acid sequence variability and higher dN/dS ratios within the

structural gene region (Fig. 3). Through calculation of a segregation score in

the program TreeOrder Scan in the SIMMONICS sequence editor package

(version 1.6; http://www2.warwick.ac.uk/fac/sci/bio/research/devans/

bioinformatics/simmonics/), grouping by virus type was observed throughout

the structural region, a zone strictly demarcated by the 5’-UTR/VP0 and

S/NS (VP1/2A) gene boundaries, the latter also being characterized by a

dramatic decline in amino acid sequence variability and dN/dS ratio. These

observations for HPeV show striking similarities to the pattern of sequence

diversity and recombination frequency in other picornaviruses and other

mammalian, nonenveloped, positive-stranded RNA viruses (Simmonds,

2006). As a unifying hypothesis for these disparate observations, the

dramatically higher dN/dS ratio in the structural gene region, combined with

its much higher amino acid sequence divergence compared with the rest of

the genome, provides further evidence for positive selection operating on the

exposed outer surface of the virus. Immune-mediated selection may drive

changes in its antigenicity and, combined with changes in receptor use (such

as the deletion of the RGD motif in HPeV), ultimately generate new

serotypes refractory to immunity to previously encountered HPeV variants.

As proposed previously (Simmonds, 2006), the resulting high amino acid

sequence divergence and biological incompatibility may be the key factor,

thus preventing the occurrence of recombination in the structural gene

region. A second, smaller increase in amino acid diversity and dN/dS ratios

was also observed within the 3AB region, although whether this reflects

positive selection or less constraint on amino acid sequence change for

these amino acids, and for those in the homologous region in HEVs, is

unknown. Patterns of amino acid sequence divergence, location of

breakpoints and codon usage in parechoviruses revealed in this and

previous studies (Simmonds, 2006) thus closely mirror those of other

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152

picornavirus groups in which extensive recombination between serotypes

has been documented.

This is the first systematic survey of recombination frequencies and temporal

dynamics in parechoviruses, and has generated comparative sequence data

of VP1 and 3Dpol regions from several HPeV isolates from three different

geographical locations. Although there is a limited number of HPeV isolates

characterized genetically to date, the limited HPeV dataset used showed a

specific evolutionary trend that is also found in other picornaviruses.

Recombination may play a major role in the evolution of this virus genus,

and was found to occur with similarly rapid temporal dynamics as HEVs.

However, more full-length data are needed to study recombination within

HPeVs further. The data presented provide further knowledge for studying

the molecular evolution and epidemiology of HPeVs and a basis for in vitro

pathogenesis studies, particularly between HPeV3 and other HPeV types.

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Benschop, K. S. M., Schinkel, J., Luken, M. E., van den Broek, P. J. M.,

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Comprehensive Full Length Sequence

Analyses of Human Parechoviruses;

Diversity and Recombination.

K. S. M. Benschop1+, M. de Vries2+, R. P. Minnaar1,

G. Stanway3, L. van der Hoek2, K. C. Wolthers1,

P. Simmonds4

Manuscript in preparation

1 Lab.of Clinical Virology and

2 Experimental Virology, Dept. of Medical

Microbiology Academic Medical Center, Amsterdam. 3

Dept.of Biological Sciences, University of Essex, Colchester, UK. 4

Virus Evolution Group, Centre for Infectious Diseases, University of

Edinburgh, Edinburgh, UK.

+ Both authors contributed equally to the study

157

Comprehensive Full Length Sequence Analyses of Human

Parechoviruses; Diversity and Recombination

Human parechoviruses (HPeVs) are highly prevalent pathogens among

very young children. Although originally classified into 2 serologically

distinct types, HPeV1 and -2, recent analyses of variants collected

worldwide have revealed the existence of 12 further types classified

genetically by sequence comparisons of complete genome sequences

or the capsid gene, VP1. To investigate the nature of HPeV evolution,

its population dynamics and recombination breakpoints, we generated

18 full length genomic sequences of the most commonly circulating

genotypes, HPeV1 and -3, collected over a time span of 14 years from

the Netherlands. In total, we analyzed 35 sequences by inclusion of

previously published full length sequences. Analysis of contemporary

strains of HPeV1 and those most similar to the prototype strain

(Harris), showed HPeV1 variants to fall into two genetically distinct

clusters much more divergent from each other than observed within

other HPeV types. Future classification criteria for HPeVs may require

modification to accommodate the occurrence of variants with

intermediate degrees of diversity within type.

Recombination was frequently observed among HPeV1, -4, -5, and -6

but was much more restricted among HPeV3 strains. Recombination

sites were identified by segregation analysis and through identification

of positions of phylogeny violations on pairwise comparison of

different genome regions. Favoured sites for recombination were found

to flank the capsid region and further sites were found within the

nonstructural region, P2. In contrast to other HPeV types, the majority

of the HPeV3 sequences remained monophyletic across the genome, a

possible reflection of its lower diversity and potentially more recent

emergence than other HPeV types, or biological and/or epidemiological

constraints that limit opportunities for co-infections with potential

recombination partners.

INTRODUCTION

Human parechoviruses (HPeVs) are members of the family Picornaviridae

and are classified within the Parechovirus genus (34). HPeVs contain a

single stranded RNA of positive polarity and are approximately 7,300

nucleotides in length. The RNA encodes for a large polyprotein within a

single open reading frame flanked by 5’ and 3’ untranslated regions (5’ and

Chapter 10

158

3’ UTRs). The polyprotein is post-translationally cleaved into 3 structural

proteins (VP0, VP3 and VP1 encompassing P1) and 7 non-structural (NS)

proteins in P2 (2A to 2C) and P3 (3A to 3D) domains. Recent advances in

molecular screening and sequencing have led to the rapid identification and

characterization of several new HPeV types (3,5,8,14,41). Currently, a total

of 14 genotypes (HPeV1-14) have been identified and classified based on a

minimum of 25 to 30% nucleotide divergence threshold (approximately 15%

amino acid divergence) in the VP1 gene (8,26). Of these, HPeV1 and 3 have

been identified as the most commonly circulating strains in Europe and

elsewhere (5,35,38,41). They have predominantly been isolated from very

young children and have been associated with a range of diseases ranging

from mild gastrointestinal and respiratory symptoms to severe diseases,

such as neonatal sepsis and meningitis (2,6,33,39,43). Clinical studies have

indicated HPeV3 to be the predominant type associated with the severe

symptoms in neonates and to be the main type infecting the central nervous

system (CNS) (5,39,43).

Phylogenetic analyses of full length genomes show the genomes of many

HPeV types to be highly mosaic consistent with the occurrence of frequent

recombination events (3,8,19,44). The exception is HPeV3, where all

sequences available to date (three complete genome sequences and 43

paired VP1/3Dpol region sequences) cluster together throughout the

genome (7,42).

In the current study, we have generated 18 additional full length sequences

of the predominant HPeV genotypes and their variants, HPeV1 and HPeV3,

identified over a time span of 14 years. Our work doubles the size of the

HPeV complete sequence database and this extended dataset allowed a

more robust analysis of the relationship between HPeV divergence and

recombination frequency, and the positions where breakpoints have

occurred.

MATERIALS AND METHODS

Samples

In total, 18 HPeV strains (10 HPeV1 and 8 HPeV3) were selected for full

length sequencing (Table 1). Ten strains (6 HPeV1 and 4 HPeV3) were

obtained between 2001 and 2007 and were previously characterized based

on VP1 genotyping (5,6). Eight additional HPeV strains (4 HPeV1 and 4

HPeV3) isolated between 1993 and 1994 were included. Samples were (re-)

cultured on Vero and tertiary monkey kidney (tMK) cells as previously

described (6).

Diversity and recombination among human parechoviruses

159

Table 1. Full length sequences.

HPeV

type

Strain Year of

isolation

Sub-

classification

Ass.

number

Ref.

HPeV1 Harris 1956 Clade H S45208 (34)

BNI_788st 2003 Clade C EF051629 (9)

7555312 2003 Clade C FM178558 (44)

Picobank/HPeV1/a 2000 Clade C FM242866 (42)

152478 2001 Clade C CQ183018 This study




K54-94 1994 Clade C CQ183024 This study




2007-863 2007 Clade H CQ183034 This study

452568 2004 Clade H CQ183035 This study

HPeV2 Williamson 1956 AF055846 (12)

HPeV3 Can82852-02 2002 AJ889918 (1)

A308-99 1999 AB084913 (14)

152037 2001 CQ183026 This study

251360 2002 CQ183027 This study

450936 2004 CQ183028 This study

651689 2006 CQ183029 This study

K8-94 1994 CQ183033 This study

K11-94 1994 CQ183030 This study

K12-94 1994 CQ183031 This study

K20-94 1994 CQ183032 This study

HPeV4 T75-4077 1975 AM235750 (3)

K251176-02 2002 DQ315670 (8)

FUK2005-123 2005 AB433629 (40)

HPeV5 CT86-6760 1986 AJ005695 (24)

T92-15 1992 AM235749 (3)

HPeV6 NII561-2000 2000 AB252582 (41)

BNI 67-03 2003 EU024629 (4)

2005-823 2005 EU077518 (10)

HPeV7 Pak5045 2007 EU556224 (19)

HPeV8 Br/217/2006 2006 EU716175 (11)

Full length sequencing

Culture supernatant was extracted through automated extraction,

MagnaPure (Roche). cDNA synthesis was done as described previously (6).

Full length sequences were generated by primer walking strategy or

VIDISCA, combined with primer walking (10,37) (Table 1). Partially

overlapping fragments of 600 to 1500 nucleotides were amplified and

purified out of agarose gel, before sequencing through Big Dye terminator

reaction kit. Partial fragment were assembled using Vector NTI software

Advance 10 (Invitrogen) and manually edited.

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160

Nucleotide sequence alignments and diversity and distance

measurements

Sequences were aligned by nucleotide and codon based identity

implemented in ClustalW within the Simmonics Sequence Editor software

package (version 1.6; http://www2.warwick.ac.uk/fac/sci/bio/research/

devans/bioinformatics/Simmonics, 31). All full length sequences available

from GenBank were included in the alignment (Table 1), along with the

following P1 sequences (type): HPeV4: AB433630, AB434673; HPeV1:

AB112485, AB112486, AB112487, EU024630, EU024632, EU024633,

EU024634, EU024635, EU024636; HPeV3: AB112484, AB112483,

AB112482 and VP1 sequences: HPeV5: AB443848, EU077511; HPeV1:

AB443802, AB443830, AB443809, AB443817, AB443814; HPeV6:

AB300942. Nucleotide sequence numbering was based on the reference

sequence from HPeV1 Harris isolate (S45208). Sequence distances within

and between types were calculated using the program Sequence Distance

within the Simmonic Editor package with sequence groups assigned to types

1-8. Sequence diversity was computed as mean pair-wise Jukes and Cantor

(JC) corrected distances between nucleotide or p-distances for amino acid

sequences.

Phylogenetic and recombination analysis

Phylogenetic trees were constructed with MEGA3.1 (18) based on JC

distances (15). One thousand bootstrap replicates were analyzed.

Recombination analysis was automated within the Simmonics Sequence

Editor software and phylogenetic incongruencies were recorded across the

genome by use of the TreeOrder scan program (30). Sequence scans were

run using a sliding window of 252 nucleotides (81 codons) and an increment

of 9 nucleotides (3 codons).

Nucleotide accession numbers

The full lengths sequences have been deposited in GenBank under the

accession numbers GQ183018-GQ183035.

RESULTS

Full length sequence determination

Each of the 18 genomes sequenced was approximately 7200 nucleotides in

length. Eight of the 10 HPeV1 strains clustered with other contemporary

HPeV1 variants on phylogenetic comparison of capsid gene sequences and

were collected over 2 periods from 1993 and 1994 and from 2002 to 2005.


161

Table 2. Nucleotide and amino acid divergence between and within types based on the VP1

gene.

% Nucleotide divergence

(mean; range)

% Amino acid divergence

(mean; range)

Between groups (n=8) 31.7% (28.9%-35.9%) 26.4% (22.3%-33.4%)

Within groups 10.8% (4.4%-16.1%) 3.5% (1.6%-5.3%)

Together with 3 previously published full length sequences from 2000 and

2003 (HPeV1 BNI788St (9), 7555312 (44), and PicoBank/HPeV1/a (42), the

data set comprised 11 full length contemporary strains of HPeV1

(designated clade C, Fig. 1 and Table 1) collected over a period of 12 years.

These variants differed substantially from the HPeV1 Harris strain from

1956. Two other HPeV 1 variants collected in 2004 and 2007 clustered

loosely with the Harris strain, a grouping we have labeled as clade H (Fig. 1

and Table 1).

Among the 8 HPeV3 strains collected over the same 12 year time span as

the HPeV1 variants, seven were closely similar to each other and the 2

prototype strains (HPeV3 A308-99 (14) and CAN82852-03 (1)). However, we

also characterized an HPeV3 strain (651689, (5)) that was more divergent in

structural gene sequences from other HPeV3 variants (Table 1). The 10

HPeV1, the 8 HPeV3 sequences and the 17 previously published full length

HPeV sequences from GenBank used for phylogenetic analysis are shown

in Table 1.

Sequence relationships in the capsid region

Phylogenetic analyses of the 35 sequences within the capsid encoding

region (Figure 1, VP0, VP3 and VP1) identified five major clusters (HPeV1, -

3, -4, -5, and -6) and 3 single sequence branchings (HPeV2, 7, and 8),

corresponding to the first 8 of the 14 HPeV types currently described.

Phylogenetic grouping of HPeV3, -4, -5 and -6 variants into type-specific

clades was bootstrap supported in each structural gene. For HPeV1, there

was a clear separation of the HPeV1 group into 2 clusters with contemporary

strains falling into the designated clade C and a looser grouping of Harris-

like variants designated clade H (Table 1; Fig. 1). Bootstrap supported

grouping was only observed within VP0 and VP1 (>70%). Despite their

separate groupings, the two clades showed 9.6% amino acid divergence in

the VP1 gene, lower than the amino acid divergence between previously

designated HPeV types (mean 26.4%, Table 2), although with a distinct

distribution of pairwise distances greater than those observed within other

HPeV types (Fig. 2B). Mean nucleotide sequence divergence between the

two HPeV1 clades of 23.5% was substantially greater than between variants

Chapter 10

162

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EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

10

01

00

100

84

10

0

100

10

0

100

96

87

95

10

0

100

10095

99

10

0

0.0

5

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 2

52

58

1

HP

EV

1 5

501

63

HP

EV

1 1

52

478

HP

EV

1 7

55

53

12

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54-9

4

HP

EV

1 K

63

-94

HP

EV

1 4

50

34

3

HP

EV

1 2

00

7-8

63

HP

EV

1 H

AR

RIS

HP

EV

1 4

52

568

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILLIA

MS

ON

HP

EV

8 B

R/2

17/2

00

6

HP

EV

4 K

251

176

-02

HP

EV

4 F

UK

20

05

-12

3

HP

EV

4 T

75

-4077

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

7 P

AK

504

5

HP

EV

3 6

51

689

HP

EV

3 4

509

36

HP

EV

3 2

51

36

0

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 1

520

37

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12

-94

HP

EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11

-94

100

10

0

100

99

92

100

100

10

0

99

93

91

96

73

81

10

0

99

89

100

76

10

0

0.0

5

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 2

525

81

HP

EV

1 1

52

478

HP

EV

1 7

55

53

12

HP

EV

1 5

501

63

HP

EV

1 4

50

343

HP

EV

1 2

00

7-8

63

HP

EV

1 H

AR

RIS

HP

EV

1 4

525

68

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

4 K

25

11

76-0

2

HP

EV

4 T

75

-4077

HP

EV

4 F

UK

20

05-1

23

HP

EV

8 B

R/2

17/2

00

6

HP

EV

7 P

AK

504

5

HP

EV

3 6

51

689

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 K

12

-94

HP

EV

3 A

30

8-9

9

HP

EV

3 4

509

36

HP

EV

3 1

52

037

HP

EV

3 K

8-9

4

HP

EV

3 C

AN

82

853

-01

HP

EV

3 2

51

360

10

0

99

10

0

100

10

0

10

0

98

100

97

9880

89

98

96

10

0

99

10

0

97

75

0.0

5H

PE

V3

450

936

HP

EV

3 2

51

360

HP

EV

3 1

52

03

7

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 A

30

8-9

9

HP

EV

3 K

8-9

4

HP

EV

3 K

12-9

4

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

1 2

00

7-8

63

HP

EV

7 P

AK

50

45

HP

EV

3 6

51

68

9

HP

EV

4 K

251

176

-02

HP

EV

1 4

52

568

HP

EV

1 7

55

531

2

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

200

5-1

23

HP

EV

5 T

92

-15

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

1 4

50

343

HP

EV

1 5

50

163

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 H

AR

RIS

HP

EV

1 1

52

478

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

5 C

T-8

6-6

760

HP

EV

6 B

NI6

7-0

3

10

0

92

70

71

91

100

73

94

94

95

84

85

98

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

360

HP

EV

3 1

52

037

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 K

8-9

4

HP

EV

3 A

308

-99

HP

EV

3 K

12

-94

HP

EV

3 K

20-9

4

HP

EV

3 K

11

-94

HP

EV

1 K

129

-93

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54-9

4

HP

EV

1 K

63-9

4

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

4 K

25

117

6-0

2

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

504

5

HP

EV

4 F

UK

200

5-1

23

HP

EV

1 2

00

7-8

63

HP

EV

1 4

52

56

8

HP

EV

1 7

55

53

12

HP

EV

4 T

75

-4077

HP

EV

5 T

92-1

5

HP

EV

5 C

T-8

6-6

76

0

HP

EV

6 B

NI6

7-0

3

HP

EV

1 4

50

34

3

HP

EV

1 H

AR

RIS

HP

EV

1 1

524

78

HP

EV

1 5

50

16

3

HP

EV

8 B

R/2

17/2

00

6

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILL

IAM

SO

N

10

0

10

0

90

10

0

10

0

90

76

99

79

99

1009

8

92

87

98

10

0

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

513

60

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 1

52

037

HP

EV

3 K

8-9

4

HP

EV

3 A

30

8-9

9

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

3 K

12

-94

HP

EV

1 2

525

81

HP

EV

1 1

52

47

8

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 4

52

568

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

1 7

55

53

12

HP

EV

1 4

503

43

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

50

45

HP

EV

4 K

25

11

76-0

2

HP

EV

1 2

00

7-8

63

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 5

50

163

HP

EV

8 B

R/2

17/2

00

6

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

HP

EV

1 H

AR

RIS

HP

EV

2 W

ILL

IAM

SO

N

99

10

0

77

91

98

10

0

989

4

86

85

96

99

78

97

0.0

2

HP

EV

3 C

AN

828

53-0

1

HP

EV

3 2

51

36

0

HP

EV

3 4

50

936

HP

EV

3 1

520

37

HP

EV

3 K

8-9

4

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12

-94

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

1 1

52

478

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 4

50

34

3

HP

EV

1 2

52

581

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

1a

HP

EV

4 K

25

11

76-0

2

HP

EV

1 2

00

7-8

63

HP

EV

1 5

50

163

HP

EV

4 F

UK

20

05-1

23

HP

EV

5 T

92-1

5

HP

EV

5 C

T-8

6-6

76

0

HP

EV

1 4

52

568

HP

EV

1 7

555

312

HP

EV

3 6

516

89

HP

EV

7 P

AK

504

5

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

8 B

R/2

17/2

006

HP

EV

4 T

75-4

077

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

99

10

0

10

0

10

0

96

10

0

96

87

90

84

10

0

1007

5

9086

0.0

2

C H

C H

C H

5’U

TR

VP

0V

P3

VP

1

2A

2B

2C

3A

HP

EV

1 K

129

-93

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54-9

4

HP

EV

1 K

63-9

4

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 7

55

53

12

HP

EV

1 5

50

16

3

HP

EV

1 B

NI-

788

St

HP

EV

1 2

525

81

HP

EV

1 1

52

478

HP

EV

1 4

50

34

3

HP

EV

4 K

25

117

6-0

2

HP

EV

5 T

92

-15

HP

EV

8 B

R/2

17/2

006

HP

EV

4 F

UK

20

05-1

23

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

HP

EV

4 T

75

-4077

HP

EV

6 B

NI6

7-0

3 HP

EV

1 4

52

56

8

HP

EV

1 2

00

7-8

63

HP

EV

3 6

51

689

HP

EV

3 1

520

37

HP

EV

3 2

513

60

HP

EV

3 4

50

936

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 A

30

8-9

9

HP

EV

7 P

AK

50

45

HP

EV

3 K

8-9

4

HP

EV

3 K

12

-94

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

1 H

AR

RIS

HP

EV

2 W

ILLIA

MS

ON

HP

EV

5 C

T-8

6-6

76

0

99

10

0

93

81

99

99

93

98

95

91

98

8073

73

0.0

1

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 1

524

78

HP

EV

1 7

55

53

12

HP

EV

1 2

52

581

HP

EV

1 5

50

16

3

HP

EV

1 4

503

43

HP

EV

1 2

00

7-8

63

HP

EV

1 H

AR

RIS

HP

EV

1 4

52

568

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

6 B

NI6

7-0

3

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

8 B

R/2

17/2

00

6

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

4 F

UK

20

05-1

23

HP

EV

4 T

75

-40

77

HP

EV

4 K

25

11

76-0

2

HP

EV

7 P

AK

50

45

HP

EV

3 6

51

689

HP

EV

3 A

30

8-9

9

HP

EV

3 4

509

36

HP

EV

3 2

51

360

HP

EV

3 C

AN

828

53-0

1

HP

EV

3 1

52

03

7

HP

EV

3 K

12

-94

HP

EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

10

01

00

100

84

10

0

100

10

0

100

96

87

95

10

0

100

10095

99

10

0

0.0

5

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 2

52

58

1

HP

EV

1 5

501

63

HP

EV

1 1

52

478

HP

EV

1 7

55

53

12

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54-9

4

HP

EV

1 K

63

-94

HP

EV

1 4

50

34

3

HP

EV

1 2

00

7-8

63

HP

EV

1 H

AR

RIS

HP

EV

1 4

52

568

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILLIA

MS

ON

HP

EV

8 B

R/2

17/2

00

6

HP

EV

4 K

251

176

-02

HP

EV

4 F

UK

20

05

-12

3

HP

EV

4 T

75

-4077

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

7 P

AK

504

5

HP

EV

3 6

51

689

HP

EV

3 4

509

36

HP

EV

3 2

51

36

0

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 1

520

37

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12

-94

HP

EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11

-94

100

10

0

100

99

92

100

100

10

0

99

93

91

96

73

81

10

0

99

89

100

76

10

0

0.0

5

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 2

525

81

HP

EV

1 1

52

478

HP

EV

1 7

55

53

12

HP

EV

1 5

501

63

HP

EV

1 4

50

343

HP

EV

1 2

00

7-8

63

HP

EV

1 H

AR

RIS

HP

EV

1 4

525

68

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

4 K

25

11

76-0

2

HP

EV

4 T

75

-4077

HP

EV

4 F

UK

20

05-1

23

HP

EV

8 B

R/2

17/2

00

6

HP

EV

7 P

AK

504

5

HP

EV

3 6

51

689

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 K

12

-94

HP

EV

3 A

30

8-9

9

HP

EV

3 4

509

36

HP

EV

3 1

52

037

HP

EV

3 K

8-9

4

HP

EV

3 C

AN

82

853

-01

HP

EV

3 2

51

360

10

0

99

10

0

100

10

0

10

0

98

100

97

9880

89

98

96

10

0

99

10

0

97

75

0.0

5H

PE

V3

450

936

HP

EV

3 2

51

360

HP

EV

3 1

52

03

7

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 A

30

8-9

9

HP

EV

3 K

8-9

4

HP

EV

3 K

12-9

4

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

1 2

00

7-8

63

HP

EV

7 P

AK

50

45

HP

EV

3 6

51

68

9

HP

EV

4 K

251

176

-02

HP

EV

1 4

52

568

HP

EV

1 7

55

531

2

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

200

5-1

23

HP

EV

5 T

92

-15

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

1 4

50

343

HP

EV

1 5

50

163

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 H

AR

RIS

HP

EV

1 1

52

478

HP

EV

6 N

II5

61

-20

00

HP

EV

6 2

00

5-8

23

HP

EV

5 C

T-8

6-6

760

HP

EV

6 B

NI6

7-0

3

10

0

92

70

71

91

100

73

94

94

95

84

85

98

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

360

HP

EV

3 1

52

037

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 K

8-9

4

HP

EV

3 A

308

-99

HP

EV

3 K

12

-94

HP

EV

3 K

20-9

4

HP

EV

3 K

11

-94

HP

EV

1 K

129

-93

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54-9

4

HP

EV

1 K

63-9

4

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

4 K

25

117

6-0

2

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

504

5

HP

EV

4 F

UK

200

5-1

23

HP

EV

1 2

00

7-8

63

HP

EV

1 4

52

56

8

HP

EV

1 7

55

53

12

HP

EV

4 T

75

-4077

HP

EV

5 T

92-1

5

HP

EV

5 C

T-8

6-6

76

0

HP

EV

6 B

NI6

7-0

3

HP

EV

1 4

50

34

3

HP

EV

1 H

AR

RIS

HP

EV

1 1

524

78

HP

EV

1 5

50

16

3

HP

EV

8 B

R/2

17/2

00

6

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILL

IAM

SO

N

10

0

10

0

90

10

0

10

0

90

76

99

79

99

1009

8

92

87

98

10

0

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

513

60

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 1

52

037

HP

EV

3 K

8-9

4

HP

EV

3 A

30

8-9

9

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

3 K

12

-94

HP

EV

1 2

525

81

HP

EV

1 1

52

47

8

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 4

52

568

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

1 7

55

53

12

HP

EV

1 4

503

43

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

50

45

HP

EV

4 K

25

11

76-0

2

HP

EV

1 2

00

7-8

63

HP

EV

5 T

92

-15

HP

EV

5 C

T-8

6-6

76

0

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 5

50

163

HP

EV

8 B

R/2

17/2

00

6

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

HP

EV

1 H

AR

RIS

HP

EV

2 W

ILL

IAM

SO

N

99

10

0

77

91

98

10

0

989

4

86

85

96

99

78

97

0.0

2

HP

EV

3 C

AN

828

53-0

1

HP

EV

3 2

51

36

0

HP

EV

3 4

50

936

HP

EV

3 1

520

37

HP

EV

3 K

8-9

4

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12

-94

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

1 1

52

478

HP

EV

1 K

12

9-9

3

HP

EV

1 K

15

0-9

3

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 4

50

34

3

HP

EV

1 2

52

581

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

1a

HP

EV

4 K

25

11

76-0

2

HP

EV

1 2

00

7-8

63

HP

EV

1 5

50

163

HP

EV

4 F

UK

20

05-1

23

HP

EV

5 T

92-1

5

HP

EV

5 C

T-8

6-6

76

0

HP

EV

1 4

52

568

HP

EV

1 7

555

312

HP

EV

3 6

516

89

HP

EV

7 P

AK

504

5

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

8 B

R/2

17/2

006

HP

EV

4 T

75-4

077

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61

-2000

HP

EV

6 2

00

5-8

23

99

10

0

10

0

10

0

96

10

0

96

87

90

84

10

0

1007

5

9086

0.0

2

C H

C H

C H


163

3C

3D

3’U

TR

3B

HP

EV

1 2

52

581

HP

EV

3 1

520

37

HP

EV

3 A

30

8-9

9

HP

EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 4

509

36

HP

EV

3 2

513

60

HP

EV

3 K

12

-94

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

1 2

00

7-8

63

HP

EV

4 K

251

176

-02

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

504

5

HP

EV

1 4

52

56

8

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 K

12

9-9

3

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 7

555

312

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 K

15

0-9

3

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

5 T

92-1

5

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

HP

EV

1 5

501

63

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 4

503

43 H

PE

V1 1

52

47

8

HP

EV

5 C

T-8

6-6

76

0

HP

EV

6 N

II56

1-2

000

HP

EV

6 2

005

-82

38

0

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

36

0

HP

EV

3 C

AN

82

853

-01

HP

EV

3 1

52

03

7

HP

EV

3 K

8-9

4

HP

EV

3 K

12-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 A

308

-99

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ban

k/H

Pe

V/1

a

HP

EV

1 4

52

56

8

HP

EV

4 K

25

11

76

-02

HP

EV

7 P

AK

50

45

HP

EV

3 6

51

689

HP

EV

1 7

555

312

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 2

00

7-8

63

HP

EV

1 1

524

78

HP

EV

6 N

II56

1-2

000

HP

EV

6 2

00

5-8

23

HP

EV

5 C

T-8

6-6

760

HP

EV

1 4

503

43

HP

EV

1 5

501

63

HP

EV

8 B

R/2

17/2

006

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

5 T

92

-15

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

10

0

10

0

100

97

10

0

94

10

0

10

0

97

97

82

99

10

0

82

73

100

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

36

0

HP

EV

3 K

8-9

4

HP

EV

3 C

AN

828

53-0

1

HP

EV

3 1

52

03

7

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12-9

4

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788S

t

HP

eV

1 P

ico

Ban

k/H

Pe

V/1

a

HP

EV

1 2

007

-863

HP

EV

7 P

AK

50

45

HP

EV

4 K

25

117

6-0

2

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

4 F

UK

20

05

-12

3

HP

EV

1 4

525

68

HP

EV

4 T

75-4

077

HP

EV

1 7

55

531

2

HP

EV

1 5

50

163

HP

EV

3 6

516

89

HP

EV

6 N

II5

61-2

000

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILLIA

MS

ON

HP

EV

5 T

92

-15

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 H

AR

RIS

HP

EV

1 1

52

478

HP

EV

6 B

NI6

7-0

3

HP

EV

1 4

50

34

3

HP

EV

5 C

T-8

6-6

76

0

10

0

10

0

10

0

96

77

81

10

0

99

99

84

10

0

99

79

0.0

2

HP

EV

3 4

509

36

HP

EV

3 2

513

60

HP

EV

3 1

520

37

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 K

8-9

4

HP

EV

3 A

308

-99

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

3 K

12

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

Pe

V/1

a

HP

EV

1 K

150

-93

HP

EV

1 K

129

-93

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 5

50

16

3

HP

EV

1 4

52

568

HP

EV

4 F

UK

200

5-1

23

HP

EV

4 T

75

-4077

HP

EV

1 7

555

312

HP

EV

4 K

25

11

76

-02

HP

EV

1 2

007

-86

3

HP

EV

7 P

AK

50

45

HP

EV

5 C

T-8

6-6

76

0

HP

EV

3 6

51

689

HP

EV

8 B

R/2

17/2

00

6

HP

EV

6 B

NI6

7-0

3

HP

EV

6 N

II5

61-2

000

HP

EV

6 2

00

5-8

23

HP

EV

1 4

503

43

HP

EV

1 H

AR

RIS

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

1 1

52

478

HP

EV

5 T

92

-15

99

84

79

95

70

0.0

5

HP

eV

6H

Pe

V7

HP

eV

3,

19

99

-200

6

HP

eV

3,

19

94

HP

eV

1,

cla

de

H

HP

eV

1,

cla

de

C,

20

00-2

00

6

HP

eV

4

HP

eV

5

HP

eV

1, cla

de C

, 1

99

3 a

nd

19

94

HP

eV

2

HP

eV

8

3C

3D

3’U

TR

3B

HP

EV

1 2

52

581

HP

EV

3 1

520

37

HP

EV

3 A

30

8-9

9

HP

EV

3 K

8-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 4

509

36

HP

EV

3 2

513

60

HP

EV

3 K

12

-94

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

1 2

00

7-8

63

HP

EV

4 K

251

176

-02

HP

EV

3 6

51

68

9

HP

EV

7 P

AK

504

5

HP

EV

1 4

52

56

8

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ba

nk/H

PeV

/1a

HP

EV

1 K

12

9-9

3

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 7

555

312

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 K

15

0-9

3

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

5 T

92-1

5

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

HP

EV

1 5

501

63

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 4

503

43 H

PE

V1 1

52

47

8

HP

EV

5 C

T-8

6-6

76

0

HP

EV

6 N

II56

1-2

000

HP

EV

6 2

005

-82

38

0

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

36

0

HP

EV

3 C

AN

82

853

-01

HP

EV

3 1

52

03

7

HP

EV

3 K

8-9

4

HP

EV

3 K

12-9

4

HP

EV

3 K

20-9

4

HP

EV

3 K

11-9

4

HP

EV

3 A

308

-99

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

Ban

k/H

Pe

V/1

a

HP

EV

1 4

52

56

8

HP

EV

4 K

25

11

76

-02

HP

EV

7 P

AK

50

45

HP

EV

3 6

51

689

HP

EV

1 7

555

312

HP

EV

4 T

75

-40

77

HP

EV

4 F

UK

20

05-1

23

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

1 2

00

7-8

63

HP

EV

1 1

524

78

HP

EV

6 N

II56

1-2

000

HP

EV

6 2

00

5-8

23

HP

EV

5 C

T-8

6-6

760

HP

EV

1 4

503

43

HP

EV

1 5

501

63

HP

EV

8 B

R/2

17/2

006

HP

EV

2 W

ILL

IAM

SO

N

HP

EV

5 T

92

-15

HP

EV

1 H

AR

RIS

HP

EV

6 B

NI6

7-0

3

10

0

10

0

100

97

10

0

94

10

0

10

0

97

97

82

99

10

0

82

73

100

0.0

2

HP

EV

3 4

50

93

6

HP

EV

3 2

51

36

0

HP

EV

3 K

8-9

4

HP

EV

3 C

AN

828

53-0

1

HP

EV

3 1

52

03

7

HP

EV

3 A

30

8-9

9

HP

EV

3 K

12-9

4

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788S

t

HP

eV

1 P

ico

Ban

k/H

Pe

V/1

a

HP

EV

1 2

007

-863

HP

EV

7 P

AK

50

45

HP

EV

4 K

25

117

6-0

2

HP

EV

1 K

12

9-9

3

HP

EV

1 K

150

-93

HP

EV

1 K

54

-94

HP

EV

1 K

63

-94

HP

EV

4 F

UK

20

05

-12

3

HP

EV

1 4

525

68

HP

EV

4 T

75-4

077

HP

EV

1 7

55

531

2

HP

EV

1 5

50

163

HP

EV

3 6

516

89

HP

EV

6 N

II5

61-2

000

HP

EV

6 2

00

5-8

23

HP

EV

2 W

ILLIA

MS

ON

HP

EV

5 T

92

-15

HP

EV

8 B

R/2

17/2

00

6

HP

EV

1 H

AR

RIS

HP

EV

1 1

52

478

HP

EV

6 B

NI6

7-0

3

HP

EV

1 4

50

34

3

HP

EV

5 C

T-8

6-6

76

0

10

0

10

0

10

0

96

77

81

10

0

99

99

84

10

0

99

79

0.0

2

HP

EV

3 4

509

36

HP

EV

3 2

513

60

HP

EV

3 1

520

37

HP

EV

3 C

AN

82

85

3-0

1

HP

EV

3 K

8-9

4

HP

EV

3 A

308

-99

HP

EV

3 K

20

-94

HP

EV

3 K

11

-94

HP

EV

3 K

12

-94

HP

EV

1 2

525

81

HP

EV

1 B

NI-

788

St

HP

eV

1 P

ico

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Fig

ure

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Unro

ote

d p

hylo

genetic a

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PeV

based o

n p

ostr

ansla

tionally

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egio

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cale

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ence.

Chapter 10

164

within each genotypic group (mean 10.8%, Table 2; Fig. 2A), yet lower than

observed between other types (31.7%, Table 2; Fig. 2A). The degree of

sequence divergence between capsid region sequences correlated closely

with difference in years of isolation (Fig. 1). For example, HPeV 1

contemporary strains isolated in the early 1990s were found to be distinct

from the more recently isolated stains and clustered separately within the

HPeV1 contemporary clade. However, HPeV3 strains isolated in the early

1990s were found to be quite similar to the more recently isolated stains

(nucleotide similarity 95.6%), indicative of a more direct line of descent over

the 12 year observation period (ie. the 1994 variants were more closely

related to the common ancestor of recently isolated HPeV3 strains), a

hypothesis supported by differences in the shape of HPeV3 and HPeV1 sub-

trees.

Figure 2. Distributions of

nucleotide (upper graph) and

amino acid sequence (lower

graph) pairwise p distances

measured in the VP1 region

within HPeV types (unfilled

boxes), between types (filled

grey), and the subset of

comparisons between clades C

and H within HPeV (filled black).


165

Phylogenetic relationship of HPeV in different genome regions

By comparison of VP1 and 3Dpol sequences, we previously showed that

sequence divergence in the structural gene and, the difference in years

between the isolation dates of the HPeV1, -4, -5, and, -6 strains, correlated

with a greater frequency of recombination. This was demonstrated by

grouping HPeV variants into a number of phylogenetically distinct clades in

the 3Dpol region (7). With the full length sequences, we were able to extend

this analysis with HPeV variants collected over a longer observation period

and to determine positions between VP1 and 3Dpol where recombination

occurred.

All HPeV strains that were closely related to each other (VP1 divergence

<2.5%) remained monophyletic throughout the NS region (Fig. 1, 2A-2C, 3A-

3D), including 9 from 10 of the HPeV3 isolates. The more divergent HPeV3

strain (651689, VP1 divergence 14%) lost its phylogenetic clustering with

other HPeV3 variants in 2A and throughout the rest of the NS region (Fig. 1).

Analysis of the segregation of sequences by type across HPeV genomes

showed a 100% or near to a 100% segregation within the capsid (zero or

low y-axis values; Fig. 3). The capsid region was sharply demarcated at its 5’

and 3’ ends, with 5’UTR and the NS region sequences showing high values,

indicative of recombination. Small violations of the type specific grouping

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

122

368

608

862

1123

1393

1645

1915

2182

2443

2674

2923

3184

3451

3721

3961

4228

4498

4762

4996

5245

5515

5782

6040

6310

6580

6850

7120

se

gre

ga

tio

n

Genome position

HPeV1, clade C

HPeV1, clade HHPeV3

HPeV4HPeV5HPeV6

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

122

368

608

862

1123

1393

1645

1915

2182

2443

2674

2923

3184

3451

3721

3961

4228

4498

4762

4996

5245

5515

5782

6040

6310

6580

6850

7120

se

gre

ga

tio

n

Genome position

HPeV1, clade C

HPeV1, clade HHPeV3

HPeV4HPeV5HPeV6

HPeV1, clade C

HPeV1, clade HHPeV3

HPeV4HPeV5HPeV6

Figure 3. Segregation scans across the HPeV genome using a window size of 252 nt

with an increment of 9 nt. Segregation scores are plotted according to their position in

the genome and are represented between 0 (perfect segregation) and 1 (no

segregation). A schematic scheme of the genome is given for visualization purposes.

The arrows are colour coded (see inset) and indicate the position at which specific

clades broke up.

Chapter 10

166

could also be observed within the VP3 gene in the capsid-encoding region

and was found to be solely caused by a lack of monophylogeny of the two

HPeV1 clades (clade C and H). This in turn was consistent with the

observed lack of bootstrap support for HPeV1 variants in the VP3

phylogenetic tree (Fig. 1). Reassignment of the Harris-like clade into a

different sequence group from the C clade restored 100% segregation within

the capsid-encoding region (data not shown).

The sharp demarcation of segregation scores at each end of the capsid

region (Fig. 3) is consistent with the existence of frequent recombination

breakpoints at the structural gene boundaries. However, identification of the

precise positions of breakpoints is complicated by the absence of the

sequences of both recombination parents of putative recombinant strains.

For example, the sequence of the HPeV variant that recombined with the

divergent 651689 HPeV3 isolate is unrepresented in the dataset. As a result,

breakpoint assignment can only be made by recording positions where

phylogenetic groupings change, although necessarily this method of analysis

does not indicate which of the two separated lineages underwent the

recombination event and which remained non-recombinant.

Changes in phylogenetic groupings can be identified by recording tree

positions and boundaries of phylogenetically supported clades of the 35

complete genome sequences using output from the TreeOrder scan

program. Using a window size of 252 bases and an increment of 9 bases,

changes in phylogeny were principally located at the structural gene

boundaries, consistent with the segregation analysis (Fig. 3). The

recombination breakpoints were spread over the 5’UTR-P1 junction for

HPeV1, -3, -4, -5, and -6. At the P1-P2 junction, recombination breakpoints

were also dispersed, starting already at the 3’end of the VP1 gene. This

included the break-up within the HPeV1 group into separate clusters, as well

as those of HPeV4 and HPeV3 (Fig. 3). Within our dataset, breakpoints in

HPeV5 and 6 were found in the 2C protein, corresponding to the second rise

in segregation values (Fig. 3).

The position where recombination events occurred can also be visualized by

calculation of frequencies of phylogeny violations between trees constructed

from different sequence fragments in the genome. Using the same fragment

size (252 bases) and increment (9 bases), violation scores were consistently

low on comparison of different fragments of the capsid gene (shaded dark

blue in Fig. 4; ie. trees constructed from VP0, VP3 and VP1 were largely


167

congruent), while higher scores were scored across the capsid/NS gene

boundary as well as within the NS region (eg. between P2 and P3) and

5’UTR, the latter observations are consistent with the existence of more

recombination breakpoints.

DISCUSSION

In this study we obtained 18 new complete genome sequences of HPeV

types 1 and 3, allowing a comprehensive analysis of intra- and inter-type

diversity of HPeV and the positions and constraints on recombination.

Phylogenetic comparison of HPeV1 strains identified two distinct clusters.

The diversity seen by the separate clustering of clade C to the original Harris

strain was already observed in previous studies (2,4,6,42). With only one

strain defining clade H, data was limited to warrant a separate classification

of the 2 variants. Divergence within the VP1 gene between the two clades of

23% approached the threshold defining HPeV types of 25% (8,26). The

diversity resembles that seen for enterovirus 71, showing a 15-22%

nucleotide divergence (approximately 8-13% amino acid divergence) within

VP4 between 3 major genogroups (22,23). Similarly, echovirus 11 (16) and

echovirus 30 (29), were found to contain specific clusters within the

genotype. However the criteria to define a sub-cluster are not specifically

defined and are often based on clustering and divergence within different

genes. Here we show a distinctive distribution of pairwise distances within

Figure 4. Phylogenetic

compatibility between different

genome regions. Matrix shows

phylogenetic compatibility scores

between trees generated using a

window size of 252 nt with an

increment of 9 nt and are

recorded in colour (see inset

scale). A 60% bootstrap was

used to define clades. The

HPeV1 group was analyzed as 2

separate clades.

Chapter 10

168

the VP1 gene that extends through the entire capsid gene (data not shown),

justifying a sub-classification of the HPeV genotype rather than a separate

classification into new genotypes. The distinction between the two clades

was specifically found within the VP3 gene. The VP3 protein contains a

majority of the proposed antigenic determinants found on the capsid surface.

It is possible that sequence change in HPeV1 underlying this diversification

was driven by immune escape mechanisms, ultimately to produce

serologically distinct descendants able to circulate independently from each

other. Nonetheless, strains identified in clade C could be neutralized by

antisera directed against the Harris strain (2, and Benschop et al, manuscript

in preparation), consistent with the much lower amino acid sequence

divergence (10% and 8.4% in VP1 and P1 respectively) than between types

(26% and 24%).

Recombination between HPeV types was frequently observed within the

nonstructural region among HPeV1, 4, 5, and 6 strains, similar to many

picornaviruses (20,21,25,27,28,30). However, we found a loss of type-

specific segregation in only one of the HPeV3 sequences (strain 651689),

indicating while HPeV3 sequences are able to recombine, it occurs less

frequently than in other genotypes. In this specific case, the high divergence

in P1 and therefore greater temporal (evolutionary) separation between this

isolate and other HPeV3 strains are likely contributory factors for a

recombination event to have occurred in the past (7). However, we did not

observe any recombination among other HPeV3 strains with a similar degree

of temporal separation. In marked contrast, most HPeV1 variants in the C

clade collected in the same time span of 12 years showed evidence for

recombination; ie. those isolated in 1993 and 1994 did not remain

segregated with strains isolated seven years later, in 2001 (152478) and

2002 (252581). The lower observed frequency of recombination observed in

HPeV3 sequences may reflect on biological constraints such as difference in

cellular tropism, spread to different tissues limiting the opportunities for

recombination to occur.

The most frequent sites for recombination flanked the capsid region, and

could readily be identified by the segregation scans and bootscan analysis

(9,44). These sites mirror those observed for other picornaviruses such as

enteroviruses (21,25,32), and aphthoviruses (13,36). Within this study we

were able to map these sites more precisely for HPeV. The recombination

breakpoints identified in the current study occurred at several positions

around the P1-P2 junction, starting already at the C terminal end of the VP1

gene, confirming the findings by Williams et al. (42). This VP1 region


169

contains the Arg-Gly-Asp (RGD receptor binding domain, and recombination

at this position can thus have major consequences for receptor usage and

tropism of different HPeV types and variants (5,42). Two breakpoints were

found near the P2-P3 junction, within the 2C protein, for HPeV5 and HPeV6,

an observation which is not uncommon among picornaviruses (13,32). Thus,

the segregation of HPeV5 and -6 was found to carry over to a large portion

of the P2 non structural region, although we cannot rule out that this is due

to a sampling effect of only 2 HPeV5 strains and 3 HPeV6 strains.

These obvious crossover points flanking the capsid gene and near the P2-

P3 boundary are relatively conserved among picornaviruses facilitating

recombination through template switching during replication (17,20).

Alternatively, recombination at other sites (ie. within functional protein

domains) may be more likely to produce non-viable or less fit viruses

preventing their fixation in the population.

The data presented here, addresses the need for clear classification criteria

that accommodates the additional tier of variability within some types,

especially in a time when new types are rapidly being identified.

Recombination in parechoviruses occurs frequently, similar to other

picornaviruses in its dynamics and positions of breakpoints. Remarkably,

there were differences in recombination frequency between HPeV3 and

other HPeV types and may underlie the clinical differences observed

between them.

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Summary and Discussion

Summary

179

Summary

Human Parechoviruses (HPeV) have predominantly been associated with

mild gastrointestinal and respiratory symptoms. Occasionally, more severe

symptoms were described to be associated with HPeVs. Nonetheless these

viruses were considered irrelevant for routine diagnostics in comparison to

human enterovirus (HEV) infection, which are the major viral cause of

neonatal sespis and meningitis. This thesis was started with a search for

HPeVs in cell cultures, which lead to the finding that HPeV3 infections are

associated with more severe disease than HPeV1 infections. From our

studies it became clear that HPeV3 is clinically different from other HPeV

infections. Not only do we show HPeV3 to be clinically different, but HPeV3

is also found to exhibit certain biological and molecular traits different from

other HPeV types.

Chapter 1 is a general introduction to our current knowledge of HPeV

infections. When first described in 1956, HPeV1 and -2 infections were

considered mild. It took almost half a century, before a third HPeV type was

described. Already this virus was molecularly distinct and was found to lack

the critical epitope for integrin binding (Arginine-Glycine-Aspartic Acid,

RGD). HPeVs exhibit a similar cytopathogenic effect (CPE) in cell culture as

enteroviruses. As the 5’UTR of HPeV is different than that for enteroviruses,

HEV specific PCRs will not detect HPeV infections, leading to a miss- or

underdiagnoses. From 303 HEV positive cultures between 2000 and 2005,

37 cultures (12%) could be identified as either HPeV1 (n=27) or HPeV3

(n=10) (chapter 2). Clinical characteristics were scored by questionnaires

and infections with HPeV3 were found to be significantly more often

associated with severe symptoms and with a younger age in comparison to

infections with HPeV1. We hypothesized that HPeV3 has a different cell

tropism, in comparison to HPeV1, ruled by its absence of the RGD motif

which could indicate an RGD independent cell entry mechanism.

To screen more efficiently for HPeV infections, we developed a rapid HPeV-

specific real-time PCR with a specific primer pair and a single degenerate

probe that could detect all known HPeV types at the time (n=6) (chapter 3).

This method was evaluated for sensitivity and specificity by analysing all

known HPeV types as well as other common human picornaviruses such as

enteroviruses, rhinoviruses and hepatitis A virus. The assay reached a

sensitivity of 75 copies per ml clinical sample, comparable to our HEV

specific PCR, and was highly specific. Infections with HPeV are clinically

indistinguishable from HEV infection. Since both viruses can be associated

Chapter 11

180

with the same array of clinical symptoms, we introduced the HPeV real time

assay in parallel to the HEV assay for diagnostic purposes.

Retrospective screening using the newly developed PCRs on CSF samples

from children <5 years lead to an increase of 31% more positive samples

than when screened for HEV alone (chapter 4). The study involved 761 CSF

samples obtained between 2004 and 2006. From the 761 CSF samples

prospectively screened for HEV infection, 716 CSF samples were available

for retrospective screening of HPeV infection. We found 33 HPeV infections

(4.6%). In comparison, we found 108 HEV infections (14.1%). Children

infected with HPeV were found to remain in hospital for a mean of 7 days of

which 82% received antibiotics for 5.7 days as a result of a negative HEV

infection diagnosed at the time. The study described in chapter 4 points out

the clinical relevance of the implementation of the HPeV PCR and shows

HPeV as a second viral cause for neonatal sepsis and meningitis. Adequate

diagnostics is essential for good paediatric care and to avoid unnecessary

hospital stays and treatment.

Within this study, HPeV prevalence varied over the years, with only one

positive infection in 2005. In comparison, HEV infections of the central

nervous system (CNS) remained constant over the years. That the variability

might be due to a specific HPeV type exhibiting neurotropic characteristics

was supported by our screening of stool samples, the same period (2004 to

2006), where stool obtained from children < 5 years were directly typed by

PCR and sequencing (chapter 5). In 2005, the year we observed a low

prevalence of HPeV in CSF, we did not identify any HPeV3 infections. Thus,

these data supported our hypothesis that HPeV3 may infect different cell

types, such as neural cells, with a more severe outcome.

Screening of stool samples obtained from children less than 5 years of age

showed a high percentage of children to be positive for HPeV (n=225,

16.3%). This was comparable to the percentage HEV positive children

(n=253, 18.4%). When all age groups were included, as in the analysis of

stool samples from 2007 and 2008 (chapter 7), a lower prevalence was

found for both HEV and HPeV. In these studies, HPeV1 was found to be the

predominant strain, followed by HPeV3. HPeV4, -5, and -6 were identified

less frequently, although the HPeV4 prevalence was quite high in 2007. To

determine the clinical manifestations of the new HPeV types (4-6),

questionnaires were used to score for different clinical parameters, such as

age, hospital stay, and disease symptoms. Infection with HPeV4, -5, and -6

were found to be predominantly associated with mild symptoms (chapter 6)

as seen for HPeV1 and -2. Only three children with HPeV4 infection were

found to suffer from neonatal sepsis or meningitis.

Summary

181

By direct genotyping we have identified four strains that lacked the RGD

motif. The strains could not be cultured. One strain could be typed as HPeV1

and the other three were characterized as HPeV5. These types should

contain the RGD motif, but instead of the RGD motif at the 3’end, the strains

contained a different specific consensus sequence at that site (chapter 5).

The insertion of the specific sequence found in the four strains could indicate

a second RGD-independent pathway.

A new HPeV type, which also could not be cultured, was identified by direct

genotyping. The strain was designated HPeV14, since it was discovered

after the identification of HPeV7 to -13. None of the new HPeV types (7 to

14) that were all identified by PCR were found to have the RGD motif.

Interestingly, we found three HPeV1 strains to be most closely related to the

Harris strain identified in 1956. The Harris strain was not identified in any of

the other recent studies and was thought to cease circulation.

The value of direct stool screening and genotyping was assessed in chapter

7. A higher percentage of HPeV and HEV positive samples was identified by

PCR in comparison to culture. This was found to be significantly correlated

to the CT value in PCR. In addition more samples could be typed by

genotyping rather than by serotyping. From the 6 cell lines used (HT29

(colon carcinoma), RD (rhabdomyosarcoma), Vero (African green monkey

kidney), tMK (tertiary monkey kidney), Hel (human embryonic lung), and

A549 (lung carcinoma)), both HPeVs and enteroviruses grew efficiently on

the HT29 cell line. Specific types were found to grow exclusively on certain

cell lines, such as Coxsackie A viruses on RD cells and HPeV3 on A549 and

vero cells, supporting our hypothesis of a different cell tropism for HPeV3

The second part of the thesis describes the identification and

characterization of HPeV4 (chapter 8). The criteria for classification we used

for this new HPeV genotype were based on those formulated by Oberste et

al, (2004) for enteroviruses. Nowadays, those criteria are considered to be

suitable to type HPeV strains. From this study, we describe for the first time

the occurrence of recombination to play a role in HPeV evolution. To study

this further, we compared two different genomic regions (VP1 and 3Dpol)

from 37 isolates obtained between 2000 and 2006 (chapter 9).

Recombination was frequently observed between types 1, 4, 5, and 6. The

likelihood of recombination was found to be correlated to the VP1

divergence and difference in years of isolation between the strains,

resembling that of enterovirus species B. The greater the divergence and

temporal separation, the higher the likelihood of recombination was found to

occur. Interestingly, no recombination was observed for HPeV3.

Chapter 11

182

In chapter 10, we generated 18 additional full length sequences of the

commonly circulating types 1 and 3. In total, we analyzed 35 full length

sequences, including previously published sequences. As shown in the

previous study (chapter 9), recombination was frequently observed for

HPeV types 1, 4, 5, and 6. Recombination was also observed for HPeV3,

indicating that HPeV3 recombination is possible. However, in comparison to

the other HPeV types, this was observed to be much more restricted

(chapter 10), possibly reflected by its different cell tropism, limiting co-

infection to occur.

Sequence scans across the genome showed the major recombination

breakpoints to flank the capsid region. Additional recombination sites were

found more downstream for HPeV5 and -6. In contrast to previous studies

depicting recombination among HPeV based on Bootscan which relies on

these parental strains, not known for HPeV, our studies provide statistical

support for the occurrence of recombination by comparison of the strains to

one another and were able to depict recombination sites more precisely per

type.

This analysis also showed a loss of perfect type-specific segregation within

the capsid region that was linked to HPeV1. During our first study, we found

the HPeV1 strains identified within our study to form a separate cluster from

the HPeV1 strain isolated in 1956. However, only one Harris strain was

known at the time. In chapter 10, we were able to analyze the diversification

of the 2 clusters with two additional strains, most closely related to the Harris

strain. Divergence analyses between and within the different type specific

clades showed a distinct distribution of divergence and pair-wise distances

of this group. The divergence between the two groups was found to be

slightly lower than that found between other types, but greater than the

divergence within other HPeV groups. The data provide statistical power to

classify the HPeV1 group in at least two subtypes and suggests extension of

the classification criteria (chapter 8) to include the characterization of highly

diverse types.

In chapter 12, the results of this thesis are placed in perspective of our

current knowledge of HPeV and a direction towards future research is given.

General discussion

185

General discussion

The identification of HPeV3 in 2004 and its association with neonatal

sepsis made it particularly clear that HPeVs can be related to severe

disease in young infants. New parechoviruses continue to be

identified. However, little is known about the clinical and molecular

characteristics of this expanding group of viruses. In our studies, we

show that distinct clinical and molecular differences exist between

HPeV3 and other HPeV types. Furthermore, we demonstrate the clinical

relevance of HPeVs and the need for specific diagnostic methods. The

studies presented here have contributed to knowledge on this, as yet,

small group of viruses, have changed the view regarding their clinical

relevance and have aided in the development of ongoing and future

studies regarding the pathogenesis and treatment of HPeV.

EPIDEMIOLOGY

HPeVs are highly prevalent viruses as is illustrated by high seroprevalence

rates found in different parts of the world (24,32,37,38,40). HPeV1,

previously classified as an enterovirus, was reported as 1 of the 6 most

prevalent enteroviruses in Finland (21). More than 99% of the Finish adults

are seropositive for HPeV1 by the age of 1 year indicating HPeV1 infection is

common in young children (3,18,19,39, and chapters 3, and 5). Identified as

the second predominant strain, at least in Europe (39, and chapters 3, and

5), the seroprevalence data of HPeV3 would be quite high but lower in

comparison to HPeV1. In Japan, the seroprevalence data for HPeV3 was

found to be 78% among adults, even though HPeV3 was first identified in

Japan in 1999 (23) which would mean that the strain already circulated long

before it was first identified. In the Netherlands, we were able to find the

strain to already circulate in 1994 (chapter 10) at similar frequencies

reported in Japan (48)) and nowadays in Europe, suggesting that

seroprevalence data within Europe would be of similar proportions as in

Japan.

The high seroprevalence seen for HPeV1 suggests that the majority of

infants should be protected from HPeV1 infection early in life due to maternal

antibodies. However, Ehrnst and Eriksson suggested that the presence of

maternal HPeV1 antibodies did not always protect against infection (10).

Nevertheless, the children infected with HPeV1 were older than 6 months

(1,42, and chapter 2), suggesting that neonates indeed were protected

Chapter 12

186

against infection with HPeV1. In contrast, children infected with HPeV3

generally were not older than 1 month (1,42,48, and chapter 2), despite the

high seroprevalence in adults, which may indicate a lack of protection from

maternal antibodies against HPeV3. One might consider that previous

infections by HPeV3 are serologically distinct from current infections, failing

to protect neonates from recent infections. However the low divergence

between HPeV3 strains collected in recent years and more than 15 years

ago (chapter 10), thus not support a genetic drift towards these serologically

distinct variants, which would be expected based on the 2 year cycle seen

for HPeV3 (18,42, and chapters 4, 5, and 7) as seen for several

enteroviruses (31,34). However, if indeed this HPeV3 protection is failing,

the virus should be commonly found among adults as well, as a result of re-

infection. However this is not the case; several studies that include samples

obtained from adults show that HPeV is almost exclusively found in young

children (3,18,19,38,39, and chapter 7). So why are children more

susceptible than adults? A possible clue may lie in the biological differences

between the different HPeV types or between children and adults.

HPeVs, FROM MILD TO SEVERE

Infections with HPeVs were previously found to be relatively mild

(10,11,24,38). More so than seen for enteroviruses as shown by the study of

Grist et al (14), which have predominantly been associated with severe

central nervous system (CNS) associated symptoms (14,25,38).

With the identification of HPeV3, the view that HPeV are mild infections

changed. Although infection with HPeV1 had occasionally been associated

with paralysis (13) and encephalitis (27), infection with HPeV3 seems more

frequently associated with more severe symptoms (5,18,42-44, and chapter

2). Moreover, this type was found to be related to severe morbidity and

sequelae of infection ranging from seizures to learning disabilities at a later

age (43). Recent data showed HPeV3 to be the predominant HPeV type

detected in CSF (18,43). Based on the high percentage of HPeV found in

CSF of children with CNS associated diseases and also neonatal sepsis,

HPeVs can be considered as another major viral cause of these diseases

(chapter 4).

Remarkably, despite the detection of HPeV in CSF of children with such

severe disease symptoms that indicate infection of the CNS, the CSF cell

counts and protein levels were not significantly increased (44). This has also

been found for enteroviruses (8,36,44,45). In children with detectable HPeV

in the CSF, symptoms of sepsis-like illness were more frequently observed

General discussion

187

than CNS-associated disease such as meningitis or encephalitis (chapters 2

and 4). It could therefore be that the presence of HPeV in the CSF was due

to a “leaky” blood-brain-barrier in neonates, without overt infection of the

brain or meninges and therefore no cell reaction in the CSF. Unfortunately,

data on the viral load in plasma are lacking to support this notion.

Notably, pleocytosis was also not observed in the children that did exhibit

signs of CNS infections. An interesting hypothesis was proposed by Volpe

(47), who suggested that HPeV3 infections could result in intracellular

binding of toll like receptor (TLR) 8, previously found to be involved in the

host immune response against HPeV1 (41), that can lead to the release of

reactive oxygen and nitrogen and pro-inflammatory cytokines that are toxic

to neural cells. This is an interesting theory that could potentially explain why

we do not observe a cell reaction within CSF. Remarkably, TLR8 is

specifically distributed in axonal perturbations and only in the developing

nervous system. This implies that HPeV infection within the CNS at a very

young age, leading to neural cell death, can be detrimental for the

development of the child, as seen by Verboon-Macieleck et al. (43), but also

explains why we specifically observe the severe HPeV3 infections in very

young infants.

HPeV3, THE ODD VIRUS OUT

The severe clinical manifestations of HPeV3 infection in comparison to other

types points out to a difference in cell tropism between the types. In vitro

data have already implied a difference in cell tropism to exist (1,48, and

chapter 7). Thus, one of the most important questions in parechovirus

research is what cells HPeV3 specifically may target and how the virus

enters the cells, and whether this contributes to its more severe

pathogenicity in comparison to other HPeV types. In this respect, the

absence of the (Arginine-Glycine-Aspartic Acid) RGD motif in HPeV3 is

intriguing. The RGD motif is known to bind to integrins (12,22) and was

found to be crucial for HPeV1 receptor binding (6). Its absence in HPeV3

would thus indicate a different receptor usage that is RGD independent.

The RGD motif has been shown to play a role in the pathogenesis of

Coxsackie A Virus (CAV) 9 and echovirus 9 infections (7,15,33,51,54) and it

was shown that both viruses can enter a specific cell via an RGD-

independent mechanism. But in contrast to what we observe for HPeV3, the

absence of the RGD motif was not found to be related to severe disease. In

fact, the direct opposite was observed. In vivo, the RGD-containing

echovirus 9 strain (Barty) was found to be pathogenic for newborn

Chapter 12

188

mice, while the RGD-negative echovirus 9 strain (Hill) was not (9,53-55).

Also CAV9 mutants without an RGD motif were found to be less pathogenic

in these mice (16,17). A possible clue for the difference between these 2

viruses and HPeV can be found in in vitro studies which show CAV9 RGD-

negative mutants to infect different cells than HPeV3 (1,20,48, and chapter

7). It thus seems probable that the viruses use different RGD independent

pathways.

Interestingly, studies on echovirus 9 (9), using both new born mice and older

mice depicted a differential pathogenicity between these mice. While the

Barty strains could induce a paralytic response in newborn mice, this

response remained absent in older mice, despite a significant increase in

viral titres within the affected tissues. This differential pathogenicity was also

seen for CAV9 (15,17).

An interesting notion was made by Harvala et al (15) that could explain this

differential pathogenicity; it was found that the alpha(v)beta(3) integrin, a

host cell receptor used by both virus was down regulated during mice

development (4,30). If translated to a human infection, where various

receptors can also be down or upregulated during development, a shift of

specific cell receptor expression necessary for HPeV3 entry would support

the observation why HPeV3 infections are rarely or never seen in older

children and adults even when the humoral protection against HPeV3 might

be lacking.

CONCLUDING REMARKS: HPEV tropism unravelled, a step toward

specific antiviral therapy against picornaviruses

Picornaviruses cause more than 6 billion infections per year and have a

significant clinical impact on global health care. As a major viral cause of

CNS-associated disease and neonatal sepsis in children, HEV and HPeV

infections lead to severe morbidity in children.

With the exception of poliovirus, effective anti-viral therapy or vaccines are

still lacking for these viruses.

Antiviral agents based on steps in the picornavirus life cycle may be

considered. As the initial event in the replication cycle of picornavirus is

attachment of the viral capsid protein to a cell surface molecule or receptor,

a major target for antiviral therapy could be the inhibition of the viral capsid

function. The experimental drug pleconaril was developed to bind to the viral

capsid protein VP1 and inhibits viral adsorption. Experience with pleconaril in

infants with severe HEV infection is limited and virological and clinical

General discussion

189

efficacy of this drug were not always demonstrated (2,17,26,33,35,50,54).

Although the drug should be effective against 98% of the most commonly

circulating types, an explanation for the efficacy differences of the drug could

be that the composition of the pocket within the capsid protein VP1, where

the pleconaril compound should bind, may differ between types and type

specific entry inhibitors may be more beneficial.

Unravelling specific viral factors involved in the infectious cycle, that can

influence pathogenesis, are pivotal in the development of antiviral drugs. A

few pathogenesis studies have implicated different viral factors to influence

pathogenesis (28,49). Studies on CAV9 and echovirus 9 (29,46,52) found

the capsid protein to play a major role in influencing cell tropism and

pathogenesis, whereas studies on enterovirus 71 showed the replication

capacity and also evolutionary factors such as recombination to define

pathogenesis. However, the occurrence of over 100 non polio-HEV

serotypes with a large diversity of clinical syndromes makes it difficult to

study viral factors that may play a role in defining pathogenesis of one type

in comparison to another. Up till now, no correlation could be found between

receptor usage of different types in relation to cell tropism, and specific

disease entities.

HPeVs form a clinically well defined group with distinct differences in

biological characteristics. These factors render this group of viruses ideal to

study cell tropism in relation to disease severity, which could be a key in

investigating cell entry inhibitors as antiviral agents, and to learn more about

picornavirus pathogenesis.

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Chapter 12

194

55. Zimmermann, H., H. J. Eggers, A. Zimmermann, W. Kraus, and B.

Nelsen-Salz. 1995. Complete nucleotide sequence and biological

properties of an infectious clone of prototype echovirus 9. Virus Res.

39:311-319.

Appendices

Samenvatting voor niet ingewijden

List of publications

Curicullim Vitae

Dankwoord/Acknowledgements

Samenvatting

voor niet ingewijden


199

Virussen

Een virus bestaat met name uit genetisch materiaal, het genoom, omhuld

door een eiwitmantel (Figuur 1A), de kapsel of capside genoemd. Sommige

virussen hebben om de capside een tweede eiwitmantel genaamd envelop.

Het genetische materiaal kan RNA of DNA zijn en kan enkelstrengs of

dubbelstrengs zijn. Dit is in tegenstelling tot andere organismen, zoals de

mens, waar het genetisch materiaal bestaat uit dubbelstrengs DNA.

Omdat virussen compleet afhankelijk zijn van een gastheer om te

vermenigvuldigen, worden ze beschouwd als niet-levend. Na binding van het

virus aan de gastheercel via een eiwit op de gastheercel (receptor), kan

deze de cel binnen dringen. Het virus gebruikt eiwitten aanwezig in de

gastheercel en ook eigen eiwitten, om te vermenigvuldigen. Hierbij wordt het

erfelijk materiaal vermenigvuldigd en nieuwe eiwitten worden aangemaakt,

waaruit nieuwe virusdeeltjes opgebouwd kunnen worden. Deze verlaten de

cel en kunnen weer nieuwe cellen infecteren. Welke cel of orgaan het virus

infecteert, is afhankelijk van de aanwezigheid van de receptor op de cel,

maar ook van het type cel en de eiwitten aanwezig in die cel (“celtropisme”).

Het uiteindelijke infectieproces en de schade die het virus hiermee aanricht,

soms te zien aan de ziektesymptomen in de gastheer, wordt de

pathogenese van het virus genoemd.

Figuur 1. Schematische weergave van

het genetisch materiaal (A) en het de

vorm van een virus (B) van de familie

Picornaviridae.

A

B


200

Evolutie

Virussen kennen een grote variatie. Zo komen er heel veel typen voor van

een specifiek virus en kan elk type virus weer variaties van zichzelf hebben.

Deze variaties ontstaan door fouten die gemaakt zijn tijdens het

replicatieproces. Deze fouten kunnen positief zijn voor het virus zodat er een

“sterker” virus ontstaat. Ze kunnen ook een negatief effect hebben (minder

sterk virus) of geen effect hebben (neutrale fouten). Replicatiefouten komen

het meest voor bij RNA virussen en spelen een grote rol bij de diversiteit van

deze virussen.

Virussen van dezelfde klasse kunnen delen van het genoom uitwisselen en

een nieuwe viruscombinatie maken (“recombinatie”). Het

recombinatieproces is een kansproces en is afhankelijk van een aantal

factoren. Zo moeten twee virusstammen dezelfde cel kunnen infecteren,

waarna tijdens het repliceren genoomsegmenten van beide virussen

verwisseld kunnen worden. Verder kunnen er andere factoren een rol spelen

die dit kansproces beïnvloeden of juist tegenhouden. Wat deze factoren zijn

is nog niet duidelijk.

Zowel variaties in het genoom (“mutaties”) als recombinatie kunnen grote

invloed hebben op de circulatie (epidemiologie) en pathogenese van het

virus. Het bepalen van de genetische code van een virus (fylogenie) en het

onderzoeken wat de evolutie en recombinatie drijft is van belang om te

weten wat er circuleert. Deze kennis is nodig zodat een passend beleid kan

worden uitgevoerd tijdens eventuele uitbraken en epidemieën. Welke

varianten en welke typen virussen circuleren, kan wisselen per mens, land,

of continent.

Humaan Parechovirussen (HPeVs)

HPeVs behoren tot de grote Picornavirus-familie (Hoofdstuk 1, figuur 1).

Picornavirussen zijn kleinste (pico staat voor klein) en oudste RNA virussen

bekend. Het HPeV-genoom (figuur 1B) is ongeveer 7300 nucleotiden, die de

genetische code vormen voor 10 eiwitten. Drie van deze eiwitten (VP0-VP3-

VP1) vormen samen de capside, en de zeven andere eiwitten, zijn nodig

voor het vermenigvuldigingsproces van het virus (figuur 1).

HPeVs werden voorheen geclassificeerd als humane enterovirussen. Er zijn

meer dan 100 enterovirustypen bekend. Enterovirussen zijn de voornaamste

oorzaak van virale hersenvliesontsteking. Vroeger werden enterovirussen

geclassificeerd op basis van hun vermogen te repliceren in humane en apen

cellijnen en op het bindend vermogen van hun eiwitmantel aan antilichamen

(onderdeel van het immuunsysteem) die per type verschillen. Tegenwoordig

worden ze geclassificeerd op basis van genetische diversiteit, in 4 groepen;


201

A, B, C (waartoe het poliovirus behoort) en D. De genetische classificatie

heeft geleid tot de reclassificatie van enkele enterovirussen, waaronder

echovirus 22 en 23, nu bekend als Humaan Parechovirussen (HPeV1 en -2).

Op dit moment zijn er 14 HPeV typen bekend.

HPeV1 en -2 werden voornamelijk geassocieerd met milde symptomen,

waaronder luchtweg infecties en infecties in het maag-darmkanaal. Af en toe

werd HPeV1 ook gezien bij verlammingen en ontstekingen in het centraal

zenuwstelsel, maar veel minder in vergelijking tot de enterovirussen.

Opvallend was dat HPeVs vrijwel alleen bij kinderen voorkomen. In 2004

werd een derde HPeV type ontdekt en bleek het type een belangrijk motief

voor receptorbinding te missen in het genoom. Dit motief slaat op de 3

aminozuren (bouwstenen) die op één van de capside vormende eiwitten,

VP1, gevonden wordt (Arginine (R), Glycine (G) en Aspartaat zuur (D),

RGD) en die zeer van belang zijn voor infectie van HPeV1. De afwezigheid

van dit motief duidt op een verschil in receptorgebruik.

Dit proefschrift

HPeV3 werd voor het eerst ontdekt bij een Japans kindje met verlammingen.

Het virus werd later gevonden bij drie Canadese kindjes met neonatale

sepsis. Dit is een aandoening bij pasgeborenen waarbij de kindjes koorts

hebben en ziek zijn met verhoogde ademhaling en hartslag. In onze studie

toonden wij aan dat kinderen met een HPeV3 infectie vaker neonatale

sepsis en infecties van het centraal zenuwstelsel hebben dan kinderen die

geïnfecteerd waren met HPeV1. Ook waren kinderen met een HPeV3

infectie vele malen jonger dan kinderen met een HPeV1 infectie (1,5 maand

en 6 maanden respectievelijk). Deze studie bracht ons tot de hypothese dat

de ernst van een HPeV3 infectie veroorzaakt wordt door het gebruik van een

ander receptor (RGD onafhankelijk) waardoor dit type in staat is ander

weefsel te infecteren zoals het centraal zenuwstelsel (ander celtropisme).

Voor het eerst werd duidelijk dat HPeVs klinisch relevant zijn en

routinediagnostiek werd essentieel. HPeVs kunnen, net als enterovirussen,

worden aangetoond door middel van viruskweek maar zijn niet van elkaar te

onderscheiden. Met deze techniek duurde het gemiddeld twee weken voor

er kan worden aangetoond of de patiënt positief of negatief is. Voor een

snelle diagnostiek hebben wij een test ontwikkeld op basis van de

genetische verschillen tussen HPeV en enterovirussen om onderscheid te

maken tussen de twee virusgroepen. Met behulp van deze test vonden we

een substantieel deel van de kinderen positief voor HPeV zowel bij het

onderzoeken van ruggenmergvloeistof als poep. Van alle positieve

poepmonsters werd het type bepaald door de genetische code van het virus


202

te ontcijferen. Helaas konden we van de positieve ruggenmergvloeistof niet

bepalen welk type infectie de kindjes hadden opgelopen.

Door het type te bepalen in de poep monsters, ontdekten we dat HPeV3

alleen te vinden was in de “even” jaren en dus elke 2 jaar te ontdekken was.

De andere typen (HPeV1, -4, -5, en -6) werden vrijwel elk jaar gevonden.

Interessant was dat ditzelfde tweejarig patroon overeenkwam met het aantal

HPeV positieve patientjes met neonatale sepsis en centraal zenuwstelsel

infecties. In de even jaren vonden we vrij veel HPeV positieve patientjes met

neonatale sepsis en centraal zenuwstelsel infecties, terwijl in de oneven

jaren dit opvallend genoeg bleef bij enkele patientjes. Op basis hiervan

konden we concludeerden dat deze infecties voornamelijk veroorzaakt

worden door HPeV3. Dit werd later bevestigd door andere

onderzoeksgroepen, die direct het virustype konden bepalen uit

ruggenmergvloeistof.

Het hoge percentage HPeV positieve kinderen met neonatale sepsis en

centraal zenuwstelsel infecties, illustreerde dat HPeVs de tweede belangrijke

oorzaak zijn van neonatale sepsis en hersenvliesontsteking bij kinderen, na

enterovirussen.

Verder speculeerden we dat snelle en gevoelige diagnostiek zou kunnen

leiden tot het staken van antibioticatherapie en een kortere opnameduur.

Bij het opkweken van de verschillende HPeV typen bleek HPeV3 één van de

moeilijk te kweken typen te zijn. Het duurde gemiddeld drie weken voor een

positieve kweek verkregen werd en het type bleek ook nog alleen te groeien

op een select aantal soorten cellijnen. Deze observatie ondersteunde onze

hypothese dat HPeV3 waarschijnlijk een ander celtropisme heeft in

vergelijking tot andere typen.

Doordat HPeV3 een afwijkend celtropisme heeft, is de kans op

dubbelinfectie van HPeV3 met een ander HPeV type zeer klein. Tijdens het

vermenigvuldigingsproces zal er dus weinig sprake zijn van het uitwisselen

van genetisch materiaal (recombinatie), waarbij recombinatie nauwelijks zal

voorkomen tussen HPeV3 en andere HPeV typen. Dit zagen wij dan ook

terug in onze evolutie studies, waaruit bleek dat het genetische materiaal

van HPeV3 over vrijwel de gehele lengte (figuur 1A) onveranderd was.

Samenvattend zijn HPeVs klinisch relevant en is onderzoek naar deze

virussen van groot belang. Dit proefschrift toont en benadrukt de verschillen

tussen HPeV3 en ander HPeV typen. De verschillen zijn zowel biologisch als

klinisch waar te nemen.


203

Onze hypothese dat HPeV3 een ander tropisme heeft, dat leidt tot ernstigere

symptomen bij zeer jonge kindjes kan komen door het gebruik van een

ander receptor (RGD onafhankelijk). Dit zal aan de hand van verdere studies

onderzocht worden.


207

Faria N. R., M. de Vries, F. J. van Hemert, K. Benschop, L. van der Hoek. 2009. Rooting human parechovirus evolution in time. BMC Evolutionary Biol. In press.

Pajkrt D., K. S. M. Benschop,

B. Westerhuis, R. Molenkamp, L.

Spanjerberg, K. C. Wolthers. 2009. Clinical characteristics of human

parechoviruses infections 4-6 in young children. P. Ped. Inf. Dis. J. In press.

Benschop K. S. M. & K. C. Wolthers. 2008. Humane parechovirussen: niet

te missen. Ned. Tijdsch. v. Med. Microbiol. 4:8-11

Benschop K, G. Stanway G, and K. Wolthers. 2008. New Human

Parechoviruses: six and counting. Chapter 4, In Scheld M (ed.), Emerging

Infections. ASM Press, Washington DC.

Harvala, H., I. Robertson, E. C. William Leitch, K. Benschop, K. C.




Wolthers, K. C., K. S. Benschop, J. Schinkel, R. Molenkamp, R. M.

Bergevoet,I. J. Spijkerman, H. C. Kraakman, and D. Pajkrt. 2008. Human

parechoviruses as an important viral cause of sepsislike illness and

meningitis in young children. Clin.Infect.Dis. 47:358-363.

Benschop, K., X. Thomas, C. Serpenti, R. Molenkamp, and K. Wolthers.

2008.High prevalence of human Parechovirus (HPeV) genotypes in the

Amsterdam region and identification of specific HPeV variants by direct

genotyping of stool samples. J.Clin.Microbiol. 46:3965-3970.

Benschop, K. S., C. H. Williams, K. C. Wolthers, G. Stanway, and P.

Simmonds. 2008. Widespread recombination within human parechoviruses:

analysis of temporal dynamics and constraints. J.Gen.Virol. 89:1030-1035.

Benschop, K., R. Molenkamp, A. van der Ham, K. Wolthers, and M.

Beld.2008. Rapid detection of human parechoviruses in clinical samples by

real-time PCR. J.Clin.Virol. 41:69-74.

Fanoy E.B., H.R. van Doorn, K.S. Benschop, C.J. Schinkel, M. Beld, D.

Pajkrt, E.E. Hagebeuk. 2007. Verworven parese van de arm bij een


208

zuigeling met enterovirusinfectie: mogelijk polio?. Tijdschrift voor

Infectieziekten. 3:104-109.

Benschop, K. S. M., J. Schinkel, M. E. Luken, P. J. M. van den Broek, M.

F. C. Beersma, N. Menelik, H. W. M. van Eijk, H. L. Zaaijer, C. M. J. E.

VandenBroucke-Grauls, M. G. H. M. Beld, and K. C. Wolthers. 2006.

Fourth Human Parechovirus Serotype. Emerg.Infect.Dis. 12:1572-1575.

Benschop, K. S., J. Schinkel, R. P. Minnaar, D. Pajkrt, L. Spanjerberg, H.

C. Kraakman, B. Berkhout, H. L. Zaaijer, M. G. Beld, and K. C. Wolthers.

2006.Human parechovirus infections in Dutch children and the association

between

serotype and disease severity. Clin.Infect.Dis. 42:204-210.

Submitted for publication

K.S.M. Benschop, M. de Vries, R. Minnaar, G. Stanway, L. van der Hoek,

K.C. Wolthers, P. Simmonds. Comprehensive full length sequence

analyses of human parechoviruses; diversity and recombination.

Curriculum Vitae

Curriculum Vitae

211

Kimberley Benschop was born on May 3rd 1982 in Paramribo, Surinam. In

1993, she moved to Curacao, where she started “atheneum”. Halfway (1997)

she moved to the Netherlands and graduated in 2000 in Delft from Stanislas

College Westplantsoen.

After graduating, she went to study Biomedical Science at the Vrije

Universiteit, in Amsterdam. She soon became interested in virology when

doing a course in Microbiology. In 2003, she finished her Bachelor with a

traineeship at the department of Pathology, working on developing a typing

assay for coutaneous Human Papilloma Virus. She did her first Master’s

traineeship at the department of Medical Microbiology, Leiden University

Medical Center where she worked on host cell interactions during Hepatitis

C Virus infection. In 2005, she finished her Masters at the department of

Medical Microbiology, Laboratory of Clinical Virology at the Academic

Medical Center were she did a second Master’s traineeship on the

epidemiology and transmission of enteroviruses and HPeV.

After receiving her Master’s degree Biomedical Science she continued her

research on HPeV as a PhD project at the same department. In 2007, she

made a 3 month work visit to the University of Essex, Colchester and to the

University of Edinburgh, Edinburgh, under the supervision of Prof. dr. G.

Stanway and Prof. dr. P. Simmonds. There she studied HPeV evolution. The

results of her PhD project are written in this thesis. In 2009 she will continue

this research as a post-doc at the Academic Medical Center.

Dankwoord

Acknowledgements


215

Daar houd je hem dan vast, HET boekje. Zoals velen weten, onderzoek doe

je nooit alleen. Daarom wil ik graag mijn begeleiders, co-auteurs, collega’s,

vrienden en familie allemaal bedanken voor hun begeleiding, inzet, en

ondersteuning. Ongetwijfeld ben ik iemand vergeten, sorry daarvoor.

Op de eerste plaats wil ik bedanken:

Katja, een kei van een co-promotor, een leukere en betere begeleiding had

ik me niet kunnen wensen. Ik heb veel van je geleerd. Dit boekje is ook van

jouw.

Christina en Menno, mijn promotoren. Dank je dat ik de kans kreeg om dit

onderzoek te starten, en ook door te zetten als postdoc.

Natuurlijk wil ik ook bedanken:

Richard, voor de begeleiding van het fundamentele deel van dit onderzoek.

Janke, een fylogenie-wizz, voor de fylogenetische ondersteuning. Marcel,

dank voor de begeleiding en de moleculaire basis. Hans, dank voor de leuke

discussies.

Rene, Sylvie, Xiomara (mijn paranimf), Nienke en Alwin, dank voor de steun

bij en input in mijn experimenten en de leuke en gezellige etentjes. Mijn

studenten, Camile, Brenda, Sara en ook Xiomara, jullie hebben mooi werk

geleverd! Jos, je bent een enorme steun in de diagnostiek. Hetty v. E.,

Gerrit, en Karen, dank voor het opkweken en typeren van al die parecho’s

en entero’s. Wat waren de lijsten lang en compleet, dankzij de mooie queries

van Frits. Marga, dank voor de hulp en steun in de administratie en Hetty B.,

dank voor de hulp bij het regelen van mijn promotie. Ook de medewerkers

van de MDU, serologie en research bacteriologie, dank je voor alle hulp en

steun.

De mensen van K3, bedankt voor de gezelligheid. Bill, Georgios and your

group, loved our meetings and discussions; en ook Lia en je groep, een

alliantie in HPeV.

Glyn, Peter, Heli, Ģigdem, and Kate (UK); and Sisko, Petri, and Timo

(Finland). Thank you for the wonderful discussions and collaborations. Of

course, I cannot forget Nigel and his family, thank you for the lovely talks.

Mijn vrienden, Ruud en Laura, Jan Jakob en Esme, Richard en Janneke,

Tom, Kris, Darran en Sabine, Martien en Ingrid, Petra (mijn paranimf) en

York, Yasmin en Nita, de 3 J’s (Jos, Jos en Jurgen). Dank voor de leuke en

gezellige avondjes. Ook, Ingrid, Judith, Alies en Anouk (het kookclubje),

dank je voor de nodige kookafleiding en leuke gesprekken.


216

De laatste paragraaf is voor mijn familie. De speciale mensen die er ervoor

gezorgd hebben dat ik dit heb kunnen doen. Mam en Frank. Ik ben er trots

op jullie dochter te zijn. Ik heb dit niet kunnen volbrengen zonder de

discipline die jullie me meegaven. Mona, Eli en Ian, broers en een zus om

naar op te kijken. Joop en Ingrid, de leukste schoonouders die je maar kan

wensen. Opa Ton, Oma Henny, Oma Rita en Oma Titi, allerleukste

grootouders. Dennis, Linda en Pedro, leukere en gezelligere zwagers en

schoonzus, vind je nergens.

Shawn en Veronique, mijn mooiste, schattigste en leukste neefje en nichtje.

Veronique, je staat er prachtig op. Ook Gwen, Ami en Chaia, ver weg in

Suriname, maar dichtbij in ons hart.

En dan de belangrijkste persoon, Mark, mijn man, mijn confidant, mijn beste

vriend, mijn chauffeur, ik zou hier niet hebben gestaan zonder jouw liefde en

steun. En natuurlijk de ritjes van en naar het AMC. Dat je zelfs op zaterdag

heen en terug voor me reed, toen ik mijn rijbewijs nog niet had, want ik had

het verkeerde bestand meegenomen. Je bent een klasse apart. We wonen

nu al 3 jaar samen in Haarlem en gaan de volgende fase in ons leven in aan

de Spaansevaart. Verhuizen en promoveren is niemand aan te raden, maar

je nam zoveel mogelijk het geregel rond de verhuizing uit mijn handen zodat

ik mij kon focussen op mijn promotie. Thanks.

Kimberley

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