MAJOR ARTICLE
Specific Association of Human Parechovirus Type 3
with Sepsis and Fever in Young Infants, as Identified
by Direct Typing of Cerebrospinal Fluid Samples
H. Harvala,1 I. Robertson,2 T. Chieochansin,1,3 E. C. McWilliam Leitch,2 K. Templeton,1 and P. Simmonds2
1
Specialist Virology Centre, Royal Infirmary of Edinburgh, and 2Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh,
United Kingdom; 3Biomedical Science Centre of Excellence in Clinical Virology, Chulalongkorn University and Hospital, Bangkok, Thailand
Background. Human parechoviruses (HPeVs), along with human enteroviruses (HEVs), are associated with
neonatal sepsis and meningitis. We determined the relative importance of these viruses and the specific HPeV types
involved in the development of central nervous system–associated disease.
Methods. A total of 1575 cerebrospinal fluid (CSF) samples obtained during 2006 –2008 were screened for HPeV
by means of nested polymerase chain reaction. All samples for which results were positive were typed by sequencing
of viral protein (VP) 3/VP1. Screening for HEV was performed in parallel, as was detection of HPeV in respiratory and
fecal surveillance samples, to identify virus types circulating in the general population.
Results. HPeV was detected in 14 CSF samples obtained exclusively from young infants (age, ⬍3 months) with
sepsis or pyrexia. The frequency of detection of HPeVs varied greatly by year, with the highest frequency (7.2%) noted
in 2008 exceeding that of HEVs. Direct typing of CSF samples revealed that all infections were caused by HPeV type
3, a finding that is in contrast to the predominant circulation of HPeV1 in contemporary respiratory and fecal
surveillance samples.
Conclusion. HPeV was a significant cause of severe sepsis and fever with central nervous system involvement in
young infants, rivaling enteroviruses. The specific targeting of young infants by HPeV type 3 may reflect a difference
in tissue tropism between virus types or a lack of protection of young infants by maternal antibody consequent to the
recent emergence of HPeV.
Human parechoviruses (HPeVs) are small, nonenveloped, single-stranded, positive-sense RNA viruses
within the Parechovirus genus of the Picornaviridae family. To date, HPeVs have been classified into 6 different
types. HPeV type 1 and HPeV type 2 (previously known
as echoviruses 22 and 23, respectively) were first isolated
in 1956 [1] and have been associated with gastrointestinal and respiratory tract symptoms, as well as with occasional cases of encephalitis and flaccid paralysis [2–5].
HPeV type 3 (HPeV3) was first described in 2004; the
Received 6 November 2008; accepted 23 December 2008; electronically published 12 May 2009.
Potential conflicts of interest: none reported.
Presented in part: Scottish Diagnostic Virology Group meeting, Glasgow, United
Kingdom, 12 November 2008 (oral presentation).
Financial support: none reported.
Reprints or correspondence: Peter Simmonds, Centre for Infectious Diseases,
University of Edinburgh, Summerhall, Edinburgh, EH9, 1QH, United Kingdom
([email protected]).
The Journal of Infectious Diseases 2009; 199:1753– 60
© 2009 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2009/19912-0007$15.00
DOI: 10.1086/599094
strain was isolated from a stool sample obtained from a
1-year-old female Japanese infant who had transient paralysis with fever and diarrhea [6], as well as from stool
samples obtained from neonates with sepsis [7], leading
many investigators to propose an association between
HPeV3 and sepsislike illness in young children [7–10].
The role of HPeV type 4, HPeV type 5, and HPeV type 6
(HPeV6) as human pathogens is still poorly understood;
HPeV type 4 and HPeV6 have been implicated as a cause
of diarrhea in children [11–13].
Most HPeVs have been identified by typing of cellcultured virus isolated from fecal or nasopharyngeal aspirate (NPA) specimens. Reflecting the much lower viral
loads noted in cerebrospinal fluid (CSF) samples, there
are only 2 descriptions of propagation of HPeV from
these samples [14]. One isolate was HPeV3, which is
associated with fever, and the other was HPeV6, which
was recovered from an individual with Reye syndrome.
The inability to directly type HPeV in clinical samples
has complicated previous attempts to associate certain
variants with different disease presentations, such as
HPeV3 and Sepsis in Young Infants
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demonstrations of associations between HPeV3 infections and
central nervous system (CNS)–related disease [9, 10, 15].
To address this issue, and to assess the importance of HPeV
and human enteroviruses (HEVs) in childhood sepsis and meningitis, we screened for HPeV in diagnostic CSF samples collected between 2006 and 2008, by use of highly sensitive nested
polymerase chain reaction (PCR) analysis. We also compared
the frequencies of detection of HPeV with those of HEV. Other
neuropathogenic viruses (e.g., herpes simplex virus types 1 and 2
[HSV1 and HSV2, respectively] and varicella-zoster virus
[VZV]) were also the subject of routine screening tests. HPeVpositive samples were typed by amplification and sequencing of
the viral protein (VP) 3/VP1 region [16]. We observed a specific
association of HPeV3 infections with CNS-related disease in
very young children (age, ⬍3 months); this finding contrasted
with the widespread circulation of HPeV1, HPeV3, and HPeV6
in contemporary respiratory and fecal surveillance samples.
MATERIALS AND METHODS
Test specimens. CSF samples referred to the Specialist Virology Centre, Royal Infirmary of Edinburgh, Edinburgh, United
Kingdom, during the period from 3 January 2006 through 18
August 2008 (representing ⬎90% of all CSF samples obtained
during the period) were anonymized and deposited in the Specialist Virology Centre CSF sample archive. Although the samples were anonymized before testing, epidemiologic and demographic information was retained while patient confidentiality
was strictly protected (in accordance with a protocol approved
by the Lothian Regional Ethics Committee [protocol 2002/4/
36]) [16, 17]. Data comprised the age group (with the youngest
group [age, 0 –3 months] subsequently referred to as “young
infants”), partial postcode, recorded symptoms or clinical information, source of referral, month of sample collection, and results of other virologic testing of the sample.
A total of 2295 anonymized respiratory samples that were collected from 1525 different individuals (792 males and 733 females) between January 2008 and June 2008 were included in the
study, screened in pools of 10, and resolved by splitting, as described elsewhere [16]. Screening tests were also performed on
pooled fecal surveillance samples referred for bacteriologic
screening and collected in March, June, and September 2008 in
Edinburgh, although identification of individual positive samples was precluded.
HPeV detection and typing. RNA was extracted from 200
␮L of pooled/individual specimens (i.e., CSF and NPA samples
and clarified fecal supernatant) and placed into 40 ␮L of Trisethylenediaminetetraacetic acid buffer, as described elsewhere
[16]. Samples were screened for HPeV RNA sequences in pools
of 3 (for CSF samples) or 10 (for NPA samples and fecal supernatant), by means of reverse-transcription (RT) nested PCR performed with 5' untranslated region (5' UTR) primers, as de1754
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scribed elsewhere [16]. Positive pools were split into individual
components and were retested to identify positive samples. Routine PCR testing for HSV1, HSV2, VZV, and enteroviruses was
performed on individual CSF samples, by use of an assay described elsewhere [18].
Positive samples were amplified in the VP3/VP1 junction region and sequenced between nucleotide positions 2182 and 2437
to identify HPeV types, as described elsewhere [16]. Nucleotide
sequences from the VP3/VP1 region were assigned GenBankaccession numbers FJ847944 –FJ848013.
RESULTS
Study group. A total of 1575 CSF samples obtained from 1480
different individuals (770 males and 710 females) were predominantly referred from very young children (age, ⬍3 months)
(30% of samples), middle-aged adults (age, 37– 65 years) (25%
of samples), and older patients (age, ⬎65 years) (25% of samples). Routine screening detected HSV1 in 14 samples, HSV2 in
7 samples, VZV in 5 samples, and enteroviruses in 93 samples
(overall frequency of virus detection, 7%).
Frequency of HPeV and enterovirus infections. The sensitivity of the screening RT-PCR using 5' UTR primers for the
detection of HPeV RNA has been established elsewhere [16].
Screening of the 1575 CSF samples detected HPeV RNA in a total
of 14 individual specimens obtained from 14 different study subjects: 3 (0.7%) of 440 samples were obtained in 2006, 0 in 2007,
and 11 (7.2%) of 153 in 2008. In comparison, enteroviruses were
detected in a total of 93 specimens: 23 (4.6%) of 502 in 2006, 49
(7.3%) of 673 in 2007, and 21 (3.8%) of 551 in 2008. All HPeVpositive samples (14 samples obtained from 14 study subjects)
identified using 5' UTR screening primers could be amplified in
the VP3/VP1 region (table 1). All sequences clustered closely
with HPeV3 (accession numbers AJ889918 and AB084913)
(figure 1).
Epidemiologic and clinical associations between HPeV and
enterovirus infections. All children infected with HPeV were
young infants (age, ⬍3 months); the frequency of detection of
HPeV in this subject group was 2.9% (14 of 480), whereas enterovirus infections were widely distributed among most age
groups (figure 2). Although the frequency of enterovirus infections was also high in young infants (37 [7.1%] of 518 study
subjects), it was lower than that found in adults (26 [11.8%] of
219 study subjects 21–36 years of age). The highest frequency of
VZV and HSV1 CNS-associated infections was seen in study
subjects ⬎65 years of age, whereas HSV2 infections were predominantly noted in the younger adult age group (21–36 years;
data not shown). Although a higher proportion of the HPeVinfected study subjects were male (8 [57.1%] of 14 subjects), they
were not significantly overrepresented in the whole study group
(770 [52.0%] of 1480; P ⫽ .59, by Fisher’s exact test). Similarly,
Table 1.
results.
Patient demographic and clinical characteristics and parechovirus typing
Year of collection,
specimen
2006
CSF-721/06
CSF-874/06
CSF-1112/06
2008
CSF-2147/08
CSF-2162/08
CSF-2169/08
CSF-2273/08
CSF-2288/08
CSF-2307/08
CSF-2327/08
CSF-2369/08
CSF-2387/08
CSF-2389/08
CSF-2426/08
Age,
months
Sex
Month
⬍3
⬍3
⬍3
F
M
F
Apr
July
Nov
Sepsis
Sepsis
Fever
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
⬍3
F
M
M
M
M
F
M
M
M
F
F
Apr
Apr
Apr
June
June
June
June
July
July
July
Aug
Sepsis
Neonatal
Neonatal
Sepsis
Neonatal
Sepsis
Sepsis
Fever
Sepsis
Sepsis
Neonatal
Diagnosis
sepsis
fever
sepsis
sepsis
Subject
location
Virus
type
GCW
GCW
GCW
HPeV3
HPeV3
HPeV3
GCW
PHDU
GCW
GCW
PITU
GCW
GCW
GCW
GCW
GCW
PITU
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
HPeV3
NOTE. CSF, cerebrospinal fluid; F, female; GCW, general children ward; HPeV3, human parechovirus type 3; M, male; NNU, neonatal unit; PHDU, pediatric high-dependency unit; PITU, pediatric
intensive care unit.
more HEV infections were seen in males (51 [55%] of 93 study
subjects).
There were marked temporal changes in the incidence of
HPeV over the study period (figure 2). HPeV was detected in 3
(2.0%) of 120 and 11 (7.2%) of 153 young infants during 2006
and 2008, respectively, but it was not detected in 2007. This variability was much greater than that noted for enterovirus infections of the CNS in the same age group, which had relatively
constant annual detection frequencies (11 [7.0%] of 158 in 2006,
15 [7.7%] of 193 in 2007, and 11 [6.6%] of 167 in 2008) (figure
2). However, these figures conceal major changes in the different
age groups infected between years, as well as marked changes in
incidence in different quarters.
Clinical presentation of HPeV infection. Referral information, including the source of the samples and the reported
symptoms, for subjects with HPeV-positive samples was compared with that for subjects with samples positive for enteroviruses, VZV, HSV1, and HSV2. HPeV3 infection was most commonly associated with sepsis (in 80% of cases), with the
remaining cases referred with unspecific fever (figure 3 and table
1). Although the presentation of enterovirus infections in young
infants was similar to that of HPeV infections (with sepsis in
30% of cases and with fever in 40% of cases), several cases of
meningitis were also recorded. Furthermore, enterovirus and
HSV1 infections in study subjects ⬎1 year of age were associated
with meningitis and headache. In our study group, encephalitis
was associated only with HSV1 or VZV infections, and it was not
documented for any subjects from whom HPeV or enterovirus
isolates were recovered.
Comparison of HPeV types detected in the CSF with circulating virus populations. To determine whether the HPeV3
variants detected in CSF samples were typical of virus circulating
in the general population over the study period, we compared
frequencies of detection, age distributions of infection, and phylogenetic relationships of HPeV types recovered from CSF with
types recovered from contemporary respiratory diagnostic specimens in 2007 [16] and 2008 and from those recovered from
fecal surveillance samples in 2008 (figures 1 and 4).
The exclusive detection of HPeV3 in CSF samples was not
reflected in other sample types. For example, in 2008, HPeV3
accounted for only 36%– 40% of total HPeV infection variants
detected in respiratory and fecal samples, in which HPeV1 predominated (P ⬍ .001 and P ⬍ .001, respectively) (figure 4A).
However, consistent with the absence of any diagnosed cases of
parechovirus-associated sepsis in 2007 and reflecting substantial
variability in the circulation of this type between years, HPeV3
was completely absent from respiratory samples in 2007 (during
which time HPeV1 and HPeV6 were detected).
The targeting of very young children by HPeV3 with sepsis
(figure 2) was reflected in respiratory samples, for which detection of HPeV3 was again almost entirely confined to samples
obtained from the 0- to 3-month age group (figure 4B). In contrast, infections with HPeV1 and HPeV6 were detected in older
age groups, although infections due to these types were invariably detected in subjects ⬍5 years of age in both study years. Of
the 4 subjects with HPeV3-positive CSF samples who had a respiratory sample collected at the same time, 3 had an HPeVpositive result for both samples, consistent with the systemic
HPeV3 and Sepsis in Young Infants
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Figure 1. Phylogenetic comparisons of the viral protein (VP) 3/VP1 region sequences amplified from human parechovirus (HPeV)–positive specimens
(circles) with all available nonidentical published sequences (labeled according to GenBank accession number). Trees were constructed by neighborjoining of maximum composite likelihood (nucleotide) pairwise distances, with bootstrap resampling done to demonstrate the robustness of groupings;
values 肁70% are shown. Multiple positive (respiratory [Resp]) samples obtained from the same individual were invariably identical, and only the first
sample collected is shown. Paired arrows denote human parechovirus variants from the 3 subjects with paired positive cerebrospinal fluid (CSF) and
respiratory samples. Fec, fecal; HPeV1–HPeV6, HPeV types 1– 6.
Figure 2. Age distributions of human parechovirus (HPeV)–infected (A) and human enterovirus (HEV)–infected (B) study subjects, subdivided by
referral year (2006, 2007, and 2008). The numbers above the bars denote total numbers of subjects with positive specimens. Dotted lines denote mean
frequencies of detection of HPeV and HEV for the whole study group.
disseminated parechovirus infections associated with sepsis in
young infants. Phylogenetic analysis confirmed that HPeV3
variants in paired samples were identical or close to identical to
each other (figure 1).
Phylogenetically, HPeV1 and HPeV3 variants recovered from
different sample types were interspersed (figure 1), with there
being no evidence for HPeV variants specifically associated with
respiratory, enteric, or CNS infections. These observations argue
against the existence of a specifically pathogenic (clonal) strain
that might account for the development of sepsis in neonates
infected with HPeV3.
HPeV3 variants recovered in 2006 and 2008 showed substantially less genetic diversity than did HPeV1 variants recovered in
those years. Mean pairwise Jukes-Cantor– corrected distances of
0.024 between HPeV3 sequences were less than one-third of the
distances between HPeV1 sequences (0.086), and they indicate a
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Figure 3. Comparison of recorded symptoms of and/or diagnoses for
study subjects with human parechovirus (HPeV)–positive samples with
those found in association with infection due to other viruses detected
during screening of cerebrospinal fluid (CSF) specimens. HSV1, herpes
simplex virus type 1; HSV2, herpes simplex virus type 2; VZV, varicellazoster virus; ⬍3 months, subjects aged ⬍3 months; 肁3 months of age,
subjects 肁3 months of age.
more recent origin for HPeV3 variants circulating in Edinburgh
over the study period. These observations are consistent with the
phylogenetic position of HPeV3 sequences as direct descendants
of the original A308 isolate recovered in 1999 (accession number
AB084913) (figure 1).
DISCUSSION
Detection and typing of HPeV. Although enteroviruses are a
well-known cause of aseptic meningitis and severe neonatal sepsis, the incidence and clinical associations of HPeV infections are
less well established. Our observation that, in the first half of
2008, the frequency of HPeV infections (7.2%; all of which were
due to HPeV3) was actually greater than that of enterovirus infections (6.6%; caused by 8 different serotypes) directly demonstrates that severe HPeV infections can at least episodically represent a significant cause of sepsis and CNS-related disease in
young infants. These findings, in agreement with those of recently performed studies [7, 9, 10, 15], strongly support the introduction of routine HPeV screening of CSF specimens. The
introduction of such screening would indeed significantly increase the rate of positive results of screening CSF specimens.
Over the study period, screening detected enteroviruses, HSV1,
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Harvala et al.
HSV2, and VZV in 6.8% of samples obtained from children ⬍5
years of age; the addition of screening for HPeV increased our
rate of detection by more than one-quarter, to 8.6%.
Although several RT-PCR– based methods for the detection
of HPeV in CSF samples have been described elsewhere [15,
19 –22], to our knowledge, this is the first systematic study to
apply direct HPeV type identification to CSF specimens. As a
result, we were able to show an absolute association of HPeV
type 3 with CNS-related infection in young infants, an association that could only be indirectly inferred from epidemiologic
data and reliance on other sample types in previous studies [9,
10, 15]. Given the marked differences between HPeV types in
terms of epidemiologic profile and disease associations, typing
has important value in the diagnosis and clinical assessment of
childhood sepsis and CNS-related diseases in neonates and infants.
HPeV epidemiologic profile and disease associations. There
were striking differences in seasonal distribution, age distribution,
and clinical outcomes between HPeV types. In common with findings from previous studies [12, 16, 23], HPeV1 was the most common type detected in non-CSF samples, with similar incidences
noted in 2007 and 2008, reflecting the likely continuous circulation
of this HPeV type throughout the 2000s [9, 12–14, 16, 23, 24]. In
contrast, on the basis of our data from CSF screening tests, HPeV3
circulated only in 2006 and 2008, a biannual cycle consistent with
previous reports of much higher frequencies of HPeV3 infections
occurring in even-numbered years between 2000 and 2006, and was
virtually absent in intervening years [10, 20]. The factors underlying
the periodicity of HPeV3 transmission are unclear, although distinct epidemic patterns of circulation have been described for several enteroviruses. Regular sharp increases in reported cases are observed every 3–5 years, in association with the emergence of new
genetic lineages [25–27]. Whether this phenomenon underlies the
biannual cycle of HPeV3 is unclear. In the short VP3/VP1 sequence
obtained in the current study, there was no evidence for a systematic
difference between variants recovered from different years; most
HPeV3 variants could be classified into 2 lineages, both of which
circulated equally in 2008 and one of which contained variants from
2006 (figure 1).
HPeV3 also differed from HPeV1 (and HPeV6) in terms of
the age range of the subjects who they infected and in terms of
outcomes of infection. Although both the latter types were rare
or absent in neonates and infants ⬍3 months of age, HPeV3
specifically targeted this age group (20 of 22 subjects), with only
2 infections noted in respiratory samples obtained from older
children (age, 1–2 years). Although HPeV1 and HPeV6 infections detected in respiratory samples were not specifically associated with respiratory or other recorded disease manifestations
[16], HPeV3 infections in young infants were associated with
severe disease (all 14 subjects with CSF-positive samples had
sepsis or fever). Moreover, of the 6 study subjects with HPeV3positive respiratory samples in this age group, 3 had CSF samples
Figure 4. A, Comparison of human parechovirus (HPeV) types in different samples, shown as proportions of total samples. Differences in distributions
were assessed using 3 ⫻ 2 exact contingency tables, with significant differences (P ⬍ .05) underlined. B, Age distributions of HPeV type 1 (HPeV1),
HPeV type 3 (HPeV3), and HPeV type 6 (HPeV6) infections. Data for respiratory samples obtained during 2007 were derived from [18]. CSF, cerebrospinal
fluid.
that were referred (all 3 of which were found to be HPeV3 positive), an observation that underlines the high morbidity associated with these infections in this age group. However, in contrast
to the findings of earlier studies [28, 29], no HPeV-infected
young infants in the present study presented with encephalitis.
Mechanisms underlying HPeV type–associated differences.
Two different possible explanations could account for the marked
HPeV3-associated differences in the epidemiologic profile and
morbidity [6, 9, 10]. HPeV3 differs from other types by not encoding an RGD (arginine-glycine-aspartic acid) motif in VP1 [6, 8],
which has been suggested to enable HPeV1 and presumably other
HPeV types to use integrins as virus receptors [30, 31]. The use of
different receptors may thus confer upon HPeV3 a different cellular
tropism, thereby accounting for its greater pathogenicity and neurotropism. However, this hypothesis awaits direct experimental
confirmation.
The alternative explanation is based on the premise that frequencies of past infection with HPeV3 in adults may be lower
than those of other HPeV types; therefore, neonates and young
infants are not protected by maternal antibody during the criti-
cal period after birth when their immature immune systems fail
to prevent systemic spread, leading to more severe disease outcomes. Although the seroprevalence (68%) of HPeV3 among
women of childbearing age in Japan [6] is indeed lower than the
seroprevalence (96%–98%) recorded for HPeV1 [32, 33], no
data are available regarding the seroprevalence of HPeV3 in Europe, where the greater pathogenicity of this type in neonates is
best documented. In further exploring this hypothesis, measurement of past frequencies of infection due to other HPeV types
would also be of value.
Interestingly, neither hypothesis accounts for the observation
that HPeV3 infections were almost entirely confined to young
infants. Neither putative differences in cellular tropism nor a
lack of protection by maternal antibody early in life would preclude transmission between older children, as observed for other
HPeV types. A possible epidemiologic clue is the observation in
the current study of a highly restricted variability of HPeV3 compared with HPeV1 variants characterized in the present study
and in isolates recovered in Canada [7] and with other HPeV
types based on available published sequences (figure 1). AlHPeV3 and Sepsis in Young Infants
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1759
though the rate of substitution for the analyzed region of VP3/
VP1 of HPeV has not been determined, rates between 4 ⫻ 10 ⫺3
and 14 ⫻ 10 ⫺3 substitutions per site per year typify structural
gene regions of other picornaviruses (e.g., enteroviruses and
aphthoviruses, as reviewed in [34]). If these reflect HPeV, a conservative (minimum) estimate of the substitution rate of
5 ⫻ 10 ⫺3 substitutions per site per year predicts only 9 years of
divergent sequence drift between Edinburgh variants and the
originally described HPeV3 isolate (A308/99) from Japan from
1999, an estimate that closely fits the difference in sample dates.
These dates support the hypothesis for extremely recent global
spread of HPeV3 into naive human populations and may therefore account for the lack of maternal protection of neonates specific to this HPeV type. Population structures and evolutionary
histories of different HPeV types can be reconstructed by coalescent methods [35] and can allow the association between emergence and pathogenicity to be further explored.
We thank Peter McCullough, Julie White, Mary Notman, Eleanor Leslie,
and Carol Thomson for providing samples, data, and other virus testing
results from the respiratory sample archive. We also thank Elly Gaunt for
help with sample extraction, pooling, and archive storage of respiratory
specimens.
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