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Human parechovirus-3 infection in children, South Korea
Journal of Clinical Virology 58 (2013) 194–199
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Human parechovirus-3 infection in children, South Korea夽 Tae-Hee Han a , Ju-Young Chung b,∗ , Su Jeong You b , Jeong-Lim Youn b , Gyu-Hong Shim b a b
Department of Laboratory Medicine, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, Republic of Korea Department of Pediatrics, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 March 2013 Received in revised form 19 May 2013 Accepted 28 May 2013 Keywords: Human parechovirus Meningitis sepsis Klasse virus Enterovirus Children
a b s t r a c t Background: Human parechoviruses (HPeVs) have recently been recognized as important viral pathogens causing sepsis-like illness and meningitis in children, but the data on these infections in Korea is limited. Klassevirus is emerging as a novel etiologic agent of acute gastroenteritis, but its role in meningitis remains unclear. Objectives: To understand the epidemiology of HPeVs and klassevirus in sepsis-like illness and meningitis through the detection and typing of the virus in cerebrospinal fluid (CSF) samples. Study design: One hundred and eighty-three CSF samples collected from 183 patients ranging in the age group 1 day to 15 years were tested by using a RT-PCR assay for HPeV, EV and klassevirus. Amplification products of the VP3/VP1 and 3D region of the HPeV, and VP1 region of the EV were sequenced to identify the type. Results: A total of 12 HPeV positive samples (6.5%) were detected from 183 CSF samples and all the samples were typed as HPeV-3. EVs were detected in 39 patients (21.3%) in which echovirus 25 and CVA6 were frequently detected, but mixed infection of HPeV-3 and EV was not observed. Klassevirus was not detected in the study population. Most of the HPeV-3 positive patients were under 3 months of age. HPeV-3 infection was detected mostly in the summer season. The VP3/VP1 gene of the 12 Korean strains clustered most closely to the Japan strain (AB759192) and the 3D gene of the Korean strains also clustered to the Japan strain, which showed no evidence of recombination. Conclusions: To our knowledge, this is the first report on the detection of HPeV-3 from CSF samples in Korea, which suggests the necessity of routine screening for this virus in young infants with sepsis-like illness and meningitis. © 2013 Elsevier B.V. All rights reserved.
1. Background Human parechovirus-1 (HPeV-1) and HPeV-2 were originally isolated from children with acute gastroenteritis (GE) and described as echovirus 22 (EV22) and EV23, but were classified into a new genus Parechovirus on the basis of studies revealing distinct molecular and biological characteristics.1 Recently, HPeVs were classified into 16 types including partial genome sequences2 and they showed highly diverse genotypes suggesting high rates of recombination.3 Infection with HPeVs is known to cause mild GE and respiratory disease.4–6 However, the role of HPeV-1 infection in GE is uncertain due to the frequent detection of this virus in stool samples from healthy children.7,8 In contrast, HPeV-3 infection showed increasing evidence of a specific role in severe neonatal diseases such as sepsis and meningitis.9–12 In Korea, HPeV-1 and
夽 This study was partially supported by the research grant (2011) by Seoul National University Department of Pediatrics Research Center. ∗ Corresponding author. Tel.: +82 02 950 1073; fax: +82 02 938 4109. E-mail address: [email protected] (J.-Y. Chung). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.05.023
HPeV-4 have been detected in stool samples from children with GE,6 but data for the epidemiology of HPeV-3 in cerebrospinal fluid (CSF) continue to be unavailable. Klassevirus has been identified in children with GE as a new picornavirus in 200913 and has been suggested as a possible etiologic agent in GE,14,15 but its role in sepsis-like illness and meningitis is uncertain. 2. Objectives The purpose of this study was to detect and characterize HPeVs and klassevirus in CSF samples from children with sepsis-like illness and meningitis in Korea. 3. Study design A total of 183 cerebrospinal fluid (CSF) collected from children under the age of 16 years admitted to Sanggyepaik Hospital, Seoul, between January 2011 and September 2012, were included in this study. Medical records of the study population were reviewed to collect data on the clinical manifestations and laboratory findings.
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199
CSF pleocytosis was defined using the criteria in a previous study16 ; CSF WBC counts >22 mm−3 under 4 weeks of age, >15 mm−3 in 4–7 weeks of age, >5 mm−3 over 7 weeks of age. All the CSF samples were tested for common bacterial pathogens by routine microbiological methods.17 The study protocol was approved by the institutional review board of the Sanggyepaik Hospital, Inje University. Total RNA was extracted from 200 L of the CSF samples by using a QIAamp viral RNA kit (Qiagen, GmbH, Hilden, Germany). Each RNA extract (5 L) was mixed with RT-mix and the RT reactions were performed at 42 ◦ C for 60 min, and 95 ◦ C for 5 min, followed by soaking at 4 ◦ C. To detect the VP1 region of EV, nested RT-PCR assays were performed using the following primers 224 (5 -GCIA TGY TIG GIA CIC AYR T-3 ), 222 (5 -CIC CIG GIG GIA YRW ACA T-3 ), AN89 (5 -CCA GCA CTG ACA GCA GYN GAR AYN GG-3 ), and AN88 (5 -TAC TGG ACC ACC TGG NGG NAY RWA CAT-3 ), as described previously.18 To detect the 5 -NCR region of HPeVs, nested RT-PCR assays were performed with the primers 5 -GGG TGG CAG ATG GCG TGC CAT AA-3 (outer sense, nt253-275), 5 -CCT RCG GGT ACC TTC TGG GCA TCC-3 (outer antisense, nt583-560), 5 -AYA CAG CCA TCC TCT RGT AAG TTT G-3 (inner sense, nt313-339), and 5 -GTG GGC CTT ACA ACT AGG TTT G-3 (inner antisense, nt556-534) under the following reaction conditions: 30 cycles of 30 s at 94 ◦ C, 30 s at 50 ◦ C, and 90 s at 72 ◦ C, combined with a final extension of 7 min, as described previously.10 To detect the VP3/VP1 gene of HPeV, RT-PCR was performed using modified primers5 : The first reaction was performed using the primers 2090 (5 -GAY AAT GCY ATM TAY ACW ATY TGT GA-3 ) and 2523 (5 -ACW GTR AAR ATR TCH ACA TTS ATD G-3 ). The second reaction was performed using the primers; 2159 (5 -TTY TCM ACH TGG ATG MGG AAR AC-3 ) and 2523 (5 -ACW GTR AAR ATR TCH ACA TTS ATD G-3 ). To detect the 3D gene of HPeV, the first reaction was performed using modified primers19,20 ; OL1502 modified (5 -GTN TAY AGR ATG ATH ATG ATG GA-3 , nt6419-6443) and 3D ParEcho-R modified (5 -CAA RYG ADT CTG CCA TRA YAC-3 , nt6914-6894). The second reaction was performed using OL1502 modified (5 -GTN TAY AGR ATG ATH ATG ATG GA-3 , nt6419-6443), and 3DR6891 (5 -TGC AGY YTC TCT GGI TCI ATY TC-3 , nt6891-6869) primers. To detect klassevirus, nested PCR was performed using the following primers: LG0118, LG0117, KL3DF, and KL3DR for the 3D region and LG0119, LG0136, KLVPF, and KLVPR for the viral protein (VP) 0/VP1 gene, as described.13 The amplicon was purified using QIAquick (Qiagen) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, USA). Sequencing products were resolved with an ABI 3730 XL autoanalyzer (Applied Biosystems). Nucleotides sequences were aligned using BioEdit v7.0 and presented in a topology tree, prepared in MEGA 4.1. The nucleotide sequences identified in this study have been deposited in the GenBank database under the accession numbers: VP3/VP1 region (KC196278-196289) and partial 3D region (KC196299-196308).
4. Results Of the 183 children who were hospitalized with sepsis-like illness or meningitis, median age was 3 months and the age distribution was as follows; 88 (48.0%) for <3 months, 24 (13.1%) for 3–11 months, and 71 (38.8%) for 1–15 years. The sex ratio of the study population was 1.3:1 (male, 104 patients; female, 79 patients). The clinical diagnoses were sepsis-like illness for 97 children, and meningitis for 86. In 183 CSF samples, which were collected from January 2011 to September 2012, 12 samples (6.5%) were found positive for HPeVs, 39 (21.3%) samples positive for EV, and none (0%) was positive for klassevirus. Mixed infection with both HPeV and EV was not
195
Fig. 1. Seasonal distribution of human parechovirus (HPeV) and enterovirus (EV) infections in children with sepsis like illness between January 2011 and September 2012.
detected. Typing of 12 HPeV positive samples by sequencing the VP3/VP1 gene revealed that all the isolated Korean strains belonged to HPeV-3. Most of the HPeV-3 samples were detected in the summer season of 2011 (June, 4 patients; July, 4 patients; August, 2 patients), but only 1 HPeV-3 positive was detected in 2012 as shown in Fig. 1. Most (91.6%, 11/12) of the HPeV-3 positive patients were under 3 months of age, ranging from 3 days to 4 months old (average, 2 months; median, 2 months) (Table 1). The sex ratio of the HPeV-3 positive patients was 1:1.4 (male: female). The most frequent symptoms were fever (100%, 12/12), gastrointestinal symptoms (25%, 3/12), respiratory symptoms (58%, 7/12), and skin rash (8%, 1/12). The mean CSF white blood cell (WBC) and CSF protein was 1 mm−3 and was 41.8 mg dL−1 , respectively (Table 2), but CSF pleocytosis was not found in HPeV-3 positive patients. The mean peripheral WBC count of HPeV-3 group was 12,160 mm−3 (range, 3990–19380 mm−3 ); leukopenia (3990 mm−3 ) in 1 patient and leukocytosis (16160–19380 mm−3 ) in 5 patients. Thrombocytopenia was not noted in HPeV-3 positive patients. Concurrent acute pyelonephritis was found in 2 patients and lower respiratory tract infections were found in 2. The mean hospital stay of the HPeV-3 group was 5.7 days similar to that of the EV group (5.6 days). None of the HPeV-3 positive patients required hospitalization in the intensive care unit (ICU) and all patients were discharged without complications. In this study, EV was frequently found in infants under the age of 3 months (64%, 25/39), but was also detected in other age groups, although HPeV-3 was only detected in children under 4 months of age (Table 1). In 2012, seasonal distribution of HPeV-3 infection and EV infection was similar, which showed a peak in the summer. Molecular typing of EV was performed based on the VP1 gene, which revealed Echo 25 in 15 patients, CVA6 in 8 patients, Echo 6 in 4 patients, Echo18 in 3 patients, CVA4 in 3 patients, CVB2 in 3 Table 1 Human prechovirus-3 (HPeV-3) and enterovirus (EV) detection in CSF samples. Age
Positive samples Total
HPeV <3 months 3–11 months 1–15 years Total
11/88 1/17 0/78 12/183 (6.5%)
HEV <3 months 3–11 months 1–15 years Total
25/88 8/17 6/78 39/183 (21.3%)
196
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199 KC196287 CSF131/2011/KR KC196288 CSF133/2011/KR KC196286 CSF123/2011/KR KC196280 CSF083/2011/KR KC196278 CSF066/2011/KR KC196279 CSF080/2011/KR KC196281 CSF085/2011/KR KC196282 CSF091/2011/KR KC196284 CSF097/2011/KR KC196283 CSF093/2011/KR KC196285 CSF111/2011/KR 81
88 KC196289 CSF170/2012/KR 82 AB759192 HPeV3/S/1517-Yamagata/2011/JP
H Pe V 3
AB759207 HPeV3/S/1585-Yamagata/2011/JP HQ897628 HPeV3/CSF-08-2389/2008/UK AB759202 HPeV3/A/1678-Yamagata/2011/JP AB759205 HPeV3/A/1924-Yamagata/2011/JP 99
AJ889918 HPeV3/A/Can82853-01/2002/CA GQ183028 HPeV3/A/450936/2004/NL HQ897616 HPeV3/CSF-06-0648/2006/UK AB084913 HPeV3/A/A308/1999/JP AB668029 HPeV3/W/1361K-162589-Yamagata/2008/JP 100 AB668032 HPeV3/W/1443-175790-Yamagata/2008/JP GQ183029 HPeV3/Q/651689/2006/NL
HQ897632 HPeV3/CSF-08-2556/2010/UK EF051629 HPeV1B/AD/BNI-788St/2003/DE 99
FM242866 HPeV1B/AD/PicoBank/2000/FI AJ005695 HPeV2/AH/Williamson/1956/USA S45208 HPeV1A/AM/Harris/1956/USA
83
EU024629 HPeV6/AL/BNI-67/2003/DE AB252582 HPeV6/BD/NII561-2000/2000/JP EU716175 HPeV8/AT/BR/217/2006/BR 96
HPeV2 HPeV1A
GQ183034 HPeV1A/AD/2007-863/2007/NL 99
HPeV1B
AB433629 HPeV4/AE/Fuk2005-123/2005/JP AM235750 HPeV4/AF/T75-4077/USA EU556224 HPeV7/R/PAK5045/2007/PK AM235749 HPeV5/AQ/T92-15/1992/USA HQ696576 HPeV5/AW/BR/77/2006/BR
HPeV6 HPeV8 HPeV4 HPeV7 HPeV5
0.05
Fig. 2. Phylogenetic analysis of the VP3/VP1 region (206 bp) of parechovirus. The tree was constructed using the neighbor-joining method with Kimura two-parameter estimation. The bootstrap values from 1000 replicates are present on each branch. The Korean strains (KC196278-196289) are presented in boldface.
patients, Echo 9 in 2 patients, and CVA10 in 1 patient. The sex ratio of the EV positive patients showed male predominance (male: female, 2:1), which was different with that of HeV-3 positive patients. In the EV positive patients, CSF pleocytosis was not observed in 33% (1/3) of infants aged 0–27days, 30% (3/10) of infants aged 28–55 days, 30% (8/26) of infants aged over 56 days–15 years. Fig. 2 shows the phylogenic tree of the VP3/VP1 gene of the 12 Korean strains, which showed clustering of all Korean strains to HPeV-3. Korean strains clustered closely with the Japan strain HPeV3/A/1678-Yamagata/2011/JP(AB759192), and the VP1 gene similarity between those strains ranged from 98.9% to 99.6% (the amino acid similarity: 100%). To determine the presence of recombination among the different HPeV types, we performed RT-PCR for the 2 different genes (3D gene and VP3/VP1 gene) of each HPeV-3 positive sample. The similarity of nucleotide sequences of isolated Korean HPeV-3 stains was 96.9–98%, and amino acid similarities were 100%, when compared to those of AB759192. The 3D region of all the Korean strains was also clustered to HPeV-3 in Fig. 3. The
similarity of nucleotide sequences of isolated strains was 97.8%, and amino acid similarities were 100% compared to those of AB759192, which showed no evidence of recombination. 5. Discussion HPeVs show a worldwide distribution, but there have been few studies on HPeV infection in Korea.6 To our knowledge, this is the first report on the detection and characterization of HPeV-3 in children with meningitis and sepsis-like illness in Korea. HPeV-3 is known to be one of the most important etiologic agents in neonatal sepsis and severe CNS infections such as meningitis and encephalitis. However, clinical suspicion of HPeV-3 infection in adults could be necessary when an outbreak of epidemic mylagia occurs.21 In a recent experiment,22 HPeV-3 replicated effectively in neural cell lines and was difficult to neutralize with specific antibodies associated with the lack of an arginine–glycine–aspartic acid motif, which is essential for HPeV-1 receptor binding.23 PCR assays for HPeV-3
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199
197
KC196303 CSF093/2011/KR KC196304 CSF097/2011/KR KC196302 CSF085/2011/KR KC196301 CSF083/2011/KR 100 KC196300 CSF080/2011/KR KC196299 CSF066/2011/KR KC196305 CSF111/2011/KR 97
KC196306 CSF123/2011/KR KC196307 CSF131/2011/KR
A
KC196308 CSF133/2011/KR 83
AB759202 HPeV3/A/1678-Yamagata/2011/JP AJ889918 HPeV3/A/Can82853-01/2002/CA
83
GQ183028 HPeV3/A/450936/2004/NL GQ183026 HPeV3/A/152037/2001/NL
97
GQ183033 HPeV3/A/K8-94/1994/NL AB084913 HPeV3/A/A308/1999/JP GQ183022 HPeV1B/M/K129-93/1993/NL GQ183024 HPeV1B/M/K54-94/1994/NL
99
FM178558 HPeV1B/D/7555312/2003/NL HQ384373 HPeV1/K/Seoul0742 3D HQ384375 HPeV1/K/Seoul1142 3D
95
EU556224 HPeV7/R/PAK5045/2007/PK GQ183029 HPeV3/Q/651689/2006/NL DQ315670 HPeV4/P/K25117602/2006/NL AB759207 HPeV3/S/1585-Yamagata/2011/JP 100 AB759192 HPeV3/S/1517-Yamagata/2011/JP AM235750 HPeV4/AF/T75-4077/1975/USA AB433629 HPeV4/AE /Fuk2005-123/2005/JP AB759185 HPeV3/W/1352-Yamagata/2008/JP
100 88
AB759186 HPeV3/W/1355-Yamagata/2008/JP HQ384372 HPeV1/X/Seoul0564 3D 100
HQ384374 HPeV1/X/Seoul0806 3D
FM242866 HPeV1B/AD/PicoBank/2000/FI EF051629 HPeV1B/AD/BNI-788St/2003/DE
100
AJ005695 HPeV2/AH/Williamson/1956 GQ183021 HPeV1B/AJ/550163/2005/NL GQ183018 HPeV1B/AK /152478/2001/NL S45208 HPeV1A/AM/Harris/1956/USA
D K R Q
P S AF AE Y
X
AD AH AJ AK AM
EU024629 HPeV6/AL/BNI-67/2003/DE HQ696577 HPeV6/AY /BR/104/2006/BR AM235749 HPeV5/AQ/T92-15/1992/USA 100
M
AB252582 HPeV6/BD/NII561-2000/2000/JP EU077518 HPeV6/BD/2005-823/2005/NL AF055846 HPeV5/BB /CT86-6760/1986/USA
AL AY AQ BD BB
EU716175 HPeV8/AT/BR/217/2006/BR 93
HQ696572 HPeV1B/AX/BR30/2006/BR HQ696575 HPeV5/BR/53/2006/BR HQ696576 HPeV5/AW/BR77/2006/BR 99
HQ696570 HPeV1B/AS /BR21/2006/BR HQ696571 HPeV1B/AS /BR27/2006/BR GQ183020 HPeV1B/AU/450343/2004/NL HQ696573 HPeV1A/AV /BR114/2006/BR
BR AW AS AU AV
0.05
Fig. 3. Phylogenetic analysis of partial 3D (456 bp) of parechovirus. The tree was constructed using the neighbor-joining method with Kimura two-parameter estimation. The bootstrap values from 1000 replicates are presented on each branch. The Korean strains (KC196299-196308) are presented in boldface.
are recommended as a rapid and effective diagnostic method because this genotype is difficult to grow in conventional cell lines. In this study, the detection rate of HPeV-3 in CSF samples (6.5%) was higher than that was observed in the other recent studies,24–26 which may be due to the differences in both the study population and the primers used. HPeV-3 was mostly detected among young children under the age of 3 months, which is consistent with previous studies.9–11,25 In contrast to previous studies describing
male predilection (57.1–80%) to HPeV-3 infection,10,11,25 we found a slight predilection toward female, a finding also reported in a British study.24 In this study, CSF pleocytosis was not found in 43% (17/39) of children with EV infection and 91.6% (11/12) of children with HPeV-3 infection, which may be due to immature immunologic functions in young infants.27 These results indicate that the evaluation for EV or HPeV-3 is necessary in young infants although CSF profiles showed non-pleocytosis.27,28
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T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199
Table 2 Clinical characteristics and laboratory profiles of HPeV-3 and EV positive patients. Variable Age (months) Mean Median (range) Duration of hospital stays (days) Mean Median (range) Clinical diagnosis Sepsis-like illness Meningitis Symptom Fever Seizure Skin rash Gastrointestinal symptoms Respiratory tract symptoms CSF Cell counts, mean no. of cells mm−3 (range) Glucose level (mg dL−1 ) Protein level (mg dL−1 ) Peripheral blood WBC counts, cells mm−3 (range) Platelet, k mm−3 (range)
Funding
HPeV-3 positive (n = 12)
EV positive (n = 39)
This study was partially supported by the 2011 Seoul National University Department of Pediatrics Research Grant.
1.8 2 (0–4)
22.2 2 (0–108)
Competing interests
5.7 4 (3–10)
5.6 5 (3–18)
12/12 (100%) 0/12 (0%)
30/39 (76.9%) 9/39 (23.1%)
12/12 (100%) 0/12 (0%) 1/12 (8.3%) 3/12 (25.0%) 7/12 (58.3%)
39/39 (100%) 3/39 (7.7%) 5/39 (12.8%) 6/39 (15.3%) 7/39 (17.9%)
1(1–3)
167(0–1310)
52.3 41.8 12,160 (3990–19380) 421 (267–619)
50.5 56.0 10,869 (2640–19960) 384 (179–553)
The authors declare no conflict of interests. Ethical approval
In the present study, peripheral blood WBC counts were over 10,000 cells mm−3 in 41.6% of patients with HPeV-3 infection and in 56.4% of patients with EV infection, which was different from the observations of previous studies on leukopenia.12,25 It is interesting that a majority of the HPeV3 infections was found in the summer of 2011in Korea, which is in contrast with what is found in Europe (summer of the even years) but follows the American pattern (summer of uneven years).9–11,24,27 The clinical course of HPeV-3 infection was favorable and no patient required intensive care, similar to the observations of recent studies10,24,25 despite the suggestion of a possible association between severe CNS infections and HPeV-3.12,29,30 Skin rash was described as a frequent manifestation in up to 80% of people with HPeV-3 infection11,31 and the distinctive palmar–plantar erythema in young infants with fever was suggested as an important clue of HPeV-3 infections.31 However, we detected with skin rash only in 1 patient, which is similar to the results from a Dutch study showing that other symptoms, except gastrointestinal symptoms, have no association with the presence of a specific serotype.32 In this study, the clinical manifestations of HPeV-3 positive patients were similar to those of EV positive patients, which was described in previous studies.9 In previous Korean studies,33–36 outbreaks of aseptic meningitis by various serotypes of EV such as Echo6, Echo 9, Echo11, Echo13, Echo 18, Echo 25, Echo30, and EV 71 were reported. In this study, Echo 25 (15 patients) was most frequently detected, which showed also epidemic in Chungnam area in 2006.36 Klassevirus is closely related to the Aichi virus in the genus Kobuvirus, and is suggested to be associated with GE.13–15 According to the results of this study, klassevirus could not be considered as one of the etiologic agents in sepsis-like illness in young infants, although further studies are needed to confirm these findings. The 3D gene and the VP3/VP1 gene of Korean HPeV-3 strains did not show recombination and clustered with the genes of the recently identified Japan strains (Figs. 2 and 3). These findings are consistent with those of a previous study showing lower diversity of HPeV-3 than of HPeV-1, which results in monophyletic sequences in the majority of HPeV-3 sequences.3 In conclusion, we detected HPeV-3 in CSF samples from young infants with sepsis-like illness and meningitis in Korea, which suggests that routine screening for HPeV-3 in CSF samples from young infants is necessary in clinical practices evaluating sepsis.
The Institutional Review Board of the Sanggyepaik Hospital approved the study protocol. Parents of all participating children gave their informed consents. References 1. 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. 2. Human parechovirus. http://www.picornaviridae.com/parechovirus/hpev/hpev.htm 3. Benschop KS, de Vries M, Minnaar RP, Stanway G, van der Hoek L, Wolthers KC, et al. Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination. J Gen Virol 2010;91:145–54. 4. Benschop K, Thomas X, Serpenti C, Molenkamp R, Wolthers K. 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 2008;12:3965–70. 5. Harvala H, Robertson I, McWilliam Leitch EC, Benschop K, Wolthers KC, Templeton K, et al. Epidemiology and clinical associations of human parechovirus respiratory infections. J Clin Microbiol 2008;46:3446–53. 6. Han TH, Kim CH, Park SH, Chung JY, Hwang ES. Detection of human parechoviruses in children with gastroenteritis in South Korea. Arch Virol 2011;156:1471–5. 7. Tapia G, Cinek O, Witso E, Kulich M, Rasmussen T, Grinde B, et al. Longitudinal observation of parechovirus in stool samples from Norweigian infants. J Med Virol 2008;80:1835–42. 8. Kolehmainen P, Oikarinen S, Koskiniemi M, Simell O, IIonen J, Knip M, et al. Human parechoviruses are frequently detected in stool of healthy Finnish children. J Clin Virol 2012;54:156–61. 9. Wolthers KC, Benschop KS, Schnikel J, Lenkamp R, Bergevoet RM, Spijkerman IJ, et al. Human parechoviruses as an important viral cause of sepsis-like illness and meningitis in young children. Clin Infect Dis 2008;47:358–63. 10. Harvala H, Robertson I, Chieochansin T, McWilliam Leitch EC, Templeton K, Simmonds P. Specific association of human parechovirus type 3 with sepsis and fever in young infants, as identified by direct typing of cerebrospinal fluid samples. J Infect Dis 2009;199:1753–60. 11. Selvarangan R, Nzabi M, Selvaraju SB, Ketter P, Carpenter C, Harrison CJ. Human parechovirus 3 causing sepsis-like illness in children from Midwestern United States. Pediatr Infect Dis J 2011;30:238–42. 12. Schuffenecker I, Javouhey E, Gillet Y, Kugner B, Billaud G, Floret D, et al. Human parechovirus infections, Lyon, France, 2008–2010: evidence for severe cases. J Clin Virol 2012;54:337–41. 13. Han TH, Kim CH, Chung JY, Park SH, Hwang ES. Klassevirus infection in children with acute gastroenteritis, South Korea. Emerg Infect Dis 2010;16:1623–5. 14. Holtz LR, Fink Einer SR, Zhao G, Kirkwood CD, Girones R, Pipas JM, et al. Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread. Virol J 2009;6:86. 15. Li L, Victoria J, Kappor A, Blinkova O, Wang C, Babrzadeh F, et al. A novel picornavirus associated with gastroenteritis. J Virol 2009;83:12002–6. 16. Bonadio WA, Stanco L, Bruce R, Barry D, Smith D. Reference values of normal cerebrospinal fluid in infants ages 0–8 weeks. Pediatr Infect Dis J 1992;11:589–91. 17. Lee JI, Chung JY, Han TH, Song MO, Hwang ES. Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis 2007:196–994. 18. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol 2006;44:2698–704. 19. Williams CH, Panayiotou M, Girling GD, Peard CI, Oikarinen S, Hyouty H, et al. Evolution and conservation in human parechovirus genomes. J Gen Virol 2009;90:1702–12. 20. 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 2006;42:204–10. 21. Mizuta K, Kuroda M, Kurimura M, Yahata Y, Sekizuka T, Aoki Y, et al. Epidemic myalgia in adults associated with human parechovirus type 3 infection, Yamagata, Japan, 2008. Emerg Infect Dis 2012;18:1787–93. 22. Westerhuis BM, Koen G, Wildenbeest JG, Pajkrt D, de Jong MD, Benschop K, et al. Specific cell tropism and neutralization of human parechovirus types 1 and 3: implications for pathogenesis and therapy development. J Gen Virol 2012;93:2363–70.
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199 23. Boonyakiat Y, Hughes PJ, Ghazi F, Stanway G. Arginine–glycine–aspartic acid motif is critical for human parechovirus 1 entry. J Virol 2001;75: 10000–4. 24. Harvala H, McLeish N, Kondracka J, McIntyre CL, Leitch E, Templeton K, et al. Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in Edinburgh: HPeV type 3 identified as the common Picornavirus type. J Med Virol 2011;83: 889–96. 25. Walters B, Penaranda S, Nix WA, Oberste MS, Todd KM, Katz BZ, et al. Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area. J Clin Virol 2011;52:187–91. 26. de Crom SCM, Obihara CC, van Loon AM, Argilagos-Alvarez AA, Peters MF, van Fruth AM, et al. Detection of enterovirus RNA in cerebrospinal fluid: comparison of two molecular assays. J Virol Methods 2012;179:104–7. 27. Seiden JA, Zorc JJ, Hodinka RL, Shah SS. Lack of cerebrospinal fluid pleocytosis in young infants with enterovirus infections of the central nervous system. Pediatr Emerg Care 2010;26:77–81. 28. Yun KW, Cho EH, Chen DS, Lee J, Choi CW, Hwang H, et al. Enteroviral meningitis without pleocytosis in children. Arch Dis Child 2013;97:874–8. 29. Biovin. Human parechovirus types 1, 2, and 3 infections in Canada. Emerg Infect Dis 2006;12:969–75.
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30. Sedmak G, Nix WA, Jentzen J, Haupt TE, Davis JP, Bhattacharyya S, et al. Infant deaths associated with human parechovirus infection in Wisconsin. Clin Infect Dis 2010;50:357–61. 31. Shoji K, Komuro H, Miyata I, Myairi I, Saitoh A. Dermatologic manifestations of human parechovirus type 3 infection in neonates and infants. Pediatr Infect Dis J 2012 [Epub ahead]. 32. van der Sanden S, de Bruin E, Vennema H, Swanink C, Koopmans M, van der Avoort H. Prevalence of human parechovirus in the Netherlands in 2000–2007. J Clin Microbiol 2008;46:2884–9. 33. Lee KY, Burgner D, Lee HS, Hong JH, Lee MH, Kang JH, et al. The changing epidemiology of pediatric aseptic meningitis in Daejeon, Korea from 1987 to 2003. BMC Infect Dis 2005;5:97. 34. Cheon DS, Lee J, Lee KB, Lee S, Park K, Ahn J, et al. Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002. J Med Virol 2004;73:439–42. 35. Jeong EJ, Lee JH, Kim MS, Bae GR, Jung C, Lee K, et al. Molecular characterization of enteroviruses detected in Gyeong-Ju and Po-Hang provinces of Korea in 2003. Arch Virol 2010:1707–12. 36. Baek K, Park K, Jung E, Chung E, Park J, Choi H, et al. Molecular and epidemiological characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006. J Microbiol Biotechnol 2009;19:1055–64.
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Human parechovirus-3 infection in children, South Korea夽 Tae-Hee Han a , Ju-Young Chung b,∗ , Su Jeong You b , Jeong-Lim Youn b , Gyu-Hong Shim b a b
Department of Laboratory Medicine, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, Republic of Korea Department of Pediatrics, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 March 2013 Received in revised form 19 May 2013 Accepted 28 May 2013 Keywords: Human parechovirus Meningitis sepsis Klasse virus Enterovirus Children
a b s t r a c t Background: Human parechoviruses (HPeVs) have recently been recognized as important viral pathogens causing sepsis-like illness and meningitis in children, but the data on these infections in Korea is limited. Klassevirus is emerging as a novel etiologic agent of acute gastroenteritis, but its role in meningitis remains unclear. Objectives: To understand the epidemiology of HPeVs and klassevirus in sepsis-like illness and meningitis through the detection and typing of the virus in cerebrospinal fluid (CSF) samples. Study design: One hundred and eighty-three CSF samples collected from 183 patients ranging in the age group 1 day to 15 years were tested by using a RT-PCR assay for HPeV, EV and klassevirus. Amplification products of the VP3/VP1 and 3D region of the HPeV, and VP1 region of the EV were sequenced to identify the type. Results: A total of 12 HPeV positive samples (6.5%) were detected from 183 CSF samples and all the samples were typed as HPeV-3. EVs were detected in 39 patients (21.3%) in which echovirus 25 and CVA6 were frequently detected, but mixed infection of HPeV-3 and EV was not observed. Klassevirus was not detected in the study population. Most of the HPeV-3 positive patients were under 3 months of age. HPeV-3 infection was detected mostly in the summer season. The VP3/VP1 gene of the 12 Korean strains clustered most closely to the Japan strain (AB759192) and the 3D gene of the Korean strains also clustered to the Japan strain, which showed no evidence of recombination. Conclusions: To our knowledge, this is the first report on the detection of HPeV-3 from CSF samples in Korea, which suggests the necessity of routine screening for this virus in young infants with sepsis-like illness and meningitis. © 2013 Elsevier B.V. All rights reserved.
1. Background Human parechovirus-1 (HPeV-1) and HPeV-2 were originally isolated from children with acute gastroenteritis (GE) and described as echovirus 22 (EV22) and EV23, but were classified into a new genus Parechovirus on the basis of studies revealing distinct molecular and biological characteristics.1 Recently, HPeVs were classified into 16 types including partial genome sequences2 and they showed highly diverse genotypes suggesting high rates of recombination.3 Infection with HPeVs is known to cause mild GE and respiratory disease.4–6 However, the role of HPeV-1 infection in GE is uncertain due to the frequent detection of this virus in stool samples from healthy children.7,8 In contrast, HPeV-3 infection showed increasing evidence of a specific role in severe neonatal diseases such as sepsis and meningitis.9–12 In Korea, HPeV-1 and
夽 This study was partially supported by the research grant (2011) by Seoul National University Department of Pediatrics Research Center. ∗ Corresponding author. Tel.: +82 02 950 1073; fax: +82 02 938 4109. E-mail address: [email protected] (J.-Y. Chung). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.05.023
HPeV-4 have been detected in stool samples from children with GE,6 but data for the epidemiology of HPeV-3 in cerebrospinal fluid (CSF) continue to be unavailable. Klassevirus has been identified in children with GE as a new picornavirus in 200913 and has been suggested as a possible etiologic agent in GE,14,15 but its role in sepsis-like illness and meningitis is uncertain. 2. Objectives The purpose of this study was to detect and characterize HPeVs and klassevirus in CSF samples from children with sepsis-like illness and meningitis in Korea. 3. Study design A total of 183 cerebrospinal fluid (CSF) collected from children under the age of 16 years admitted to Sanggyepaik Hospital, Seoul, between January 2011 and September 2012, were included in this study. Medical records of the study population were reviewed to collect data on the clinical manifestations and laboratory findings.
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199
CSF pleocytosis was defined using the criteria in a previous study16 ; CSF WBC counts >22 mm−3 under 4 weeks of age, >15 mm−3 in 4–7 weeks of age, >5 mm−3 over 7 weeks of age. All the CSF samples were tested for common bacterial pathogens by routine microbiological methods.17 The study protocol was approved by the institutional review board of the Sanggyepaik Hospital, Inje University. Total RNA was extracted from 200 L of the CSF samples by using a QIAamp viral RNA kit (Qiagen, GmbH, Hilden, Germany). Each RNA extract (5 L) was mixed with RT-mix and the RT reactions were performed at 42 ◦ C for 60 min, and 95 ◦ C for 5 min, followed by soaking at 4 ◦ C. To detect the VP1 region of EV, nested RT-PCR assays were performed using the following primers 224 (5 -GCIA TGY TIG GIA CIC AYR T-3 ), 222 (5 -CIC CIG GIG GIA YRW ACA T-3 ), AN89 (5 -CCA GCA CTG ACA GCA GYN GAR AYN GG-3 ), and AN88 (5 -TAC TGG ACC ACC TGG NGG NAY RWA CAT-3 ), as described previously.18 To detect the 5 -NCR region of HPeVs, nested RT-PCR assays were performed with the primers 5 -GGG TGG CAG ATG GCG TGC CAT AA-3 (outer sense, nt253-275), 5 -CCT RCG GGT ACC TTC TGG GCA TCC-3 (outer antisense, nt583-560), 5 -AYA CAG CCA TCC TCT RGT AAG TTT G-3 (inner sense, nt313-339), and 5 -GTG GGC CTT ACA ACT AGG TTT G-3 (inner antisense, nt556-534) under the following reaction conditions: 30 cycles of 30 s at 94 ◦ C, 30 s at 50 ◦ C, and 90 s at 72 ◦ C, combined with a final extension of 7 min, as described previously.10 To detect the VP3/VP1 gene of HPeV, RT-PCR was performed using modified primers5 : The first reaction was performed using the primers 2090 (5 -GAY AAT GCY ATM TAY ACW ATY TGT GA-3 ) and 2523 (5 -ACW GTR AAR ATR TCH ACA TTS ATD G-3 ). The second reaction was performed using the primers; 2159 (5 -TTY TCM ACH TGG ATG MGG AAR AC-3 ) and 2523 (5 -ACW GTR AAR ATR TCH ACA TTS ATD G-3 ). To detect the 3D gene of HPeV, the first reaction was performed using modified primers19,20 ; OL1502 modified (5 -GTN TAY AGR ATG ATH ATG ATG GA-3 , nt6419-6443) and 3D ParEcho-R modified (5 -CAA RYG ADT CTG CCA TRA YAC-3 , nt6914-6894). The second reaction was performed using OL1502 modified (5 -GTN TAY AGR ATG ATH ATG ATG GA-3 , nt6419-6443), and 3DR6891 (5 -TGC AGY YTC TCT GGI TCI ATY TC-3 , nt6891-6869) primers. To detect klassevirus, nested PCR was performed using the following primers: LG0118, LG0117, KL3DF, and KL3DR for the 3D region and LG0119, LG0136, KLVPF, and KLVPR for the viral protein (VP) 0/VP1 gene, as described.13 The amplicon was purified using QIAquick (Qiagen) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, USA). Sequencing products were resolved with an ABI 3730 XL autoanalyzer (Applied Biosystems). Nucleotides sequences were aligned using BioEdit v7.0 and presented in a topology tree, prepared in MEGA 4.1. The nucleotide sequences identified in this study have been deposited in the GenBank database under the accession numbers: VP3/VP1 region (KC196278-196289) and partial 3D region (KC196299-196308).
4. Results Of the 183 children who were hospitalized with sepsis-like illness or meningitis, median age was 3 months and the age distribution was as follows; 88 (48.0%) for <3 months, 24 (13.1%) for 3–11 months, and 71 (38.8%) for 1–15 years. The sex ratio of the study population was 1.3:1 (male, 104 patients; female, 79 patients). The clinical diagnoses were sepsis-like illness for 97 children, and meningitis for 86. In 183 CSF samples, which were collected from January 2011 to September 2012, 12 samples (6.5%) were found positive for HPeVs, 39 (21.3%) samples positive for EV, and none (0%) was positive for klassevirus. Mixed infection with both HPeV and EV was not
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Fig. 1. Seasonal distribution of human parechovirus (HPeV) and enterovirus (EV) infections in children with sepsis like illness between January 2011 and September 2012.
detected. Typing of 12 HPeV positive samples by sequencing the VP3/VP1 gene revealed that all the isolated Korean strains belonged to HPeV-3. Most of the HPeV-3 samples were detected in the summer season of 2011 (June, 4 patients; July, 4 patients; August, 2 patients), but only 1 HPeV-3 positive was detected in 2012 as shown in Fig. 1. Most (91.6%, 11/12) of the HPeV-3 positive patients were under 3 months of age, ranging from 3 days to 4 months old (average, 2 months; median, 2 months) (Table 1). The sex ratio of the HPeV-3 positive patients was 1:1.4 (male: female). The most frequent symptoms were fever (100%, 12/12), gastrointestinal symptoms (25%, 3/12), respiratory symptoms (58%, 7/12), and skin rash (8%, 1/12). The mean CSF white blood cell (WBC) and CSF protein was 1 mm−3 and was 41.8 mg dL−1 , respectively (Table 2), but CSF pleocytosis was not found in HPeV-3 positive patients. The mean peripheral WBC count of HPeV-3 group was 12,160 mm−3 (range, 3990–19380 mm−3 ); leukopenia (3990 mm−3 ) in 1 patient and leukocytosis (16160–19380 mm−3 ) in 5 patients. Thrombocytopenia was not noted in HPeV-3 positive patients. Concurrent acute pyelonephritis was found in 2 patients and lower respiratory tract infections were found in 2. The mean hospital stay of the HPeV-3 group was 5.7 days similar to that of the EV group (5.6 days). None of the HPeV-3 positive patients required hospitalization in the intensive care unit (ICU) and all patients were discharged without complications. In this study, EV was frequently found in infants under the age of 3 months (64%, 25/39), but was also detected in other age groups, although HPeV-3 was only detected in children under 4 months of age (Table 1). In 2012, seasonal distribution of HPeV-3 infection and EV infection was similar, which showed a peak in the summer. Molecular typing of EV was performed based on the VP1 gene, which revealed Echo 25 in 15 patients, CVA6 in 8 patients, Echo 6 in 4 patients, Echo18 in 3 patients, CVA4 in 3 patients, CVB2 in 3 Table 1 Human prechovirus-3 (HPeV-3) and enterovirus (EV) detection in CSF samples. Age
Positive samples Total
HPeV <3 months 3–11 months 1–15 years Total
11/88 1/17 0/78 12/183 (6.5%)
HEV <3 months 3–11 months 1–15 years Total
25/88 8/17 6/78 39/183 (21.3%)
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T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199 KC196287 CSF131/2011/KR KC196288 CSF133/2011/KR KC196286 CSF123/2011/KR KC196280 CSF083/2011/KR KC196278 CSF066/2011/KR KC196279 CSF080/2011/KR KC196281 CSF085/2011/KR KC196282 CSF091/2011/KR KC196284 CSF097/2011/KR KC196283 CSF093/2011/KR KC196285 CSF111/2011/KR 81
88 KC196289 CSF170/2012/KR 82 AB759192 HPeV3/S/1517-Yamagata/2011/JP
H Pe V 3
AB759207 HPeV3/S/1585-Yamagata/2011/JP HQ897628 HPeV3/CSF-08-2389/2008/UK AB759202 HPeV3/A/1678-Yamagata/2011/JP AB759205 HPeV3/A/1924-Yamagata/2011/JP 99
AJ889918 HPeV3/A/Can82853-01/2002/CA GQ183028 HPeV3/A/450936/2004/NL HQ897616 HPeV3/CSF-06-0648/2006/UK AB084913 HPeV3/A/A308/1999/JP AB668029 HPeV3/W/1361K-162589-Yamagata/2008/JP 100 AB668032 HPeV3/W/1443-175790-Yamagata/2008/JP GQ183029 HPeV3/Q/651689/2006/NL
HQ897632 HPeV3/CSF-08-2556/2010/UK EF051629 HPeV1B/AD/BNI-788St/2003/DE 99
FM242866 HPeV1B/AD/PicoBank/2000/FI AJ005695 HPeV2/AH/Williamson/1956/USA S45208 HPeV1A/AM/Harris/1956/USA
83
EU024629 HPeV6/AL/BNI-67/2003/DE AB252582 HPeV6/BD/NII561-2000/2000/JP EU716175 HPeV8/AT/BR/217/2006/BR 96
HPeV2 HPeV1A
GQ183034 HPeV1A/AD/2007-863/2007/NL 99
HPeV1B
AB433629 HPeV4/AE/Fuk2005-123/2005/JP AM235750 HPeV4/AF/T75-4077/USA EU556224 HPeV7/R/PAK5045/2007/PK AM235749 HPeV5/AQ/T92-15/1992/USA HQ696576 HPeV5/AW/BR/77/2006/BR
HPeV6 HPeV8 HPeV4 HPeV7 HPeV5
0.05
Fig. 2. Phylogenetic analysis of the VP3/VP1 region (206 bp) of parechovirus. The tree was constructed using the neighbor-joining method with Kimura two-parameter estimation. The bootstrap values from 1000 replicates are present on each branch. The Korean strains (KC196278-196289) are presented in boldface.
patients, Echo 9 in 2 patients, and CVA10 in 1 patient. The sex ratio of the EV positive patients showed male predominance (male: female, 2:1), which was different with that of HeV-3 positive patients. In the EV positive patients, CSF pleocytosis was not observed in 33% (1/3) of infants aged 0–27days, 30% (3/10) of infants aged 28–55 days, 30% (8/26) of infants aged over 56 days–15 years. Fig. 2 shows the phylogenic tree of the VP3/VP1 gene of the 12 Korean strains, which showed clustering of all Korean strains to HPeV-3. Korean strains clustered closely with the Japan strain HPeV3/A/1678-Yamagata/2011/JP(AB759192), and the VP1 gene similarity between those strains ranged from 98.9% to 99.6% (the amino acid similarity: 100%). To determine the presence of recombination among the different HPeV types, we performed RT-PCR for the 2 different genes (3D gene and VP3/VP1 gene) of each HPeV-3 positive sample. The similarity of nucleotide sequences of isolated Korean HPeV-3 stains was 96.9–98%, and amino acid similarities were 100%, when compared to those of AB759192. The 3D region of all the Korean strains was also clustered to HPeV-3 in Fig. 3. The
similarity of nucleotide sequences of isolated strains was 97.8%, and amino acid similarities were 100% compared to those of AB759192, which showed no evidence of recombination. 5. Discussion HPeVs show a worldwide distribution, but there have been few studies on HPeV infection in Korea.6 To our knowledge, this is the first report on the detection and characterization of HPeV-3 in children with meningitis and sepsis-like illness in Korea. HPeV-3 is known to be one of the most important etiologic agents in neonatal sepsis and severe CNS infections such as meningitis and encephalitis. However, clinical suspicion of HPeV-3 infection in adults could be necessary when an outbreak of epidemic mylagia occurs.21 In a recent experiment,22 HPeV-3 replicated effectively in neural cell lines and was difficult to neutralize with specific antibodies associated with the lack of an arginine–glycine–aspartic acid motif, which is essential for HPeV-1 receptor binding.23 PCR assays for HPeV-3
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199
197
KC196303 CSF093/2011/KR KC196304 CSF097/2011/KR KC196302 CSF085/2011/KR KC196301 CSF083/2011/KR 100 KC196300 CSF080/2011/KR KC196299 CSF066/2011/KR KC196305 CSF111/2011/KR 97
KC196306 CSF123/2011/KR KC196307 CSF131/2011/KR
A
KC196308 CSF133/2011/KR 83
AB759202 HPeV3/A/1678-Yamagata/2011/JP AJ889918 HPeV3/A/Can82853-01/2002/CA
83
GQ183028 HPeV3/A/450936/2004/NL GQ183026 HPeV3/A/152037/2001/NL
97
GQ183033 HPeV3/A/K8-94/1994/NL AB084913 HPeV3/A/A308/1999/JP GQ183022 HPeV1B/M/K129-93/1993/NL GQ183024 HPeV1B/M/K54-94/1994/NL
99
FM178558 HPeV1B/D/7555312/2003/NL HQ384373 HPeV1/K/Seoul0742 3D HQ384375 HPeV1/K/Seoul1142 3D
95
EU556224 HPeV7/R/PAK5045/2007/PK GQ183029 HPeV3/Q/651689/2006/NL DQ315670 HPeV4/P/K25117602/2006/NL AB759207 HPeV3/S/1585-Yamagata/2011/JP 100 AB759192 HPeV3/S/1517-Yamagata/2011/JP AM235750 HPeV4/AF/T75-4077/1975/USA AB433629 HPeV4/AE /Fuk2005-123/2005/JP AB759185 HPeV3/W/1352-Yamagata/2008/JP
100 88
AB759186 HPeV3/W/1355-Yamagata/2008/JP HQ384372 HPeV1/X/Seoul0564 3D 100
HQ384374 HPeV1/X/Seoul0806 3D
FM242866 HPeV1B/AD/PicoBank/2000/FI EF051629 HPeV1B/AD/BNI-788St/2003/DE
100
AJ005695 HPeV2/AH/Williamson/1956 GQ183021 HPeV1B/AJ/550163/2005/NL GQ183018 HPeV1B/AK /152478/2001/NL S45208 HPeV1A/AM/Harris/1956/USA
D K R Q
P S AF AE Y
X
AD AH AJ AK AM
EU024629 HPeV6/AL/BNI-67/2003/DE HQ696577 HPeV6/AY /BR/104/2006/BR AM235749 HPeV5/AQ/T92-15/1992/USA 100
M
AB252582 HPeV6/BD/NII561-2000/2000/JP EU077518 HPeV6/BD/2005-823/2005/NL AF055846 HPeV5/BB /CT86-6760/1986/USA
AL AY AQ BD BB
EU716175 HPeV8/AT/BR/217/2006/BR 93
HQ696572 HPeV1B/AX/BR30/2006/BR HQ696575 HPeV5/BR/53/2006/BR HQ696576 HPeV5/AW/BR77/2006/BR 99
HQ696570 HPeV1B/AS /BR21/2006/BR HQ696571 HPeV1B/AS /BR27/2006/BR GQ183020 HPeV1B/AU/450343/2004/NL HQ696573 HPeV1A/AV /BR114/2006/BR
BR AW AS AU AV
0.05
Fig. 3. Phylogenetic analysis of partial 3D (456 bp) of parechovirus. The tree was constructed using the neighbor-joining method with Kimura two-parameter estimation. The bootstrap values from 1000 replicates are presented on each branch. The Korean strains (KC196299-196308) are presented in boldface.
are recommended as a rapid and effective diagnostic method because this genotype is difficult to grow in conventional cell lines. In this study, the detection rate of HPeV-3 in CSF samples (6.5%) was higher than that was observed in the other recent studies,24–26 which may be due to the differences in both the study population and the primers used. HPeV-3 was mostly detected among young children under the age of 3 months, which is consistent with previous studies.9–11,25 In contrast to previous studies describing
male predilection (57.1–80%) to HPeV-3 infection,10,11,25 we found a slight predilection toward female, a finding also reported in a British study.24 In this study, CSF pleocytosis was not found in 43% (17/39) of children with EV infection and 91.6% (11/12) of children with HPeV-3 infection, which may be due to immature immunologic functions in young infants.27 These results indicate that the evaluation for EV or HPeV-3 is necessary in young infants although CSF profiles showed non-pleocytosis.27,28
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Table 2 Clinical characteristics and laboratory profiles of HPeV-3 and EV positive patients. Variable Age (months) Mean Median (range) Duration of hospital stays (days) Mean Median (range) Clinical diagnosis Sepsis-like illness Meningitis Symptom Fever Seizure Skin rash Gastrointestinal symptoms Respiratory tract symptoms CSF Cell counts, mean no. of cells mm−3 (range) Glucose level (mg dL−1 ) Protein level (mg dL−1 ) Peripheral blood WBC counts, cells mm−3 (range) Platelet, k mm−3 (range)
Funding
HPeV-3 positive (n = 12)
EV positive (n = 39)
This study was partially supported by the 2011 Seoul National University Department of Pediatrics Research Grant.
1.8 2 (0–4)
22.2 2 (0–108)
Competing interests
5.7 4 (3–10)
5.6 5 (3–18)
12/12 (100%) 0/12 (0%)
30/39 (76.9%) 9/39 (23.1%)
12/12 (100%) 0/12 (0%) 1/12 (8.3%) 3/12 (25.0%) 7/12 (58.3%)
39/39 (100%) 3/39 (7.7%) 5/39 (12.8%) 6/39 (15.3%) 7/39 (17.9%)
1(1–3)
167(0–1310)
52.3 41.8 12,160 (3990–19380) 421 (267–619)
50.5 56.0 10,869 (2640–19960) 384 (179–553)
The authors declare no conflict of interests. Ethical approval
In the present study, peripheral blood WBC counts were over 10,000 cells mm−3 in 41.6% of patients with HPeV-3 infection and in 56.4% of patients with EV infection, which was different from the observations of previous studies on leukopenia.12,25 It is interesting that a majority of the HPeV3 infections was found in the summer of 2011in Korea, which is in contrast with what is found in Europe (summer of the even years) but follows the American pattern (summer of uneven years).9–11,24,27 The clinical course of HPeV-3 infection was favorable and no patient required intensive care, similar to the observations of recent studies10,24,25 despite the suggestion of a possible association between severe CNS infections and HPeV-3.12,29,30 Skin rash was described as a frequent manifestation in up to 80% of people with HPeV-3 infection11,31 and the distinctive palmar–plantar erythema in young infants with fever was suggested as an important clue of HPeV-3 infections.31 However, we detected with skin rash only in 1 patient, which is similar to the results from a Dutch study showing that other symptoms, except gastrointestinal symptoms, have no association with the presence of a specific serotype.32 In this study, the clinical manifestations of HPeV-3 positive patients were similar to those of EV positive patients, which was described in previous studies.9 In previous Korean studies,33–36 outbreaks of aseptic meningitis by various serotypes of EV such as Echo6, Echo 9, Echo11, Echo13, Echo 18, Echo 25, Echo30, and EV 71 were reported. In this study, Echo 25 (15 patients) was most frequently detected, which showed also epidemic in Chungnam area in 2006.36 Klassevirus is closely related to the Aichi virus in the genus Kobuvirus, and is suggested to be associated with GE.13–15 According to the results of this study, klassevirus could not be considered as one of the etiologic agents in sepsis-like illness in young infants, although further studies are needed to confirm these findings. The 3D gene and the VP3/VP1 gene of Korean HPeV-3 strains did not show recombination and clustered with the genes of the recently identified Japan strains (Figs. 2 and 3). These findings are consistent with those of a previous study showing lower diversity of HPeV-3 than of HPeV-1, which results in monophyletic sequences in the majority of HPeV-3 sequences.3 In conclusion, we detected HPeV-3 in CSF samples from young infants with sepsis-like illness and meningitis in Korea, which suggests that routine screening for HPeV-3 in CSF samples from young infants is necessary in clinical practices evaluating sepsis.
The Institutional Review Board of the Sanggyepaik Hospital approved the study protocol. Parents of all participating children gave their informed consents. References 1. 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. 2. Human parechovirus. http://www.picornaviridae.com/parechovirus/hpev/hpev.htm 3. Benschop KS, de Vries M, Minnaar RP, Stanway G, van der Hoek L, Wolthers KC, et al. Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination. J Gen Virol 2010;91:145–54. 4. Benschop K, Thomas X, Serpenti C, Molenkamp R, Wolthers K. 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 2008;12:3965–70. 5. Harvala H, Robertson I, McWilliam Leitch EC, Benschop K, Wolthers KC, Templeton K, et al. Epidemiology and clinical associations of human parechovirus respiratory infections. J Clin Microbiol 2008;46:3446–53. 6. Han TH, Kim CH, Park SH, Chung JY, Hwang ES. Detection of human parechoviruses in children with gastroenteritis in South Korea. Arch Virol 2011;156:1471–5. 7. Tapia G, Cinek O, Witso E, Kulich M, Rasmussen T, Grinde B, et al. Longitudinal observation of parechovirus in stool samples from Norweigian infants. J Med Virol 2008;80:1835–42. 8. Kolehmainen P, Oikarinen S, Koskiniemi M, Simell O, IIonen J, Knip M, et al. Human parechoviruses are frequently detected in stool of healthy Finnish children. J Clin Virol 2012;54:156–61. 9. Wolthers KC, Benschop KS, Schnikel J, Lenkamp R, Bergevoet RM, Spijkerman IJ, et al. Human parechoviruses as an important viral cause of sepsis-like illness and meningitis in young children. Clin Infect Dis 2008;47:358–63. 10. Harvala H, Robertson I, Chieochansin T, McWilliam Leitch EC, Templeton K, Simmonds P. Specific association of human parechovirus type 3 with sepsis and fever in young infants, as identified by direct typing of cerebrospinal fluid samples. J Infect Dis 2009;199:1753–60. 11. Selvarangan R, Nzabi M, Selvaraju SB, Ketter P, Carpenter C, Harrison CJ. Human parechovirus 3 causing sepsis-like illness in children from Midwestern United States. Pediatr Infect Dis J 2011;30:238–42. 12. Schuffenecker I, Javouhey E, Gillet Y, Kugner B, Billaud G, Floret D, et al. Human parechovirus infections, Lyon, France, 2008–2010: evidence for severe cases. J Clin Virol 2012;54:337–41. 13. Han TH, Kim CH, Chung JY, Park SH, Hwang ES. Klassevirus infection in children with acute gastroenteritis, South Korea. Emerg Infect Dis 2010;16:1623–5. 14. Holtz LR, Fink Einer SR, Zhao G, Kirkwood CD, Girones R, Pipas JM, et al. Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread. Virol J 2009;6:86. 15. Li L, Victoria J, Kappor A, Blinkova O, Wang C, Babrzadeh F, et al. A novel picornavirus associated with gastroenteritis. J Virol 2009;83:12002–6. 16. Bonadio WA, Stanco L, Bruce R, Barry D, Smith D. Reference values of normal cerebrospinal fluid in infants ages 0–8 weeks. Pediatr Infect Dis J 1992;11:589–91. 17. Lee JI, Chung JY, Han TH, Song MO, Hwang ES. Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis 2007:196–994. 18. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol 2006;44:2698–704. 19. Williams CH, Panayiotou M, Girling GD, Peard CI, Oikarinen S, Hyouty H, et al. Evolution and conservation in human parechovirus genomes. J Gen Virol 2009;90:1702–12. 20. 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 2006;42:204–10. 21. Mizuta K, Kuroda M, Kurimura M, Yahata Y, Sekizuka T, Aoki Y, et al. Epidemic myalgia in adults associated with human parechovirus type 3 infection, Yamagata, Japan, 2008. Emerg Infect Dis 2012;18:1787–93. 22. Westerhuis BM, Koen G, Wildenbeest JG, Pajkrt D, de Jong MD, Benschop K, et al. Specific cell tropism and neutralization of human parechovirus types 1 and 3: implications for pathogenesis and therapy development. J Gen Virol 2012;93:2363–70.
T.-H. Han et al. / Journal of Clinical Virology 58 (2013) 194–199 23. Boonyakiat Y, Hughes PJ, Ghazi F, Stanway G. Arginine–glycine–aspartic acid motif is critical for human parechovirus 1 entry. J Virol 2001;75: 10000–4. 24. Harvala H, McLeish N, Kondracka J, McIntyre CL, Leitch E, Templeton K, et al. Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in Edinburgh: HPeV type 3 identified as the common Picornavirus type. J Med Virol 2011;83: 889–96. 25. Walters B, Penaranda S, Nix WA, Oberste MS, Todd KM, Katz BZ, et al. Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area. J Clin Virol 2011;52:187–91. 26. de Crom SCM, Obihara CC, van Loon AM, Argilagos-Alvarez AA, Peters MF, van Fruth AM, et al. Detection of enterovirus RNA in cerebrospinal fluid: comparison of two molecular assays. J Virol Methods 2012;179:104–7. 27. Seiden JA, Zorc JJ, Hodinka RL, Shah SS. Lack of cerebrospinal fluid pleocytosis in young infants with enterovirus infections of the central nervous system. Pediatr Emerg Care 2010;26:77–81. 28. Yun KW, Cho EH, Chen DS, Lee J, Choi CW, Hwang H, et al. Enteroviral meningitis without pleocytosis in children. Arch Dis Child 2013;97:874–8. 29. Biovin. Human parechovirus types 1, 2, and 3 infections in Canada. Emerg Infect Dis 2006;12:969–75.
199
30. Sedmak G, Nix WA, Jentzen J, Haupt TE, Davis JP, Bhattacharyya S, et al. Infant deaths associated with human parechovirus infection in Wisconsin. Clin Infect Dis 2010;50:357–61. 31. Shoji K, Komuro H, Miyata I, Myairi I, Saitoh A. Dermatologic manifestations of human parechovirus type 3 infection in neonates and infants. Pediatr Infect Dis J 2012 [Epub ahead]. 32. van der Sanden S, de Bruin E, Vennema H, Swanink C, Koopmans M, van der Avoort H. Prevalence of human parechovirus in the Netherlands in 2000–2007. J Clin Microbiol 2008;46:2884–9. 33. Lee KY, Burgner D, Lee HS, Hong JH, Lee MH, Kang JH, et al. The changing epidemiology of pediatric aseptic meningitis in Daejeon, Korea from 1987 to 2003. BMC Infect Dis 2005;5:97. 34. Cheon DS, Lee J, Lee KB, Lee S, Park K, Ahn J, et al. Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002. J Med Virol 2004;73:439–42. 35. Jeong EJ, Lee JH, Kim MS, Bae GR, Jung C, Lee K, et al. Molecular characterization of enteroviruses detected in Gyeong-Ju and Po-Hang provinces of Korea in 2003. Arch Virol 2010:1707–12. 36. Baek K, Park K, Jung E, Chung E, Park J, Choi H, et al. Molecular and epidemiological characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006. J Microbiol Biotechnol 2009;19:1055–64.