Isolation and identification of a novel human parechovirus

Miyabi Ito1, Teruo Yamashita1, Hideaki Tsuzuki1, Naokazu Takeda2 and Kenji Sakae1

1 Department of Microbiology, Aichi Prefectural Institute of Public Health, Nagare 7-6, Tsuji-machi, Kita-ku, Nagoya, Aichi 462-8576, Japan
2 Department of Virology II, National Institute of Infectious Disease, Tokyo, Japan

Correspondence
Miyabi Ito
itorymi{at}crest.ocn.ne.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A cytopathic agent (A308/99) was isolated using Vero cells from a stool specimen of a 1-year-old patient with transient paralysis. The agent was approximately 28 nm in diameter with a distinct ultrastructure resembling the virus particle of an enterovirus. It could not be neutralized by antisera against human picornaviruses such as human enterovirus, Aichi virus or human parechovirus. The virion contained three capsid proteins with molecular masses of 38, 30·3 and 30 kDa. Determination of the complete nucleotide sequence of A308/99 revealed that the nucleotide and deduced amino acid sequences were closely related to those of human parechoviruses. When 11 regions encoding the structural and non-structural proteins were compared, A308/99 had between 75 and 97 % and 73 and 97 % nucleotide identity with human parechovirus type 1 (HPeV-1) and type 2 (HPeV-2), respectively. The most distinctive divergence was seen in VP1, which had 74·5 % and 73·1 % nucleotide identity with HPeV-1 and HPeV-2, respectively. Viruses related to A308/99 were also isolated from three patients with gastroenteritis, exanthema or respiratory illnesses. A308/99 and these other three isolates had no arginine–glycine–aspartic acid (RGD) motif, which is located near the C terminus of VP1 in HPeV-1 and HPeV-2. A seroepidemiological study revealed that the prevalence of A308/99 antibodies was low (15 %) among infants but became higher with age, reaching more than 80 % by 30 years of age. These observations indicate that A308/99 is genetically close to, but serologically and genetically distinct from, HPeV-1 and HPeV-2 and accordingly can be classified as third serotype of human parechovirus.

The complete nucleotide sequence of the A308/99 strain has been deposited in DDBJ, EMBL and GenBank under accession no. AB084913. The GenBank accession nos of A317/99, A354/99, A628/99, A1086/99, A942/99 and A10987/00 reported in this paper are AB112482AB112487.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Picornaviruses form a diverse group of small, non-enveloped, single-stranded RNA viruses, which include important pathogens of humans and animals (Stanway, 1990). They have been classified into nine genera based on acid lability, serology and their molecular characteristics (King et al., 2000). Parechoviruses, which have recently been assigned to a sixth picornavirus genus, are considered to be composed of human parechovirus type 1 (HPeV-1) and type 2 (HPeV-2). These two viruses were formerly classified as echovirus 22 and echovirus 23, respectively (Stanway & Hyypiä, 1999). HPeV-1 is often isolated from children with diarrhoea and gastroenteritis. Previous studies on the epidemiology of HPeV-1 infection have demonstrated that gastroenteritis, respiratory infections, encephalitis and flaccid paralysis are the most common symptoms observed in this virus infection (Ehrnst & Eriksson, 1993, 1996; Figueroa et al., 1989; Koskiniemi et al., 1989; Birenbaum et al., 1997).

In this study, a novel cytopathic agent isolated from a patient with transient paralysis was examined. The virus, designated A308/99, did not react with representative antisera to human picornaviruses. A308/99 was characterized genetically by RT-PCR coupled with amplicon sequencing and comparison with a database of human picornavirus nucleotide sequences. Molecular cloning followed by complete nucleotide sequence analysis suggested that A308/99 could be classified as a new member of the genus Parechovirus. A serological study of the prevalence of neutralizing antibodies against this new virus suggested that A308/99 infection occurs primarily during early childhood and that a majority of children are infected with this virus before entering elementary school (<6 years old) in Aichi Prefecture, Japan.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patient's samples and virus isolation.
In August 1999, a 1-year-old girl with transient paralysis, a high fever (38·3 °C) and diarrhoea was hospitalized in a regional general hospital in Aichi Prefecture, Japan. As a virus infection was suspected, the faeces, a throat swab, cerebrospinal fluid and acute-phase serum were collected within a few days of the onset of illness. Convalescent-phase serum was collected 2 weeks after the acute-phase samples were obtained.

A stool specimen was prepared as a 10 % homogenate with veal-infusion broth containing 0·5 % BSA (fraction V; Sigma), 200 units penicillin ml-1 (Banyu Pharmaceutical) and 200 µg streptomycin ml-1 (Meiji Seika). The stool and throat swab specimens were centrifuged at 10 000 g for 20 min and their supernatants were used for virus isolation with Vero, HeLa and RD cells. The isolate was designated A308/99.

Other isolates.
A317/99, A354/99 and A628/99 were also isolated from patients with gastroenteritis, exanthema and respiratory illness, respectively. These isolates were neutralized with anti-A308/99 serum but not with anti-HPeV-1 or HPeV-2 sera. A1086/99, A942/99 and A10987/00 were also isolated from patients with gastroenteritis and respiratory illnesses. These were identified as being similar to HPeV-1 by neutralization tests.

Physico-chemical properties.
The stability of A308/99 was examined by standard methods (Hamparian, 1979). A308/99 was incubated in 10 % chloroform for 10 min at room temperature to evaluate its stability in organic solvents, and in at pH 3·5 for 3 h at room temperature to evaluate its stability in acid. Determination of the nature of the nucleic acid of the virus was performed by incubating A308/99 in the presence of 20 mM 5-iodo-2'-deoxyuridine (IUDR) (Ketler et al., 1962), a DNA virus-specific inhibitor, with poliovirus type 1 and herpes simplex virus type 1 as the controls. Seroconversion of the patient against A308/99 was determined by a neutralizing test utilizing acute- and convalescent-phase sera.

Purification of virus and preparation of immune sera.
A308/99 was plaque-purified twice in Vero cells and a stock virus was prepared. Prototype strains of HPeV-1 (Harris strain) and HPeV-2 (Williamson strain) were obtained from the National Institute of Infectious Diseases, Japan, and grown in HeLa and Vero cells, respectively. These viruses were stored at -70 °C until the neutralizing test was done. A308/99 and HPeV-1 virus particles were purified by CsCl followed by sucrose density gradient centrifugation and used for an electron microscopic study and SDS-PAGE. Purified A308/99 virion was also used for molecular cloning. Immune sera were obtained from guinea pigs that had been subcutaneously injected with purified A308/99. Intersecting serum pools and type-specific antisera to human enteroviruses HPeV-1 and HPeV-2 prepared using horses or guinea pigs were supplied by the National Institute of Infectious Diseases, Japan.

For the serological study, 207 blood specimens were obtained from 115 children (aged 7 months to 19 years) who were hospitalized at other regional general hospitals. A308/99-like virus was not isolated from the specimens of these 115 children. Ninety-two blood samples from healthy blood donors (aged over 20 years) at the Aichi Red Cross Blood Center were collected after obtaining their informed consent. Neutralizing antibody titres were measured using 100 TCID50 A308/99 per 25 µl as a challenge virus.

Molecular cloning.
Virus RNA was extracted from the purified virion with TRIZOL LS reagent (Invitrogen Life Technologies) followed by isopropanol precipitation, as previously reported (Yamashita et al., 2000). One µg of the RNA was converted into cDNA with a mixture of random pd(N)9 (Takara) and oligo(dT)15 primers (Promega) using Moloney murine leukaemia virus reverse transcriptase (Invitrogen Life Technologies). Double-stranded cDNA preparation and cloning into the pBR322 vector (Invitrogen Life Technologies) by a dC/dG tailing method was done as described previously (Supanaranond et al., 1992). Clones containing inserts were identified by agarose gel electrophoresis of the plasmid DNA digested with PstI. Inserts were subcloned into the PstI site of the pUC19 vector. The nucleotide sequences of five clones that cover 45·5 % of the genome were determined. A308/99-specific oligonucleotides were synthesized on the basis of the nucleotide sequences near the ends of each clone and used as the primers for RT-PCR. Oligo(dT)33 was used to obtain clones encoding the extreme 3' end of the genome. Six clones were obtained with the pGEM-T vector (Promega) and sequenced to close the gaps between the pUC19 cDNA clones. Clones encoding the extreme 5' end of the genome were obtained using a 5'RACE kit (Invitrogen Life Technologies).

RT-PCR for human parechoviruses.
Based on the nucleotide sequences of A308/99, HPeV-1 (Harris strain) and HPeV-2 (Williamson strain), the following primers were constructed: a forward primer E23P1 (5'-CCGYAGGTAACAAGWGACAT-3') and a reverse primer HPV-N1 (5'-TAGGGGATACATARGTCRGCYT-3') were prepared to amplify 810 bp between the 5'untranslated region (5'UTR) and VP0; and, a forward primer HPV-N1-S (5'-AGGCTGACCTATGCATCCCCTA-3') and a reverse primer RGD-R (5'-CGACATCAGACCAGCAGT-3') were prepared to amplify 2030 bp between VP0 and 2A of the human parechoviruses. Amplification was performed in a thermal cycler (Takara). After an initial denaturation at 94 °C for 5 min, 35 cycles of amplification were carried out consisting of denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C and extension for 1 min at 72 °C, followed by a final incubation for 7 min at 72 °C. The amplified fragments were introduced into a pGEM-T vector and the DNA sequence was determined using a SequiTherm LongRead Cycle Sequencing Kit-LC (Epicentre Technologies) and a model 4000 automated DNA sequencer (Li-Cor).

Three internal primers for sequencing, 308P1 (5'-ATGGATACTTGATTGGGCAGC-3'), 308N1 (5'-ACGTCACATACCAAGCTCTAC-3') and 308N1-P (5'-GTAGAGCTTGGTATGTGAGACGT-3'), were designed on the basis of a partial A308/99 strain gene sequence determined in this study. The other three internal primers for sequencing, E22P1 (5'-TYAAGTGGTCAKCWACAACWGC-3'), E22N1 (5'-CCATGGCCTTGTTTTGGAAA-3') and E22N1-P (5'-TTTCCAAAACAAGGYCATGG-3'), were also designed on the basis of HPeV-1 gene sequences in the database. The nucleotide sequence was determined at least twice in both directions. The alignments were performed with the PILEUP program in the Genetics Computer Group (GCG) package. The phylogenetic trees were constructed with neighbour-joining (NJ) methods using Clustal X. The following nucleotide sequences were obtained from GenBank: poliovirus type 1 (Polio1), J02281; coxsackievirus B1 (CV-B1), M16560; human rhinovirus 1 (HRV-1B), D00239; human rhinovirus 2 (HRV-2), X02316; human rhinovirus 14 (HRV-14), K02121; encephalomyocarditis virus (EMCV), M81861; Theiler murine encephalomyelitis virus (TMEV), M20301; foot-and-mouth disease virus type O (FMDV-O), X00871; hepatitis A virus (HAV), M14707; HPeV-1 Harris strain, L02971; HPeV-2 Williamson strain, AJ005695; HPeV-2 Connecticut/86-6760 strain, AF055846; Ljungan Virus (LV), AF327920; equine rhinitis A virus (ERAV), X96870; equine rhinitis B virus 1 (ERBV-1), X96871; Aichi virus (AiV), AB010145; and porcine teschovirus 1 (PTV-1), AJ011380.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus isolation
A cytopathic agent was isolated from a stool specimen using Vero cells and serially passaged with HeLa cells. It was successfully titrated using Vero cells grown in microplates and found to have a titre of 103–104 TCID50 per 25 µl. No cytopathic agent was isolated from a throat swab and cerebrospinal fluid specimens. The isolated virus was further plaque purified twice and a virus stock was prepared. This virus stock showed an extensive cytopathic effect (CPE) 3–4 days after inoculation of Vero cells. The virus titre of the stock was 103–104 TCID50 per 25 µl and the virus was designated A308/99. This virus was not cytopathic in RD cells and was not pathogenic to newborn mice when inoculated intracerebrally. A308/99 could not be neutralized by antisera against the prototype strains of human parechoviruses, human enteroviruses (20 units) and the Aichi virus. In addition, antiserum to A308/99 could not neutralize any prototype strains of human parechoviruses, human enteroviruses or the Aichi virus. Titres of the cross neutralization between A308/99, HPeV-1, HPeV-2 and Polio 1 are given in Table 1.


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Table 1. Cross-neutralization test using virus-infected HeLa or Vero cells

 
Biological and physico-chemical characteristics
Addition of 20 mM IUDR to the culture medium did not affect the CPE, indicating that the isolate A308/99 has an RNA genome. A308/99 was stable when incubated in the presence of 10 % chloroform and was also stable for 3 h when incubated under acidic conditions (pH 3·5). A308/99 was resistant to treatment at 50 °C for 30 min, but labile following treatment at 60 °C for 30 min.

The purified virion was found to be approximately 28 nm in diameter following negative staining with 2 % phosphotungstic acid (pH 7·0) and had morphology similar to that of human enteroviruses. When the purified A308/99 virion was analysed by SDS-PAGE, it revealed three capsid proteins with molecular masses of 38, 30·3 and 30 kDa. These properties are closely related to those of human parechoviruses (Fig. 1) and suggested that A308/99 could be a member of this group.



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Fig. 1. SDS-PAGE analysis of HPeV-1 and A308/99 polypeptides in purified virions. The migration of HPeV-1 capsid polypeptides (VP0, 38 kDa; VP1, 30·5 kDa; VP3, 30 kDa) and A308/99 capsid polypeptides (VP0, 38 kDa; VP1, 30·3 kDa; VP3, 30 kDa) are indicated by arrows on the right. The numbers on the left indicate molecular sizes (kDa) of standard proteins.

 
Genetic analysis of A308/99
After obtaining 11 cDNA clones spanning the entire genome by molecular cloning and RT-PCR as described in the Methods, complete nucleotide sequences were determined. It was found that the RNA genome of A308/99 consisted of 7321 nucleotides, excluding a poly(A) tract. Following a 5'UTR of 699 nucleotides, a large open reading frame encoding a potential polyprotein precursor of 2177 amino acids was found, which was followed by 91 nucleotides of the 3'UTR and a poly(A) tail. It was revealed that the genome organization of A308/99 is analogous to that of picornaviruses and the overall nucleotide and predicted amino acid similarities with HPeV-1 were 77·6 % and 86·8 % and with HPeV-2 were 77·2 % and 84·7 %, respectively (Table 2). The amino acid similarities of the capsid proteins VP0, VP3 and VP1 between A308/99 and HPeV-1 (Harris strain) and HPeV-2 (Williamson and Connecticut strain) were between 68·8 and 78·2 %. These values are of a similar order of magnitude to those obtained when poliovirus type 1 is compared with poliovirus type 2 or type 3. The comparison of the deduced amino acid sequences of the P1 regions between A308/99 and HPeV-1 and HPeV-2 are shown in Fig. 2. The amino acid sequences in the {beta}-barrel structure of HPeV-1 (Stanway et al., 1994) were well conserved in A308/99. The variable regions of A308/99 were found to be located in similar locations at amino acid positions 14–39 of VP0, the N terminus of VP3 and the C terminus of VP1, as also seen in HPeV-1 and HPeV-2 (Ghazi et al., 1998; Stanway et al., 1994). It should be noted that the arginine–glycine–aspartic acid (RGD) motif, which is located close to the C terminus of VP1 of HPeV-1 and HPeV-2, was not present in the VP1 sequence of A308/99. Neither could an RGD motif be observed in RT-PCR products from the clinical sample of the patient positive for A308/99.


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Table 2. Comparisons of RNA and amino acids between A308/99, HPeV-1 and HPeV-2

HPeV-1, Harris strain; HPeV-2W, Williamson strain; HPeV-2C, Connecticut strain (86-6760).

 


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Fig. 2. Alignment of the predicted amino acid sequence of A308/99 capsid proteins, VP0, VP3 and VP1, and the corresponding polypeptides of HPeVs with the known three-dimensional virus particle structures. Regions of secondary structure are underlined, and amino acids in the {beta}-barrels common to all four sequences are shown in bold. The RGD motif of VP1 C-terminal is highlighted in grey.

 
Phylogenetic trees based on P1 and 2C3CD proteins are depicted in Fig. 3(a, b). This confirmed that the A308/99 strain was clearly more closely related to the HPeVs than to any other members of the picornavirus genera. These results indicated that A308/99 can be classified into the same genus as HPeV-1 and HPeV-2. This conclusion was further supported by the fact that the amino acid sequences of the non-structural proteins of A308/99 were very similar to those of the HPeVs.



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Fig. 3. Phylogenetic trees showing the relationship between A308/99 and representatives of other picornavirus genera based on amino acid differences in capsid proteins (P1 region) (a) and the 2C3CD region (b). The trees were constructed with NJ methods using Clustal X. Branch lengths are proportional to genetic distance, indicated by the scale bar. The following nucleotide sequences were obtained from GenBank: Polio1, J02281; CV-B1, M16560; HRV-1B, D00239; HRV-2, X02316; HRV-14, K02121; EMCV, M81861; TMEV, M20301; FMDV-O, X00871; HAV, M14707; HPeV-1 Harris strain, L02971; HPeV-2 Williamson strain, AJ005695; HPeV-2 Connecticut/86-6760 strain, AF055846; LV, AF327920; ERAV, X96870; ERBV-1, X96871; AiV, AB010145; PTV-1, AJ011380.

 
Among the non-structural proteins, amino acid sequences of the 2B, 3C and 3D regions were well aligned with the corresponding regions of HPeVs (Table 2). Some of the non-structural proteins of parechoviruses are known to contain common motifs such as GX2GXGK(S/T) and DDLXQ in 2C proteins, GXCG in 3Cpro proteins and KDELR, PSG, YGDD and FLKR in 3Dpol proteins (Ghazi et al., 1998). Analysis of the complete nucleotide sequences revealed that A308/99 contained all of these motifs. Possible cleavage sites of the deduced amino acid sequence of A308/99 were almost identical to other HPeVs. The exceptions were seen at the cleavage site of VP0 and VP3 (N/G) and at the VP1/2A cleavage sites (E/S) of A308/99. The cleavages were N/A, T/A and N/S (VP0/VP3) and Q/S (VP1/2A) in the other HPeVs.

The 5'UTR of A308/99 was revealed as a potential internal ribosome entry sequence, probably involved in the cap-independent translation of the viral RNA, and lacking the poly(C) tract that is seen in HPeV-1 (Ghazi et al., 1998; Hyypiä et al., 1992; Oberste et al., 1998).

The predicted secondary structure of the 5'UTR of A308/99 created using the FOLD program by Zuker and Turner (http://mfold2.wustl.edu) was, however, different from the stem–loop domain E of HPeV-1 and HPeV-2 as reported by Ghazi et al. (1998) using the same FOLD program (data not shown).

Genetic analysis for P1 regions of other isolates identified as A308/99 and HPeV-1
The A308/99-related viruses (A317/99, A354/99 and A628/99) and HPeV-1 isolates (A1086/99, A942/99 and A10987/00) were examined by RT-PCR using two primer sets. The primer sets used were able to amplify products from all six isolates at the regions between the C terminus of the 5'UTR and the N terminus of 2A, and the nucleotide sequences of these P1 regions were determined. A dendrogram based on the P1 proteins of HPeV-1s, HPeV-2C, HPeV-2W, A308/99 and these six isolates is shown in Fig. 4. Four genetic groups could be defined among the HPeVs, which consisted of HPeV-1 isolates, HPeV-2C, HPeV-2W and the A308/99 isolates. A high degree of P1 amino acid similarity was seen, including the lack of the RGD motif, between A308/99 and the related viruses (A317/99, A354/99 and A628/99).



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Fig. 4. Phylogenetic tree showing the relationship between A308/99, the other isolates, HPeV-1 and HPeV-2 based on amino acid differences in capsid proteins (P1 region).

 
Antibody to A308/99 strain
We determined the neutralizing antibody titre of sera from the original patient. The titre was 1 : 32 in the acute phase, which increased to 1 : 512 in the convalescent phase, clearly indicating that the girl was infected with A308/99.

A serological study was performed using sera collected from 207 individuals aged from 7 months to over 40 years. A titre of 1 : 8 was considered to be positive for antibody against A308/99. Out of the 207 samples, 139 (67 %) were found to be antibody positive against A308/99. The lowest seroconversion rate (15 %) was found in the youngest age group (from 7 months to 1 year old, n=20) in our study. The rate of seropositivity increased to 45 % in the 2–3-year-old group and to 85 % in the 4–6-year-old group. In the other age groups, the positive rate varied from 56·5 to 91·3 % (Table 3).


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Table 3. Prevalence of A308/99 antibody in different age groups

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although A308/99 caused apparent CPE on Vero and HeLa cells, this virus could not be neutralized with any antisera against human picornaviruses. A dendrogram using the complete nucleotide sequence of A308/99 and other picornaviruses revealed that this virus was similar to the human parechoviruses HPeV-1 and HPeV-2. Therefore, A308/99 was considered to be a possible new serotype of HPeV. A previous report has indeed described the characterization of HPeV-1 variants using a neutralization test (Schnurr et al., 1996) and they may be related to such a virus as A308/99. However, there is no genetic information regarding these isolates. The phylogenetic tree using the P1 region of the prototype and some of our isolates of HPeV showed that there were four genetically distinct groups within the HPeVs, consisting of HPeV-1s, HPeV-2C, HPeV-2W and the A308/99 group. Classification of enteroviruses according to the amino acid sequence of the VP1 protein, which serves as the antigenic determinant, is known to closely relate to that of the serotype (Muir et al., 1998). If, as we surmise, there are two kinds of HPeV-2 genotypes, the A308/99 group can be thought of as third serotype of human parechoviruses.

Amino acid sequences of the predicted antigenic sites and cleavage sites were conserved in A308/99, having the same sequence and cleavage sites as HPeV-1 and HPeV-2. It is noteworthy, however, that A308/99 does not have an RGD motif, which is located near the C terminus of VP1 in both HPeV-1 and HPeV-2. This RGD motif is known to participate in cell-to-cell and cell-to-matrix interactions in general, whilst the virus may use the RGD motif for recognizing and attaching to host cells (Stanway et al., 1994; Hynes, 1992; Ruoslahti & Pierschbacher, 1987). This RGD motif is also found in the main antigenic determinant of FMDV VP1 and is known to function as part of the virus receptor-binding site for binding to target cells (Baranowski et al., 2000). Notably, an antiserum against this motif was shown to have neutralizing activity against HPeV-1 in cell culture (Joki-Korpela et al., 2000). The importance of an RGD motif in HPeV-1 has been shown by observations following mutagenesis. In reverse genetics experiments in which RGD was changed to RGE (arginine–glycine–glutamic acid), only revertant RGD virus was recovered, and deletion of GD was lethal and showed no reversion (Boonyakiat et al., 2001). Biochemical and genetic approaches performed in FMDV, CV-A9, echovirus 9 (EV-9) and HPeV-1 have established the role of the RGD motif in internalization of these viruses (Hughes et al., 1995; Zimmermann et al., 1997; Fox et al., 1989). However, isolates of CV-A9 and EV-9 are viable without the RGD motif and, moreover, are also able to grow efficiently in some cell types (Hughes et al., 1995; Zimmermann et al., 1995, 1997). These observations suggest that there are at least two entry pathways for these viruses, one RGD-dependent and one RGD-independent. The absence of this motif in A308/99 indicates, however, that A308/99 infects human cells using a receptor other than that used by HPeV-1 and HPeV-2.

A seroepidemiological study in Finland showed a low prevalence (20 %, 2/10) of HPeV-1 antibody in out-patient infants aged less than 1 year old, but this became higher with age, reaching 89 % (8/9) in children aged between 1 and 2 years old. A prevalence rate of 97 % (29/30) was reported in blood donation volunteer adults aged over 18 years old (Joki-Korpela & Hyypiä, 1998).

It is noteworthy that the results of our seroepidemiological study regarding the presence of A308/99 antibody was basically the same as that for HPeV-1 performed in Finland. Our study revealed that A308/99 infection occurs during early infancy life (<12 months old) and that the majority of children (85 %) have been infected with this virus before entering elementary school (<6 years old) in the Aichi Prefecture, Japan. The high proportion of seropositive children may indicate that there had recently been an outbreak of infection with this virus. Most A308/99 infections, like many other enteroviruses, appear to be asymptomatic; this could be one reason why isolation and identification of this virus has escaped detection for such a long time.

The major clinical symptoms of HPeV-1 and HPeV-2 infections have been reported to be gastrointestinal and respiratory, with fewer central nervous system (CNS) symptoms (Birenbaum et al., 1997; Ehrnst & Eriksson, 1993; Nakao & Miura, 1970; Sato et al., 1972; Hynes, 1992). HPeV-1 has also been associated with an outbreak of acute flaccid paralysis in Jamaica in 1986 (Figueroa et al., 1989) and with encephalitis in a 5-month-old boy in Finland (Koskiniemi et al., 1989). The pathogenicity of A308/99 is, however, not clear at this moment. We have isolated A308/99 from a patient with transient paralysis, high fever and diarrhoea, and infection with this virus was confirmed by seroconversion of the patient after recovery from all pre-existing disease signs and symptoms. Accordingly, A308/99 may be considered to have a pathogenicity similar to other HPeVs such as gastroenteritis and disorders in the CNS. It is evident that there is a need for more epidemiological information about the known parechoviruses as well as about A308/99-like viruses to establish the relationship between infection with these viruses and subsequent development of specific disease(s). We believe that information obtained from the present study will be useful for exploring the biological environment of HPeV.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 25 June 2003; accepted 20 October 2003.



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