Isolation and characterization of novel human parechovirus from clinical samples.

Using Vero cells, we isolated a virus (NII561-2000) from a cerebrospinal fluid specimen of a 1-year-old girl with Reye syndrome. The determined amino acid sequence of the virus indicated that the isolate was a human parechovirus (HPeV), a member of Picornaviridae. Neutralization test showed that the NII561-2000 virus had distinct antigenicity to HPeV-1, HPeV-2, and HPeV-3, and that the sequence was distinct from these types as well as from HPeV-4 and HPeV-5. Thus, we propose the virus (NII561-2000) as the prototype of HPeV-6. We isolated 10 N II561-2000-related viruses, 14 HPeV-1, 16 HPeV-3, and 1 HPeV-4 of 41 HPeVs from various clinical samples collected in Niigata, Japan. Clinical symptoms of the persons infected with the NII561-2000-related viruses were infectious gastroenteritis, rash, upper respiratory tract infection, and paralysis, in addition to Reye syndrome in the 1-year-old girl.

**********

Human parechovirus (HPeV) is a small, nonenveloped RNA virus with a single-stranded genuine of positive polarity, [approximately equal to] 7.3 kb in length; it is a member of the Picornaviridae family (1-3). On the basis of serologic and genetic studies, HPeV has been found to have 5 types, HPeV-1, HPeV-2, HPeV-3, HPeV-4, and HPeV-5, with 76.0%-80.9% similarity at the nucleotide level and 84.7%-90.0% similarity at the amino acid level (1,4-7). HPeV infections are commonly observed in general populations. For example, [approximately equal to] 20% of healthy children in Finland have antibodies against HPeV- 1, and the percentage is as high as 97% in adults (8). In addition, these viruses are frequently isolated from patients with various human diseases, such as gastroenteritis, encephalitis, flaccid paralysis, and respiratory infections, and they are thought to be associated with these diseases (2,3,5,8,9).

We report a 1-year-old girl who died with Reye syndrome, which is characterized as an acute, noninflammatory encephalopathy with hepatic dysfunction and fatty infiltration of the viscera; the syndrome is frequently associated with an antecedent viral infection, such as influenza or varicella (10-12). Inoculation of a cerebrospinal fluid (CSF) specimen from the patient into Vero cells identified a virus (NII561-2000) with similar properties to HPeVs. The nucleotide sequence of this virus showed it was closely related to HPeVs, especially HPeV-1, with 79.5% nucleotide and 90.7% amino acid (aa) similarities. Moreover, mutual neutralization assay showed that NII561-2000 has distinct antigenicity to HPeV-1, HPeV-2, and HPeV-3. In addition, the NII561-2000 virus was genetically distinct from HPeV-4 and HPeV-5. Thus, we propose that NII561-2000 is the prototype of HPeV-6.

Materials and Methods

Cell Lines and Culture Conditions

Eight adherent cell lines, MDCK, Caco-2, RD-18S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1, were used in our study. In brief, MDCK originated from a kidney of a normal adult cocker spaniel, Caco-2 was from a primary colorectal adenocarcinoma, RD-18S was from a rhabdomyosarcoma, Veto was from the kidney of a normal adult African green monkey, HeLa was from a cervical adenocarcinoma, HEp-2 was from an epidermoid carcinoma of the larynx, LLC-MK2 was from a kidney of a normal adult rhesus monkey, and BSC-1 was from a kidney of a normal adult African green monkey. These cell lines were cultured in Eagle minimum essential medium with 6 mmol/L L-glutamine, 1.1 g/L sodium bicarbonate, antimicrobial agents (0.2 g/L gentamicin, 0.25 g/L amphotericin B), and 8% fetal bovine serum at 37[degrees]C under 5% carbon dioxide.

Virus Isolation and Purification

MDCK, Caco-2, RD-18S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1 cells were cultured on 24-well plates for 3 days. Then, these cells were inoculated with a CSF specimen (100 [micro] L per well) from a patient with a diagnosis of Reye syndrome and cultured at 33[degrees]C for 2 weeks. To check for cytopathic effect (CPE), we examined the cells under a light microscope. To purify the virus particles, we mixed 900 mL culture fluid of Vero cells inoculated with the NII561-2000 virus with 20 g NaCl and 76 g polyethylene glycol #6,000 at 4[degrees]C overnight. Next, the sample was centrifuged at 5,000 rpm for 30 min, and the pellets were suspended in 4.5 mL of phosphate buffer (pH 7.2). After treatment with 4.5 mL of chloroform at 4[degrees]C for 5 min, the sample was centrifuged at 2,500 rpm for 20 min. The supernatant was centrifuged at 120,000 x g for 24 h in cesium chloride (CsC1) solution at an initial density of 1.34 g/mL, and the virion-containing fraction was collected and used for viral RNA isolation.

Molecular Cloning

Double-stranded eDNA was synthesized from 5 [micro]g of the viral RNA from the viruses purified by the CsC1 density gradient ultracentrifugation method described above. The nucleotide sequences of the cDNAs from randomly picked-up bacterial colonies transfected with the eDNA-containing plasmids were determined by using the Big Dye sequencing kit (Applied Biosystems, Foster City, CA, USA). Two cDNAs isolated contained part of the NII561-2000 gene. To isolate the 3' eDNA fragment of the isolated NII561-2000 cDNAs, the 3' rapid amplification of eDNA ends (RACE) was performed by using the RNA PCR Kit (AMV) according to the instructions provided by the supplier (TaKaRa, Kyoto, Japan). The primers used for 3' RACE were 35F-out (forward primer for the first PCR; 5'-GAT GCG GAA AAC TGC TGG ACA C-Y), 35F-in (forward primer for the second PCR; 5'-TGC CAA ATT TTT CTG CCC TAC TG-3'), and M13M4 (reverse primer for the first and second PCR; 5'-GTT TTC CCA GTC ACG AC-3'). Herculase Hotstart DNA Polymerase (Stratagene, La Jolla, CA, USA) was used. To isolate the 5' eDNA fragment of the isolated NII561-2000, the eDNA fragment containing part of the 5' untranslated region (UTR) and capsid precursor protein VP0 was amplified by PCR from the cDNA prepared from the NII561-2000 virus with degenerate primers corresponding to this region. The degenerate primers used were E23P1 (forward primer; 5'-CCG YAG GTA ACA AGW GAC AT-Y) (5) and 35R (reverse primer; 5'-TCT CAG CAC TAA TGA CCC TC-Y). To further extend the sequence information of 5' UTR of the NII561-2000 virus, the 5' RACE was performed with 5' RACE System for Rapid Amplification of eDNA Ends (Version 2.0), by using the instructions provided by the supplier (Invitrogen, San Diego, CA, USA). The primers used for 5' RACE were AAP (forward primer for the first PCR; 5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3') HPeV-GSP2 (reverse primer for the first PCR; 5'-AGA TGC ATC ATC TGC GAC TC-3'), UAP (forward primer for the second PCR; 5'-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3'), and HPeV-GSP (reverse primer for the second PCR; 5'GCC ATG TCT GCA ATG CTC TT-3'). Taq polymerase (Biotech International, Bentley, Western Australia, Australia) was used as a polymerase. Because 5' RACE did not reach to the 5' end of the NII561-2000 virus eDNA, the 5' end eDNA fragment of the NII561-2000 virus was amplified from the NI1561-2000 viral RNA by reverse transcription-PCR (RT-PCR). The primers used for RT-PCR were HPeV-head (forward primer; 5'-TTT GAA AGG GGT CTC CT-3') and HPeV-mid (reverse primer; 5'-CAT AAG TTC CAC AAG CGT GG-3') HPeV-head primer was designed as a conserved 5' end sequence of the HPeVs 5' UTR.

Neutralization Test

Twenty-five microliters (100 median tissue culture doses; 50% tissue infective dose [[TCID.sub.50]]) of the indicated viruses and 25 [micro]L of the serially diluted antisera (an initial 10-fold dilution and then 2-fold serial dilutions) were mixed in a 96-well plate and incubated at 37[degrees]C for 2 h. Then suspended Veto cells (100 [micro]L/well) were added into these wells. Three to 10 days later the CPE of Vero cells was checked by light microscopy. To prepare the antiserum against the NII561-2000 virus, the virus particles grown in Vero cells were purified by CsC1 centrifugation as described above. By using this purified virus, we prepared antiserum by Nippon Biotest Laboratory (Tokyo, Japan). In brief, the purified viruses were subcutaneously injected into rabbits 3 x every 2 weeks. After we checked the antiviral titer by the Ouchterlony method, blood was collected from the vaccinated rabbits.

Comparisons of VPO Amino Acid Sequences of Clinical Isolates

To determine the VP0 amino acid sequences of viruses isolated from clinical samples with cultured cell lines, we extracted the viral RNA from culture supernatant of cells injected with the clinical samples by using the High Pure Viral RNA Kit (Roche, Mannheim, Germany). cDNA was synthesized from 8 [micro]L of the viral RNA by using 1 U Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 20 U of recombinant RNAs in ribonuclease inhibitor (Promega, Madison, WI, USA). The 810-bp fragment containing part of 5' UTR and VP0 was amplified by PCR, using 5 [micro]L of eDNA in a 50-[micro]L reaction mixture containing 50 mmol/L KC1, 10 mmol/L Tris/HCl (pH 8.5), 2.5 mmol/L [MgC1.sub.2], 0.2 mmol/L of each deoxynucleoside triphosphates, 50 pmol of each primer, and 1 U of Taq polymerase (Biotech International). The amplification reaction consisted of 30 cycles at 95[degrees]C for 30 s, at 59[degrees]C for 30 s, and at 72[degrees]C for 1 min. The primer set used was E23P1 as a forward primer and HPV-N1 (5'-TAG GGG ATA CAT ARG TCR GCY T-3') as a reverse primer (5).

Phylogenic Tree Analysis

The bootstrap values were calculated from P 1 amino acid sequences of parechoviruses with the CLUSTAL X software program (13), and the phylogenetic tree of these P1 amino acid sequences was constructed by using the neighbor-joining method. The nucleotide sequences of the following parechoviruses were obtained from GenBank: and their accession numbers were L02971 for HPeV-1 (Harris strain), AJ005695 for HPeV-2 (Williamson strain), AB084913 for HPeV-3(A308/99), AJ889918 for HPeV-3 (Can82853-01), DQ315670 for HPeV-4 (K251176-02), AM235750 for HPeV-4 (T75-4077), AF055846 for HPeV-5 (CT86-6760), AM235749 for HPeV-5 (T92-15), and AF327920 for Ljungan virus (LV). The GenBank/EMBL/ DDBJ accession numbers of NII428-2000, NII2392-2001, NII2667-2001, NII2694-2001, NII2729-2001, and NII561-2000 are AB252577-AB252582.

Results

Virus Isolation

In January 2000, a 1-year-old girl with croup and high fever (39.6[degrees]C) was hospitalized in a regional general hospital in Niigata Prefecture, Japan. The infant died, and her condition was diagnosed as Reye syndrome after postmortem pathologic examination. To identify the pathogenic agent, we added a CSF specimen collected before death to 8 cell lines: MDCK, Caco-2, RD-18S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1 cells. Only Vero cells inoculated with the specimen exhibited a CPE. The CPE titer of the culture fluid was [10.sup.5]-[10.sup.6] [TCID.sub.50] per 25 [micro]L against Vero cells. Electron microscopic examination detected typical enteroviruslike virions in the culture fluid of the specimen (round, no envelop, [approximately equal to] 30 nm in diameter). Taken together, these results suggested that the CSF from the patient contained a virus, which we refer to here as NII561-2000.

Physical and Antigenic Properties

Neutralization tests were performed to examine whether the NII561-2000 agent is related to known viruses. The NII561-2000 virus infection of Vero cells was not neutralized by a pool of enterovirus typing antisera or 3 HPeV typing antisera [(HPeV-1, HPeV-2, and HPeV-3) (A308/99)] (Table 1). Conversely, the rabbit antiserum to the NII561-2000 virus did not neutralize the infection of prototype strains of echovirus (serotypes 1-6, 9, 11-15, 17-21, 24-27, 29, 30, and 33), enteroviruses (serotypes 68 and 69), and 3 HPeVs, while it efficiently inhibited the infection of the NII561-2000 virus. These results suggest that the NII561-2000 agent is distinct from the examined known enteroviruses and HPeVs.

We next examined the sensitivity of the virus to 5-iodo-2'-deoxyuridine (IUDR) (14). The NII561-2000 virus, treated with 10-4.5 [micro]mol/L IUDR, was injected into Vero cells. The IUDR treatment did not affect the CPE in Vero cells, indicating that the virus has the RNA genome (data not shown). We next examined the acid-stability and thermostability of the NII561-2000 virus. The virus was treated at pH 3.0 for 3 h at room temperature, but the infectivity of the treated virus to Vero cells was little affected. Incubation of the virus at 50[degrees]C for 30 min reduced the infectivity, while incubation at 50[degrees]C for 1 h in the presence of 1 mol/L Mg[Cl.sub.2] did not reduce the infectivity. These results indicate that the NII561-2000 virus has similar properties to human enteroviruses and HPeVs.

Genetic Analysis of NII561-2000

To determine the nucleotide sequence of the NII561-2000 virus, the viral RNA was extracted from the purified virus. The partial nucleotide sequences of cDNA clones derived from this viral RNA were determined. BLAST (www.ncbi.nlm.nih.gov/blast) search identified that the nucleotide sequences of 2 isolated cDNAs showed high similarity with that of HPeV-2. By using these cDNAs as a starting material, the cDNAs containing the 5' portion and 3' portion of the NII561-2000 gene were isolated by RT-PCR with degenerated primers, 5' RACE and 3' RACE. The determined nucleotide sequence of NII561-2000 was 7,347 nt in length, excluding a poly (A) tract. Following a 709-nt 5' UTR, a long open reading frame encoded a putative polyprotein precursor of 2,182 aa, which was followed by an 89-nt 3' UTR. To verify the nucleotide sequence of the determined NII561-2000 genome, RT-PCR with a set of primers and RNA sample extracted from the virus-infected Vero cells was carried out. The nucleotide sequences of the NII561-2000 genome obtained by using this RT-PCR method were perfectly matched with those of the originally determined sequences (data not shown).

Phylogenetic tree analysis with complete P1 (VP0, VP3, and VP1) amino acid sequences showed that the NII561-2000 virus was most similar to HPeV-1/HPeV-2 (Figure, panel A). The nucleotide sequence and amino acid similarities of NII561-2000 virus to HPeV-1 were 79.5% and 90.7%, those with HPeV-2 were 77.1% and 87.3%, those with HPeV-3 (A308/99) were 76.7% and 85.9%, those with HPeV-4 were 77.1% and 88.2%, and those with HPeV-5 (CT86-6760) were 77.0% and 86.9%, respectively (Table 2). Thus, the NII561-2000 virus is the most similar to HPeV-1 at the amino acid level. The VP1 capsid gene of NII561-2000 was the most divergent (33.8% at nucleotide level) from that of HPeV-5 (CT86-6760).

[FIGURE OMITTED]

HPeVs have 9 polyprotein cleavage sites: VP0/VP3, VP3/VP1, VP1/2A, 2A/2B, 2B/2C, 2C/3A, 3A/3B, 3B/3C, and 3C/3D (Table 3). Comparison of these polyprotein cleavage sites among 6 HPeVs showed that those in VP3/ VP1, 2A/2B, 2B/2C, 2C/3A, 3A/3B, 3B/3C, and 3C/3D were conserved among all 6 HPeVs. The cleavage site in VP1/2A was identical among HPeV-1, HPeV-2, HPeV-4, HPeV-5, and NII561-2000 but not HPeV-3. The cleavage site in VP0/VP3 of NII561-2000 was identical to that of HPeV-3 but not to those of others.

The NII561-2000 virus, HPeV-1, HPeV-2, HPeV-4, and HPeV-5, but not HPeV-3, had an RGD (arginine-glycine-aspartic acid) motif at the C terminus of VP1 (15). The RGD motif may be used for an entry receptor of these HPeVs to attach, penetrate, or both, into host cells. Mutant HPeV-1 viruses with 2 aa deletions in the RGD motif showed little infectivity, while an RGD-to-RGE (arginine-glycine-glutamic acid) change showed reduced infectivity, and the resultant viruses possessed a rescued RGD. In addition, mutations at the +1 and +2 positions downstream from the RGD motif produced small virus-inducing plaques, and an M-to-P change at +1 position was lethal (15). The amino acids (+1 and +2 positions) downstream from the RGD motif of NII561-2000 were identical to those of HPeV-1.

The N terminal ends of VP4 of many picornaviruses are myristoylated, and they have a consensus myristoylation motif (16). HPeVs, including NII561-2000, lacked a myristoylation motif in the corresponding VP0 sequence. Myristoylation of capsid proteins is suggested to play a role in virion assembly. Thus, the virion assembly mechanism of HPeVs, including NII561-2000, might be distinct from other picornaviruses, including poliovirus.

Isolation of HPeVs from Clinical Samples

By using cultured cells (Caco-2, RD-I8S, Vero, HeLa, HEp-2, LLC-MK2, and BSC-1), we have isolated 8,195 CPE-inducing agents from 13,656 clinical samples (stool, throat swab, and CSF) collected between 1991 and 2005, at Niigata, Japan (Table 4, Table 5). The CPE morphologic features and CPE-inducing cell types suggested that 1,521 isolates are likely to be enteroviruses, HPeVs, rhinoviruses, or other viruses, and the others are likely to be influenza viruses or adenoviruses. Neutralization tests that used antisera against enteroviruses and HPeV-1 showed that 1,365 were enteroviruses and 12 were HPeV-1, and the remaining 144 viruses were not neutralized by these agents. RT-PCR that used degenerate primers against HPeVs, enteroviruses, and rhinoviruses identified 29 HPeVs, 72 enteroviruses, and 26 rhinoviruses, respectively. The remaining 17 viruses were not identified by these methods. Thus, a total of 41 HPeVs were isolated from the samples collected in Niigata.

Phylogenetic tree analysis showed 14 HPeV-1, 16 HPeV-3, and 1 HPeV-4, but no HPeV-2 and HPeV-5 among the examined 41 HPeVs. In addition, 10 viruses, including NII561-2000, formed a distinctive tree from those of the other HPeVs (Figure, panel B). Of note, in our search from 1991 through 2005, the NII561-2000-related viruses were isolated only in 2000 (3 cases) and 2001 (7 cases); HPeV-1 were isolated in 1991, 1998, 2001, 2004, and 2005; HPeV-3 were isolated in 1998, 1999, 2000, 2001, 2004, and 2005; and HPeV-4 was isolated only in 1993 (Table 4). The clinical symptoms of the patients infected with NII561-2000-related viruses were gastroenteritis, respiratory symptoms, rash, and flaccid paralysis in addition to Reye syndrome (Table 5), and these disease categories were similar to those of other HPeVs (2,3).

Discussion

In this study, we isolated a novel HPeV (NII561-2000) from a 1-year-old girl with Reye syndrome and determined the nucleotide sequence. Nucleotide sequence analysis and mutual neutralization test indicated that the NII561-2000 virus was distinct from 5 known HPeVs (Figure, panel A) (17). Thus, we propose that the NII561-2000 virus is the prototype of HPeV-6.

The NII561-2000 virus was originally isolated from a patient with Reye syndrome, an acute noninflammatory encephalopathy characterized by an antecedent viral infection, such as influenza or varicella (10-12). The significance of the NII561-2000 virus in this syndrome is not clear, because our samples did not include any other samples from this patient. We also isolated 9 NII561-2000-related viruses from clinical samples collected from other patients. The clinical symptoms of the persons infected with NII561-2000--related viruses were gastroenteritis, upper respiratory tract infection, rash, and flaccid paralysis. These disease categories of the NII561-2000-related virus are similar to those of other HPeVs, but the pathologic roles of the NII561-2000 viruses in these diseases, including Reye syndrome, need further etiologic and biologic studies.

In our search at Niigata from 1991 through 2005, the NII561-2000 and related viruses were isolated only in 2 consecutive years (2000 and 2001), HPeV-1 were isolated in 5 years, and HPeV-3 were isolated in 6 years. The genetic variations of these NII561-2000-related viruses were small (Figure, panel B). These results suggest that a 2-year outbreak of NII561-2000-related virus may have occurred in Niigata, Japan.

Thirteen of 16 HPeV-3 isolated in Niigata were from the patients <3 years of age, consistent with the previous report (18). Sepsislike illness and central nervous system involvement were more frequently reported in children infected with HPeV-3 than HPeV-1 (18). Consistent with those findings, the HPeV-3 infections in Niigata included 2 aseptic meningitis patients, whereas no such illness was associated with HPeV-1.

HPeV-4 was isolated from a 5-year-old patient with lymphadenitis in Niigata (Table 5). Thus, this virus is prevalent and is likely to be pathogenic in at least 3 countries. The diseases associated with HPeV-4 in the Netherlands and the United States were fever and TORCH (toxoplasmosis; other infections; namely, hepatitis B, syphilis, herpes zoster, rubella, cytomegalovirus, and herpes simplex virus infections) (6,19). Further analysis is required to establish an association of HPeV-4 with these diseases.

The degenerate primer set we used here was originally developed by Ito et al. (5) to amplify 3 known HPeV cDNAs. Here, we successfully amplified the NII561-2000 viral cDNA and HPeV-4 from culture supernatants of the infected cells, indicating that this primer set can amplify the cDNA fragments of at least 5 HPeVs from culture supernatants of infected cells. Thus, this primer set is a useful tool to determine the subtypes of HPeVs.

Acknowledgments

We thank Dr. Hiromu Yoshida for providing HPeV-1, HPeV-2, and monkey antisera against HPeV-1 and HPeV-2 and Dr. Miyabi Ito for providing HPeV-3 and guinea pig antiserum against HPeV-3.

References

(1.) Hyypia T, Horsnell C, Maaronen M, Khan M, Kalkkinen N, Auvinen P, et al. A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci U S A. 1992;89:8847-51.

(2.) Joki-Korpela P, Hyypia T. Parechoviruses, a novel group of human picornaviruses. Ann Med. 2001;33:466-71.

(3.) Stanway G, Joki-Korpela P, Hyypia T. Human parechoviruses--biology and clinical significance. Rev Med Virol. 2000;10:57-69.

(4.) Ghazi F, Hughes PJ, Hyypia T, Stanway G. Molecular analysis of human parechovirus type 2 (formerly echovirus 23). J Gen Virol. 1998;79:2641-50.

(5.) Ito M, Yamashita T, Tsuzuki H, Takeda N, Sakae K. Isolation and identification of a novel human parechovirus. J Gen Virol. 2004;85:391-8.

(6.) Benschop KSM, Schinkel J, Luken ME, van den Broek PJM, Beersma MFC, Menelik N, et al. Fourth human parechovirus serotype. Emerg Infect Dis. 2006;12:1572-5.

(7.) Oberste MS, Maher K, Pallansch MA. Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses. Virus Res. 1998;56:217-23.

(8.) Joki-Korpela P, Hyypia T. Diagnosis and epidemiology of echovirus 22 infections. Clin Infect Dis. 1998;27:129-36.

(9.) Abed Y, Bovin G. Human parechovirus infections in Canada. Emerg Infect Dis. 2006;12:969-75.

(10.) Casteels-Van Daele M, Van Geet C, Wouters C, Eggermont E. Reye syndrome revisited: a descriptive term covering a group of heterogeneous disorders. Eur J Pediatr. 2000;159:641-8.

(11.) Centers for Disease Control. Reye syndrome surveillance--United States, 1989. MMWR Morb Mortal Wkly Rep. 1991;40:88-90.

(12.) Reye RD, Morgan G, Baral J. Encephalopathy and fatty degeneration of the viscera. A disease entry in childhood. Lancet. 1963;91:749-52.

(13.) Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876-82.

(14.) Tamm I, Eggers HJ. Specific inhibition of replication of animal viruses. Science. 1963;142:24-33.

(15.) 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.

(16.) Chow M, Newman JF, Filman D, Hogle JM, Rowlands DJ, Brown F. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. 1987;327:482-6.

(17.) AL-Sunaidi M. Williams CH, Hughes PJ, Schunurr DP, Stanway G. Analysis of a new human parechovirus allows the definition of parechovirus types and the identification of RNA structural domains. J Virol. 2007;81:1013-21.

(18.) Benschop KSM, 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.

(19.)Schnurr D, Dondero M, Holland D, Connor J. Characterization of echovirus 22 variants. Arch Virol. 1996;141:1749-58.

Kanako Watanabe, * ([dagger]) Masayasu Oie, * Masaya Higuchi, * Makoto Nishikawa, ([dagger]) and Masahiro Fujii *

* Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; and ([dagger]) Niigata Prefectural Institute of Public Health and Environmental Sciences, Niigata, Japan

Mrs Watanabe is a graduate student at the Niigata University Graduate School of Medical and Dental Sciences. Her research interests include molecular biology and molecular epidemiology of enterovirus infection.

Address for correspondence: Masahiro Fujii, Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Niigata 951-8510, Japan; email: fujiimas@med. niigata-u.ac.jp

Table 1. Neutralization activities of anti-HPeV antibodies using
Vero cells *
                                         Virus

                        NII561-
Antiserum                 2000                    HPeV-1

NII561-2000               160                       10
HPeV-1 ([dagger])         <10        [greater than or equal to] 1,280
HPeV-2                    <10                       <10
HPeV-3                    <10                       <10

                                         Virus

Antiserum                HPeV-2                   HPeV-3

NII561-2000               <10                       <10
HPeV-1 ([dagger])         <10                       <10
HPeV-2                    160                       <10
HPeV-3                    <10        [greater than or equal to] 1,280

* HPeV, human parechovirus.

([dagger]) The HPeV strains used were Harris strain for HPeV-1,
Williamson strain for HPeV-2, and A308/99 for HPeV-3.

Table 2. Comparisons of nucleotide and amino acid sequences among
NII561-2000, HPeV-1, HPeV-2, HPeV-3, HPeV-4,and HPeV-5 *

                       Nucleotide similarity (% amino acid
                           similarity) with NII561-2000

Sequence     HPeV-1 ([dagger])          HPeV-2               HPeV-3

5'UTR               85.3                 87.3                 86.9
VP0             73.6 (78.2)          76.0 (80.9)          70.6 (72.3)
VP3             74.4 (84.7)          73.0 (80.2)          67.0 (74.1)
VP1             71.7n(80.3)          67.3 (72.2)          71.1 (75.3)
2A              80.8 (90.7)          77.1 (88.0)          81.0 (85.3)
2B              78.6 (97.5)          77.8 (97.5)          82.0 (97.5)
2C              84.7 (97.3)          76.4 (86.9)          79.2 (92.7)
3A              85.7 (96.6)          77.4 (88.0)          78.0 (87.2)
3B              81.4 (80.0)          72.9 (80.0)          73.3 (75.0)
3C              84.2 (99.0)          82.7 (99.0)          82.3 (98.0)
3D              83.2 (95.9)          83.7 (95.5)          83.9 (95.7)
3'UTR               81.6                 88.5                 83.9
ORF             79.5 (90.7)          77.1 (87.3)          76.7 (85.9)

                 Nucleotide similarity (% amino
                     acid similarity) with
                         NII561-2000

Sequence           HPeV-4               HPeV-5

5'UTR               88.5                 86.9
VP0             72.6 (81.0)          73.1 (77.2)
VP3             70.9 (78.0)          68.7 (73.1)
VP1             70.0 (71.9)          66.2 (70.2)
2A              77.3 (91.2)          76.2 (88.7)
2B              79.8 (97.5)          78.7 (99.2)
2C              78.7 (92.4)          79.2 (93.6)
3A              75.5 (89.7)          78.6 (83.8)
3B              66.7 (85.0)          70.0 (85.0)
3C              82.5 (98.0)          80.5 (99.0)
3D              84.2 (96.8)          87.2 (97.0)
3'UTR               84.9                 93.2
ORF             77.1 (88.2)          77.0 (86.9)

* HPeV, human parechovirus; UTR, untranslated region; ORF, open
reading frame.

([dagger]) The HPeV strains used were Harris strain for HPeV-1,
Williamson strain for HPeV-2, A308/99 for HPeV-3, K2511876-02
for HPeV-4, and CT86-6760 for HPeV-5.

Table 3. Amino acid sequences of protein cleavage sites of HPeVs

Virus             VP0/VP3    VP3/VP1     VP1/2A

NII561-2000         N/G        Q/N        Q/S
HPeV-1 *            N/A        Q/N        Q/S
HPeV-2              T/A        Q/N        Q/S
HPeV-3              N/G        Q/N        E/S
HPeV-4              N/N        Q/N        Q/S
HPeV-5              N/S        Q/N        Q/S

Virus              2A/2B      2B/2C      2C/3A

NII561-2000         Q/G        Q/G        Q/T
HPeV-1 *            Q/G        Q/G        Q/T
HPeV-2              Q/G        Q/G        Q/T
HPeV-3              Q/G        Q/G        Q/T
HPeV-4              Q/G        Q/G        Q/T
HPeV-5              Q/G        Q/G        Q/T

Virus              3A/3B      3B/3C      3C/3D

NII561-2000         E/R        Q/R        Q/G
HPeV-1 *            E/R        Q/R        Q/G
HPeV-2              E/R        Q/R        Q/G
HPeV-3              E/R        Q/R        Q/G
HPeV-4              E/R        Q/R        Q/G
HPeV-5              E/R        Q/R        Q/G

* The human parechovirus (HPeV) strains used were Harris strain
for HPeV-1, Williamson strain for HPeV-2, A308/99 for HPeV-3,
K2511876-02 for HPeV-4, and CT86-6760 for HPeV-5.

Table 4. Numbers of isolated HPeVs in 1991-2005 at Niiqata, Japan *

                                  HPeV subtype

Year        Type 1          Type 3          Type 4          Type 6

1991           1               0               0               0
1992           0               0               0               0
1993           0               0               1               0
1994           0               0               0               0
1995           0               0               0               0
1996           0               0               0               0
1997           0               0               0               0
1998           1               1               0               0
1999           0               3               0               0
2000           2               2               0               3
2001           4               5               0               7
2002           0               0               0               0
2003           0               0               0               0
2004           5               2               0               0
2005           1               3               0               0
Total         14              16               1              10

         No. isolated    No. examined    No. isolated
Year         HPeVs          samples         viruses

1991           1              303             255
1992           0              142             53
1993           1              166             50
1994           0              151             55
1995           0              167             116
1996           0              133             54
1997           0              277             84
1998           2             1,607           1,117
1999           3             2,118           1,203
2000           7             1,974           1,127
2001          16             1,699            989
2002           0             2,783           1,787
2003           0              986             597
2004           7              584             329
2005           4              566             379
Total         41            13,656           8,195

* HPeV, human parechovirus.

Table 5. Diseases associated with HPeVs isolated 1991-2005
in Niigata, Japan *

                              HPeV
Strain                      subtype                       Specimen

NII1099-91                     1                           Stool
NII1852-98                     1                        Throat swab
NII2534-2000                   1                           Stool
NII2632-2000                   1                           Stool
NII36-2001                     1                           Stool
NII2647-2001                   1                        Throat swab
NII2715-2001                   1                           Stool
NII2726-2001                   1                           Stool
NII196-2004                    1                           Stool
NII197-2004                    1                           Stool
NII198-2004                    1                           Stool
NII940-2004                    1                        Throat swab
NII1056-2004                   1                           Stool
NII751-2005                    1                           Stool
NII7-98                        3                           Stool
NII2486-99                     3                           Stool
NII2701-99                     3                        Throat swab
NII2927-99                     3                           Stool
NII2319-2000                   3                           Stool
NII2825-2000                   3                        Throat swab
NII2335-2001                   3                        Throat swab
NII2573-2001                   3                        Throat swab
NII2735-2001                   3                           Stool
NII2826-2001                   3                        Throat swab
NII2930-2001                   3                        Throat swab
NII1018-2004                   3                            CSF
NII1063-2004                   3                           Stool
NII414-2005                    3                        Throat swab
NII562-2005                    3                        Throat swab
NII572-2005                    3                        Throat swab
NII370-93                      4                           Stool

NII428-2000                    6                           Stool
NII561-2000                    6                            CSF
NII2819-2000                   6                        Throat swab
NII2392-2001                   6                           Stool
NI12485-2001                   6                        Throat swab
NII2589-2001                   6                        Throat swab
NII2611-2001                   6                           Stool
NII2667-2001                   6                           Stool
NII2694-2001                   6                        Throat swab
NII2729-2001                   6                           Stool

Strain                  Clinical symptom                    Sex

NII1099-91              Gastroenteritis                      F
NII1852-98          Hand-foot-mouth disease                  M
NII2534-2000        Fever of unknown origin                  M
NII2632-2000            Gastroenteritis                      M
NII36-2001              Gastroenteritis                      M
NII2647-2001   Upper respiratory tract infection             M
NII2715-2001            Gastroenteritis                      M
NII2726-2001            Gastroenteritis                      F
NII196-2004             Gastroenteritis                      M
NII197-2004             Gastroenteritis                      M
NII198-2004             Gastroenteritis                      M
NII940-2004                Bronchitis                        M
NII1056-2004            Gastroenteritis                      F
NII751-2005             Gastroenteritis                      M
NII7-98                 Gastroenteritis                      F
NII2486-99                    Rash                           F
NII2701-99                    Rash                           F
NII2927-99             Aseptic meningitis                    M
NII2319-2000   Upper respiratory tract infection             M
NII2825-2000   Upper respiratory tract infection             M
NII2335-2001                  Rash                           F
NII2573-2001                  Rash                           F
NII2735-2001           Aseptic meningitis                    F
NII2826-2001   Upper respiratory tract infection             M
NII2930-2001                Myositis                         M
NII1018-2004        Fever of unknown origin                  F
NII1063-2004            Gastroenteritis                      M
NII414-2005          Influenzalike illness                   M
NII562-2005          Influenzalike illness                   F
NII572-2005          Influenzalike illness                   M
NII370-93                Lymphadenitis                       M

NII428-2000             Gastroenteritis
NII561-2000              Reye syndrome
NII2819-2000                  Rash
NII2392-2001           Flaccid paralysis
NI12485-2001   Upper respiratory tract infection
NII2589-2001            Gastroenteritis
NII2611-2001            Gastroenteritis
NII2667-2001            Gastroenteritis
NII2694-2001                  Rash
NII2729-2001            Gastroenteritis

Strain                       Age, y                     Cell line(s)

NII1099-91                     1                           BSC-1
NII1852-98                     <1                      CaCo2, RD-18S
NII2534-2000                   <1                          CaCo2
NII2632-2000                   1                        RD-18S, Vero
NII36-2001                     1                           BSC-1
NII2647-2001                   1                           RD-18S
NII2715-2001                   <1                      CaCo2, RD-18S
NII2726-2001                   1                           CaCo2
NII196-2004                    9                          LLC-MK2
NII197-2004                    <1                         LLC-MK2
NII198-2004                    <1                         LLC-MK2
NII940-2004                    <1                   CaCo2, RD-18S, Vero
NII1056-2004                   <1                         LLC-MK2
NII751-2005                    <1                          CaCo2
NII7-98                        1                           BSC-1
NII2486-99                     <1                           Vero
NII2701-99                     1                            Vero
NII2927-99                     <1                           Vero
NII2319-2000                   <1                          BSC-1
NII2825-2000                   1                       Vero, LLC-MK2
NII2335-2001                   <1                      Vero, LLC-MK2
NII2573-2001                   <1                      Vero, LLC-MK2
NII2735-2001                   <1                      Vero, LLC-MK2
NII2826-2001                   <1                      Vero, LLC-MK2
NII2930-2001                   8                        CaCo2, Vero
NII1018-2004                   <1                      Vero, LLC-MK2
NII1063-2004                   <1                      CaCo2, LLC-MK2
NII414-2005                    4                       Vero, LLC-MK2
NII562-2005                    35                         LLC-MK2
NII572-2005                    5                       Vero, LLC-MK2
NII370-93                      5                       RD-18S, Vero,
                                                          LLC-MK2
NII428-2000                    1                            Vero
NII561-2000                    1                            Vero
NII2819-2000                   2                           BSC-1
NII2392-2001                   1                           CaCo2
NI12485-2001                   <1                          RD-18S
NII2589-2001                   8                           RD-18S
NII2611-2001                   3                        CaCo2, Vero
NII2667-2001                   6                            HeLa
NII2694-2001                   2                       CaCo2, RD-18S
NII2729-2001                   2                           BSC-1

* HPeV, human parechovirus; CSF, cerebrospinal fluid.