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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
|
|
|
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 stemloop 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).
|
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 23-year-old group and to 85 % in the 46-year-old group. In the other age groups, the positive rate varied from 56·5 to 91·3 % (Table 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (arginineglycineglutamic 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Birenbaum, E., Handsher, R., Kuint, J., Dagan, R., Raichman, B., Mendelson, E. & Linder, N. (1997). Echovirus type 22 outbreak associated with gastro-intestinal disease in a neonatal intensive care unit. Am J Perinatol 14, 469473.[Medline]
Boonyakiat, Y., Hughes, P. J., Ghazi, F. & Stanway, G. (2001). Arginine-glycine-aspartic acid motif is critical for human parechovirus1 entry. J Virol 75, 1000010004.
Ehrnst, A. & Eriksson, M. (1993). Epidemiological features of type 22 echovirus infection. Scand J Infect Dis 25, 275281.[Medline]
Ehrnst, A. & Eriksson, M. (1996). Echovirus type 23 observed as a nosocomial infection in infants. Scand J Infect Dis 28, 205206.[Medline]
Figueroa, J. P., Ashley, D., King, D. & Hull, B. (1989). An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22. J Med Virol 29, 315319.[Medline]
Fox, G., Parry, N. A., Barnett, P. V., McGinn, B., Rowlands, D. J. & Brown, F. (1989). The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). J Gen Virol 70, 625637.[Abstract]
Ghazi, F., Hughes, P. J., Hyypiä, T. & Stanway, G. (1998). Molecular analysis of human parechovirus type 2 (formerly echovirus 23). J Gen Virol 79, 26412650.[Abstract]
Hamparian, V. V. (1979). Rhinoviruses. In Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 5th edn, pp. 535575. Edited by E. H. Lennette & N. J. Schmidt. Washington, DC: American Public Health Association.
Hughes, P. J., Horsnell, C., Hyypiä, T. & Stanway, G. (1995). The coxsackievirus A9 RGD motif is not essential for virus infectivity. J Virol 69, 80358040.[Abstract]
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 1125.[Medline]
Hyypiä, T., Horsnell, C., Maaronen, M., Khan, M., Kalkkinen, N., Auvinen, P., Kinnunen, L. & Stanway, G. (1992). A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci U S A 89, 88478851.[Abstract]
Joki-Korpela, P. & Hyypiä, T. (1998). Diagnosis and epidemiology of echovirus 22 infections. Clin Infect Dis 26, 129136.
Joki-Korpela, P., Roivainen, M., Poyry, T. & Hyypiä, T. (2000). Antigenic properties of human parechovirus 1. J Med Virol 81, 17091718.
Ketler, A., Hamparian, V. V. & Hilleman, M. R. (1962). Characterization and classification of ECHO 28-rhinovirus-coryzavirus agents. Proc Soc Exp Biol Med 110, 821831.
King, A. M. Q., Brown, F., Christian, P. & 8 other authors (2000). Picornaviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 657673. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Koskiniemi, M., Paetau, R. & Linnavuori, K. (1989). Severe encephalitis associated with disseminated echovirus 22 infection. Scand J Infect Dis 21, 463466.[Medline]
Muir, P., Kammerer, U., Koru, K., Mulders, M. N., Doyry, T., Weissbrich, B., Kandolf, R., Cleator, G. M. & Vanloon, A. M. (1998). Molecular typing of enteroviruses: current status and future requirements. The European Union concerted action on virus meningitis and encephalitis. Clin Microbiol Rev 1, 202227.
Nakao, T. & Miura, R. (1970). ECHO virus type 22 infection in a premature infant. Tohoku J Exp Med 102, 6168.[Medline]
Oberste, M. S., Maher, K. & Pallansch, M. A. (1998). Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enterovirus. Virus Res 56, 217223.[CrossRef][Medline]
Ruoslahti, E. & Pierschbacher, M. D. (1987). New perspectives in cell adhesion: RGD and integrins. Science 238, 491493.[Medline]
Sato, N., Sato, H., Kawana, R. & Matumoto, M. (1972). Ecological behaviour of 6 coxsackie B and 29 echo serotypes as revealed by serologic survey of general population in Aomori, Japan. Jap J Med Sci Biol 25, 355368.[Medline]
Schnurr, D., Dondero, M., Holland, M. & Connor, J. (1996). Characterization of echovirus 22 variants. Arch Virol 141, 17491758.[Medline]
Stanway, G. (1990). Structure, function and evolution of picornavirus. J Gen Virol 71, 24832501.[Medline]
Stanway, G. & Hyypiä, T. (1999). Parechoviruses. J Virol 73, 52495254.
Stanway, G., Kalkkinen, N., Roivainen, M., Ghazi, F., Khan, M., Smyth, M., Meurman, O. & Hyypiä, T. (1994). Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group. J Virol 68, 82328238.[Abstract]
Supanaranond, K., Takeda, N. & Yamazaki, S. (1992). The complete nucleotide sequence of a variant of coxsackievirus A24, an agent causing acute hemorrhagic conjunctivitis. Virus Genes 6, 149158.[Medline]
Yamashita, T., Sugiyama, M., Tsuzuki, H., Sakae, K., Suzuki, Y. & Miyazaki, Y. (2000). Application of a polymerase chain reaction for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans. J Clin Microbiol 38, 29552961.
Zimmermann, H., Eggers, H. J., Kraus, W. & Nelsen-Salz, B. (1995). Complete nucleotide sequence and biological properties of an infectious clone of prototype echovirus 9. Virus Res 39, 311319.[CrossRef][Medline]
Zimmermann, H., Eggers, H. J., Kraus, W. & Nelsen-Salz, B. (1997). Cell attachment and mouse virulence of echovirus 9 correlate with an RGD motif in the capsid protein VP1. Virology 233, 149156.[CrossRef][Medline]
Received 25 June 2003;
accepted 20 October 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |