1 Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden
2 Centre National de Reference des Enterovirus, Lyon, France
Correspondence
Lars Magnius
lars.magnius{at}smi.ki.se
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ABSTRACT |
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The complete VP1 sequences of EV-74, EV-77 and EV-78 and the partial VP1 sequences of the other isolates are deposited in GenBank with accession nos AY208081AY208120.
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INTRODUCTION |
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The 65 HEV types are classified into five species, HEV-A to HEV-D and poliovirus (PV), based on sequence data and analysis of the VP1 region (King et al., 2000). HEV-A consists of CV (coxsackievirus)-A2 to CV-A8, CV-A10, CV-A12, CV-A14, CV-A16 and EV (enterovirus)-71. All CV-B viruses, CV-A9, EV-69, EV-73 and all echoviruses form HEV-B; CV-A1, CV-A11, CV-A13, CV-A15, CV-A17 to CV-A22 and CV-A24 form HEV-C; and EV-68 and EV-70 form HEV-D. This classification is based on previous subdivisions of enteroviruses into four genetic groups after analysis of the VP2, 3D and VP1 regions, as well as complete genomes (Dahllund et al., 1995
; Pulli et al., 1995
; Huttunen et al., 1996
; Pöyry et al., 1996
; Oberste et al., 1999a
, b
).
EV-69, EV-70 and EV-71 were described 28 years ago (Melnick et al., 1974). Since then no new types have been reported until EV-73 was identified by molecular techniques in California and Oman (Oberste et al., 2001
) and as imports into Sweden from South and East Asia (Norder et al., 2002
). One reason for this is the labour-intensive conventional methods for establishing new enterovirus types, including the production of hyperimmune antisera and cross-testing against previous prototypes. Serological typing may be hampered by failure to neutralize the virus with the antisera used due to the isolate containing multiple types, genetic changes of the virus or conceivably by the encounter of a prime strain or a previously undescribed type. The correlation between the enterovirus VP1 sequence and serotype has been shown in several recent studies, and genetic typing of enteroviruses by complete or partial sequencing of the VP1 region may now considerably simplify the typing (Oberste et al., 1999a
, b
, 2000
; Caro et al., 2001
; Casas et al., 2001
; Norder et al., 2001
).
Within the VP1 region, the BC loop has been shown to be important for the reactivity of type-specific antibodies and for CV-B4, substitutions of residues 84 and 85 in the BC loop abolish neutralizing reactivity to the virus (McPhee et al., 1994). Little is known about the exact antigenic binding sites of most enterovirus types, although for polioviruses, CV-B4 and CV-A9 the neutralization epitopes have been mapped in the exposed structures on the virion, mainly in the loop structures connecting the
-strands of the capsid proteins (Reimann et al., 1991
; Mateu et al., 1995
; Pulli et al., 1998
). Antigenic differences between different enterovirus types could thus be assumed to be due to amino acid variations in the exposed regions of the capsid, mainly the BC loop of the VP1 protein and the adjacent
-B region, which may be exposed on the surface of the virion (Muckelbauer et al., 1995
). Substitutions resulting in conformational changes of the BC loop have also been shown to be important for host adaptation of polioviruses and rhinoviruses (Lentz et al., 1997
). In this study, the N-terminal part of the VP1 region including the BC loop was sequenced for 43 enterovirus isolates that were not readily neutralizable with antisera to all types in order to investigate whether the isolates were already-known types divergent in this region or whether some of them were new.
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METHODS |
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Non-neutralizable isolates were typed by sequencing the N-terminal part of VP1. These isolates were filtered and further typing was performed using twofold concentrated intersecting LBM pools or high-titre monovalent antisera directed against the type obtained by sequencing.
Four isolates were obtained from sludge in a water-treatment plant, 39 isolates were from children aged 8 days to 9 years and one isolate was from a 28-year-old man. The patients had a broad range of symptoms, with gastroenteritis being the most common symptom (Table 1). The materials used for virus isolation were mainly stools, vesicles and nasal or throat swabs (Table 1
).
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RNA extraction, reverse transcription and PCR.
Infected cell cultures with full cytopathic effect were treated with 200 µg proteinase K ml-1 and SDS to a final concentration of 0·5 % in 10 mM Tris/HCl, pH 8·0, for 2 h at 37 °C. Nucleic acids were extracted with phenol, followed by ethanol precipitation, and were dissolved in purified water (Millipore, Water Purification Systems). cDNA was synthesized using 5 µl RNA, 1 U Superscript II reverse transcriptase (Gibco BRL), 20 U Recombinant RNAsin Ribonuclease inhibitor (Promega) and 1 nmol of random hexamer primers (Roche Diagnostics). Amplification of the VP1 region was performed using 5 µl cDNA in a 50 µl reaction mixture containing 50 mM KCl, 10 mM Tris/HCl, pH 8·5, 2·5 mM MgCl2, 0·15 mM of each deoxynucleoside triphosphates, 20 pmol of each primer and 1·2 U Taq polymerase (Applied Biosystems). Four sets of primers were used; three sets have been described previously (Norder et al., 2001) and the fourth set used sense primers 187, 188 and 189 and antisense primer 222, as detailed in Oberste et al. (2000)
. The complete VP1 region of three divergent isolates was amplified and sequenced with the antisense primer ent110 and the sense primers above (Norder et al., 2001
). The amplification reactions were performed for 40 cycles with denaturation for 15 s at 94 °C, annealing for 15 s at 52 or 56 °C and elongation for 40 s at 72 °C.
Template purification and sequencing.
The amplified products were purified from excess dNTPs and primers using GFX PCR DNA and the Gel Band Purification Kit (Amersham Pharmacia Biotech). Sequencing was performed in both directions with 0·033 pmol purified template and 4 pmol of the primers used in the PCR. Cycle sequencing was carried out with fluorescent-labelled dideoxy-chain terminators and the reagents in the BigDye sequencing kit (Applied Biosystems).
Sequence analysis.
The deduced sequences of the VP1 protein were aligned with previously published sequences (Norder et al., 2001) and sequences from GenBank. The number of amino acid differences between the sequences was calculated with the MEGA program package, version 1.02 (Kumar et al., 1993
). Genetic distances were calculated using Dayhoff PAM matrix in the Protdist program in the PHYLIP 3.53 program package (Felsenstein, 1993
). Dendrograms were constructed using the UPGMA and neighbour-joining algorithms in the Neighbor program (Felsenstein, 1993
).
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RESULTS |
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HEV-A
CV-A16 was the most prevalent type and only one isolate had a substitution between residues 92 and 108 of the VP1 protein, encompassing the BC loop (Fig. 1a). Six out of seven EV-71 isolates had at least one amino acid substitution in this region not present in typable isolates. Two CV-A14 isolates had Thr99 or Ala99 while the prototype had Val99 (Fig. 1a
). All the isolates clustered with their respective prototype in the dendrogram (Fig. 2
a).
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HEV-D
The isolate 98/30259-37/99 was most similar to EV-68, with which it formed a separate cluster in the dendrogram (Fig. 2a). Four of eleven amino acid substitutions for 98/30259 compared with EV-68 were within the BC loop (not shown).
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DISCUSSION |
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Two types were predominant among the isolates CV-A16 and EV-71. These types thus seem to have diverged more from their prototypes than other types. All EV-71 isolates had substitutions within the BC loop and two isolates also had substitutions within the -B region. All but one of the CV-A16 isolates, on the other hand, were identical to the prototype within these regions, but differed at amino acid 108, which most probably resides within the
-C region of the VP1 protein. This might indicate that the BC loop might be less important in defining neutralization of CV-A16 or that the substitution of residue 108 might reorientate the BC loop and affect its antigenic properties. Variant CV-A16 strains resulting in untypable isolates may be more common in Europe, since this was the predominant type in our study, whereas it was represented by only two out of 55 untypable isolates in a similar study in the USA (Oberste et al., 2000
). In that study, EV-71 and E-18 were also common types, represented by six and four isolates, respectively. The Lys86Glu substitution in the BC loop of the EV-71 isolates may be important for neutralization, since this residue is also expressed in EV-71 isolates from Asia, for which typing by neutralization had failed (Singh et al., 2000
). Besides CV-A16 and EV-71, the typing of E-18 was also associated with difficulties. However, this was overcome by filtration, as has been reported for E-4 strain Pesascek (Wallis & Melnick, 1967
).
In the compared region, constituting around 40 % of VP1, eight strains diverged by more than 15 % from the most similar prototype, a divergence that, within the complete VP1 region, would be compatible with a new serotype. Two of these isolates diverged by 19·4 % from the most related prototype, E-15, and were not neutralizable with high-titre monovalent antiserum. Cross-reactivities between related types may, however, occur. We could not exclude the possibility that these were prime strains of E-15. Five other isolates were also not neutralizable; two of these were most similar to CV-A13/18, but were derived from sludge, which could be the reason for typing problems. The remaining three isolates were found to be highly divergent from all previously described enterovirus types by molecular typing, although one of the isolates, W553-130/99, showed high genetic similarity to a strain with the proposed designation EV-74, identified in the USA, where strains with the suggested designations EV-75 and EV-76 have also now been identified (S. Oberste & M. Pallansch, personal communication). The lowest amino acid divergence for the complete VP1 region between these two strains compared with that for all prototypes was 29 %, which is considerably more than the minimum 15 % amino acid sequence divergence in this region proposed to indicate a new type (Oberste et al., 2001). According to this criterion, two of the isolates represent new HEV-B members, which we have tentatively designated EV-77 and EV-78. Hence, sequencing and analysis of the VP1 region enable the typing of serologically untypable enterovirus isolates, as well as the identification of new types. Using the accepted criterion, these two enterovirus strains were confirmed to constitute two new types, showing the value of this approach.
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ACKNOWLEDGEMENTS |
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Received 10 June 2002;
accepted 14 November 2002.