Sequencing of ‘untypable’ enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types

Helene Norder1, Lotte Bjerregaard1, Lars Magnius1, Bruno Lina2, Michèle Aymard2 and Jean-Jacques Chomel2

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 ‘untypable’ isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19·4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15·7–18·2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24·2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.

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.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human enterovirus (HEV) infections are associated with a broad spectrum of clinical features including myocarditis, aseptic meningitis and encephalitis (Grist et al., 1978), although they are often mild or asymptomatic (Melnick, 1997). Aseptic meningitis and respiratory illness may be caused by most enteroviruses, whereas coxsackie virus A (CV-A) and CV-B viruses are the main causes of herpangina and myocarditis, respectively (Modlin, 1995).

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 {beta}-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 {beta}-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.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus isolates.
Forty-four virus strains were first isolated on either MRC5 (Biomérieux), BGM (ATCC), HEp-2 (Cincinnati), or L20B (NIBSC) cell cultures in France between 1996 and 2001. The strains, for which typing by neutralization had failed, were further propagated on GMK cells, human embryonal lung fibroblasts or RD cells. Neutralization attempts had been performed with 30–300 TCID50 with 25 µl of each intersecting LBM pool A–H for typing echovirus, CV-B1 to CV-B6, PV-1 to PV-3, CV-A7, A9 and A16. The LBM intersecting pools J–P for typing CV-A viruses were used when neutralization failed with the intersecting A–H pools.

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).


View this table:
[in this window]
[in a new window]
 
Table 1. Designation of investigated isolates, patient data, isolation materials and results from comparisons of 120 residues of the VP1 protein with the most similar prototype

CSF, cerebrospinal fluid; BAL, broncho-alveolar lavage. NA, not applicable.

 
The VP1 region was also amplified and sequenced for another 13 isolates of CV-A16, EV-71, echovirus (E)-3, E-13, E-17, E-18 and E-25, where there had been no typing difficulties.

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).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
All 44 ‘untypable’ isolates could be typed by VP1 sequencing and phylogenetic analysis. One isolate was identified as HRV (human rhinovirus)-1B, while enterovirus types of all four species were identified among the other isolates, with HEV-A and HEV-B dominating (18 isolates of each; Table 1). Six isolates belonged to HEV-C, while one isolate was EV-68 within HEV-D. The virus type obtained by molecular typing could be verified for all patient isolates apart from six by neutralization using LBM pools or high-titre monovalent antisera or after filtration (Table 1).

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. 2a).




View larger version (136K):
[in this window]
[in a new window]
 
Fig. 1. Deduced amino acid sequence of 120 residues covering the BC loop of 56 isolates, 43 of which were ‘untypable’, and sequences from GenBank aligned to the corresponding prototype. (a) Alignments for isolates within HEV-A and HEV-C. (b) Alignments for isolates within HEV-B. The BC loop is boxed. -, Identity; *, sequence data missing; #, ‘untypable’ strain.

 



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 2. UPGMA dendrogram based on 120 N-terminal residues of the VP1 protein for 165 enterovirus strains belonging to HEV-A to HEV-D. The isolates typed by sequencing in this study are shown in bold. (a) Section of the dendrogram with the 52 strains belonging to HEV-A, HEV-C and HEV-D. (b) Section of the dendrogram with the 113 HEV-B strains. EV-74 and the two new virus types, EV-77 and EV-78, are indicated by arrows.

 
HEV-B
Five of the 18 HEV-B isolates were E-18. These could be neutralized after filtration and had no amino acid substitutions in common within the BC loop that were not present in typable isolates (Fig. 1b). One divergent E-13 isolate, 97/20062-33/99, typable with monovalent antiserum, had ten unique amino acid substitutions not present in the most similar E-13 strain, four of which were in the BC loop (Fig. 1b). The other typed HEV-B isolates also had two to five amino acid substitutions between residues 80 and 105 compared with their respective prototype, with which they clustered in the dendrogram (Fig. 2b). Two isolates, W179-45/01 and W181-46/01, diverged by only 1·6 % from each other and formed a separate cluster on the same branch as E-2 and E-15, although they were more similar to E-15 by pairwise comparison. These two isolates were not neutralizable with high-titre monovalent antiserum, and four of the 20 amino acid substitutions, which differed between the two isolates and the E-15 prototype, were within the BC loop. Three isolates that were not neutralizable, W543-122/99, W137-126/99 and W553-130/99, diverged by more than 24 % from all prototypes (Table 1). The two former split off separately from the same branch as E-26 in the dendrogram, from which the two isolates showed least divergence, 30 % and 29 %, respectively (Fig. 2b). W553-130/99 formed a deep bifurcation from the E-18 branch (Fig. 2b). Due to the high divergence from all prototypes, the complete VP1 regions were sequenced for all three isolates and compared with the complete VP1 regions of all prototypes, including the EV-74 prototype (Fig. 3). As a result, W553-130/99 was found to be closely related to the EV-74 prototype, while W543-122/99 and W137-126/99 remained unique (Fig. 3). The length of the putative VP1 protein was 290 and 284 residues for the latter two isolates, respectively.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. UPGMA dendrogram based on the sequences of the complete VP1 protein of 74 enterovirus strains belonging to HEV-A to HEV-D. EV-74 and the two new virus types, EV-77 and EV-78, are indicated by arrows.

 
HEV-C
Four of the six HEV-C isolates were most similar to the CV-A13 and A18 prototypes, with which they also clustered in the dendrogram. Only 210 nucleotides of the N-terminal VP1 region could be sequenced for two of these isolates, W184-125/99 and W200-124/99. In a dendrogram (not shown) based on 210 nucleotides, they were found to be on the same branch as the CV-A13 and A18 prototypes. All four were most similar to CV-A13 and A18 by pairwise comparisons and had 13 unique substitutions in the BC loop. In addition, the two non-neutralizable isolates shared an amino acid deletion in the BC loop (Fig. 1a).

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).


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Despite rather high genetic divergence for some isolates, all but six of the isolates could be classified into one of the 65 known HEV types by sequencing the N-terminal VP1 region. Comparison of 120 amino acids with the prototype strains showed that most of the substitutions were within or close to the BC loop for the majority of the ‘untypable’ isolates compared with their respective prototypes and other strains, irrespective of species, suggesting that this loop is important for the reactivity of neutralizing antibodies.

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 {beta}-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 {beta}-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.


   ACKNOWLEDGEMENTS
 
We are grateful to Drs Steve Oberste and Mark Pallansch for sharing with us the VP1 sequences of EV-74, EV-75 and EV-76 prior to publication.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Caro, V., Guillot, S., Delpeyroux, F. & Crainic, R. (2001). Molecular strategy for serotyping of human enteroviruses. J Gen Virol 82, 79–91.[Abstract/Free Full Text]

Casas, I., Palacios, G. F., Trallero, G., Cisterna, D., Freire, M. C. & Tenorio, A. (2001). Molecular characterization of human enteroviruses in clinical samples: comparison between VP1, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing. J Med Virol 65, 138–148.[CrossRef][Medline]

Dahllund, L., Nissinen, L., Pulli, T., Hyttinen, V.-P., Stanway, G. & Hyypiä, T. (1995). The genome of echovirus 11. Virus Res 35, 215–222.[CrossRef][Medline]

Felsenstein, J. (1993). PHYLIP: phylogeny inference package, version 3.52c. University of Washington, Seattle, Washington.

Grist, N. R., Bell, E. J. & Assaad, F. (1978). Enterovirus in human disease. Prog Med Virol 24, 114–157.[Medline]

Huttunen. P., Santti. J., Pulli, T. & Hyypiä, T. (1996). The major echovirus group is genetically coherent and related to coxsackie B viruses. J Gen Virol 77, 715–725.[Abstract]

King, A. M. Q., Brown, F., Christian, P. & 8 other authors (2000). Picornaviridae. In Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses, pp. 657–673. Edited by M. H. V. Van Regenmortel, C. M. Fauquet, D. H. L. Bishop & 9 others. San Diego: Academic Press.

Kumar, S., Tamura, K. & Nei, M. (1993). MEGA: Molecular Evolutionary Genetics Analysis version 1.02. Pennsylvania State University, Pennsylvania.

Lenz, K. N., Smith, A. D., Geisler, S. C. & 9 other authors (1997). Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of the three poliovirus serotypes. Structure 5, 961–978.[Medline]

McPhee, F., Zell, R., Reimann, B. Y., Hofschneider, P. H. & Kandolf, R. (1994). Characterization of the N-terminal part of the neutralizing antigenic site I of coxsackievirus B4 by mutation analysis of antigen chimeras. Virus Res 34, 139–151.[Medline]

Mateu, M. G. (1995). Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus Res 38, 1–24.[CrossRef][Medline]

Melnick, J. L. (1997). Enteroviruses, polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Fields Virology, pp. 655–712. Edited by B. N. Fields, D. N. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Melnick, J. L., Tagaya, I. & von Magnus, H. (1974). Enteroviruses 69, 70, and 71. Intervirol 4, 369–370.[Medline]

Modlin, J. F. (1995). Coxsackieviruses, echoviruses and newer enteroviruses. In Principles and Practice of Infectious Diseases, 4th edn, pp. 1620–1632. Edited by G. L. Mandell, J. E. Bennet & R. Dolin. New York: Churchill Livingstone.

Muckelbauer, J. K., Kremer, M., Minor, I., Diana, G., Dutko, F. J., Groarke, J., Pevear, D. C. & Rossmann, M. G. (1995). The structure of coxsackievirus B3 at 3·5 Å resolution. Structure 3, 653–667.[Medline]

Norder, H., Bjerregaard, L. & Magnius, L. (2001). Homotypic echoviruses share aminoterminal VP1 sequence homology applicable for typing. J Med Virol 63, 35–44.[CrossRef][Medline]

Norder, H., Bjerregaard, L. & Magnius, L. (2002). Open reading frame sequence of an Asian enterovirus 73 strain reveals that the prototype from California is recombinant. J Gen Virol 83, 1721–1728.[Abstract/Free Full Text]

Oberste. M. S., Maher. K., Kilpatrick, D. R., Flemister, M. R., Brown, B. A. & Pallansch, M. A. (1999a). Typing of human enteroviruses by partial sequencing of VP1. J Clin Microbiol 37, 1288–1293.[Abstract/Free Full Text]

Oberste, M. S., Maher, K., Kilpatrick, D. R & Pallansch, M. A. (1999b). Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 73, 1941–1948.[Abstract/Free Full Text]

Oberste, M. S., Maher, K., Flemister, M. R., Marchetti, G., Kilpatrick, D. R. & Pallansch, M. A. (2000). Comparison of classic and molecular approaches for the identification of ‘untypeable’ enteroviruses. J Clin Microbiol 38, 1170–1174.[Abstract/Free Full Text]

Oberste, M. S., Schnurr, D., Maher, K., al-Busaidy, S. & Pallansch, M. A. (2001). Molecular identification of new picornaviruses and characterization of a proposed enterovirus 73 serotype. J Gen Virol 82, 409–416.[Abstract/Free Full Text]

Pöyry, T., Kinnunen, L., Hyypiä, T., Brown, B., Horsnell, C., Hovi, T. & Stanway, G. (1996). Genetic and phylogenetic clustering of enteroviruses. J Gen Virol 77, 1699–1717.[Abstract]

Pulli, T., Koskimies, P. & Hyypiä, T. (1995). Molecular comparison of coxsackie A virus serotypes. Virology 212, 30–38.[CrossRef][Medline]

Pulli, T., Roivainen, M., Hovi, T. & Hyypiä, T. (1998). Induction of neutralizing antibodies by synthetic peptides representing the C terminus of coxsackievirus A9 capsid protein VP1. J Gen Virol 79, 2249–2253.[Abstract]

Reimann, B.-Y., Zell, R. & Kandolf, R. (1991). Mapping of a neutralizing antigenic site of coxsackie B4 by construction of an antigen chimera. J Virol 65, 3475–3480.[Medline]

Singh, S., Chow, V. T. K., Chan, K. P., Ling, A. E. & Poh, C. L. (2000). RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus 71 strains from Asia. J Virol Methods 88, 193–204.[CrossRef][Medline]

Wallis. A. & Melnick, J. L. (1967). Virus aggregation as the cause of non-neutralizable persistent fraction. J Virol 1, 478–488.[Medline]

Received 10 June 2002; accepted 14 November 2002.