1 Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA
2 Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, CA, USA
3 Instituto Nacional de Enfermedades Infectiosos ANLIS Carlos Malbran, Buenos Aires, Argentina
4 Department of Virology, Swedish Institute for Disease Control, Solna, Sweden
5 Institute of Public Health, Dhaka, Bangladesh
6 Centre National de Référence des Entérovirus, Lyon, France
7 Department of Health, Queen Mary Hospital, Hong Kong Special Administrative Region, People's Republic of China
8 National Polio Laboratory, Baghdad, Iraq
9 Department of Laboratories, Directorate General of Health Affairs, Ministry of Health, Muscat, Oman
Correspondence
M. Steven Oberste
soberste{at}cdc.gov
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are AY556057AY556070.
Present address: VaxGen Inc., 347 Oyster Point Blvd, Suite 102, South San Francisco, CA 94080, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In previous studies, we used analysis of partial or complete VP1 nucleotide sequences as a surrogate for antigenic typing to distinguish enterovirus serotypes (Oberste et al., 1999b, 2000
). This method was used to identify a new enterovirus serotype, EV73, from among enterovirus isolates that were deemed untypable by classical identification methods (Oberste et al., 2001
). Isolation of additional strains of EV73 from Bangladesh, India, Korea and Oman (Oberste et al., 2001
; Norder et al., 2002
; M. S. Oberste, unpublished data) confirmed its worldwide circulation. Three additional new proposed types, EV76 (M. S. Oberste, unpublished data), EV77 and EV78 (Norder et al., 2003
; Bailly et al., 2004
), were also identified by similar methods. The discovery of these new types suggested that numerous additional enterovirus types await identification.
In this study, 14 enterovirus isolates were characterized as members of two new types. Isolates within each of these two types were significantly different from one another and from all other known enterovirus serotypes, based on sequences that encode either the VP1 capsid protein or the entire capsid region. We propose that these isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Isolates were further characterized by sequencing of the complete capsid (P1) region. RT-PCR primers were designed to anneal to sites that encode amino acid motifs that are highly conserved among members of the species Human enterovirus B (HEV-B) (Oberste et al., 2004). Specific, non-degenerate primers were designed from preliminary sequences to close gaps between the original PCR products. Complete capsid sequences were determined by the primer-walking method. PCR products were purified for sequencing by the use of a High Pure PCR product purification kit (Roche). Both strands were sequenced by automated methods, by the use of fluorescent dideoxy chain terminators (Applied Biosystems). The complete genome sequences of the candidate prototype strains, USA/CA75-10213 and USA/OK85-10362, were also determined by the strategy of degenerate primer RT-PCR and sequencing by primer-walking.
Sequence analysis.
Nucleotide and deduced amino acid sequences of candidate EV74 and EV75 isolates were compared with one another and with those of all HEV-B serotypes by using the programs GAP and DISTANCES (Wisconsin Package, version 10.2; Accelrys). Nucleotide sequences were aligned by using the PILEUP program (Wisconsin Package) and were adjusted manually to conform to the optimized alignment of deduced amino acid sequences. Phylogenetic relationships were inferred from the aligned nucleic acid sequences by the neighbour-joining method, implemented in the programs DNADIST and NEIGHBOR (PHYLIP version 3.57; University of Washington, Seattle, WA, USA), by using the Kimura two-parameter substitution model (Kimura, 1980) and a transitiontransversion ratio of 10 (Oberste et al., 2004
). Support for specific tree topologies was estimated by bootstrap analysis with 1000 pseudoreplicate datasets. Branch lengths in consensus trees were calculated by the maximum-likelihood quartet-puzzling method, using the nucleotide substitution model of Tamura & Nei (1993)
, as implemented in Tree-Puzzle 5.0 (Strimmer & von Haeseler, 1996
).
Nucleotide sequence accession numbers.
The sequences that are described here have been deposited in GenBank under accession nos AY556057AY556070. The data have also been furnished to the Picornavirus Study Group of the International Committee on Taxonomy of Viruses (ICTV) in support of a proposal to establish the new types EV74 and EV75. The Picornavirus Study Group has agreed to act as registrar for new enterovirus types (G. Stanway, personal communication). For registration (and reservation of the next available number), the complete VP1 sequence must be submitted to the Study Group and must be <70 % identical to the VP1 sequences of all serotypes and proposed types that have been registered previously. The contact for registration is Dr Glyn Stanway (stanwg@essex.ac.uk), chair of the Picornavirus Study Group.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The French EV74 isolate and the United States EV74 and EV75 isolates were obtained from patients that were diagnosed with a variety of illnesses, including respiratory tract infections, neonatal disease and unspecified central nervous system disease (Table 1). Additional isolates of both EV74 and EV75 were obtained from stool specimens of children who presented with AFP during poliovirus surveillance activities in southern and western Asia. Non-polio enteroviruses are often isolated from the stools of children with AFP (Gear, 1984
; Grist & Bell, 1984
; Melnick, 1984
; Chaves et al., 2001
; Kapoor et al., 2001
; Oberste et al., 2001
; Santos et al., 2002
; Grimwood et al., 2003
), but isolation of a ubiquitous infectious agent from a non-sterile site is not sufficient to imply causality. On the other hand, it is likely that at least some non-polio AFP cases are attributable to infection with non-polio enteroviruses, as they have occasionally been isolated directly from neural tissues of paralysed patients (Chumakov et al., 1979
). Molecular identification of new enterovirus serotypes provides a tool to assist in the epidemiological investigation of AFP cases that are associated with non-polio enterovirus infection.
Whilst the non-translated regions and non-structural proteins may influence enterovirus replication and translation, the serotype-specific properties of an enterovirus are encoded in the viral capsid. Enteroviruses have been identified by the antigenic properties of the capsid for over 50 years (Bodian et al., 1949) and, more recently, by the partial sequences of their capsid-encoding genes (Oberste et al., 1999c
, 2003b
). The antigenic properties of the capsid are also critical for host immunity, as the host immune response is serotype-specific and the humoral response to capsid antigens is both necessary and sufficient for protection against most systemic enterovirus disease. Specific sites on the capsid surface also control receptor specificity and virus binding to the host cell. Receptor-binding specificity can contribute to cell and tissue tropism, which affect the distribution of virus replication sites within the infected host. Once the virus enters a host cell, the presence or absence of specific host-cell proteins may further restrict replication in a given tissue or cell type.
In previous studies, we and others have shown that complete or partial VP1 sequence correlates completely with antigenic typing by neutralization assay and that it may serve as a molecular surrogate for traditional serotyping methods (Oberste et al., 1999b, c
, 2000
, 2001
; Caro et al., 2001
; Casas et al., 2001
; Norder et al., 2001
). We have also recommended specific criteria for the interpretation of VP1 sequence data when a VP1 sequence is compared to reference sequences for all human enterovirus serotypes: (i) a partial or complete VP1 nucleotide sequence identity of
75 % (85 % amino acid sequence identity) between a clinical enterovirus isolate and serotype prototype strain may be used to establish the serotype of the isolate, on the provision that the second highest score is <70 %; (ii) a best-match nucleotide sequence identity of <70 % may indicate that the isolate represents an unknown (that is, new) serotype; and (iii) a sequence identity between 70 and 75 % indicates that further characterization is required before the isolate can be identified firmly (Oberste et al., 2000
, 2001
, 2003b
).
We recognize that the term serotype implies identification by antigenic means (usually by neutralization in the case of the human enteroviruses). The term genotype might be more acceptable to describe an identification that has been made by molecular methods, but this term has been used widely to discriminate among lineages of several enterovirus serotypes that have been defined genetically, including the polioviruses and coxsackieviruses A9 and B4 (Rico-Hesse et al., 1987; Mulders et al., 2000
; Santti et al., 2000
). The term genogroup has also been used in this way (Brown et al., 1999
; Oberste et al., 2003a
). We suggest that the term serotype should be maintained in the description of isolates that have been identified by using the VP1 sequence as a surrogate marker for antigenic type, until a consensus can be reached among enterovirologists on a more appropriate term. We also recommend that molecular typing, by complete sequencing of VP1 and application of these criteria, should be recognized as a substitute for the extensive cross-neutralization studies that have traditionally been required for the establishment of new enterovirus serotypes. Finally, we recommend that a complete capsid (P1) sequence should be determined for the strain that is proposed as the prototype of a new enterovirus serotype and, preferably, also for other isolates that are included in the original description of the serotype. Complete genome sequences, if made available, will facilitate additional studies (e.g. on the evolution of related viruses within a species). These additional data will allow for further validation by the virology community and contribute to refinement of molecular-typing criteria.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bodian, D., Morgan, I. M. & Howe, H. A. (1949). Differentiation of types of poliomyelitis viruses. III. The grouping of fourteen strains into three basic immunological types. Am J Hyg 49, 234245.
Brown, B. A., Oberste, M. S., Alexander, J. P., Jr, Kennett, M. L. & Pallansch, M. A. (1999). Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol 73, 99699975.
Brown, B., Oberste, M. S., Maher, K. & Pallansch, M. A. (2003). Complete genomic sequencing shows that polioviruses and members of human enterovirus species C are closely related in the noncapsid coding region. J Virol 77, 89738984.
Caro, V., Guillot, S., Delpeyroux, F. & Crainic, R. (2001). Molecular strategy for serotyping of human enteroviruses. J Gen Virol 82, 7991.
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 VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products. J Med Virol 65, 138148.[CrossRef][Medline]
Chaves, S. S., Lobo, S., Kennett, M. & Black, J. (2001). Coxsackie virus A24 infection presenting as acute flaccid paralysis. Lancet 357, 605.[CrossRef][Medline]
Chumakov, M., Voroshilova, M., Shindarov, L. & 16 other authors (1979). Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol 60, 329340.[Medline]
Gear, J. H. S. (1984). Nonpolio causes of polio-like paralytic syndromes. Rev Infect Dis 6 (Suppl. 2), S379S384.[Medline]
Grimwood, K., Huang, Q. S., Sadleir, L. G., Nix, W. A., Kilpatrick, D. R., Oberste, M. S. & Pallansch, M. A. (2003). Acute flaccid paralysis from echovirus type 33 infection. J Clin Microbiol 41, 22302232.
Grist, N. R. & Bell, E. J. (1984). Paralytic poliomyelitis and nonpolio enteroviruses: studies in Scotland. Rev Infect Dis 6 (Suppl. 2), S385S386.[Medline]
Kapoor, A., Ayyagari, A. & Dhole, T. N. (2001). Non-polio enteroviruses in acute flaccid paralysis. Indian J Pediatr 68, 927929.[Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111120.[Medline]
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. 657678. 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.
Melnick, J. L. (1984). Enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis. Rev Infect Dis 6 (Suppl. 2), S387S390.[Medline]
Mulders, M. N., Salminen, M., Kalkkinen, N. & Hovi, T. (2000). Molecular epidemiology of coxsackievirus B4 and disclosure of the correct VP1/2Apro cleavage site: evidence for high genomic diversity and long-term endemicity of distinct genotypes. J Gen Virol 81, 803812.
Norder, H., Bjerregaard, L. & Magnius, L. O. (2001). Homotypic echoviruses share aminoterminal VP1 sequence homology applicable for typing. J Med Virol 63, 3544.[CrossRef][Medline]
Norder, H., Bjerregaard, L. & Magnius, L. O. (2002). Open reading frame sequence of an Asian enterovirus 73 strain reveals that the prototype from California is recombinant. J Gen Virol 83, 17211728.
Norder, H., Bjerregaard, L., Magnius, L., Lina, B., Aymard, M. & Chomel, J.-J. (2003). 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. J Gen Virol 84, 827836.
Oberste, M. S., Maher, K., Kennett, M. L., Campbell, J. J., Carpenter, M. S., Schnurr, D. & Pallansch, M. A. (1999a). Molecular epidemiology and genetic diversity of echovirus type 30 (E30): genotypes correlate with temporal dynamics of E30 isolation. J Clin Microbiol 37, 39283933.
Oberste, M. S., Maher, K., Kilpatrick, D. R., Flemister, M. R., Brown, B. A. & Pallansch, M. A. (1999b). Typing of human enteroviruses by partial sequencing of VP1. J Clin Microbiol 37, 12881293.
Oberste, M. S., Maher, K., Kilpatrick, D. R. & Pallansch, M. A. (1999c). Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 73, 19411948.
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, 11701174.
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, 409416.
Oberste, M. S., Nix, W. A., Kilpatrick, D. R., Flemister, M. R. & Pallansch, M. A. (2003a). Molecular epidemiology and type-specific detection of echovirus 11 isolates from the Americas, Europe, Africa, Australia, southern Asia and the Middle East. Virus Res 91, 241248.[CrossRef][Medline]
Oberste, M. S., Nix, W. A., Maher, K. & Pallansch, M. A. (2003b). Improved molecular identification of enteroviruses by RT-PCR and amplicon sequencing. J Clin Virol 26, 375377.[CrossRef][Medline]
Oberste, M. S., Maher, K. & Pallansch, M. A. (2004). Evidence for frequent recombination within species Human enterovirus B based on complete genomic sequences of all thirty-seven serotypes. J Virol 78, 855867.
Pallansch, M. A. & Roos, R. P. (2001). Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Fields Virology, 4th edn, pp. 723776. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. Philadelphia, PA: Lippincott Williams & Wilkins.
Rico-Hesse, R., Pallansch, M. A., Nottay, B. K. & Kew, O. M. (1987). Geographic distribution of wild poliovirus type 1 genotypes. Virology 160, 311322.[Medline]
Santos, A. P., Costa, E. V., Oliveira, S. S., Souza, M. C. & Da Silva, E. E. (2002). RT-PCR based analysis of cell culture negative stools samples from poliomyelitis suspected cases. J Clin Virol 23, 149152.[CrossRef][Medline]
Santti, J., Harvala, H., Kinnunen, L. & Hyypiä, T. (2000). Molecular epidemiology and evolution of coxsackievirus A9. J Gen Virol 81, 13611372.
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964969.
Tamura, K. & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10, 512526.[Abstract]
Received 30 March 2004;
accepted 19 July 2004.