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, USA1
Viral and Rickettsial Diseases Laboratory, California Department of Health Services, Berkeley, CA 94704, USA2
Department of Laboratories, Directorate General of Health Affairs, Ministry of Health, 113 Muscat, Oman3
Author for correspondence: Steven Oberste. Fax +1 404 639 4011. e-mail soberste{at}cdc.gov
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Abstract |
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Introduction |
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The accepted approach to classification requires the investigator to generate antisera against each potentially new serotype and to perform reciprocal cross-neutralization testing, using a complete panel of prototype strains and antisera (Committee on Enteroviruses, 1962 ). The effect of this labour-intensive approach on the pace of enterovirus discovery was recognized as early as 1962, when a leader in the field remarked, new human enteroviruses are still being discovered. The rate of making new discoveries has slowed, probably only because the labours involved in establishing new serotypes are now so very great! (Wenner, 1962
). Since that time, only nine new human enterovirus serotypes have been identified (Melnick et al., 1974
; Panel for Picornaviruses, 1963
; Rosen & Kern, 1965
; Rosen et al., 1973
; Schieble et al., 1967
) and none have been identified in the last 25 years.
Recognizing the technical difficulties and limitations inherent in the classic approach to enterovirus identification, we have developed rapid molecular techniques for enterovirus typing (Oberste et al., 2000 , 1999a
, b
). In the present study, we describe a comparison of immunological and molecular methods for the characterization and classification of a group of related human enteroviruses isolated over a period of 40 years and propose the classification of these isolates as members of a new human enterovirus serotype.
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Methods |
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Immune sera were prepared against CA55-1988 and CA78-1480 in hamsters and against CA644454 in monkeys. Antisera were standardized with all other enterovirus serotypes, as previously described (Schnurr et al., 1996 ), and used to assess the antigenic relationships of CA55-1988, CA64-4454, CA78-1480 and OMA95-6498 by neutralization, using standard methods (Grandien et al., 1989
).
Physical characterization.
The California isolates were tested for stability to acid by incubation of the virus in pH 3·0 buffer for 1 h at 4 °C and inoculation of cell cultures after adjustment to pH 7 (Gwaltney et al., 1989 ). The isolates were purified by three terminal dilutions and further characterized by determination of size and morphology using electron microscopy, stabilization to heat by divalent cations, determination of genome type (DNA or RNA) and measurement of ether stability. The Oman isolates were not physically characterized.
Molecular characterization of viruses.
Viral RNA extraction, RTPCR, nucleotide sequencing and sequence analysis were performed as described previously (Oberste et al., 2000 , 1999a
). For initial molecular characterization, viral RNA from each isolate was amplified by RTPCR, using VP1 primers 012-011, 040-011, 187-222, 188-222 and 189-222, as described previously (Oberste et al., 2000
, 1999a
). The partial VP1 sequences were compared with a database of all complete enterovirus VP1 sequences (Oberste et al., 1999b
) and other picornavirus VP1 sequences that are available in the GenBank database, as described previously (Oberste et al., 2000
, 1999a
), to determine whether the isolates were genetically related to any known picornavirus serotype. Sequences spanning the 3' portion of the 5'NTR to the 5' end of VP2 were determined as described previously (Oberste et al., 1998
). Complete capsid sequences were determined using the primer-walking method to amplify and sequence the region between the 5'-NTR-VP4-VP2 and VP1 sequences. The complete genome sequence of CA55-1988 was also determined by primer walking.
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Results |
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Molecular serotyping and genetic relationships
The complete VP1 sequence (867 nucleotides) of each of the isolates was determined and compared with those of the human enterovirus prototype strains and of other picornaviruses. In all cases, the nearest taxa were viruses in the Enterovirus genus, and the highest pair-wise nucleotide sequence identity score was between 67 and 68% (Table 3). The highest scoring serotypes were members of enterovirus cluster B, which contains the coxsackie B viruses, the echoviruses, coxsackievirus A9 and EV69. Scores for comparisons with available human rhinovirus sequences were between 47 and 52%, similar to the scores for comparisons with cluster A enteroviruses (Table 3
). Scores for comparisons with picornaviruses of all other genera were below 44%. For the complete VP1 sequence comparisons, a highest identity score below 70% indicates that the query sequence is heterologous in serotype to all sequences in the database (Oberste et al., 2000
, 1999a
, b
), suggesting that the California and Oman isolates are unclassified members (a new serotype) of cluster B. The complete VP1 sequence of OMA95-6499 was identical to that of OMA95-6498, and therefore OMA95-6499 was not further characterized genetically.
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Discussion |
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Presumably, enteroviral structural diversity, and hence the number of possible serotypes, is constrained by the ability of different primary capsid sequences to form a functional viral particle capable of interacting with and infecting a susceptible host cell. However, the large number of enterovirus serotypes suggests that the viable conformation space may be very large, so that many more serotypes may remain to be discovered, either as historical, previously untyped enteroviruses, or as newly emerging serotypes. In either case, the molecular methods described here will be invaluable in rapidly identifying and characterizing these agents. For example, national enterovirus surveillance figures indicate that 3% of all enterovirus isolates for the years 1970 to 1983 (Strikas et al., 1986 ), and 3·8% of isolates for 1993 to 1996 (Centers for Disease Control and Prevention, 1997
), were reported by state public health laboratories as untyped enterovirus. In our own collection, more than 12% of isolates from the period 1962 to 1997 were originally reported as untypeable (Centers for Disease Control and Prevention, 1999
). This higher figure is partially explained by the fact that the Centers for Disease Control and Prevention is a reference centre and may be expected to receive those isolates that were most difficult to type in the primary clinical or public health laboratory. Our recent pilot study suggests that VP1 sequencing and database comparison will easily identify the vast majority of untypeable isolates as variants of known serotypes (Oberste et al., 2000
). In that study, we identified one potentially new serotype, represented by four isolates from three states, isolated between 1985 and 1987, and those isolates could be analysed by methods similar to those outlined in the present study. Broadly reactive VP1-specific primers are available to rapidly screen untypeable isolate collections for potentially novel serotypes (Oberste et al., 2000
, 1999a
).
Practical criteria must be established before molecular sequence information can be applied routinely to picornavirus identification. A partial or complete VP1 nucleotide sequence identity of at least 75% (minimum 85% amino acid sequence identity) between a clinical enterovirus isolate and serotype prototype strain may be used to establish the serotype of the isolate (Oberste et al., 2000 , 1999a
, b
). These criteria also appear to apply to comparisons among isolates of foot-and-mouth-disease virus (Vosloo et al., 1992
), but a study directly comparable to the enterovirus studies has not yet been performed. A best-match nucleotide sequence identity of between 70 and 75% or a second-highest score of greater than 70% may provide a tentative identification, pending confirmation by other means, such as neutralization with monospecific antisera (Oberste et al., 2000
) or more extensive sequencing. A best-match nucleotide sequence identity below 70% (less than 85% amino acid sequence identity) may indicate that the isolate represents an unknown serotype. Sequencing of the complete capsid-coding region may be useful in confirming this result, but complete capsid sequences are available for less than half of the known enterovirus serotypes, limiting the utility of complete capsid sequence comparisons. More extensive characterization, possibly including complete genome sequences, may be required for viruses that appear to represent previously unknown genera (Hyypiä et al., 1992
; Marvil et al., 1999
; Niklasson et al., 1999
; Yamashita et al., 1998
). Due to the high frequency of recombination among picornaviruses (King, 1988
; Kopecka et al., 1995
; Santti et al., 1999
), sequence information from non-capsid regions is of little value in characterizing new serotypes within known genera.
On the basis of the antigenic and molecular comparisons presented here, we propose that CA55-1988, CA64-4454, CA78-1480, OMA95-6498 and OMA95-6499 be recognized as isolates of a new human enterovirus serotype, enterovirus 73 (EV73), subject to approval by the appropriate taxonomic authority, and that CA55-1988 be designated as the prototype strain. RTPCR coupled with amplicon sequencing is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes. We propose this method as a general approach for the molecular classification of newly discovered picornaviruses and for the reclassification of known serotypes. In light of the increased use of molecular sequence data in virus classification (Calisher et al., 1995 ; Mayo & Pringle, 1998
; Van Regenmortel et al., 1997
) and the correlation of picornavirus capsid sequence data with classic antigenic typing, we further recommend that molecular data be given increased weight in the classification of picornaviruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 17 July 2000;
accepted 27 October 2000.