Department of Virology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden1
Author for correspondence: Lars Magnius. Fax +46 8 32 83 30. e-mail lars.magnius{at}smi.ki.se
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Abstract |
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Introduction |
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The junction between the P1 region, and the P2 and P3 regions, may be the site for recombination events, which do not change the serotype or affect important virus functions. Such recombinants between wild-type and vaccine strains of polioviruses, as well as between different enterovirus types belonging to the same species, have been described (Furione et al., 1993 ; Santti et al., 1999
; Guillot et al., 2000
).
The Enterovirus genus comprises 65 types, which are assigned to four genomic groups or species designated human enterovirus (HEV-A through D), based on degree of similarity within the VP2 region (Pöyry et al., 1994 , 1996
; Dahllund et al., 1995
; Pulli et al., 1995
; Huttunen et al., 1996
; Oberste et al., 1999a
). Coxsackie A virus 28, 10, 12, 14 and 16 and enterovirus (EV) 71 belong to HEV-A. All echo- and coxsackie B (CB) viruses, coxsackie A9 virus, EV69 and EV73 form HEV-B. Coxsackie A virus 1, 11, 13, 15, 1722 and 24 form group HEV-C. Poliovirus 13 also cluster within HEV-C, although they have distinct receptor usage and unique clinical features of infection. HEV-D is formed by EV68 and EV70 (Pöyry et al., 1996
; Hyypiä et al., 1997
; Oberste et al., 1999b
).
Classical typing of enteroviruses relies on virus neutralization in cell cultures with pools of type-specific polyclonal antisera followed up by neutralization with monospecific antisera. Recently, enterovirus strains belonging to the same type have been shown to share sequence similarity within the VP1 region (Oberste et al., 1999a , b
, 2000
; Norder et al., 2001
). Although, the exact molecular counterpart, i.e. the type-specific amino acid residues of the capsid proteins that define the different enterovirus serotypes, remains to be elucidated, this sequence similarity enables molecular typing of enteroviruses (Oberste et al., 1999a
, b
; Norder et al., 2001
). VP1 sequencing has also been applied to strains not neutralized by available antisera to investigate whether these are just divergent or represent putative new types (Oberste et al., 2000
).
A new enterovirus type within HEV-B designated enterovirus 73 (EV73) was recently proposed (Oberste et al., 2001 ). We report here on three enterovirus isolates with Asian origins showing >91% sequence identity to EV73. The complete polyprotein of an isolate originating from Korea was deduced by sequencing and compared with that of the EV73 prototype. Part of the P2 region for all three strains was also compared with that of the prototype.
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Methods |
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Molecular methods.
RNA extraction and reverse transcription were performed as described (Norder et al., 2001 ). The VP1 region was amplified by PCR with primers 76C/62C and sequenced in both directions (Norder et al., 2001
). The complete ORF of the genome of 2776/82 was amplified with primers 6N (sense; 5' GTACCTTTGTGAGCCTGTT 3') and 3PR (antisense; 5' TAWMGRARAATTTACCCCYAC 3') with the Expand Long Template PCR System, according to the manufacturer (Roche Diagnostics). The amplificate was sequenced by primer walking in both directions using cycle sequencing with fluorescein-labelled dideoxy-chain terminators and the reagents in the BigDye sequencing kit (Applied Biosystems). Primers used in the sequencing reactions are available upon request.
The P2 region of isolates 57/99 and 22/00 was amplified with primers 296 (sense; 5' TGGGNGGHGARGGNGTIGTIGGNTT 3') and 1471 (antisense; 5' TGACNGGRCARCACTCNTCRTCACA 3'). The amplificate was sequenced in both directions with the primers used in the PCR and primer 1295 (5' GCATYYTGTTYACBTCHCCRTTYGT 3') as sequencing primers.
Sequence analysis.
The VP1 sequences of the three isolates were aligned with those of 127 enterovirus strains belonging to HEV-A through D. The sequence of the complete P1 region for 2776/82 was aligned with those of 54 enterovirus strains retrieved from GenBank. The sequence obtained of the P2 and P3 regions of strain 2776/82 as well as 1188 nucleotides of the P2 region of strains 57/99 and 22/00 were aligned with the corresponding sequence of 51 enterovirus strains retrieved from GenBank. The number of nucleotides and predicted amino acid differences were calculated with the Mega program package, version 1.02 (Kumar et al., 1993 ). Genetic distances were calculated using the Kimura two-parameter method for the nucleotide sequences and the Dayhoff PAM matrix for the deduced amino acid sequences. The dendrograms were constructed using the UPGMA and neighbour-joining algorithms in the PHYLIP 3.57 program package (Felsenstein, 1993
). Bootstrap anaysis was performed on 1000 replicas using the programs Seqboot and Consense (Felsenstein, 1993
). Similarity and boot-scanning analyses were performed using the SimPlot program (Lole et al., 1999
; Ray, 1997
) and a boot-scanning program package using the PHYLIP package for phylogenetic analysis (Salminen & Cobb, 1998
). The sequences were also analysed using the Recombinant Identification Program (RIP) (Siepel & Korber, 1995
).
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Results |
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In a dendrogram based on part of the P2 region, nucleotides 35934780, isolates 2776/82, 57/99 and 22/00 formed a separate cluster, while CA55-1988 formed a cluster with CB3 strain Woodruff (Fig. 4). In the compared region isolates 2776/82, 57/99 and 22/00 diverged by 3·9 to 7·5% from each other, by 20·6 to 21·3% from CA55-1988, and 17·2 to 21·3% from other HEV-B members.
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Discussion |
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The P2 and P3 regions of 2776/82 showed a high divergence from that of CA55-1988. While 2776/82 was divergent from all HEV-B members sequenced so far in these regions, CA55-1988 was most similar to CB3. The difference between the EV73 strains was also evident in the compared part of the P2 region, where the three Asian strains formed a separate cluster, while the prototype strain CA55-1988 clustered with CB3. The P2 and P3 regions may vary significantly between homotypic strains due to recombinations involving different serotypes (Furione et al., 1993 ; Santti et al., 1999
; Guillot et al., 2000
). Thus the CA55-1988 strain seems to be a recombinant between a parental EV73 strain and CB3. Since CA78-1480 and OMAN95 cluster with CA55-1988 in the P1 region, it would be of interest to characterize the P2 regions of the former strains, and in particular OMAN95, to elucidate if the recombination event occurred before or after CA55-1988 like strains were imported to California.
The finding that CA55-1988 is a recombinant virus has relevance with regard to CA64-4454 being a prime strain. CA64-4454 cannot have been formed by genetic drift from CA55-1988, since only the latter strain should be recombinant, considering that none of our three Asian isolates in the same genetic subcluster as CA64-4454 was recombinant. The two subclusters of EV73 may represent two different genotypes, the geographical distribution of which should be further investigated and include limited sequencing within VP1 and P2 regions. This will also elucidate if the recombinant strain has spread outside California. Further analysis of EV73 strains will reveal whether the recombination event occurred in the United States or in Asia, and will elucidate the evolutionary history of this virus. The future description of recombinants for other enteroviruses may therefore be important to understand the evolution and origins of these viruses in general.
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Footnotes |
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References |
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Felsenstein, J. (1993). PHYLIP: phylogeny inference package, version 3.52c. University of Washington, Seattle, Washington, USA.
Furione, M., Guillot, S., Otelea, D., Balanant, J., Candrea, A. & Crainic, R. (1993). Polioviruses with natural recombinant genomes isolated from vaccine associated paralytic poliomyelitis. Virology 196, 199-208.[Medline]
Grandien, M., Forsgren, M. & Ehrnst, A. (1989). Enterovirus and reovirus. In Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections , pp. 513-569. Edited by E. H. Lennette & N. J. Schmidt. Washington, DC:American Public Health Association.
Guillot, S., Caro, V., Cuervo, N., Korotkova, E., Combiescu, M., Persu, A., Combiescu, A., Delpeyroux, F. & Crainic, R. (2000). Natural genetic exchanges between vaccine and wild poliovirus strains in humans. Journal of Virology 74, 8434-8443.
Huttunen, P., Santii, J., Pulli, T. & Hyypiä, T. (1996). The major echovirus group is genetically coherent and related to coxsackie B viruses. Journal of General Virology 77, 715-725.[Abstract]
Hyypiä, T., Hovi, T., Knowles, N. J. & Stanway, G. (1997). Classification of enteroviruses based on molecular and biological properties. Journal of General Virology 78, 1-11.
Kumar, S., Tamura, K. & Nei, M. (1993). Molecular Evolutionary Genetics Analysis Version 1.02. Pennsylvania State University, Pennsylvania, USA.
Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. Journal of Virology 73, 152-160.
Norder, H., Bjerregaard, L. & Magnius, L. O. (2001). Homotypic echoviruses share aminoterminal VP1 sequence homology applicable for typing. Journal of Medical Virology 63, 35-44.[Medline]
Oberste, M. S., Maher, K. & Pallansch, M. A. (1999a). Molecular phylogeny of all human enterovirus serotypes based on comparison of sequences at the 5' end of the region encoding VP2. Virus Research 58, 35-43.
Oberste, M. S., Maher, K., Kilpatrick, D. R., Flemister, M. R., Brown, B. A. & Pallansch, M. A. (1999b). Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. Journal of Virology 73, 1941-1948.
Oberste, M. S., Maher, K., Flemister, M. R., Marchetti, G., Kilpatrick, D. R. & Pallansch, M. A. (2000). Comparison of classic and molecular approches for the identification of untypable' enteroviruses. Journal of Clinical Microbiology 38, 1170-1174.
Oberste, M. S., Schnurr, D., Maher, K., al-Busaidy, S. & Pallansch, M. A. (2001). Molecular identification of a new picornavirus and characterization of a proposed enterovirus 73 serotype. Journal of General Virology 82, 409-416.
Pöyry, T., Hyypiä, T., Horsnell, C., Kinnunen, L., Hovi, T. & Stanway, G. (1994). Molecular analysis of coxsackie A16 reveals a new genetic group of enteroviruses. Virology 202, 982-987.[Medline]
Pöyry, T., Kinnunen, L., Hyypiä, T., Brown, B., Horsnell, C., Hovi, T. & Stanway, G. (1996). Genetic and phylogenetic clustering of enteroviruses. Journal of General Virology 77, 1699-1717.[Abstract]
Pulli, T., Koskimes, P. & Hyypiä, T. (1995). Molecular comparison of coxsackie A virus serotypes. Virology 211, 30-38.
Ray, S. C. (1997). SimPlot for Windows 95/NT, version 1.2.2 (distributed by author). http://sray.med.som.jhmi.edu/RaySoft.
Salminen, M. O. & Cobb, W. (1998). Boot scanning package for Unix/Linux version 1. National Public Health Institute, Helsinki, Finland.
Santti, J., Hyypiä, T., Kinnunen, L. & Salminen, M. (1999). Evidence of recombination among enteroviruses. Journal of Virology 73, 8741-8749.
Siepel, A. C. & Korber, B. T. (1995). Scanning the database for recombinant HIV-1 genomes. Human Retroviruses and AIDS Compendium III, 3560.
Received 12 November 2001;
accepted 14 March 2002.