| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Department of Medical Microbiology, Laboratory of Clinical Virology, Academic Medical Center, Amsterdam, The Netherlands
2 Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
3 Virus Evolution Group, Centre for Infectious Diseases, University of Edinburgh, Edinburgh EH9 1AJ, UK
Correspondence
K. S. M. Benschop
k.s.benschop{at}amc.uva.nl
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU170302–EU170304 and AM933167–AM933171 (VP1) and EU170312–EU170340 and AM933159–AM933166 (3Dpol).
A supplementary table showing HPeV sequence data is available with the online version of this paper.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The parechovirus genome is approximately 7400 nt in length and encodes a large polyprotein within a single open reading frame flanked by 5' and 3' untranslated regions (UTRs). The polyprotein is cleaved post-translationally into three structural proteins (VP0, VP3 and VP1) and seven non-structural proteins (2A–2C and 3A–3D).
There are now six HPeV types classified based on their genetic sequences. HPeV1 and 2 were originally assigned to the genus Enterovirus as echovirus serotypes 22 and 23, respectively. However, these two viruses were shown to be genetically distinct from the entire genus Enterovirus and also from other genera within the family Picornaviridae, prompting their current classification as members of the genus Parechovirus along with Ljungan viruses isolated from rodents (Stanway et al., 2005). A further HPeV variant, originally described as ‘type 2’ (CT86-6760; Oberste et al., 1998), was shown to be different from the prototype HPeV2 sequence (Ghazi et al., 1998) and was reclassified as type 5 (Al-Sunaidi et al., 2007). Novel HPeV types have subsequently been classified as HPeV3 (Ito et al., 2004), HPeV4 (Al-Sunaidi et al., 2007; Benschop et al., 2006a) and HPeV6 (Watanabe et al., 2007). HPeVs have predominantly been isolated from young infants and are commonly associated with mild gastrointestinal and respiratory symptoms, but more severe conditions, such as paralysis (Figueroa et al., 1989; Ito et al., 2004), neonatal sepsis (Benschop et al., 2006a; Boivin et al., 2005) and bronchiolitis (Abed & Boivin, 2006), have also been reported. Although HPeVs are considered to be a widespread pathogen, they remain largely undiagnosed due to the lack of HPeV-specific screening tools, such as RT-PCR. Therefore, little is known about the spread and pathogenicity of these viruses.
Previous analysis of novel HPeV types has indicated that recombination might play a role in the evolution of HPeV (Al-Sunaidi et al., 2007; Benschop et al., 2006b). The occurrence of recombination may have a profound impact on the spread and pathogenicity of RNA viruses in a population (Kendal, 1987; Minor, 1992; Robertson et al., 1995). Recombination has been documented extensively in human enteroviruses and was found to play a major role in the evolution of these viruses (Lindberg et al., 2003; Lukashev, 2005; Oberste et al., 2004a, b; Santti et al., 1999; Simmonds & Welch, 2006).
To study the likelihood and dynamics of recombination within parechoviruses, we analysed several HPeV sequences obtained from two distant regions within the genome (VP1 and 3Dpol). In total, 37 HPeV isolates were obtained from the Netherlands (n=29), California (n=6) and Finland (n=2). A full listing of the HPeV sequences used is available as Supplementary Table S1 in JGV Online. Twenty-nine samples were sequenced and typed previously based on the VP1 region and submitted to GenBank under the accession numbers DQ172416, DQ172418, DQ172420–DQ172421, DQ172424–DQ172428, DQ172430, DQ172432–DQ172433, DQ172435–DQ172446, DQ172448, DQ172451 (Dutch isolates containing six-digit numbers, prefixed with NL; Benschop et al., 2006a), AM234724, AM234726 and AM234728 (Californian isolates; Al-Sunaidi et al., 2007; Schnurr et al., 1996). The remaining samples were typed within the VP1 region as described previously (Al-Sunaidi et al., 2007; Benschop et al., 2006b). To determine partial 3Dpol sequences of each HPeV isolate, RNA was extracted by using a QIAamp Viral RNA mini kit according to the manufacturer's instructions (Qiagen). The extracted RNA was reverse-transcribed and amplified by using a SuperScript III One-Step RT-PCR kit (Invitrogen) according to the manufacturer's instructions. The following primers were designed within the conserved region within the 3Dpol sequence by aligning all known HPeV types: OL1502 (5'-GTGTACAGGATGATCATGATGGA-3', nt 6419–6441) and OL1501 (5'-CTTAGTCAAACACCATGGGCA-3', nt 7253–7233), where nucleotide numbering is relative to that of the HPeV1 Harris strain (GenBank accession no. S45208). Amplicons were isolated by agarose-gel electrophoresis and purified by using a QIAquick gel extraction kit according to the manufacturer's instructions (Qiagen), using a spin-column protocol. DNA was eluted in 50 µl water. BigDye Terminator sequencing reactions were performed by GeneService, Cambridge, UK (http://www.geneservice.co.uk).
The HPeV VP1 and 3Dpol sequences were aligned by using CLUSTAL W (Thompson et al., 1994) and edited manually by using the SIMMONICS sequence editor (version 1.6; http://www2.warwick.ac.uk/fac/sci/bio/research/devans/bioinformatics/simmonics/). Neighbour-joining phylogenetic trees based on the nucleotide sequence were constructed separately for the VP1 and 3Dpol regions of the HPeV genome by using the MEGA 3.1 software package (Kumar et al., 2004) with Jukes–Cantor (J-C)-corrected distances (Jukes & Cantor, 1969) (Fig. 1). Sequence data were bootstrap-resampled 1000 times to determine robustness of the observed grouping; branches supported by >70 % of replicate trees are indicated. All available full-length sequences for HPeV were obtained from GenBank and were included in the analysis: HPeV1 strains Harris (S45208) and BNI-788St (EF051629); HPeV2 strain Williamson (AJ005695); HPeV3 strains A308-99 (AB084913) and Can82853-01 (AJ889918); HPeV4 strains K251176-02 (DQ315670) and T75-4077 (AM235750); HPeV5 strains CT86-6760 (AF055846) and T92-15 (AM235749); HPeV6 strains NII561-2000 (AB252582) and BNI-67/03 (EU024629).
|
). However, within the 3Dpol region (706 nt), a breakdown of the type-specific segregation was observed; sequences in this region of HPeV1, 4, 5 and 6 frequently failed to group according to type. The majority of recently isolated HPeV1 strains showed 3Dpol sequences distinct from that of the prototype HPeV1 strain Harris, first isolated in 1956. As an initial indication of the time-related nature of the recombination events in HPeV, isolates that clustered closely together in the VP1 region (pairwise-compared J-C distances of <0.0601) almost invariably remained together in the 3Dpol region. When a greater VP1 divergence between HPeV1 isolates was observed, a loss of segregation was consistently observed in the 3Dpol region. These findings are consistent with the previously observed incongruent phylogenetic relationships within the non-structural region of HPeV4 and 5 (Al-Sunaidi et al., 2007; Benschop et al., 2006b).
To test formally whether the loss of segregation between variants within each of the parechovirus types was related to their degree of evolutionary and epidemiological separation (as indicated by their divergence in VP1), members of the same HPeV type were classified further by their bootstrap-supported phylogenetic groupings within the 3Dpol region. Subsequent pairwise comparison of sequences recorded their VP1 sequence divergence (evolutionary separation) and whether the two variants remained clustered in 3Dpol. The likelihood of recombination (i.e. separate grouping of two types in the 3Dpol region) increased steadily with VP1 sequence divergence (Fig. 2a), indicative of time-related recombination comparable to that observed for human enterovirus species A and B sequences (HEV-A, -B; Simmonds & Welch, 2006). Although different HPeV isolates were obtained from different geographical locations and over different collection periods, measurement of VP1 divergence provided an independent measure of their temporal separation from each other, and thus provided a robust comparator with recombination frequency. Furthermore, this independent measure of divergence time allowed us to compare the dynamics of recombination directly with those of HEV-A and -B.
|
) containing newly published, full-length sequences from 2006–2007. A full listing of the HEV sequences used is available as Supplementary Table S1 in JGV Online. It was not possible to perform a parallel analysis of recombination frequency in HEV-C or HEV-D, because of a lack of published complete-genome sequences of epidemiologically independent isolates (although there are several available HEV-C sequences, many are from vaccine-derived poliovirus strains). The dynamics of recombination for HPeV variants were remarkably similar to those of the larger HEV-B sequence dataset (Fig. 2a, black and white bars). This similarity extended to a second comparison of recombination frequency and differences in isolation dates (Fig. 2b), an alternative measure of temporal separation of isolates, but one that does not take geographical separation into account. Consistent with previous findings, the time course (measured by both VP1 divergence and isolation-time differences) of recombination of HEV-A variants was substantially slower than that of HEV-B. Separate analysis of specific HPeV and HEV types showed a similar pattern of increase in recombination when types were analysed within a species (data not shown).
Despite the detection of frequent recombination events in parechoviruses, recombination was never observed among HPeV3 sequences (Fig. 1). Although the majority of the HPeV3 isolates were isolated within the same year (see Supplementary Table S1, available in JGV Online) and clustered tightly together, a measurable proportion of recombination was detected among HPeV1, 4, 5 and 6 types that were similarly divergent in VP1 (approx. one-fifth of pairwise comparisons where VP1 divergence <0.025 showed recombination). In addition, 50 % of non-HPeV3 variants isolated in the same year were recombinant, compared with a frequency of zero for HPeV3 (Figs 1 and 2). These observations suggest possible biological constraints that limit HPeV3 recombination events. In HEVs and other picornavirus genera, the high degree of sequence divergence between species in the non-structural region previously appeared to be the main factor limiting inter-species recombination (Simmonds, 2006). However, analysis of all available full-length sequences (n=11; Fig. 3) showed that HPeV3 was similarly divergent from other parechovirus types (green line) as the latter were from each other (red line). The only exception was the slightly greater sequence divergence between HPeV3 and other types at the C terminus of VP1. This very local region of greater divergence corresponds to the part of the HPeV3 VP1 sequence where the RGD integrin-binding sequence is absent. As the RGD motif in other HPeV types was found to be critical for replication (Boonyakiat et al., 2001), its absence in HPeV3 suggests the use of a different (non-integrin) receptor for entry. Different receptor use can result in a change in cellular tropism and might account for different clinical outcomes observed in HPeV infections, such as more severe disease and central nervous system involvement (Benschop et al., 2006a; Boivin et al., 2005; Ito et al., 2004). Infection of different cell types in vivo might additionally reduce the opportunity for recombination between HPeV3 and other HPeV types to occur, and therefore potentially account for the failure to detect such recombinants in our genetic survey.
|
). Through calculation of a segregation score in the program TreeOrder Scan in the SIMMONICS sequence editor package (version 1.6; http://www2.warwick.ac.uk/fac/sci/bio/research/devans/bioinformatics/simmonics/), grouping by virus type was observed throughout the structural region, a zone strictly demarcated by the 5'-UTR/VP0 and S/NS (VP1/2A) gene boundaries, the latter also being characterized by a dramatic decline in amino acid sequence variability and dN/dS ratio. These observations for HPeV show striking similarities to the pattern of sequence diversity and recombination frequency in other picornaviruses and other mammalian, non-enveloped, positive-stranded RNA viruses (Simmonds, 2006). As a unifying hypothesis for these disparate observations, the dramatically higher dN/dS ratio in the structural gene region, combined with its much higher amino acid sequence divergence compared with the rest of the genome, provides further evidence for positive selection operating on the exposed outer surface of the virus. Immune-mediated selection may drive changes in its antigenicity and, combined with changes in receptor use (such as the deletion of the RGD motif in HPeV), ultimately generate new serotypes refractory to immunity to previously encountered HPeV variants. As proposed previously (Simmonds, 2006), the resulting high amino acid sequence divergence and biological incompatibility may be the key factor, thus preventing the occurrence of recombination in the structural gene region. A second, smaller increase in amino acid diversity and dN/dS ratios was also observed within the 3AB region, although whether this reflects positive selection or less constraint on amino acid sequence change for these amino acids, and for those in the homologous region in HEVs, is unknown. Patterns of amino acid sequence divergence, location of breakpoints and codon usage in parechoviruses revealed in this and previous studies (Simmonds, 2006) thus closely mirror those of other picornavirus groups in which extensive recombination between serotypes has been documented. This is the first systematic survey of recombination frequencies and temporal dynamics in parechoviruses, and has generated comparative sequence data of VP1 and 3Dpol regions from several HPeV isolates from three different geographical locations. Although there is a limited number of HPeV isolates characterized genetically to date, the limited HPeV dataset used showed a specific evolutionary trend that is also found in other picornaviruses. Recombination may play a major role in the evolution of this virus genus, and was found to occur with similarly rapid temporal dynamics as HEVs. However, more full-length data are needed to study recombination within HPeVs further. The data presented provide further knowledge for studying the molecular evolution and epidemiology of HPeVs and a basis for in vitro pathogenesis studies, particularly between HPeV3 and other HPeV types.
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Al-Sunaidi, M., Williams, Ç. H., Hughes, P. J., Schnurr, D. P. & Stanway, G. (2007). Analysis of a new human parechovirus allows the definition of parechovirus types and the identification of RNA structural domains. J Virol 81, 1013–1021.
Benschop, K. S., Schinkel, J., Minnaar, R. P., Pajkrt, D., Spanjerberg, L., Kraakman, H. C., Berkhout, B., Zaaijer, H. L., Beld, M. G. & Wolthers, K. C. (2006a). Human parechovirus infections in Dutch children and the association between serotype and disease severity. Clin Infect Dis 42, 204–210.[CrossRef][Medline]
Benschop, K. S. M., Schinkel, J., Luken, M. E., van den Broek, P. J. M., Beersma, M. F. C., Menelik, N., van Eijk, H. W. M., Zaaijer, H. L., VandenBroucke-Grauls, C. M. J. E. & other authors (2006b). Fourth human parechovirus serotype. Emerg Infect Dis 12, 1572–1575.[Medline]
Boivin, G., Abed, Y. & Boucher, F. D. (2005). Human parechovirus 3 and neonatal infections. Emerg Infect Dis 11, 103–105.[Medline]
Boonyakiat, Y., Hughes, P. J., Ghazi, F. & Stanway, G. (2001). Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry. J Virol 75, 10000–10004.
Figueroa, J. P., Ashley, D., King, D. & Hull, B. (1989). An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22. J Med Virol 29, 315–319.[Medline]
Ghazi, F., Hughes, P. J., Hyypiä, T. & Stanway, G. (1998). Molecular analysis of human parechovirus type 2 (formerly echovirus 23). J Gen Virol 79, 2641–2650.[Abstract]
Ito, M., Yamashita, T., Tsuzuki, H., Takeda, N. & Sakae, K. (2004). Isolation and identification of a novel human parechovirus. J Gen Virol 85, 391–398.
Jukes, T. & Cantor, C. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kendal, A. P. (1987). Epidemiologic implications of changes in the influenza virus genome. Am J Med 82, 4–14.[Medline]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lindberg, A. M., Andersson, P., Savolainen, C., Mulders, M. N. & Hovi, T. (2003). Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species. J Gen Virol 84, 1223–1235.
Lukashev, A. N. (2005). Role of recombination in evolution of enteroviruses. Rev Med Virol 15, 157–167.[CrossRef][Medline]
Minor, P. D. (1992). The molecular biology of poliovaccines. J Gen Virol 73, 3065–3077.
Oberste, M. S., Maher, K. & Pallansch, M. A. (1998). Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses. Virus Res 56, 217–223.[CrossRef][Medline]
Oberste, M. S., Maher, K. & Pallansch, M. A. (2004a). Evidence for frequent recombination within species human enterovirus B based on complete genomic sequences of all thirty-seven serotypes. J Virol 78, 855–867.
Oberste, M. S., Penaranda, S. & Pallansch, M. A. (2004b). RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses. J Virol 78, 2948–2955.
Robertson, D. L., Sharp, P. M., McCutchan, F. E. & Hahn, B. H. (1995). Recombination in HIV-1. Nature 374, 124–126.[Medline]
Santti, J., Hyypiä, T., Kinnunen, L. & Salminen, M. (1999). Evidence of recombination among enteroviruses. J Virol 73, 8741–8749.
Schnurr, D., Dondero, M., Holland, D. & Connor, J. (1996). Characterization of echovirus 22 variants. Arch Virol 141, 1749–1758.[CrossRef][Medline]
Simmonds, P. (2006). Recombination and selection in the evolution of picornaviruses and other mammalian positive-stranded RNA viruses. J Virol 80, 11124–11140.
Simmonds, P. & Welch, J. (2006). Frequency and dynamics of recombination within different species of human enteroviruses. J Virol 80, 483–493.
Stanway, G. & Hyypiä, T. (1999). Parechoviruses. J Virol 73, 5249–5254.
Stanway, G., Brown, F., Christian, P., Hovi, T., Hyypiä, T., King, A. M. Q., Knowles, N. J., Lemon, S. M., Minor, P. D. & other authors (2005). Family Picornaviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 757–778. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Watanabe, K., Oie, M., Higuchi, M., Nishikawa, M. & Fujii, M. (2007). Isolation and characterization of novel human parechovirus from clinical samples. Emerg Infect Dis 13, 889–895.[Medline]
Received 4 October 2007;
accepted 7 January 2008.
This article has been cited by other articles:
|
|
 |
|
  C. H. Williams, M. Panayiotou, G. D. Girling, C. I. Peard, S. Oikarinen, H. Hyoty, and G. Stanway Evolution and conservation in human parechovirus genomes J. Gen. Virol., July 1, 2009; 90(7): 1702 - 1712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tolf, M. Gullberg, E. S. Johansson, R. B. Tesh, B. Andersson, and A. M. Lindberg Molecular characterization of a novel Ljungan virus (Parechovirus; Picornaviridae) reveals a fourth genotype and indicates ancestral recombination J. Gen. Virol., April 1, 2009; 90(4): 843 - 853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zoll, J. M. D. Galama, and F. J. M. van Kuppeveld Identification of Potential Recombination Breakpoints in Human Parechoviruses J. Virol., April 1, 2009; 83(7): 3379 - 3383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Benschop, X. Thomas, C. Serpenti, R. Molenkamp, and K. Wolthers High Prevalence of Human Parechovirus (HPeV) Genotypes in the Amsterdam Region and Identification of Specific HPeV Variants by Direct Genotyping of Stool Samples J. Clin. Microbiol., December 1, 2008; 46(12): 3965 - 3970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Harvala, I. Robertson, E. C. McWilliam Leitch, K. Benschop, K. C. Wolthers, K. Templeton, and P. Simmonds Epidemiology and Clinical Associations of Human Parechovirus Respiratory Infections J. Clin. Microbiol., October 1, 2008; 46(10): 3446 - 3453. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
