MediCity Research Laboratory and Department of Virology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland1
Department of Epidemiology and Health Promotion, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland2
Haartman Institute, Department of Virology, PO Box 21, FIN-00014 University of Helsinki, Helsinki, Finland3
Author for correspondence: Juhana Santti. Fax +358 2 2513303. e-mail juhana.santti{at}utu.fi
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Abstract |
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Introduction |
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Enteroviruses, like other RNA viruses, have a high mutation rate due to the lack of proofreading activity during genome replication. It has been estimated that approximately one mutation is generated per newly synthesized genome (Drake et al., 1993 ). In addition to mutations, recombination is involved in enterovirus evolution. Homologous recombination, which is considered to take place by copy choice (strand switching) (Kirkegaard & Baltimore, 1986
), has been demonstrated between PVs of vaccine- and wild-type origin (Cammack et al., 1988
; Furione et al., 1993
). Recent phylogenetic analysis of complete enterovirus genomes provided evidence suggesting that recombination also occurs between other enteroviruses, and that intraspecies recombination is a relatively frequent event in the evolution of enterovirus genomes (Santti et al., 1999
).
The high mutation rate of enteroviruses enables detailed molecular epidemiological studies based on comparison of relatively short regions of the genome. A 150 nucleotide (nt) sequence at the junction of the capsid and nonstructural proteins (90 nt encoding the VP1 capsid protein and 60 nt encoding the 2A protease) has been extensively used in molecular epidemiological studies of PVs, resulting in a large sequence database (Rico-Hesse et al., 1987 ; Zheng et al., 1993
; Huovilainen et al., 1995
; Kew et al., 1995
; Mulders et al., 1995
; Chezzi et al., 1997
; Morvan et al., 1997
; Fiore et al., 1998
). The term genotype, which is defined as a cluster of genetically related viruses with less than 15% nucleotide divergence across the 150 nt sequence, has been used for discrimination of genetic lineages of PVs, whereas less than 2% nucleotide divergence between strains has been considered to indicate direct epidemiological linkage (Rico-Hesse et al., 1987
). It has been demonstrated that within each PV serotype the independent genotypes usually show a distinct geographical distribution, but that occasionally long-distance importation of PV strains occurs. In addition to PVs, the molecular epidemiology of CAV24 variant and enterovirus 70, pathogens which have caused large outbreaks of acute haemorrhagic conjunctivitis, has been studied by partial sequencing (Ishiko et al., 1992
; Takeda et al., 1994
). Furthermore, regression analysis of genetic distances between isolates has enabled estimation of the time of emergence of these viruses. Partial genomic sequencing has also been applied to the molecular epidemiological analysis of CBV1 (Zoll et al., 1994
), CBV4 (Hughes et al., 1993
), CBV5 (Kopecka et al., 1995
) and enterovirus 71 (Brown et al., 1999
), as well as EV30 (Gjøen et al., 1996
) which is characteristically involved in epidemics of meningitis.
Analysis of the CAV9 prototype strain (Griggs) revealed that, despite its CAV-like pathogenicity in newborn mice, it is genetically more closely related to CBVs than to other CAVs (Chang et al., 1989 ; Pulli et al., 1995
). However, when compared to CBVs, the CAV9 genome has an apparent insertion of approximately 15 amino acids at the C terminus of the VP1 capsid protein. This insertion contains an RGD (arginine-glycine-aspartic acid) tripeptide, which is known to be the cell attachment motif of a number of adhesive extracellular matrix, blood and cell surface proteins, and is recognized by several integrins (Ruoslahti & Pierschbacher, 1987
; Ruoslahti, 1996
). In further studies it was demonstrated that the RGD motif, which was found to be fully conserved among five clinical CAV9 strains isolated in the UK over a period of 25 years (Chang et al., 1992
), mediates CAV9 attachment to
v
3 integrin (Roivainen et al., 1991
, 1994
). However, studies on trypsin-treated virus and virus mutants, which lack the RGD motif, indicated that the virus is able to use an RGD-independent pathway in cell entry (Roivainen et al., 1991
; Hughes et al., 1995
). The C-terminal region of the VP1 capsid protein was also shown to be antigenic by using a peptide-scanning technique, but it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sites of the motif (Pulli et al., 1998a
, b
).
In this study, 35 clinical isolates of CAV9, collected from different geographical regions during the last five decades, were subjected to molecular analysis. To explore phylogenetic relationships between the isolates in various genomic regions and to analyse variation, in particular at the C terminus of the VP1 protein, three regions, one covering the hypervariable part of the 5'NCR, VP4 gene and part of the VP2 capsid protein-coding region, another covering the junction region of the VP1 capsid protein and 2A protease genes, and the third encoding part of the 3D polymerase, were selected for RTPCR amplification and direct sequencing.
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Methods |
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A 652 bp amplicon representing the 5'NCR, VP4 and VP2 capsid protein-coding regions (nt 5471198; numbering according to the CAV9 Griggs strain; Chang et al., 1989 ) was obtained using primers 4+ (sense; 5' TACTTTGGGTGTCCGTGTTTC) and 5-n (antisense; 5' GGBAAYTTCCACCACCANCC). The VP1/2A junction region of the genomes was amplified using primers VP1+ (sense; 5' ACCAGAGCTTGGGTGCCGCG) and 2A- (antisense; 5' ACAACACCTTCNCCNCCCAT), which produce a 560 bp amplicon (nt 31803739). Two specimens (Net/1/64 and Net/1/79) that did not react with these primers were amplified using primers VP1+n (sense; 5' AACCCCAGCRTNTTYTGGAC) and 2A-n (antisense; 5' GTCTCTRTTRTAATCYTCCCA), which give rise to a 516 bp PCR product (nt 29463461). Primers 3D+ (sense; 5' TTTGAYTACWCWGGNTATGATGC) and 3D- (antisense; 5' WGSRTTCTTKGTCCATC) were used to amplify nt 66547184 (531 bp), which encode part of the 3D polymerase. The amplicons were purified using the Qiaex II Gel Extraction Kit (Qiagen) and sequenced with fluorescent dye-labelled terminators using an automated DNA Sequencer (Applied Biosystems) according to the manufacturers instructions. Primers 5-n, VP1+, VP1+n and 3D+ were used in the sequencing reactions. A schematic representation of the amplification and sequencing strategy is shown in Fig. 1
.
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Results |
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Genotypes found in the analysis of the VP1/2A junction region
PV genotypes have been defined as clusters of related strains with <15% nucleotide divergence in a 150 nt long sequence at the junction region between the VP1 capsid protein and the 2A protease genes (Rico-Hesse et al., 1987 ). By comparing an analogous region of the CAV9 genome (nt 32583407), the studied isolates and the Griggs strain could be assigned to 12 genotypes, which were designated IXII (Roman numerals) in the chronological order of the isolation of the earliest virus strain in each genotype (Fig. 2
). The maximum nucleotide divergence between the isolates was 34%, which was seen between strains Net/1/67 and US-MD/1/88. The prototype Griggs strain, which was isolated in the early 1950s, was the only representative of genotype I. Genotype II included the earliest isolates from the Netherlands from 19591967, which share a minimum of 89% nucleotide identity, and, in addition, an isolate from Denmark from 1971, which exhibits on average 88% identity with the Dutch isolates. Strains Net/1/59 and Net/1/61 share 98% identity in this genomic region, which is indicative of a direct epidemiological linkage between the isolates. Genotype III was represented by one isolate (Net/1/64) which is distant from all the other strains analysed. An isolate from the Netherlands from 1971 forms genotype IV. It shows 84% nucleotide identity to a geographically and temporally distant isolate from Thailand from 1993 (genotype X).
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The largest number of virus strains included in the study originated from Finland. Ten of the isolates, from 19931997, represent genotype XI. Strains isolated within each year share a minimum identity of 98% which, again, indicates direct epidemiological linkage. The only isolate from 1995 in this genotype is most closely related to the two isolates from 1993 sharing on average 97% nucleotide identity with these strains. Another isolate from Finland from 1995 represents genotype XII, which is distant from genotype XI but closer to the genotype VIII strains.
Phylogenetic analysis of the isolates
Phylogenetic grouping in the VP4/VP2 region.
To investigate the evolutionary relationships between the CAV9 isolates in the VP4/VP2-coding region, a neighbour-joining tree based on analysis of nt 7441154 (Griggs strain) of the CAV9 strains together with 11 representatives of other cluster B enteroviruses was constructed (Fig. 3). The two strains of CBV3 and CBV4 as well as EV9 clustered together with high bootstrap values. A group consisting of all the CAV9 isolates was supported by a bootstrap value of 61% whereas a subgroup containing all the other CAV9 isolates except the Net/1/64 strain was slightly better supported (74%). In general, virus strains within the CAV9 group segregated similarly to that observed in the analysis of the 150 nt interval at the VP1/2A junction region (Fig. 2
). Virus groups representing genotypes V, VI, IX and XI were supported by high bootstrap values. However, in the case of genotype II, only the relationship between the Dutch isolates was highly supported (100%; data not shown) whereas the whole group was supported in 63% of the bootstrap replicates. The bootstrap value for the group of strains representing genotype VIII was low (42%; data not shown); only clustering of strains Fin/1/88 and Net/1/81 was significantly supported. Of the remaining genotypes which were all represented by a single isolate, a monophyletic group consisting of genotype IV and X isolates was highly supported. In addition, statistically significant clustering was seen between genotypes I and VI. The maximum nucleotide divergence between the CAV9 isolates was 22%, which was observed between Net/1/64 and Can/1/85 as well as Net/1/67 and Fin/2/93.
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3D region.
To examine the evolutionary relationships of the CAV9 isolates at the 3'-terminal part of the genome, sequence encoding part of the 3D polymerase (nt 67207160 in Griggs strain) was determined from 29 isolates which represented 10 of the 12 genotypes recognized in the analysis of the VP1/2A region. Phylogenetic analysis of the CAV9 strains together with other representatives of cluster B viruses revealed a strikingly different grouping pattern compared to that obtained in the analysis of the other coding regions (Fig. 3). The CAV9 isolates did not cluster together, but they were instead interspersed among the other strains studied. In addition, none of the five genotypes, which were represented by more than one strain, formed monophyletic groups. In contrast, some groups, which contained both CAV9 isolates as well as other members of the enterovirus genus, were highly supported. The analysis also detected the close relationship between the Griggs prototype strain, CBV1, CBV3W and EV9H, that has been reported previously (Santti et al., 1999
). The maximum nucleotide divergence between the CAV9 isolates in this genome region was 23% which was seen between strains Net/1/71 and US-MS/1/78.
Amino acid sequence diversity among the isolates
The deduced amino acid sequences of the VP4/VP2, VP1/2A and 3D regions of the studied CAV9 sequences were aligned with the prototype Griggs strain together with eight representatives of other cluster B viruses as well as three antigenic variants (VP1/2A alignment is shown in Fig. 4). The VP4/VP2 amino acid sequences were highly conserved among these virus strains. Most of the amino acid differences in the VP4 capsid protein, which in picornaviruses is positioned internally in the virion, were concentrated on a relatively short region of the polypeptide between codons 1624. Variation in the N terminus of the VP2 capsid protein was limited to 19 of the 68 sites.
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Substitutions in the 2A region were relatively evenly distributed (Fig. 4). Histidine at position 21 and aspartate at position 39, which together with a cysteine residue form the catalytic triad of the entero- and rhinovirus 2A proteases (Bazan & Fletterick, 1988
; Yu & Lloyd, 1991
), were fully conserved. Little variation was seen in the 3D polymerase (data not shown). The YGDD sequence, which has been hypothesized to be at or near the catalytic site of the molecule (Kamer & Argos, 1984
; Jablonski et al., 1991
), was fully conserved among the isolates.
Analysis of the hypervariable region
The hypervariable region, covering approximately 100 nt preceding the polyprotein initiation codon, is the most variable part of the enterovirus genome (Toyoda et al., 1984 ; Rivera et al., 1988
). For example, the nucleotide sequences of the three PV Sabin strains differ from each other by about 50% in this region (Toyoda et al., 1984
). It has been suggested that this segment serves as a spacer region between the internal ribosome entry site and the polyprotein initiation codon; the ribosome scans through this region for the initiation site. The lack of stable secondary structures and initiation codons in this region are consistent with this hypothesis (Rivera et al., 1988
).
A dendrogram illustrating the relationships of the CAV9 strains in the hypervariable region (corresponds to nt 655743 of the Griggs strain) is shown in Fig. 5. The virus grouping was highly similar to that observed in the analysis of the 150 nt sequence at the VP1/2A junction region. The maximum nucleotide divergence between the isolates was 51%, which was seen between strains Net/1/67 and US-NC/1/83 as well as Net/1/67 and Hon/1/77. Despite the high overall diversity, strains within each VP1/2A region genotype shared >85% nucleotide identity. The strain Den/1/71 was an exception as it showed only 58% nucleotide identity to other genotype II isolates. In addition, strain Net/1/78 was slightly more distant from genotype V strains in this region (75% identity) than in the VP1/2A region (85% identity) whereas genotype VIII and XII viruses shared 85% nucleotide identity in this region.
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Discussion |
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Phylogenetic analysis of the CAV9 isolates together with 11 representatives of other cluster B enteroviruses revealed that the CAV9 strains formed a monophyletic group in the VP4/VP2 and VP1/2A regions but not in the 3D region. In the VP1/2A region, the CAV9 group was supported by a bootstrap value of 88% while in the VP4/VP2 region the group was found in 61% of the 1000 bootstrap replicates. These findings are consistent with previous observations that the serotype specificity is reflected in the capsid protein-coding sequence but not necessarily in other parts of the genome (Zoll et al., 1994 ; Kopecka et al., 1995
; Santti et al., 1999
). Furthermore, these results correlate well with the findings of Oberste et al. (1998
, 1999
), which indicate that the VP1 sequence represents the serotype identity better than the sequence of the VP4/VP2 junction region does.
The CAV9 isolates could be divided into 12 genotypes using the criteria designated for PVs (Rico-Hesse et al., 1987 ) that strains sharing >85% identity in a 150 nt sequence at the VP1/2A junction region belong to the same genotype (Table 2
). While most of the strains within each genotype showed geographical clustering, the analysis also showed evidence of long-distance importation of virus strains. In fact, three out of the six genotypes which were represented by more than one strain contained isolates both from Europe and the Americas. This finding suggests that individual CAV9 genotypes are frequently found over a wide geographical region, although more precise evaluation of geographical distribution of CAV9 genotypes cannot be made from this relatively limited data. For example, it cannot be concluded that genotype XI was geographically restricted to Finland since the study material did not contain any isolates from this time from any other country. It is also difficult to estimate the number of CAV9 genotypes which co-circulate at a given time. However, at least four genotypes were present in the Netherlands from 1978 to 1981. Appearance of several co-circulating genotypes is a typical phenomenon, at least for PVs in endemic countries with low vaccine coverage (Huovilainen et al., 1995
), but probably also occurs in other enteroviruses. Reliable estimation of the evolutionary rate of enterovirus genomes is usually complicated by the existence of multiple co-circulating lineages. In rare cases, when the founder virus has been identified, such calculations have, however, been possible. The VP1/2A sequence of PV type 1 introduced into the Andean region of South America in 1980 was estimated to have evolved at a nearly constant rate of 9x10-3 nt substitutions/site/year (Kew et al., 1995
). Accordingly, the maximum time for the existence of a given genotype has been evaluated to be approximately 17 years. Interestingly, all CAV9 genotypes found in the present study followed this rule.
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The different phylogenetic grouping of the CAV9 isolates in the 3D region compared to other genomic regions could most easily be explained by the occurrence of multiple recombination events between members of cluster B. This is supported by a recent analysis of 17 complete enterovirus genomes belonging to cluster B which detected several regions of higher than average sequence homology between individual virus strains, suggesting that intraspecies recombination occurs relatively frequently in the evolution of enterovirus genomes (Santti et al., 1999 ). This analysis also suggested that most of the putative recombination breakpoints between different serotypes were located at the region encoding the nonstructural proteins. It needs to be kept in mind, however, that the number of completely sequenced enterovirus genomes is still limited and restricted mainly to prototype strains isolated decades ago. More extensive analysis of different isolates may reveal currently undiscovered genetic relationships between and within the genetic clusters.
Previous studies have indicated that the RGD motif found within the C-terminal extension of the VP1 capsid protein of CAV9 (Chang et al., 1989 ) is functionally significant and plays a role in early viruscell interactions, because RGD-containing oligopeptides block CAV9 infectivity (Roivainen et al., 1991
). Moreover, it has been demonstrated that the RGD motif mediates CAV9 attachment to
v
3 integrin in GMK cells (Roivainen et al., 1994
). Sequence analysis of five temporally highly distinct CAV9 clinical isolates from the UK demonstrated that the motif was fully conserved (Chang et al., 1992
). The analysis also identified similarity of the VP1 extension of CAV9 to part of the precursor of human transforming growth factor
1, suggesting that the virus might have acquired this additional sequence by heterologous recombination with cellular sequences.
Previous investigations have also shown that infectivity of CAV9 is not completely abolished by removal of the RGD motif by trypsin (Roivainen et al., 1991 ) or by mutagenesis (Hughes et al., 1995
), which indicates that the virus is able to use an RGD-independent pathway in its entry into the host cell. In the present study, the RGD triplet was found to be fully conserved among 35 CAV9 clinical isolates which suggests that the motif, although not essential for virus viability in cell culture conditions, plays a vital role in replication of the virus in humans. The amino acid residues surrounding the RGD triplet were highly variable among the isolates reflecting a high degree of structural flexibility. As already noted by Chang et al. (1992)
, the situation resembles that seen in foot-and-mouth disease virus (FMDV), another integrin-binding picornavirus, in which the RGD motif is in a surface-accessible location and forms part of the major antigenic site of the virion (Acharya et al., 1989
; Fox et al., 1989
; Berinstein et al., 1995
). The RGD motif of FMDV has been shown to interact directly with some antiviral neutralizing antibodies (Verdaguer et al., 1995
) whereas analysis of CAV9 suggested that antibodies bind most strongly to amino acid residues surrounding the RGD motif while the motif itself is poorly immunogenic (Pulli et al., 1998a
, b
).
In conclusion, the present analysis of the CAV9 isolates provides an overview of the molecular epidemiology and evolution of CAV9. While most strains within each genotype were found to cluster geographically, the analysis also detected long-distance importation of virus strains. Analysis of sequences derived from three different genomic regions demonstrated that the capsid protein but not the nonstructural protein-coding region correlates with the serotype. In addition, the invariant nature of the RGD sequence provides further evidence for its importance in the clinical pathogenesis of CAV9 infection.
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Acknowledgments |
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Footnotes |
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Received 13 July 1999;
accepted 25 January 2000.