Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia1
Division of Virology, TVW Telethon Institute for Child Health Research, Perth, WA, Australia2
Author for correspondence: K. B. Chua. Fax +60 3 7958 4844. e-mail chuakb{at}ummc.edu.my
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Abstract |
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Introduction |
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With the exception of the polioviruses, distinct clinical entities associated with particular enterovirus serotypes are rare, although several defined clinical entities caused by specific enterovirus serotypes have been well described (Melnick, 1996 ). One of the best-known examples is hand, foot and mouth disease (HFMD), a febrile papulovesicular eruption most frequently associated with coxsackievirus A16 (CA16) infection. Several other serotypes within the human enterovirus A species [CA4, CA5, CA9 and CA10 and enterovirus 71 (EV71)] have also been reported to cause HFMD (Melnick, 1996
).
HFMD is endemic in Malaysia. In 1997, a large outbreak of HFMD resulted in the deaths of 41 children due to acute brainstem encephalitis (Lum et al., 1998a , b
). Both CA16 and EV71 were isolated from cases of HFMD, but EV71 was the only virus isolated from fatal cases (Lum et al., 1998a
, b
). A similar outbreak of HFMD, with several fatal cases, occurred in peninsular Malaysia during the latter part of 2000. In addition to CA16 and EV71, echovirus 7 (Eo7) was also isolated from cases of HFMD seen at the University of Malaya Medical Centre (UMMC) during this outbreak. In the past, Eo7 has been implicated as a cause of mild febrile exanthematous diseases in children, although there have been several earlier reports of fatal infections (Madhavan & Sharma, 1969
; Andersson et al., 1975
; Wreghitt et al., 1989
; Ho-Yen et al., 1989
). However, during the 2000 HFMD outbreak, Eo7 was isolated from several fatal cases of encephalomyelitis (Lum et al., 2001
). Moreover, to our knowledge, Eo7 infection has not previously been reported in association with HFMD, although there has been a report of maculopapular eruption with distribution confined mainly to the hands and feet (Ho-Yen et al., 1989
). In this paper, we compare the complete nucleotide sequences of the Eo7 UMMC strain, isolated from the cerebrospinal fluid of a child with HFMD and fatal encephalomyelitis, and the prototype Eo7 (Wallace strain), isolated from the stool of an asymptomatic child (Ramos-Alvaraz & Sabin, 1954
). A detailed analysis of genetic differences between Eo7-UMMC and Eo7-Wallace is presented, including comparison of nucleotide and deduced amino acid sequences and secondary structure analysis in the 5' UTR. Finally, the phylogenetic relationship of Eo7 to other human enteroviruses is presented.
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Methods |
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Virus RNA isolation, genome amplification and sequencing.
Total RNA was extracted from the infected RD cell pellet using the Qiagen RNeasy Mini kit and the eluted total RNA was quantified using the GeneQuant II RNA/DNA Calculator (Pharmacia). The complete nucleotide sequences of a number of enteroviruses, Eo1 (accession no. AF029859), Eo5 (NC_002601), Eo6 (NC_001657), Eo9 (NC_001656), Eo11 (X80059), Eo30 (NC_000873), coxsackievirus B (CB) 1 (NC_001472), CB2 (NC_000881), CB3 (U57056), CB4 (NC_001360), CB5 (AF114383), CB6 (NC_002003), CA9 (NC_002347), CA16 (NC_001612), CA21 (NC_001428), CA24 (D90457), EV70 (NC_001430), EV71 BrCr strain (EV71-BrCr) (NC_001769), EV71-MS (U22522), poliovirus type 1 (PV1) (K01392), poliovirus type 2 (PV2) (NC_002058) and poliovirus type 3 (PV3) (M12197), were obtained from GenBank.
The complete nucleotide sequences of these enteroviruses were aligned using the Clone Manager 5/Align Plus-4 software (S&E Software). Fifteen pairs of degenerate primers, covering the predicted entire genomic nucleotide sequence, were synthesized based on the relatively conserved nucleotide regions of these enteroviruses (Table 1). Segments of the complete Eo7 genome were amplified by RTPCR with the extracted viral RNA as the template for the reaction. The PCR products were sequenced by ABI Prism Big-Dye dideoxy chain-termination cycle sequencing (Pharmacia) using the respective degenerate forward and reverse primers and analysed on an ABI 377 automatic sequencer (Applied Biosystems). The derived sequences of all segments were assembled by using the Clone Manager 5/Align Plus-4 software. Overlapping virus-specific primers were then synthesized, based on the sequence information derived from the degenerate primers, to cover regions of the degenerate primer sites. Further virus genome sequences were obtained by RTPCR by using the virus-specific primers and the extracted RNA as the template. Thus, each nucleotide within the viral genome was sequenced at least four times.
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Single-stranded cDNA synthesis reactions were performed by reverse transcription (Thermoscript RT kit) using the most distal primer (CR). The RNA templates were removed by alkaline lysis and the single-stranded cDNA was extracted by precipitation with isopropanol, 3 M sodium acetate and glycogen. The precipitate was dissolved in 20 µl TrisHCl (pH 8·0) after two washes in 80% ethanol. Ligation of each single-stranded cDNA to the anchor (DT88) was carried out overnight at 30 °C in a 13 µl reaction: 10 µl cDNA, 1·3 µl 10x ligation buffer, 0·3 µl T4 RNA ligase and 1·4 µl DT88 anchor (4 pmol/µl).
Amplification of the ligated 5' terminus of the viral genome (3' terminus of cDNA) was performed in a 50 µl reaction consisting of 44 µl Platinum PCR Supermix (Gibco BRL), 2 µl anchor primer (DT89, 20 pmol/µl), 2 µl primer BR (20 pmol/µl) and 2 µl of the ligated product. A standard 35-cycle PCR amplification was performed with an annealing step of 55 °C for 30 s, following which a second PCR (hemi-nested) was performed using DT89 and primer AR under the same cycling conditions.
The presence of the amplified hemi-nested PCR product was confirmed by agarose gel electrophoresis. The appropriately sized product was purified by gel extraction using the QIAquick gel purification kit (Qiagen); between 30 and 40 ng of purified product was sequenced.
Nucleotide and deduced amino acid sequence analysis.
Sequence data derived from the cDNA fragments generated by RTPCR and 5' RACE were manipulated using the Clone Manager 5/Align Plus 4 software. Multiple sequence alignments were undertaken using a standard linear scoring matrix with the following parameter settings: mismatch penalty of 1, open gap penalty of 4, extended gap penalty of 1 and similarity significance value cut-off of 60%. A homology search was conducted using the BLAST server at the National Center for Biotechnology Information (National Library of Medicine, Bethesda, MD, USA).
Multiple sequence alignments and a neighbour-joining phylogram were generated using the Clustal X program (Higgins & Sharp, 1989 ). Phylogenetic analysis was conducted using PROTDIST and KITSCH programs in the PHYLIP software package, version 3.5c (Felsenstein, 1993
). The most likely evolutionary relationships were determined by evaluation of trees from 1000 randomized resampling cycles. Bootstrap verification of the resulting phylogenetic tree was performed by analysis of 1000 bootstrapped pseudo-replicates using the SEQBOOT, NEIGHBOR and CONSENSE programs of the PHYLIP package. Phylogenetic trees were constructed using TreeView (Page, 1996
). The RNA secondary structures of the 5' and 3' UTRs were constructed using RNAStructure version 3.5 (Mathews et al., 1999
).
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Results |
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Sequence comparison between the genomes of Eo7-Wallace and Eo7-UMMC
The sequences of the 5' UTRs of Eo7-Wallace and Eo7-UMMC were aligned using Clone Manager 5/Align Plus 4 software (Fig. 1a). Two regions with higher sequence variation (underlined) were noted; region one extends from nt 89 to 168 and region two corresponds to the last 100 nt (643742) of the 5' UTR. Within the first more-variable region, Eo7-UMMC has a 2-base (AT) deletion at positions 102 and 103 but has an extra base (A) at position 127, compared with the 5' UTR sequence of Eo7-Wallace (Fig. 1a
, bold and italic). The degree of nucleotide identity between the two 5' UTRs is shown in Table 2
.
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An alignment of the deduced polyprotein sequences of Eo7-Wallace and Eo7-UMMC is shown in Fig. 2. The initiation site for the synthesis of the viral polyprotein and the predicted cleavage sites for the individual peptides were based on homology with other enteroviruses and with the established sequence motifs of enterovirus protein-cleavage sites (Pallansch et al., 1984
; Kozak, 1986
; Pincus et al., 1986
; Jenkins et al., 1987
). The cleavage sites and individual peptides are indicated by downward-pointing arrows immediately above the cleavage site of the aligned sequences (Fig. 2
). The amino acid motifs (Fig. 2
, in bold) involved in the cleavage of the polyprotein are identical for Eo7-Wallace and Eo7-UMMC and the various viral peptides are of equal lengths. The degree of nucleotide and amino acid sequence identity between the two viruses is shown in Table 2
. Overall, the degree of identity between the two ORFs is 80 (nt) and 96 (aa) %. As expected, the P3 region (nt 82%; aa 97%) had the highest level of identity, followed by the P1 (nt 78%; aa 95%) and P2 (nt 79%; aa 95%) regions. Of the individual proteins, 3C had the highest level of identity (98%) and 2A had the lowest level of identity (92%).
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Discussion |
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Analysis of the complete nucleotide and deduced amino acid sequence of the UMMC strain confirmed its original identification in neutralization tests as Eo7 (Lum et al., 2001 ). Sequence homology studies based on alignment of the complete genome and sub-genomic regions showed clearly that Eo7-UMMC is not a recombinant virus. However, the data indicate that the sequence of Eo7-UMMC has drifted significantly from that of the prototype Eo7 (Wallace) strain in the 47 years that separate the isolation of the two strains. Comparison of the Eo7-UMMC sequence with available sequence data from other recent Eo7 strains shows that it is closely related to recent isolates of Eo7 (94% nucleotide sequence identity), with the greatest sequence identity being to a recent Eo7 isolate from Spain (GenBank accession no. AF252186; data not shown). The VP1 sequence of Eo7-Wallace reported here differs from that reported by Oberste et al. (1999
a) (accession no. AF081324) by three nucleotides (one amino acid; methionine to valine). By contrast, our Eo7-Wallace VP1 sequence exactly matches the VP1 gene sequence of Eo7-Wallace reported by J. L. Bailly and others (unpublished accession no. AJ241426), suggesting that the AF081324 VP1 sequence of Eo7-Wallace may have been inaccurate or derived from a clone that was genetically diverged from that sequenced as AJ241426 and by ourselves.
An interesting result has emerged from comparison of the putative RNA secondary structure of the UTRs of Eo7-UMMC and Eo7-Wallace. The secondary structures of both the 5' and 3' UTRs of Eo7-UMMC fold at a thermodynamically lower energy level than Eo7-Wallace and are presumably more stable. The extent to which the neurovirulence phenotype of Eo7 may be influenced by a thermodynamically more stable 5' UTR secondary structure requires further study. Interestingly, similar analysis of the folding energy levels of the putative 5' UTR secondary structures of poliovirus 1 Mahoney [accession no. J02281, G=-242·0 kcal/mol (-57·8 kJ/mol)], Sabin 1 [accession no. J02282,
G=-246·7 kcal/mol (-58·9 kJ/mol)] and a neurovirulent isolate [accession no. AJ132961,
G=-250·8 kcal/mol (-59·9 kJ/mol)], poliovirus 2 Lansing [accession no. M12197,
G=-251·7 kcal/mol (-60·1 kJ/mol)] and Sabin 2 P712 Ch 2ab [accession no. X00595,
G=-246·2 kcal/mol (-58·8 kJ/mol)] and poliovirus 3 P3/Leon/37 [accession no. K01392,
G=-275·4 kcal/mol (-65·8 kJ/mol)] and Sabin 3 [accession no. AJ293918,
G=-239·0 kcal/mol (-57·1 kJ/mol)] has shown that the 5' UTR secondary structures of the neurovirulent or wild strains are at a thermodynamically lower energy level than those of the attenuated Sabin strains, with the exception of the poliovirus 1 Mahoney strain (data not shown). Furthermore, the 5' UTR RNA secondary structure of Eo7-UMMC assumed a configuration that is quite distinct from that of Eo7-Wallace. The UTR of the enterovirus genome is known to form secondary, and possibly higher order, RNA structures. Biochemical and genetic data have been used to support a model of the secondary structural motifs within the 5' UTR of poliovirus (Skinner et al., 1989
; Pilipenko et al., 1989
). Secondary structural models of the 5' UTR of CB1 (Iizuka et al., 1987
), CB3, CB4 (Skinner et al., 1989
), CA9 (Chang et al., 1989
) and swine vesicular disease virus (Inoue et al., 1989
; Seechurn et al., 1990
) are also generally consistent with this hypothesis. The enterovirus 5' UTR contains an internal ribosome entry site (IRES) that plays a major role in the control of virus replication and translation and possibly other virus functions; the IRES is considered to result from the formation of RNA secondary structures within the 5' UTR. The following observations, among others, appear to support this concept. Some natural or engineered mutations in the 5' UTR affect the translation activity of poliovirus RNA in vitro (Svitkin et al., 1985
; Pelletier et al., 1988
; Bienkowska-Szewczyk & Ehrenfeld, 1988
; Minor, 1992
) and the efficiency of virus replication (Racaniello & Meriam, 1986
; Kuge & Nomoto, 1987
; Johnson & Semler, 1988
; Trono et al., 1988a
, b
). These findings undoubtedly point to an important functional significance of the picornavirus 5' UTR. The deviation of the Eo7-UMMC 5' UTR from that of the prototype Eo7 may have contributed to the phenotypic changes observed during the 2000 HFMD outbreak in peninsular Malaysia. However, the extent to which the changes in primary structure of the Eo7-UMMC 5' UTR may lead to changes in its secondary structure and influence pathogenesis and neurovirulence remain speculative. A study of the translation efficiency in vitro of infectious cDNA clone-derived recombinant viruses expressing the 5' UTR of Eo7-Wallace on the Eo7-UMMC genetic background and the pathogenic potential of the recombinant viruses in transgenic mice expressing the receptor for Eo7 (decay-accelerating factor, CD55) are ongoing.
To our knowledge, Eo7 infection has not previously been associated with HFMD. In the 2000 HFMD outbreak in peninsular Malaysia, Eo7 was isolated from both fatal and non-fatal cases (Lum et al., 2001 ). Our analysis failed to find any evidence for the UMMC strain having originated through recombination of Eo7 with CA16, EV71 or another member of the species human enterovirus A. This finding is supported by the topology of the polyprotein-based phylogenetic tree, which showed clearly that Eo7-UMMC and Eo7-Wallace belong to the species human enterovirus B. Initial speculation for the unusual phenotypic manifestation of HFMD due to Eo7-UMMC was based on its close genetic similarity to CA9 (Table 3
), an enterovirus known to give rise to vesicular eruptions (Melnick, 1996
). However, as shown in Table 3
, the genome of Eo7-Wallace actually has slightly closer similarity than that of Eo7-UMMC to that of CA9. Thus, the molecular features of Eo7-UMMC that contribute to its unusual phenotype remain largely unknown. However, it is possible that changes in the 5' UTR may have contributed to its dermatotropism and neurovirulence.
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Acknowledgments |
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Footnotes |
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References |
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Received 5 June 2001;
accepted 6 August 2001.