Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes
© Wen et al.; licensee BioMed Central Ltd. 2013
Received: 14 June 2012
Accepted: 2 April 2013
Published: 11 April 2013
Hand, foot and mouth diseases (HFMD) caused by enterovirus 71(EV71) presents a broad spectrum of clinical manifestations ranging from mild febrile disease to fatal neurolocal disease. However, the mechanism of virulence is unknown.
We isolated 6 strains of EV71 from HFMD patients with or without neurological symptoms, and sequenced the whole genomes of the viruses to reveal the virulence factors of EV71.
Phylogenetic tree based on VP1 region showed that all six strains clustered into C4a of C4 sub-genotype. In the complete polypeptide, 298 positions were found to be variable in all strains, and three of these positions (ValP814/IleP814 in VP1, ValP1148/IleP1148 in 3A and Ala P1728/Cys P1728/Val P1728 in 3C) were conserved among the strains with neurovirulence, but variable in strains without neurovirulence. In the 5′-UTR region, it showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. The secondary structure prediction of 5′-UTR sequences showed that two of three strains without neurovirulence (SDLY11 and SDLY48) were almost the same, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each other. SDLY107 (a fatal strain) was found different from other strains on four positions (CP241/TP241, AP571/TP571, CP579/TP579 in 5′-UTR and TP7335/CP7335 in 3′-UTR).
The three positions (ValP814/IleP814 in VP1, ValP1148/IleP1148 in 3A and Ala P1728/Cys P1728/Val P1728 in 3C), were different between two phenotypes. These suggested that the three positions might be potential virulent positions. And the three varied positions were also found to be conserved in strains with neurovirulence, and variable in strains without neurovirulence. These might reveal that the conservation of two of the three positions or the three together were specific for the strains with neurovirulence. Varation of secondary structure of 5′-UTR, might be correlated to the changes of viral virulence. SDLY107 (a fatal strain) was found different from other strains on four positions, these positions might be related with death.
KeywordsEnterovirus 71 Virulent determinant Hand, foot and mouth disease
Hand, foot and mouth diseases
Reverse transcription-polymerase chain reaction
Open reading frame
Internal ribosome entry site
Dulbecco’s modified Eagle’s medium
Supplemented with 10% fetal bovine serum.
Enterovirus 71 (EV71) belongs to the Enterovirus genus of the family Picornaviridae. It is one of the pathogens that are associated with hand, foot and mouth disease (HFMD). In most cases, EV71 infections are generally mild. However, this virus has also been implicated to cause severe neurological manifestations including aseptic meningitis, polio-like paresis and possibly fatal encephalitis .
Since 1969, when EV71 was first isolated in California, USA , EV71 associated outbreaks have been reported worldwide [3–10]. In recent years, it has gained more attention as there is an upward trend in the prevalence of EV71 in Asia . EV71 infection is a serious threat to the health of infants and young children; therefore, it is necessary to understand the mechanism of central nervous system involvement. Zheng et al. reported nucleotide differences in 5′-UTR between strains isolated from patients with and without neurological symptom, and proposed that such variation may be correlated with different clinical presentations . Shih-Cheng Chang reported that a significant amino acid change was observed in more than one of high virulent strains . Melchers et al. suggested that point mutations in 3′-UTR can result in a lethal phenotype . All these points were located in different regions of the genome, therefore, it is necessary to search for potential points associated with neurovirulence in complete genome.
Virus identification and segmented amplification
Sequence analysis of the genomes
The sequences of the six strains were desposited in GenBank (GenBank accession number JX244182, JX244183, JX244184, JX244185, JX244186, JX244187). The genomes of strains SDLY11, SDLY48, SDLY96 and SDLY107 were all 7405 bp in length, whereas strains SDLY1 and SDLY153 were 7408 bp in length. All six strains had one ORF which encoded a polypeptide of 2193 amino acids.
The nucleotide and amino acid homology of the six strains with reference strains(nucleotide/amino acid)
Analysis of polyprotein
The polyprotein consists in three regions : P1 containing capsid proteins (VP ~ VP4), P2 and P3 containing non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D) which are crucial for virus replication.
Complete genome sequences of 31 strain used in this study
Strains with neurovirulence
Strains without neurovirulence
Isolated in this study
Isolated in this study
Isolated in this study
Isolated in this study
Isolated in this study
Isolated in this study
Significant positions of polyprotein of 31 strains
Strains with neurovirulence
Strains without neurovirulence
Analysis of 5′-UTR
5′-UTR sequences of 31 strains were aligned. It showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. No position was found statistical significantly different between strains with and without neurological symptom. Whereas SDLY107 (a fatal strain) was found different from other strains on three positions (CP241/TP241, AP571/TP571, CP579/TP579), suggesting that these positions might be related to death.
Analysis of 3′-UTR
The 3′-UTR of EV71 was a highly conserved region and point mutations in the 3′-UTR could result in a lethal phenotype . Alignment of 3′-UTR sequences of 31 strains by BioEdit 7.09 software did not reveal significant position associated with virulence. However, SDLY107 (a fatal strain) was found different from other strains on position TP7335/CP7335, suggesting that this position might be correlated to death.
EV71 is one of the most virulent enteroviruses and can cause mortality in children . Defining virulent positions on molecular level is considered as one of the most important aspects of disease prevention. In our study, complete genomes of six EV71 strains with different clinical phenotypes were sequenced and analyzed. Together with other strains isolated in Shandong in recent years, the six strains clustered into C4a of C4 sub-genotype .
At present, molecular neurovirulence determinant of EV71 remains unclear, though virulence factors of other enteroviruses have been reported. Nucleotide 480, 481 and 472 on 5′-UTR of poliovirus were identified as neurovirulence determinants of poliovirus [19–21]. Minetaro et al. reported that mutation of the EV71 standard strain BrCr in 5′-UTR showed attenuated neurovirulence in the cynomolgus monkey model . In this study, insertions and deletions were frequently found in 5′-UTR region. Two of three EV71 strains (SDLY11 and SDLY48) from patients without neurovirulence had almost the same secondary structure of 5′-UTR, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each another. In IRES element, domain III and II were relatively conserved regions, however, domainI, IV and V are very variable. These suggest that variation of the secondary structure of the 5′-UTR, especially domainI, IV and V might be correlated to the virulence. When aligned the strain isolated from a fatal patient (SDLY107) with other five strains, three position of 5′-UTR (CP241/TP241, AP571/TP571, CP579/TP579) might be related to the virulence.Li et al. reported that four amino acids (GlyP710/GlnP710/ArgP710 and GluP729) in the DE and EF loop of VP1, one (LysP930) in the surface of protease 2A were potentially associated with EV71 virulence . In our study, three positions, ValP814/IleP814 in VP1, ValP1148/IleP1148 in 3A and Ala P1728/Cys P1728/Val P1728 in 3C, were different between two phenotypes. These results suggest that three positions are potential virulent positions. The position 814 locates in C-terminal part of the VP1 protein which locates on the surface of the virus, mediates the initiation of infection by binding to receptors on the host membrane . C-terminal part of the VP1 protein were supposed to be capable of eliciting neutralizing antibodies against EV71 . Variations in VP1 region may influence the ability of the virus binding to host cell and eliciting neutralizing antibodies. Protein 3A plays a role in inhibiting cellular protein secretion and mediating presentation of membrane proteins during viral infection. Variations in 3A region may affect the process of viral infection. Protein 3C can cleave numerous factors and regulators that are associated with cellular DNA-dependant RNA polymerase I, II and III, and may be involved in the virus-induced blockage of host transcription. Variations in 3C region may affect activity of RNA polymerase and host cellular transcription. The three positions were conserved in strains with neurovirulence, and variable in strains without neurovirulence. These also reveales that the conservation of two of the three positions or the three together maybe specific for the strains with neurovirulence.
The 3′-UTR is a highly conserved domain and mutations in the 3′-UTR may cause change of phenotype. However, in our study, analysis of nucleotides of 3′-UTR showed no virulence associated nucleotides.
To test our aforementioned findings, site-directed mutagenesis need to be performed on these positions in the future study, and infectious cDNA clones with different potential virulent positions need to be constructed and evaluated at ex vivo and in vitro.
Cells and viruses
EV71 strains SDLY1, SDLY11, SDLY48, and SDLY96 were isolated from stool samples of four patients without neurovirulence. SDLY107, SDLY153 were isolated from anal swabs samples of two patients. Among these strains, SDLY1, SDLY11 and SDLY48 were isolated from patients with mild symptoms. SDLY96 and SDLY153 were isolated from patients with neurological symptom, and SDLY107 was isolated from a fatal patient. All six patients were from Linyi City, Shandong Province, China. Human rhabdomyosarcoma (RD) cells were maintained in DMEM supplemented with 10% FBS. Viruses were propagated on RD cells to increase the titer for use in subsequent assays.
RNA extraction and virus identification
Total virus RNAs were extracted from EV71-infected cell culture supernatants using a RNA extraction kit (OMEGA) following the manufacture′s instructions. Virus types were identified by One-Step RT-PCR described previously .
Segmented amplification of the complete genomes
Primers used for amplifying the genome
Name of primer
The nucleotide sequences of six complete genomes and the derived amino acid sequences were analyzed by BioEdit 7.09 software. The genotype and subgenotype were determined by comparing sequences with reference strains from GenBank. The secondary structures of 5′-UTR and 3′-UTR were predicted by RNA structure 4.0 software. The phylogenetic tree was constructed using MEGA 4 software based on the nucleotide sequences of the complete VP1 region.
This study was approved by the ethical committees of School of Public Health, Shandong University, Jinan, Shandong 250012, China (permit number 20080301). Written consents were obtained from all children′s parents involved in the study.
The research is supported by National Natural Science Foundation of China (30901265); Natural Science Foundation of Shandong Province (ZR2010CQ030); Innovation Foundation of Young Talent, School of Public Health, Shandong University(GGWS200801). We thank Shandong medical equipment quality supervision and inspection center, Shandong Linyi City people's hospital for support to conduct this study.
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