Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes

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 [1].

Since 1969, when EV71 was first isolated in California, USA [2], 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 [11]. 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 [12]. Shih-Cheng Chang reported that a significant amino acid change was observed in more than one of high virulent strains [13]. Melchers et al. suggested that point mutations in 3^′-UTR can result in a lethal phenotype [14]. 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.

EV71 is one of the most virulent enteroviruses and can cause mortality in children [1]. 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 [18].

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 [22]. 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 (C^P241/T^P241, A^P571/T^P571, C^P579/T^P579) might be related to the virulence.Li et al. reported that four amino acids (Gly^P710/Gln^P710/Arg^P710 and Glu^P729) in the DE and EF loop of VP1, one (Lys^P930) in the surface of protease 2A were potentially associated with EV71 virulence [23]. In our study, three positions, Val^P814/Ile^P814 in VP1, Val^P1148/Ile^P1148 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 [24]. C-terminal part of the VP1 protein were supposed to be capable of eliciting neutralizing antibodies against EV71 [25]. 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.