Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand, foot, and mouth disease (HFMD) in children. EV71 infection can lead a wide spectrum of clinical disease ranging from mild febrile illness to more severe conditions, such as HFMD, poliomyelitis–like paralysis and neurogenic-pulmonary oedema. It was first reported in California in 1969. Later, many countries reported the EV71 epidemic in different areas and was first reported in Wuhan, China in 1995[8, 9]. In 2008, a large HFMD epidemic caused by EV71 infection, occurred in China resulting in about 25 000 cases and 34 deaths. From 2009 to 2010, the number of EV71 infection cases and deaths has increased gradually and the EV71 outbreaks have become an important public health issue in China.
EV71 is a single stranded, positive-sense RNA virus with a genome approximately 7.5 kB in length, has untranslated regions at both the 5' and 3'ends and a single polyprotein that divided into three different genomic regions. The P1 gene region encodes for the structural proteins VP1 to VP4. The P2 and P3 genomic regions encode for non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D. The VP1 is the most external and is immunodominant among the viral capsid proteins, it is located mainly on the virion surface and is the source of many major neutralization sites. Several studies have indicated the potential of the VP1 protein to act as an antigen in both diagnosis and subunit vaccine development against EV71[5, 15]. Based on the VP1 nucleotide sequence the enterovirus 71 is divided into three genogroups A, B, and C. Most EV71 isolates belong to either genogroups B or C, which are each further divided into ﬁve genotypes or subgenogroups, B1–B5 and C1–C5. EV71 genogroup C has become dominant in Europe in recent years with genotypes C1 and C2 occurring in United Kingdom, and genotypes C1 and C4 occuring in Austria and Germany[1, 2, 6, 11]. The BrCr isolate from USA was believed to be the only A subtype, but recently additional strains belonging to the A subgenogroup have been isolated in China; this is also the first time that the other subtypes have been reported in China. Although many isolates have been cloned, there have been no reports about P1 sequences from Gansu province. In this study the P1 sequence of different strains from Gansu were cloned. For 55F, one of the strains the VP1 protein was expressed. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.
After second generation blind passages a pathogenic effect (CPE) could be observed when the specimens were passaged in Vero cells, and were visible when cultured for 24 h. Different strains had the almost same CPE time in Vero cells, illustrating that the all strains could grow well in the selected cells. The infectious titers of the strains were all higher than 106 CCID50/mL, plaque-purification showed it can form the uniform plaque and there were no significant different on the plaque, but the size of uniform plaque were different, suggested that the stains have different virulence.
The p1 region of ten isolates from Gansu were cloned (Fig. 1), they were all 2586 nucleotide (nt) in length, the same as that of the full-length region of other EV71 strains. It shared 94.1%-99.9% nucleotide homology with reference strains, and 98.6% to 99.8 at the amino acid level(Table 1). Although there were no insertions or deletions in the p1 gene of all strains, mutations were found among the different isolates. 11x and 11y, which from the same fatal case, nucleotide and amino acid homologies of p1 were 99.9% and 99.8%, and it shared the highest similarity with strain SHENZHEN08 China HFMD that was also isolated from a fatal case. Strains 51F, 55F, 0901, 0902, 0907 and 0917 that were from the other infected cases shared the highest nucleotide and amino acid homologies with the lanzhou/01 strain, 46F and 47F were highly homologies but had low homologies with other strains.
Figure 1. Amplified product of P1.M: 2000 bp marker, 1-10: different EV71 strains, 11X, 11Y, 46F, 47F, 51F, 55F, 901, 902, 903 and 917.
Table 1. The nucleotides and amino acids homology of p1 genes compare the isolates from Gansu to other strains
The p1 nucleotide phylogenetic tree showed that all the strains from Gansu clustered into a separtate group, but in the tree generated from the amino acid sequence, the strains were clustered into 2 subgroups, differentiating strains 11X and 11Y from the others. In order to determine the subtype of the Gansu isolates, a phylogenetic tree based on the vp1 genes was constructed. The resulted showed all isolates from gansu are C4 subtype (Fig. 2). The tree based on the p1 genes also revealed the same result (Fig. 3). Another tree was constucted based on the p1 amino acid sequence and the results showed that all the strains analyzed were divided into three groups with, the isolates from Gansu belonging to group Ⅰ and Ⅱ (Fig. 4). The vp1 phylogenetic tree showed that all the strains from Gansu were C4b subtype, but belonged to a different branch. It suggests that the source of infection were from different placelocations, and that at least three kinds of EV71 infections are prevanlent in Gansu. From both the vp1 and p1 nucleotides phylogenetic tree it can be seen that fatal strains 11X and 11Y have a close relationship with fatal strain SHENZHEN08 China HFMD, and we speculate that these strains have the same virulence signal. Amino acids sequence alignment showed there were many mutations among the different strains. The VP1 amino acid A to V mutation at position 170 appears to be a marker for a neurovirulent lineage, and it is possible that this mutation is a virulence determinant[12, 13]. However the VP1 at the position of 11x and 11y which from severe neurological fatal cases were also A, so this hypothesis needs to be tested. Alignment of the EV71 p1 gene showed there are 17 nucleotides conserved among the 11x, 11y and SHENZHEN08 China HFMD strains but that are distinct from the other strains, whether these nucleotides are associated with virulence should also be investigated.
Recombinant plasmid pET32-VP1 was digested with SalI and BamH I producing two fragments of 5400 bp (pET32a) and 891 bp. PCR identification confirmed the presence of the 891 bp fragment (Fig. 5). To obtain the expressed recombinant protein, the time course expression of recombinant plasmid pET32a-VP1 was induced by IPTG ranging from 0.5-1 mmol at 2, 3, 4, 5 and 6 h respectively. SDS-PAGE revealed that the best conditions were obtained for IPTG with 1 mmol at 6 h. The expressed VP1 protein had a molecular weight of 55 KDa as expected (Fig. 6). Western blot showed the expressed protein was able to bind immunologically to anti-EV71 serum (Fig. 7). VP1 protein had been expressed with different expression systerm[4, 16], if were used as diagnostic antibody, prokaryotic expression would be the best choice because of the high expression levels, but it is difficult to expressed the entire VP1 protein. In this research, the fusion protein with His-tag from pET32a and entire VP1 were expressed, the result revealed recombinant protein was expressed with high levels and reacted specifically with the EV71 patient antibody. The recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. This indicated that the protein has a good immunogenicty and would serve as an experiment basis for EV71 diagnosis research.
Figure 5. Identification of recombinant plasmid by PCR and restriction enzyme. M, 2500 ladder marker; 1, recombinant plasmid PET32a(+)/VP1; 2, identification of 5400bp fragment by restriction enzyme digestion; 3, identification of 891bp fragment by restriction enzyme digestion.
Figure 6. SDS-PAGE of recombinant VP1 protein. M, low protein marker; 1, after inducing with IPTG; 2, without IPTG treatment.