Enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family and is the major cause of Hand, foot, and mouth disease (HFMD) in children. EV71 infection can be accompanied by a series of syndromes with or without central nervous system involvement, including herpangina, aseptic meningitis, poliomyelitis-like paralysis, and possibly fatal encephalitis (14). Since the initial description of EV71, outbreaks of infection with this virus have been reported periodically in Europe, America, and the Asia-Paciﬁc region. Since 1997, EV71 outbreaks with fatalities have been reported in Asia-Paciﬁc countries. Subsequent smaller EV71-associated epidemics have been reported almost annually. During 2008, a large HFMD epidemic, caused mostly by EV71 infection, occurred in China, resulting in about 25, 000 cases and 34 deaths. The EV71 outbreaks have been and continue to be 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. The single long open reading frame (ORF) encodes a polyprotein of about 2194 amino acids. There are four capsid proteins: VP1, VP2, VP3, and VP4. 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. VP1 has also been used for phylogenetic analyses (13). At present, EV71 can be divided into three distinct genogroups (A, B, and C). Most EV71 isolates belong to genogroups B or C, which are each further divided into ﬁve subgenogroups, B1-B5 and C1-C5 (10). The evolution of viral genomes may lead to an altered spectrum of diseases caused by the virus. For the present study, nine EV71 isolates from pediatric patients in Fuyang, China, were collected for determination of the complete VP1 gene sequence. These isolates were then investigated by sequence alignment, molecular phylogenetic analyses, and analysis of neutralizing epitopes.
Clinical specimens were collected from patients with HFMD in Fuyang, China. Basic data for the nine patients are listed in Table 1.
Table 1. Clinical diagnosis of patients from whom enterovirus 71 was isolated
Vero cells were cultured in five tissue culture flasks in 25 cm2 of minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS). A 1 mL throat swab sample was inoculated into Vero cells. After contact for 1 h at 37℃, the samples were removed, and MEM supplemented with 2% FCS was added. Within three passages, samples that induced an EV71-specific cytopathic effect (CPE) were considered positive for virus isolates. Samples that did not induce CPE after three passages in Vero cells were considered negative. Supernatants from the inoculated wells showing CPE were harvested and tested by neutralization using the antisera set from the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College (IMB). The supernatant was extracted and used for the microneutralization test.
Virus RNA was extracted from 140μL of virus culture supernatant using a QIAmp Viral RNA kit (Qiagen, USA) according to the manufacturer's instructions. RT-PCR was performed with the primer pair listed below using an Access Quick TMRT-PCR System (Promega, USA) in 50-μL reactions. The cycling conditions consisted of an initial denaturation at 45℃ for 45 min, then 94℃ for 5 min followed by 35 cycles of 94℃ for 30 s, 52℃ for 30 s, and 72℃ for 2 min, and a final extension step for 10 min at 72℃ in a thermal cycler. PCR products (910 bp in length) were visualized by agarose gel electrophoresis. These amplified products were gel-purified and then cloned into a PMD18-T vector (Takara Dalian Co., Japan) according to standard procedures. Recombinant plasmids that had been confirmed by PCR were sequenced by the Sangon Corporation. Primers were: P1: 5'-CATCCAGGGAGATAGGGTGGCA-3' (2430-2451); P2: 5'-TCCAAATTTCCCAAGAGTGGTG-3' (3319-3341).
The sequencing results were analyzed and compared using the MEGA4.1 software package (Rainbow Technologies, INC) to define the genetic variation and the relationships with other strains obtained from GenBank.
A genotype isolates: BrCr (AB204853), BrCr-TR (AB204852). B genotype isolates: 26M-USA-4-99 (EU364841), 26M-USA-4-99.1 (EU376005), 26MUSA-4-99.CHO (EU376004), 5511-SIN-00 (AY125988), SB-12736-SAR-03 (DQ341362), 5666-SIN-002209 (AF352027), 5865-SIN-000009 (AF316321). C1 genotype isolates: J115-MAL-01 (DQ341360), 804NO-03 (DQ452074), 1M-AUS-12-00 (DQ341361). C2 genotype isolates: 6F-AUS-6-99 (DQ381846), 7FAUS-6-99 (DQ341357; Tainan-4643-98 (AF304458), Tainan-6092-98 (AF304459), Tainan-5746-98 (AF304457), C3 genotype isolates: 03-KOR-00 (DQ341356), 06-KOR-00 (DQ341355). C4 genotype isolates: Fuyang31 (EU913470), Fuyang5 (EU913467), Fuyang23 (EU8125150), Fuyang22 (EU913466), Fuyang26 (EU913468), Fuyang49 (EU913471), Fuyang44 (EU913469), Fuyang.Anhui.P.R.C-17.08-1 (EU703812), Fuyang. Anhui.P.R.C-17.08-1 (EU703814), FY-08 (EU697901), Fuyang. Anhui.P.R. C-17.08-1 (EU703813), F1-CHN-00 (AB115490), SHZH98 (AF302996), 804-NO-03 (DQ452074), SHZH03 (AY465356), 1431-Yamagata-07 (AB433881), 1571Yamagata-07 (AB433883), AnHui-HeFei (EU697903).
RNA extraction, RT-PCR, and Sequencing
The strain/isolate names and GenBank accession numbers
After three passages, all nine samples induced the EV71-specific CPE in vero cells and the supernatants from the inoculated wells showing CPE could be neutralized by the IMB anti-EV71 sera set via the cell culture-neutralization test. When ampliﬁed using EV71-speciﬁc primers via RT-PCR, each sample showed a clear fragment of 910 bp in size. So EV71 was detected in samples from the nine patients.
The nucleotide sequences of the ampliﬁed fragments revealed that nine EV71 strains isolated were over 98% homologous. The isolates were also 88%-90% homologous to other C subgenogroups (C1, C2, and C3) and 93%-100% homologous to members of the C4 subgenogroup. Dong et al. proposed that a level of 15% sequence diversity be used as a cutoff for the delineation of new genotypes and a 9% cutoff for the delineation of subgenogroups (7). So the Fuyang isolates and the other Chinese isolates belonged to genotype C4. SHZH98 had the most divergent VP1 sequences among C4 subgenogroup. The nine EV71 strains with other enteroviruses are displayed in a phylogenetic tree constructed by the neighbor-joining method (Fig. 1). Those strains are clustered in three distinct lineages (genotypes), designated A, B, and C. Genotype A contains a single member, BrCr, the EV71 prototype, and differs from all other isolates by 16.4%-19.7%. Genotype B was isolated from 1972 to 1997 in the United States, Australia, Colombia, and Malaysia Genotype C includes viruses from the United States, Australia, China, Canada, and mainland Malaysia. Genotype C is further divided into four subgenogroups. In addition, C at nucleotide position 65 was conserved among the Fuyang strains, while A was conserved within other isolates. Therefore, C at nucleotide position 65 appears to be a marker for Fuyang strains.
Figure 1. Phylogenetic analysis of 35 EV71 strains found worldwide based on complete VP1 gene sequence.The phylogenetic tree was constructed by the neighbor-joining method with MEGA version 4.1 software, and the reliabilities indicated at the branch nodes were evaluated using 500 bootstrap replications.
The EV71 isolates from patients in Fuyang were 99%-100% homologous to one another at the amino acid level. The isolates were also 96%-97% homologous to other C subgenogroups (C1, C2, and C3) and 97%-100% homologous to members of the C4 subgenogroup. SHZH98 had the lowest homology to C4 (97%), and we found that the deduced sequence of the SHZH98 strain exhibited the largest genetic distance from other C4 members (Fig. 2 and Table 2). In Table 2, we also found that the evolutionary divergence values between sequences of the same genogroups were low, but these values were high between sequences of other genogroups (A and B). This data was identical to the result produced using the neighbor-joining method. The Fuyang isolates were 95%-97% homologous to other genogroups. In addition, the histidine (H) at amino acid position 22 was conserved among the Fuyang strains (Fig. 3), while the lysine (K) at position 43 and the alanine(A) at position 58 were conserved within genogroup C. Therefore, an H at position 22 appears to be a marker for Fuyang strains.
Figure 2. Phylogenetic analysis of 35 EV71 strains found worldwide based on complete VP1 amino acid sequence. The phylogenetic tree was constructed by the neighbor-joining method with MEGA version 4.1 software, and the reliabilities indicated at the branch nodes were evaluated using 500 bootstrap replications.
Table 2. Estimates of Evolutionary Divergence between Sequences
Figure 3. Comparison of putative part VP1protein aa 1-120 regions of EV71 Fuyang strains and other isolated strains.
The VP1 protein is the major viral neutralization determinant and thus has a high degree of antigenic and genetic diversity that correlates with the viral serotype. Homologous recombination has not been shown to occur within the VP1 gene. Some amino acid substitutions in the VP1 gene were observed between Fuyang strains and other strains. So far, known epitopes recognized by neutralizing antibodies against EV71 are located in the VP1 protein in amino acid regions 91-106 (the BC loop) and 208-222 (SP70) (6, 8). In the deduced VP1 protein sequence of the Fuyang strains (Fig. 3), the BC loop was different from the BC loops of other strains (6F-AUS-6-99, AnHui-HeFei, BrCr, BrCr-TR, F1-CHN-00, Fuyang24, and SHZH98). In contrast, SP70 was identical to those from other isolated strains (data not shown).