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J Clin Microbiol. 2010 April; 48(4): 1276–1282.
Published online 2010 February 17. doi:  10.1128/JCM.02384-09
PMCID: PMC2849586

Molecular Identification and Analysis of Nonserotypeable Human Enteroviruses[down-pointing small open triangle]

Abstract

Conventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at a reference laboratory from 1979 to 2007; these common serotypes were identified using one sense and three antisense primers and a set of 80 serotype-specific probes in VP1 (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). In this study, one HEV-specific primer pair, two probes in the 5′ untranslated region (UTR), and a new set of 80 serotype-specific probes in VP1 were designed. First, we successfully applied the modified RT-PCR-RLB (using two HEV-specific probes and two sets of serotype-specific probes) to synchronously detect the 5′ UTR and VP1 regions of 131/132 isolates previously studied (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). Then, this method was used to identify 73/92 nonserotypeable HEV isolates; 19 nonserotypeable isolates were hybridized only with HEV-specific probes. The VP1 region of 92 nonserotypeable HEV isolates was sequenced; 73 sequences corresponded with one or both RLB results and 19 (not belonging to the 20 most common genotypes) were identified only by sequencing. Two sets of serotype-specific probes can capture the majority of strains belonging to the 20 most common serotypes/genotypes simultaneously or complementarily. Synchronous detection of the 5′ UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, especially nonserotypeable isolates.

Human enteroviruses (HEVs), members of the genus Enterovirus, family Picornaviridae, are common human pathogens associated with a wide spectrum of disease, ranging from asymptomatic infection to serious illness, such as aseptic meningitis, meningoencephalitis, myocarditis, and acute flaccid paralysis, especially in children (36, 52) and immunocompromised patients (37). The original classification of HEVs consisted of polioviruses (PV), coxsackieviruses A (CVA) and B (CVB), and echoviruses (E), based on biological activity and disease. Conventional approaches to detecting and characterizing HEVs are based on the time-consuming and labor-intensive procedures of viral isolation in cell culture and neutralization with mixed hyperimmune equine serum pools and specific monovalent polyclonal antisera for confirmation (14, 19, 24, 40). This approach fails to identify and characterize some clinical isolates (i.e., nonserotypeable isolates) due to aggregation of virus particles, the existence of viral mixtures (3), undiscovered serotypes/genotypes (5, 13, 27, 29, 30, 33, 45, 46), recombination within the capsid region (9), or the emergence of variants that are antigenically different from the prototype strains used for the production of the antisera in the 1960s (25).

The HEV genome is a single positive-stranded RNA molecule of approximately 7,500 nucleotides which is unique because it is covalently linked at the 5′ end to a protein called VPg (viron protein, genome linked) (11). The 5′ untranslated region (UTR), the most conserved region of the enterovirus genome, contains the internal ribosome entry site, which directs translation of the mRNA by internal ribosome binding (10, 43). The genome encodes four structural proteins, VP1 to VP4: VP1, VP2, and VP3 are located at the surface of the viral capsid and are exposed to immune pressure, whereas VP4 is located inside the capsid (25). The capsid protein VP1 contains the majority of the neutralization epitopes. Current classification of the more than 100 HEV serotypes (http://www.picornaviridae.com) divides HEVs into four species (HEV-A, HEV-B, HEV-C, and HEV-D) based on genome organization, sequence similarity, and biological properties (37). At present, molecular typing methods largely depend on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3′ half of the VP1 gene (37). Comparison of the VP1 sequence with databases of VP1 sequences of HEV prototype and variant strains (33) allows genotype assignation. As molecular techniques for enterovirus typing are becoming increasing available, new enteroviruses continue to be identified. Although other molecular methods that assist serotype-specific identification have been described (2, 8, 18, 44, 47, 48), non-sequencing-based molecular methods to identify more HEV serotypes/genotypes are generally limited.

The most frequent 20 HEV serotypes (HEV-A [CVA16 and EV71], HEV-B [E3, -6, -7, -9, -11, -14, -17, -18, and -30; CVB1, -2, -3, -4, and -5; and CVA9], and HEV-C [PV1, -2, and -3]) identified at a reference laboratory in New South Wales (NSW), Australia, accounted for 87.1% (5,558/6,383) of all HEVs referred for typing from 1979 to 2007. However, 3.6% (227/6,383) of HEVs could not be serotyped by neutralization.

We have previously described an RT-PCR-based reverse line blot (RLB) hybridization assay to characterize common HEVs (53), where VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then used to genotype 132 HEV isolates, including the 37 previously sequenced isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeating conventional serotyping.

Molecular identification and analysis of nonserotypeable HEV isolates is relevant for epidemiological surveillance and the determination of relationships between serotypes/genotypes and diseases and to assist in the discovery of new serotypes/genotypes. We developed a modified HEV RT-PCR-RLB assay as a quick, accurate, and efficient approach to characterizing nonserotypeable HEV isolates, complemented by sequencing of VP1 genes.

MATERIALS AND METHODS

Viruses.

Our laboratory database contained 6,383 HEV isolates, including 227 nonserotypeable isolates, collected between 1979 and 2007. Isolates were obtained by inoculation of clinical specimens, including specimens in which HEV had been detected by real-time fluorescence PCR (39), into cultures of monkey kidney cells (primary, secondary, or cell lines) and various continuous cell lines, including A549 (human lung carcinoma), MRC-5 (human embryonic lung fibroblast), and RD (human rhabdomyosarcoma) cells. Prior to 2002, the titer of each cell culture with enterovirus cytopathic effect (CPE) was first determined, and then a neutralization assay was performed in tissue culture tubes by using intersecting pools of hyperimmune horse sera (National Institute of Allergy and Infectious Diseases, Bethesda, MD) against 100 50% tissue culture infective doses (TCID50) of each isolate. If necessary, further identification was performed using a monovalent horse antiserum corresponding to the serotype identified by the intersecting pools. After 2002, group-specific monoclonal pools and/or type-specific monoclonal antibodies (Chemicon, Temecula, CA) were used as a rapid identification alternative, and neutralization was performed on isolates that were not identified. HEV isolates that were not identifiable by standard seroneutralization procedures were labeled as “nonserotypeable” isolates. Virus isolates were stored as unpurified cell culture supernatants at −70°C.

In this study, 224 HEV clinical isolates, including 132 isolates previously studied (53) and 92 nonserotypeable isolates (from 1990 to 2007) randomly selected from 227 nonserotypeable HEV isolates were tested by RT-PCR-RLB.

HEV-specific primer and probe design for 5′ UTR.

To design HEV-specific primers and probes, the 5′ UTR sequences of 83 HEV prototype strains (37) (not available for EV78 and EV96) plus EV92 (GenBank accession no. EF667344), EV94 (GenBank accession no. DQ916376), EV98 (GenBank accession no. AB426608), EV107 (GenBank accession no. AB426609), PV Sabin strain 1 (GenBank accession no. V01150), PV Sabin strain 2 (GenBank accession no. X00595), and PV Sabin strain 3 (GenBank accession no. X00596) were aligned using the ClustalW tool provided on Biomanager (http://biomanager.info/). This allowed the design, in the highly conserved regions of the 5′ UTR, of one primer pair (5UTR-Sn and 5UTR-Ab) for PCR amplification and two probes (5UTR-Sp1 and 5UTR-Sp2) for RLB hybridization to cover all known HEV serotypes (see Table S1 in the supplemental material). Primer and probe sequences were evaluated using the Sigma DNA Calculator (Sigma, http://www.sigma-genosys.com/calc/DNACalc.asp) and synthesized by Sigma-Aldrich (Sydney, NSW, Australia).

Previous primers and previous set of serotype-specific probes for VP1.

Previously designed primers (VP1-All-Sn, VP1-A-Ab, VP1-B-Ab, and VP1-C-Ab) for VP1 PCR amplification and 80 serotype-specific probes (i.e., four serotype-specific probes [probes 1 to 4] for each of 20 common HEV serotypes/genotypes) for RLB hybridization were used (53).

New set of serotype-specific probes designed for VP1.

To confirm the RLB results on all 224 HEV isolates (especially the 92 nonserotypeable isolates) using the initial set of 80 serotype-specific probes (53) and to compare the efficiency of two sets of serotype-specific probes, a new set of 80 serotype-specific probes (i.e., four serotype-specific probes [probes 5 to 8] [see Table S1 in the supplemental material] for each of 20 common HEV serotypes/genotypes) were designed in other relatively conserved regions of VP1 for use in RLB according to the methods and principles described previously (53). Probe sequences were evaluated using the Sigma DNA Calculator (Sigma, http://www.sigma-genosys.com/calc/DNACalc.asp), and the probe specificity was confirmed by a BLASTn search in Biomanager. All probes were synthesized by Sigma-Aldrich (Sydney, NSW, Australia).

RT-PCR for RLB.

RT-PCR was performed as described previously (53). HEV-specific 5′ UTR PCR (using 5UTR-Sn and 5UTR-Ab) and VP1 PCR (using VP1-All-Sn, VP1-A-Ab, VP1-B-Ab, and VP1-C-Ab) for 224 HEV clinical isolates, including 92 nonserotypeable isolates, were performed separately using a Qiagen HotStarTaq DNA polymerase kit (Qiagen, Clifton Hill, Victoria, Australia) under the following conditions: 95°C for 15 min; 35 cycles of 94°C for 30 s, 42°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and a hold at 22°C. For HEV-specific 5′ UTR PCR, the PCR mixture contained the following: 2 μl template cDNA, 0.3 μl each of 5UTR-Sn (100 pmol μl−1) and 5UTR-Ab (100 pmol μl−1), 1 μl deoxynucleoside triphosphates (dNTPs) (2.5 mM each dNTP), 2.5 μl 10× PCR buffer [containing Tris-Cl, KCl, (NH4)2SO4, 15 mM MgCl2; pH 8.7 (20°C)], 0.2 μl Qiagen HotStarTaq DNA polymerase (5 units μl−1), and water to 25 μl. For VP1 PCR, the PCR mixture contained the following: 2 μl template cDNA; 1 μl VP1-All-Sn (100 pmol μl−1); 0.5 μl each of VP1-A-Ab, VP1-B-Ab, and VP1-C-Ab (100 pmol μl−1); 2.4 μl dNTPs (2.5 mM each dNTP); 3 μl 10× PCR buffer; 4.2 μl MgCl2 (25 mM); 0.2 μl Qiagen HotStarTaq DNA polymerase (5 units μl−1); and water to 30 μl. Then, the two PCR products were mixed together (55 μl) for RLB hybridization.

RLB hybridization assay.

Two sets of membranes were employed. The first set consisted of two membranes, each of which was bound with 42 probes (two HEV-specific probes and four previously designed serotype-specific probes [probes 1 to 4, as described in reference 53] for each of 10 common serotypes/genotypes). The second set was also made up of two membranes, each of which was bound with 42 probes (two HEV-specific probes and four newly designed serotype-specific probes [probes 5 to 8; see Table S1 in the supplemental material] for each of 10 common serotypes/genotypes) (Fig. (Fig.1).1). For 224 isolates, each isolate was detected by two sets of membranes separately. The RLB assay was performed as described previously, with minor modifications (16, 17). The mixed PCR products (55 μl) for each isolate were halved over one set of membranes (two membranes). The RLB procedure was described previously (53).

FIG. 1.
Representative RT-PCR-RLB results for 42 clinical isolates, including 40 nonserotypeable isolates and the positive control CVA9 (See Table S1 in the supplemental material for description and targets of probes listed on the left). The first membrane (shown ...

RT-PCR, sequencing, and analysis of VP1 genes for 92 nonserotypeable HEV clinical isolates.

The complete or partial VP1 genes of 92 nonserotypeable HEV isolates were sequenced and analyzed. The methods for RT-PCR, sequencing, and analysis of complete VP1 genes for 86 isolates were as described previously (53). To sequence and analyze partial VP1 genes for each of another 6 isolates (Table (Table1)1) (due to poor quality of complete VP1 sequencing results), PCR was performed using a Qiagen HotStarTaq DNA polymerase kit (Qiagen, Clifton Hill, Victoria, Australia) with the following cycling conditions: 95°C for 15 min; 35 cycles of 94°C for 30 s, 42°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and a hold at 22°C. The PCR mixture contained the following: 2 μl template cDNA, 0.3 μl each of primer 292 (100 pmol μl−1) and primer 222 (100 pmol μl−1) (32), 1 μl dNTPs (2.5 mM each dNTP), 2.5 μl 10× PCR buffer, 0.2 μl Qiagen HotStarTaq DNA polymerase (5 units μl−1), and water to 25 μl. The approach for sequencing of partial VP1 genes was as described previously (53), except for the addition of sequencing primers 292 and 222. Then, the genotype of each of these 6 isolates was determined by pairwise comparison of the partial VP1 sequence with a database containing complete VP1 sequences for the prototype and variant strains of all known HEV serotypes (33). Using criteria described previously (33), strains of homologous serotypes/genotypes can be easily distinguished from heterologous serotypes/genotypes and new serotypes/genotypes can be identified.

TABLE 1.
RT-PCR-RLB and sequencing genotyping results for 92 nonserotypeable HEV clinical isolates

Nucleotide sequence accession numbers.

Eighty new complete or partial VP1 sequences for 92 nonserotypeable isolates have been deposited in GenBank with accession nos. FJ868337 to FJ868371 and GU142867 to GU142911 (Table (Table11).

RESULTS

5′ UTR primer and probe design.

The 5′ UTR sequences of all known 87 prototype strains, as well as PV Sabin strains 1, 2, and 3, were aligned to allow the design of one primer pair (5UTR-Sn and 5UTR-Ab) and two probes (5UTR-Sp1 and 5UTR-Sp2) in highly conserved regions of the 5′ UTR (see Table S1 in the supplemental material). The primer and probe lengths were 18 to 22 bases, and the melting temperatures (Tm) were 58.62 to 73.44°C.

Design of new set of VP1 probes.

To confirm the RLB results on all 224 HEV isolates (especially the 92 nonserotypeable isolates) using the initial set of 80 serotype-specific probes (53) and to compare the efficiency of two sets of serotype-specific probes, another, new set of 80 serotype-specific probes (see Table S1 in the supplemental material) were designed in other relatively conserved regions of VP1 for use in RLB according to the methods and principles described previously (53). The probe lengths were 18 to 28 bases, and melting temperatures (Tm) were 58.02 to 66.62°C. The numbers of VP1 sequences aligned for probe design for each serotype/genotype are shown in Table S1 in the supplemental material. For the four probes per serotype/genotype, the proportions of strains covered among all strains within the serotype/genotype ranged from 71.6 to 100% (see Table S1 in the supplemental material). These data demonstrate that four probes per serotype/genotype of the new set are also capable of capturing the majority of the serotype/genotype strains.

RT-PCR-RLB results for 132 serotyped HEV clinical isolates.

HEV-specific 5′ UTR PCR (using 5UTR-Sn and 5UTR-Ab) and VP1 PCR (using VP1-All-Sn, VP1-A-Ab, VP1-B-Ab, and VP1-C-Ab) for 132 previously serotyped HEV clinical isolates were performed separately, and then the two PCR products were mixed together for RLB hybridization. The first membrane of each of the two sets contained four probes for CVA9; one previously sequenced CVA9 isolate (GenBank accession no. FJ868282) was used as the positive control to determine the locations of the first and the last samples of the RLB readout on the film (Fig. (Fig.1).1). Similarly, one previously sequenced E6 isolate (GenBank accession no. FJ868294) was chosen as the positive control for the second membrane of each of the two sets (not shown).

The RLB results showed that the 5′ UTR PCR products of all 132 serotyped isolates could be hybridized with two HEV-specific 5′ UTR probes on both sets of membranes. Meanwhile, after hybridization with two sets of serotype-specific probes in VP1 regions, both RLB results were concordant with the serological identification for 131 corresponding isolates, which meant that at least one probe among the four serotype-specific probes detected each isolate (not shown). One isolate (that could only be hybridized with two HEV-specific probes), previously identified as E14 by serotyping but negative by both RLBs, was confirmed as E25 by VP1 sequencing (GenBank accession no. FJ868313) and by repeating the conventional serotyping. These results correspond with our previous results for detection of the VP1 region (53).

The RLB assay generally cannot distinguish sequences with a one-base difference. As noted regarding our previous results (see Fig. Fig.11 in reference 53), in the current work some isolates also showed more than one positive dot on the membrane because some serotype/genotype-specific probes for some serotypes/genotypes contained sequences with only one base difference from each other. However, given the specificity of the probes, any one among four probes per serotype/genotype can confirm the specific serotype/genotype of each isolate.

RT-PCR-RLB results for 92 nonserotypeable HEV clinical isolates.

HEV-specific 5′ UTR PCR and VP1 PCR for 92 previously nonserotypeable HEV clinical isolates were performed separately, and then the two PCR products were mixed together for RLB hybridization. One CVA9 isolate (GenBank accession no. FJ868282) (Fig. (Fig.1)1) and one E6 isolate (GenBank accession no. FJ868294) were used as positive controls as described above.

The 5′ UTR PCR products of all 92 nonserotypeable isolates could be hybridized with two HEV-specific 5′ UTR probes on both sets of membranes, which confirmed that these nonserotypeable isolates were HEVs (Fig. (Fig.1).1). For the RLB using a previous set of 80 serotype-specific probes, each of 72/92 isolates was confirmed as a single genotype, while one isolate (belonging to one of the 20 common serotypes) was not detected (Table (Table1).1). For the RLB using the new set of 80 serotype-specific probes, each of 71 isolates could be identified as a single genotype (Fig. (Fig.1),1), whereas two isolates (belonging to 2 of the 20 common serotypes) were negative (Table (Table1).1). In total, two sets of RLB serotype-specific probes confirmed the genotypes for 73 nonserotypeable isolates, accounting for 79.3% (73/92) of all nonserotypeable isolates tested. Nineteen nonserotypeable isolates were not identified with either set of serotype-specific probes.

Two sets of serotype-specific probes identified 12 common genotypes among the 92 nonserotypeable isolates (Table (Table1),1), including HEV-A (CVA16 and EV71) and HEV-B (CVA9; CVB2 and -4; and E6, -7, -9, -11, -14, -18, and -30) genotypes. They could not detect 9 uncommon genotypes, including HEV-B (E1, -5, -13, -16, and -25) and HEV-C (CVA11, -20, -21, and -24) genotypes (see details below). In HEV-A, five and four isolates were genotyped as CVA16 and EV71, respectively. In HEV-B, E18 and E30 (containing 15 isolates, respectively) were the most prevalent genotypes among the nonserotypeable isolates.

Sequencing results for VP1 genes of 92 nonserotypeable HEV clinical isolates.

To further confirm and compare the accuracy of the two RLB results using the two sets of serotype-specific probes and to identify those nonserotypeable HEV isolates which were negative with both RLB serotype-specific probe sets, the complete or partial VP1 genes of 92 nonserotypeable isolates were also sequenced and analyzed (Table (Table1).1). Of these 92 isolates, the complete VP1 genes of 86 and partial VP1 genes of 6 isolates (belonging to uncommon genotypes) were sequenced: 21 genotypes (12 common genotypes plus 9 uncommon genotypes) were obtained. Of all 92 sequencing results, 73 corresponded with one or both RLB results; these 73 isolates belonged to the group of 20 common genotypes. Sequencing analysis of three isolates that could only be identified with one set of serotype-specific probes demonstrated that at least a two-base difference existed between the VP1 sequence and four serotype-specific probes for each of these isolates, which confirmed the accuracy and reliability of the RLB assay (Table (Table1).1). Another 19 isolates, which belonged to 9 uncommon genotypes and were not detected by either set of serotype-specific probes, could only be genotyped by VP1 sequencing. Interestingly, this included three genotypes (CVA11, -20, and -21) not previously identified in our laboratory. The VP1 sequences from the 92 nonserotypeable isolates clustered with their respective prototypes in the dendrogram (not shown).

DISCUSSION

Neutralization is a time-consuming and labor-intensive method for typing HEVs. Although a variety of molecular technique-based methods have been developed for the identification of HEV serotypes/genotypes (6, 7, 26, 28), the diversity of HEVs complicates genotyping. The design of HEV-specific primers and probes in the 5′ UTR (after alignment of 5′ UTR sequences of all known prototype strains) to target members of all known HEV species is practical and reasonable (12). Therefore, 5′ UTR HEV-specific primers and probes can be used as an effective tool to confirm whether an isolate is an HEV, and the design of generic assays lowers the risk of missing newly emerging or still unrecognized variants (49). In this study, two RT-PCR-RLB assays using two sets of serotype-specific probes for the VP1 region not only demonstrated excellent concordance with serotyping by neutralization for those isolates previously serotyped as belonging to the group of 20 common serotypes/genotypes but also corresponded with VP1 sequencing for previously nonserotypeable isolates belonging to the 20 common genotypes. For each of three nonserotypeable isolates only identified by one set of serotype-specific probes, sequencing showed that there was at least a two-base difference between the VP1 sequence and four serotype-specific probes; however, two sets of serotype-specific probes were capable of capturing these isolates complementarily. Meanwhile, sequencing and analysis of VP1 genes enables the genotyping of serologically nonserotypeable HEV isolates not identified by the RT-PCR-RLB assay. In view of the high proportion of serotyped (87.1%; 5,558/6,383) and nonserotypeable (79.3%; 73/92) isolates belonging to the 20 common HEV serotypes, in practice, either of the two sets of serotype-specific probes can be applied first to identify HEV isolates. Then, for isolates which are negative with the first set of serotype-specific probes, the other set can be employed. For those isolates not detected by either set, VP1 sequencing can be used as a complementary method.

The application of RT-PCR-RLB to the HEV genotyping as described here enables the simultaneous screening of up to 43 isolates with a turnaround time (including RNA extraction, RT-PCR, and RLB hybridization) of about one and a half working days. The preparation of one set of RLB membranes (two membranes) before hybridization takes less than 3 h, and they can be reused at least 20 times, based on prior experience. These features afford this technique a significant cost and time advantage over conventional serotyping methods, which are expensive and time consuming (usually at least 5 to 7 days), especially for those isolates finally regarded as nonserotypeable.

Microarray techniques for the detection and characterization of human pathogens have been already applied in clinical practice in some countries as part of standard laboratory diagnosis and monitoring of infectious diseases. Susi et al. (47) developed a straightforward assay for the rapid typing of HEVs using oligonucleotide arrays in microtiter wells. In their study, 10 probes for 10 different HEV reference strains were designed and employed. They used serotype consensus oligonucleotide probes for CVA9 to detect 25 CVA9 isolates. In combination with microarray techniques, our strategy could potentially be extended to cover more than 100 HEV serotypes with high efficiency and accuracy in the near future.

Interestingly, three genotypes (CVA11, -20, and -21) were identified for the first time in our laboratory by VP1 sequencing. The molecular typing system based on nucleotide sequencing of partial or complete VP1 genes can be used as a system for genotyping HEVs, especially those nonserotypeable isolates belonging to uncommon serotypes/genotypes.

Failure of serotyping due to aggregation can be overcome by ultrafiltration or treatment with sodium deoxycholate or chloroform, reducing agents, or a nonionic detergent prior to neutralization (15, 51). Plaque purification or limiting dilution can resolve the presence of a virus mixture (28). VP1 sequencing and analysis can be applied to discover new or previously unrecognized HEV serotypes. The detection of antigenic variants, especially among certain HEV serotypes (e.g., E30 and CVB), requires additional antisera not generally available to most clinical laboratories (28).

Norder et al. (27) compared 120 amino acids covering the VP1 BC loop of isolates with the prototype strains and demonstrated that most of the substitutions, compared with the sequences of their respective prototypes and other strains, were within or close to the BC loop for the majority of the untypeable isolates, which indicates that the BC loop is important for the reactivity of neutralizing antibodies. In our study, 21 genotypes (HEV-A [CVA16 and EV71], HEV-B [CVA9; CVB2 and -4; and E1, -5, -6, -7, -9, -11, -13, -14, -16, -18, -25, and -30], and HEV-C [CVA11, -20, -21, and -24]) were identified among 92 nonserotypeable isolates. In HEV-A, nonserotypeability of EV71 isolates may be due to substitutions within the BC loop and within the β-B region (27), while for nonserotypeable CVA16 isolates, the substitution of amino acid residue 108 might reorient the BC loop and affect its antigenic properties (27). Besides EV71 and CVA16, the serotyping of E18 was also difficult. This may be overcome by filtration (27). In addition, for CVB4, substitutions for residues 84 and 85 in the BC loop may be responsible for the abolition of neutralizing reactivity to the virus (23).

Predicted structural changes caused by mutations in the 5′ UTR and VP1 capsid have provided further insight into how enteroviruses evolve to cause a diverse spectrum of diseases (38, 50). Sandager et al. (41) revealed that postviral myopathy develops from a complex interplay between viral determinants in the 5′ UTR and VP1 capsid and have uncovered similarities between genetic determinants that cause viral myopathy and those involved in pathogenesis of other enteroviral diseases. Analysis of enterovirus prototype strains and clinical isolates has suggested that recombination is a frequent event in enterovirus evolution and that it generally occurs among viruses of the same species, except in the 5′ UTR (1, 4, 20, 21, 22, 31, 34, 35, 42, 45).

This work presents synchronous detection of the 5′ UTR and VP1 region by RT-PCR-RLB, which is especially beneficial for the identification of nonserotypeable HEV isolates. Genotyping by RT-PCR-RLB complements traditional serotyping and VP1 sequencing and provides quick, accurate, and cost-effective identification of 20 common serotypes/genotypes. Furthermore, the ease and low cost of this strategy will improve the diagnosis and epidemiological investigation of enteroviral infections and outbreaks where other typing methods are unavailable.

Conclusions.

A variety of factors can interfere with the identification of HEVs using serological methods, which can be overcome or resolved by various methods. In this study, two sets of serotype-specific probes are shown to be capable of capturing the majority of the serotype/genotype strains belonging to 20 common serotypes/genotypes, either simultaneously or complementarily. The VP1 sequencing results for nonserotypeable isolates confirmed the accuracy and reliability of RT-PCR-RLB. Synchronous detection of the 5′ UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, which is especially beneficial for the detection of nonserotypeable HEV isolates. In combination with microarray techniques, our strategy could potentially be extended to cover current more than 100 HEV serotypes with high efficiency and accuracy in the near future.

Supplementary Material

[Supplemental material]

Acknowledgments

Fei Zhou is supported by an Australian National Health and Medical Research Council (NHMRC) postgraduate scholarship, a CIDM Public Health postgraduate scholarship, and a Westmead Medical Research Foundation postgraduate scholarship.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 February 2010.

Supplemental material for this article may be found at http://jcm.asm.org/.

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