RESEARCH Molecular Classification of Enteroviruses Not Identified by Neutralization Tests Hideyuki Kubo,* Nobuhiro Iritani,* and Yoshiyuki Seto* We isolated six viruses from patients diagnosed with aseptic meningitis or hand, foot, and mouth disease. The cytopathic effect of these viruses on cultured cells was like that of enteroviruses. However, viral neu- tralization tests against standard antisera were negative. Phylogenetic analysis with the complete VP4 nucleotide sequences of these 6 viruses and 29 serotypes of enteroviruses classified 3 of the viruses as serotype echovirus type 18 (EV18) and 3 as serotype human enterovirus 71 (HEV71). These results were confirmed by remicroneutralization tests with HEV-monospecific antisera or an additional phylogenetic analysis with the complete VP4 nucleotide sequences. Phylogenetic analysis with complete VP4 genes is more useful than neutralization tests with enterovirus serotype-specific antisera in identifying enterovirus serotypes. T naviridae includes the human pathogens that cause a wide he human enterovirus (HEV) genus of the family Picor- spectrum of acute disease, including hand, foot, and mouth disease (1), aseptic meningitis (2,3), encephalitis (3-6), and neonatal sepsislike disease (7,8). Sixty-four serotypes of HEV have been recognized antigenically by neutralization tests with anti-HEV antibodies (9). HEVs have long been classified on the basis of serotype-specific antisera in virus neutralization tests (1,10), the only method available for serotyping HEVs. However, virus neutralization is both labor- and time-inten- sive, and antigenic variants in many serotypes of HEV can affect test results (1). The HEV genome comprises a 5' nontranslated region (NTR), a long open reading frame that encodes a protein of approximately 2,100 amino acid residues, a short 3' NTR, and a polyadenylated tail. The polyprotein is co- and post-transla- tionally cleaved to yield four structural proteins: VP4, VP2, VP3, and VP1 (1). Recently, attempts have been made to clas- sify the HEV serotypes by using the partial nucleotide sequences of the HEV genomes (i.e., the 5' NTR [11-13], the VP4-VP2 junction [14-16], and VP1 [17-20]). Methods for molecular classification of HEVs should not only identify the serotypes rapidly but also detect antigenic variant strains or new serotypes. A new serotype of HEV has recently been identified by comparing the complete VP1 nucleotide sequences; its proposed name is HEV73 (19). To investigate the HEV serotypes of six HEV-like viruses that were not neutralized by standard HEV typing sera, we determined the complete VP4 nucleotide sequences of these 6 viruses and 21 HEV antigenically defined serotypes, then per- formed phylogenetic analysis with another 8 HEV serotypes available from GenBank. The classifications of the untypeable viruses were confirmed by using HEV-monospecific antisera or an additional phylogenetic analysis with the VP4 *Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan sequences. The molecular classification of HEV with the com- plete VP4 sequences is useful for identifying the HEV sero- types. Methods Virus Isolation and the Neutralization Test The clinical specimens were injected into Vero, RD-18S, or MA104 cells to isolate viruses. All cells were grown in min- imum essential medium (MEM) containing 10% fetal bovine serum (FBS) and maintained in MEM containing 1% to 2% FBS after being added to 48-well plates (Sumitomo Bakelite, Tokyo, Japan). The cells were incubated for 1 week, after which culture fluids were passaged and incubated for another week. Cultured cells showing cytopathic effects were regarded as virus isolation-positive and, together with the culture super- natant, were harvested and stored at -80°C before use. To sero- type the viruses, microneutralization tests were performed with antiserum pools of Lim and Benyesh-Melnick (21) (Denka Seiken, Tokyo, Japan) or in-house monospecific immune sera against coxsackie virus A10 (CAV10), CAV16, and HEV71, respectively. Viruses Of the six viruses that could not be identified by the neu- tralization tests described above (Table 1), strains OC/0071, OC/0073, and OC/00272 were isolated from patients diag- nosed with aseptic meningitis by using RD-18S cells. OC/ 00219, OC/00260, and OC/00261 were isolated from patients diagnosed with hand, foot, and mouth disease or aseptic men- ingitis by using Vero cells. No sera from these patients was available for analysis. Twenty-one serotypes were isolated and identified in our laboratory during 1995-2000 (Table 2); these strains were used in the experiments. For additional investiga- tions of HEV71, we used eight HEV71 strains isolated and identified in our laboratory (Table 3). 298 Emerging Infectious Diseases • Vol. 8, No. 3, March 2002