RESEARCH States in 1970 and 1987, respectively (25), four in Malaysia in 1997 (15), one in Singapore in 1998 (28), eight in Taiwan in 1998 (15,28), two in the United Kingdom in 1999, and one in China (year unknown). The HEV71 strains were clustered in three distinct genotypes, designated A, B, and C. The genotype nomenclature of HEV71 strains for phylogenetic analyses based on the VP1 (17,25,28) and VP4 (29) nucleotide sequences has been reported, and the results (Figure 2) were consistent with previous findings. Among the HEV71 strains that were identified in our laboratory, only OC/99-Ikeda was classified in genotype C. Seven of eight strains identified in our laboratory by neutralization tests were classified in geno- type B; five of these had the same VP4 nucleotide sequence. OC/00219, OC/00260, and OC/00261 were also classified in this genotype. This result demonstrated that strains OC/00219, OC/00260, and OC/00261 were HEV71 serotypes. Comparison of the Complete VP1 Nucleotide and Deduced Amino Acid Sequences of HEV71 Strains Strains OC/00219, OC/00260, and OC/00261 were classi- fied in HEV71 by the phylogenetic analysis, although these viruses were not neutralized by monospecific anti-HEV71 sera. Because the VP1 protein contains a number of important neutralization sites (1,30), we determined the complete VP1 nucleotide sequences and compared the deduced amino acid sequences of OC/00219, OC/00260, and OC/00261. OC/ 00168 used as a prototype strain for HEV71 was also analyzed because this strain was neutralized by anti-HEV71/BrCr serum; moreover, its VP4 gene was the same as that of OC/ 00219 (Figure 2). The complete VP1 nucleotide sequences of these strains were 891 nt long, and the deduced amino acid sequences were 297 amino acids long. The differences of VP1 nucleotide sequences were 4 to 42 nt (0.4% to 4.7%), and the difference of deduced amino acid sequences was one amino acid (0.3%) among these viruses. The differences of VP1 nucleotide sequences between OC/00168 and OC/00219 were 4 nt (0.4%), and the deduced VP1 amino acid sequences of these strains were the same. The VP1 nucleotide and deduced amino acid sequences of OC/00260 and OC/00261 were Table 4. Results of re-microneutralization tests with human enterovi- rus (HEV) monospecific antiserum, Osaka, Japan, 2000 HEV monospecific antiserum Strain OC/0071 OC/0073 OC/00219 OC/00260 OC/00261 OC/00272 Isolated cells RD-18S RD-18S Vero Vero Vero RD-18S Predicted HEV Serotypea EV18b EV18 HEV71 HEV71 HEV71 EV18 Anti- EV18 + + ND ND ND + Anti- HEV71/ BrCr ND ND -- -- -- ND aSerotypes were predicted from the phylogenetic analysis in Figure 1. b EV18 = echovirus 18; ND = Test not done. Anti- HEV71/C7 ND ND -- -- -- ND 301 Figure 1. Phylogenetic analysis based on the human enterovirus (HEV) VP4 nucleotide sequences. The phylogenetic tree was constructed by the neighbor-joining method as implemented in CLUSTAL X program (version 1.63b). The marker denotes a measurement of the relative phylogenetic distance. The VP4 sequences of eight HEV serotypes described below are available from GenBank. The strain name and accession number are shown in parentheses: HEV1 (Bryson, AF250874), HEV12 (Travis, NC 001810), echovirus 26 (EV26, Coro- nel, AF117697), EV29 (JV-10, AF117698), coxsackie virus A2 (CAV2; Epsom/14448/99, AJ2296215), CAV21 (Coe, NC 001428), CAV24 (EH24/70, D90457), and HEV70 (J670/71, D00820). Phylogenetic Analysis of HEV71 Strains To establish whether OC/00219, OC/00260, and OC/ 00261 belong to HEV71, we used another phylogenetic analy- sis based on the complete VP4 nucleotide sequences of various HEV71 strains (Figure 2). In this analysis, we examined eight HEV71 strains isolated and identified in our laboratory from 1996 to 2000 (Table 3). All these HEV71 strains except for OC/9632 were identified by microneutralization tests with anti HEV71/BrCr serum (data not shown). Of the HEV71 strains available from GenBank, two were isolated in the United Emerging Infectious Diseases • Vol. 8, No. 3, March 2002