by anti- HEV71/BrCr and anti-HEV71/C7 sera, both available as standard monospecific anti HEV71 serum in Japan. These results also indicate that phylogenetic analysis with the VP4 sequences of HEVs can identify the serotypes in the same way as neutralization tests with HEV serotype-specific antisera. We are now preparing antiimmune sera against OC/00219, OC/ 00260, and OC/00261, respectively, to confirm antigenically that these are the prime strains of HEV71 neutralized by anti- HEV71/BrCr serum. Oberste et al. have shown that HEV VP1 nucleotide sequences correlate with antigenically defined serotypes and have demonstrated the utility of VP1 sequences as a molecular surrogate for antigenic type (19,35). They have also shown that the VP1 sequences have a better correlation with HEV serotypes than the 5' NTR or the VP4-VP2 junction (36). The phylogenetic analysis based on the VP4 sequences we have described also correlates well with HEV serotypings by anti- immune sera. We used 21 HEV serotypes antigenically defined in our laboratory and another 8 strains available from GenBank as prototype strains in this analysis. We do not know whether 29 serotypes are sufficient for the phylogenetic analy- sis of HEV, as there are >60 serotypes. The good result of HEV phylogenetic classification based on the VP4 sequences might depend on the prototype numbers (29 of 64 serotypes) that we used. Ishiko et al., who performed HEV phylogenetic analyses based on VP4 sequences (23), used 45 HEV sero- types as prototype strains and obtained a phylogenetic tree similar to ours (Figure 1) except for a difference in the proto- type strain numbers. Another phylogenetic analysis based on the VP4 sequences in this article was performed against the HEV71 strains (Figure 2). For this analysis, the HEV71 strains were clustered in three distinct genotypes, and the nomencla- ture was almost the same as for the HEV71 analyses based on the VP1 nucleotide sequences (17,25). Recently, Chu et al. also reported the appropriateness of the phylogenetic analysis with the VP4 sequences for the molecular epidemiology of HEV71 outbreak in Taiwan in 1998 (29). These results suggest that the phylogenetic analysis based on the VP4 nucleotide sequences is also useful as a molecular surrogate for antigenic HEV serotyping. The analysis was more convenient based on the VP4 sequences than the VP1 sequences, since the com- plete VP4 sequence is 207 nt and the complete VP1 sequences RESEARCH are 834 to 951 nt (35), although the 3' third of the VP1 sequence of 365 nt was used (32). The VP4 nucleotide sequences of OC/99-Hanasaka and OC/00272 were identical, but the results of neutralization assays were different. OC/99-Hanasaka was easily neutralized by HEV pooled sera against EV18, but OC/00272 was not. The same results were observed for strains OC/00168 and OC/ 00219 HEV71, i.e., the results of their neutralization tests dif- fered in spite of the VP4 sequence identity. These results indi- cate that the VP4 nucleotide sequences are highly conserved even though the neutralizable epitopes are antigenic variants. We compared the VP4 nucleotide and deduced amino acid dif- ferences of HEV71 strains, BrCr (25), E1387 (15), OC/9632, OC/99-Ikeda, OC/0078, OC/00219, and OC/00260. HEV71 genotypes indicated 1 to 37 nt (0.5% to 17.9%) differences. However, we found no amino acid differences (100% identity) (Table 6). Complete homology of the HEV71 VP4-deduced amino acid sequences has also been described (20,29), and Singh et al. demonstrated amino acid substitutions in the VP2 and VP3 regions, with the greatest variation in VP1 (20). These results indicate that VP4 is the most stable protein; accordingly, VP4 genes will be suitable for the molecular iden- tification of HEV serotypes in the future. VP4 is not exposed on the outer surface of the capsid, and no neutralizable epitopes appear to exist in VP4. On the other hand, VP1, VP2, and VP3 are outer capsid proteins and con- tain neutralizable epitopes (37,38). A number of important neutralization epitopes may exist on VP1 (1,30,39). To con- firm the important neutralization sites on VP1, we compared the deduced VP1 amino acid sequences of HEV71 strains OC/ 00168, OC/00219, OC/00260, and OC/00261. OC/00168 was neutralized by anti-HEV71/BrCr serum, while OC/00219, OC/ 00260, and OC/00261 were not. Comparison of the deduced VP1 amino acid sequences showed that no mutated residues on the VP1 region corresponded to the result of the neutraliza- tion tests. This result indicates that either the important neu- tralization epiotopes for anti-HEV71/BrCr serum do not exist on the VP1 protein, or the epitopes are specifically masked in the cases of OC/00219, OC/00260, and OC/00261. Further analysis against the VP2 and VP3 regions of these strains should allow interpretation of these findings. Table 6. Number of nucleotide and deduced amino acid differences between the VP4 genes of human enterovirus 71 strains,a Osaka, Japan, 2000 OC/9632 OC/0078 OC/00219 OC/00260 OC/99-Ikeda BrCr E1387 OC/9632 OC/0078 OC/00219 OC/00260 OC/99-Ikeda BrCr 0 0 0 0 0 0 E1387 33 0 0 0 0 0 aNucleotide numbers are given above the diagonal and amino acid numbers below it. 37 10 0 0 0 0 36 7 13 0 0 0 37 8 14 1 0 0 32 7 15 10 11 0 34 33 37 37 36 34 Emerging Infectious Diseases • Vol. 8, No. 3, March 2002 303