RESEARCH aVP1 nucleotide sequence was obtained from GenBank (accession no. U22521) and translated into the deduced amino acid sequence. pools (1,10). Since >60 serotypes of HEV are known to infect humans (1,19), the HEV serotype is almost impossible to iden- tify by using monospecific antiserum from the first microneu- tralization test. Furthermore, the neutralization test is labor- intensive and time-consuming, requiring several weeks. As an alternative, identification based on nucleotide sequences has been used successfully in several laboratories (15,19,20, 23,29,31-35). To investigate the serotypes of the six untype- able HEV-like viruses that were not neutralized by the stan- dard HEV antisera, we used phylogenetic analyses based on the complete VP4 nucleotide sequences of HEVs and were able to determine the serotype of each virus in the light of these results. OC/0071, OC/0073, and OC/00272 were thought to be EV18 strains by the phylogenetic analysis (Figure 1) and were neutralized by the monospecific anti-EV18 serum. These results indicate that the phylogenetic analysis based on the VP4 nucleotide sequence is consistent with the result of the microneutralization tests using the serotype-specific sera. OC/ 00219, OC/00260, and OC/00261 were thought to be HEV71 strains by the same analysis (Figure 1), but these viruses were not neutralized by the two monospecific anti-HEV71 sera. The phylogenetic analysis based on the HEV71 VP4 sequences confirmed that these viruses were HEV71 strains belonging to genotype B (Figure 2). We considered that OC/00219, OC/ 00260, and OC/00261 were all HEV71 strains not neutralized Table 5. Differences in deduced VP1 amino acid sequences (aa 1- 297) of human enterovirus 71 strains BrCr, OC/00168, OC/00219, OC/00260, and OC/00261, Osaka, Japan, 2000 Amino acid position BrCra OC/00168 OC/00219 OC/00260 OC/00261 Lys Pro Pro Asp Lys Ala Lys Arg Asp Asp Pro Ser Ser Glu Ser Ile Ser Asp Arg Gln Gln Asn Glu Thr Glu Glu Glu Glu Gln Leu Thr Lys Pro Val Ala Asn Figure 2. Phylogenetic analysis based on the human enterovirus 71 (HEV71) VP4 nucleotide sequences. The phylogenetic tree was con- structed as described in the Figure 1 legend. The genotypes of the HEV71 cluster are denoted on the right. The VP4 sequences of 18 HEV71 strains available from GenBank are denoted by strain name, followed by the country and year isolated. Abbreviations for countries are as follows: US, United States; ML, Malaysia; CH, China; UK, United Kingdom; TW, Taiwan; and SG, Singapore. The accession numbers are as follows: BrCr; U22521, Tainan/6092/98; AF304459, 1425a/98/tw; AF176044, NCKU9822; AF136379, TW/2086/98; AF119796, 5142/98; AB037251, TW/2272/98; AF119795, HO106/98; AB037252, Epsom/ 815/99; AJ296213, SHZH9; AF302996, KED005; AB051334, E1387; AB051313, 13/Sin/98; AF251358, SK036; AB051333, KED60; AB051335, SK026; AB051332, MS/7423/87; U22522, Epsom/10620/ 99; and AJ296214. identical (data not shown). The deduced VP1 amino acid sequences of these four strains were compared with that of BrCr (Table 5). There were 18 amino acid (6%) differences between BrCr and other strains. The different amino acid posi- tions of strains OC/00168, OC/00219, OC/00260, and OC/ 00261 against BrCr were the same. Any mutated residues dis- tinguishable BrCr and OC/00168 from OC/00219, OC/00260, and OC/00261 were not recognized in the VP1 amino acid sequences. Discussion The serotype identification of HEVs has been performed by microneutralization tests using standard HEV antiserum 18 22 30 31 43 58 98 145 164 167 172 183 184 244 246 249 275 282 302 Emerging Infectious Diseases • Vol. 8, No. 3, March 2002 Strain Arg Gln Gln Asn Glu Thr Glu Glu Glu Glu Gln Leu Thr Lys Pro Val Ala Asn Arg Gln Gln Asn Glu Thr Glu Glu Glu Glu Gln Leu Thr Lys Pro Val Ala Ser Arg Gln Gln Asn Glu Thr Glu Glu Glu Glu Gln Leu Thr Lys Pro Val Ala Ser