RESEARCH Strain OC/9632 OC/99-Ikeda OC/0078 OC/0080 OC/0096 OC/00114 OC/00125 OC/00168 Table 3. Characteristics of eight human enterovirus (HEV)71 strains, as antigenically defined, Osaka, Japan, 2000 Specimen Date of sampling Clinical symptoms Age(years) NAa 6 1 5 0 6 4 Throat swab Stool Stool Stool CSF CSF Throat swab Throat swab 4/11/1996 9/11/1999 5/18/2000 5/9/2000 5/25/2000 5/31/2000 6/7/2000 6/21/2000 HFMD HFMD, AM HFMD, AM Diarrhea Diarrhea, AM Fever Encephalitis Herpangina Isolated cells MA104 Vero Vero Vero Vero Vero Vero Vero a = Not available; HFMD = Hand, foot, and mouth disease; AM = aseptic meningitis; CSF = cerebrospinal fluid. anti-HEV71/C7 sera; both supplied by the National Institute of Infectious Diseases, Japan) were performed to confirm the serotype of the untypeable strains from the first microneutral- ization assay. Complete VP1 Nucleotide Compared with Deduced Amino Acid Sequences of HEV71 Strains The complete VP1 nucleotide sequences of HEV71 strains OC/00168, 0C/00219, OC/00260, and OC/00261 were deter- mined by the same procedure described above, except for the primers. The primers used for the analysis of VP1 nucleotide sequence were 71F2399 (5' -AGAAYTTYACCAT- GAAACTG-3' relative to nt 2380-2399 in the genome of HEV71 MS/7423/87 strain [25]; the nucleotide positions of the following are also relative to this strain: 71F2793 (5'- AGACATAACTGGYTACGCCAC-3' nt 2774-2793) and 71F3042 (5'-CATGTCACCYGCGAGCGCTT-3' nt 3023- 3042) as the forward, 71R2712 (5'-CTACCAARCCTGC- CCTACTG-3' nt 2693-2712), 71R3066 (5'-GGTACCCGTCG- TAAAACCAC-3' nt 3047-3066) and 71R3376 (5'- AAGTTGCCCACGTAGATGGC-3' nt 3357-3376) as the reverse. The VP1 nucleotide sequence of HEV71 BrCr strain (25) was obtained from GenBank. Sequencer software (ver- sion 3.0; Hitachi Software) was used for determination and comparison of the complete VP1 nucleotide and deduced amino acid sequences of these HEV71 strains. Results Determination of Complete VP4 Nucleotide Sequences of HEVs During May to July 2000, six viruses submitted to our lab- oratory (OC/0071, OC/0073, OC/00219, OC/00260, OC/ 00261, and OC/00272) could not be neutralized by standard pools of HEV typing sera and three antimonospecific sera (Table 1). However, the cytopathic effects of these viruses on RD-18S or Vero cells were all HEV-like (data not shown). To identify the serotypes of these untypeable HEV-like viruses by a method other than the neutralization assay, we determined the complete VP4 nucleotide sequences of all 6 strains and another 21 HEV serotypes identified in our laboratory over the past 6 years. The 3' end of the VP4 gene of each virus was determined from the deduced amino acid sequences as described (26,27). The complete VP4 nucleotide sequences of all HEV strains used in this study were 207 nt long, and the deduced amino acid sequences of all VP4 proteins were 69 amino acids long (data not shown). Phylogenetic Analysis of HEVs A phylogenetic tree was constructed based on the complete VP4 nucleotide sequences of the 6 HEV-like untypeable strains, the 21 HEV serotypes identified in our laboratory as prototype strains, and another 8 HEV serotypes available from the GenBank database (Figure 1). The 29 different HEV sero- types defined antigenically were clustered in four distinct lin- eages, as described (23). Three of the six untypeable strains (OC/0071, OC/0073, and OC/00272) were classified nearest to EV18. The VP4 nucleotide sequences of strains OC/0071 and OC/0073 were identical. The VP4 gene sequence of OC/00272 was the same as that of OC/99-Hanasaka, which was used as a prototype strain for EV18. The nucleotide sequences of these two clusters differed by 5 nt, but the deduced amino acid sequences were the same (data not shown). The other three untypeable strains (OC/00219, OC/00260, and OC/00261) were classified nearest to HEV71. The VP4 sequence of OC/ 00219 was the same as that of OC/00168, which was used as a prototype strain for HEV71. The VP4 nucleotide sequences of OC/00260 and OC/00261 were identical. The difference between these two clusters was 11 nt. The deduced amino acid sequences were the same (data not shown). Remicroneutralization Tests According to the results of the phylogenetic analysis based on the complete VP4 nucleotide sequences, remicroneutraliza- tion tests were performed. Microneutralization tests using the monospecific immune serum for EV18 were done against OC/ 0071, OC/0073, and OC/00272, and this serum neutralized these viruses. The same tests, using the two species of mono- specific immune serum, anti-HEV71/BrCr and anti-HEV71/ C7, were performed against OC/00219, OC/00260, and OC/ 00261, but neither serum neutralized the viruses (Table 4). 300 Emerging Infectious Diseases • Vol. 8, No. 3, March 2002