HEV serotype PV1 PV2 PV3 EV3 EV6 EV7 EV9 EV11 EV16 EV18 EV25 EV30 CAV9 CAV16 CBV1 CBV2 CBV3 CBV4 CBV5 CBV6 HEV71 Strain OC/00417 OC/00138 OC/99355 OC/00467 OC/99350 OC/96221 OC/00129 OC/98535 OC/95378 OC/99- Hanasaka OC/00263 OC/97633 OC/96234 OC/00351 OC/00364 OC/99284 OC/97620 OC/00362 OC/00223 OC/00325 OC/00168 0 0 0 7 0 7 3 3 1 7 0 1 4 0 0 6 1 0 0 2 RESEARCH Isolated cells Vero Vero Vero RD-18S RD-18S MA104 RD-18S RD-18S MA104 RD-18S RD-18S RD-18S RD-18S Vero Vero RD-18S RD-18S Vero Vero Vero Vero Table 1. Unidentified enterovirus strains and patient information, Osaka, Japan, 2000 Table 2. Characteristics of 21 human enterovirus (HEV) serotypes antigenically defined, Osaka, Japan, 1995–2000 Strain OC/0071 OC/0073 OC/00219 OC/00260 OC/00261 OC/00272 Patient age (years) Date of Specimen sampling Clinical symptoms 2 2b 0 0 0c 6 Stool CSF Throat swab Throat swab Stool 5/11/2000 5/11/2000 7/7/2000 7/18/2000 7/18/2000 Stool 7/18/2000 AMa AM HFMD HFMD, AM HFMD, AM AM Isolated cells RD-18S RD-18S Vero Vero Vero RD-18S aAM = aseptic meningitis; CSF = cerebrospinal fluid; HFMD = hand, foot, and mouth disease. bSame patient as OC/0071. cSame patient as OC/00260. RNA Extraction and Reverse Transcription Viral RNAs were extracted from the cell-culture superna- tants by using ISOGEN-LS (Nippon Gene, Tokyo, Japan). cDNAs were synthesized with an Omniscript Reverse Tran- scriptase Kit (QIAGEN K.K., Tokyo, Japan) according to the manufacturer’s instructions. The primers used for the synthesis were EVP-2 (5'-CCTCCGGCCCCTGAATGCGGCTAAT-3' relative to nt 444-468 in the genome of Poliovirus (PV) Sabin 1 strain) (22) and OL68-1 (5'-GGTAAYTTCCACCAC- CANCC-3' relative to nt 1178-1197 of Sabin 1), as described (23). Polymerase Chain Reaction Amplification of cDNAs Polymerase chain reaction (PCR) was performed by using 2 µL of each cDNA in a 50-µL reaction mixture containing 1.5 U of Taq DNA polymerase (Takara Shuzo, Shiga, Japan), 20 pmol of EVP-2 primer, and 20 pmol of OL68-1 primer. Each reaction was incubated in a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA) according to the fol- lowing protocol: 5 minutes at 95°C, 40 cycles of 95°C for 30 seconds, 68°C for 30 seconds, 72°C for 1 minute, and then at 72°C for 5 minutes. After the appearance of approximately 750 bp-specific amplified fragments was confirmed by agar- ose gel electrophoresis, the amplicons were purified with a QIAquick PCR purification kit (QIAGEN). DNA Sequence Analysis Approximately 100 ng of purified amplicon was used in the reaction with the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems), and DNA sequenc- ing was performed by using an ABI PRISM 310 DNA sequencer (Applied Biosystems). All DNA sequencings were performed on both strands using EVP-4 (5'-CTACTTTGGGT- GTCCGTGTT-3' relative to nt 541-560 in the genome of PV Sabin 1 strain) as the forward primer and OL68-1 as the reverse primer (23). Sequencer software (version 3.0; Hitachi Software, Tokyo, Japan) was used to determine the approxi- mately 600-bp nucleotide sequence spanning 5' NTR to one a CSF = Cerebrospinal fluid; = not available. third of VP2 (including all of VP4), translate nucleotide sequence to amino acid sequence, and decide the complete VP4 coding sequence of each virus. Phylogenetic Analysis A phylogenetic tree based on the complete VP4 nucleotide sequence was constructed by the neighbor-joining method (24) as implemented with the CLUSTAL X program (version 1.63b, December 1997; http://www-igbmc.u-strasbg.fr/Bio- Info/ClustalX/). The reliability of the neighbor-joining tree was estimated by bootstrap analysis with 1,000 pseudorepli- cate datasets. The complete VP4 sequences of eight HEV sero- types not isolated in our laboratory were obtained from GenBank and included in the HEV analysis. Complete VP4 nucleotide sequences of another 18 HEV71 strains were obtained from GenBank and used in the phylogenetic analysis. Remicroneutralization Tests According to results of HEV phylogenetic analysis with the complete VP4 nucleotide sequences, remicroneutralization tests using monospecific antiserum against echovirus 18 (EV18; Denka Seiken), or HEV71 (anti-HEV71/BrCr and Emerging Infectious Diseases • Vol. 8, No. 3, March 2002 299 Age (years) Specimen Throat swab 10/13/2000 Throat swab 8/31/2000 Date of sampling 6/10/2000 11/8/1999 11/13/2000 11/8/1999 7/22/1996 6/8/2000 9/23/1998 9/11/1995 11/8/1999 7/17/2000 9/29/1997 8/2/1996 9/6/2000 9/11/1999 9/19/1997 9/8/2000 7/7/2000 8/8/2000 Nasal mucus Throat swab Throat swab Stool Stool Stool CSF Stool Stool Stool Stool CSFa CSF Stool CSF Stool CSF Throat swab Throat swab 6/21/2000