Twenty throat swab samples were obtained from the Center for Disease Control and Prevention (CDC) of Jilin, China. Each sample was resuspended in 3 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal bovine serum. The samples were subsequently stored at −80°C until further use. Vero cells in DMEM containing 2% fetal bovine serum were inoculated with 200 µl of resuspended sample and incubated at 37°C in a 5% CO₂ atmosphere. The cells were observed daily for cytopathic effects (CPE). At least two passages were performed before the culture was regarded as negative for CPE. Following complete CPE, the cells were freeze/thawed three times and subsequently centrifuged at 8,000 × g at 4°C for 15 min. The supernatant was collected and stored at −80°C.

Ten-fold serial dilutions (10⁻¹ to 10⁻¹⁰) were made for each virus. Each dilution of virus was inoculated into 8 wells of a 96-well plate containing Vero cells. The plates were incubated at 37°C for 7 days. Viral titers were calculated using the Reed-Muench method (9).

Total RNA was extracted from the infected cell suspension using the Qiagen Viral RNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The extracted RNA was used as a template for RT-PCR amplification using Takara RT-PCR kit version 3.0. The enterovirus universal primers and specific primers of the VP1 genes for CVB3, CVA16 and EV71 are listed in Table I. RT was performed at 42°C for 30 min followed by a denaturation step at 95°C for 5 min. The cycling conditions for enterovirus specific PCR were as follows: Initial denaturation at 94°C for 1 min, 40 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 90 sec, and a final extension at 72°C for 10 min. The thermocycling conditions for CVB3 were as follows: Initial denaturation at 94°C for 2 min, 30 cycles at 94°C for 30 sec, 55°C for 45 sec and 72°C for 45 sec, and a final elongation step at 72°C for 8 min. RT-PCR products were gel purified and sent for sequencing at the Sangon Biotech Institute Co., Ltd. (Shanghai, China).

The GenBank database was searched for similar sequences using the BLAST program. Nucleotide and deduced amino acid sequences of the viruses were aligned using the BioEdit 7.0.5.3 software (10) and subsequently compared with each other and with the reference sequences of homologous CVB3 retrieved from the GenBank database. Phylogenetic trees were generated using Mega 6.0 (11) and the maximum likelihood method and Kimura two-parameter model. Bootstrapping was performed with 1,000 replicates.

Twenty throat swabs were collected from patients who presented with symptoms of FRI (temperature ≥37.5°C and a rash on the face, neck or trunk) between April and June 2013 in three different areas of Jilin, China. All the samples were provided by the Jilin Provincial CDC. The 20 samples were all positive for enterovirus by RT-PCR. When specific primers for CVB3, CVA16 and EV71 were used, only five samples were positive for CVB3. The other 15 samples were negative for these three viruses. Specific details of the five positive samples (JL-01, JL-02, JL-03, JL-04 and JL-05) are shown in Table II.

The titers of JL-01, JL-02, JL-03, JL-04 and JL-05 were 10^6.5, 10^6.4, 10^7.5, 10^6.5 and 10^6.5 tissue culture infectious dose₅₀/100 µl, respectively, indicating a strong ability to infect Vero cells.

The amplified VP1 fragments of CVB3 were 772 base pairs. Comparisons of the VP1 gene fragments indicated that the five CVB3 viruses were between 99.99–100% identical at the nucleotide and amino acid levels. In addition, all the isolates were 99.99% identical at the nucleotide and amino acid levels to the CVB3/MKP and CVB3/Macocy viruses, which have been reported to induce myocarditis and central nervous system disease (12,13), respectively. The phylogenetic analysis using the VP1 sequences (Fig. 1) showed that the five CVB3 isolates were genetically distinct from the CVB3/96HN.3/SD (JQ364844) and CVB3/01318/SD (JQ364847) viruses, which have been isolated from Shandong and are known to cause aseptic meningitis (14). DQ000164 and AF169026, which were isolated from Harbin and Beijing in 2005 and 1999, are located on the same branch with the five CVB3 isolates in the present study.