An epidemic of aseptic meningitis due to coxsackievirus B5 in Nara prefecture, Japan: an epidemiological analysis by PCR-RFLP.

*Corresponding author: Mailing address: Department of Virology, Nara Prefectural Institute for Hygiene and Environment, Ohmoricho 57-6, Nara 630-8131, Japan. Tel: +81-742-20-2887, Fax: +81-742-27-0634, E-mail: [email protected] The epidemics of aseptic meningitis in the summer season are caused mainly by enteroviruses. A surveillance study in Japan showed that more than 70% of cases of viral meningitis were caused by serotypes 30, 9, 7, and 6 echoviruses and serotype 5 group B coxsackieviruses (CB5) (1). In 2001, we encountered an epidemic of aseptic meningitis, gastroenteritis and herpangina due to CB5 in the central areas of Nara Prefecture. The first case was reported from Kashihara City in June 25 and was a 7-year-old female patient. In the epidemic, a total of 46 CB5 isolates were obtained from 57 aseptic meningitis patients. The epidemic appeared to consist of a local outbreak at Sakurai City area and sporadic outbreaks involving the whole central area of Nara Prefecture. In order to determine whether or not CB5 viruses causing local and sporadic epidemics were genetically different, we conducted a PCR-RFLP and sequencing analysis of the virus isolates. Cerebrospinal fluids were obtained from patients in Saiseikai-Chuwa Hospital, Saiseikai-Gose Hospital, Mimuro Hospital, and Kokuho-Central Hospital. Virus was isolated by using HEp-2, RD-18S, and MA cells; the virus developed CPE in HEp-2 cells in 2-3 days after inoculation. The isolated viruses were identified by neutralization assay with specific antibodies (Denka-Seiken Co., Tokyo). RT-PCR of the VP1-2C region (about 1500 bp) was carried out according to Caro et al.’s protocol (2). RFLP analysis was conducted on 50 μl of PCR products after DdeI (C↓TNA), HpaII (C↓CGG) or HaeIII (GG↓CC) restriction enzyme (Invitrogen, Carlsbad, Calif., USA) digestions. DNA sequences were determined by using a sequencing kit (Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing Kit, Amersham Biosciences, Buckinghamshire, UK). As a standard strain, RNA from Faulkner strain was used. The geographical distribution of the patients is shown in Figure 1. On one hand, the disease affected 27 patients in Sakurai City in a short, local outbreak (From July 17 to August 5), while 19 patients occurred in wide areas involving Kashihara City, Yamatotakada City, and Gose City over a long period (from June 25 to November 19), on the other. The age of the patients ranged from 3 months to 10 years and the highest incidence was found in the 5-year-old children’s group. The male: female ratio was about 2: 1. All of the patients had a fever lasting for 5.9 days in average. Thirty-four of 46 patients (74%) had nausea or vomiting, and 27 (59%) had Fig. 1. Map of the distribution of meningitis patients in the central of Nara Prefecture.