Enterovirus 71 (EV71) is one of the major etiologic agents of hand, foot and mouth disease (HFMD). The surveillance data indicate that EV71 infection follows an epidemic mode of transmission, causing large outbreaks and then becoming quiescent for a few years. We investigated the genetic diversity of a total of 121 EV71 strains isolated from patients with HFMD in Fukushima, Japan, from 1983 to 2003 and compared their genetic relation with the 164 EV71 strains isolated in the world using phylogenetic analysis based on the VP4 sequence. We observed EV71-related HFMD outbreaks in Fukushima in 1984, 1987, 1990, 1993, 1997, 2000 and 2003. Phylogenetic reconstruction of EV71 strains isolated in Fukushima demonstrated 8 genetically distinct clusters, including 6 subgroups previously designated as B-1, B-2 and 3, B-4, C-1, C-2, and C-3 and 2 subgroups newly designated as B-5 and C-4. Additional 2 indistinct clusters belonged to genogroup C and were named C-U1 and C-U2. Of those subgroups, B-1, C-U1, C-U2, C-2, B4, and C-4 and B-5 dominantly related to epidemics that occurred in the years 1984, 1987 and 1990, 1993, 1997, 2000 and 2003, respectively. EV71 strains derived from each outbreak in Fukushima formed a single cluster with those isolated during almost the same time period in other area of Japan and in other countries. Our results suggested that the repeated EV71 outbreaks might be the result of the worldwide transmission of the newly introduced genetically divergent EV71 strains.

To clarify the chronologic genetic diversity of coxsackievirus A16 (CV-A16) strains associated with hand, foot, and mouth disease (HFMD) epidemics in a restricted area and their genetic relation with those isolated in other areas, we investigated the genetic diversity of the 129 CV-A16 strains associated with HFMD epidemics in Fukushima, Japan, from 1983 to 2003, and compared their genetic relation to 49 CV-A16 strains isolated in other areas of Japan and in China by using phylogenetic analysis based on the VP4 sequences. Phylogenetic reconstruction of the CV-A16 strains isolated in Fukushima from 1983 to 2003 demonstrated three distinct genetically divergent clusters related to HFMD epidemics that occurred from 1984 to 1994 (including the 1985 and 1991 outbreaks), HFMD epidemics from 1987 to 1998 (including the 1988 and 1998 outbreaks), and HFMD epidemics from 1995 to 2003 (including the 1995 and 2002 outbreaks). CV-A16 strains isolated during each period in Fukushima formed a single cluster with those isolated during essentially the same time period in other areas of Japan and in China. Our results demonstrated that prevalent CV-A16 strains causing HFMD in Fukushima, Japan, genetically changed twice during 21 epidemics, and changes were also observed in the CV-A16 strains causing HFMD epidemics in other areas. We concluded that repeated outbreaks of CV-A16-related HFMD in Japan were caused, in part, by the introduction of genetically changed CV-A16 strains, which might be transmitted overseas.

Two patients with subacute sclerosing panencephalitis (SSPE) were treated safely and effectively with high doses of intravenous ribavirin combined with intraventricular alpha interferon. The ribavirin concentrations maintained in the serum and cerebrospinal fluid were higher than those which inhibit SSPE virus replication in vitro and in vivo.

Aims: To evaluate the prognostic predictive values of cytochrome c, cytokines, and other laboratory measurements in serum collected during neurological onset in acute encephalopathy with multiple organ failure. Methods: In addition to general laboratory examinations, the concentrations of cytochrome c (apoptosis marker) and cytokines (inflammatory markers) were measured in serum samples collected at the initial phase in 29 patients with acute encephalopathy. The obtained values were evaluated as predictors for the development of severe encephalopathy. Results: Cytochrome c, tumour necrosis factor α (TNF-α), interleukin 6 (IL-6), soluble TNF-receptor 1 (sTNF-R1), and aspartate aminotransferase (AST) concentrations at the initial phase were high and correlated well with patient outcome. High concentrations of serum cytochrome c (>45 ng/ml), sTNF-R1 (>2000 pg/ml), AST (>58 IU/dl), IL-6 (>60 pg/ml), and TNF-α (>15 pg/ml) predicted an unfavourable prognosis (sequelae and death) at 93%, 79%, 82%, 77%, and 60%, respectively. The specificity of those markers was 100%, 89%, 83%, 100%, and 100%, respectively. Conclusions: Serum cytochrome c is the most sensitive and specific predictor for the development of severe encephalopathy at the initial phase. Results suggest that this marker might be used to guide decisions regarding the start of the initial treatment and further intensive care.

Ribavirin is a broad-spectrum antiviral drug with inhibitory activity against many RNA viruses, including measles virus. Five patients with subacute sclerosing panencephalitis (SSPE) were treated with ribavirin by intraventricular administration. Although there were transient side effects attributed to ribavirin, such as drowsiness, headache, lip and gingival swelling, and conjunctival hyperemia, intraventricular ribavirin therapy was generally safe and well tolerated. The cerebrospinal fluid (CSF) ribavirin concentration decreased, as described by a monoexponential function, after a single intraventricular dose. There was considerable interindividual variability, however, in the peak level and half-life. We aimed to adjust the individual dose and frequency of intraventricular administration based on the peak level and half-life of ribavirin in the CSF in order to maintain the CSF ribavirin concentration at the target level. Clinical effectiveness (significant neurologic improvement and/or a significant decrease in titers of hemagglutination inhibition antibodies against measles virus in CSF) was observed for four of five patients. For these four patients, CSF ribavirin concentrations were maintained at a level at which SSPE virus replication was almost completely inhibited in vitro and in vivo, whereas the concentration was lower in the patient without clinical improvement. These results suggest that intraventricular administration of ribavirin is effective against SSPE if the CSF ribavirin concentration is maintained at a high level. Intraventricular ribavirin therapy should be pursued further for its potential use for patients with SSPE and might be applied in the treatment of patients with encephalitis caused by other RNA viruses.

Objective.: To clarify the relationship between enteroviral infection and febrile seizures. Study Design.: Cerebrospinal fluid (CSF), serum, throat swab, and rectal swab samples were collected for virologic examination from 67 children with febrile seizures from April 1997 to March 1999. Those samples were examined for the presence of enterovirus using cell culture and 2 polymerase chain reaction (PCR) methods. Results.: No enterovirus was isolated from cell culture of CSF, throat swab, or rectal swab samples. All samples were screened for the presence of enteroviral sequences using a sensitive PCR method (PCR-Fukushima). We obtained positive results from 14 of 67 CSF samples, 10 of 62 serum samples, 12 of 64 throat swab samples, and 13 of 64 rectal swab samples. Of 21 patients in whom febrile seizures had developed during the summer months (June through August), 13 (61.9%) had positive PCR results in the CSF. Forty-seven of the 49 samples with a positive result using PCR-Fukushima were reexamined independently for the presence of the enteroviral genome using another PCR method (PCR-Mitsubishi). PCR-Mitsubishi had slightly lower sensitivity than PCR-Fukushima but identified genotypes of enterovirus by subsequent sequence analysis of the PCR products. The presence of the enteroviral genome was confirmed in 39 of the samples (83.0%). In 8 of the 9 enteroviruses detected in the CSF and/or serum samples using PCR-Mitsubishi, the genotypes were identified as coxsackieviruses group A, which are usually difficult to isolate using cell culture methods. Conclusions.: These findings proved that the causative agents of febrile illness associated with seizures in summer were primarily enteroviruses, especially coxsackieviruses group A, and that febrile seizures might be caused by enteroviral infection in the central nervous system.