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During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

A novel neutralization assay for human group C rotavirus (CHRV) was developed by using a reverse passive hemagglutination (RPHA) test for endpoint determination. In this assay, the neutralization (N)-RPHA test, serial twofold dilutions of sera were mixed with a solution of CHRV that yielded an RPHA test titer of 8 at 3 days after infection. The mixtures were incubated at 37 degrees C for 1 h and were inoculated onto CaCo-2 cell monolayers in a 96-well microplate. Maintenance medium containing 100 microgram of pancreatin per ml was placed in each well. The plate was sealed with sticky plastic film and was incubated at 37 degrees C for 3 days under continuous rotation. Then, the RPHA test titer of each well was determined. The neutralization titer was expressed as the reciprocal of the maximum dilution of the serum that exhibited a fourfold (75%) or greater reduction in the RPHA test titer (8 to 2 or less). Seroconversion of neutralizing antibody was demonstrated by this method in four sets of paired serum specimens from patients with diarrheal disease caused by CHRV. The seroprevalence of CHRV in the general population in Okayama Prefecture was 26.8% by immunofluorescence and 25.5% by the N-RPHA test. The N-RPHA test described here is the first system used to assay for a neutralization antibody against CHRV and is applicable in both clinical and epidemiological settings.

Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.