A total of 224 nasopharyngeal swab specimens were collected from patients who had been clinically diagnosed with ARI with fever and/or cough and/or rhinorrhea at 3 pediatric clinics in Yamagata and Sendai between December 2007 and July 2008, as described previously for the evaluation of a newly developed rapid antigen detection kit using immunochromatography compared with real-time RT-PCR [
12]. The specimens were collected and used for the previous and present studies, after getting the informed consent from the patients or their guardians. These studies were approved by the Institutional Review Board of Sendai Medical Center, National Hospital Organization Japan, on Feb. 29, 2008 (Reference No CHI-19-33). Of these specimens, 166 were from children aged under 5 years, 43 were from children aged between 5 and 9 years, 7 were from children aged between 10 and 15 years and 8 were from patients > 15 years. Each specimen was placed immediately in a tube containing 3 ml of transport medium [
11,
12] and transported at 4°C to the Department of Microbiology, Yamagata Prefectural Institute of Public Health for virus isolation and real-time RT-PCR. Prior to specimen inoculation for virus isolation, 200 μl of the specimen was transferred to a 1.5 ml microtube and stocked at -80°C until application to real-time RT-PCR assay. Virus isolation was carried out by the 96-well modified microplate method including Vero E6 and other five cell lines [
10,
11]. We observed the plates two or three times per week for CPEs for 4 weeks without passage or medium change [
10]. When a hMPV-like CPE was observed, second passage, identification and genotyping was carried out as described previously [
10]. The method and results of real-time RT-PCR assay with a TaqMan probe using an ABI Prism 7500 Fast real-time PCR system (Applied Biosystems) were described previously [
12]. Primers and a probe targeting the hMPV N gene were designed based on the reports of Maertzdorf et al. [
13] and Bonroy et al. [
14]. Statistical analysis was performed by Fisher's exact probability test.