We used 96-well tissue culture plates (Greiner Bio-one, Frickenhausen, Germany) vertically and prepared two rows of each cell line as described previously (9, 11). Beginning in 2008, we also prepared HMV-II cell lines as separate 96-well tissue culture plates and inoculated the specimens onto them, mainly to isolate HPIVs (12, 13). After centrifugation of the specimens at 1500 g for 20 min, we inoculated 75 μL of supernatant directly into two wells

of each cell line. We stored the remainder of each specimen at −80 C. We centrifuged the inoculated plates at 450 g for 20 min, incubated them at 33 C in a 5% CO2 incubator and assessed them for CPE for 14 days, except MK-8669 datasheet for the Vero E6 cell lines, which we observed for approximately

one month without changing the medium to isolate human metapneumovirus (11). When we observed a CPE or hemagglutination test and/or found a hemadsorption test to be positive using guinea pig erythrocytes (0.8%), we performed virus identification SAHA HDAC order using a hemadsorption inhibition test, RT-PCR and sequence analysis as described previously (9, 12). With regard to HPIVs, we isolated 1033 (6.1%) HPIV1–3 strains, comprising 305 HPIV1 (1.8%), 154 HPIV2 (0.9%) and 574 HPIV3 (3.4%) strains, from the 16,962 specimens we obtained during the study period. After we introduced the HMV-II cell line, the annual virus isolation frequencies of HPIV1–3 increased from 1.6 to 7.9% between 2002 and 2008 and from 9.4 to 10.8% between 2009 and 2011. Figure 1 shows monthly numbers of HPIV1–3 isolates. HPIV1 was uncommon in winter but quite commonly isolated between April and October. Further, although we isolated HPIV2 year-round, we recovered 55% of isolates between September and December. For HPIV3, we recovered 86% of isolates between May and July, but none between November and February, indicating that HPIV3 infections have clear seasonality. Figure 2 shows a breakdown of HPIV1–3 infections Protirelin by age. For HPIV3, 53.5% of the children were younger than 2 years

and the proportion decreased with age apart from the ≥ 10 years age group. In contrast, we found the highest percentage of HPIV1 and HPIV2 infections in the 2–4 years (2.4–2.7%) and 3–5 years (1.1–2.0%) age groups, after which the percentage of infections generally decreased with age. Regarding the clinical diagnosis of patients with HPIV1, HPIV2 and HPIV3 infections, 236 (77.4%), 123 (79.9%), and 458 patients (79.8%) were diagnosed with upper respiratory infections such as rhino-pharyngitis, respectively; 25 (8.2%), 11 (7.1%), and 13 (2.3%) with croup, respectively; 32 (10.5%), 18 (11.7%), and 63 (11.0%) with lower respiratory infections such as bronchitis, bronchiolitis, and pneumonia; and the rest with other diseases including viral exanthema.