Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

Nakamura, Shota; Yang, Cheng-Song; Sakon, Naomi; Ueda, Mayo; Tougan, Takahiro; Yamashita, Akifumi; Goto, Naohisa; Takahashi, Kazuo; Yasunaga, Teruo; Ikuta, Kazuyoshi; Mizutani, Tetsuya; Okamoto, Yoshiko; Tagami, Michihira; Morita, Ryoji; Maeda, Norihiro; Kawai, Jun; Hayashizaki, Yoshihide; Nagai, Yoshiyuki; Horii, Toshihiro; Iida, Tetsuya; Nakaya, Takaaki

We analyzed unlinked, anonymous samples in the Osaka Prefectural Institute of Public Health. The samples were nasopharyngeal aspirates and stools (n = 3 and 5, respectively) isolated during 2005-2007 in Osaka, Japan. Seasonal influenza A virus (Flu) in nasopharyngeal aspirates from 3-to 7-year-old children was detected by a rapid diagnostic kit (immunochromatography) using Flu-specific antibodies. In 2006/2007, a large-scale norovirus outbreak occurred in Osaka, Japan, mainly infecting patients in nursing homes and welfare facilities (53%), hospitals (27%), kindergartens (15%), and elementary and junior high schools (5%) [26] . #N1 to #N3 samples were collected during this outbreak. #N1 sample was derived from a hospitalized patient and #N2 and #N3 samples were derived from patients in a welfare facility in 2006 (October to December). #N4 sample was a kindergarten student when an outbreak occurred at the elementary school of its elder brother and sister. In contrast to these four cases of putative human-to-human transmission, #N5 sample was oyster-associated. #N4 and #N5 samples were collected in May 2005 and January 2006, respectively. Diagnosis of norovirus infection was based on RT-PCR [26] . The collected stool was suspended with an equal amount of PBS and was centrifuged at 15,000 rpm for 10 min. The supernatants (0.25 ml) were used for RNA isolation. This study was approved by the ethical review committees of the RIMD, Osaka University, Osaka Prefectural Institute of Public Health, National Institute of Infectious Diseases, and RIKEN.