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

HCoV-HKU1, which was recently identified as the fifth human coronavirus [27] , was detected in one specimen (#N4). A total of 14 reads from 18,014 reads matched to the HCoV-HKU1 virus, with four regions being detected (Table 4 ). Epidemiological studies have reported that HCoV-HKU1 was found in the nasopharyngeal aspirates of 10/418 (2.4%) studied patients with community- Figure 2 . Pyrosequencing using the GS FLX system. Amplified cDNA was used as a template for GS FLX analysis. A 70675 PicoTiterPlate device (gasket for 16 regions) was divided into 2 regions each for 8 samples. Obtained data were then subjected to a data analysis pipeline, as described in the Methods section. Comparison of the organisms from which the best matches for the sequences was shown. doi:10.1371/journal.pone.0004219.g002 acquired pneumonia [28] , and that HCoV-HKU1 could be detected in respiratory and stool samples from children and adults. Studies have also reported a 9-month-old patient who was coinfected with HCoV-HKU1 and influenza C virus [29] . We showed here that a 5-year-old child was co-infected with HCoV-HKU1 and norovirus, although the relationship between these two viruses and/ or the relationship between pathogenesis (enteric tract illness) and coinfection of these two viruses remains unknown. Other human coronaviruses (OC-43, 229E, and NL63) were not detected from these fecal samples or from the nasal samples with RT-PCR (data not shown). Human endogenous retrovirus K (HERV-K)-derived sequences was also detected in patient #N4, who was 3 years old (Table 1) . HERV-K is the name given to an approximately 30million-year-old family of endogenous retroviruses present at .50 copies per haploid human genome [30] .