In some sporadic cases and outbreaks of gastroenteritis at Toyama Institute of Health, the causative pathogens have not been identified using standard methods such as RT-PCR. Here, we performed metagenomics using next-generation sequencing (NGS) to investigate a suspected case of foodborne gastroenteritis that occurred in Toyama Prefecture in April 2013, the pathogen of which had not been identified. Approximately 2–3 million reads were obtained from each specimen, and 18–845 reads of the nucleotide sequence of sapovirus (SaV) were identified in all specimens. These SaV reads were similar to a part of the genome sequence of sapovirus strain Hu/Nagoya/NGY-1/2012/JPN (SaV NGY-1), belonging to genotype GV.2, first reported in a foodborne gastroenteritis outbreak that occurred in Aichi Prefecture in April 2012. We generated specific primers for SaV NGY-1 and performed a nested RT-PCR. The amplicons were obtained from all six samples, and their sequences were identical and revealed to be 99.8% identical to that of SaV NGY-1. We thus concluded that SaV GV.2 was the causative agent in the present case. SaV GV.2 strains were detected in raw sewage but not clinical specimens collected during the corresponding period of the outbreak in Toyama, suggesting that the SaV GV.2 strains had circulated among inhabitants with subclinical infections. In conclusion, our findings demonstrated that NGS is useful for the detection of a causative virus in fecal specimens collected from a gastroenteritis outbreak.
In the present study, behavior of norovirus during bread manufacturing process was analyzed by using murine norovirus strain 1 (MNV-1). Firstly, the transfer of MNV-1 during bread manufacturing process was investigated. MNV-1 was inoculated to various surfaces and then transferred to other surfaces by contacting repeatedly; slicer to bread dough, bread dough to latex glove, and latex glove to sliced loaf. As a result, MNV-1 was transferrable between various surfaces and bread products. Additionally, the protecting effect of sucrose and milk fat, ingredients of pastries, on the survivability of MNV-1 during heating was investigated. MNV-1 was inoculated into pancake mix with different concentration of sucrose or milk fat and then heated at 60℃ for 5 min. The results indicated that sucrose and milk fat contained in high concentrations in the dough/mix ingredients of pastries have a protective effect on the survivability of MNV-1. The data obtained in this study suggested that breads that had not been recognized as a risk of bacterial food poisoning could be a risk of norovirus-associated gastroenteritis if the failure of employee operation occurred in the bread manufacturing process.