Effect of High Pressure Processing on a Wide Variety of Human Noroviruses Naturally Present in Aqua-Cultured Japanese Oysters.

The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.

Effect of High-Pressure Processing on Human Noroviruses in Laboratory-Contaminated Oysters by Bio-Accumulation.

The contamination of oysters with human noroviruses poses a human health risk, since oysters are often consumed raw. In this study, human norovirus genogroup II was allowed to bio-accumulate in oysters, and then the effect of high-pressure processing (HPP) on human noroviruses in oysters was determined through a polymerase chain reaction (PCR)-based method with enzymatic pretreatment to distinguish infectious noroviruses. As a result, oysters could be artificially contaminated to a detectable level of norovirus genome by the reverse transcription-PCR. Concentrations of norovirus genome in laboratory-contaminated oysters were log normally distributed, as determined by the real-time PCR, suggesting that artificial contamination by bio-accumulation was successful. In two independent HPP trials, a 1.87 log10 and 1.99 log10 reduction of norovirus GII.17 genome concentration was observed after HPP at 400 MPa for 5 min at 25°C. These data suggest that HPP is a promising process of inactivation of infectious human noroviruses in oysters. To our knowledge, this is the first report to investigate the effect of HPP on laboratory-contaminated noroviruses in oysters.

Human sapovirus classification based on complete capsid nucleotide sequences.

The genetically diverse sapoviruses (SaVs) are a significant cause of acute human gastroenteritis. Human SaV surveillance is becoming more critical, and a better understanding of the diversity and distribution of the viral genotypes is needed. In this study, we analyzed 106 complete human SaV capsid nucleotide sequences to provide a better understanding of their diversity. Based on those results, we propose a novel standardized classification scheme that meets the requirements of the International Calicivirus Scientific Committee. We believe the classification scheme and strains described here will be of value for the molecular characterization and classification of newly detected SaV genotypes and for comparing data worldwide.

Detection of Human Parechoviruses in Clinical and Municipal Wastewater Samples in Miyagi, Japan, in 2012-2014.

In order to study the epidemiology of human parechovirus (HPeV) infections and to evaluate the feasibility of environmental surveillance, we analyzed 281 stool samples, 265 nasopharyngeal swab samples, and 79 municipal wastewater samples for HPeV. The samples were collected in Miyagi Prefecture, Japan, between April 2012 and March 2014. HPeV was detected by reverse-transcription-PCR targeting the partial 5'-untranslated region and was genotyped by sequencing the capsid VP1 region. Seven stool samples (2.5%) and 1 nasopharyngeal swab sample (0.4%), all of which were from children under 2 years old, and 14 wastewater samples (18%) were positive for HPeV. Clear seasonality was observed: all positive samples were collected between July and December during the study period. All strains detected in the stool and wastewater samples had genotype HPeV1, and the strain from the nasopharyngeal swab sample had genotype HPeV6. A phylogenetic analysis revealed that all HPeV1 strains from the stool samples cluster together with those from the wastewater samples, indicating that the HPeV1 strains circulating in human populations can also be detected in municipal wastewater.

Pyrosequencing Analysis of Norovirus Genogroup II Distribution in Sewage and Oysters: First Detection of GII.17 Kawasaki 2014 in Oysters.

Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.

Norovirus pathway in water environment estimated by genetic analysis of strains from patients of gastroenteritis, sewage, treated wastewater, river water and oysters.

In this study, Norovirus (NV) capsid gene was detected from patients of gastroenteritis, domestic sewage, treated wastewater, river water and cultivated oysters in geographically close areas where all of samples were collected. In order to improve recovery efficiency of NVs from oysters, a new method using a spallation apparatus was developed. As a result, 18 of 30 oysters (60%) were positive for NV gene, while 7 of 30 (23%) oysters from the same sampling point were positive with the conventional ultracentrifugal method between November 2003 and February 2004. These results indicate that our new method exhibits the higher efficiency of recovering NVs than the conventional ultracentrifugal method. Six of 8 samples (75%) of river water were positive for NV gene between November 2003 and February 2004. Furthermore, 8 of 9 samples (89%) of treated wastewater and all 9 samples of sewage were positive for NV gene in the same period. These results indicated that treated wastewater would be one of the main sources for NV pollution in this area. The phylogenetic analysis in isolated NV capsid genes was conducted, in which high identities of gene sequences between NVs from patients, domestic sewage, river water and cultivated oysters were observed. These results implied that there would be a geographically associated circulation of NVs between human and cultivated oysters via water environment. It would be important to quantitatively analyze the moving pathway of NVs, which directly link to the development of a new scheme for preventing water environment and cultivated oysters from NV contamination.

Detection of Sapovirus in oysters.

SaV sequences which are either genetically identical or similar were detected from oysters, feces from gastroenteritis patients, and domestic wastewater samples in geographically close areas. This is the first report of the detection of SaV in oysters which meet the legal requirements for raw consumption in Japan.

Sapovirus in water, Japan.

Sapoviruses are etiologic agents of human gastroenteritis. We detected sapovirus in untreated wastewater, treated wastewater, and a river in Japan. A total of 7 of 69 water samples were positive by reverse transcription-PCR. Phylogenetic analysis of the viral capsid gene grouped these strains into 4 genetic clusters.

Persistence of caliciviruses in artificially contaminated oysters during depuration.

The fate of calicivirus in oysters in a 10-day depuration was assessed. The norovirus gene was persistently detected from artificially contaminated oysters during the depuration, whereas feline calicivirus in oysters was promptly eliminated. The prolonged observation of norovirus in oysters implies the existence of a selective retention mechanism for norovirus within oysters.

Human sapovirus in clams, Japan.

Human sapovirus was detected in 4 of 57 clam packages by reverse transcription-PCR and sequence analysis. This represents the first finding of sapovirus contamination in food. Closely matching sequences have been detected in stool specimens from patients with gastroenteritis in Japan, which indicates a possible food-to-human transmission link.

Genetic variation in the conservative gene region of Norovirus genogroup II strains in environmental and stool samples.

Noroviruses (NoVs) have been one of leading etiological agents for infectious gastroenteritis over the world. Gastroenteritis caused by NoVs is prevalent in winter season, and the contamination of the water environment with NoVs in the epidemic cold season is frequently reported. In contrast, the number of gastroenteritis patients and NoVs in the water environment are reduced during the nonepidemic summer season, and the year-round fate of NoVs has remained to be elucidated. In this study, we collected nucleotide sequences of NoV genogroup II (GII) from domestic sewage, sewage sludge, treated wastewater, river water, and stool samples of gastroenteritis patients in geographically close areas. Phylogenetic analysis of the obtained NoV gene revealed that six out of seven isolates from environmental samples and 10 out of 11 isolates from stool samples belong to genotype 3 (NoV GII.3) or 4 (NoV GII.4), which have been prevalent throughout the world. Genetic distances between the conservative gene region of NoV GII.4 variants implied that genetically diverse strains are likelyto occur in environmental samples. The evaluation of the evolutionary change of NoV gene obtained from environmental samples would make it possible to elucidate the year-round fate of NoVs.