Human sapovirus propagation in human cell lines supplemented with bile acids.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log10-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

GII.17 norovirus infections in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan during two decades.

Norovirus (NoV) is a major cause of viral gastroenteritis, and GII.4 has been the predominant genotype worldwide since the mid-1990s. During the 2014 to 2015 winter, a rare genotype, NoV GII.17, emerged and became prevalent mainly in East Asia. Over the past two decades, NoV molecular surveillance in Osaka City, Japan, has revealed that NoV GII.17 was detected for the first time in February 2001 and that NoV GII.17-associated outbreaks remarkably increased during the 2014 to 2015 season, with higher incidence recorded in January to March 2015. Genetic analysis indicated that 28 GII.17 outbreak strains were closely related to the novel GII.P17-GII.17 variants represented by the Kawasaki308/2015/JP strain, similar to that in other regions. Statistical analysis showed that NoV GII.17 infections were more common in adults than GII.3 and GII.4 infections, suggesting that the affected adults most likely did not have antibodies against NoV GII.17 and the novel GII.17 variant had recently appeared. Regarding transmission, food was one of the most important factors involved in the spread of NoV GII.17 among adults; 61% of GII.17 outbreaks were foodborne, with oysters being the most common vehicle. Interplay between pathogens, hosts, and environmental factors was considered to be important in the 2014 to 2015 NoV GII.17 epidemic.

Comparison between patients with norovirus-related gastroenteritis and asymptomatic carriers with respect to distribution of antibody-complexed viral particles and intestinal flora.

Asymptomatic carriers have a major influence on the spreading of norovirus infections. The objective of this study was to examine the characteristics of patients and asymptomatic carriers affected by norovirus-related community gastroenteritis outbreaks. No significant difference between the two groups was observed in terms of the number of norovirus-antibody complexes with respect to total numbers. Principal coordinates analysis of the intestinal flora based on ß-diversity analysis, revealed a different bacterial composition between patients and asymptomatic carriers, particularly regarding the genera Pseudomonas, Bacteroides, and Erwinia, as well as the Ruminococcaceae family. Although the proportional changes between these intestinal microorganisms were not sufficient to explain gastroenteritis symptoms, they represent possible markers shared by asymptomatic norovirus carriers.

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.

Detection of gastroenteritis viruses among pediatric patients in Hiroshima Prefecture, Japan, between 2006 and 2013 using multiplex reverse transcription PCR-based assays involving fluorescent dye-labeled primers.

Multiplex reverse transcription (RT)-polymerase chain reaction (PCR)-based assays involving fluorescent dye-labeled primers were modified to detect 10 types of gastroenteritis viruses by adding two further assays to a previously developed assay. Then, these assays were applied to clinical samples, which were collected between January 2006 and December 2013. All 10 types of viruses were effectively detected in the multiplex RT-PCR-based assays. In addition, various viral parameters, such as the detection rates and age distributions of each viral type, were examined. The frequency and types of mixed infections were also investigated. Among the 186 virus-positive samples, genogroup II noroviruses were found to be the most common type of virus (32.7%), followed by group A rotaviruses (10.6%) and parechoviruses (10.3%). Mixed infections were observed in 37 samples, and many of them were detected in patients who were less than 2 years old. These observations showed that the multiplex RT-PCR-based assays involving fluorescent dye-labeled primers were able to effectively detect the viruses circulating among pediatric acute gastroenteritis patients and contributed to the highly specific and sensitive diagnosis of gastroenteritis. J. Med. Virol. 89:791-800, 2017. © 2016 Wiley Periodicals, Inc.

Next-Generation Sequencing Analysis of the Diversity of Human Noroviruses in Japanese Oysters.

To obtain detailed information on the diversity of infectious norovirus in oysters (Crossostrea gigas), oysters obtained from fish producers at six different sites (sites A, B, C, D, E, and F) in Japan were analyzed once a month during the period spanning October 2015-February 2016. To avoid false-positive polymerase chain reaction (PCR) results derived from noninfectious virus particles, samples were pretreated with RNase before reverse transcription-PCR (RT-PCR). RT-PCR products were subjected to next-generation sequencing to identify norovirus genotypes in oysters. As a result, all GI genotypes were detected in the investigational period. The detection rate and proportion of norovirus GI genotypes differed depending on the sampling site and month. GII.3, GII.4, GII.13, GII.16, and GII.17 were detected in this study. Both the detection rate and proportion of norovirus GII genotypes differed depending on the sampling site and month. In total, the detection rate and proportion of GII.3 were highest from October to December among all detected genotypes. In January, the detection rates of GII.4 and GII.17 reached the same level as that of GII.3. The proportion of GII.17 was relatively lower from October to December, whereas it was the highest in January. To our knowledge, this is the first investigation on noroviruses in oysters in Japan, based on a method that can distinguish their infectivity.

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.

Detection and genetic characterization of human enteric viruses in oyster-associated gastroenteritis outbreaks between 2001 and 2012 in Osaka City, Japan.

Enteric viruses are an important cause of viral food-borne disease. Shellfish, especially oysters, are well recognized as a source of food-borne diseases, and oyster-associated gastroenteritis outbreaks have on occasion become international occurrences. In this study, 286 fecal specimens from 88 oyster-associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections. All virus-positive patients were over 18 years old. The most common enteric virus in outbreaks (96.6%) and fecal specimens (68.9%) was norovirus (NoV), indicating a high prevalence of NoV infection associated with the consumption of raw or under-cooked oysters. Five other enteric viruses, aichiviruses, astroviruses, sapoviruses, enteroviruses (EVs), and rotavirus A, were detected in 30.7% of outbreaks. EV strains were characterized into three rare genotypes, coxsackievirus (CV) A1, A19, and EV76. No reports of CVA19 or EV76 have been made since 1981 in the Infectious Agents Surveillance Report by the National Infectious Diseases Surveillance Center, Japan. Their detection suggested that rare types of EVs are circulating in human populations inconspicuously and one of their transmission modes could be the consumption of contaminated oysters. Rapid identification of pathogens is important for the development of means for control and prevention. The results of the present study will be useful to establish an efficient approach for the identification of viral pathogens in oyster-associated gastroenteritis in adults.

Sealing ability of various endodontic sealers with or without ethylenediaminetetraacetic acid (EDTA) treatment on bovine root canal.

This study investigated the wettability and consistency of various endodontic sealers, both inorganic and organic, and evaluated their sealing ability of root canals using the single-cone obturation technique, with and without ethylenediaminetetraacetic acid (EDTA) treatment. Bovine root canals were endodontically prepared and filled in preparation for the dye penetration test with toluidine blue solution. All sealers exhibited contact angles similar to or lower than dentin and displayed superior consistency. Among the sealers, organic sealers used without EDTA treatment showed reduced dye penetration compared to inorganic sealers. However, some inorganic and organic sealers showed dye penetration in the sealer and dentin of root canals subjected to EDTA treatment. In conclusion, the single-cone obturation technique, combined with these endodontic sealers, achieved close contact with root canal dentin due to their wettability and consistency. However, the sealing ability of certain sealers was influenced by EDTA treatment.

[A mechanism of norovirus pandemic based on comprehensive genome analysis].

Norovirus GII.4 is a major etiological agent of acute viral gastroenteritis worldwide. We examined GII.4 evolution using 277 near-full-length GII.4 genome sequences from human stool specimens collected at 20 sites in Japan between May 2006 and March 2010. We found outbreaks of 8 monophyletic GII.4 subtypes, among which a single subtype, termed 2006b, had continually predominated (222/277: 80.7%). Four of the 8 GII.4 subtypes were chimera viruses of recently prevalent GII.4 subtypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF) 1 and ORF 2 (P<0.0001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII.4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsid were preferentially positioned on the outer surface loops of the protruding P2 domain. These data and computer-assisted structural study of NoV capsid protein are compatible with a model of antigenic drift with tuning of the structure-functions of multiple proteins for the survival strategy of GII.4 2006b variant.

Divergent evolution of norovirus GII/4 by genome recombination from May 2006 to February 2009 in Japan.

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P

[Current topics on inactivation of norovirus].

Human norovirus is the most important foodborne virus in Japan. According to the statistics of food poisoning by the Ministry of Health, Labour, and Welfare (MHLW), the number of patients infected with norovirus has accounted for half of all the patients with food poisoning in recent years. One of the most important measures for the control of infectious diseases is establishing of techniques for inactivating pathogens. For the prevention of food poisoning caused by norovirus, MHLW recommends that foods be subjected to heat treatment at 85 degrees C for 1 min or more; moreover, it recommends the use of sodium hypochlorite to inactivate (disinfect) this virus. However, application of these treatments is not always feasible because heat results in denaturation and sodium hypochlorite can be toxic to the human body and can cause discoloration. Therefore, it is necessary to develop and improve the efficacy of disinfectants and physiochemical treatments against the virus. Human norovirus cannot be propagated in cell culture or in a small animal. This matter is the greatest hindrance for testing the stability of this virus in environments or for evaluating the efficacy of disinfectants, heat treatment, pH treatment, ultraviolet or gamma irradiation, high hydrostatic pressure treatment, and other methods for the inactivation of the virus. Hence, some viruses such as human enterovirus, feline calicivirus, or mouse norovirus have been used as surrogates of human norovirus. The data on inactivation and stability of surrogate viruses are exclusively used as the data of human noroviruses. In recent years, some attempts to distinguish between infectious and noninfectious virus particles by genetic methods such as polymerase chain reaction have been made. These methods include pretreatments by RNase for digesting viral RNAs from non-intact or destroyed virus particles, or addition of a reagent such as ethidium monoazide for inhibiting PCR amplification of viral RNAs from them, before RNA extraction. Non-intact virus particles, which may represent virus particles with some damage (s) in the structural protein(s), are not necessarily synonymous with non-infectious virus particles. However, the results of methods using these treatments, compared to the results of traditional methods without these treatments, seem to be more correlated to the amount of the infectious virus particles. Although many disinfectants or physiochemical treatments have been reported, traditional techniques such as removal of virus particles by washing in running water, heat treatment, or disinfection by sodium hypochlorite are still important control measures. Establishment of control measures for human norovirus and successful propagation of the virus in cell culture are strongly desired.

Effects of different bonding systems with various polymerization modes and root canal region on the bond strength of core build-up resin composite.

Purpose The aim of this study was to clarify the effects of different bonding systems (BSs) with various polymerization modes and root canal regions on the bond strength of core build-up resin composite to dentin.Methods Post cavities were prepared in the roots of 54 bovine teeth. Three types of BS with various polymerization modes (light, chemical, and dual-cure) were applied to the walls of the cavities, which were subsequently filled with core build-up resin composite, and stored in 37°C water for 7 days. Each tooth was then sectioned perpendicular to the long axis of the tooth into 9-disk from the coronal to the apical side. Bond strengths were measured on two-thirds of the disks, while dye penetration was examined in the remaining third.Results Statistical analysis revealed significant differences between the bond strengths of BSs with different polymerization modes, indicating chemical-cured BS had higher bond strength than light-cured BS. The chemical-cured BS group showed cohesive failure in both resin composite and dentin regardless of the root canal region, while adhesive failure was observed in the coronal region for dual-cured BS and in the apical region for light-cured BS. Dye penetration was significantly more at the bonding interface at the apical region of the light-cured BS.Conclusions Chemical-cured BS displayed a greater bond strength than light-cured BS. Cohesive failure was observed in both core build-up resin and dentin, indicating that the integration of tooth structure with resin composite was effective for retaining the resin core and sealing the root canal.

Detection of sapoviruses and noroviruses in an outbreak of gastroenteritis linked genetically to shellfish.

Norovirus (NoV) and sapovirus (SaV) are important pathogens of human gastroenteritis. Compared to NoV, the transmission route of SaV is unclear. An outbreak of gastroenteritis occurred at a restaurant in June 2008, and SaV and NoV were detected in fecal specimens from 17 people who ate at the restaurant and one asymptomatic food handler and also in stripped shellfish and liquids remaining in the shellfish packages by reverse transcription-polymerase chain reaction (RT-PCR) and/or real-time RT-PCR. Nucleotide sequencing analysis of the RT-PCR products corresponding to the partial capsid region revealed 99.3-100% identities for SaV and 98.6-99.3% identities for NoV among the digestive diverticulum of the frozen stripped shellfish (Ruditapes philippinarum), "Asari," the package liquid, and feces from symptomatic or asymptomatic guests. These results suggested a link between the consumption of contaminated shellfish and clinical features in the patients. While the transmission of NoV by shellfish has been reported, this report shows that SaV can also be transmitted by shellfish.

Detection of multiple sapovirus genotypes and genogroups in oyster-associated outbreaks.

This report describes multiple viruses in stool specimens from oyster-associated gastroenteritis. Eleven outbreaks of oyster-associated gastroenteritis were examined for enteric viruses between January 2002 and March 2006 in Japan. Multiple norovirus genotypes were detected in all outbreaks; moreover, kobuvirus, sapovirus, and astrovirus were also detected in 6, 3, and 1 of the 11 outbreaks, respectively. Notably, multiple sapovirus genogroups were detected in the stool specimens from subjects in two oyster-associated gastroenteritis outbreaks.

Improved bond performance of a dental adhesive system using nano-technology.

Since adhesive technology was introduced into dental field, metal-based restoration has been gradually replaced by metal-free restoration. Using the adhesive technology, minimum invasive technique has been possible in daily clinical practice as well as esthetic tooth-colored restorations have become very popular all over the world.One of the current issues of the dental adhesive is durability of bond between tooth structure and adhesive resin. Several approaches to overcome the issues have been carried out. Self-etching approach is believed to create durable bond because demineralization of superficial tooth surface is very shallow. Other approach is to utilize the inhibitor of enzymes which are suggested to catalyze the decomposition of resin composites and are always secreted within the oral environment.In the present study, Colloidal Platinum Nanoparticles (CPN) was applied before the application of 4-META/MMA-TBB resin cement as the third possibility to prolong the durability of bond. This implies that the use of the CPN solution would create higher conversion at the interface compared with conventional bonding procedures.

Molecular epidemiology of noroviruses detected in food handler-associated outbreaks of gastroenteritis in Japan.

Twelve outbreaks of food handler-associated gastroenteritis between November 2002 and March 2006 in Japan were examined for norovirus (NoV) using RT-PCR and sequence analysis. NoV was detected in 77 of 81 customers and 45 of 104 food handlers. Identical NoV sequences were detected in patients and food handlers in each outbreak.

Statistical analysis of attack rate in norovirus foodborne outbreaks.

Norovirus (NoV), which causes foodborne gastroenteritis outbreaks, is one of the important viruses in public health. We statistically analyzed the attack rate in foodborne outbreaks caused by NoV. The attack rate in 95 oyster-associated outbreaks was significantly higher than that in 195 food handler-associated outbreaks (P=0.007). The difference in the number of NoV genotypes implicated is considered to be an important factor for this difference. The attack rate in 20 outbreaks associated only with GII/3 was higher than that in 143 other outbreaks (P=0.247), while the attack rate in 27 outbreaks associated only with GII/4 was lower than that in 136 other outbreaks (P=0.004), suggesting that GII/4 NoVs cause asymptomatic infection more frequently than do other NoV genotypes. Our results suggest that differences in implicated foods, susceptibility of the host to NoV infection, and pathogenicity of NoVs may influence the attack rate in NoV foodborne outbreaks.

Detection of human enteric viruses in Japanese clams.

A total of 57 clam packages that were collected from supermarkets and fish markets from 11 different sites in western Japan between 8 December 2005 and 6 September 2006 were examined for human enteric viruses (i.e., norovirus, Aichi virus, rotavirus, adenovirus, hepatitis A virus, and astrovirus), using PCR and reverse transcription PCR. Sixty-one percent of the packages were contaminated with one type of virus, 9% had two different types of viruses, 28% had three different types of viruses, and 9% had at least four different types of viruses. Thirty-one (54%) of 57 packages were contaminated with noroviruses. Norovirus genogroup I and genogroup II sequences were detected in 24 and 23 packages, respectively, and these sequences belonged to nine genogroup I and eight genogroup II genotypes. Aichi viruses were found in 19 (33%) of 57 packages, and these belonged to genogroup A. Rotaviruses (group A) were detected in 14 (42%) of 33 of packages and 9 of 14 rotavirus-positive packages contained two or more rotavirus genogroup types. Adenoviruses (Ad40 and Ad41) were detected in 17 (52%) of 33 packages. One of the 57 (2%) packages was positive with hepatitis A virus (subtype IA). Astrovirus was not detected in any of the packages. This is the first study to detect such a high level of contamination in Japanese clams. These results represent an important finding because the Japanese clams were considered suitable for human consumption. Further studies are needed to determine the health risks associated with eating these highly contaminated clams.