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Background: Human norovirus (HuNoV) is a major cause of enteric infectious gastroenteritis and is classified into several genotypes based on its capsid protein amino acid sequence and nucleotide sequence of the polymerase gene. Among these, GII.4 is the major genotype worldwide. Epidemiological studies have highlighted the prevalence of GII.2. Although recent advances using human tissue- and induced pluripotent stem cell (iPSC)-derived intestinal epithelial cells (IECs) have enabled in vitro replication of multiple HuNoV genotypes, GII.2 HuNoV could replicate only in tissue-derived IECs and not in iPSC-derived IECs., Methods: We investigated the factors influencing GII.2 HuNoV replication in IECs, focusing on histo-blood group antigens. We also assessed the immunogenicity of GII.2 virus-like particles (VLPs) and their ability to induce neutralizing antibodies. Antibody cross-reactivity was tested to determine whether GII.2 VLPs could neutralize other HuNoV genotypes, including GII.4, GII.3, GII.6, and GII.17., Results: Our findings indicated that GII.2 HuNoV replication in vitro requires the presence of both H and B antigens. Moreover, GII.2 VLPs generated neutralizing antibodies effective against both GII.2 and GII.4 but not against GII.3, GII.6, or GII.17. Comparatively, GII.2 and GII.17 VLPs induced broader neutralizing responses than GII.4 VLPs., Conclusions: The findings of this study suggests that GII.2 and GII.17 VLPs may be advantageous as HuNoV vaccine candidates because they elicit neutralizing antibodies against the predominant GII.4 genotype, which could be particularly beneficial for infants without prior HuNoV exposure. These insights will contribute to the development of effective HuNoV vaccines., Competing Interests: Potential conflicts of interest. Y. Y. and H. K. are the cofounders and directors of HanaVax, which provided research funding to S. S. Y. Y., H. K., and S. S. have filed a patent application related to the content of this article. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

We investigated the molecular epidemiology of human norovirus (HuNoV) in all age groups using samples from April 2019 to March 2023, before and after the COVID-19 countermeasures were implemented. GII.2[P16] and GII.4[P31], the prevalent strains in Japan before COVID-19 countermeasures, remained prevalent during the COVID-19 pandemic, except from April to November 2020; in 2021, the prevalence of GII.2[P16] increased among children. Furthermore, there was an increase in the prevalence of GII.4[P16] after December 2022. Phylogenetic analysis of GII.P31 RdRp showed that some strains detected in 2022 belonged to a different cluster of other strains obtained during the present study period, suggesting that HuNoV strains will evolve differently even if they have the same type of RdRp. An analysis of the amino acid sequence of VP1 showed that some antigenic sites of GII.4[P16] were different from those of GII.4[P31]. The present study showed high infectivity of HuNoV despite the COVID-19 countermeasures and revealed changes in the prevalent genotypes and mutations of each genotype. In the future, we will investigate whether GII.4[P16] becomes more prevalent, providing new insights by comparing the new data with those analyzed in the present study.

Sapoviruses, like noroviruses, are single-stranded positive-sense RNA viruses classified in the family Caliciviridae and are recognized as a causative pathogen of diarrhea in infants and the elderly. Like human norovirus, human sapovirus (HuSaV) has long been difficult to replicate in vitro. Recently, it has been reported that HuSaV can be replicated in vitro by using intestinal epithelial cells (IECs) derived from human tissues and cell lines derived from testicular and duodenal cancers. In this study, we report that multiple genotypes of HuSaV can sufficiently infect and replicate in human-induced pluripotent stem cell-derived IECs. We also show that this HuSaV replication system can be used to investigate the conditions for inactivation of HuSaV by heat and alcohol, and the effects of virus neutralization of antisera obtained by immunization with vaccine antigens, under conditions closer to the living environment. The results of this study confirm that HuSaV can also infect and replicate in human normal IECs regardless of their origin and are expected to contribute to future virological studies.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens (e.g., norovirus) in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab (VHH, nanobody) against norovirus on the cell surface of Lactobacillus , a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall-anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection.

Several organophosphorus compounds such as organophosphate pesticides (OPPs) and trialkylphosphates (TAPs) are suspected to inhibit cholinesterase activities, to affect endocrine systems or to possibly be carcinogenic. To evaluate their adverse effects on health with chronic exposure in the general population, especially in children, we measured the household exposure to OPPs and TAPs by Japanese children via all exposure pathways and the contribution of indoor air quality. First-morning void urine was collected from subjects aged 6 to 15 years (n = 132), and airborne organophosphorus compounds were sampled in the subject's bedroom for 24 h. Airborne levels of nine OPPs and three TAPs and their urinary metabolites were determined. No significant correlations were detected for any compounds between their airborne concentrations and the urinary excretion amounts of their corresponding metabolites. The estimated daily intakes were as follows (median, μg/kg b.w./d): chlorpyrifos, 0.042; diazinon, 0.067; tri-n-butylphosphate, 0.094. The 95th percentiles of the intakes for fenthion, fenitrothion and the above three compounds did not exceed their reference limit values, although one subject had a daily intake of tri-n-butylphosphate that was about twice its reference limit value. The concentration levels of the urinary metabolite of tri-n-butylphosphate in our subjects tended to be higher than those for children in many other countries. The fractions of the amounts absorbed by inhalation to the amounts absorbed via all of the exposure pathways was only 2.3 % (median) for tri-n-butylphosphate. Inhalation did not seem to contribute very much as an absorption pathway of the organophosphorus compounds in these Japanese children while they were at home. The exposure amounts of OPPs were not suggested to be high enough to adversely affect the health of these children at present on the basis of their daily intakes compared to their reference limit values., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

(1) Background: General infection control measures have been implemented at the societal level against COVID-19 since the middle of 2020, namely, hand hygiene, universal masking, and social distancing. The suppressive effect of the social implementation of general infection control measures on pediatric infections has not been systematically assessed. (2) Methods: We addressed this issue based on publicly available data on 11 pediatric infections reported weekly by sentinel sites in Osaka and Iwate prefectures in Japan since 2010. We obtained the 5-year average for 2015-2019 and compared it to the weekly report for 2020-2021. (3) Results: The rate of 6 of the 11 pediatric infections decreased significantly during 2020-2021, regardless of the magnitude of the prevalence of COVID-19 in both areas. However, only RSV infection, one of the six infections, was endemic in 2021. Exanthem subitum was not as affected by COVID-19 countermeasures as other diseases. (4) Conclusions: The social implementation of infectious disease control measures was effective in controling certain infectious diseases in younger age groups, where compliance with the countermeasures should not be as high as that of adults.

p-Dichlorobenzene (DCB) and naphthalene (NP) used as moth repellents in indoor environments are suspected to be carcinogenic. To evaluate their adverse effects on health with chronic exposure in the general population, especially children, we need to know their amounts absorbed by the body and the relationships between their amounts and air quality in residences. At present, little is known worldwide about them. This study examined the daily intakes of DCB and NP by Japanese children via all exposure pathways and the contribution of indoor air quality to the intakes. First-morning void urine samples from the subjects aged 6 to 15 years and air samples in their bedrooms were collected. Airborne NP and DCB and their urinary metabolites were measured. Significant correlations were detected between their airborne concentrations and the urinary excretion amounts of their corresponding metabolites. The absorption amounts of DCB and NP by inhalation of the children while at home were calculated to be 26 and 2.0 ng/kg b.w./h, respectively, as median values. The daily intake was estimated to be 2.4 and 0.90 μg/kg b.w./d (median), respectively. The fractions (median) of inhalation absorption amounts to overall absorption amounts for DCB and NP were 30% and 5%, respectively. In children living in residences where the indoor air concentrations of these compounds were more than half the level of each guideline value for indoor air quality, the main exposure route for their absorption was considered to be inhalation while at home. The indoor concentrations of DCB exceeded the lifetime excess cancer risk level of 10 -4 in 22% of the residences and 10 -3 in 9% of them. Our findings indicate the need to further reduce airborne concentrations of DCB in Japanese residences to prevent its adverse effects on the health of Japanese children., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children <5 years of age with AGE. Participants in 16 countries across 6 continents used standardized protocols for dual typing (genotype and polymerase type) and uploaded 1,325 dual-typed sequences to the NoroSurv web portal during 2016-2020. More than 50% of submitted sequences were GII.4 Sydney[P16] or GII.4 Sydney[P31] strains. Other common strains included GII.2[P16], GII.3[P12], GII.6[P7], and GI.3[P3] viruses. In total, 22 genotypes and 36 dual types, including GII.3 and GII.20 viruses with rarely reported polymerase types, were detected, reflecting high strain diversity. Surveillance data captured in NoroSurv enables the monitoring of trends in norovirus strains associated childhood AGE throughout the world on a near real-time basis.

Several synthetic pyrethroids are suspected to have carcinogenicity or reproductive toxicity. However, there is little knowledge about indoor air pollution in residences or the extent of intake by the residents of the newly developed pyrethroids, transfluthrin, profluthrin, and metofluthrin, although they have been widely used indoors as mosquito repellents and mothproof repellents in recent years. In the present study, the household exposure to pyrethroids through all exposure pathways and the contribution of inhalation pathway in Japanese children were examined by measuring urinary pyrethroid metabolites in the children and the airborne pyrethroids in their residences. Urine excreted first after waking up was collected from subjects aged 6 to 15 years (n = 132), and airborne pyrethroids were sampled in the subjects' bedrooms for 24 h. Nineteen pyrethroids and their nine urinary metabolites were measured. Their daily intakes estimated were as follows (median, ng/kg b.w./d): bifenthrin, 56; transfluthrin, 22; metofluthrin, 11; profluthrin, 0.86. The contribution rates of the amounts absorbed by inhalation to the amounts absorbed via all of the exposure pathways while at home tended to decrease in the following order: profluthrin (median 15%) ≈ transfluthrin (14%) > metofluthrin (1%) > bifenthrin (0.1%). Transfluthrin was considered to be the most notable pyrethroid as an indoor air pollutant. Our study demonstrated widespread exposure to transfluthrin, metofluthrin, profluthrin, and bifenthrin in a sample of Japanese children.

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sasou, Yuki, Kurokawa, Sato, Goda, Uchida, Matsumoto, Sagara, Watanabe, Kuroda, Sakon, Sugiura, Nakahashi-Ouchida, Ushijima, Fujihashi and Kiyono.)

Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Some phthalates, which are used mainly as plasticizers, are suspected to be endocrine disruptors. In the present study, daily intakes of phthalates by Japanese children through all exposure pathways and the contribution of indoor air quality to the intakes were examined by measuring urinary phthalate metabolites in the children and the airborne phthalates in their residences. Spot urine samples excreted first after waking up in the morning were collected from the subjects aged 6 to 15 years (n = 132), and airborne phthalates were sampled in the subjects' bedrooms for 24 h. Eight airborne phthalates and their urinary metabolites were determined by gas chromatography/mass spectrometry. The daily intakes of the phthalates estimated were as follows (median, μg/kg b.w./day): dimethyl phthalate (DMP), 0.15; diethyl phthalate (DEP), 0.42; diisobutyl phthalate (DiBP), 1.1; di-n-butyl phthalate (DnBP), 2.2; dicyclohexyl phthalate (DcHP), 0.026; benzylbutyl phthalate (BBzP), 0.032; di(2-ethylhexyl) phthalate (DEHP), 6.3. The 95th percentile (21 μg/kg b.w./day) of the DEHP intakes exceeded the reference doses (RfD, 20 μg/kg b.w./day) of the US Environmental Protection Agency (EPA). DEHP was suggested to be the most notable phthalate from the perspective of adverse effects on the health of Japanese children. On the other hand, DcHP and di-n-hexyl phthalate were not considered to be very important as indoor air pollutants and as internal contaminants for the children. The contribution rates of the amounts absorbed by inhalation to the amounts absorbed via all of the exposure pathways were only 7.9, 4.4, 6.6, 3.2, 0.22, and 1.0% as the median for DMP, DEP, DiBP, DnBP, BBzP, and DEHP, respectively. Therefore, inhalation did not seem to contribute very much as an absorption pathway of the phthalates for Japanese children while at home.

Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both VP1 and RNA-dependent RNA polymerase ( RdRp ) regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013-2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 VP1 region diverged around 1949, while the ancestral GII.P17 RdRp region diverged around 2010. The evolutionary rates of the VP1 and RdRp regions were estimated at ~2.7 × 10 -3 and ~2.3 × 10 -3 substitutions/site/year, respectively. The phylogenetic distances of the VP1 region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the RdRp region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years., (Copyright © 2019 Matsushima, Mizukoshi, Sakon, Doan, Ueki, Ogawa, Motoya, Tsukagoshi, Nakamura, Shigemoto, Yoshitomi, Okamoto-Nakagawa, Suzuki, Tsutsui, Terasoma, Takahashi, Sadamasu, Shimizu, Okabe, Nagasawa, Aso, Ishii, Kuroda, Ryo, Katayama and Kimura.)

Residents of long-term care facilities for the elderly are vulnerable to health care-associated infections. However, compared to medical institutions, long-term care facilities for the elderly lag behind in health care-associated infection control and prevention. We conducted a epidemiologic study to clarify the current status of infection control in long-term care facilities for the elderly in Japan. A questionnaire survey on the aspects of infection prevention and control was developed according to SHEA/APIC guidelines and was distributed to 617 long-term care facilities for the elderly in the province of Osaka during November 2016 and January 2017. The response rate was 16.9%. The incidence rates of health care-associated infection outbreaks and residents with health care-associated infections were 23.4 per 100 facility-years and 0.18 per 1,000 resident-days, respectively. Influenza and acute gastroenteritis were reported most frequently. Active surveillance to identify the carrier of multiple drug-resistant organisms was not common. The overall compliance with 21 items selected from the SHEA/APIC guidelines was approximately 79.2%. All facilities had infection control manuals and an assigned infection control professional. The economic burdens of infection control were approximately US$ 182.6 per resident-year during fiscal year 2015. Importantly, these data implied that physicians and nurses were actively contributed to higher SHEA/APIC guideline compliance rates and the advancement of infection control measures in long-term care facilities for the elderly. Key factors are discussed to further improve the infection control in long-term care facilities for the elderly, particularly from economic and social structural standpoints., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Seven foodborne norovirus outbreaks attributable to the GII.P17-GII.17 strain were reported across Japan in 2017, causing illness in a total of 2,094 persons. Nori (dried shredded seaweed) was implicated in all outbreaks and tested positive for norovirus. Our data highlight the stability of norovirus in dehydrated food products.

The number of person-to-person transmitted norovirus cases (n = 4,712) in school children in Osaka, Japan, during 2016/17 was the largest since 2012/13. Norovirus outbreaks were reported by 101 schools including 53 nursery schools (1,927 cases), 18 kindergartens (1,086 cases) and 30 elementary schools (1,699 cases). The dominant genotype among outbreaks was GII.P16-GII.2 (57.4%; 58/101), followed by GII.P2-GII.2 (8.9%; 9/101) and GII.P7-GII.6 (5.9%; 6/101). GII.4 was not detected despite dominance in previous years.

In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10 -3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

During the 2016-17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010-12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

Rotavirus vaccines have been successful in controlling severe diarrhea and have decreased deaths of young children globally. Rotarix and RotaTeq are the two currently available live-attenuated rotavirus vaccines. The vaccine virus can grow in a recipient's gut and spread from the vaccinee to naïve individuals. The potential for the emergence of revertant viruses is a concern with live-attenuated vaccines. We identified a previously healthy infant with severe acute gastroenteritis that was positive for rotavirus in a non-endemic season. A whole genome sequencing revealed that all of the viral genome segments were highly similar to those of the Rotarix virus, with the exception of five amino acid mutations in viral genes that could be associated with virulence. The younger sibling of this patient was administered Rotarix before the onset of disease in this patient, although no gastrointestinal symptoms were reported. Epidemiological data, circumstantial evidence, and the genome analysis suggest that the vaccine virus was transmitted from the vaccinee to the patient., Conclusion: This is a severe acute gastroenteritis case most probably attributed to the secondary infection of Rotarix-related virus without underlying diseases. The importance of molecular surveillance of rotavirus infections is discussed. What is Known: • The live-attenuated rotavirus vaccines, Rotarix and RotaTeq, have been successful in controlling severe diarrhea and have decreased deaths of young children globally. • Attenuated vaccine virus can grow in a recipient's gut and spread to naïve individuals and may revert to cause secondary symptomatic infections. What is New: • This is the first report describing a Rotarix-associated secondary infection resulting in severe acute gastroenteritis in an infant without underlying diseases. • Amino acid mutations that might contribute to viral pathogenesis were identified by whole genome sequencing.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

The RNA-dependent RNA polymerase ( RdRp ) and capsid ( VP1 ) genes of 51 GII.2 human norovirus (HuNoV) strains collected during the period of 2004-2015 in Japan were analyzed. Full-length analyses of the genes were performed using next-generation sequencing. Based on the gene sequences, we constructed the time-scale evolutionary trees by Bayesian Markov chain Monte Carlo methods. Time-scale phylogenies showed that the RdRp and VP1 genes evolved uniquely and independently. Four genotypes of GII.2 (major types: GII.P2-GII.2 and GII.P16-GII.2) were detected. A common ancestor of the GII.2 VP1 gene existed until about 1956. The evolutionary rates of the genes were high (over 10 -3 substitutions/site/year). Moreover, the VP1 gene evolution may depend on the RdRp gene. Based on these results, we hypothesized that transfer of the RdRp gene accelerated the VP1 gene evolution of HuNoV genotype GII.2. Consequently, recombination between ORF1 (polymerase) and ORF2 (capsid) might promote changes of GII.2 antigenicity.

A total of 1,141 rotavirus-negative stool specimens collected from diarrheic children in 4 distinct regions under sentinel surveillance in Thailand between 2006 and 2008 were examined by reverse-transcription (RT)-PCR for norovirus (NoV) and sapovirus (SaV). Three hundred 3 specimens (26.6%) were positive for NoV, with 34 and 269 belonging to genogroup I (GI) and genogroup II (GII), respectively. Twelve specimens (1.1%) were positive for SaV. Mixed infections were found in 5 specimens: 3 samples indicated the presence of both NoV GI and GII, and 2 samples indicated the presence of both NoV GII and SaV. Analysis of the monthly distribution of NoV and SaV revealed that NoV GII was clustered between September and February, while NoV GI was detected mainly in June and July; SaV was found in May, June, and July. In addition, 3 outbreaks of acute gastroenteritis at 2 junior high schools in Phichit and Bangkok, and at a university in Phitsanulok, Thailand in 2006 were found to have been caused by NoV infection. Sequence analysis of NoVs from sporadic cases and outbreaks showed them to be genotypes GII.4 and GII.6.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.

Rotaviruses C (RVCs) circulate worldwide as an enteric pathogen in both humans and animals. Most studies of their genetic diversity focus on the VP7 and VP4 genes, but the complete genomes of 18 human RVCs have been described in independent studies. The genetic background of the Far East Asian RVCs is different than other human RVCs that were found in India and Bangladesh. Recently, a RVC detected in 2010 in South Korea had genetic background similar to the Indian-Bangladeshi RVCs. This study was undertaken to determine the whole genome of eight Japanese RVCs detected in 2005-2012, and to compare them with other human and animal global RVCs to better understand the genetic background of contemporary Far East Asian RVC. By phylogenetic analysis, the human RVCs appeared to be distinct from animal RVCs. Among human RVCs, three lineage constellations had prolonged circulation. The genetic background of the Far East Asian RVC was distinguished from Indian-Bangladeshi RVC as reported earlier. However, we found one Japanese RVC in 2012 that carried the genetic background of Indian-Bangladeshi RVC, whereas the remaining seven Japanese RVCs carried the typical genetic background of Far East Asian RVC. This is the first report of the Indian-Bangladeshi RVC in Japan. With that observation and the reassortment event of human RVCs in Hungary, our study indicates that the RVCs are spreading from one region to another., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5' ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Human norovirus is a major cause of viral acute gastroenteritis worldwide. However, the transition of endemic norovirus genotypes remains poorly understood. The characteristics of natural immunity against norovirus are unclear because few studies have been performed in the natural infection setting. This prospective 10-year surveillance study of acute gastroenteritis in the province of Osaka, Japan, revealed that norovirus spread shows temporal, geographic, and age group-specific features in the humans. Genogroup II genotype 4 (GII.4) was detected in most sporadic pediatric cases, as well as in foodborne and nursing home outbreaks, respectively. The dominant genotypes in outbreaks at childcare facilities and schools shifted every season and involved GI, GII.2, GII.3, GII.4, and GII.6. Evidence at both the facility and individual levels indicated that genotype-specific herd immunity lasted long enough to influence the endemic norovirus genotype in the next season. Thus, norovirus circulates through human populations in a uniquely dynamic fashion., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Norovirus is a major etiologic agent in worldwide outbreaks of gastroenteritis associated with food as well as person-to-person transmission. The ubiquitous nature of Norovirus necessitates simple and rapid detection methods with high accuracy and sensitivity. To this end, several investigators have evaluated the usefulness of commercial reverse-transcription loop-mediated isothermal amplification (RT-LAMP) kits for detecting Norovirus genogroups I (GI) and II (GII). In previous studies, the conventional Loopamp kit for Norovirus GII showed a relatively high detection rate, while that for Norovirus GI showed a relatively low detection rate. In the present study, clinical Norovirus specimens were used to compare the detection rate of a modified Loopamp kit for Norovirus GI with the rates of the conventional Loopamp kit for Norovirus GI and an "in-house" RT-LAMP GI primer set, methods which had a high detection rate. Results from the present study showed that the modified Loopamp kit for Norovirus GI had a higher detection rate for two viral genotypes (GI.3, GI.11). On comparison with an "in-house" GII primer set using genotype GII.4 viruses circulating recently, the detection rate by the Loopamp kit for Norovirus GII was found to be higher, with a 98% detection rate. These results indicate the applicability of the modified LAMP kit for GI and the conventional LAMP kit for GII for detection of Noroviruses in clinical samples., ((c) 2009 Wiley-Liss, Inc.)

With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a "next-generation" parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1-0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 microg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298-32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome-derived, 20-460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484-15,260 reads of norovirus sequence (78-98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.

Sapovirus (SaV) is a causative agent of gastroenteritis. On the basis of capsid protein (VP1) nucleotide sequences, SaV can be divided into 5 genogroups (GI-GV), of which the GI, GII, GIV, and GV strains infect humans. SaV is uncultivable, but expression of recombinant VP1 in insect cells results in formation of viruslike particles (VLPs) that are antigenically similar to native SaV. In this study, we newly expressed SaV GII and GIV VLPs to compare genetic and antigenic relationships among all human SaV genogroups. Hyperimmune antiserum samples against VLPs reacted strongly with homologous VLPs. However, several antiserum samples weakly cross-reacted against heterologous VLPs in an antibody ELISA. Conversely, an antigen ELISA showed that VLPs of SaV in all human genogroups were antigenically distinct. These findings indicate a likely correspondence between SaV antigenicity and VP1 genogrouping and genotyping.

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range.

We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.