Sequence-Specific Capture of Oligonucleotide Probes (SCOPE): a Simple and Rapid Microbial rRNA Quantification Method Using a Molecular Weight Cutoff Membrane.

A method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, a molecular weight cutoff membrane (MWCOM) was used for the separation of fluorescence-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof of concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using a spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria, Archaea, and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced more reliable and coherent results. Thus, the SCOPE method allows simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions. IMPORTANCE Microorganisms play integral roles in the Earth's ecosystem. Microbial populations and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it provides information only on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate, and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial populations in complex ecosystems. This method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.

Detection of Rotavirus Vaccine Strains in Oysters and Sewage and Their Relationship with the Gastroenteritis Epidemic.

Rotavirus is one of the major causes of infectious gastroenteritis among infants and children, and live attenuated vaccines for rotavirus A (RVA), namely, Rotarix and RotaTeq, have recently become available in Japan. Rotavirus is known to be excreted from patients and accumulated in oysters similar to norovirus; however, the vaccine strains in aquatic environments or oysters have not yet been analyzed. In this study, we focused on wild-type RVA, which is highly important in considering the risk of infectious diseases. We quantified total RVA, Rotarix, and RotaTeq strains in oyster and sewage samples collected between September 2014 and July 2016 to assess the contamination levels of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of total RVA. The positive rates of wild-type RVA, Rotarix, and RotaTeq in oysters were 54, 14, and 31%, respectively. These rates were comparable to those of wild-type RVA (57%) and RotaTeq (35%) in sewage; however, Rotarix was not detected in any sewage samples. The comparison of viral concentrations in oysters and sewage suggested more efficient accumulation of the vaccine strains in oysters than the wild-type RVA. The concentration of wild-type RVA in oysters was significantly correlated with that in sewage with a lag time of -6 to 0 weeks which is required for viral transportation from wastewater treatment plants to oysters. On the other hand, no significant correlation was observed between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases, implying the existence of asymptomatic RVA-infected individuals.IMPORTANCE We quantified rotavirus A (RVA), Rotarix, and RotaTeq strains in oyster and sewage samples during two gastroenteritis seasons and revealed the exact contamination of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of RVA. The concentration of wild-type RVA was significantly correlated between oysters and sewage, although no significant correlation was seen between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases. This finding suggested the existence of asymptomatic patients and that monitoring of rotavirus vaccine strain could be useful to understand the trend of wild-type RVA and rotavirus outbreak in detail. We believe that our study makes a significant contribution to the literature because it reports the detection of rotavirus vaccine strains in oysters.

Environmental Surveillance of Norovirus Genogroups I and II for Sensitive Detection of Epidemic Variants.

Sewage samples have been investigated to study the norovirus concentrations in sewage or the genotypes of noroviruses circulating in human populations. However, the statistical relationship between the concentration of the virus and the number of infected individuals and the clinical importance of genotypes or strains detected in sewage are unclear. In this study, we carried out both environmental and clinical surveillance of noroviruses for 3 years, 2013 to 2016. We performed cross-correlation analysis of the concentrations of norovirus GI or GII in sewage samples collected weekly and the reported number of gastroenteritis cases. Norovirus genotypes in sewage were also analyzed by pyrosequencing and compared with those identified in stool samples. The cross-correlation analysis found the peak coefficient (R = 0.51) at a lag of zero, indicating that the variation in the GII concentration, expressed as the log10 number of copies per milliliter, was coincident with that in the gastroenteritis cases. A total of 15 norovirus genotypes and up to 8 genotypes per sample were detected in sewage, which included all of the 13 genotypes identified in the stool samples except 2. GII.4 was most frequently detected in both sample types, followed by GII.17. Phylogenetic analysis revealed that a strain belonging to the GII.17 Kawasaki 2014 lineage had been introduced into the study area in the 2012-2013 season. An increase in GI.3 cases was observed in the 2015-2016 season, and sewage monitoring identified the presence of GI.3 in the previous season (2014-2015). Our results demonstrated that monitoring of noroviruses in sewage is useful for sensitive detection of epidemic variants in human populations.IMPORTANCE We obtained statistical evidence of the relationship between the variation in the norovirus GII concentration in sewage and that of gastroenteritis cases during the 3-year study period. Sewage sample analysis by a pyrosequencing approach enabled us to understand the temporal variation in the norovirus genotypes circulating in human populations. We found that a strain closely related to the GII.17 Kawasaki 2014 lineage had been introduced into the study area at least 1 year before its appearance and identification in clinical cases. A similar pattern was observed for GI.3; cases were reported in the 2015-2016 season, and closely related strains were found in sewage in the previous season. Our observation indicates that monitoring of noroviruses in sewage is useful for the rapid detection of an epidemic and is also sensitive enough to study the molecular epidemiology of noroviruses. Applying this approach to other enteric pathogens in sewage will enhance our understanding of their ecology.

Human G3P[4] rotavirus obtained in Japan, 2013, possibly emerged through a human-equine rotavirus reassortment event.

Two novel G3P[4] rotavirus strains were detected from children with acute diarrhea in Sendai, Japan, identified as a G3-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotype constellation by whole-genome sequence analysis. The VP7 gene of the two strains displayed the highest nucleotide sequence identity (91 %) and showed a close genetic relationship (99 % bootstrap value) to an equine rotavirus reported in India. The other gene segments were related to human group A rotaviruses. This report suggests a possible reassortment event between human and equine rotaviruses.

[Infectivity Titers of Each Component of the Influenza Virus in the Live Vaccine Purchased from a Parallel Import Distributing System].

Currently in Japan, the only approved influenza vaccine is the inactivated vaccine which is injected subcutaneously. On the other hand, there is a live vaccine available elsewhere in the world. Flumist, an intranasal influenza live vaccine which contains four strains of infectious viruses, has been used in the United States for more than 10 years; the vaccine has been found effective in clinical trials, while it has some limitations such as those on subjects for the administration, strict storage conditions, relatively short expiration date etc. It is not yet approved in Japan, but available through personal import by some medical institutions, and prescribed based on the decision of the doctor. However, in Japan, there is no checking system whether the vaccine contains appropriate amounts of infectious viruses or not. In the present study, we purchased 2013-14 and 2014-15 years' lots of Flumist from a parallel importer and measured the amount of infectious viruses of each component of them using the focus assay. Consequently, for type A influenza viruses, the titers of both of H1N1pdm09 and H3N2 viruses in the 2013-14's lot were 1/30 of the lower limit of those shown in the package insert and 1/10 in 2014-15's lot, while those of type B viruses, both of B/Massachusetts and B/Brisbane viruses marginally cleared the lower limit. The digital PCR analysis showed that the absolute genome copy numbers of type A viruses were 1/10 of those of type B viruses. The relatively higher titer of B/Massachusetts also gradually decreased over time during its storage at 4°C and finally reached the lower limit at about one week before the expiration date. In case it is approved officially in the future to be used in Japan, some studies will be required to elucidate the minimum viral titers of the components necessary for effective live vaccine. In addition, there should be a system to check the titer during the distribution process in Japan.

Genetic diversity and molecular characterization of enteroviruses from sewage-polluted urban and rural rivers in the Philippines.

Despite the vast distribution and expansive diversity of enteroviruses reported globally, indicators defining a complete view of the epidemiology of enteroviruses in tropical countries such as the Philippines are yet to be established. Detection of enteroviruses in the environment has been one of the markers of circulating viruses in a community. This study aimed to bridge the gap in the epidemiology of enteroviruses in the Philippines by providing an overview of the occurrence of enteroviruses in both urban and rural rivers. Molecular detection directed at the VP1 region of the enterovirus genome was performed on 44 grab river water samples collected from April to December 2009. The majority of the enterovirus serotypes detected were clustered with human enterovirus C species (HEV-C; 21/42), followed by HEV-B (12/42) and HEV-A (9/42). Porcine enterovirus 9 was also found in 12 out of 44 water samples. Phylogenetic analysis indicated that the viruses detected were closely related, if not all forming a monophyletic clade, with those enteroviruses detected previously from acute flaccid paralysis cases in the country. The clustering of environmental and human enterovirus strains implies that the circulation of these strains were associated with river contamination. This study gives further evidence of the environmental persistence of enteroviruses once they are shed in feces and likewise, provides additional data which may help in understanding the epidemiology of enteroviruses in humans, highlighting the need for more studies on the potential public health risks linked with enteroviruses found in the environment and their eventual clinical consequences in the country.

Dose-response assessment for influenza A virus based on data sets of infection with its live attenuated reassortants.

Reported data sets on infection of volunteers challenged with wild-type influenza A virus at graded doses are few. Alternatively, we aimed at developing a dose-response assessment for this virus based on the data sets for its live attenuated reassortants. Eleven data sets for live attenuated reassortants that were fit to beta-Poisson and exponential dose-response models. Dose-response relationships for those reassortants were characterized by pooling analysis of the data sets with respect to virus subtype (H1N1 or H3N2), attenuation method (cold-adapted or avian-human gene reassortment), and human age (adults or children). Furthermore, by comparing the above data sets to a limited number of reported data sets for wild-type virus, we quantified the degree of attenuation of wild-type virus with gene reassortment and estimated its infectivity. As a result, dose-response relationships of all reassortants were best described by a beta-Poisson model. Virus subtype and human age were significant factors determining the dose-response relationship, whereas attenuation method affected only the relationship of H1N1 virus infection to adults. The data sets for H3N2 wild-type virus could be pooled with those for its reassortants on the assumption that the gene reassortment attenuates wild-type virus by at least 63 times and most likely 1,070 times. Considering this most likely degree of attenuation, 10% infectious dose of H3N2 wild-type virus for adults was estimated at 18 TCID50 (95% CI = 8.8-35 TCID50). The infectivity of wild-type H1N1 virus remains unknown as the data set pooling was unsuccessful.

Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus.

BACKGROUND: Water contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies. RESULTS: A gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure. CONCLUSIONS: EVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.

Development of an effective method for recovery of viral genomic RNA from environmental silty sediments for quantitative molecular detection.

Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol-chloroform-isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest rate of recovery (geometric mean of 11%, with a geometric standard deviation of 0.02; n = 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest rate of PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River, Miyagi, Japan, and its estuary from November 2007 to April 2009, and the genomic RNAs of enterovirus and human norovirus in these samples were quantified by reverse transcription (RT)-quantitative PCR (qPCR). The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 × 10(2) and 2.4 × 10(2) copies/g (wet weight), respectively. This is the first report to obtain quantitative data for a human pathogenic virus in a river and in estuarine sediments using RT-qPCR.

Mechanism and kinetics of ligand exchange between ferric citrate and desferrioxamine B.

The kinetics of ligand exchange between ferric citrate and desferrioxamine B (DFB) was investigated at pH 8.0 and high citrate/Fe molar ratios (500-5000) with particular attention given to understanding the precise mechanism of ligand exchange. Ferric citrate complexes present in a test solution and therefore involved in the reaction with the incoming ligand (DFB) were initially examined by evaluating ferric citrate speciation on the basis of published thermodynamic constants. The speciation analysis indicated that mononuclear (mono- and dicitrate) ferric complexes are the major species responsible for the ligand exchange with DFB under the conditions examined in the present work. Given the tendency of DFB to adjunctively associate with the ferric citrate complexes, we propose a kinetic model containing the following three mechanisms: (i) direct association of DFB to the ferric dicitrate complex prior to any dissociation of citrate molecules from the Fe center, (ii) adjunctive association of DFB toward ferric monocitrate complex following dissociation of one molecule of citrate from the parent complex, and (iii) complexation of hydrated Fe by DFB after sequential dissociation of two molecules of citrate from the Fe center. Overall rates for the ligand exchange were determined by spectrophotometrically monitoring the formation of ferrioxamine B. Further analysis in quantifying the rate of each mechanism by use of published and determined rate constants of relevant elemental reactions suggested that the first and second mechanisms were significant under our experimental conditions where [Cit] ≫ [DFB] with the relative importance of these two pathways depending on citrate concentration.

Oxygen and superoxide-mediated redox kinetics of iron complexed by humic substances in coastal seawater.

Complexes with terrestrially derived humic substances represent one of the most reactive pools of dissolved Fe in natural waters. In this work, redox kinetics of Fe-humic substance complexes (FeL) in simulated coastal seawater were investigated using chemiluminescence techniques with particular attention given to interactions with dioxygen (O2) and superoxide (O2•-). Although rate constants of FeIIL oxidation by O2 (5.6-52 M-1 · s-1) were 4-5 orders of magnitude less than those for O2•- (6.9-23 × 105 M-1 · s-1),O2 is likely to outcompete O2•- for FeIIL oxidation in coastal seawaters where steady-state O2•- concentrations are generally subnanomolar. Rate constants for FeIIIL reduction by O2•- of 1.8-5.6 × 104 M-1 · s-1 were also determined. From the balance of FeIIL oxidation rates and O2•- -mediated FeIIIL reduction rates, steady-state FeIIL concentrations were estimated to be in the subpicomolar to picomolar range, which is generally lower than measured in situ Fe(II) concentrations under relevant conditions. This suggests that (i) processes other than O2•- -mediated reduction (such as photochemical ligand-to-metal charge transfer) may be responsible for Fe(II) formation, (ii) the in situ ligands differ significantly from the humic substances used in this work, and/ or (iii) the influence of other environmental factors such as pH and temperature on Fe redox kinetics may have to be considered.

Development of a dose-response model for SARS coronavirus.

In order to develop a dose-response model for SARS coronavirus (SARS-CoV), the pooled data sets for infection of transgenic mice susceptible to SARS-CoV and infection of mice with murine hepatitis virus strain 1, which may be a clinically relevant model of SARS, were fit to beta-Poisson and exponential models with the maximum likelihood method. The exponential model (k= 4.1 x l0(2)) could describe the dose-response relationship of the pooled data sets. The beta-Poisson model did not provide a statistically significant improvement in fit. With the exponential model, the infectivity of SARS-CoV was calculated and compared with those of other coronaviruses. The does of SARS-CoV corresponding to 10% and 50% responses (illness) were estimated at 43 and 280 PFU, respectively. Its estimated infectivity was comparable to that of HCoV-229E, known as an agent of human common cold, and also similar to those of some animal coronaviruses belonging to the same genetic group. Moreover, the exponential model was applied to the analysis of the epidemiological data of SARS outbreak that occurred at an apartment complex in Hong Kong in 2003. The estimated dose of SARS-CoV for apartment residents during the outbreak, which was back-calculated from the reported number of cases, ranged from 16 to 160 PFU/person, depending on the floor. The exponential model developed here is the sole dose-response model for SARS-CoV at the present and would enable us to understand the possibility for reemergence of SARS.

Spatial genetic structure of the invasive tree Robinia pseudoacacia to determine migration patterns to inform best practices for riparian restoration.

The black locust Robinia pseudoacacia (Robinieae, Fabaceae) is a common invasive riparian tree in Japan. There are less effective management strategies to remove the tree from the riparian area because of its quickly established high population. We investigated the expansion patterns of R. pseudoacacia through sympatric (i.e. between high- and low-water channel (HWC/LWC) within a study site) and allopatric (i.e. along river corridor) dispersal in the Tama River (Tokyo, Japan). Four microsatellites were used to examine the effects of gene flow on six populations in three sites. These subpopulations showed small genetic distance (i.e. no barrier or slightly limited) and genetically mixed population structure. It indicated that both sympatric and allopatric dispersals were active. Many migrants were younger individuals (i.e. <5 years old) and were found in the LWC area. Thus, the LWC could receive more migrants than the HWC through both types of dispersals. In addition, our age and genetic structure analyses reveal that recruited individuals likely settled immediately after the clearing project of R. pseudoacacia through sympatric dispersal. It appears that the migration by allopatric dispersal occurred following this. For the effective management of R. pseudoacacia, migrants should be removed regularly following initial removal of invaders during site restoration.

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa.

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.

Weekly Variation of Rotavirus A Concentrations in Sewage and Oysters in Japan, 2014-2016.

Concentrations of rotavirus A, in sewage and oysters collected weekly from September 2014 to April 2016 in Japan, were investigated using RT-qPCR; results showed up to 6.5 log10 copies/mL and 4.3 log10 copies/g of digestive tissue (DT) in sewage and oysters, respectively. No correlation was found between rotavirus concentration in sewage and oysters and cases of rotavirus-associated gastroenteritis.

Biodilution of heavy metals in a stream macroinvertebrate food web: evidence from stable isotope analysis.

Analysis of carbon (delta13C) and nitrogen (delta15N) stable isotopes provides an increasingly important means of understanding the complex trophic structure of macroinvertebrate communities in streams. We coupled a stable isotope approach with a contaminant analysis of six metals (Pb, Ag, Zn, Hg, Cu, As) to trace the accumulation and dilution of metals from an abandoned mine across trophic levels of the benthic community in Ginzan Creek, Japan. The delta15N signature increased with trophic level, with mean increases of 4.70 per thousand from producers to primary consumers and 3.06 per thousand from primary to secondary consumers. Tissue Pb and Ag concentrations were negatively correlated with delta15N, indicating biodilution of both metals through the food web. Although macroinvertebrate taxon body mass was negatively correlated with tissue metal concentration at several sites, it did not increase with trophic level (as delta15N) in any of the sites, suggesting that changes in body mass were not the cause of biodilution. Our findings suggest invertebrates at higher trophic levels may exhibit increasingly efficient excretion of metals. Autotrophic epilithon (mean delta13C= -21.3 per thousand) had a much higher concentration of mined metals than did riparian vegetation (mean delta13C= -29.3 per thousand); nonetheless, a carbon-mixing model indicated that taxa feeding on autochthonous carbon sources did not accumulate more metal than allochthonous feeders. It is likely that the notably high metal concentration of allochthonous FPOM plays an important role in the trophic transfer of metals. Our data suggest the strong potential for stable isotope analysis to enhance our understanding of metal transfer through stream macroinvertebrate food webs.

Trophic structure of stream macroinvertebrate communities revealed by stable isotope analysis.

The trophic structure of a macroinvertebrate community was investigated using carbon and nitrogen stable isotopes analysis (delta(13)C and delta(15)N) at three longitudinal sites along the main channel of the Hirose River, Japan, and at an outlet site of the tributary reservoir. Along the main channel, primal food supply gradually changed from allochthonous to autochthonous source. The percentage of autochthonous origin of the macroinvertebrate community calculated by a carbon-mixing model (=38-83%) increased with that of suspended fine particulate organic matter (FPOM = 21-67%), a main resource for filtering-collectors that dominated the communities. The lowest percentage of autochthonous in the lake outlet community (=21%) was a result of the notably low percentage autochthonous of FPOM due to the decomposition of allochthonous coarse particulate organic matter (e.g., leaf) strained in the reservoir. Predators had higher mean delta(15)N than non-predators in three sites. The average enrichments of delta(15)N signature per trophic level for all sites were 3.82 per thousand from producers to first consumers and 1.24 per thousand from first to second consumers. Our data suggest the strong potential for stable isotope analysis to enhance our understanding of the trophic structure of stream macroinvertebrate community.

Weekly variations in norovirus genogroup II genotypes in Japanese oysters.

Increased levels of norovirus contamination in oysters were reportedly associated with a gastroenteritis epidemic occurring upstream of an oyster farming area. In this study, we monitored the norovirus concentration in oysters weekly between November 2014 and March 2015 and investigated the statistical relationship between norovirus genogroup II (GII) concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. A peak correlation coefficient (R = 0.76) at a time lag of +1 week was observed between the number of gastroenteritis cases and norovirus GII concentrations in oysters, indicating that oyster contamination is correlated with the number of gastroenteritis cases with a 1-week delay. Moreover, weekly variations in norovirus GII genotypes in oysters were evaluated using pyrosequencing. Only GII.3 was detected in November and December 2014, whereas GII.17 and GII.4 were present from January to March 2015. GII.17 Kawasaki 2014 strains were detected more frequently than GII.4 Sydney 2012 strains in oyster samples, as previously observed in stool and sewage samples collected during the same study period in Miyagi, Japan. Our observations indicate that there is a time lag between the circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters.

Cellular proteins of Microcystis aeruginosa inhibiting coagulation with polyaluminum chloride.

Cyanobacterial growth in semi-closed water areas such as reservoirs brings about a coagulation inhibition in a drinking water treatment system, but the inhibitory substances and mechanisms involved have yet to be elucidated. In this study, proteins having a high affinity with polyaluminum chloride (PACl) were isolated from organic substances produced by Microcystis aeruginosa with the affinity chromatography technique. Both extracellular organic matter (EOM) and cellular organic matter (COM) disturbed the flocculation of suspended kaolin with PACl, but it was likely that nonproteinous substances in EOM cause the reduction of coagulation effciency. In contrast, proteins in COM were obtained as possible inhibitory substances for the coagulation with PACl. These proteins could consume PACl in the coagulation process due to the formation of chelate complexes between these inhibitory proteins and the coagulant. The consumption of PACl by cyanobacterial proteins could be one of the important causes of the increase in coagulant demand.

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.