Eiji Haramoto

Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan

ABSTRACT The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% ± 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.

Detection of Noroviruses in Tap Water in Japan by Means of a New Method for Concentrating Enteric Viruses in Large Volumes of Freshwater

ABSTRACT A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H2SO4 (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.

Real-time PCR detection of adenoviruses, polyomaviruses, and torque teno viruses in river water in Japan.

The prevalence of DNA viruses in water from the Tamagawa River, Japan was quantitatively surveyed for 6months, from April to September 2003. A total of 18 river water samples were subjected to virus concentration method using an electronegative membrane, followed by DNA extraction and direct quantitative real-time polymerase chain reaction (qPCR) for DNA viruses. Adenoviruses of serotypes 40 and 41 were detected most frequently in the river water samples tested (61.1%), at a concentration ranging from 3.16x10(3) to 1.38x10(5) copies/l, followed by JC polyomaviruses (11.1%) and torque teno viruses (5.6%). No sample was positive for BK polyomaviruses. In addition, for selective detection of virus particles, adenoviruses 40 and 41 were tested with qPCR combined with an immunomagnetic separation technique; they were detected in only 16.7% of the samples, showing a concentration ranging from 7.42x10(2) to 4.24x10(4) copies/l. This study is significant since it is the first study to demonstrate the prevalence of polyomaviruses in water samples in Japan and to use immunomagnetic separation qPCR to detect adenovirus particles in aquatic environments.

Quantitative analysis of human enteric adenoviruses in aquatic environments

Aims:  The aim of this study was to determine human adenoviruses (HuAdVs) in aquatic environments by real‐time polymerase chain reaction (PCR).

Recovery of human norovirus from water by virus concentration methods.

The aim of this study was to evaluate the applicability of virus concentration methods to detect human norovirus (HuNoV) in water. One conventional virus concentration method using an electropositive filter (1MDS-method) and two methods developed by our research group using an electronegative filter (Mg-method and Al-method) were subjected to recovery tests of the HuNoV strain GII.4, which was obtained from a diarrhea patient, and poliovirus (PV) type 1 inoculated into 5 kinds of water samples. The mean recovery yields of HuNoV by the Mg-method, determined by real-time PCR, were 186%, 80%, 167%, 15%, and 39% for MilliQ water, tap water, bottled water, river water, and pond water, respectively (n=2 each), which were generally comparable to those of PV. A similar trend was observed for the Al-method (n=8 in total), suggesting that both Mg- and Al-methods can be appropriate for concentrating HuNoVs from water samples. Unlike these two methods, no clear correlation was found between the recovery of HuNoV and PV by the 1MDS-method (n=6 in total). This study is significant because it provides observations on the use of virus concentration methods for the detection of uncultivable HuNoV in water samples.

Quantitative detection of sapoviruses in wastewater and river water in Japan

Aims: To detect sapoviruses at a wastewater treatment plant (WWTP) and in a river in Japan, quantitatively.

Detection of genogroup IV norovirus in wastewater and river water in Japan.

Aims:  To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV).

Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treatment plant in Japan.

Aims:  To investigate the prevalence, seasonality and genotype distribution of human noroviruses (NoVs) in wastewater in Japan.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

Prevalence and Genetic Diversity of Aichi Viruses in Wastewater and River Water in Japan

ABSTRACT Aichi virus (AiV) genomes were detected in 12 (100%) influent and 11 (92%) effluent wastewater and 36 (60%) river water samples. Among 260 strains identified, 255 were genotype A and 5 were genotype B. This is the first report describing the molecular characterization of AiVs in aquatic environments in Japan.