Identification of novel norovirus polymerase genotypes from pediatric fecal samples collected between the year 1997 and 2000 in Japan.

We analyzed 46 pediatric fecal samples collected between the years 1997 and 2000 to retrospectively evaluate the norovirus strains circulating during that era and to identify possible re-emergence patterns. From the tested fecal samples, we detected GII.1, GII.3, GII.4 (95/96-US) and GII.6 strains. Most importantly, two novel polymerase genotypes (GI.PNA4 and GII.PNA7) were detected during the study. Two possible recombinant strains (GII.6[P7] and GII.3[P29]) were identified and SimPlot analysis confirmed that GII.6[P7] is a recombinant strain. The study emphasizes the importance of retrospective evaluation of human fecal samples in obtaining a better understanding of norovirus circulation, re-emergence and evolution.

Pepper mild mottle virus as a process indicator at drinking water treatment plants employing coagulation-sedimentation, rapid sand filtration, ozonation, and biological activated carbon treatments in Japan.

To assess the potential of pepper mild mottle virus (PMMoV) as a viral process indicator, its reduction through coagulation-sedimentation (CS) and rapid sand filtration (RSF) were compared with those of Escherichia coli, previously used viral indicators, and norovirus genotype II (NoV GII; enteric virus reference pathogen) in a bench-scale experiment. PMMoV log10 reductions in CS (1.96 ±â€¯0.30) and RSF (0.26 ±â€¯0.38) were similar to those of NoV GII (1.86 ±â€¯0.61 and 0.28 ±â€¯0.46). PMMoV, the most abundant viruses in the raw water, was also determined during CS, RSF, and advanced treatment processes at two full-scale drinking water treatment plants under strict turbidity management over a 13-month period. PMMoV was concentrated from large-volume water samples (10-614 L) and quantified by Taqman-based quantitative polymerase chain reaction. The PMMoV log10 reduction in CS (2.38 ±â€¯0.74, n = 13 and 2.63 ±â€¯0.76, n = 10 each for Plant A and B) and in ozonation (1.91 ±â€¯1.18, n = 5, Plant A) greatly contributed to the overall log10 reduction. Our results suggest that PMMoV can act as a useful treatment process indicator of enteric viruses and can be used to monitor the log10 reduction of individual treatment processes at drinking water treatment plants due to its high and consistent copy numbers in source water.

Virus Particle Detection by Convolutional Neural Network in Transmission Electron Microscopy Images.

A new computational method for the detection of virus particles in transmission electron microscopy (TEM) images is presented. Our approach is to use a convolutional neural network that transforms a TEM image to a probabilistic map that indicates where virus particles exist in the image. Our proposed approach automatically and simultaneously learns both discriminative features and classifier for virus particle detection by machine learning, in contrast to existing methods that are based on handcrafted features that yield many false positives and require several postprocessing steps. The detection performance of the proposed method was assessed against a dataset of TEM images containing feline calicivirus particles and compared with several existing detection methods, and the state-of-the-art performance of the developed method for detecting virus was demonstrated. Since our method is based on supervised learning that requires both the input images and their corresponding annotations, it is basically used for detection of already-known viruses. However, the method is highly flexible, and the convolutional networks can adapt themselves to any virus particles by learning automatically from an annotated dataset.

Development of a high resolution melting analysis for detection and differentiation of human astroviruses.

Human astroviruses (AstVs), the common causes of viral gastroenteritis, consist of 8 different sero- or genotypes in which a variety of subtypes have been found. In the present study, a rapid and high-throughput method for detection and sequence-discrimination of AstVs by high resolution melting (HRM) analysis was developed. A newly designed primer set for the assay targeting ORF1b-ORF2 junction region of AstVs successfully reacted with all 8 serotypes of AstVs and allowed genotyping using their amplicons. The HRM assay consists of intercalating dye based real time quantitative PCR (qPCR) and melting curve analysis. The qPCR assay was sensitive enough to detect 1.0×10(1) copies/reaction of AstV serotypes. However, 1.0×10(3) copies/reaction of AstVs gene was required to obtain a sequence-specific difference curve, indicating that pre-amplification is necessary to apply the assay to samples containing low numbers of AstVs. AstVs in clinical specimens were subjected to the HRM assay after pre-amplification. The strains possessing same nucleotide sequences at the target region showed an identical difference curve and those possessing different nucleotide sequences showed a distinguishable difference curve. The newly developed HRM assay is an effective technique for screening of AstVs to quantify and discriminate the strains.

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.

Development of a rapid high-throughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses.

We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories.

Development of sample storage methods for detecting enteric viruses in environmental water.

In a field survey of enteric viruses, water samples collected sometimes need to be stored for a long duration before analysis is performed. The aim of this study was to develop an appropriate sample storage method for detecting viruses in environmental water. Three types of sample storage methods were evaluated using MilliQ water, pond water, and treated sewage inoculated with poliovirus and norovirus: (i) storage followed by the full concentration procedure, (ii) filtration and storage followed by the remaining concentration procedure, and (iii) the full concentration procedure before storage. The recovery of norovirus on day 0 of inoculation was 110.2+/-38.2% (n=2), which was comparable to that of poliovirus. During sample storage for up to 13 days, virus recovery showed different patterns of decrease, depending on the storage method, sample type, and storage temperature. Among the three methods tested, the method of storing the eluted samples was judged to be most appropriate for detection of viruses from water samples. This method does not require any special equipment and can be easily adopted in field surveys, especially in developing countries.

Application of an automated specimen search system installed in a transmission electron microscope for the detection of caliciviruses in clinical specimens.

To evaluate the performance of an automated specimen search system in the detection of caliciviruses such as Norwalk-like viruses and Sapporo-like viruses, a suitable negative staining method was developed and the viruses were examined using the system installed in a transmission electron microscope (TEM). Clear images of the viruses were obtained by staining with 2% uranyl acetate at pH 4.0 as compared with 2% phosphotungstic acid staining at any pH. When the image parameter of 30+/-6 nm for the diameter of a single virus-like particle of 2% uranyl-acetate-stained Norwalk-like virus was set on the automated specimen search system, 95% of the virus-like particles that were counted by the conventional TEM technique were detected. The system was used to detect Norwalk-like viruses in five semipurified stool samples in which Norwalk-like viruses had already been detected by reverse transcription-polymerase chain reaction assay and conventional electron microscopy. The positive detection rate for Norwalk-like viruses, which had been counted by the conventional technique, ranged from 56.2 to 77.9% using this system. Our findings indicate that the automated specimen search system installed in a TEM is suitable for the detection of caliciviruses in semipurified stool samples. The system is useful for clinical diagnosis without the need for operator intervention.