Characterization of Natural Organic Substances Potentially Hindering RT-PCR-Based Virus Detection in Large Volumes of Environmental Water.

Quantitative detection of pathogenic viruses in the environmental water is essential for the assessment of water safety. It is known that some of natural organic substances interfere with virus detection processes, i.e., nucleic acid extraction and reverse transcription-PCR. Such substances are carried over into a sample after virus concentration. In this study, inhibitory substances in coastal water samples were characterized in view of their effects on efficiency of virus detection and property as organic matters. Among 81 samples tested, 77 (95%) showed low recoveries (<10%) of spiked murine norovirus. These recovery rates were correlated with the levels of organic matter present in virus concentrates as measured by ultraviolet absorbance at 254 nm (r = -0.70 - -0.71, p < 0.01). High-performance gel chromatography and fluorescence excitation-emission matrix spectroscopy revealed that organic fractions in the 10-100 kDa size range, which were not dominant in the original samples, and those possessing humic acid-like fluorescence properties were dominant in virus concentrates. The inhibitory effect was more pronounced during summer. Substances originating from seawater seemed to cause a more pronounced effect than those originating from wastewater. Our data highlight the previously unknown characteristics of natural inhibitory substances and are helpful in establishing an effective sample purification technique.

Comparative Evaluation of Real-Time PCR Methods for Human Noroviruses in Wastewater and Human Stool.

Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently.

Pepper mild mottle virus as an indicator and a tracer of fecal pollution in water environments: comparative evaluation with wastewater-tracer pharmaceuticals in Hanoi, Vietnam.

We analyzed pepper mild mottle virus (PMMoV) in 36 samples taken from surface water, wastewater, groundwater, tap water and bottled water in Hanoi, Vietnam. We then compared the occurrence and fates of PMMoV with pharmaceuticals and personal care products (PPCPs), which are known wastewater tracers. PMMoV was detected in 94% of the surface water samples (ponds, water from irrigated farmlands and rivers) and in all the wastewater samples. The PMMoV concentration ranged from 5.5×10(6)-7.2×10(6)copies/L in wastewater treatment plant (WWTP) influents, 6.5×10(5)-8.5×10(5)copies/L in WWTP effluents and 1.0×10(4)-1.8×10(6)copies/L in surface water. Among the sixty PPCPs analyzed, caffeine and carbamazepine had high detection rates in surface water (100% and 88%, respectively). In surface water, the concentration ratio of PMMoV to caffeine remained unchanged than that in WWTP influents, suggesting that the persistence of PMMoV in surface water was comparable to that of caffeine. The persistence and the large concentration ratio of PMMoV in WWTP influents to the method detection limit would account for its ubiquitous detection in surface water. In comparison, human enteric viruses (HEV) were less frequently detected (18-59%) than PMMoV in surface water, probably because of their faster decay. Together with the reported high human feces-specificity, our results suggested that PMMoV is useful as a sensitive fecal indicator for evaluating the potential occurrence of pathogenic viruses in surface water. Moreover, PMMoV can be useful as a moderately conservative fecal tracer for specifically tracking fecal pollution of surface water. PMMoV was detected in 38% of the groundwater samples at low concentrations (up to 19copies/L). PMMoV was not detected in the tap water and bottled water samples. In groundwater, tap water and bottled water samples, the occurrence of PPCPs and HEV disagreed with that of PMMoV, suggesting that PMMoV is not suitable as an indicator or a tracer in those waters.

Distribution of marine birnavirus (MABV) in marine organisms from Okinawa, Japan, and a unique sequence variation of the VP2/NS region.

Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25 degrees C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.