Improved real-time quantitative reverse transcription PCR detection of norovirus following removal of inhibitors.

Human norovirus (HuNoV) is an important enteric virus that can cause large gastroenteritis outbreaks via the fecal-oral route from contaminated water and produce. Real-time quantitative reverse transcription PCR (RT-qPCR) is the only method to apply the routine detection of HuNoV in various samples, however, inhibitors present in the samples can affect the accuracy and sensitivity of RT-qPCR results. Here, we suggest an inhibitor-removal treatment for two types of noroviruses using two commercial kits. Two types of water sample (surface and seawater) and four types of produce (green onions, lettuces, radishes, and strawberries) were evaluated. The recovery efficiencies of noroviruses in water samples clearly increased in surface and seawater samples with the inhibitor-removal treatment compared to untreated samples. Moreover, murine norovirus-1 was well recovered from the four types of produce with the inhibitor-removal treatment. The mean recovery efficiencies of HuNoV genogroup II genotype 4 in lettuces and strawberries were also increased in the treated samples. Therefore, we suggest that the inhibitor-removal treatment could be useful for improving the accuracy and sensitivity of RT-qPCR methods for noroviruses in water and produce.

Male-Specific and Somatic Coliphage Profiles from Major Aquaculture Areas in Republic of Korea.

Human and animal feces are important sources of various types of microbial contamination in water. Especially, enteric viruses, the major agents of waterborne infection, can attain long-term survival in water environments due to their strong resistance to various environmental factors including pH, salinity, and temperature. Coliphages are promising viral indicators for fecal contamination in water environments. Here, we investigated the seasonal and spatial distribution of male-specific and somatic coliphages in surface water and seawater at three major aquaculture areas, including Goseong Bay, Aphae Island, and Gomso Bay, in Republic of Korea over a period of 1 year. We selected 6 surface water and 14 seawater sampling sites for each study area and collected a total of 480 water samples from March 2014 to February 2015. Overall, surface water samples contained higher occurrences of coliphages than seawater samples. The high coliphage concentrations were detected in spring (March to May 2014). The differences in geographical features and patterns in land usage of the three aquaculture areas may have affected the coliphage concentration and occurrence. Moreover, environmental factors such as cumulative precipitation were strongly correlated with coliphage concentrations. Therefore, we suggest that further longitudinal studies on coliphage concentrations and distributions should be performed to support the application of coliphages in tracking fecal contamination in water.

Use of coliphages to investigate norovirus contamination in a shellfish growing area in Republic of Korea.

A number of severe norovirus outbreaks due to the consumption of contaminated shellfish have been reported recently. In this study, we evaluated the distribution of coliphage densities to determine their efficacy as fecal indicators of enteric viruses, including noroviruses, in water samples collected from a shellfish growing area in Republic of Korea over a period of approximately 1 year. Male-specific and somatic coliphages in water samples were analyzed using the single agar layer method, and norovirus genogroups I and II, which infect mainly humans, were analyzed using duplex reverse transcription quantitative PCR. Male-specific and somatic coliphages were detected widely throughout the study area. Several environmental parameters, including salinity, precipitation, temperature, and wind speed were significantly correlated with coliphage concentrations (P < 0.05). Moreover, the concentrations of male-specific coliphages were positively correlated with the presence of human noroviruses (r = 0.443; P < 0.01). The geospatial analysis with coliphage concentrations using a geographic information system revealed that densely populated residential areas were the major source of fecal contamination. Our results indicate that coliphage monitoring in water could be a useful approach to prevent norovirus contamination in shellfish.

Comparison of Swab Sampling Methods for Norovirus Recovery on Surfaces.

Human noroviruses (HuNoVs) can be easily transferred by the contacts of humans or fomites. Swab sampling methods are widely used for recovering HuNoVs from small surfaces of various fomites or hard-to-reach locations and swab sampling conditions are important for the accurate detection of HuNoVs, which have a low infectious dose and relatively long persistence under a range of environmental conditions. Therefore, to determine the suitable swab sampling method for recovering HuNoVs from various surfaces, we evaluated combinations of four swab materials (cotton, microdenier polyester [a type of microfiber], polyurethane foam, and rayon) and three elution buffer solutions (phosphate-buffered saline [PBS], PBS with 0.2% Tween-80, and 3% beef extract-50 mM glycine [pH 9.5]). First, we inoculated HuNoVs or murine noroviruses (MuNoVs), the surrogate of HuNoVs, onto test coupons (10 × 10 cm) consisting of three common surface materials (high-density polyethylene, stainless steel, and wood). Coupons were swabbed using a combination of each swab material and elution buffer, and the viral recovery was measured by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) or plaque assay. By RT-qPCR, we confirmed that the cotton swab-PBS and microdenier polyester-PBS combinations had recovery efficiencies greater than 80% for viruses on plastic and stainless steel surfaces. The cotton swab-PBS combination had the highest recovery efficiency on all surface materials via the plaque assay. Therefore, a cotton or a microdenier polyester swab with PBS could be a useful method for sampling HuNoVs on various surfaces.

Host-Specific Bacteroides Markers-Based Microbial Source Tracking in Aquaculture Areas.

Various waterborne pathogens originate from human or animal feces and may cause severe gastroenteric outbreaks. Bacteroides spp. that exhibit strong host- or group-specificities are promising markers for identifying fecal sources and their origins. In the present study, 240 water samples were collected from two major aquaculture areas in Republic of Korea over a period of approximately 1 year, and the concentrations and occurrences of four host-specific Bacteroides markers (human, poultry, pig, and ruminant) were evaluated in the study areas. Host-specific Bacteroides markers were detected widely in the study areas, among which the poultry-specific Bacteroides marker was detected at the highest concentration (1.0-1.2 log10 copies L-1). During the sampling period, high concentrations of host-specific Bacteroides markers were detected between September and December 2015. The host-specific Bacteroides marker-combined geospatial map revealed the up-to-downstream gradient of fecal contamination, as well as the effects of land-use patterns on host-specific Bacteroides marker concentrations. In contrast to traditional bacterial indicators, the human-specific Bacteroides marker correlated with human specific pathogens, such as noroviruses (r=0.337; P<0.001). The present results indicate that host-specific Bacteroides genetic markers with an advanced geospatial analysis are useful for tracking fecal sources and associated pathogens in aquaculture areas.