Development of a Virus Concentration Method and Its Application to Detection of Enterovirus and Norwalk Virus from Coastal Seawater

Abstract

We developed a new procedure for concentration of enteric viruses from water using a negatively charged membrane. Rinsing the membrane with 0.5 mM H 2 SO 4 (pH 3.0) in order to elute cations prior to viral elution with 1 mM NaOH (pH 10.5) promoted poliovirus recovery yields from 33 to 95% when applied to pure water and 38 to 89% when applied to natural seawater from Tokyo Bay, Japan, respectively. This method showed average recovery yields of spiked poliovirus of 62% (n ‫؍‬ 8) from 1 liter of artificial seawater. This method showed higher recovery yields (>61%) than that of the conventional method using positively charged membrane (6%) when applied to seawater. This method is also free from beef extract elution, which has an inhibitory effect in the subsequent viral genome detection by reverse transcription-PCR. Naturally occurring Norwalk viruses from 2 liters of Tokyo Bay water in winter and infectious enteroviruses from 2 liters of recreational coastal seawater in summer were detected by using this viral concentration method.To determine the public health risk caused by human enteric viruses in water, a reliable, sensitive, and practical method for detecting small concentrations of viruses is needed. Concentrating viruses in water by adsorption to and subsequent elution from a positively charged membrane (38) is currently considered to be one of the most useful methods (3). This method has been applied to tap water (25, 39), groundwater (1), river water (22, 23), lake water (23), secondarily treated sewage (36) or marine water (29). The virus concentrations are determined by conventional plaque assays (22,23,25,36,38,39). However, the recoveries from seawater are not always high enough because of low adsorption of viruses to the positively charged membrane due to the influence of salts (24). Most of the enteric viruses are known to adsorb to a negatively charged membrane in the presence of Mg 2ϩ (40,43) or other multivalent cations, or under acid conditions (37), while the recovery of viruses is not always easy.According to the infectious disease weekly reports from the National Institute of Infectious Disease, Tokyo, Japan, infection with enteroviruses is common in the summer. The use of recreational seawater is suspected as one of the main pathways of infection. On the other hand, the outbreaks of Norwalk viruses have been occurring often in winter, and the consumption of molluscan shellfish fecally contaminated in the harvesting seawater has been suspected to be one of the main pathways of these viruses. Hence, the viral contamination of seawater is one of the important issues from the epidemiological point of view. The occurrence of these viruses in oysters or other seafood has been widely reported (4,8,10,18), while the fate of viruses in seawater is unknown and the levels of the virus have not been quantitatively discussed (9).In recent years, the PCR method has been used to detect enteric viruses in environmental samples (16, 19-21, 28, 31, 42). PCR is one of the most-sensitive methods available for ...

Wastewater Analysis Indicates that Genetically Diverse Astroviruses, Including Strains Belonging to Novel Clades MLB and VA, Are Circulating within Japanese Populations

“…During the 6-month study period, a total of 24 samples were collected from four locations in the treatment train: influent, after secondary treatment, after chlorination, and after sand filtration (effluent). The samples (100 ml for the influent samples and 1,000 ml for each of the other samples) were concentrated using an electronegative membrane (type HA; diameter, 90 mm; pore size, 0.45 m; Millipore, Tokyo, Japan) and a centrifugal ultrafiltration device (Centriprep YM-50; Millipore) to obtain a final volume of approximately 0.7 ml, as previously described (29). As a process control, 2.2 ϫ 10 7 genome copies of murine norovirus (MNV) were inoculated into 140 l of the concentrate.…”

First description of Adenovirus, Enterovirus, Rotavirus and Torque teno virus in water samples collected from the Arroio Dilúvio, Porto Alegre, Brazil

“…Putative viral particles present on the samples were concentrated using an adsorption-elution method with negatively charged membranes (HA, Millipore, USA), as described previously by Katayama et al (2002) with minor modifications. Briefly, 500 mL of each water sample was mixed with 0.3 g MgCl 2 and pH adjusted to 5.0 with 10% HCl .…”

Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

“…Our protocol differed from that of Katayama et al (13) in a few ways that we think are more appropriate for our laboratory. First, we did not FIG.…”

“…The concentrate was then subjected to conventional RT-PCR (9,11,22) or quantitative RT-PCR (qRT-PCR) analysis (4, 13). Katayama et al (13) reported that negatively charged filters were efficient (Ͼ61%) in capturing viruses from relatively small volumes of seawater for qRT-PCR and for virus cultivation. This appeared to be a promising protocol for routine virus assays.…”

“…However, our attempt to reproduce that method in our laboratory was prevented by the lack of availability (in the United States) of the proprietary extraction kit described in that report; we also noted that the concentrations of poliovirus they used in their seeding experiments (300 to 770 PFU/ml) were very high compared to what one might see in field samples, leading to questions about recovery at realistic field concentrations. We modified the Katayama et al (13) protocol to include a readily available extraction kit, different qRT-PCR parameters that we tested in a previous study to avoid inhibition (1), and a range of test virus concentrations at levels more realistic of realworld field samples. We also tested this modified protocol with both freshwater and seawater samples and compared the cellulose mixed-ester filter retention with the retention of more porous and freely flowing glass fiber filters used in a previous enterovirus field study (1).…”