An Improved Method for Detecting Viruses in Water Leah Fohl Villegas US EPA/Office of Research and Development (ORD)/ National Exposure Research Laboratory (NERL)/Cincinnati, OH Background Enteroviruses,such as echovirus and coxsackievirus, have been implicated in numer- ous waterborne disease outbreaks of gastroenteritis worldwide. However, compre- hensive occurrence studies of enteroviruses in drinking water matrices are limited, in part because of the lack of available methods that are rapid, sensitive, and able to detect live infectious virus.To address this issue, the US EPA has included echoviruses and coxsackievi- ruses on the microbial contaminant candidate list (CCL).To provide the research needed to support regulation decisions for these CCL viruses, our efforts have focused on developing methods for the rapid detection of live virus. To achieve this goal we have developed a technique which integrates cell culture and molecular assays into one method. This inte- grated cell culture/polymerase chain reaction method (ICC/PCR) approach uses molecular tools to An electron micrograph of an rapidly detect infectious enterovirus, enterovirus Current Detection Methods for Enterovirus in Water: Molecular-Based Assays Rapid Sensitive Does not determine if the virus present is infectious Often the assay is inhibited by environmental matrices Culture-Based Assays Detects any viable virus that can grow in cell culture Requires several days to weeks No one cell line grows all viruses Not all virus growth can be visualized by cell culture alone Integrated Cell Culture & Molecular Method Sample Collection & Elution Step 1: Sample Collection and Elution The method begins by filtering 200 liters of surface water through a 10-inch 1MDS filter. Any virus present in the sample will be adsorbed by electrostatic interac- tions with the filter. These viruses are then eluted from the filter using 1.6 liters of 1.5% w/v beef extract at pH 9.5. Step 2: Concentration The viral particles are extracted from the eluate by adding celite. The celite/virus complex is subsequently collected by filtration and the virus is eluted from the celite using sodium phosphate at a high pH. The sample is further concentrated by an ultra-centrifugation step, and the virus containing pellet is resuspended in 0.5ml of PBS+ 0.2% BSA. Step 3: Tissue Culture The entire concentrated sample is then inoculated into two culture tubes containing BGM cell monolayers, and incubated for 1-7 days at 37°C. At daily time points, one of the two infected cell culture tubes undergoes a quick freeze/thaw to release the virus from the cells, and cell lysate aliquots are taken to test for viral nucleic acid. The remaining cell culture lysate is used to inoculate a new BGM monolayer to allow for additional growth. Step 4: Detection Aliquots of the cell lysate are tested for the presence of viral nucleic acid using both conventional reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR). Identical pan-enterovirus primer sets were used for both molecular assays and a pan-enterovirus specific TaqMan probe was used for the qRT-PCR. Conventional PCR products are resolved in an agarose gel and hybridization with a viral specific probe in order to determine the specific- ity of the amplicon;in contrast, the qPCR assay gives an immediate measure of the amount and specificity of the PCR product. Results and Conclusions Table 1. Tests of the integrated cell culture/PCR method using spiked reagent water samples PFU/tube* Cell Culture Alone CPEt ICC/PCR method qRT-PCR RT-PCR 50 20 10 5 2 3 days 4 days 3-4 days 3-6 days 4-8 days 24 hours 24 hours 24 hours 48 hours 48 hours 24 hours 48 hours 24 hours 72 hours 72 hours * Each tube spiked with plaque forming units (PFU) of poliovirus t Cytopathic effect (CPE) refers to cell lysis by viral replication Table 2. Tests of the ICC/PCR method using surface water samples Cell Culture Alone ICC/PCR method PFU/tube* CPEt aRT-PCR 400 (2) 2 days 24 hours 200 (4) 2 days 24 hours 100 (4) 2-3 days 24 hours 50 (4) 3 days 24 hours 20(4) 3-4 days 24 hours 10(4) 4-6 days 24 hours 6(4) 4-6 days 24-48 hours * Each tube spiked with plaque forming units (PFU) of poliovirus and number of trials is in parentheses t Cytopathic effect (CPE) refers to cell lysis by viral replication Using the integrated cell culture/PCR method allows for detection of 6-10 infectious virus units in 24-48 hours verses the 4-6 days required for detection by cell culture alone. Similar results were observed for poliovirus and other enteroviruses (echovirus and coxsackievirus A9, data not shown). qRT-PCR is more sensitive than conventional RT-PCR, also qRT-PCR is faster and less labor intensive than conventional RT-PCR Cytotoxicity and PCR inhibition has not been found to be deleterious to this method and can be addressed easily without large decreases in sensitivity and speed Our results demonstrate that incorporating a qRT-PCR step allows the detection of live infectious virus before infection is visible in cell culture alone. The sensitivity of the method is comparable to cell culture and molecular assays alone Serial passage of the sample during cell culture allows for early detection without loss of any of the concentrated water sample Disclaimer: This poster does not necessarily reflect EPA policy. Mention of trade names or commercial products does not constitute an endorsement. Contact: Leah Fohl Villegas Post-Doctoral Fellow US EPA/ORD/NERL 26 W. Martin Luther King Drive Cincinnati, OH 45268 513-569-7886 villegas.leah@epa.gov epascienceforum Your Health • Your Environment • Your Future ------- |