mm mm®©mn(§WK] m G, Shay Fout1, Ann C. Grirnm1, Daniei R. Dahling1, Jennifer L. Cashdollar1, Christina M, Newport1, Sandhya Parshionikar2, Sandra Wiiiian-True2 1 National Exposure Research Laboratory, Cincinnati, Ohio; 20akridge Postdoctoral Trainees, Cincinnati, Ohio Emerging Tec Environmental Issue: WATERBORNE DISEASE (1920-2000) 350 300 250 200 150 100 50 0 OUTBREAKS PER DECADE 1920s 1930s 1940s 1950s 1960s 1970s 1980s 1990s 2000s ¦ Waterborne disease outbreaks occur every year ¦Annual average is 23 outbreaks with 19,000 cases ¦Over half of the outbreaks are caused by viruses ¦ The actual disease burden from waterborne viruses is unknown because of a lack of detection methods. Collaborative Approach: A partnership of scientists and engineers from ORD, the Office of Ground Water and Drinking Water's Technical Support Center (TSC) and the American Water Works Association Research Foundation (AWWARF) was formed to improve every aspect of standard virus sampling procedures and to develop a new molecular virus detection method. The steps that are needed to detect viruses in water are as follows: 1. Sample Collection: Alignment of Enterovirus RNA Sequences The emerging tools of molecular genomics and bioinfomatics was used to develop the new method. 2. Filter Processing Results Ground Water: The virus method developed under the partnership between ORD, TSC and AWWARF was used to examine 29 ground water wells on a monthly basis for 12 months. Results showed that 16% of 321 groundwater samples and 72% of 29 groundwater sites were positive for viruses. Virus in water are collected on filters Viruses are removed from filters with over 90% recovery. 3. Virus Purification 4. Virus Detection RT - PCR Analysis of Samples 1 234 567 BP Virus are purified and concentrated over 500,000 times with greater than a 70% recovery. A new method was developed to detect major groups of waterborne viruses. RESULTS Virns % Positive Samples Sites Enteroviruses 5 38 Reo virus 10 62 Rotaviruses 0 0 Hepatitis A virus 1 14 Norwalk virus 3 21 Total 16 72 Surface Water: A second partnership was developed between ORD and the U.S. Geological Survey to test the methods using stream waters from five rural to urban sites across the U.S. The method proved effective for surface water with all sites being positive for viruses as shown below. Rotavirus Hepatitis A virus NorwaLk virus Hepatitis A virus Poliovirus Site Land Population Use Density3 Percent Virus Enterovirus Positive Samples Rotavirus HAV EIWA Rural" 16 100 0 0 KANA SANT Rural" 88 Rurald 544 33 100 0 0 100 33 LERI Urban6 2849 100 67 67 PUGT Urban 15540 100 33 0 1 Agricultur a Agricultu ' Site includ per square kilometer and confined animal facilities as and septic tanks near site es some agricultural contribution mm Outbreaks: A third partnership was developed between ORD, EPA Region VIII, CDC and state Departments of Health to investigate two groundwater-related outbreaks of disease in the state of Wyoming in 2001. The method proved valuable for outbreak investigations in that norovirus strains were detected in the groundwater and shown to be identical to patient strains. The two outbreaks were caused by different norovirus gene groups. Calicivirus Viruses in Group: Norovirus Genogroup I Norwalk Genogroup II Snow Mountain Nucleic Acid: Single-Stranded Disease: Gastroenteritis Impact: ¦ The virus detection method is available to collect virus occurrence data needed to support the development of regulations to protect the American public. ¦ The virus detection method is available to support exposure studies designed to determine the disease burden caused by waterborne viruses. ¦ Data from using the virus detection method will lead to improvement of drinking and recreational water quality. ¦ The method is adaptable to testing for new viruses in water, such as the recently emerging SARS corona- virus. Partnering to Protect Human ------- |