Waterborne enteric viruses are transmitted via the fecal-oral route and have been isolated from various types of water ranging from sewage to tap water. Water matrices characterized by low levels of organic material (e.g. clean surface water and tap water) contain fewer numbers of viruses than sewage and wastewater effluents. A number of methods have been developed to concentrate, elute (recover), and re-concentrate viruses from water. The goal of this dissertation is two-fold. An extensive review of the literature is provided in Appendix A that focuses on method development in the three aforementioned areas. A review of this detail has not been conducted in over two decades, and as such will contribute to the fields of water quality and environmental virology. Second, a novel and inexpensive method for the concentration of viruses (MS2 coliphage, poliovirus 1, echovirus 1, Coxsackievirus B5, and adenovirus 2) is presented in Appendix B. The method uses a new electropositive filter (comprised of nanoalumina fibers) for the capture of viruses from 20-L volumes of dechlorinated tap water. Average filter retention efficiencies for each of the viruses was ≥ 99%. Viruses that are adsorbed to filters must then be recovered (eluted). A number of inorganic solutions were evaluated for this purpose, the most effective being a moderately alkaline (pH 9.3) glycine buffered-polyphosphate solution. Secondary reconcetration of the eluates was performed using an optimized ultrafiltration method (Centricon Plus-70, Millipore, Billerica, MA), and achieved final concentrates volumes of 3.3 ± 0.3 mL. Total method efficiencies meeting the project recovery goal of ≥ 50% were obtained for each of the tested viruses except for MS2 coliphage at high input titers (45 ± 15%) and adenovirus 2 (14 ± 4%). Appendix C provides the Standard Operating Procedures, sample calculations, and detailed data for the experiments conducted. Appendix D details the steps taken towards optimizing the secondary concentration procedure in effort to meet the 50% recovery goal.

En pilotanläggning för ultrafiltering testas nu i Norrvattens reningsprocess för att undersöka ifall den kan användas som en tredje mikrobiologisk barriär i reningsprocessen. Målet med detta projekt är att testa membranets kapacitet att filtrera bort viruspartiklar men även membranet generella reduktionsförmåga för andra mikrobiologiska och kemiska kontamineringar. För att hitta lämpliga kandidater att använda sig av för att mäta reduktionskapaciteten av membranet har en litteraturstudie samt experimentell testning av råvattnet genomförts. OD mätningar på bakteriekulturer samt plackbildandeenheter (PBE) har undersökt för att se om bakteriofager kan finnas i proven. Ungefär 9000 L av ingående och utgående vatten från ultrafilteringen har koncentrerats med hjälp av ett elektropositivt filter som senare har eluerats och ultracentrifugerats. Pellet från ultracentrifugeringen har testat för virusdetektion med hjälp av PCR, qPCR samt PBE. TOC och absorbansmätningar har också genomförts på ingående och utgående vatten från ultrafiltermembranet. Slutligen utfördes ett bänkskaleexperiment för att undersöka hur väl filtret reducerade MS2 fager i utgående vatten. Den inledande testningen visade att plantviruset PMMoV och Pseudomonas fager kan vara bra kandidater att använda sig av för att mäta virusreduktionen över ultrafiltermembranet. När elueringen från ultrafiltreringen testades indikerades en minskad DNA koncentrationen över ultrafiltermembranet med hjälp av Qubit-mätningar. Testningen visade även indikation på att PMMoV reduceras över membranet samt att Pseudomonas fager kan finnas i vattnet. TOC och absorbansmätningarna visade en konstant reduktion över membranet. I bänkskaleexperiment borde enlig teori alla fager stoppas av membranet eftersom viruset är större än porstorleken 20 nm, dock visade experimentell testning på att fager även fanns i utgående vatten från filteringen. Resultat av studien indikerar att mikrobiologiska och kemiska kontamineringar tas bort av membranet, dock för att bestämma den exakta virusreduktionen över membranet och ifall alla kontamineringar större än filters porstorlek (20 nm) tas bort kräver vidare testning.  E. coli fager, som i Livsmedelverket nya restriktioner används för att undersöka mikrobiologiska risker i vattenreningsprocesser, har också testats under studien på vattnet utan positiva utslag. Det kan därför vara av intresse att även undersöka andra fager, så som Pseudomonas fager för att kontrollera dem mikrobiologiska riskerna med vattenrening. / The present study was investigating the effectiveness of the ultrafiltration membrane as third biological barrier in Norrvattens drinking water treatment process, using a pilot scale model. This project aims to test the viral reduction capability of the membrane but also to remove other microbiological and chemical contaminants. To find suitable candidates for measuring the reduction capability, literature research has been performed as well as experimental testing of the raw water coming into the treatment plant and the backwash water from the membrane. Bacterial growth analysis using optical density (OD) measurements and plaque forming unit (PFU) has been performed to investigate the presence of bacteriophages. Approximately 9000 L of incoming and outgoing water from the ultrafiltration membrane has been concentrated using an electropositive membrane which then was eluted and ultracentrifuged. The pellet from ultracentrifugation has been tested for viral detection with PCR, qPCR and plating. TOC and absorbance measurement was also performed on the ingoing and outgoing water from the ultrafiltration pilot plant. Finally, a bench-scale experiment was performed using MS2-spiked water to investigate how well the filter reduced MS2 phages in the outgoing water.  The initial testing of the raw and backwash water showed that the plant virus Pepper Mild Mottle Virus (PMMoV) and Pseudomonas phages may be good candidates to use when evaluating the ultrafiltration membrane. When testing the eluate from the ultrafiltration pilot plant a reduction was seen in the starting DNA concentration when comparing the inlet and outlet water to the ultrafiltration pilot plant. The testing gave indications of a reduction of PMMoV and presence of Pseudomonas phages. The bench-scale experiment was hypothesized to stop all viral particles since according to theory the virus should be stopped by the membrane due to its pore size, but experimental testing indicated viruses in the outgoing water from the membrane as well. TOC and absorbance measurements showed a constant reduction over the membrane. The result of the study indicates that microbiological and chemical contaminants are removed by the filter, however, to determine the exact viral reduction potential of the filter and if all contaminant over the size of 20 nm is removed further testing is required.  No indications were seen for Escherichia coli (E. coli) phages in the water throughout the study, which in Livsmedelverket’s (The National Food Agency) new regulations is used for determining the microbiological risks in water treatment processes. It may be of interest to investigate the possibility to also look for other type of phages to determine the microbiological risks, for example Pseudomonas phages which has been seen in this study.

Water quality, and therefore human health, may be significantly affected by the presence of pathogenic enteric microorganisms derived for improper disposal of wastewater to aquatic environments. Detection of waterborne viruses is complex due to the difficulties in concentrating the sample and then in detecting the virus by cell culture or molecular techniques. Methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years. The U.S. Environmental Protection Agency requires the use of 1MDS electropositive filters for concentrating enteric viruses from water; however, these filters are expensive for routine viral monitoring. The NanoCeram® filter, an electropositive cartridge filter, has been proposed as a new alternative for large volumes of water. The objective of the study was: to evaluate the effectiveness of NanoCeram® filters for the concentration of poliovirus-1 from wastewater samples and compare to 1MDS cartridge filters. This study suggested that NanoCeram® filters are a viable alternative to the use of 1MDS filters for viral monitoring in surface waters and wastewaters. L. monocytogenes outbreaks with Latin-style soft cheese have been well-documented; however, more information to characterize the human health risk associated with the consumption of queso fresco (QF) using unpasteurized milk is needed. The objectives of the study were: i) to evaluate the prevalence of Listeria, Escherichia coli, Salmonella and fecal coliforms in QF obtained from markets in the northwestern state of Sinaloa, Mexico, and ii) to address the human health impact associated with the consumption of QF contaminated with L. monocytogenes using quantitative microbial risk assessment (QMRA). The study suggested that QF produced in Culiacan, Sinaloa, Mexico have microbial loads above the maximum values recommended by the Official Mexican Regulations; and QMRA can be used to interpret microbial contamination data for impacts on public health.