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Viruses in raw sewage and their potential to indicate fecal pollution in coastal environments

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Title:
Viruses in raw sewage and their potential to indicate fecal pollution in coastal environments
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Symonds, Erin M
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University of South Florida
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Subjects / Keywords:
Water quality
Fecal-associated pathogenic viruses
Viral diversity
Microbial indicators
Picobirnaviruses
Dissertations, Academic -- Marine Science -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: The presence of pathogenic viruses in coastal environments is an important tool in evaluating water quality and health risks. Millions of viruses are excreted in fecal matter and bacterial indicators do not correlate with the presence of pathogenic viruses. Enteroviruses have been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter could be used to indicate fecal pollution. The purpose of this research is to develop a baseline understanding of the diversity of viruses found in raw sewage and to assess their presence in the marine environment. PCR was used to detect adenoviruses, herpesviruses, hepatitis B viruses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picobirnaviruses, reoviruses, rotaviruses, and sapporoviruses in raw sewage collected from throughout the United States and from five marine environments ranging in their proximity to dense human populations. Adenoviruses, noroviruses, pepper mild mottle viruses, and picobirnaviruses were detected in raw sewage but absent in the marine environment, making these viruses potential indicators of fecal pollution in marine environments. These viruses were also found in many of the final effluent samples. Pepper mild mottle viruses may be useful for source tracking fecal contamination since it was consistently found in human sewage and is not expected in the feces of other animals due to its dietary origin. Furthermore, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments.
Thesis:
Thesis (M.S.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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by Erin M. Symonds.
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Title from PDF of title page.
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Document formatted into pages; contains 64 pages.

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Viruses Found in Raw Sewage and Their Potential to Indicate Fecal Pollution in Coastal Environments by Erin M. Symonds A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor: Mya Breitbart, Ph.D. John H. Paul, Ph.D. Dale W. Griffin, Ph.D. Date of Approval: June 16, 2008 Keywords: Water Quality, Fecal-Associated Pathogen ic Viruses, Viral Diversity, Microbial Indicators, Picobirnaviruses Copyright 2008, Erin M. Symonds

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ACKNOWLEDGEMENTS I am extremely grateful for the guidance and suppo rt that I have received from Dr. Mya Breitbart, my major professor, throughout the c ourse of my degree. She is an outstanding advisor whose creativity, patience, goo d humor, and dedication facilitated the formation and execution of this study. Funding for this work was provided by the U.S. Environmental Protection Agency (Grant # X7-9646550 7-0). I would also like to thank the members of my committee, Dale Griffin and John Paul, for their advice, expertise, and support throughout this research as well as in the revision of my thesis. Special thanks to members of the Breitbart Lab (Cam ille Daniels, Dawn Goldsmith, Terry Ng, Kim Pause, and Karyna Rosario) for their knowledge, advice, and support throughout this project. Their willingness to help ensured successful collection of samples. Without their support in the lab, the comp letion of this project would have been much more difficult. Thanks to Mike Gray (USGS) for his expertise in phylogenetics. I would like to recognize several parties for their assistance in collecting samples for this project. Thanks to Kim Ritchie and Eric Ba rtels of Mote Marine Laboratory as well as the crew of the R/V Bellows for their suppo rt in collecting samples. Thanks to Dale Griffin, Valerie Harwood and her lab, the Rosa rio Cora family, Gabe Vargo, and the Suncoast Seabird Sanctuary for supplying samples. F inally, I would like to express gratitude to all of the wastewater treatment facili ties for supplying wastewater samples. The completion of my thesis would not have happened without the support of my family and friends. Thanks to all of you who listen ed over the last couple of year.

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT v INTRODUCTION 1 PROJECT OBJECTIVES 8 MATERIALS AND METHODS 9 Concentration of Viruses in Wastewater Samples 9 Wastewater Viral Concentration Efficiency 10 Identification of Targeted Viruses 11 Characterizing the Diversity of Picobirnaviruses in Raw Sewage 18 Determination of Assay Sensitivity 20 Isolation of Viruses from Large-scale Seawater Samp les 20 Seawater Viral Isolation Methods Comparison 25 Determination of Viral Stability in Seawater 2 7 RESULTS 29 Viral Detection in Raw Sewage 29 Viral Detection in Final Effluent 30 PCR Assay Sensitivity 30 Wastewater Viral Concentration Efficiency 31 Diversity of Picobirnaviruses in Raw Sewage 33 Viral Isolation and Detection in Large-Scale Seawat er Samples 37 Seawater Viral Isolation Methods Comparison 39 Stability of Pepper Mild Mottle Virus and Picobirna virus in Seawater 41 DISCUSSION 43 Viruses Detected in Raw Sewage 43

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ii Viruses Detected in Final Effluent 44 Wastewater Viral Concentration Efficiency 46 Diversity of Picobirnaviruses in Raw Sewage 46 Viral Isolation and Detection in Large-Scale Seawat er Samples 48 Seawater Viral Isolation Methods Comparison 48 Stability of Pepper Mild Mottle Virus and Picobirna virus in Seawater 49 Limitations 50 Potential of PMMoV and Picobirnaviruses as Water Qu ality Indicators 51 CONCLUSIONS 54 REFERENCES 55

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iii LIST OF TABLES Table 1 Viruses under investigation in raw sewage, final effluent, and the marine environment. 5 Table 2 Date and location of raw sewage and final effluent sampling from municipal wastewater treatment facilities. 10 Table 3 Primer nucleotide sequences used to PCR amp lify viruses from 10 viral groups. 12 Table 4 Characteristics of viral families found in raw sewage (35). 24 Table 5 Primer nucleotide sequences used for PCR am plification of poliovirus (38). 27 Table 6 Summary table of the targeted viruses detec ted in 12 raw sewage samples collected from throughout the coastal Unit ed States (two samples came from Florida). 29 Table 7 Summary table of the targeted viruses dete cted in 12 final effluent samples collected from 11 different coasta l states. 30 Table 8 The sensitivities of each PCR assay employ ed in this study, which is identical to previous studies. 31 Table 9 The top BLASTN hits of cloned pi cobirnavirus PCR products from U.S. raw sewage samples. 34 Table 10 The concentration of viruses (viral-like particles/milliliter) at each step of the concentration and purification pro cess for each seawater sample. 38 Table 11 Direct counts of virus-like parti cles (VLP) per milliliter of seawater for each step in the TFF Method’s concent ration and purification protocol with samples from the three spiking experiments. 39 Table 12 Summary of wastewater treatment processes at each of the wastewater treatment facilities identified by loca tion. 45

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iv LIST OF FIGURES Figure 1 Sites of large-scale seawater sampling in Florida. 22 Figure 2 Agarose gel electrophoresis (2%) of adeno virus PCR products from the raw sewage spiking experiment. 32 Figure 3 Agarose gel electrophoresis (2%) of adenovirus PCR products from the final effluent spiking experiment. 32 Figure 4 A condensed, neighbor joining (Jukes-Cant or model) phylogenetic tree of a ~200 bp segment of the picobi rnavirus RNA-dependent RNA polymerase gene from 207 U.S. raw sewage sequences. 35 Figure 5 Rarefaction analyses of the picobirnaviru s sequences from U.S. raw sewage. 37 Figure 6 Agarose gel electrophoresis (2%) showing the effectiveness of the TFF Method (lanes 2-8) and the Public Health Me thod (lanes 9-15) from the first spiking experiment using polio viruses. 40 Figure 7 Agarose gel electrophoresis (2%) showing t he effectiveness of the TFF Method (lanes 2-8) and the Public Health Me thod (lanes 9-15) from the second spiking experiment using aden oviruses. 40 Figure 8 Agarose gel electrophoresis (2%) showing the effectiveness of the TFF Method (lanes 2-8) and the Public Health Me thod (lanes 9-15) from the third spiking experiment with an ove rnight incubation using adenoviruses. 41 Figure 9 Agarose gel electrophoresis (2%) depictin g picobirnavirus PCR products (~200 bp) from two experiments assessing th e stability of picobirnaviruses from raw sewage in seawater. 42 Figure 10 Agarose gel electrophoresis (2%) of PMMo V PCR products from two experiments assessing the stability of PMM oV from raw sewage in seawater. 42

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v VIRUSES FOUND IN RAW SEWAGE AND THEIR POTENTIAL TO INDICATE FECAL POLLUTION IN COASTAL ENVIRONMENTS Erin M. Symonds ABSTRACT The presence of pathogenic viruses in coastal envir onments is an important tool in evaluating water quality and health risks. Millions of viruses are excreted in fecal matter and bacterial indicators do not correlate with the presence of pathogenic viruses. Enteroviruses have been used to identify fecal poll ution in the environment; however, other viruses shed in fecal matter could be used to indicate fecal pollution. The purpose of this research is to develop a baseline understan ding of the diversity of viruses found in raw sewage and to assess their presence in the mari ne environment. PCR was used to detect adenoviruses, herpesviruses, hepatitis B vir uses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picob irnaviruses, reoviruses, rotaviruses, and sapporoviruses in raw sewage collected from thr oughout the United States and from five marine environments ranging in their proximity to dense human populations. Adenoviruses, noroviruses, pepper mild mottle virus es, and picobirnaviruses were detected in raw sewage but absent in the marine env ironment, making these viruses potential indicators of fecal pollution in marine e nvironments. These viruses were also found in many of the final effluent samples. Pepper mild mottle viruses may be useful for source tracking fecal contamination since it was co nsistently found in human sewage and is not expected in the feces of other animals due t o its dietary origin. Furthermore, this

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vi research uncovered previously unknown sequence dive rsity in human picobirnaviruses. This baseline understanding of viruses in raw sewag e and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments.

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1 INTRODUCTION Nearly 40% of Americans live in coastal environment s and they produce approximately 1.0x1010 gallons of wastewater per day. The majority of tre ated wastewater, as well as untreated sewage, drains int o the marine environment (2). Millions of viruses and bacteria are excreted in hu man fecal matter (6, 17, 98) and current methods of sewage treatment do not always effective ly remove these organisms (90-92, 94). In addition to treated wastewater discharge, o ther point sources of fecal pollution include the following: combined sewer overflows, mu nicipal storm sewer systems, concentrated animal feeding operations, and meat-, fish-, and shellfish-processing facilities (33). While point sources of fecal pollution can have sig nificant impacts to the health of coastal environments, non-point sources of fecal pollution can pose an even bigger threat due to difficulties in identifying and mitig ating the source of pollution. In 2002, nearly half of the pollution sources responsible fo r beach closures in the United States were from unknown and non-point sources (33). Run-o ff, farm animals, wildlife, septic systems, swimmers, and faulty sanitary sewer lines are all examples of non-point sources of fecal contamination (31, 33). Non-point source f ecal pollution is a major problem in areas such as the Florida Keys, where the community relies on the use of ~25,000 septic tanks (some of which are outdated), ~5-10,000 illega l cesspits, and ~1,207 aerobic treatment facilities (34, 70). Additionally, the pe rmeable nature of Florida’s soils and limestone bedrock combined with the use of inapprop riate wastewater disposal systems

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2 increases the risk of surface and ground water cont amination with fecal-associated microorganisms (65, 78). As a means to understand the fate of wastewater in the Florida Keys, several viral tracer studies were conducted in the late 1990s. Vi ral tracers from a domestic septic tank were shown to move into surrounding canals and coas tal waters in as little as 3.5 hours and 23 hours respectively (76, 77). In the case of injection wells, viral tracers were found in groundwater and marine waters within 8 hours and 10 – 53 hours respectively after seeding (78). An additional study investigating the movement of viral tracers from injection wells and septic tanks revealed that it w as possible for the viral tracers to move from the on-site disposal systems into neighboring canals and out to coastal marine surface waters at a rate of 1.7 to 57.5 meters per hour for septic systems and 66.8 to 141 meters per hour for injection wells (76). These stu dies convincingly demonstrated that septic systems and injection wells can be non-point sources of wastewater pollution in the Florida Keys. Due to the high pathogen loads and nutrient concent rations present in raw sewage, fecal pollution poses a threat to environmental and human health. Marine water containing excess nutrients from wastewater polluti on, can cause hypoxic events, eutrophication, a decline in seagrass and coral ree f biomass, as well as a reduction in finfish and shellfish populations (52). Many epidem iological investigations have shown an increased risk of disease in people who have swa m in polluted waters identified by high concentrations of fecal bacteria (2, 26, 37, 5 1, 74). Furthermore, ingestion of seafood exposed to fecal pollution has also been sh own to increase one’s risk of disease (29, 87). The coastal environment is an important e conomic resource with tourism,

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3 recreational activities, and commercial fishing gen erating significant revenues. As a result of the known health risks associated with sw imming in polluted waters and consuming seafood contaminated by wastewater, the U S government passed legislation to address this problem. The Beaches Environmental Assessment and Coastal He alth Act, enacted in 2000, holds each state responsible for monitoring and rep orting the presence of fecal pollution in all recreational waters via indicator organisms (Public Law 106-284). An ideal indicator organism of fecal pollution, as defined b y the Environmental Protection Agency (EPA), should posses several qualities (33). First, detection of the organism should be simple and affordable. Second, the organism should not be present in the environment naturally in the absence of pollution. Third, the organism should be present in polluted waters in concentrations correlated with the amount of fecal pollution. Finally, the indicator organism should have a decay rate compara ble to that of the pathogen of concern. Currently, the US EPA mandates the use of bacterial indicators, such as fecal coliforms and enterococci, to assess water quality. Although monitoring of these bacteria is simple and inexpensive, it has been shown that f ecal-associated bacteria are not ideal indicators of fecal pollution for several reasons. One unsuitable characteristic of bacterial indicators is their ability to live in sediments (1 9, 36, 62). If bacterial indicators are able to survive in sediments, then their re-suspension i nto the water column can mask correlations between their concentrations and the e xtent of current fecal pollution (i.e., false positives). Furthermore, it is also likely th at the die-off rate of bacterial indicators does not match that of fecal associated pathogens, like viruses.

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4 Another unfavorable characteristic of current bacte rial indicators is their inability to predict or correlate with the presence of pathogeni c viruses associated with wastewater (45, 46, 96). Human pathogenic viruses associated w ith feces are generally more robust than enteric bacteria and are not as easily elimina ted by current methods of wastewater treatment (50, 96). A California study showed that adenoviruses are more resilient to tertiary wastewater treatment and ultraviolet-disin fection than are bacterial indicators of fecal pollution (90). Additionally, adenoviruses an d noroviruses have been detected in treated wastewater discharge in Europe (91, 94). Si nce concentrations of fecal coliforms and enterococci inadequately detect fecal pollution and therefore inaccurately depict the risks to human health, many have proposed the use o f an alternative viral indicator of wastewater contamination (39, 46, 69). Over the last ten years researchers have investigat ed the distribution and abundance of enteric viruses in Florida as a means of underst anding fecal pollution and public health risks. Enteric viruses describe a group of eukaryot ic viruses (including viruses belonging to the Adenoviridae Caliciviridae Picornaviridae and Reoviridae families) transmitted via the fecal-oral route. These viruses have been f ound throughout the marine environment (i.e. in seawater, sponge, and coral mu cus samples) adjacent to the Florida Keys (28, 39, 44, 45, 64, 96). They have also been detected in seawater and freshwater samples from Sarasota, Florida (65). Despite the id entification of pathogenic viruses within the coastal environment of Florida, the pres ence of other, potentially hazardous viruses transmitted via the fecal-oral route still remains unknown because they have yet to be studied. The occurrence of emerging diseases in humans and marine organisms appears to be rising and some of these diseases may be caused by human-associated fecal

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5 flora (48, 71, 73, 85). While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the deve lopment of an accurate viral indicator of sewage contamination is needed for enhanced wate r quality monitoring. To develop a comprehensive water quality indicator protocol, it is first necessary to establish a broad, baseline understanding of the ma ny pathogenic, eukaryotic viruses in raw sewage. Currently no broad baseline data exists on the presence of eukaryotic viruses in raw sewage in the United States. Several studies have identified adenoviruses, noroviruses, reoviruses, rotaviruses, and other ent eroviruses (e.g. polioviruses, coxsackie viruses, echo-viruses) in untreated raw sewage in A ustralia, Europe and South Africa (30, 54, 66, 91, 92, 94). Since no comprehensive study h as been executed to identify the prevalence of many different eukaryotic viruses in raw sewage from the United States, the first objective of this research was to identif y the presence of ten viral groups known to be transmitted via the fecal-oral route in raw s ewage samples collected throughout the country. TABLE 1. Viruses under investigation in raw sewage, final ef fluent, and the marine environment Viral FamilyVirus Type Adenoviridae Human Adenoviruses, A F dsDNA Caliciviridae Noroviruses & Sapporoviruses ssRNA (+ strand) Hepadnaviridae Hepatitis B Viruses reverse transcribing Herpesviridae Human Herpesviruses dsDNA Papillomaviridae Human Papillomaviruses dsDNA Paramyxoviridae Morbilliviruses ssRNA (strand) Mammal Reoviruses Rotaviruses, Group A Unassigned Pepper Mild Mottle Viruses ssRNA Unclassified Human Picobirnaviruses dsRNA dsRNA Reoviridae The presence of the viral families listed in Table 1 was analyzed in raw sewage samples collected from around the United States. Al l of the viral families under

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6 investigation are known to infect humans except pep per mild mottle viruses, which infects plants The majority of these viral families, excluding st udies of Adenoviridae, Caliciviridae, and Reoviridae have not been studied in sewage despite their fec al-oral transmission. Picobirnaviruses and pepper mild mott le viruses (PMMoV) have been detected in individual fecal samples (13, 83, 95, 9 8); however, their presence has never been analyzed in collective waste nor have they bee n proposed as markers of fecal pollution. Although PMMoV are plant pathogens, these viruses w ere most abundant in a metagenomic survey of RNA viruses in human feces (9 8). PMMoV is a rod-shaped single-stranded RNA virus that infects ornamental p eppers. Through human consumption of processed pepper products (ie. hot sauce), the P MMoV is excreted in human feces on the order of nearly one billion viruses per gram dr y weight of feces. Interestingly, these viruses are stable enough to survive passage throug h the gut and remain infectious to plants (98). The prevalence of PMMoV in human feces regardless of its importance for human health, makes it a potential indicator of fec al pollution. After determining which pathogenic viruses were fou nd in raw sewage, their presence in final effluent and the marine environme nt was determined. Since accurate indicators would not be found in the marine environ ment in the absence of pollution, the natural presence of these potential viral markers i n seawater samples ranging in their exposure to human populations was determined. The results of this research will have important applications to water quality monitoring programs mandated by the US EPA throughout the country. Characterizing the presence and diversity of a broad range of viral families suspected to be found in feces in ra w sewage from around the United States

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7 will narrow the list of possible viral indicators o f fecal pollution. The conclusions of this research will identify viruses that can potentially be used to indicate wastewater pollution in coastal environments. This discovery phase is a critical first step before rapid and sensitive assays can be developed for these indicat or viruses.

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8 PROJECT OBJECTIVES Current bacterial indicators of fecal pollution ina dequately assess the extent of fecal pollution in recreational waters; accordingly, many have proposed the use of viruses to indicate fecal pollution in the environment. Before a viral indicator of fecal pollution can be identified, many factors must be investigated to include the prevalence of different viral types in sewage, their correlation to the pre sence of other pathogens, and their behavior in coastal environments. This study aims t o begin to assess the use of viruses as indicators of fecal contamination. The objectives o f this study are: To determine the presence of ten types of viruses t hat transmit via the fecal-oral route in raw sewage collected from 11 states in the U.S. To characterize the diversity of picobirnaviruses i n raw sewage collected from 11 states in the U.S. To determine the presence of the viruses that were detected in raw sewage in the following sample types: o Final effluent collected from 11 states. o Seawater samples collected from locations ranging i n their proximity to dense human populations. To investigate the stability of picobirnaviruses an d PMMoV in seawater.

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9 MATERIALS AND METHODS Concentration of Viruses in Wastewater Samples A total of 12 raw sewage and 12 final effluent samp les were collected from wastewater treatment facilities in the United State s as listed in Table 2. For each sample, 10 ml was filtered through a 0.45 m m polyether sulfone membrane filter cartridge (Sterivex; Millipore, Billerica, MA, USA) to separa te large bacteria and other particles from viruses. The filtrate was then concentrated us ing a centrifugal concentration device (Centriplus YM-50; Millipore, Billerica, MA, USA) t o a volume less than one milliliter. Samples were further concentrated to less than 200 m l using another centrifugal concentration device (Microcon Ultracel YM-30; Mill ipore, Billerica, MA, USA). Nucleic acid was extracted from 200 m l of filtered, concentrated sample using the MinElute Virus Spin Kit (QIAGEN , Valencia, CA, US A). Immediately following nucleic acid extraction, cDNA was synthesized from the extracted RNA using First Strand Synthesis Superscript III Reverse Transcript ion Kit (Invitrogen Carlsbad, CA, USA) with random hexamers. Whole genome amplication using GenomiPhi V2 (GE Healthcare, Piscataway, NJ, USA) was utilized for e very raw sewage and final effluent sample as a means of increasing the concentration o f total DNA and cDNA prior to virus identification.

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10 TABLE 2. Date and location of raw sewage and final effluent sampling from municipal wastewater treatment facilities. DateLocationAbbreviation 11/5/2007AlabamaAL 10/18/2007CaliforniaCA11/14/2007Connecticut CT11/30/2007FloridaFL 12/8/2006Florida KeysFL K 11/12/2007LouisianaLA 11/5/2007MaineME 11/24/2007MarylandMD11/13/2007New JerseyNJ11/13/2007North CarolinaNC11/13/2007OregonOR 11/8/2007WashingtonWA Wastewater Viral Concentration Efficiency Known quantities of adenoviruses were added to a ra w sewage and a final effluent sample in order to determine the efficiency of the methods employed to concentrate viruses from raw sewage and final effluent. Twenty milliliters of raw sewage and final effluent were autoclaved for 15 minutes at 121 C as a means of eliminating the presence of adenovirus particles while minimizing the altera tion of particulates in the sample. Ten milliliters of raw sewage and final effluent were s piked to a final concentration of 8.48x105 Adenovirus-20 particles per milliliter. The remain ing ten milliliters of autoclaved raw sewage and final effluent were not s piked; rather, they served as a negative control throughout the viral concentration nucleic acid isolation, and viral detection processes. Viruses from all four samples were concentrated using the methods previously described for raw sewage and final efflu ent. Adenoviruses were assayed for in 1:10 serial dilutions of extracted DNA from spik ed and control raw sewage and final effluent using nested PCR using previously publishe d primers in Table 3 and conditions (3).

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11 Identification of Targeted Viruses To identify the presence and diversity of the targe ted viruses in raw sewage, PCR or RT-PCR for each of the targeted viral groups was executed using previously published primers and conditions described in the following s ubsections for each viral family. All PCR products were visualized using agarose gel (2%) electrophoresis stained with ethidium bromide. If no positive PCR products were amplified, the presence of inhibitors was analyzed by assaying serially diluted (to 10-4) sample DNA spiked with positive control. Positive PCR products with one distinct b and were purified using the UltraClean PCR Clean-up Kit (MO BIO Laboratories, Inc., Carlsb ad, CA, USA). If more than one band was present, the UltraClean Gelspin Kit (MO BI O Laboratories, Inc., Carlsbad, CA, USA) was utilized to gel-purify the positive PCR pr oduct of the correct size. All purified positive PCR products were sequenced with their res pective primers. Sequences were trimmed using Sequencher (Gene Codes Corporation, A nn Arbor, MI, USA) and the identity of the positive PCR products were confirme d by comparing the sequence against the GenBank non-redundant database using BLASTN (4, 5). If BLASTN searches did not result in sequence identities to the targeted virus then TBLASTX searches were executed in an attempt to detect weaker sequence similaritie s to known sequences in GenBank.

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12 TABLE 3. Primer nucleotide sequences used to PCR am plify viruses from 10 viral groups. Note that mixed bases represent the following nucleotides: H (A, C, or T), M (A or C), R (G or A), S (G or C), V (G, C, or A), W (A or T), and Y (T or C). Note that I h as base-paring bias to C, A, G, and then T. Target VirusesPrimer NamePrimer Sequence (5'-3')Re ference AV-A1 (+)GCC GCA GTG GTC TTA CAT GCA CAT C AV-A2 (-)CAG CAC GCC GCG GAT GTC AAA GT AV-B1 (+)GCC ACC GAG ACG TAC TTC AGC CTG AV-B2 (-)TTG TAC GAG TAC GCG GTA TCC TCG CGG TC P290 (+)GAT TAC TCC AAG TGG GAC TCC AC P289 (-)TGA CAA TGT AAT CAT CAC CAT A HBS-1 (+)ATC AGG ATT CCT AGG ACC C HBS-R1 (-)AGG ACA AAC GGG CAA CAA C HBS-11 (+)GCG GGG TTT TTC TTG TTG AC HBS-R11 (-)GAA CCA ACA AGA AGA TGA GGC FP1 (+)GAY TTY GCI AGY YTI TAY CCFP2 (+)TCC TGG ACA AGC AGC ARI YSG CIM TIA A FR1 (-)GTC TTG CTC ACC AGI TCI ACI CCY TT FP3 (+)TGT AAC TCG GTG TAY GGI TTY ACI GGI GT RP4 (-)CAC AGA GTC CGT RTC ICC RTA IAT upstreamATG TTT ATG ATC ACA GCG GT downstreamATT GGG TTG CAC CAC TTG TC FAP59TAA CWG TIG GIC AYC CWT ATTFAP64CCW ATA TCW VHC ATI TCI CCA TC PicoB25 (+)TGG TGT GGA TGT TTC PicoB43 (-)ART GYT GGT CGA ACT T PMMV-F AAC CTT TCC AGC ACT GCG PMMV-R GCG CCT ATG TCG TCA AGA CT L1.rv5 (+)GCA TCC ATT GTA AAT GAC GAG TCT G L1.rv6 (-)CTT GAG ATT AGC TCT AGC ATC TTC TG L1.rv7 (+)GCT AGG CCG ATA TCG GGA ATG CAG L1.rv8 (-)GTC TCA CTA TTC ACC TTA CCA GCA G RV1 (-)GTC ACA TCA TAC AAT TCT AAT CTA AG RV2 (+)CTT TAA AAG AGA GAA TTT CCG TCT GRV3 (+)TGT ATG GTA TTG AAT ATA CCA C RV4 (-)ACT GAT CCT GTT GGC CAW CC 57 Human Adenoviruses, A F 3 Noroviruses & Sapporoviruses Hepatitis B Viruses 608343 Rotaviruses, Group A 61 Human Picobirnaviruses 40 Papillomaviruses Mammal Reoviruses PMMoV 98 Herpesviruses 1211 Morbilliviruses Adenoviruses were identified using a nested primer set (Table 3) that can detect 47 adenovirus serotypes infecting humans (3) and ha s been previously used to detect adenoviruses in the environment (39). These primers target the adenovirus hexon gene and have a sensitivity of 100 targets. Adenovirus-2 0 DNA, extracted from viral particles (number VR-1097 ATCC, Manassas, VA, USA), was used as a positive control. For the first round of PCR, a total 50 m l reaction mixture contained 2 m l of sample DNA, 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3 50.0 mM KCl, 1.1 mM

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13 MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), an additional 0.4mM MgCl2, 0.25 mM each dNTP, 1 m M AV-A1 primer, 1 m M AV-A2 primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, M O, USA). The second PCR reaction was identical to the first, except the tem plate was 5 m l of the first round product and primers were replaced with 1 m M AV-B1 and 1 m M AV-B2. The amplicon of the first reaction was 300 bp and 143 bp for the second reaction. Both reactions were incubated for 4 min at 94oC, followed by 40 cycles of [92 oC for 30 sec, 60 oC for 30 sec, and 72 oC for 1 min], followed by incubation at 72 oC for 5 min. One primer pair capable of amplifying the RNA polym erase of noroviruses and sapporoviruses was used in this study (Table 3) (57 ). Human norovirus genotype II cDNA, extracted and reverse transcribed from Freon extracted norovirus genotype II from stool, was used as a positive control. This pr imer pair produced an amplicon of 319 bp for noroviruses and 331 bp for sapporoviruses. T he 50 m l PCR reaction mixture was composed of 2 m l of target cDNA, 1X REDTaq PCR Reaction Buffer (10 .0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an additional 0.4 mM MgCl2, 100 m g/ml BSA, 0.25 mM each dNTP, 1 m M of each primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldric h St. Louis, MO, USA). The reaction mixture was incubated for 3 min at 94oC, followed by 40 cycles of [94 oC for 30 s, 49 oC for 80 s, and 72 oC for 1 min], and a final extension of 72 oC for 10 min. Nested PCR targeting the S gene was used to analyze the presence of hepatitis B virus (Table 3) (60). The initial amplicon had a le ngth of 310 bp and the final amplicon was 241 bp. Human hepatitis B DNA (number 45020 ATC C, Manassas, VA) was used as a positive control. Both rounds of PCR had 50 m l reaction volumes. The initial round

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14 of PCR contained 2 m l of target DNA, 1X REDTaq PCR Reaction Buffer (10. 0 mM TrisHCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an additional 0.4 mM MgCl2, 0.25 mM each dNTP, 1 m M HBS-1 primer, 1 m M HBS-R1 primer, and 1 U REDTaq DNA Polymerase (Sig ma-Aldrich St. Louis, MO, USA). The final round of PCR was the same as th e initial round, except the template was 2 m l of initial PCR product and the initial primers we re replaced with 1 m M HBS-11 and 1 m M HBS-R11. The PCR conditions, used for both rounds of PCR, were as follows: 5 min at 95oC, followed by 30 cycles of [95 oC for 30 s, 55 oC for 40 s, and 72 oC for 40 s]. A degenerate, nested primer set was used to detect a range of herpesviruses, including eight human strains (Table 3) (12). This nested reaction targeted the DNA polymerase and yielded an initial amplicon of 800 b p and a final amplicon between 215 bp and 235 bp. Human herpesvirus simplex 1 DNA was used as a positive control. The final volume of both PCR reactions was 50 m l. The first PCR reaction contained 1 m l of extracted DNA, 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), 0.25 mM each dNTP, 1 m M FP1 primer, 1 m M FP2 primer, 1 m M FR1 primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA) The second PCR reaction was identical to the first, except the template was 5 m l of the first round product and primers were replaced with 1 m M FP3 and 1 m M RP2. Both reactions were incubated for 2 min at 94oC, followed by 55 cycles of [94 oC for 20 s, 46 oC for 30 s, and 72 oC for 30 s], followed by incubation at 72 oC for 10 min.

PAGE 23

15 An assay able to detect 87% of human papillomavirus es using a pair of degenerate primers was utilized to screen samples ( Table 3) (40). These primers targeted the L1 gene and amplified a 478 bp product with a s ensitivity ranging from 1-10 copies. Human papillomavirus 11 DNA (number 45151 ATCC, Ma nassas, VA) was used as a positive control. The total reaction mixture, conta ining 2 m l of target DNA, was 50 m l and had final concentrations of 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an additional 1.4 mM MgCl2, 0.25 mM each dNTP, 1 m M of each primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, M O, USA). The PCR reaction was incubated for 10 min at 94oC, followed by 45 cycles of [94 oC for 1 min, 50 oC for 1 min, and 72 oC for 1 min], followed by incubation at 72 oC for 5 min. One primer set, amplifying a region of the phosphop rotein gene, was used to detect the presence of morbilliviruses and produced a 429 bp amplicon (Table 3) (11). Measles virus cDNA, extracted and reverse transcrib ed from Measles virus Edmonston strain particles (number VR-24 ATCC, Manassas, VA) was used as a positive control. Two m l of cDNA was added to a 48 m l PCR reaction mixture of 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM K Cl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an ad ditional 0.4 mM MgCl2, 1 m M of each primer, 0.25 mM each dNTP, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA). The PCR reaction underwent 35 cycles of [94 oC for 1.5 min, 25 oC for 2 min, and 72 oC for 2 min], followed by incubation at 72 oC for 7 min. The presence of reoviruses and rotaviruses, both me mbers of the Reoviridae family, were analyzed in this study. The details of the primer sets used in PCR

PAGE 24

16 amplification of each of these groups is found in T able 3. The reovirus PCR assay, consisting of a nested reaction targeting the L1 ge ne, yielded a 344 bp amplicon (61). This assay was able to detect one copy of 44 reovir us isolates from human, bovine, and murine strains. Human reovirus cDNA, extracted and reverse transcribed from human reovirus type 1 strain Lang (number VR-230 ATCC, M anassas, VA), was used as a positive control. For the primary PCR reaction, 2 m l of target cDNA was added to the 50 m l reaction mixture with final concentrations of the f ollowing components: 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an ad ditional 1.4 mM MgCl2, 0.25 mM each dNTP, 1 m M L1.rv5 primer, 1 m M L1.rv6 primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA). Th e secondary PCR reaction was carried out by adding 1 m l of product from the primary PCR reaction to a 25 m l reaction mixture containing: 1X REDTaq PCR Reaction Buffer ( 10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), an additional 0.9 mM MgCl2, 0.25 mM each dNTP, 1 m M L1.rv7 primer, 1 m M L1.rv8 primer, and 1 U REDTaq DNA Polymerase (Sigma-Aldric h St. Louis, MO, USA). Both primary and secondary reactions were initially incubated for 1 min at 94oC, followed by 35 cycles of [94 oC for 20 s, 50 oC for 30 s, and 72 oC for 30 s], followed by incubation at 72 oC for 10 min. Rotaviruses were detected using the primer set desc ribed in Table 3. This nested PCR assay targeted the VP7 gene of group A rotaviru ses and produced an initial 1059 bp amplicon and a final amplicon of 346 bp (43). It ha s been previously used to detect

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17 rotaviruses in river water. Simian rotavirus SA-11 cDNA, extracted and reverse transcribed from viral particles extracted from tis sue, was used as a positive control. The initial PCR reaction had 2 m l of target cDNA and a total volume of 50 m l with the following concentrations: 1X REDTaq PCR Reaction Bu ffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), an additional 0.4 mM MgCl2, 1 m M R1 primer, 1 m M R2 primer, 0.25 mM each dNTP, 2 m g/ml BSA, and 1 U REDTaq DNA Polymerase (Sigma-Aldr ich St. Louis, MO, USA). The initial PCR reaction was incubated for 1 min at 94oC, followed by 25 cycles of [94 oC for 30 s, 55 oC for 30 s, and 72 oC for 1 min], followed by incubation at 72 oC for 3 min. Then 2 m l of product from the initial reaction was added to the final PCR reaction. The final 50 m l PCR reaction was composed of the following: 1X RE DTaq PCR Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM K Cl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an ad ditional 2.4 mM MgCl2, 1 m M R3 primer, 1 m M R4 primer, 0.25 mM each dNTP, 2 m g/ml BSA, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA). Th e final PCR reaction was incubated for 1 min at 94oC, followed by 40 cycles of [94 oC for 30 s, 55 oC for 30 s, and 72 oC for 30 s], followed by incubation at 72 oC for 3 min. PMMoV were detected using primers that amplify a 20 1 bp region of the helicase gene (98). Two microliters of cDNA was added to a 5 0 m l final volume PCR reaction mixture of 1X REDTaq PCR Reaction Buffer (10.0 mM T ris-HCl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), 1 m M of each primer, 0.25 mM each dNTP, and 1 U REDTaq DNA Polym erase (Sigma-Aldrich St. Louis, MO, USA). The PCR reaction underwent the fol lowing: incubation at 95 oC for 5

PAGE 26

18 min, followed by 40 cycles of [94 oC for 1 min, 50 oC for 45 sec, and 72 oC for 1 min], and an incubation at 72 oC for 5 min. Raw sewage was used as a positive cont rol. A portion of the genomic segment 2 of genotype I hu man picobirnaviruses ranging from 123 to 246 bp was amplified using the primer set described in Table 3 (83). Genotype I human picobirnaviruses were targeted for this study since genotype II picobirnaviruses are rarely found in human populati ons (13). These primers have been previously used to detect genotype I picobirnavirus es in feces (13). Raw sewage was used as a positive control. The PCR reaction had a final volume of 50 m l and had 2 m l of target cDNA, 1X REDTaq PCR Reaction Buffer (10.0 mM Tris-H Cl pH 8.3, 50.0 mM KCl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA ), an additional 0.9 mM MgCl2, 1 m M of each primer, 0.25 mM each dNTP, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA). Th e PCR reaction was incubated for 2 min at 94 oC, followed by 40 cycles of [94 oC for 1 min, 49 oC for 2 min, and 72 oC for 3 min], followed by incubation at 72 oC for 7 min. Characterizing the Diversity of Picobirnaviruses in Raw Sewage In order to gain a deeper understanding of the dive rsity of picobirnaviruses in raw sewage, all positive PCR products were cloned into pCR4-TOPO (Invitrogen Carlsbad, CA, USA) and transformants were screened for insert s by PCR with the M13F and M13R primers. UltraClean PCR Clean-up Kit (MO BIO Labora tories, Inc., Carlsbad, CA, USA) was used to purify PCR products of the proper size. Positive PCR products were sequenced using M13F. All sequences were trimmed us ing Sequencher (Gene Codes Ann Arbor, MI, USA) and the identity of the positiv e PCR products were confirmed by

PAGE 27

19 comparing the sequence against the GenBank non-redu ndant database using BLASTN and TBLASTX (4, 5). All sequences with insignificant identities (E-valu es > 0.001 and/or identities less than 50 bp in length) to known picobirnaviruses wer e removed. All of the sequences from raw sewage were de-replicated to 99% sequence ident ity with gaps using the online program FastGroup II (97). The de-replication proce ss and the representative sequences for each group were visually confirmed. In addition to de-replicating sequences, FastGroup II also executed Chao1 (24, 25) and raref action analyses (49, 53), measurements of diversity (97). The phylogenetic re lationship of the picobirnaviruses detected in raw sewage to known picobirnaviruses wa s identified through aligning dereplicated raw sewage sequences with known sequence s from GenBank. These known sequences from GenBank had the best BLASTN identity to at least one of the sequences from raw sewage. All sequences were trimmed to ~200 bp. Since reference sequence S and T were less than 200 bp, they were not included in the alignment. Alignments were executed using ClustalX v.1.8 (89). The resulting a lignment was verified visually for accuracy and then imported into MEGA v. 4 for phylo genetic analyses (88). The phylogenetic analysis of cloned picobirnavirus PCR products from raw sewage was executed similar to previous picobirnavi rus studies (13, 83). Prior to creating a phylogenetic tree, the average pairwise Jukes-Can tor distance was calculated in order to determine the appropriateness of creating a neighbo r joining tree. Since the average pair wise Jukes-Cantor distance was less than 1.0 (0.356 ), a neighbor joining phylogenetic tree was calculated using a Jukes-Cantor model alon g with bootstrap analyses for 2000

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20 replicates. Branches with insignificant bootstrap v alues ( 50) were condensed to create the final phylogenetic tree. Determination of Assay Sensitivity The amount of nucleic acid in the cleaned up PCR po sitive control after completion of PCR amplification was quantified using a NanoDro p ND-1000 (NanoDrop Technologies , Wilmington, DE, USA). If the PCR re action was nested, then the positive control from the first round of PCR amplif ication was quantified. The number of targets was back-calculated using the measured conc entration of DNA and knowledge of the amplicon size using the following equation, whe re Y is the concentration of DNA measured by the Nanodrop and Z is the length of the amplicon: l targets # bp target 1 bp mol 1 bp 10 02.6 g 660 bp mol 1 ng 10 g 1 l 1 ng 239m m= Z x Y The cleaned up PCR positive control was then serial ly diluted to 1 target/ m l. The entire dilution series underwent the appropriate PCR or ne sted PCR previously described for that viral group, with the exception that only 1 m l of target DNA/cDNA was added to the reaction mixture. PCR products were visualized on a 2% agarose gel stained with ethidium bromide and the sensitivity of the assay w as determined. Isolation of Viruses from Large-scale Seawater Samp les A total of four 200 liter samples of seawater were collected from four different sites ranging in their proximity to dense human populatio ns. These environmental samples were ultimately analyzed for any viruses detected i n raw sewage. Seawater was collected from North Shore Beach (N27 46’57.42”, W82 37’26.63”), a frequently polluted Saint

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21 Petersburg City, Florida beach, on the morning of 2 5 September 2007. Saint Petersburg, located along Tampa Bay, is home to nearly 250,000 people (U.S. Census Bureau, 2006 Population Estimates) and serves as the beach close st to dense human populations in this study. Based upon water quality analyses conducted by the Pinellas County Health Department using current bacterial indicators of fe cal pollution, North Shore beach had ‘good’ concentrations of fecal coliform (<200 organ isms/100ml seawater) and enterococcus (<36 organisms/100 ml seawater) on the day of sampling. However, North Shore beach had been closed for nearly two weeks pr ior to sampling due to high concentrations of bacterial indicators. Seawater was also collected from three other locati ons. One sample was gathered from a canal with a nearby injection well in Lower Matacumbe Key, Florida (N24 51’29.26”, W80 43’40.27”) on 10 Dec 2006. This site in Lower Matac umbe is located adjacent to an on-site disposal system and surrounded by a moderate human population. In past studies, large amounts of enter ic viruses have been found this Lower Matacumbe Key sample site (45, 96). Another seawate r sample was collected from a near shore reef at Looe Key buoy #10 (about N24 33’03.16”, W81 24’27.51”) on 11 Dec 2006. Due to Looe Key’s proximity to shore, it will serve as the second furthest location from dense human population. Aboard the R/V Bellows the final seawater sample was collected 31 May 2007 offshore of the Dry Tortugas (N24 36.548’, W82 50.464’). This site is furthest from shore, closest to the Gulf St ream, and furthest from dense human populations.

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22 FIGURE 1. Sites of large-scale seawater sampling in Florida. The colored dots represent the four areas sampled: North Shore Beach (blue), Lower Matacumbe canal (red), Looe Key (yellow), and offshore the Dry Tortugas (green). Large-scale seawater samples collected from North S hore Beach, the Florida Keys, and off-shore the Dry Tortugas were concentrated fo r viral isolation using a combination of differential filtration with tangential flow fil ters (TFF), density-dependent centrifugation in cesium chloride (CsCl), and polye thylene glycol (PEG) precipitation (18, 22). All of the size-based filtration and dens ity-dependent isolation steps were chosen based upon the sizes and densities of the vi ral families under investigation (Table 4). The particles larger than the TFF pore size are concentrated into the retentate; conversely, the filtrate will contain particles sma ller than the TFF pore size. First, a 0.2 m m TFF (GE Healthcare, Piscataway, NJ) was used to r emove bacteria, eukaryotes, and other large particles from 200 liters of seawater. The 0.2 m m TFF retentate, containing Saint Petersburg, FL Lower Matacumbe, FL Looe Key, FL Dry Tortugas Decreasing proximity to dense human populations Human Influence

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23 everything > 0.2 m m, was filtered through a 0.22 m m polyether sulfone membrane filter cartridge (Sterivex; Millipore, Billerica, MA) and the filtrate was added to the 0.2 m m TFF filtrate. The bacteria and eukaryotes concentra ted into the 0.2 m m TFF retentate were collected on the 0.22 m m polyether sulfone membrane filter cartridge. Then the resulting 0.2 m m TFF filtrate, containing the viral particles, was concentrated through a 100 kDa TFF (GE Healthcare, Piscataway, NJ) to generate a final volume of <100 ml. It is possible that herpesviruse s and morbilliviruses may have been excluded from the viral concentrate by the initial 0.2 m m filtering. If either of these viruses were detected in raw sewage, then total DNA or RNA was extracted directly from the 0.2 m m retentate collected on the 0.22 m m polyether sulfone membrane filter cartridge (Sterivex; Millipore, Billerica, MA) using the Pow erSoil DNA or RNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). Pur ification of the viral concentrate and removal of potential PCR inhibitors was achieve d through loading the sample on to a cesium chloride (CsCl) density gradient. The gradie nt was ultracentrifuged at 61,120 X g (22,000 rpm in a SW40Ti rotor) at 4 C for 2 hours and the 1.2-1.5 g/ml fraction (i.e. CsCl Viral fraction) was collected to isolate the t arget viruses. The remaining CsCl density gradient (i.e. CsCl Other fraction) was als o collected to measure the effectiveness of the CsCl density gradient via epifluorescent mic roscopy. PEG precipitation was used to further concentrate e ach sample before nucleic acid extraction (86). A final concentration of 10% PEG 8 000 and 1 M NaCl was added to the viral concentrate. After the mixture was incubated overnight at 4 C, it was spun for 30 min at a temperature of 4 C and a speed of 13,000 X g. The supernatant was po ured off and the pellet was re-suspended. Two samples deviat ed slightly from the above

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24 concentration steps. For seawater samples collected from North Shore Beach and the Dry Tortugas, viruses were further concentrated using P EG precipitation before densitydependent centrifugation in cesium-chloride. Hereaf ter this method will be referred to as the “TFF Method”. An additional concentration step using centrifugal concentration filters (Centriplus YM-50; Millipore, Billerica, MA USA) was used for seawater collected from the Dry Tortugas. TABLE 4. Characteristics of viral families found in raw sewage (35). Viral FamilyVirus Size Range (nm)Density CsCl (g/cm3) Adenoviridae Human Adenoviruses, A F 70 901.30 1.37 Caliciviridae Noroviruses & Sapporoviruses 27 401.33 1.41 Hepadnaviridae Hepatitis B Viruses 42 501.25 Herpesviridae Human Herpesviruses 125 200 1.22 1.28 Papillomaviridae Human Papillomaviruses 551.34 1.35 Paramyxoviridae Morbilliviruses > 1501.23 Mammal Reoviruses Rotaviruses, Group A Unassigned Pepper Mild Mottle Viruses 35 411.38 1.40 Unclassified Human Picobirnaviruses 30 401.4 1.36 1.39 Reoviridae 60 80 To ensure purification and concentration of the vir al sample, a portion of the concentrate after each step was stained with SYBR G old nucleic acid stain (Invitrogen Carlsbad, CA, USA) and direct counts were made usin g epifluorescent microscopy (75). QIAamp MinElute Virus Spin Kits (QIAGEN , Valencia CA, USA) were used to isolate DNA and RNA from 200 m l of each of the purified, final viral concentrates In order to detect RNA viruses, cDNA was immediately s ynthesized from viral RNA using random hexamers and the First Strand Synthesis Supe rscript III Reverse Transcription Kit (Invitrogen Carlsbad, CA, USA). Whole genome amplication using GenomiPhi V2 (GE Healthcare, Piscataway, NJ, USA) was utilize d for each seawater sample from

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25 North Shore Beach, the Florida Keys, and off-shore the Dry Tortugas to increase the concentration of total DNA and cDNA prior to virus identification. Seawater Viral Isolation Methods Comparison In order to confirm and compare the effectiveness o f the large-scale seawater viral concentration, purification, and nucleic acid extra ction techniques employed in this study, four spiking experiments were completed to compare the TFF Method previously described for North Shore Beach seawater to widely accepted Public Health Method (58). Over the last 10 years, the TFF Method has been dev eloped to concentrate viruses from seawater. These methods include the use of TFF filt ers and subsequent purification and concentration steps (e.g. density-dependent centrif ugation and PEG precipitation). The TFF Method was modified for the comparison experime nts to a starting volume of seawater of 15 liters. The Public Health Method us ed in this experiment were a modified version of the Katayama et al. method and consisted of the following steps (39). Acetic acid (10%) was added to 1 liter of seawater until the sample was acidified to a pH between 3.5 and 4.0. Following acidification, the sample was filtered through a type HA, negatively charged, 0.47 mm diameter, 0.45 m m pore size membrane (Millipore, Billerica, MA). One hundred milliliters of H2SO4 (0.5mM) was filtered through the membrane and viruses were eluted using 10 ml of NaO H (1mM). The viral eluate was collected in a container with 100 m l of H2SO4 (50mM) and 100 m l of 100X Tris-EDTA (TE) buffer to neutralize the viral concentrate. Vi ral concentrates were stored at -20oC until further concentrated using a centrifugal conc entration device (Centriplus YM-50; Millipore, Billerica, MA, USA) to a volume less tha n 2 ml. For both methods, nucleic

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26 acid was extracted from 200 m l of viral concentrate using the MinElute Virus Spi n Kit (QIAGEN , Valencia, CA, USA). To detect RNA viruse s, cDNA was synthesized immediately after nucleic acid extraction using ran dom hexamers in the First Strand Synthesis Superscript III Reverse Transcription Kit (Invitrogen Carlsbad, CA, USA). A total of three spiking experiments were executed to assess the concentration efficiency of the TFF and Public Health Methods. Th ree experiments were executed to determine the methods’ efficiency at concentrating different types of viruses (DNA and RNA) as well as the methods’ ability to separate vi ruses potentially attached to particles. For the first experiment, the effectiveness of each method was tested by initially spiking water samples with a final concentration of 6.14Ex1 02 poliovirus particles per milliliter of seawater. Nested PCR, with a reported sensitivit y of about 4 viral particles, was executed using the primers in Table 5 on cDNA synth esized from each method (39). The sensitivity of this assay was confirmed by calculat ing the number of Poliovirus particles cDNA was synthesized from, serially diluting the po liovirus cDNA to 10-5 (~ 4 poliovirus particles), and completing the nested PCR reaction on each dilution. To roughly determine the efficiency of each method, the cDNA from each method was serially diluted to 10-6 and nested PCR was also carried out on each diluti on. For the first round of PCR, the total reaction volume was 2 5 m l and contained 3 m l of cDNA, 2X Promega PCR Master Mix (3 mM MgCl2, 50 U/ml TaqDNA Polymerase pH 8.5, 0.40 mM each dNTP; Promega , Madison, WI, USA), 1 m M JP UP primer, and 1 m M PAN ENT DWN primer. The first round PCR reaction underw ent 40 cycles of [95 oC for 30 s, 57.7 oC for 30 s, and 72 oC for 45 s] and an extension of 72 oC for 5 min. The second round of PCR, 25 m l, contained 1 m l of the first round PCR product, 1X REDTaq PCR

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27 Reaction Buffer (10.0 mM Tris-HCl pH 8.3, 50.0 mM K Cl, 1.1 mM MgCl2, 0.01% gelatin; Sigma-Aldrich St. Louis, MO, USA), an ad ditional 1 mM MgCl2, 1 m M PAN ENT UP primer, 1 m M JP DWN primer, 0.25 mM each dNTP, and 1 U REDTaq DNA Polymerase (Sigma-Aldrich St. Louis, MO, USA). Th e second round PCR reaction underwent 40 cycles of [95 oC for 30 s, 56.5 oC for 30 s, and 72 oC for 30 s] and an extension of 72 oC for 5 min. TABLE 5. Primer nucleotide sequences used for PCR a mplification of poliovirus (38). Primer NamePrimer Sequence 5' 3'NestedTarget Gene Amplicon JP UPTTA AAA CAG CTC TGG GGT TG PAN ENT DWNCTA ACC GGT AGG CCA PAN ENT UPCCT CCG GCC CCT GAA TG JP DWNCCG ACG AAT ACC ACT GTT A Outside Inside 600 bp154 bp untranslated region The second method efficiency comparison was complet ed following the same protocol as previously described except seawater sa mples were spiked with 4.24x104 Adenovirus-20 particles per milliliter of seawater. Nested PCR was used to identify the presence of these viruses in the extracted DNA and the 1:10 serial dilutions of this DNA for each method using previously published conditio ns and primers in Table 3 (3). The day before the third trial, 500 ml of seawater was spiked with Adenovirus-20. Following incubation overnight at room temperature while hori zontally shaking, the third trial was executed as described for the second trial. This la st trial was executed to understand if the two methods’ concentrating efficiency changed after allowing the viruses to attach to particles overnight. Determination of Viral Stability in Seawater The stability of PMMoV and picobirnaviruses in seaw ater was approximated by spiking 950 ml of seawater with 50 ml of raw sewage in a 1 liter clear plastic bottle. Two

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28 sets of experiments were executed, beginning on 6 F ebruary 2008 and 19 March 2008. For both experiments, ten milliliter samples were c ollected prior to spiking as a control (Tc). Before this bottle was placed off the seawall in Bayboro Harbor (Saint Petersburg, Florida), the sewage spiked seawater was mixed for 10 minutes and an initial 10 ml sample (To) was collected. In the first experiment, samples w ere subsequently taken on days 1 (T1), 2 (T2), 3 (T3), 4 (T4), 5 (T5), 8 (T8), and 39 (T39). During this time period, the seawater surface temperature fluctuated between 20 C and 24 C (NOAA CO-OPS station 8726520). The second experiment measured th e stability of the viruses over a similar period of time but samples were collected o n days 4 (T4), 7 (T7), 14 (T14), 21 (T21), and 28 (T28). The seawater surface temperature varied between 22 C and 27 C during this time (NOAA CO-OPS station 8726520). Nucleic acid from the viruses in each 10 ml sample was isolated the same day they were collected using the methods previously outline d for wastewater. cDNA was synthesized from the extracted RNA using First Stra nd Synthesis Superscript III Reverse Transcription Kit (Invitrogen Carlsbad, CA, USA) with random hexamers. The presence of PMMoV and Picobirnaviruses was determin ed through PCR using the methods previously described for identification in raw sewage. In the presence of PCR products of many sizes, the identity of key positiv e PCR products (control and last day seen) were confirmed through purifying PCR products and sequencing as previously described.

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29 RESULTS Viral Detection in Raw Sewage Raw sewage samples were collected from 12 wastewate r treatment facilities located in 11 different states. The nucleic acid of viruse s from 10 ml of raw sewage was isolated and analyzed for the presence of ten types of virus es (Table 6). Three types of viruses were found in 100% of the raw sewage samples: adeno viruses, picobirnaviruses, and PMMoV. Noroviruses were detected in 7 out of 12 sam ples. After whole genome amplification, papillomaviruses were identified in 2 out of 12 samples. Herpesviruses, reoviruses, rotaviruses, morbilliviruses, and hepad naviruses were not detected in any of the raw sewage samples; however, it is possible tha t they were present in concentrations below the detection limit of their assays. TABLE 6. Summary table of the targeted viruses dete cted in 12 raw sewage samples collected from throughout the coastal United States (two samples c ame from Florida). Positive identities to known targeted viruses in GenBank were those with a BLAST N hit with an E value 0.001. All positive identifications are from original nucleic acid samp les unless otherwise marked. Two asterisks illustra te that the positive identifications were from GenomiPhi am plified samples. Viral GroupRaw Sewage Adenoviruses 100% (12/12) Noroviruses & Sapporoviruses 58.3% (7/12) Hepadnaviruses 0% Herpesviruses 0% Morbilliviruses 0% Papillomaviruses ** 16.7% (2/12) Picobirnaviruses 100% (12/12) PMMoV100% (12/12) Reoviruses 0% Rotaviruses 0%

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30 Viral Detection in Final Effluent Final effluent samples were collected on the same d ay of raw sewage collection from 11 states throughout the U.S. Viruses in 10 ml of final effluent were concentrated and their nucleic acid was extracted to identify th e ten viruses under study. The results of the PCR analyses are listed in Table 7. PMMoV was d etected in all but one final effluent sample. In addition to detecting PMMoV, four other viruses were found. Picobirnaviruses was identified in a third of the final effluent sam ples, including one sample that underwent whole genome amplification prior to PCR. Three of the 12 final effluent samples were positive for adenoviruses. Finally, no roviruses and reoviruses (after whole genome amplification) were identified in only one o f the final effluent samples. TABLE 7. Summary table of the targeted viruses dete cted in 12 final effluent samples collected from 11 different coastal states. Positive identities to k nown targeted viruses in GenBank were those with a BLASTN hit with an E value 0.001. All positive identifications are from orig inal nucleic acid samples unless otherwise marked. An asterisk denotes a posi tive identification from a GenomiPhi amplified samp le. Viral GroupFinal Effluent Adenoviruses 25% (3/12) Noroviruses & Sapporoviruses 8.3% (1/12) Hepadnaviruses 0% Herpesviruses 0% Morbilliviruses 0% Papillomaviruses 0% Picobirnaviruses *33.3% (4/12) PMMoV91.7% (11/12) Reoviruses *8.3% (1/12) Rotaviruses 0% PCR Assay Sensitivity The sensitivity of the assays used to identify the viruses under investigation ranged from 1 target to 100,000 targets and was ide ntical to those previously reported (Table 8). According to the manufacturer, whole gen ome amplification increases the total amount of DNA or cDNA prior to PCR amplification up to 600-fold (GenomiPhi V2, GE

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31 Healthcare, Piscataway, NJ, USA; reviewed by (14)); thus, the sensitivity of each assay was approximately increased 100fold. TABLE 8. The sensitivities of each PCR assay employ ed in this study, which is identical to previous studies. 3 Human Adenoviruses, A F 100 1 57 Noroviruses & Sapporoviruses 10000 100 60Hepatitis B Viruses10000 100 12Herpesviruses10 1 40Papillomaviruses100 1 11Morbilliviruses10000 100 61Mammal Reoviruses1 1 43 Rotaviruses, Group A 100000 1000 98PMMoV100 1 83 Human Picobirnaviruses 1000 10 Detection Limit (targets) GenomiPhi Adjusted Detection Limit (targets) Primer ReferenceTarget Virus Wastewater Viral Concentration Efficiency The efficiency of the centrifugal concentration dev ices used to concentrate raw sewage and final effluent were evaluated by spiking autoclaved samples with a final concentration of 8.48x105 adenovirus particles/ml. Both the autoclaved un-sp iked raw sewage and final effluent samples were run in paral lel to the spiked samples and the lack of PCR product ensured the elimination of pre-exist ing adenovirus particles (far right well in Figure 2 & Figure 3). This methods experime nt produced identical results for raw sewage and final effluent. Assuming near 100% recov ery of adenovirus DNA from the nucleic acid extraction kit, a 10-3 dilution (equivalent to ~100 viruses) of the origin al

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32 DNA should have resulted in an amplicon. For both s amples, a dilution of 10-3 was the most diluted DNA sample to produce a PCR product (F igure 2 & Figure 3), which suggests that the methods used to concentrate waste water samples were successful in concentrating viruses. FIGURE 2. Agarose gel electrophoresis (2%) of adeno virus PCR products from the raw sewage spiking experiment. The numbers in each lane indicate the extent of DNA dilution prior to PCR amplification. ‘C’ represents the spiking experiment control. This con trol was autoclaved raw sewage. FIGURE 3. Agarose gel electrophoresis (2%) of adeno virus PCR products from the final effluent spiking experiment. The numbers in each lane indicate the extent of DNA dilution prior to PCR amplification. ‘C’ represents the spiking experiment control. This con trol was autoclaved raw sewage.

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33 Diversity of Picobirnaviruses in Raw Sewage Since picobirnaviruses were found in all U.S. raw s ewage samples and limited knowledge exists on this virus, the diversity of pi cobirnaviruses in raw sewage was analyzed. A total of 288 (~ 22 per location) cloned picobirnavirus PCR products of the RNA-dependent RNA polymerase gene were sequenced by Agencourt (Beverly, MA, USA) and 72% of these sequences had significant ide ntities (E-value 0.001 and identities over 50 bp) to known picobirnaviruses in GenBank. Those sequences without significant BLASTN hits also did not show significa nt TBLASTX similarities to GenBank, and were therefore not used in subsequent analyses. The significant BLASTN sequence identities ranged from 83% to 100% over re gions ranging from 56 bp to 194 bp to known human and porcine picobirnaviruses. The sequences from GenBank with the greatest identity to the sequences from raw sewage were extracted for alignment purposes (Table 9).

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34 TABLE 9. The top BLASTN hits of cloned picobirnavir us PCR products from U.S. raw sewage samples. These sequences were included in the alignment of r aw sewage sequences as a means of understanding the identity and relatedness of picobirnaviruses presen t in raw sewage. Those sequences highlighted in gr ay were too short to include in the alignment. LetterGenBank Accession NumberDescription Adbj|AB186898.1|Human Bdbj|AB193349.1|Human Cemb|AJ504794.1|HPI504794Human strain 1-HUN-01 Demb|AM419115.1|Human isolate castellon-3880 Eemb|AM706365.1|Porcine strain c10 clone 6 Femb|AM706366.1|Porcine strain c10 clone 14 Gemb|AM706367.1|Porcine strain D4 clone 1Hemb|AM706368.1|Porcine strain D4 clone 2 Iemb|AM706374.2|Porcine strain D6 clone 1 Jemb|AM706379.1|Porcine strain D6 clone 10 Kemb|AM706380.1|Porcine strain D6 clone 11 L emb|AM706397.1|Porcine strain E4 clone 14 Memb|AM706399.1|Porcine strain E4 clone 30 Ngb|AF245701.1|AF245701Human strain 2-GA-91Ogb|AF246612.1|AF246612 Human strain 1-GA-91 Pgb|AF246935.1|AF246935Human strain 202-FL-97 Qgb|AF246936.1|AF246936Human strain 203-FL-97 Rgb|AY805390.1|Human strain 745-ARG-99 Sgb|EU104359.1|Porcine strain 1a Tgb|EU104360.1| Porcine strain 2 Ugb|EU104362.1|Porcine strain 4 Prior to alignment, a total of 70 groups were crea ted from the 207 raw sewage picobirnavirus sequences by FastGroup II (97) set t o group sequences with 99% identity with gaps. While the majority of groups were compos ed of one or two sequences, seven groups contained 3 sequences or more. The de-replic ated sequences along with the top BLASTN hits (Table 9) were aligned in ClustalX v.1. 8 (89) and phylogenetic analyses were carried out in MEGA v. 4 (88). Given an averag e pair wise Jukes-Cantor distance of 0.356, a neighbor joining tree was created using th e Jukes-Cantor model with bootstrap replication of 2000. The final phylogenetic tree wi th insignificant (bootstrap value < 50) branches condensed is displayed in Figure 4.

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35 FIGURE 4. A condensed, neighbor joining (Jukes-Cant or model) phylogenetic tree of a ~200 bp segment of the picobirnavirus RNA-dependent RNA polymerase gene from 207 U.S. raw sewage sequences. Each branch represents a sequence or a group of sequence s (99% identical with gaps) depending upon the bloc k color. Every colored block is numbered and each nu mber corresponds to the adjacent table. This table explains the locations represented by each block. E ach letter represents a reference sequence from GenBank (Table 9). The pink pig next to a letter re presents porcine sequences. Letters without a pig indicate that the sequence is from a human.

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36 Three main points can be interpreted from the conde nsed phylogenetic tree and table in Figure 4. First, the high number of single sequences (blue blocks) suggest that the RNA-dependent RNA polymerase of picobirnaviruses fr om raw sewage has a great deal of sequence diversity. Furthermore, it suggests tha t more cloned PCR products would need to be sequenced to fully understand the divers ity of picobirnaviruses. The need for further sequencing is further supported by the calc ulated Chao1 value and the rarefaction curve created in FastGroup II (97). Chao1 is a mini mum estimator of the number of unique sequences (i.e. richness) (24, 25). Chao1 pr edicted a minimum of 200 unique picobirnaviruses in U.S. raw sewage but only 70 wer e sampled in this study. This suggests that more clones need to be sequenced in o rder to describe adequately the diversity of picobirnaviruses in U.S. raw sewage. The rarefaction analyses executed in FastGroupII fu rther support the need to sequence more clones to gain a complete view of pic obirnavirus sequence diversity in U.S. raw sewage (97). The rarefaction analyses plot the number of unique sequences versus the number of clones sequenced (Figure 5) (4 9, 53). Sufficient clones have been sequenced when the curve reaches an asymptote. Sinc e it is unclear where the curve would asymptote, more clones will need to be sequen ced in order to ensure a complete analysis of picobirnavirus diversity in U.S. raw se wage.

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37 FIGURE 5. Rarefaction analyses of the picobirnaviru s sequences from U.S. raw sewage. Some of the picobirnavirus sequences from raw sewag e had the greatest identities to porcine picobirnaviruses in GenBank (Table 9) an d this grouping is illustrated in the condensed phylogenetic tree. It is also interesting to note that the picobirnavirus sequences from raw sewage do not seem to have any s ort of geographic distribution based upon visual analysis. The blocks on the phylo genetic tree not colored blue show that identical (or >99% identical) sequences were r ecovered from multiple states. For example, 8 out of 10 sequences from North Carolina were unique (or <99% identical). Viral Isolation and Detection in Large-Scale Seawat er Samples Viral concentrates from all large-scale seawater sa mples were collected and purified using a combination of TFF filtration, den sity-dependent centrifugation in CsCl, and PEG precipitation. The combination of these pro tocols effectively concentrated viruses 10,000 fold from seawater samples (Table 10 ). Despite successful viral

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38 concentration, some viruses were lost in the PEG su pernatant and CsCl Other fractions (Table 10). The amount of viruses lost is approxima tely equivalent to concentrating 100% of viruses in 2.5 L of seawater. Although viru ses were lost during the concentration and purification processes, the results of the meth ods comparison experiments illustrate the effectiveness of the TFF Method at concentratin g viruses. TABLE 10. The concentration of viruses (viral-like particles/milliliter) at each step of the concentra tion and purification process for each seawater sample. The sample from which nucleic acid was extracted fr om is displayed in bold text. Lower MatacumbeLooe Key Dry TortugasNorth Shore Bea ch Original4.84E+073.06E+063.06E+069.72E+07 TFF Concentrate1.70E+105.75E+099.56E+082.24E+10 CsCl Concentrate1.93E+101.07E+105.62E+09 1.72E+11 CsCl Other5.20E+092.96E+081.37E+087.09E+09 PEG Concentrate 2.21E+111.18E+10 3.48E+092.47E+11 PEG Supernatant2.11E+085.20E+061.53E+084.81E+08 Centriplus Concentrate 5.19E+10 TFF Method Step Environmental Sample, Concentration VLP/ml All environmental samples were tested for the prese nce of PCR inhibitors and no significant inhibition was observed. None of the vi ruses under investigation were detected by PCR in any of the large-scale seawater samples, regardless of their proximity to dense human populations. Therefore, the viruses found in raw sewage (adenoviruses, noroviruses, picobirnaviruses, and PMMoV) were eith er absent in the marine environment or present at concentrations below the detection limit of the assays at the time of sampling. The presence of these viruses in raw sewage coupled with their absence in the marine environment suggests that they would be good candidates for fecal pollution indicators.

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39 Seawater Viral Isolation Methods Comparison Two methods, TFF and Public Health, for viral conce ntration were evaluated by spiking seawater samples with known concentrations of viral particles. Although three spiking experiments were executed using two differe nt types of viruses, the results of all three experiments concluded that the TFF Method was at least 10 times more effective at concentrating viruses than the Public Health Method Despite losing viruses in each step of the viral concentration and purification method (Table 11), the TFF Method was able to detect polioviruses better than the Public Healt h Method (Figure 6). Similar results were noted in the adenovirus spiking experiments (F igure 7), including the third trial that involved the incubation of adenovirus particles ove rnight (Figure 8). These experiments prove that the TFF Method effectively concentrates DNA and RNA viruses. Furthermore, the results of the third study indicate that the TF F Method can concentrate eukaryotic viruses even after they are allowed to attach to pa rticles. The sensitivities of the nested PCR assays for poliovirus and adenovirus were equiv alent to previous studies at ~ 4 virus particles and ~100 virus particles respectively (3, 38, 39). TABLE 11. Direct counts of virus-like particles (VL P) per milliliter of seawater for each step in the TFF Method’s concentration and purification protocol wi th samples from the three spiking experiments. Viruses were concentrated from seawater using TFF a nd PEG precipitation. The PEG Pellet was further purified by extracting the ‘viral’ fraction of the cesium chloride gradient after centrifugation. Viru ses in the PEG supernatant and the CsCl Other fraction were lo st. Nucleic acid was extracted from the CsCl Viral step highlighted in bold text. Spiked with Poliovirus Spiked with Adenovirus 20 Spiked with Adenovirus 20 (incubated overnight) VLP per ml VLP per ml VLP per ml Original Seawater 7.00E+06 1.22E+07 9.76E+06 TFF Viral Concentrate5.63E+094.49E+096.36E+09 PEG Pellet3.50E+102.39E+103.10E+10 PEG Supernatant6.78E+088.90E+082.68E+08 CsCl Viral1.34E+113.50E+108.04E+10CsCl Other4.31E+094.47E+082.30E+09 Spiking Experiment Trials TFF Method Steps

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40 FIGURE 6. Agarose gel electrophoresis (2%) showing the effectiveness of the TFF Method (lanes 2-8) and the Public Health Method (lanes 9-15) from the firs t spiking experiment using polioviruses. The number s in each lane indicate the extent of cDNA dilution prio r to PCR amplification. FIGURE 7. Agarose gel electrophoresis (2%) showing the effectiveness of the TFF Method (lanes 2-8) an d the Public Health Method (lanes 9-15) from the seco nd spiking experiment using adenoviruses. The numbers in each lane indicate the extent of DNA dil ution prior to PCR amplification.

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41 FIGURE 8. Agarose gel electrophoresis (2%) showing the effectiveness of the TFF Method (lanes 2-8) and the Public Health Method (lanes 9-15) from the thir d spiking experiment with an overnight incubation using adenoviruses. The numbers in each lane indica te the extent of DNA dilution prior to PCR amplification. Stability of Pepper Mild Mottle Virus and Picobirna virus in Seawater The stability of two viruses in raw sewage, PMMoV a nd picobirnaviruses, was estimated via a bottle experiment in which seawater was spiked with a known quantity of raw sewage. This bottle was placed into the water o ff the seawall and sampled intermittingly. Two trials of this basic experiment were executed and varied in their duration. The viruses were detected using PCR and g el electrophoresis. The results of these two experiments are displayed separately for picobirnaviruses (Figure 9) and PMMoV (Figure 10). The results of the picobirnaviru s stability experiments are difficult to interpret due to the non-specific amplification of nucleic acid from seawater samples (Figure 9). Based upon sequence confirmation, the f aint band observed in the un-spiked seawater (control) of the picobirnavirus experiment s is actually to the chloroplast of Ostrecoccus tauri Despite the non-specific binding of the primers a nd lack of sequence confirmation, picobirnaviruses from raw sewage clea rly remained stable in seawater no longer than 4 to 7 days.

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42 FIGURE 9. Agarose gel electrophoresis (2%) depictin g picobirnavirus PCR products (~200 bp) from two experiments assessing the stability of picobirnavir uses from raw sewage in seawater. It is easier to interpret the stability of PMMoV th an the stability of picobirnavirus due to the specificity of the primers (Figure 10). Although a light band appears in the control of experiments 1 and 2, the spiking of raw sewage is apparent in both experiments. PMMoV in raw sewage appears to be stab le in seawater for approximately a week before returning to background levels. Since o nly a single PCR product was produced, these results were not confirmed via sequ encing. FIGURE 10. Agarose gel electrophoresis (2%) of PMM oV PCR products from two experiments assessing the stability of PMMoV from raw sewage in seawater.

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43 DISCUSSION Viruses Detected in Raw Sewage The primary goal of this study was to identify poss ible viral indicators of fecal pollution for the marine environment. In order to a chieve this goal, the presence of ten viral groups, known to be shed in human feces, were analyzed via PCR in raw sewage collected from throughout the country. In summary, five different types of viruses were detected in one or more of the raw sewage samples. Noroviruses, previously found in European raw sewage (66, 91), was detected in less than 60% of the raw sewage samples; thus, their use as a marker of fecal pollution coul d potentially underestimate the extent of fecal contamination. Papillomaviruses were detected in 16.7% of raw sewage samples after whole genome amplification. Although their pr esence is interesting, papillomaviruses were found in too few samples to b e considered a potential indicator of fecal pollution. Three of these viruses, adenoviruses, picobirnaviru ses, and PMMoV, were found in 100% of the raw sewage samples. The results of this study support prior findings regarding the prevalence of adenoviruses in raw sew age (15, 42, 90, 92, 94). Furthermore, the use of adenoviruses to indicate fe cal contamination has been demonstrated by numerous studies in a variety of en vironments (1, 27, 32, 38, 55, 56, 64, 82). This study is the first of its kind to demonst rate widespread prevalence of picobirnaviruses and PMMoV in raw sewage, suggestin g that these viruses are good

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44 indicators of sewage. In addition, the detection of these viruses in the marine environment will suggest a recent contamination eve nt since neither were found in the marine environment in the absence of pollution. Interestingly, rotaviruses and reoviruses were not among those viruses detected in raw sewage samples even though their presence in ra w sewage has been documented in other countries (30) and they have been used in pri or fecal pollution studies (7, 8, 16, 21, 59, 72, 81). It is possible that these viruses were present at concentrations below the detection limit of their assays and/or that they we re not prevalent on the day of sampling. Alternatively, it is plausible that these viruses a re not abundant in the U.S. Further studies need to be completed in order to verify the absence of Reoviridae in U.S. raw sewage. Viruses Detected in Final Effluent It is important to note that the raw sewage and fin al effluent samples were collected at approximately the same time; therefore, the fina l effluents in this study do not necessarily represent the final product of the raw sewage analyzed. The wastewater treatment process for each of the wastewater treatm ent facilities is summarized in Table 12 to assist in analyzing the results of viral dete ction in final effluent. Five viral groups (adenoviruses, noroviruses, reoviruses, pepper mild mottle viruses, and picobirnaviruses) were found in 10 ml samples of final effluent. In g eneral, no correlation appears to exist between the viruses found in raw sewage and those f ound in the final effluent, regardless of wastewater treatment. Noroviruses were detected in final effluent from Lo uisiana while reoviruses were found in final effluent from New Jersey after whole genome amplification. Since noroviruses and reoviruses were detected in few or no raw sewage samples, it is not

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45 surprising that they were detected in a few final e ffluent samples. Adenoviruses were detected in the final effluent samples from Maine, Florida, and Oregon. The detection of adenoviruses in final effluent in this study suppor ts previous studies that have demonstrated the prevalence of adenoviruses in trea ted sewage (15, 27, 90, 92, 94). Picobirnaviruses were detected in a third of the fi nal effluent samples (Connecticut, Maine, Oregon, and Washington). It is important to note that the presence of chlorine has been shown to interfere with the detection of enter oviruses ( pers. com. Dale Griffin) and that residual chlorination in final effluent sample s could have interfered with the detection of viruses in this study. TABLE 12. Summary of wastewater treatment processes at each of the wastewater treatment facilities identified by location. FL K describes the Florida Keys. U.S. StatePrimary TreatmentSecondary TreatmentTerti ary Treatment ALgrit removal via screenaeration basin, 2o clarifierchlorination CAsedimentationactivated sludge systemgravity filte rs, chlorination CTsedimentationactivated sludge systemchlorination FLnoneaeration basin, 2 o clarifiersand filters, chlorination FL Kgrit removal via screenactivated sludge systemf abric filtration, UV, chlorination LAnoneactivated sludge systemfinal clarifiers, chlo rination MDsedimentationactivated sludge systemsand filters, chlorination MEsedimentationrotating biological contactorschlori nation NCgrit removal via screenactivated sludge systemdee p bed filter, chlorination NJsedimentationactivated sludge systemmultimedia fi lter, chlorination ORsedimentationactivated sludge systemchlorination WAsedimentationactivated sludge systemchlorination Of the three groups of viruses detected in 100% of the raw sewage samples, PMMoV was the only virus to be found in all final e ffluent samples except the Florida Keys. These viruses are known to be extremely stabl e and have been shown to survive food processing and passage through the gut (98). I t is reasonable to believe that PMMoV was not identified in the Florida Keys final efflue nt due to the extensive treatment process that includes fabric filtration, UV radiati on, and chlorination. Since PMMoV was

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46 detected in raw sewage and final effluent samples, its presence may not correlate with or predict the infectivity of the pathogens of concern in raw sewage. However, these viruses may be useful ultraconservative indicators of fecal contamination. Wastewater Viral Concentration Efficiency Control experiments demonstrated that the centrifug al concentration devices effectively recovered adenoviruses that were spiked into raw sewage and final effluent. No viruses were observed in the flow-through via ep ifluorescent microscopy and the expected dilution of final DNA resulted in amplicon via PCR. The use of a centrifugal concentration device is included in methods used by Public Health protocols (58). These experiments suggest that centrifugal concentration devices can be used to effectively concentrate enteric viruses from raw sewage and eff luent samples. Diversity of Picobirnaviruses in Raw Sewage Picobirnaviruses are currently an unclassified grou p of viruses even though they have been recently proposed to belong to the family Picobirnaviridae (10). As their name suggests, these viruses have a bisegmented double s tranded RNA genome (23, 83). Picobirnavirus particles are fairly small (35 nm), non-enveloped, and spherical. They have been found in the feces of a wide range of mam mals, including: humans (13, 83, 95), pigs (10), rats (80), chickens (63), calves (9 3), giant anteaters (47), rabbits (41, 67), guinea pigs (79), water foals (20), Choiques, Chine se geese, American ostriches, pelicans, donkeys, orangutans, armadillos, and gloo my pheasants (68). They are currently unculturable and their pathogenesis is unknown (23) These viruses have been implicated

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47 as possible enteric pathogens and have occasionally been associated with gastroenteritis (13, 23). Several important conclusions can be drawn from the phylogenetic analysis of the picobirnavirus sequences from raw sewage. First, i t appears that a complete understanding of the sequence diversity of picobirn aviruses remains unknown given the large number of single sequences. This is further s upported by the Chao1 and rarefaction analyses. The great diversity of genotype I human p icobirnaviruses, based on the RNAdependent RNA polymerase gene, have been previously reported (9, 13). Similarly, extensive diversity has been observed in genotype I porcine picobirnaviruses and their existence as quasispecies has been postulated (10). In order to fully grasp the sequence diversity of the RNA-dependent RNA polymerase of ge notype I human picobirnaviruses in raw sewage, it would be necessary to sequence mo re clones. It is also interesting to observe that the picobirn aviruses found in raw sewage group with both human and porcine picobirnaviruses. The strong sequence identity among human and porcine picobirnaviruses has been p reviously identified (10); however, Banyai et al. still observed separate groupings of human and porcine picobirnaviruses. While the majority of picobirnavirus sequences from raw sewage grouped with known human sequences, many raw sewage sequences grouped closely with porcine picobirnaviruses. The relatedness observed in this study among porcine and human genotype I picobirnaviruses suggests the inability of picobirnaviruses to source track fecal pollution based upon the sequence alone. Alth ough picobirnaviruses may not be suitable for identifying sources of fecal pollution they have the potential to be effective indicators of fecal pollution throughout the wastew ater treatment process as well as in

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48 coastal environments because of their widespread ab undance in raw sewage and their absence from unpolluted marine environments. Viral Isolation and Detection in Large-Scale Seawat er Samples None of the viruses that were detected in raw sewag e (human adenoviruses, noroviruses, human picobirnaviruses, and PMMoV) wer e found in any of the large-scale seawater samples, regardless of their proximity to dense human populations. Although it is possible that these viruses were present in conc entrations below the detection limit of the assays at the time of sampling, it is reasonabl e to believe their absence in the natural marine environment is due to a lack of hosts. This concept is supported by the lack of adenoviruses and other human DNA virus sequence sim ilarities to marine metagenomic sequences in the CAMERA (Community Cyberinfrastruct ure for Advanced Marine Microbial Ecology Research and Analysis) database ( 84). Since none of the viruses found in raw sewage were detected in the marine environme nt, the presence of any of these viruses (adenovirus, norovirus, picobirnavirus, and PMMoV) should identify a wastewater input. Seawater Viral Isolation Methods Comparison The TFF Method was developed for the concentration of marine bacteriophage; therefore, it is possible that they could have sele cted against the collection of the targeted eukaryotic viruses. Enteric viruses are relatively small and should have been included in the filtrate of the 0.2 m m TFF filter. However, they are known to become ass ociated with solids and could have been excluded from collection in the 0.2 m m TFF retentate (39). Additionally, it is possible that enteric viruses c ould have been lost through further

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49 concentration using PEG precipitation and CsCl dens ity-dependent centrifugation. While all of these are possible mechanisms of enteric vir al loss, the results of the methods comparison experiments suggest that the TFF Method was 10 to 1000-fold more effective than the commonly used Public Health Method at conc entrating polioviruses and adenoviruses. These results contest the status quo among environmental public health scientists who believe that TFF concentration is le ss effective than other commonly used methods (44). Despite demonstrating the usefulness of the TFF Method, it is expensive in terms of cost and time and is not recommended for r outine water quality monitoring. Stability of Pepper Mild Mottle Virus and Picobirna virus in Seawater The stability of PMMoV and picobirnavirus from raw sewage in seawater was ~1 week and less than a week, respectively. Unfortunat ely, the results of this study only approximate the stability of picobirnaviruses in se awater due to the non-specific binding of primers, the presence of light bands in the cont rol samples, the inability to sequence key PCR products, and the intermittent disappearanc e of positive PCR products. Before picobirnaviruses can be considered as a marker of f ecal pollution, it is essential that the specificity of the picobirnavirus primers be increa sed to reduce the amount of nonspecific binding observed in environmental samples. Similarly, intermittent PCR amplification made estimating the stability of PMMo V difficult. The appearance/disappearance of amplicon is likely the result of the patchiness of the seeded viruses. The stability of enteric adenoviruses has been prev iously compared to type 1 polioviruses and the hepatitis A viruses (32). Enri quez et al. found that enteric adenoviruses were considerably more stable than pol iovirus type 1 and hepatitis A

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50 viruses. Specifically, the T99 (the time it takes for 99% inactivation of the ori ginal titer), for adenovirus-40 and -41 is 77 days and 85 days in seawater, respectively (32). This is over four times longer than the 18 day T99 of poliovirus type 1 in seawater (32). Since the methods employed by Enriquez et al. are much more r obust, it is difficult to compare the stability of enteric adenoviruses in seawater to th e estimated stability of picobirnaviruses and PMMoV as outlined in this research. However, it appears that picobirnaviruses and PMMoV exhibit stability more similar to poliovirus type 1 in seawater. Limitations It is important to recognize a few shortcomings of this research. First, it is possible that any undetected or infrequently detected viruse s were present in concentrations at or below the detection limit of their assays. It is al so possible that these under-identified viruses were not prevalent on the day of sampling. Furthermore, it is important to emphasize that the results are based upon a relativ ely small volume of raw sewage, final effluent, or seawater. Additionally, the detection of any viral group in raw sewage or final effluent does not imply infectivity. This study is the first of its kind to illustrate d iversity of picobirnaviruses in raw sewage. It is important to recognize that the phylo genetic analyses in this study were dependent upon a short region of one gene. In order to obtain a clear picture of picobirnavirus phylogenetics, it would be necessary to analyze both the RNA-dependent RNA polymerase as well as the capsid protein. It is also important to remember that the primers utilized only amplify genotype I picobirnav iruses (13, 83). Furthermore, additional sequences need to be collected from huma n and animal picobirnaviruses to better understand the diversity of this viral group

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51 Potential of PMMoV and Picobirnaviruses as Water Qu ality Indicators In order to protect public health, it is necessary to identify a practical method for assessing fecal pollution in recreational waters. T his is often accomplished through the use of indicators, which are used to approximate th e presence of pathogens. The current bacterial indicators of fecal contamination are not good indicators of wastewater pollution or human health risk (19, 36, 45, 46, 62, 96). The results of this study suggest that picobirnaviruses and PMMoV may be utilized as indicators of fecal pollution since they were detected in all raw sewage samples collec ted in the United States. It is also clear that these viruses do not replicate in the ma rine environment since they were not detected in any of the seawater samples. Before pic obirnaviruses and PMMoV can be used as indicators to monitor water quality, it is important to determine several correlations to prove their utility as indicators o f fecal pollution. The characteristics of an ideal indicator are defin ed by the US EPA and include the following correlations (33). First, it would b e necessary to understand if the decay rate of the proposed indicator correlates with path ogens of concern throughout the wastewater treatment process and in the marine envi ronment. Pathogens of concern in raw sewage include viruses, bacteria, and protists. Future studies will need to determine if the presence of picobirnaviruses and PMMoV correlat es with non-viral pathogens of interest throughout the wastewater treatment proces s as well as in coastal environments. The results of this study indicate that picobirnavi ruses may correlate with pathogens of concern since they were detected in a third of final effluent samples and appear to remain stable in the marine environment f or less than a week. Given the presence of PMMoV in 91.7% of final effluent sample s and its estimated stability of one

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52 week in seawater, it is likely that this virus may overestimate the presence of sewage associated pathogens. It is possible that the utili ty of PMMoV is as an ultraconservative indicator of fecal pollution since it was found in most of the final effluent samples. While an ultraconservative indicator of fecal pollution c ould misjudge health risks, an ultraconservative indicator could indicate the poss ible presence of risk associated contaminants (i.e. pharmaceuticals, nutrients, etc) and thus has the potential to be a useful tool for environmental investigations. Another important correlation to determine is if th e proposed viral indicator concentrations correlate with the amount of fecal p ollution in the marine environment. Although it is difficult to measure the true extent of fecal pollution in the environment given the lack of correlation among the current ind icators and pathogens of interest, thorough analysis of a variety of contaminated envi ronmental sites will facilitate addressing this issue. This study has demonstrated the absence of PMMoV and picobirnaviruses from five marine sites that ranged in their proximity to human populations. It is interesting to note that PMMoV, but not picob irnaviruses, was detected using methods described for wastewater in one 20 ml Hills borough River sample collected in knee deep water adjacent to a sewage outfall pipe. This site was characterized to have poor water quality based upon current bacterial ind icators (sample & bacterial indicator data courtesy of the Harwood Laboratory, University of South Florida). Furthermore, PMMoV was detected in four out of seven 50 ml groun d water samples that contained enteroviruses (samples & enterovirus data courtesy of Dale Griffin, United States Geological Survey). These ground water samples were collected in rural areas, which rely

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53 on on-site disposal systems, outside of Tallahassee Florida. The detection of PMMoV in small volumes of water exposed to wastewater source s supports its potential use as an indicator. The most useful indicator of fecal pollution would also be able to distinguish human from animal sewage contamination. Since the m ajority of human fecal pollution comes from non-point sources, it is often difficult to identify the source of pollution. If an indicator is able to differentiate between human an d animal fecal contamination, then the ability to identify the source of pollution increas es. As a result, it is important to assess the potential of PMMoV and picobirnaviruses at sour ce tracking tools. The strong sequence identity between picobirnaviru ses from human sewage and GenBank reference sequences from pigs in this resea rch suggests the inability of picobirnaviruses to source track fecal pollution us ing sequencing alone. The lack of source tracking capability of picobirnaviruses usin g only the sequence is further supported by their prevalence in a wide range of ma mmals. Alternatively due to its dietary origin, PMMoV is not expected in animal fec es and to date has yet to be detected in animal feces ( pers. com. Karyna Rosario). The following collective fecal sa mples have tested negative for PMMoV: laughing gulls (n = 8) f rom the Suncoast Seabird Sanctuary in Pinellas County, Florida, bovine feces from a da iry farm in Puerto Rico, equine feces from a horse racetrack in Puerto Rico (samples cour tesy of the Rosario family), a rooster (n = 1), Grey Horned Owl (n = 1), Barred Owl (n = 1 ), Bald Eagle (n = 1), and Turkey Vultures (n = 2) from the Boyd Hill Nature Park (sa mples courtesy of Gabe Vargo) in St. Petersburg, Florida.

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54 CONCLUSIONS Adenoviruses, picobirnaviruses, and PMMoV were the only viruses detected in 100% of raw sewage samples collected from 11 U.S. c oastal states. Some of these viruses were also detected in a range of final effluent sam ples from the same wastewater treatment facilities but none of these viruses were detected in the marine environment in the absence of identifiable pollution. While adenov iruses are known human pathogens and have been proposed as markers of fecal pollutio n in previous studies, the results of this research demonstrate the potential use of pico birnaviruses and PMMoV as indicators of fecal pollution. Further research will be needed to determine if these candidate viruses have the necessary characteristics of a microbial w ater quality indicator.

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Symonds, Erin M.
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Viruses in raw sewage and their potential to indicate fecal pollution in coastal environments
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by Erin M. Symonds.
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[Tampa, Fla] :
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2008.
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Thesis (M.S.)--University of South Florida, 2008.
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ABSTRACT: The presence of pathogenic viruses in coastal environments is an important tool in evaluating water quality and health risks. Millions of viruses are excreted in fecal matter and bacterial indicators do not correlate with the presence of pathogenic viruses. Enteroviruses have been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter could be used to indicate fecal pollution. The purpose of this research is to develop a baseline understanding of the diversity of viruses found in raw sewage and to assess their presence in the marine environment. PCR was used to detect adenoviruses, herpesviruses, hepatitis B viruses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picobirnaviruses, reoviruses, rotaviruses, and sapporoviruses in raw sewage collected from throughout the United States and from five marine environments ranging in their proximity to dense human populations. Adenoviruses, noroviruses, pepper mild mottle viruses, and picobirnaviruses were detected in raw sewage but absent in the marine environment, making these viruses potential indicators of fecal pollution in marine environments. These viruses were also found in many of the final effluent samples. Pepper mild mottle viruses may be useful for source tracking fecal contamination since it was consistently found in human sewage and is not expected in the feces of other animals due to its dietary origin. Furthermore, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments.
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Advisor: Mya Breitbart, Ph.D.
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Water quality
Fecal-associated pathogenic viruses
Viral diversity
Microbial indicators
Picobirnaviruses
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Masters.
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